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Metoprolol and cardiac carnitine palmitoyltransferase-1 : unravelling a complex interaction in normal… Sharma, Vijay 2007

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( M E T O P R O L O L A N D C A R D I A C C A R N I T I N E P A L M I T O Y L T R A N S F E R A S E - 1 U N R A V E L L I N G A C O M P L E X I N T E R A C T I O N IN N O R M A L A N D D I A B E T I C H E A R T S by V I J A Y S H A R M A B S c (Hon), University of Edinburgh, 1998 M B C h B , University of Edinburgh, 2001 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Pharmaceut ical Sc iences ) T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A October 2007 © Vi jay Sha rma , 2007. 11 ABSTRACT Diabet ic cardiomyopathy may be initiated or compounded by the heavy rel iance of the heart on fatty ac ids and ketones as metabol ic fuels, p-blockers have been proposed to inhibit fatty acid oxidation by decreas ing the activity of the e n z y m e carnit ine palmitoyltransferase-1 (CPT-1 ) . By inhibiting fatty ac id oxidat ion, p-blockers could improve myocardial eff iciency and amel iorate the cytop lasmic accumulat ion of toxic fatty acid and g lucose intermediates. In this study, we investigated whether metoprolol improves card iac function and inhibits fatty ac id oxidation in the streptozotocin (STZ) diabetic rat, a mode l of poorly controlled type 1 diabetes. The animals were injected with 60 m g / kg S T Z and were euthanized six weeks following the induction of d iabetes. W e investigated the effects of chronic metoprolol treatment (75 mg/ kg/ day), and acute metoprolol perfusion on card iac function, substrate utilization and three major sys tems of C P T - 1 regulation: malonyl C o A levels, C P T - 1 transcription and covalent modif ications (phosphorylat ion, nitrosylation, glutathiolation, nitration). Chron ic metoprolol treatment improved card iac function in the diabet ic heart. W h e r e a s , chronic metoprolol treatment increased fatty acid oxidation in control hearts but dec reased it in diabetic hearts, acute metoprolol perfusion a lways inhibited fatty acid oxidation. Metoprolol lowered malonyl C o A levels in control hearts, and both acute metoprolol perfusion and chronic metoprolol treatment led to dec reased C P T - 1 max imum activity and dec reased C P T - 1 malonyl C o A sensitivity. C P T - 1 sensitivity was increased by ca lc ium/ ca lmodul in-dependent protein k inase phosphorylat ion and dec reased by protein k inase A -dependent phosphorylat ion in vitro. C P T - 1 activity w a s inhibited by nitrosylation and glutathiolation, and st imulated by nitration in vitro. Chron ic metoprolol treatment dec reased the binding and coactivation of peroxisome-prol i ferator receptor-y coactivator 1-a ( P G C - 1 a ) and peroxisome-prol i ferator receptor-a Ill ( P P A R - a ) , and a lso increased the binding of the repressor protein upstream stimulatory factor-2 (USF-2). In conc lus ion, metoprolol inhibited fatty acid oxidation, and acted partly by regulating malonyl C o A levels and partly by modulating the activity and malonyl C o A sensitivity of CPT -1 itself. The effects of metoprolol on CPT -1 were mediated acutely by covalent modif ications and chronical ly by inhibition of the transcriptional complex that induces CPT -1 express ion. TABLE OF CONTENTS P a g e Abstract » Tab le of Contents iv List of Tab les vii List of F igures ." viii List of S c h e m e s x List of Abbreviat ions xi Acknowledgments xiv Dedicat ion xvi I N T R O D U C T I O N 1 I: Diabet ic Card iomyopathy 1 II: Ca rd iac Metabol ism 8 III: Modulat ion of Card iac Metabol ism as a Therapeut ic Strategy 20 IV: The Benefi ts of p-Adrenergic B lockade 22 V : p-Adrenoceptor Signal l ing 24 VI: Potential L inks Between p-Adrenoceptors and Card iac Metabo l ism. 30 V l l : Spec i f ic R e s e a r c h Problem and Resea rch Strategy 33 V l l l : Work ing Hypotheses 35 IX: Objec t ives . . 36 M A T E R I A L S A N D M E T H O D S 39 I: Measurement of ex vivo Left Ventr icular Funct ion 39 (a) An ima l Treatments 39 (b) Measurement of P l a s m a Parameters 39 (c) Direct Measurement of Left Ventr icular Pressure 40 II: Measurement of ex wVo Card iac Metabol ism 40 (a) An ima l Treatments and Measurement of P l a s m a Parameters 40 (b) Measurement of Card iac Metabol ism 42 (c) Measuremen t of T issue G lycogen and Triglyceride Levels 43 (d) Measurement of T issue Malonyl C o A and Aden ine Nucleot ide Leve ls 44 (e) Measurement of T issue Nitrate/ Nitrite Levels 44 III: Measurement of K inase and Biochemica l E n z y m e Activit ies 44 (a) A M P K , P K A a n d C A M K Activit ies 44 (b) C P T - 1 A s s a y 45 (c) Acy l C o A Dehydrogenase A s s a y 46 (d) Citrate Syn thase A s s a y 47 IV: Immunoprecipitation and Measurement of Protein Express ion by Weste rn Blotting 48 (a) Overv iew of Exper imental Design 48 (b) S a m p l e Preparat ion 50 (c) S D S P A G E , Western Blotting and Dot Blotting 50 (d) Immunoprecipitation Protocol 51 (e) Dot Blotting 51 V : Funct ional Effects of C P T - 1 Covalent Modif icat ions in Isolated Mitochondr ia 52 (a) Isolation of Mitochondria 52 (b) K inase Phosphory lat ion of Isolated Mitochondria 53 (c) Peroxynitr i te D o s e - R e s p o n s e Curve in Isolated Mitochondria 54 (d) Measurement of C'PT-1 Activity, Phosphorylat ion and K inase Binding 54 VI: Identification of C P T - 1 Phosphorylat ion Si tes by L C M S / M S 54 VII: Data Ana lys is 55 R E S U L T S 56 I: Genera l Character is t ics. .'. 56 II: Funct ional and Metabol ic Effects of Chron ic Metoprolol Treatment 56 VI III: Malonyl C o A Leve ls 78 IV: C P T - 1 Activity and Malonyl C o A Sensitivity 78 V : Regulat ion of C P T - 1 Express ion 79 VI: p-Adrenoceptor Signal l ing 100 VII: C P T - 1 Covalent Modif icat ions 117 D I S C U S S I O N 137 I: Effects of Metoprolol on Card iac Funct ion and Metabol ism 137 II: C P T - 1 Activity and Regulat ion by Malonyl C o A 142 III: Regulat ion of C P T - 1 Express ion 145 IV: p-Adrenoceptor Signal l ing Pathways: Modulat ion of K inases a n d e N O S 149 V: ' N O / R N S - Induced Covalent Modif icat ions of C P T - 1 152 VI: Phosphory lat ion of C P T - 1 157 VII: S igni f icance of the Present Studies 160 VIII: Conc lus ions 164 B I B L I O G R A P H Y 168 v i i LIST OF TABLES Table P a g e 1. Genera l characterist ics and p lasma parameters at termination 57 2. Lactate production and t issue g lycogen, triglyceride and malonyl C o A levels following chronic metoprolol treatment 64 3. Myocard ia l energet ics and A M P K activity following chronic metoprolol treatment 65 4. G lyco lys is and fatty acid oxidation enzyme activities following chronic metoprolol treatment 67 5. Lactate production and t issue g lycogen, triglyceride and malonyl C o A levels following acute metoprolol perfusion 70 6. Myocard ia l energet ics and A M P K activity following acute metoprolol perfusion 71 7. T i ssue activities of P K A and C A M K 109 v i i i LIST OF FIGURES Figure P a g e 1. Mechan ica l performance of isolated perfused hearts: left ventricular pressure measurements 58 2. Mechan ica l performance of isolated perfused hearts: f low and rate-pressure product measurements 60 3. Effects of chronic in vivo metoprolol treatment on metabol ism of isolated perfused hearts 62 4. Acu te effects of metoprolol on metabol ism of isolated perfused hearts 68 5. A C C and M C D express ion and phosphorylat ion 72 6. C P T - 1 activity and malonyl C o A sensitivity 74 7. Pharmaco log ica l effects of metoprolol on C P T - 1 activity 76 8. Total C P T - 1 express ion 80 9. C P T - 1 B (Musc le Isoform) express ion 82 10. C P T - 1 A (Liver Isoform) express ion 84 11. P P A R - a , P G C 1 a , P D K - 4 express ion following chronic treatment with metoprolol 86 12. Densi tometr ic analysis of P P A R - a , P G C 1 a , P D K - 4 express ion following chronic treatment with metoprolol 88 13. U S F - 1 , U S F - 2 , M H C and S E R C A - 2 expression following chronic treatment with metoprolol 90 14. Densitometr ic analysis of U S F - 1 and U S F - 2 express ion following chronic treatment with metoprolol 92 15. Densi tometr ic analysis of M H C and S E R C A express ion following chronic treatment with metoprolol 94 16. Binding of P P A R - a , M E F - 2 A and U S F - 2 to P G C 1 a , and of M E F - 2 A and U S F - 2 to P P A R - a measured by immunoprecipitat ion 96 < ix 17. Densitometr ic analysis of P G C 1 a binding 98 18. Express ion of p-Adrenoceptor subtypes 101 19. Densitometr ic analysis of express ion of p-Adrenoceptor subtypes following chronic metoprolol treatment 103 20. Binding of G s and G i to p2-adrenoceptors 105 21. Akt Phosphory lat ion 107 22. Express ion of e N O S and i N O S 111 23. Phosphory lat ion of e N O S a t S e r 1177 and T h r 4 9 5 113 24. B iomarkers of N O and R N S 115 25. Cova lent modif ications of C P T - 1 measured by immunoprecipitat ion 118 26. Densitometr ic analysis of C P T - 1 covalent modif ications 120 27. Binding of P K A and A K A P - 1 4 9 to C P T - 1 , and phosphorylat ion state of A K A P - 1 4 9 122 28. Densitometr ic analysis of the binding of P K A and A K A P - 1 4 9 to C P T - 1 , and phosphorylat ion state of A K A P - 1 4 9 124 29. Binding of CAMK- I I to C P T - 1 126 30. Phosphory lat ion of C P T - 1 by P K A in isolated mitochondria 128 31. Phosphory lat ion of C P T - 1 by C A M K in isolated mitochondria 130 32. Phosphory lat ion of C P T - 1 by Akt in isolated mitochondria 132 33. Incubation of isolated mitochondria with peroxynitrite 134 X LIST OF SCHEMES S c h e m e P a g e 1. Mechan i sms involved in the pathogenesis of diabetic cardiomyopathy 10 2. Summary of fatty acid and g lucose metabol ism 14 3. Regulat ion of malonyl C o A levels. 18 4. p-adrenergic signal ing pathways 26 5. P roposed mechan ism of action of metoprolol 147 6. N O and RNS-med ia ted modif ications of thiol residues 154 7. Summary of the acute effects of metoprolol on malonyl C o A levels, C P T - 1 malonyl C o A sensitivity and C P T - 1 activity 166 LIST OF ABBREVIATIONS +dP/ dt Max imum Rate of Contract ion -dP/dt Max imum Rate of Relaxat ion A C C Acety l C o A Carboxy lase Acy l C o A Acy l C o E n z y m e A A D P Adenos ine diphosphate A G E A d v a n c e d Glycosylat ion Endproduct Akt Protein K inase B A M P Adenos ine monophosphate c A M P Cyc l i c A M P A N O V A Ana lys is of Var iance A M P K AMP-ac t i va ted protein k inase A N P Atrial Natriuretic Pept ide API Atmospher ic Pressure Ionization A T P Adenos ine tr iphosphate B H 4 Tetrahydrobiopterin B S A Bovine Serum Albumin C A M K Ca l c i um/ calmodul in dependent protein k inase C A P R I C O R N Carvedi lo l Post-Infarction Survival Control in Left Ventr icular Dysfunction Trial C A T Carnit ine Acyl t ransferase C D 3 6 Fatty acid t ranslocase C P T Carnit ine Palmitoyl transferase D C A Dichloroacetate DIGAMI Diabetes Insulin G lucose in Acu te Myocardia l Infarction Trial E D T A Ethylenediamine Tetraacet ic Ac id E G T A - Ethylene-glycol-bis(p-aminoethyl ether)tetraacetic Ac id E R G O - 1 Etomoxir for the Recovery of G lucose Oxidat ion Trial E R R Est rogen-Related Receptor F A C S Fatty Acy l C o A Synthase F A D H 2 F lavine Aden ide Dinucleotide F A B P Fatty Ac id Binding Protein L P L Lipoprotein L ipase G A P D H Glycera ldehyde-3-phosphate Dehydrogenase G L M - A N O V A Genera l linear model A N O V A G L P - 1 Glucagon- l ike Pept ide 1 c G M P Cyc l ic guanos ine monophosphate H E P E S 4-(2-hydroxyethyl)-1-piperazineethanesulfonic Ac id M H C Myos in Heavy Cha in H M G C o A p-hydroxy-p-methylglutaryl-CoA H P L C High-performance Liquid Chromatography IP Intraperitoneal IV Intravenous 3 - K A T 3-Ketoacyl Transferase L C M S M S Liquid Chromatography T a n d e m M a s s Spect roscopy L V D P Left Ventr icular Deve loped Pressure L V E D P Left Ventr icular End-Diasto l ic Pressure M C D Malonyl C o A Decarboxy lase M C T Monocarboxylate Transporter M E F - 2 A Myocyte enhancer factor-2A M E R I T - H F Metoprolol C R / X L Randomised Intervention Trial in Congest ive Heart Fai lure Trial M O P S 3-(N-Morphol ino)-propanesulfonic Ac id m/z M a s s to Charge Ratio N A D H Nicot inamide adenide dinucleotide N O Nitric Ox ide e N O S Endothel ia l Nitric Oxide Synthase i N O S Inducible Nitric Oxide Syn thase m t N O S Mitochondrial Nitric Oxide Synthase P D E Phosphod ies te rase P D H Pyruvate Dehydrogenase P D K P D H kinase P F K Phosphofructok inase P G C - 1 P P A R - y coactivator protein-1 P I3K Phosphat idyl inositol-3 kinase, P K A Protein K inase A P K C Protein K inase C P K G Protein K inase G P A R P Po ly (ADP-r ibose) Po lymerase P P A R Perox isome proliferator activated receptor R A G E Receptor for A G E R N A Ribonucle ic Ac id R N S React ive Nitrogen Spec ies R O S React ive Oxygen Spec ies R X R Ret inoic Ac id Receptor S E M Standard Error of the M e a n S E R C A Sarcop lasmic Ret iculum Ca lc ium A T P a s e S T Z Streptozotocin T C A cyc le Tricarboxyl ic Ac id Cyc le U S F Upstream Stimulatory Factor x i v ACKNOWLEDGMENTS First and foremost, I want to set down here my deep gratitude to Dr. John H. McNei l l , my supervisor, tutor and mentor for providing me with the opportunity to work with him over the past four years and for his unfailing encouragement , inspiration, thoughtfulness and care. I a lso wish to thank my superv isor Dr. Michae l Al lard for his advice, constant support and for his thoughtfulness and meticulous attention to my work carried out in his laboratory. I a lso owe thanks to all the members of my research committee, Dr. Roger Brownsey, Dr. Kather ine M a c L e o d , Dr. Brian Rodr igues and Dr. W a y n e Riggs for their invaluable input and constructive advice throughout the course of my P h D program. I am a lso indebted to Dr. Roger Brownsey for many stimulating and invaluable d iscuss ions , and to his technic ian, Jerzy Ku lpa, for carrying out the measurements of adenine dinucleot ides and C o A esters. S p a c e forbids me from a personal mention of many wonderful people both in the Facul ty of Pharmaceut ica l S c i e n c e s and at the iCapture center, but I must make an except ion of Ms . Violet Y u e n and thank her for her fr iendship and t ireless support over the years; I found her technical expert ise to be truly remarkable. I am a lso indebted to Mr. Richard Wambol t and Mrs. Hannah Pa rsons , two outstanding technic ians, for all the time they devoted to training and support ing me as well as for their fr iendship. I would a lso like to thank Pavan Dhil lon, a pharmacy student, who has worked diligently with me as my right-hand man and friend for three years. I have had the good fortune to work with many outstanding work study and summer students, Shah i leen Remtul la, L iza Tong , Dale Dhi l lon, Karen Win and Sherry W u , whose help and devotion to work I gratefully acknowledge. My thanks are due to Ms . Moira Greaven for her expert secretar ial ass is tance, and also to our former laboratory manager, M s . Mary Battel!, for her X V help and support. The support of the personnel in the animal care and purchase/ ordering facil it ies at U B C and St. Pau l ' s Hospital is greatly appreciated. I am grateful to Dr. Helen Burt and Mr. John Jackson for al lowing me to use their spectrophotometer to do enzyme kinetics, to Dr. Katherine Thompson and Dr. Chr is Orvig for the use of the infra-red spectrophotometer, and to Dr. J a s o n Dyck and Dr. Gary Lopaschuk at the University of Alberta for the generous gift of M C D ant ibodies. I a lso thank Dr. S u z a n n e Perry and Dr. Shouming He at the U B C Proteomics Co re Facility for carrying out the L C M S M S work. R e s e a r c h at this level is impossible without f inancial help and I wish to acknowledge the support I received from the Canad ian Institutes of Health R e s e a r c h (CIHR), the R x & D Health Foundat ion and the Canad ian Diabetes Assoc ia t ion with scholarsh ips over the course of my studies. I would like to mention my c lose friends Dr. S h o u m a Dutta, Dr. Helen Engdar and M s Barbara Buckinx for their many years of loyal fr iendship. Finally, I wish to thank my wonderful parents for their end less love and encouragement ; they have been the source of my inspiration and I love them dearly. x v i DEDICATION Dedicated to my prescient parents whose timely nudge led me to the study of medic ine and sc iences , and opened up for me a new and excit ing world to explore life's secrets in all their wonder and beauty. I pray: "Teach me, my G o d and King, In all things thee to see , A n d what I do in any thing, To do it for thee." Geo rge Herbert (1593-1662): T h e Elixir' A n d I know: " . . . exper ience is an arch wherethrough G l e a m s that untraveled world, whose margin fades For ever and ever when I move To strive, to seek, to find and not to yield." Alfred Lord Tennyson (1809-1892), 'U lysses ' 1 INTRODUCTION I. Diabetic Cardiomyopathy Card iovascu lar d i sease is the leading cause of death among diabetic patients, account ing for 8 0 % of all deaths in this group (1). Indeed, d iabetes is an independent risk factor for cardiovascular death, and mortality following myocardia l infarction is increased in diabetic patients (2-5). The most common cause of this card iovascular mortality is heart failure. The Framingham Heart study revealed that heart failure is twice as common in diabetic males and five t imes as common in diabetic females aged 45-74 years when compared to non-diabetic age-matched controls (6). Furthermore, according to the results of the U K prospect ive diabetes study, for each 1% increase in the H b A 1 C l the risk of heart failure increases by 1 5 % (7). Taken together, these data clearly establ ish that there is a link between diabetes and heart failure. The prognosis of heart failure in the context of diabetes is very poor. In the Diabetes Insulin G lucose in Acu te Myocard ia l Infarction (DIGAMI) study, a prospect ive study of first myocardia l infarctions in diabetic patients, 6 6 % of deaths in the first year following myocardia l infarction were caused by heart failure (8). Systo l ic dysfunction in diabetic patients carr ies an annual mortality of 15-20%. Three pathophysiological p rocesses account for d iabetes-associated heart failure: myocardia l i schemia, hypertension, and diabetic cardiomyopathy (the "cardiotoxic triad") (9). The first two components of this triad are not unique to diabetes, but are important pathophysiological p rocesses which diabetes can trigger, sustain and exacerbate. Diabet ic cardiomyopathy is a d isease process in which diabetes produces a direct and cont inuous myocardial insult even in the absence of ischemic, hypertensive or valvular d i sease (Scheme 1). It can act synergist ical ly with hypertension or ischemia to damage heart musc le , but can a lso cause heart failure in its own right. Diabetic cardiomyopathy was first descr ibed by Rubier et 2 al who reported four c a s e s of heart failure in normotensive diabetic patients with no ev idence of coronary artery d isease , valvular pathology or congenital heart d i sease (10). Exper imental ev idence for the ex is tence of this entity began to appear shortly thereafter, and epidemiological ev idence also suggested that an addit ional card iac insult was present in diabetic patients. However, the direct cl inical demonstrat ion of diabetic cardiomyopathy proved chal lenging until the 1990's, when a ser ies of studies using echocard iography and Doppler echocard iography documented ev idence of left ventricular hypertrophy and diastol ic dysfunct ion in both type 1 and type 2 diabetic patients independent of other risk factors (see (11) for review). Studies using Doppler ultrasound have revealed that the prevalence of diabetic cardiomyopathy is alarmingly high. Preva lence rates of >40% in young normotensive type 1 diabetic patients and 50-6 0 % in well-control led type 2 diabetic patients have been reported (12-15). In a recent case-contro l study, Bertoni et al reported that diabetes is independently assoc ia ted with the development of idiopathic cardiomyopathy (16). The clinical course of diabetic cardiomyopathy is long, and can be divided into three s tages (17). In the early stage, the cardiomyopathy presents with mild asymptomat ic diastolic dysfunction which is assoc ia ted with ultrastructural changes in t issue architecture, impaired calc ium handling, oxidative st ress and changes in card iac metabol ism. A s the d isease progresses, ev idence of left ventricular hypertrophy appears which is assoc ia ted with more severe diastol ic dysfunct ion and mild systol ic dysfunction. Card iomyocyte apoptosis and necros is , myocardia l f ibrosis, mild autonomic neuropathy and activation of the renin-angiotensin system appear at this stage. Finally, combined systol ic and diastol ic dysfunction occur which are assoc ia ted with card iac microvascular d isease , severe autonomic neuropathy and systemic sympathet ic nervous sys tem activation. This late stage is frequently assoc ia ted with hypertension and the onset of ischemia (17). The mechan isms underlying the process are poorly understood, but an overall picture is emerging. The sustained diabetic card iac insult appears to be produced by two major factors: hyperglycemia, a major 3 mediator of many diabetic compl icat ions, and a shift in energy substrate select ion by the heart (18). This d isease process impairs both pass ive and active mechan ica l properties of the myocard ium; the compl iance of the heart wall dec reases (due to increased cross-l inking of co l lagen, card iac hypertrophy and fibrosis (18; 19)), and contractility a lso decreases . Ca rd iac metabol ism in the diabetic heart differs from that in the non-diabetic hypertrophied and failing heart. It has been shown that, in the advanced s tages of d i sease , the hypertrophied and failing heart increases its rel iance on glycolysis and g lucose oxidation, although fatty acid oxidation remains the predominant fuel (20-22). The reverse is true in diabetic cardiomyopathy. Transport of g lucose into the cardiomyocyte is dependent on the Glut-4 transporter whose translocation, translation and transcription are all dec reased by d iabetes, and the constitutive Glut-1 transporter (18). The diabet ic heart is therefore less able to use g lucose as an energy source, and relies more heavily on alternative substrates such as fatty ac ids and ketones. P l a s m a fatty acid levels are increased. Fatty acid delivery to the heart from the coronary lumen by the enzyme lipoprotein l ipase (LPL) , and its subsequent uptake by fatty ac id transporters, is a lso increased (23). The heavy rel iance of the diabetic heart on fatty ac ids is harmful for two reasons: it dec reases myocardial efficiency, and it induces 'lipotoxicity'. Despi te the fact that fatty acid oxidation increases dramatically in the diabetic heart, the delivery of free fatty acids into the cardiomyocyte exceeds its capacity to metabol ise and store them. Intermediate products of fatty acid metabol ism, particularly long-chain acy l -coenzyme A ' s (Acyl CoA ' s ) , therefore accumulate in the cytoplasm (23). This effect could be exacerbated if ketone body utilisation is increased, as ketone bodies are compet ing substrates with fatty ac ids. Long chain a c y l - C o A ' s are converted into ceramides, toxic subs tances which induce reactive oxygen spec ies ( R O S ) and cardiomyocyte apoptosis (24). Interventions which select ively increase cytoplasmic fatty acid influx (overexpression of fatty 4 acid transport protein-1 or LPL ) or which select ively increase long-chain acyl C o A synthesis (overexpression of long chain acyl C o A synthetase) produce a card iac phenotype which is similar to diabetic cardiomyopathy (23). Taken together, these data provide convincing ev idence that lipotoxicity is a signif icant d i sease -inducing myocardia l injury. Fatty ac ids bind and activate perox isome proliferator-activated receptors ( P P A R ' s ) of which P P A R - a is a major isoform in the heart. P P A R - a is a transcription factor which acts as a 'lipostat', inducing genes involved in every step of fatty acid metabol ism (25). P P A R - a upregulates enzymes at every step of the fatty acid oxidation pathway, but it is the transcriptional control of mitochondrial long-chain acyl C o A uptake which has the greatest impact on the overal l fatty acid oxidation rate (26). W h e n P P A R - a is overexpressed, a phenotype similar to that seen in diabetic cardiomyopathy is induced. Furthermore, when P P A R - a is overexpressed in diabetic hearts, the phenotype produced by diabetes is worsened (23). Converse ly , deletion of P P A R - a is sufficient to protect diabetic hearts against the development of diabetic card iomyopathy (23; 27). P P A R - a activation is therefore essent ia l to the pathogenes is of diabetic cardiomyopathy. It is not clear, however, whether P P A R - a activation is a consequence or a cause of increased fatty acid oxidation. Overal l , the rel iance of the diabetic heart on fatty acid oxidation is harmful for three reasons: dec reased myocardial efficiency, lipotoxicity and P P A R - a -activation. Protein metabol ism is a lso affected by diabetes, an aspect which is rarely cons idered. Ribonucle ic acid (RNA) levels fall. Diabetes produces a marked artero-venous difference in branched chain amino acids across the myocard ium, suggest ing that protein catabol ism is increased. This would dec rease the availability of amino ac ids for translation. The combinat ion of increased protein breakdown and decreased protein synthesis would be expected to produce qualitative and quantitative dec reases in myocardial proteins (18). 5 Hyperg lycemia, coupled with the marked dec rease in g lucose oxidation, increases the formation of reactive oxygen spec ies ( R O S ) by severa l mechan isms (28). Pro longed exposure of proteins to hyperglycemia induces a ser ies of chemica l reactions which eventually lead to the irreversible formation of advanced-glycat ion end-products (AGE 's ) which act on their own receptors ( R A G E ) to increase the synthesis of diacylglycerol (29; 30). Increased de novo synthesis of diacylglycerol , either by increased flux of g lucose through the a ldose reductase/ polyol pathway or by R A G E activation, leads to activation of protein k inase C ( P K C ) isoforms and stress signal ing pathways (31; 32). Act ivat ion of these pathways increases mitochondrial R O S production, st imulates apoptosis and increases the transcription of pro-inflammatory and pro-fibrotic genes (28). In addit ion, flux of g lucose through the hexosamine biosynthetic pathway leads to O- l inked-N-acety lg lucosaminat ion and activation of transcription factors which also regulate pro-inflammatory and pro-fibrotic genes (33). Diabet ic cardiomyopathy is therefore assoc ia ted with widespread deleterious changes in fatty ac id , protein and g lucose metabol ism. However, because g lucose utilisation is dec reased in the diabetic heart, fatty acid oxidation may be a more important regulator of oxidative stress in this setting. In addit ion to the stress and P K C pathways activated by g lucose, G protein-mediated signal ing pathways (particularly Gj and G q ) activated by cel l -sur face receptors a lso contribute to the induction of pro-hypertrophic and pro-fibrotic genes (34). R h o A is a smal l molecular weight monomer ic G-protein which activates R h o K inase to induce card iac hypertrophy and f ibrosis. The R h o - A / Rho-k inase pathway is known to be activated by ai adrenoceptor st imulation, as well as endothel ium-derived vasoconstr ictors such as endothelin-1 and thromboxane A 2 . At the level of the cardiomyocyte, e lectromechanical coupl ing is impaired. Myocyte shortening and lengthening is s low because the action potential is 6 prolonged and calc ium efflux is too s low (35). Card iac contracti le protein adenos ine tr iphosphatase (ATPase ) is crucial for the generat ion of card iac force and is markedly dep ressed . In animal models, the so-cal led 'fetal gene program', which consis ts of re-expression of atrial natriuretic peptide (ANP) , p-myosin and a-act in, and decreased express ion of a-myos in and sarcop lasmic reticulum calc ium A T P a s e - 2 ( S E R C A - 2 ) in the ventricle, is induced. This shift in contracti le protein express ion from a card iac pattern to a skeletal musc le pattern is a lso observable when the pattern is a s s e s s e d functionally. The isomyosin pattern seen in the normal heart gives way to a s lower V 3 pattern in the diabetic heart (18). Total contracti le protein levels may also be dec reased because of the effects of d iabetes on protein synthesis and breakdown. A s diabetic cardiomyopathy progresses to symptomat ic heart failure, downregulat ion of myocyte-enhancer factor-2 (MEF-2) and its target genes occurs , and a more severe contracti le dysfunction ensues (36). The dec rease in S E R C A - 2 express ion seen as part of the fetal gene program, combined with a concomitant decrease in S E R C A - 2 function and N a + / C a 2 + exchanger express ion and function, disturbs cardiomyocyte calc ium handl ing. In the diabetic heart, the uptake, sarco lemmal binding and myofibrillar ca l c ium-ATPase-med ia ted intake activity are all dec reased . The calc ium sensitivity of myofi laments becomes abnormal ly high as a result of P K C activation, p-adrenergic respons iveness dec reases (28) and autonomic neuropathy dec reases sympathet ic nervous system input (18); as a result, the cardiomyocyte is cut off from systemic regulation of excitation-contraction coupl ing. A s the heart fails, the t issue renin-angiotensin sys tem, the sympathet ic nervous sys tem and other neuro-hormonal regulatory sys tems are st imulated in an effort to maintain systemic perfusion. Activation of these sys tems increases apoptos is and necrosis (via p i -adrenoceptors) (37-41), and f ibrosis (via c t r adrenoceptors) and R O S generat ion (through the t issue renin-angiotensin 7 system) (28; 42-44). This t ime-course is reflected by pathological f indings at autopsy and echocard iographic f indings. Left ventricular mass , th ickness and s ize are found to increase progressively as diabetic cardiomyopathy progresses from asymptomat ic diastol ic dysfunction to symptomatic systol ic dysfunct ion (17). The architecture of the myocardium is a lso progressively disturbed. Fibrosis is the most prominent histopathological finding in the diabetic heart and is diffuse. Per ivascu lar and interstitial f ibrosis appear during the intermediary s tages of the d i sease process. Interstitial f ibrosis dec reases card iac compl iance, but per ivascular f ibrosis has addit ional functional consequences because it breaks the connect ion between the endothel ium and the card iomyocytes, thereby impairing relaxation (45). Progress ion to systol ic dysfunction is assoc ia ted with myocyte cell death which induces replacement f ibrosis (17). Both apoptosis and necrosis are seen , but only necrotic cell death stimulates f ibrosis (17). Foca l microangiopathy is a lso present in the intermediary and advanced s tages of the d i sease and may contribute to the observed fibrosis; it is unlikely to be the so le cause of f ibrosis because , in diabetic cardiomyopathy, f ibrosis is diffuse, not focal (17). The ultrastructure of the cardiomyocyte is profoundly damaged ; cytoplasmic area is increased, with an increase in cytoplasmic lipid content (consistent with the p resence of lipotoxicity) and increased collagen-f ibre cross-sect ional area (consistent with increased col lagen cross-l inking). Disturbance of the ultrastructure disrupts mitochondria, impairing mitochondrial function and inducing oxidative stress (18). To summar ize , diabetic cardiomyopathy is initiated by hyperg lycemia and a shift in substrate select ion in favour of fatty ac ids. Both p rocesses increase oxidative stress and stimulate pro-apoptotic, pro-fibrotic and pro-inflammatory pathways. Necros is a lso occurs, stimulating further f ibrosis. Exci tat ion-contraction coupl ing is impaired by disordered calc ium handl ing, impaired A T P a s e activity and a shift in contractile protein express ion from a card iac to a skeletal musc le pattern. Excitation-contraction coupl ing is d isconnected from 8 autonomic regulation as a result of impaired (3-adrenergic respons iveness and autonomic neuropathy. T h e s e functional changes are accompan ied by extensive damage to the architecture of the myocard ium and the ultrastructure of the cardiomyocyte. The result is a d i sease process which can be initiated years before the appearance of hypertensive or ischemic d isease . Initially presenting as asymptomat ic diastolic dysfunction, the d isease progresses to combined symptomat ic systol ic and diastolic dysfunction, and the myocard ium is rendered more suscept ib le to damage from the other components of the cardiotoxic triad. Despi te recent advances in drug therapy for heart failure, this condit ion still carr ies a worse prognosis than most cancers , and is assoc ia ted with signif icant morbidity. Current therapies are directed at symptomat ic relief (diuretics, (3-adrenergic agonists, phosphodiesterase inhibitors) and attenuation of left ventricular remodell ing (angiotensin converting enzyme inhibitors, p-blockers, a ldosterone antagonists). Agents which restore the normal ba lance of card iac metabol ism could improve the mechan ica l efficiency of the myocard ium, and prevent the harmful seque lae of shifts in energy substrate select ion. This mechan ism has been proposed as a useful avenue to pursue in the identification of new drug targets for heart failure and may be particularly useful in diabetic cardiomyopathy (46-48). II. Cardiac Metabolism The heart requires a constant supply of adenos ine tr iphosphate (ATP) for muscu lar contract ion and the maintenance of ionic homeostas is (49). Under aerobic condit ions most of this A T P (>95%) is generated by mitochondrial oxidative phosphorylat ion. Oxidat ion of energy substrates is coupled to the reduction of nicot inamide adenine dinucleotide (NADH) and f lavoproteins. N A D H and f lavoproteins are then re-oxidised by oxygen, and the reducing equivalents they received are passed on to the electron transport chain which pumps protons out of the mitochondria; the result is the creation of an e lectrochemical gradient 9 consist ing of a t ransmembrane pH gradient and a membrane potential. The F i F o A T P a s e located on the inner mitochondrial membrane al lows protons to reenter the mitochondrial matrix down their concentration gradient, harness ing the electr ical potential energy of the gradient to generate A T P from A D P and Pj (50). The oxygen consumed is converted to carbon dioxide, and the reducing equivalents are eventually transferred to hydrogen and oxygen, forming water. A T P is exported in exchange for A D P by the adenine nucleotide transporter, and a cont inuous supply of Pi is maintained by the phosphate translocator. The mitochondria are precisely fixed between two T-tubules, and both A T P and its metabol ic intermediates are continuously channeled between the mitochondria, the myofibrils, the sarcop lasmic reticulum and the sa rco lemma. This exquisi te coupl ing system between oxidative phosphorylat ion and myofibril contraction al lows A T P demand to be precisely and instantly met over a wide range of work loads (see (51) for review). Al though functional coupl ing between the myofibrils and oxidative phosphorylat ion matches A T P production to A T P consumpt ion, further communicat ion is required to ensure that the supply of energy substrates responds to changes in A T P demand . This role is fulfilled by A M P activated protein k inase ( A M P K ) , which is activated by an increase in the A M P / A T P ratio (a s ignal of A T P depletion) and acts to deactivate A T P - c o n s u m i n g pathways (protein, triglyceride and glycogen synthesis) and activate ATP-p roduc ing pathways (protein, g lycogen and triglyceride catabol ism, g lucose and fatty acid uptake and oxidation) (52). The heart is an omnivorous organ which has the ability to use any energy substrate provided to it (lipids, carbohydrates, ketone bodies, amino acids); however, the normal heart der ives most of its A T P from the metabol ism of fatty ac ids and carbohydrates (53). Al though fatty acid oxidation produces more A T P , g lucose oxidation is more efficient in terms of oxygen consumpt ion. Severa l 10 SCHEME 1 M e c h a n i s m s involved in the pathogenesis of diabetic cardiomyopathy. The cardiomyopathy ar ises as a result of a decrease in heart musc le compl iance, produced by f ibrosis and increased col lagen cross- l inking, and a dec rease in contractility, produced as a result of w idespread changes in contractile signal ing pathways, card iac metabol ism and oxidative stress. Alterations in G-proteins, RhoA-Rho Kinase, PKC Myocyte Altered Cardiac Remodelling/ Apoptosis Gene Expression Hypertrophy ^ o t " " S , R h ^ \ / ft R O S •DECREASED CONTRACTILITY Metabolic 'Switch' Disordered calcium handling DIABETIC CARDIOMYOPATHY DECREASED COMPLIANCE 1T Collagen Cross Linking Myocardial Fibrosis 12 factors account for this. Firstly, to maintain a fixed A T P production rate, fatty acid oxidation requires greater oxygen consumpt ion. The A T P produced per unit oxygen is theoretically predicted to be 3.17 for g lucose but only 2.80 for palmitate (48). Second ly , fatty acids allow protons to leak across the mitochondrial membrane, uncoupl ing oxidative phosphorylat ion and wast ing oxygen (54). Indeed, fatty ac ids have recently been shown to activate mitochondrial uncoupl ing in models of type 2 diabetes (55; 56). Thirdly, fatty acids activate sarcb lemmal calc ium channels . Ca lc ium pumps must increase their activity, and therefore their A T P utilization, to compensate for the resulting increase in ca lc ium influx (57). For optimal card iac efficiency, the normal heart maintains a ba lance of 6 0 - 8 0 % fatty acid oxidation and 20 -40% pyruvate oxidation; the heart der ives approximately equal amounts of pyruvate from glycolysis and lactate metabol ism (58; 59). Ketone utilization is concentrat ion dependent; in situations where blood ketone levels rise (starvation, diabetes), ketones become a major card iac fuel (60; 61). The metabol ism of g lucose and fatty ac ids by the heart is summar ized in s c h e m e 2. G lucose-6-phosphate , the glycolytic substrate, is obtained from endogenous g lycogen stores and from exogenous g lucose taken up by the Glut-1 and Glut-4 g lucose transporters. G lucose uptake is regulated by Glut-4 ves ic le translocat ion, docking and fusion to the sarco lemma (stimulated by insulin and AMP-ac t i va ted protein kinase) and is responsible for the majority of g lucose uptake (59; 62; 63). Glyco lys is compr ises a ser ies of reactions which convert g lucose-6-phosphate to pyruvate. In the normal aerobic heart, the major fate of pyruvate is decarboxylat ion to acetyl C o A , cata lysed by the pyruvate dehydrogenase complex (PDH) in the mitochondria (64). Lactate is metabol ised to pyruvate, but pyruvate is a lso metabol ized to lactate. Under aerobic condit ions, the normal heart is a net consumer of lactate; the heart only b e c o m e s a net producer of lactate when the glycolytic flux exceeds the rate of pyruvate oxidation (59). A smal l proportion of pyruvate is metabol ized to either 13 oxaloacetate or malate in order to replenish tricarboxylic acid cyc le ( T C A cycle) intermediates, a process known as anaplerosis (65). Fatty ac ids are highly hydrophobic and are therefore transported as tr iglycerides in chylomicrons or very-low-density l ipoproteins (VLDL) , al though a smal l proportion are also transported bound to serum albumin. Fatty ac ids are re leased from chylomicrons and V L D L by lipoprotein l ipase (LPL) on the luminal sur face and are then taken up into the cardiomyocyte by fatty acid transporters such as C D 3 6 (66). Upon entry to the cytoplasm, fatty ac ids are bound by fatty acid binding protein ( F A B P ) until they are esterified to fatty acyl C o A by fatty acyl C o A synthetase ( F A C S ) . Fatty acyl C o A has two major fates: it can enter the mitochondria to be oxid ized, or it can be esterified to triglyceride and stored (24; 67). A smal l proportion of palmitoyl C o A ' s are a lso converted to ceramides (24). W h e r e a s short and medium-chain acyl C o A ' s can pass freely into the mitochondria to be oxidized, long-chain acyl C o A ' s cannot c ross the mitochondrial membrane and must be transported. This function is carr ied out by a carni t ine-dependent shuttle sys tem. In the first step, carnitine palmitoyltransferase 1 (CPT-1) converts acyl C o A to acyl-carnit ine. Th is is a major control step of overall fatty acid oxidation. Acyl-carni t ine is then transported to the mitochondrial matrix by carnitine acyl t ransferase (CAT) in exchange for free carnit ine. Finally, carnitine palmitoyltransferase 2 (CPT-2 ) reverses the initial reaction and regenerates acyl C o A (68). Acy l C o A enters the p-oxidation spiral, a ser ies of four reactions which c leaves two carbons (one acetyl C o A molecule) from the acyl C o A molecule per cycle and generates N A D H and FADH2. There are speci f ic enzymes for long-; medium- and short-chain acyl C o A ' s (69). Acety l C o A generated by carbohydrate or fatty acid oxidation (as well as from other pathways) enters the T C A cycle to generate additional reducing equivalents which drive oxidative phosphorylat ion. 14 SCHEME 2 Summary of fatty acid and g lucose metabol ism. G l u c o s e is taken up by Glut-1 and Glut-4 transporters and is converted by glycolysis to pyruvate. Pyruvate then enters the mitochondria to be oxidized, producing acetyl C o A . Fatty ac ids are l iberated from lipoproteins by L P L , and are taken up by C D 3 6 and F A B P . L C A S converts the fatty acid to a C o A ester which is then taken up by the carnit ine shuttle sys tem to the mitochondria. The fatty acyl C o A undergoes p-oxidation, removing two carbons per turn of the cycle and generating acetyl C o A . Acety l C o A , generated by either pathway, enters the T C A cycle to generate reducing equivalents (NADH) . T h e s e pass electrons to the electron transport chain which creates an electrochemical proton gradient to drive A T P synthesis. A T P synthesis is exquisitely coupled to the sys tems which create the A T P demand (abbreviat ions: L P L = lipoprotein l ipase, CD36= fatty acid t rans locase, F A B P = fatty acid binding protein, F A C S = fatty acyl C o A synthase, C P T = carnit ine palmitoyltransferase, C A T = carnitine acyl t ransferase, C o A = coenyme A , T C A cycle = tr icarboxylic acid cycle, A G E = advanced glycosylat ion endproduct, P D H = pyruvate dehydrogenase, M C T = monocarboxylate transporter, P D H = pyruvate dehydrogenase, N A D H = reduced nicotinamide adenide dinucleot ide, A T P = adenos ine tr iphosphate, A D P = adenos ine monophosphate ), Glucose [• Oxidation The Rail die Cycle - Fatty Acid ^ Oxidation Lactate 16 The rate of fatty acid oxidation is determined by the p lasma concentrat ion of fatty ac ids, the work performed by the heart, the entry of fatty ac ids into the cytoplasm, the entry of fatty ac ids into the mitochondrion, and the activity of the enzymes involved in the p-oxidation spiral (62; 70). High rates of fatty acid oxidation increase the ratios of N A D H / NAD+ and acetyl G o A / free C o A , both of which feed back and inhibit g lucose oxidation by decreas ing flux through P D H (62). High rates of fatty acid oxidation a lso increase citrate production. Citrate inhibits glycolysis by inhibiting the key glycolytic enzyme phosphofructokinase ( P F K ) . Th is is known as the Rand le cycle. Converse ly , high rates of g lycolysis and g lucose oxidation can feed back to inhibit fatty acid oxidation. At the level of C P T - 1 , fatty acid oxidation is controlled by malonyl C o A , a potent inhibitor which binds C P T - 1 on the cytosol ic s ide (68); by altering malonyl C o A levels in the cytosol ic microdomain adjacent to the site of fatty a c y l - C o A uptake, fatty acid oxidation can be controlled; tonic inhibition of C P T - 1 by malonyl C o A is a lways present, but a rise in malonyl C o A levels inhibits fatty acid oxidation while a fall rel ieves inhibition (71; 72). Two isoforms of C P T - 1 are expressed in the heart: C P T - 1 A (the isoform which predominates in the liver) and C P T - 1 B (the isoform which predominates in the heart). The sensitivity of C P T - 1 B to malonyl C o A is 30 t imes greater than that of C P T - 1 A (71; 72). The turnover of malonyl C o A in the heart is rapid. Malonyl C o A is synthesized from acetyl C o A by acetyl C o A carboxy lase ( A C C ) and is broken down to acetyl C o A by malonyl C o A decarboxy lase (MCD) (73; 74). The activity of A C C is inhibited by phosphorylat ion; the major k inase responsible is A M P K , although P K A mediates p-adrenoceptor induced A C C phosphorylat ion (73) . The mechan isms by which M C D is regulated are still unknown; it is possib le that M C D is activated by A M P K (75). Malonyl C o A levels can be increased either by stimulating A C C or by inhibiting M C D (Scheme 3). M C D is a transcriptional target of the perox isome proliferator activated receptor-a, whose actions are d iscussed below. 17 In the long term, regulation of the express ion of the enzymes , transporters and k inases that compr ise the metabol ic machinery enab les the heart to adapt more permanent ly when the stimulus to do so is susta ined. The mitochondria contain D N A which codes for 13 electron transport chain subunits (for comp lexes 1, III, IV, and V) , but all other components of the metabol ic machinery are coded for in the nuclear D N A (76). There are severa l important inter-related nuclear transcriptional regulators of metabol ism genes. The first are the perox isome proliferator activated receptors ( P P A R ) . P P A R s form heterodimers with retinoid X receptors ( R X R ) , and formation of an active P P A R / R X R complex requires binding of 9-c/s-retinoic acid to the R X R and binding of long chain fatty ac ids or an exogenous P P A R ligand to the P P A R . Upon activation, the complex t ranslocates to the nucleus and binds to P P A R response e lements ( P P R E s ) within the promoter regions of its target genes. In the heart, the major isoform is P P A R - a , and its target genes encompass the full pathway of fatty acid metabol ism from fatty acid uptake to the p-oxidation spiral, as well as pyruvate dehydrogenase kinase-4 (PDK-4), the major inhibitory k inase of P D H (77). The second important regulator is P P A R -y c o a c t i v a t o M a (PGC-1a). P G C -1a regulates the capacity of the cell to generate A T P so that, when A T P demand increases, the reserve of the metabol ic machinery can meet the demand (78; 79). To this end , the major role of PGC -1a in card iac musc le is to increase mitochondrial b iogenesis and the capacity of each mitochondrion for oxidative phosphorylat ion. PGC -1a binds and enhances the action of other transcription factors including P P A R - a (fatty acid oxidation control), myocyte-enhancer factor 2A (MEF-2A, contracti le machinery control) and orphan nuclear receptor estrogen-related receptor a (ERR - a , carbohydrate and fatty acid oxidation control, contracti le machinery control, oxidative phosphorylat ion genes) (80; 81). 18 S C H E M E 3 Regulation of malonyl CoA levels. Malonyl CoA is synthesised from cytosolic acetyl CoA by acetyl CoA carboxylase (ACC), and is broken down to acetyl CoA by malonyl CoA decarboxylase (MCD). MCD is regulated through transcription, but A C C undergoes acute regulation by inhibitory phosphorylation. P K A and A M P K both phosphorylate and inhibit A C C , decreasing malonyl CoA levels and relieving inhibition of C P T - 1 . This is the major mechanism by which CPT-1 is regulated in the heart (abbreviations: CPT-1 = carnitine palmitoyltransferase-^ PKA = protein kinase A, A M P K = adenosine monophosphate-activated protein kinase). Malonyl CoA Decarboxylase + n Acetyl CoA Malonyl CoA I f Acetyl CoA Carboxylase PKA AMPK ^ C P T - 1 FATTY ACID OXIDATION 20 111: Modulation of Cardiac Metabolism as a Therapeutic Strategy Inhibition of fatty acid uptake and oxidation, or stimulation of g lucose oxidation, would be expected to be beneficial to the diabetic heart for three reasons: improvement in myocardial efficiency, ameliorat ion of lipotoxicity and ameliorat ion of glucotoxicity. Most experimental and clinical data demonstrat ing the ef fect iveness of this therapeutic strategy pertain to non-diabetic heart failure, al though a smal l body of data a lso exists for diabetic animal models and patients. Intravenous infusion of dichloroacetate (DCA) , a drug which inhibits P D K , produces a rapid improvement in left ventricular performance in patients with heart failure (82). This suggests that dec reased flux through P D H contributes significantly to the cardiac dysfunction of heart failure. W e have previously shown that the administration of D C A also produces a marked improvement in left ventricular function in the diabetic heart (83). Increasing p lasma insulin levels can a lso st imulate g lucose oxidation both directly by increasing g lucose uptake, and indirectly by decreas ing fatty acid delivery to the heart, thereby relieving Rand le cyc le-mediated inhibition of g lucose oxidation (84). Infusion either of insulin in heart failure patients (85) or the insulinotropic peptide glucagon-l ike peptide 1 (GLP-1 ) in dogs with pacing- induced heart failure improved left ventricular function (86). In the Diabetes Mell itus Insul in-Glucose Infusion in Acu te Myocard ia l Infarction (DIGAMI) study, stimulation of g lucose oxidation reduced mortality following myocardial infarction in diabetic patients (87). Taken together, these data suggest that acute stimulation of g lucose oxidation may be beneficial to the failing heart, but more definitive clinical studies are required to confirm this. Furthermore, there are, at present, no pharmacological agents which act as chronic g lucose oxidation stimulators. A number of agents have been developed which inhibit fatty acid oxidat ion, thereby stimulating g lucose oxidation indirectly through the Rand le 21 cycle. Fatty acid oxidation inhibitors have been classif ied according to whether they produce reversible (partial fatty acid oxidation inhibitors) or irreversible inhibition. Etomoxir is an irreversible C P T - 1 inhibitor. It has been shown to improve card iac function in rats with left ventricular hypertrophy (88; 89) and diabetic cardiomyopathy (90-92). In experimental studies, etomoxir treatment improved S E R C A - 2 express ion and calc ium handling, indicating that C P T - 1 inhibition produces improvements in calc ium handling (89; 93). Inhibition of C P T -1 without concomitant inhibition of fatty acid delivery and uptake results in an accumulat ion of long-chain fatty acyl C o A ' s in the cytosol and activation of the P P A R - a / R X R complex. Rupp et al speculated that the increased S E R C A - 2 express ion induced by etomoxir is mediated by P P A R - a , noting that the regulatory region of the S E R C A - 2 gene contains a sequence similar to the P P R E (89). A smal l preliminary trial of etomoxir in patients demonstrated an improvement in left ventricular function (94). However, etomoxir produced an unacceptable rate of side-effects in the 'etomoxir for the recovery of g lucose oxidation' ( E R G O - 1 ) P h a s e II cl inical trial, and the trial was terminated early (95). Etomoxir has also been shown to induce mild myocardial hypertrophy which was prevented by feeding rats a medium-chain fatty acid diet, suggest ing that the effect is attributable to C P T - 1 inhibition (96; 97). Etomoxir has also been shown to cause oxidative stress in H e p G 2 cel ls (98). The fact that etomoxir is a potent irreversible inhibitor of C P T - 1 may explain its tendency to produce adverse effects. It is not c lear whether etomoxir-associated hypertrophy is related to excess ive P P A R - a activation, a mild lipotoxicity or the fact that chronic C P T - 1 inhibition mimics a growth-promoting anabol ic state. Perhexi l ine and oxfenicine are C P T - 1 inhibitors which are cons idered to be partial fatty acid oxidation inhibitors. No cases of card iac hypertrophy arising from either the experimental or clinical use of these agents have been reported. Perhexi l ine fell into disfavor when it was found to induce hepatotoxicity and neuropathy in a subset of patients; this toxicity is now known to be due to s low cytochrome P450 metabol ism of the drug and can be avoided by appropriate 22 dose titration (99). In a smal l randomized-control trial, perhexi l ine w a s shown to improve ejection fraction, myocardial efficiency and symptoms (100). Tr imetazidine is a partial fatty acid oxidation inhibitor which may act by inhibiting 3-ketoacyl C o A thiolase (3-KAT), the final step of the p-oxidation spiral (101). Severa l smal l cl inical trials, including one randomized control trial, have shown that tr imetazidine improves ejection fraction and symptoms in patients receiving optimal treatment for their heart failure (102). Tr imetazidine was tested in diabetic patients with i schemic heart d i sease and w a s found to significantly improve heart function (103-105). Taken together, these data indicate that inhibition of fatty acid oxidation is beneficial to the failing heart and is a mechan ism which can produce meaningful improvements in function. IV: The Benefits of P-Adrenergic Blockade Heart failure is assoc ia ted with activation of the sympathet ic nervous sys tem. The sympathet ic drive to a failing resting heart is equivalent to the max imum drive a normal heart is subjected to during severe exerc ise; spi l lover of ca techo lamines increases as much as 50-fold, producing marked elevation of card iac and sys temic catecholamine levels (106-109). Th is large response is initiated in an effort to maintain systemic perfusion, but sympathet ic activation is harmful to the failing heart, regardless of the cause of the failure, and correlates inversely with survival (110). Converse ly , the p-blocking agents bisoprolol , carvedi lol and metoprolol have been shown in large-scale randomized controlled trials to reduce heart failure mortality by a third or more (111). p-blocking drugs produce negative chronotropic and inotropic responses when administered acutely. For this reason, they were contraindicated in heart failure for many years. However, in the 1970's, p-blockers were pioneered as heart failure treatments (112), and they are now among the agents of cho ice for the treatment of heart failure (111). 23 There have been no clinical or experimental studies examining whether p-blocking agents are beneficial in diabetic cardiomyopathy. However , a number of cl inical studies have examined the effects of these agents in the context of ischemia. In patients taking p-blocker therapy post-myocardial infarction, the improvement in survival produced by treatment is greater in diabetic patients than non-diabetic patients (113). The DIGAMI study showed that diabetic patients receiving a p-blocker had a 5 0 % reduction in mortality (87), and carvedi lol produced a 2 9 % reduction in mortaility in the Carvedi lo l Post-Infarction Survival Control in Left Ventr icular Dysfunction ( C A P R I C O R N ) trial (114). Simi lar improvements in survival are a lso seen in the presence of asymptomat ic coronary artery d i sease (115). Looking at heart failure patients as a whole, more than 20 clinical trials have been publ ished covering more than 10,000 patients whose symptoms ranged from mild to severe; these studies consistently found that p-blocker therapy reduces all cause mortality by more than a third, a similar improvement to that seen following p-blocker treatment of myocardial infarction or i schemia (116-120). However, the dec rease in mortality is greater for sudden deaths than other causes . W h e n d iabetes-associated ischemic heart failure is a s s e s s e d , p-blocker therapy produces a large improvement in heart function and survival. In the Metoprolol C R / X L Randomised Intervention Trial in Congest ive Heart Fai lure ( M E R I T - H F ) trial, metoprolol produced a greater functional improvement in heart failure patients without diabetes than those with diabetes (119). In the Carvedi lo l Prospect ive Randomised Cumulat ive Survival Trial, survival was improved by more than a third in both diabetic and non-diabetic patients with severe heart failure (116). Taken together, these data indicate that the ability of p-blocker therapy to improve survival in heart failure patients is greater in the non-diabetic heart than the diabetic heart. However, the ability of p-blocker therapy to improve survival fol lowing myocardial infarction is greater in the diabetic heart than the non-diabet ic heart. This raises the possibility that p-blockers could have a mechan ism of action that is particularly beneficial to the diabetic cardiomyopathy 24 component of the cardiotoxic triad in terms of protecting against acute injury. In the context of ischemic heart failure, however, this spec ia l benefit is lost. It should be noted that, in all heart failure clinical trials to date, (3-blocker therapy had been instigated following the onset of systol ic failure in accordance with existing ev idence and guidel ines, and the diabetic patients could therefore have had more severe heart failure to begin with. The crucial difference may be the timing of the intervention. It is not known whether 3-blocker therapy can reverse the diabetic cardiomyopathy component at a much earl ier stage because this possibil i ty has not been investigated either experimental ly or clinically. Putat ive mechan isms for the chronic effect of p-blockers include antiarrhythmic effects, amelioration of cardiomyocyte hypertrophy, necrosis and apoptosis, reversal of the fetal gene program (thereby improving ca lc ium handling and force of contraction), increases in cardiac receptor density (for s o m e p-blockers including metoprolol), anti-inflammatory effects (p-blockers lower serum C-react ive protein levels) and partial restoration of card iac g lucose oxidation (37; 121). Metoprolol (122), carvedilol (123) and bucindolol (124) have all been shown to induce a switch from fatty acid to g lucose oxidation in non-diabetic patients with heart failure. Furthermore, metoprolol was shown to increase lactate uptake in heart failure patients, an effect which is consistent with an increase in carbohydrate oxidation (125). A study in dogs with microembol ism- induced heart failure revealed a potential mechan ism for this effect: C P T - 1 was inhibited by chronic treatment with metoprolol (126). Cons ider ing the hypothesis that the switch from g lucose to fatty acid oxidation plays an important role in diabetic cardiomyopathy, the ability of p-blockers to partially reverse this switch could be especial ly beneficial to the diabetic heart. V: P-Adrenoceptor Signalling In 1948, Ahlquist first demonstrated the existence of two broad subtypes of adrenoceptors: a-adrenoceptors and p-adrenoceptors (127). Two subtypes of 2 5 p-adrenoceptors, pi and p2, were identified and character ized in the late 1960's (128), whi le a third, p3, was isolated and c loned in 1989 (129). Al l three subtypes are expressed in the heart, but the major subtypes are p1 and p2, the ratio of p1: p2 being approximately 60-70%: 40-30 %, with very low p3 express ion (130). The effects of the putative p4 adrenoceptor are now bel ieved to be mediated by a low-affinity state of the pi adrenoceptor (131; 132). The receptor reserve is low because the absolute express ion levels are in the femtomolar range (50-70 fmol/ mg protein for the pi adrenoceptor) (37). The affinities of these receptors for their l igands differ: pi (adrenaline = 4 uM, noradrenal ine = 4 u M , i s o p r o t e r e n o l 0.2 uM ) , p2 (adrenaline = 0.7 u M , noradrenal ine = 26 uM, isoproterenol = 0.5 uM), p3 (adrenaline = 130 uM, noradrenal ine = 4 uM, isoproterenol = 2 uM) (133). P -adrenoceptor signaling pathways are summar ized in s c h e m e 4. p-adrenoceptors are G-protein coupled receptors. In the c lass ica l p-adrenoceptor pathway, pi and p2 adrenoceptors, acting via G s , produce an acute posit ive inotopic response mediated by increased c A M P levels and stimulation of protein k inase A (PKA) . P K A then phosphorylates several key proteins involved in ca lc ium handling and calc ium sensitivity of myofi laments. Phosphorylat ion and activation of L-type calc ium channels and ryanodine receptors increases calc ium uptake and re lease, while phosphorylat ion of phospholamban rel ieves inhibition of S E R C A , thereby increasing sarcop lasmic reticulum calc ium uptake (134-136). Finally, P K A modulates calc ium sensitivity of myofi laments through phosphorylat ion of troponin I and myosin binding protein B (137; 138). P K A a lso act ivates protein phosphatase inhibitor-1, sustaining its effects by preventing dephosphorylat ion of its targets (139). Recent ly , a major paradigm shift has occurred in adrenoceptor biology. The p-adrenoceptors are now known to form complex 's igna lomes ' which are temporal ly and spatially organized. A s ignalome can be defined as all genes , 26 SCHEME 4 p-adrenergic signal ing pathways, p i -adrenerg ic receptors activate P K A , which regulates calc ium sensitivity and calc ium handling. Pro longed activation of this receptor act ivates a harmful CAMK- I I pathway which is pro-apoptotic and induces pathological remodel ing. p2-adrenergic receptors also activate P K A , but prolonged activation causes a switch to G i signal ing which act ivates P D E 4 , inhibiting c A M P formation, and activates the cardioprotective PI3K/ Akt pathway. Desensi t izat ion of p2-adrenergic receptors by p-arrestin can recruit p38 and E R K , which protect the cell from apoptosis. p3-adrenergic receptors produce a negative inotopic effect which is mediated by N O produced via the PI3K/ Akt pathway. P2 Gs Gi cAMPh-PDE4 PI3K CAMK-II PKA Akt ERK + p38 Calcium Handling Calcium Sensitivity Pathological Remodeling Apoptosis 28 phosphorylat ion, mediated by (3-arrest ins acting together with G protein-coupled receptor k inases or P K A itself (140-142). In addit ion to receptor desensi t izat ion, proteins and l igands which are involved in the transduct ion and response to a biological s ignal . With regard to temporal organizat ion, it is wel l -establ ished that p-adrenoceptors, and most particularly the p2-adrenoceptor, desensi t ize by uncoupl ing from their G-proteins. This dissociat ion is st imulated by receptor p-adrenoceptors change their coupl ing to downst ream signal ing pathways. Pro longed activation of p i adrenoceptors c a u s e s a switch from P K A to ca lc ium/ calmodul in dependent protein k inase- l l ( C A M K II) - dependent s ignal ing, leading to CAMK-I I mediated apoptosis and pathological hypertrophy (143). In contrast, prolonged activation of p2-adrenoceptors swi tches G-protein coupl ing from G s to G i , which is cardioprotective (144). W h e r e a s p i adrenoceptor signal ing is widely d isseminated throughout the cel l , p2 adrenoceptor signal ing is compartmental ized, and the positive inotopic effect elicited by p2/ G s signal ing is therefore smal ler (145; 146). p2 adrenoceptor compartmental izat ion is partly a c h i e v e d . by the select ive enr ichment of p2 adrenoceptors in caveo lae (147; 148). It has been suggested that translocat ion of p2 adrenoceptors out of caveo lae following susta ined stimulation c a u s e s the switch from G s to G i associat ion (149). p2 adrenoceptor-G i signal ing act ivates the phosphoinositol-3 k inase (PI3K) - protein k inase B (Akt) pathway and phosphodiesterase 4 (145). Phosphod ies terase 4 increases the breakdown of c A M P generated by p1-adrenoceptor-Gs stimulation, enabl ing the p2-adrenoceptor-Gi pathway to functionally antagonize the p i -ad renocep to r -Gs pathway. The PI3K-Akt pathway protects the cardiomyocyte against apoptosis (145). Recent ly, a role for the extracellular-signal-regulated k inase ( E R K ) 1/2 in mediating p2-adrenoceptor-Gi cardioprotection has been suggested (150). Taken together, these data indicate that the coupl ing of p- adrenoceptors to downst ream signaling pathways is compartmental ized and t ime-dependent. 29 Susta ined activation of (31 adrenoceptors is harmful, whereas susta ined activation of p2 adrenoceptors could be cardioprotective. Another consequence of PI3K/ Akt activation is stimulation of nitric oxide (NO) production. N O is synthesized from the terminal guanidine nitrogen atom of the amino acid L-arginine and molecular oxygen by nitric oxide syn thase (NOS) . This p rocess requires tetrahydrobiopterin (BH 4 ) as a cofactor; without B K 4 , e N O S becomes 'uncoupled ' , and produces reactive oxygen spec ies , including peroxynitrite, instead of N O . Endothel ial nitric oxide synthase ( eNOS) is constitutively exp ressed in adult cardiomyocytes, producing physiological N O signal ing in the nanomolar range. Inducible nitric oxide synthase ( iNOS) is exp ressed in response to inflammatory stimuli (151; 152) and produces higher levels of N O , mediating pathophysiological effects (153; 154) . N O and related reactive nitrogen spec ies (e.g. peroxynitrite) covalently modify target proteins in one of three ways: nitrosylation, oxidation or nitration. Binding of N O to a protein, termed nitrosylation, is a reversible reaction and the modification produced is labile. Oxidat ion (e.g. glutathiolation of cysteine residues) or nitration of a protein (on tyrosine residues) produces more stable covalent modif icat ions (155). Tyros ine nitration, nitrosylation and oxidation can be stimulatory or inhibitory depending on the target protein and residue affected. Nitrosylation of the heme moiety of soluble guanylyl cyc lase by N O activates the enzyme, stimulating the product ion of cyc l ic 3', 5 'guanosine monophosphate ( c G M P ) from guanos ine tr iphosphate (156). Just as c A M P activates P K A , c G M P activates protein k inase G ( P K G ) isoforms. The N O / c G M P signaling pathway induces a negative inotropic effect in the heart (151). p3-adrenoceptors a lways couple to G i , activating the PI3K/ Akt pathway. p3-adrenoceptors produce a negative inotropic effect which is mediated by N O . Therefore, p2 adrenoceptor-Gi signal ing and p3 adrenoceptor-G i signal ing both stimulate N O production (157; 158). T h e effects of d iabetes on card iac p-adrenergic respons iveness have been studied for many years, but the results obtained have been confl ict ing. 30 Vad lamud i and McNei l l (159) showed a dec rease in the card iac relaxant effects without an effect on heart rate or contractility. Zo la et al (160) showed a dec rease in the chronotropic response in rabbit heart in vivo. Foy and Lucas (161) demonstrated an increased chronotropic response and a dec reased inotropic response in atria. Most recent studies report dec reased sensitivity to p-adrenergic stimulation in cardiac t issues (162; 163). The effects of d iabetes on p-receptor express ion and downstream signall ing are a lso controversial (163-167). 14 weeks but not 8 weeks of d iabetes blunted the chronotropic response to noradrenal ine, but the response to fenoterol, a select ive p2 agonist, w a s preserved (168). This suggests that p i -med ia ted responses are select ively blunted in the diabetic heart. The express ion of p i is markedly dec reased and that of p2 adrenoceptors modestly dec reased in the diabetic heart, whereas the express ion of p3 adrenoceptors is increased twofold (163). A similar increase in P3 adrenoceptor express ion has been reported in failing human hearts (169). The signi f icance of this shift in receptor subtypes towards p3 adrenoceptors remains to be determined; it is possib le that this shift contributes to card iac dysfunction by promoting a negative inotropic effect; on the other hand, a cardioprotective effect may also result if p3 adrenoceptor-mediated activation of the PI3K/ Akt pathway a lso prevents apoptosis. VI: Potential Links Between B-Adrenoceptors and Cardiac Metabolism Mechan i sms linking p-adrenergic signall ing with card iac metabol ism have not been investigated in great detail. W e therefore employed a combinat ion of 'bottom-up' (known rate-limiting enzymes) and ' top-down' (known p-adrenoceceptor pathways) approaches to unravel the pathways involved. A s d i scussed above, a previous study in microembol ism- induced heart failure demonstrated that chronic metoprolol treatment decreased the activity of C P T - 1 (126). In the heart, the major mechan ism by which C P T - 1 is regulated is through modulat ion of malonyl C o A levels. Isoproterenol has previously been shown to 31 lower malonyl C o A levels by increasing PKA-med ia ted phosphorylat ion of A C C (73). It is therefore possib le that p-adrenergic b lockade could have the opposi te effect, preventing A C C phosphorylat ion and increasing malonyl C o A levels. Recent ly, a study in isolated cardiomyocytes using activators and inhibitors of c A M P revealed that stimulation of fatty acid oxidation by contraction is P K A -dependent (170). Chron ic p-adrenergic b lockade could a lso dec rease the express ion of C P T - 1 . The express ion of C P T - 1 is controlled by P P A R - a , but the P P A R - a / R X R complex produces only modest induction of C P T - 1 when acting a lone (26; 171; 172). P G C 1 a greatly enhances C P T - 1 induction by P P A R - a , but can also induce C P T - 1 independently by binding to M E F - 2 A (173). P G C l a - m e d i a t e d express ion of C P T - 1 has been shown to be repressed in isolated card iomyocytes by upstream stimulatory factor (USF) -2 . Upstream stimulatory factors are transcription factors of the basic helix-loop-helix leucine z ipper family which bind to the E-box consensus sequence C A N N T G (174). In the heart, U S F ' s are involved in excitation-transcription coupl ing, responding to sustained increases in electrical stimulation by increasing the express ion of sarcomer ic genes such as sarcomer ic mitochondrial creatine k inase and M H C (175; 176). p-blockers, by improving function and thereby indirectly increasing electrical st imulation, could activate U S F ' s with the secondary effect that U S F - 2 represses P G C 1 a - m e d i a t e d C P T - 1 express ion . Alternatively, if p-blockers are acute fatty acid oxidation inhibitors, the activation of P P A R - a and its binding to coact ivators could be altered as a result of changes in cytoplasmic long chain fatty acid levels. In the study by Pancha l et al (126), a decrease in C P T - 1 activity was detected using the in vitro assay ; allosteric effects are typically lost during sample preparat ion, so the observed dec rease could be due to dec reased C P T - 1 express ion , or alternatively to a covalent modification of C P T - 1 itself. F e w studies have examined whether covalent modif ications of C P T - 1 occur. Phosphory lat ion of C P T - 1 A has been demonstrated in vitro (177), and the stimulation of C P T - 1 by 32 okada ic acid in hepatocytes was prevented by a specif ic inhibitor of C A M K II, indicating that C A M K II is involved in stimulation of C P T - 1 A activity (178). Phosphory lat ion of C P T - 1 B in the heart has never been demonstrated. However, activation of the sympathet ic nervous system centrally by cerulinin w a s found to stimulate C P T - 1 B activity in so leus musc le within 3 hours (179). Th is effect must have been mediated by an as-yet unidentified covalent modif ication of C P T -1B; the modif ication in question could conceivably be phosphorylat ion. It is possib le that phosphorylat ion of C P T - 1 requires the presence of other proteins present on or recruited to the outer mitochondrial membrane. Compartmental izat ion of P K A signaling in the cardiomyocyte is ach ieved in part by the action of A -k inase anchoring proteins ( A K A P s ) , a group of proteins which bind to P K A targets in order to regulate PKA-dependen t phosphorylat ion of those targets (180). Three mitochondrial A K A P ' s have been identified - A K A P 1 2 1 , D-A K A P - 1 and A K A P 1 4 9 - but functional studies of their role in the heart are awaited (180). It is possib le that mitochondrial A K A P ' s mediate, and are even essent ia l for, C P T - 1 phosphorylat ion. This possibil ity has never been invest igated. Tyros ine nitration of C P T - 1 by peroxynitrite has been shown to inhibit C P T - 1 activity following endotoxemia (181). Furthermore, superoxide, N O and peroxynitrite were all shown to inhibit C P T - 1 activity in vitro when C P T - 1 w a s co -incubated with sys tems which continuously generated these reactive oxygen and nitrogen spec ies (182). This suggests that C P T - 1 can be regulated by covalent modif icat ions mediated by nitrogen spec ies . It is likely that, in addit ion to tyrosine nitration, cysteine nitrosylation and glutathiolation a lso occur. Indeed, cyste ine-scann ing mutagenes is of the musc le isoform of C P T - 1 revealed that cysteine 305 was important for catalysis; nitrosylation or glutathiolation of this residue could conceivably increase or dec rease the catalytic activity of the enzyme. p-blockers could conceivably modulate C P T - 1 activity through nitrogen spec ies generated as a result of p2 adrenoceptor-Gi or p3 adrenoceptor -Gi 33 signal ing. However , N O is known to have more extensive effects on card iac metabol ism which must be cons idered. Firstly, N O is wel l -known to inhibit overall oxygen utilization by irreversibly inhibiting mitochondrial respiration (183; 184). Second ly , N O can alter myocardial substrate select ion. N O inhibits g lycolysis by nitrosylating and inhibiting the enzyme g lycera ldehyde-3-phosphate dehydrogenase ( G A P D H ) (185-187). In isolated hearts perfused without fatty ac ids, N O was found to inhibit Glut-4-mediated g lucose uptake and glyolytic flux (188). In the s a m e study, 8 B r - c G M P , a stable analogue of c G M P , was found to, double the activity of A C C (188). Treatment of dogs with N O S inhibitors st imulated g lucose use and reduced fatty acid oxidation, and administration of a long-acting N O donor switched metabol ism back to fatty acid use (189). T h e s e data suggest that N O is an inhibitor of g lucose use acting primarily to inhibit g lucose uptake and glycolytic flux. Modulat ion of N O levels by p-blockers could therefore affect g lucose oxidation directly. VII: Specific Research P r o b l e m and Research Strategy There is no speci f ic treatment strategy for diabetic cardiomyopathy despite its emerging importance as a cause of heart failure in diabetic patients and alarmingly high prevalence, p-blockers have been shown to improve function and survival in diabetic patients with ischemic heart d isease . However, no exper imental or cl inical studies have investigated whether p-blockers a lso improve function in diabetic cardiomyopathy. Consider ing the hypothesis that the rel iance of the diabetic heart on fatty acid oxidation is a causat ive injury of diabetic cardiomyopathy, the putative ability of p-blockers to inhibit fatty acid oxidation could be especial ly beneficial to the diabetic heart. Data obtained using fatty acid oxidation inhibitors support the idea that this is a mechan ism which can produce meaningful improvements in card iac function, p-blockers are proposed to inhibit fatty acid oxidation by inhibiting long chain acyl C o A uptake into the mitochondria, cata lysed by C P T - 1 . They could do this by severa l mechan isms : increasing malonyl C o A levels, decreas ing C P T - 1 express ion or decreas ing 34 C P T - 1 activity through covalent modif ications (phosphorylat ion, nitrosylation, glutathiolation, nitration). W e therefore undertook the present study to determine whether p-blocker treatment amel iorates diabetic cardiomyopathy by inhibiting fatty acid oxidation, and to determine the mechan ism of the effect. Three p-blockers have been shown to exert effects on cardiac metabol ism: metoprolol, carvedilol and bucindolol . Carvedi lo l and metoprolol are used clinically in the treatment of heart failure. Carvedi lo l is a non-select ive p-blocker which a lso blocks the a1-adrenerg ic receptor and, at high doses , ca lc ium channels . Metoprolol is a second generat ion p-blocker which is select ive for the pi receptor and is an inverse agonist at this receptor. W e chose to use metoprolol in the present study as it does not produce a1-adrenerg ic , ca lc ium channel blocking or antioxidant effects which would compl icate interpretation of the results. Al though metoprolol is c lassical ly regarded as being highly pi-select ive, its selectivity in intact cel ls is less than was previously supposed ; the selectivity ratio of metoprolol for p i and p2 adrenoceptors was recently shown to be 2.3 (190; 191). Metoprolol will therefore block both pi and p2 adrenoceptors at cl inical doses . The dose of metoprolol used in our studies was 75 mg / kg/ day by intraperitoneal injection. This dose is equivalent to a human dose of approximately 100 mg per day after correction for inter-species di f ferences in sur face area (the surface area of 150 mg rat is 0.025 m 2 whereas the surface area of a 60 kg human is 1.6 m 2 (192) ). Furthermore, this dose w a s wel l tolerated by the rats in our preliminary studies, and produced a demonstrable improvement in card iac function (see results). The streptozotocin (STZ) diabetic rat is a model of poorly controlled type 1 d iabetes which is assoc ia ted with a marked decrease in insulin levels. S T Z is an antibiotic synthes ised by the bacterium Streptomyces achromogenes which select ively targets and destroys the insulin-secreting p-cells of the pancreas (193; 194). The mechan ism of this cytotoxicity is incompletely understood. It is 35 bel ieved that S T Z induces D N A strand breaks through the generat ion of oxygen free radicals or carbonium ions (195; 196). A s part of the repair p rocess , poly (ADP-r ibose) po lymerase ( P A R P ) is act ivated. P A R P uses large amounts of A T P and N A D + , and the resulting depletion of these molecules impairs the synthesis and secret ion of insulin and, if sufficiently severe, triggers cell death (197-199). The induction of oxidative stress may be partly related to the ability of N O to inhibit oxidative phosphorylat ion; A D P is shunted into degradat ion pathways which produce xanthine, which via xanthine ox idase generates oxygen free radicals and uric acid (200). Intravenous (IV) or intraperitoneal (IP) injection of S T Z (>40mg/ kg) produces stable insulin-deficient diabetes, but, at low doses , sufficient insulin secret ion is preserved to enable the animals to survive without exogenous insulin. 2-4 hours following injection of S T Z , insulin secret ion is inhibited and g lucose levels rise. Over the next 2-8 hours, hypoglycemia ensues , finally giving way to susta ined hyperglycemia 24 hours following the injection (201). In our laboratory, we routinely use a dose of 60 mg/ kg. S T Z diabetic rats develop both the symptoms (hyperphagia, polyuria, polydipsia, weight loss), and compl icat ions (diabetic retinopathy, nephropathy and cardiomyopathy) of cl inical d iabetes (202-204). The diabetic cardiomyopathy of the S T Z rat c losely resembles that which is seen clinically, and appears 6 weeks following S T Z injection (202-204). S T Z rats do not develop atherosclerosis or hypertension, thereby enabl ing diabetic card iomyopathy to be studied in the absence of ischemic or hypertensive heart d i sease . VIII: Working Hypotheses It was hypothesized that: 36 1. Chron ic treatment with metoprolol would improve card iac function in the diabet ic heart by directly inhibiting fatty ac id oxidation and indirectly stimulating g lucose oxidation through the Rand le Cyc le . 2. Acu te perfusion with metoprolol directly inhibits fatty acid oxidation and indirectly st imulates g lucose oxidation through the Rand le Cyc le . 3. Acu te perfusion and chronic treatment with metoprolol dec rease phosphorylat ion of A C C , leading to an increase in malonyl C o A levels. 4. Acu te perfusion and chronic treatment with metoprolol dec rease the activity of C P T - 1 without affecting the sensitivity of C P T - 1 to malonyl C o A . 5. Chron ic treatment with metoprolol dec reases the express ion of C P T - 1 by increasing the express ion, activity and P G C 1 a - b i n d i n g of U S F - 2 . 6. Chron ic treatment with metoprolol increases the express ion of all three p-adrenoceptor subtypes without affecting G-protein associat ion. Acu te perfusion or chronic treatment with metoprolol dec rease p i - and p2-adrenoceptor s ignal ing, but accentuate p3-adrenoceptor signal ing. 7. Phosphory lat ion of C P T - 1 by P K A and C A M K II is demonstrable in isolated mitochondria and modulated by acute metoprolol perfusion in whole hearts. 8. P K A , A K A P 149 and C A M K II bind to and co- immunoprecipi tate with C P T - 1 . A K A P 149 mediates the binding of P K A to C P T - 1 . 9. Nitrosylation, glutathiolation and nitration of C P T - 1 is demonstrable in isolated mitochondria and modulated by acute metoprolol perfusion in whole hearts. IX: Objectives The first objective was to determine whether metoprolol improves card iac function in the diabetic heart and whether the functional improvement is assoc ia ted with direct inhibition of fatty acid oxidation. The second objective was to determine whether metoprolol directly inhibits fatty ac id oxidation during short-term perfusion. In a ser ies of studies, g lucose oxidation and fatty acid oxidation 37 were measured in the presence or absence of insulin. To character ise the metabol ic effects more fully, measures of g lucose and fatty acid d isposa l (lactate production, g lycogen levels, triglyceride levels) and myocardia l energet ics (tissue adenine nucleotide levels, A M P K activity) were obtained. The third objective was to determine the effect of short term metoprolol perfusion and long term metoprolol treatment on C P T - 1 activity and regulation by malonyl C o A , as well as on the activities of other key fatty acid oxidation enzymes (acyl C o A dehydrogenase, citrate synthase). W e measured C P T - 1 activity, C P T - 1 sensit ivity to malonyl C o A and t issue malonyl C o A levels. T o investigate the regulation of malonyl C o A levels, we measured M C D and A C C express ion , as well as A C C phosphorylat ion by A M P K and P K A . The fourth objective was to determine whether metoprolol treatment controls C P T - 1 express ion by increasing the express ion, activation and P G C 1 a -binding of U S F - 2 . W e also investigated whether metoprolol dec reases the binding of P G C 1 a to its coactivators. The fifth objective was to character ise the acute and chronic effects of metoprolol on the express ion and G-protein coupl ing of (3-adrenoceptors and the activation of downstream second messengers ( P K A , C A M K II, P I 3 K / A k t , NO) The sixth objective was to determine whether short-term metoprolol perfusion regulates C P T - 1 activity and malonyl C o A sensitivity through direct covalent modif ications of C P T - 1 . W e investigated whether P K A and CAMKI I directly bind to C P T - 1 , whether A K A P 1 4 9 binds to C P T - 1 and mediates P K A -binding and whether C P T - 1 is phosphorylated. W e also measured the cyste ine nitrosylation, glutathiolation and tyrosine nitration of C P T - 1 . In order to determine whether these changes were susta ined, the measurements were a lso carr ied out following chronic metoprolol treatment. The seventh objective was to measure the effects of k inase phosphorylat ion and peroxynitrite-mediated nitrosylation, glutathiolation and nitration on C P T - 1 activity and C P T - 1 sensitivity. The final 38 objective w a s to use a mass spect roscopy approach to identify phosphorylat ion sites on C P T - 1 . 39 MATERIALS AND METHODS I: Measurement of ex vivo Left Ventricular Function (a) Animal Treatments An ima ls were cared for in accordance with the guidel ines of the Canad ian Counc i l on An ima l Care . Ma le Wistar Rats (weight matched 200-220g) were purchased from Char les River Laborator ies and al lowed to accl imat ize for 1 week prior to the beginning of the study. Rats were al lowed ad libitum a c c e s s to standard rat chow and water for the duration of the study. Rats were randomly divided into four groups: control (C), control treated (CT), diabetic (D) and diabetic treated (DT). Diabetes was induced by the injection of 60mg/kg streptozotocin (STZ) into the caudal vein. One week following the induction of diabetes, treatment was commenced . The treated groups received 75 mg/ kg/ day metoprolol by intraperitoneal injection while untreated groups received an equivalent vo lume of vehicle (normal sal ine). S ix weeks following the induction of diabetes, the animals were euthanized. 5-hour fasting blood samp les were taken one week following STZ-inject ion and immediately prior to termination. For perfused groups, metoprolol was added to the perfusate in the isolated working heart preparation as descr ibed below. (b) Measurement of Plasma Parameters Five-hour fasting p lasma samp les were col lected one week following S T Z injection and immediately prior to termination. P l a s m a g lucose concentrat ion was determined using the Beckmann G lucose analyser. P l a s m a insulin was measured using the rad io immunoassay kit avai lable from Mil l ipore/ L I N C O (Bil lerica, Massachusse ts ) . P l a s m a free fatty ac ids, cholesterol and tr iglycerides were determined by colorimetric assay kits avai lable from R o c h e (Base l , 40 Switzer land). P l a s m a ketone levels were measured using the Ca rd ioChek Ana lyzer from Po lymer Technology Sys tems, Inc (Indianapolis, Indiana). (c) Direct Measurement of Left Ventricular Pressure Six weeks after S T Z injection, the rats were euthanized by an intraperitoneal injection of 60 mg/kg sodium pentobarbital. The hearts were removed, and mounted on the working heart apparatus by cannulat ion of the aorta. T h e hearts were first perfused in Langendorff mode with warm oxygenated (95% 0 2 , 5 % C 0 2 ) Chenoweth-Koe l le buffer (composit ion: 120 m M NaCI ; 5.6 m M KCI, 2.18 m M C a C I 2 , 2.1 m M MgCI 2 , 19.2 m M N a H C 0 3 , 10 m M g lucose, T e m p 37°C). Fol lowing cannulat ion of the pulmonary vein, the apparatus was switched to working heart mode so that the heart was being perfused via the pulmonary vein. The afterload was set by co lumn of H 2 0 (height=19cm). The heart was paced at 300 beats per minute. A 20-gauge needle was inserted into the left ventricle to measure left ventricular pressure via a Stratham pressure t ransducer. Fol lowing equil ibration for 10 minutes, the hearts were subjected to atrial filling pressures from 3 to 11 mm Hg. The left ventricular deve loped pressure ( L V D P ) , left ventricular end diastol ic pressure ( L V E D P ) , rate of contraction (+dP/dT) and rate of relaxation (-dP/dT) were calculated for each atrial filling pressure by a microcomputer as it col lected the data. II: Measurement of ex vivo Cardiac Metabolism (a) Animal Treatments and Measurement of Plasma Parameters For preliminary studies in which g lucose oxidation and glycolysis were measured , rats were divided into four groups (C, C T , D, DT). For the main studies in which g lucose and fatty acid oxidation were measured , rats were randomly divided into six groups: control (C), control + acute metoprolol perfusion 41 (CP) , control + chronic metoprolol treatment (CT), diabetic (D), diabet ic + acute metoprolol perfusion (DP) and diabetic + chronic metoprolol treatment (DT). The treatment protocol was the s a m e as descr ibed in sect ion I (a). The treated groups received 75 mg/ kg/ day metoprolol by intraperitoneal injection, while the remaining groups received an equivalent vo lume of vehicle (normal sal ine). In the acute metoprolol perfusion groups, isolated hearts were perfused with metoprolol ex vivo as descr ibed below. P l a s m a parameters were measured as descr ibed in sect ion I (b) for all groups. An ima l s .we re terminated six weeks following S T Z injection. T i ssue ana lyses were only undertaken in samp les that were perfused with insulin. To improve clarity, the acute and chronic effects of metoprolol on function and metabol ism are presented separately. However, acute and chronic effects were a lways investigated together in the s a m e exper iment and the controls are j the s a m e . (b) Measurement of Cardiac Metabolism In a preliminary study, the effects of chronic metoprolol treatment on g lucose oxidation and glycolysis were measured to confirm whether metoprolol improves g lucose use by the heart. Activit ies of key enzymes involved in fatty acid oxidation ( C P T - 1 , acyl C o A dehydrogenase, citrate synthase) were measured . For the main studies, the effects of acute metoprolol perfusion and chronic metoprolol treatment on g lucose oxidation and palmitate oxidation were measured . In a ser ies of studies, perfusions were carried out in the p resence or a b s e n c e of insulin to determine whether inhibition of fatty ac id oxidation by metoprolol is direct or mediated by the Rand le cycle in response to direct stimulation of g lucose oxidation. B a s e d on the findings of the preliminary study, we did not measu re acyl C o A dehydrogenase or citrate synthase activities in the main studies. 42 Measurement of cardiac metabol ism was carried out as previously descr ibed (20; 205-209). The hearts were perfused in working heart mode with Krebs-Hense le i t buffer (composit ion: 118 m M NaCI, 4.7 m M KCI, 1.2 m M K H 2 P 0 4 , 1.2 m M M g S 0 4 , 2 m M C a C I 2 , 5.5 mMol g lucose , 0.5 mMol lactate, 100 or 0 uunits insulin, 0.8 mMol palmitate bound to 3 % B S A ) in an aerobic perfusion for 60 minutes. The preload was set at 11.5 mm Hg and the afterload set at 80 mm Hg. The palmitate concentrat ion was selected based on the p lasma lipid profile. A physiological g lucose concentration was se lected. For s imul taneous measurement of g lucose and palmitate oxidation, the production of 1 4 C 0 2 and 3 H 2 0 from 1 4 C g lucose and 3 H palmitate was measured . For s imul taneous measurement of g lucose oxidation and glycolysis, the production of 1 4 C 0 2 and 3 H 2 0 from 1 4 C g lucose and 3 H g lucose was measured . Card iac output, aortic and pulmonary flow were measured by probes posit ioned upstream of the pulmonary cannu la and downst ream of the aortic cannula . P ressure was measured by a pressure t ransducer posit ioned downstream of the aortic cannula. For perfused groups ( C P , DP) , 2000ng/ ml (4.8 uM) metoprolol was added to the perfusate after 30 minutes. For other groups (C, C T , D, DT), an equivalent vo lume of vehic le w a s added . Lactate production w a s measured in the perfusate using the colorimetric lactate assay kit from BioVis ion. Fol lowing complet ion of the perfusion, t issues were immediately f lash-frozen in liquid nitrogen, weighed and stored at -70°C for further assay . (c) Measurement of Tissue Glycogen and Triglyceride Levels Glycogen levels were determined by measurement of g lycogen-der ived g lucose fol lowing extraction of g lycogen in K O H and hydrolysis in H 2 S 0 4 a s previously descr ibed (210). T issue triglyceride levels were measured by performing a chloroform-methanol extraction and redissolving the lipid pellet in phosphate-buffered sal ine ( P B S ) containing 1% Triton X . Tr iglyceride levels in the P B S Triton X solution were a s s a y e d using the colorimetric a s s a y from R o c h e (211). 43 (d) Measurement of Tissue Malonyl CoA and Adenine Nucleotide Levels Rat heart samp les that had been snap frozen in liquid nitrogen following the isolated working heart perfusions were extracted in 0 .4M perchloric acid containing 0 .5mM ethylene-glycol-bis(B-aminoethyl ether)tetraacetic ac id ( E G T A ) in a ratio of 100 mg/ ml. H P L C a s s a y s for malonyl C o A and adenine nucleot ides were carr ied out on the perchoric acid extracts. T i ssue adenine nucleotide levels were measured by gradient ion pair reversed-phase H P L C as a measure of card iac energet ics. The H P L C procedure has been descr ibed previously (212) and can be summar ized as fol lows. S a m p l e s of ac id extracts were appl ied to a C 1 8 reverse-phase co lumn via a precolumn cartridge at a flow rate of 0.5ml/min. Buffer A (25mM K H 2 P 0 4 , 6 m M tetrabutylammonium hydrogensulphate, pH 6.0, 125mM E D T A ) and buffer B (1:1 v/v mixture of buffer A and H P L C - g r a d e acetonitrile), were filtered through a 0.2 um membrane filter and helium de -gassed . After 10 minutes of isocratic elution with 9 8 % A and 2 % B, Waters curvil inear program no 3 was used , ending with a gradient of 4 5 % A and 5 5 % B after 10 minutes. This gradient was be maintained at a f low rate of 1.5 ml/min for a further 5 minutes. The co lumn was re-equil ibrated with 9 8 % A and 2 % B. The H P L C procedure for measurement of malonyl C o A levels was as fol lows (213). S a m p l e s of acid extracts were be appl ied to a C 1 8 reverse-phase co lumn via a precolumn cartridge at a flow rate of 0.5ml/min. The appl ied gradient was as fol lows: Buffer A , 0 .2M N a H 2 P 0 4 , Buffer B, 0 .25M N a H 2 P 0 4 and acetonitri le (20% v/v). At the time of sample appl icat ion, the buffer composi t ion was 2 0 % B. The gradient rose linearly to 5 7 % at 16.7 minutes, remained at 5 7 % until 18 minutes, rose to 9 0 % B by 22 minutes, and fell back to 2 0 % B by 30 minutes. C o A and C o A ester elution were detected by a flow through a monitor set at 254 nm. 4 4 (e) Measurement of Tissue Nitrate/ Nitrite Levels N O is rapidly scavenged and has a half life of 4 seconds in biological fluids. It is converted, by chemica l reactions, to nitrate and nitrite. T h e sum of nitrate and nitrite levels provides an indirect index of total N O production. T i ssue nitrate and nitrite levels were measured using the colorimetric assay avai lable from C a y m a n chemica ls (Ann Arbor, Michigan). In the first step of the assay , nitrate reductase is added to convert nitrate to nitrite. In the second step, the addit ion of the Gr iess reagents converts nitrite to a purple azo product whose abso rbance is read at 540 nm. Ill: Measurement of Kinase and Biochemical Enzyme Activities (a) AMPK, PKA and CAMK Activities Protein k inase and enzyme activities were only measured in samp les that had been perfused with insulin. The activities of A M P K , P K A and C A M K were assayed using kits avai lable from Upstate Biotechnology/ Mil l ipore (Bil lerica, Massachusse ts ) (214). The a s s a y s are based on the rate of incorporation of 3 2 P from [A,3 2P] A T P into synthetic pept ides containing speci f ic consensus sequences for the k inase of interest. ( A M P K : A M A R A peptide, sequence = A M A R A A S A A A L A R R R ; P K A : kemptide, sequence = L R R A S L G ; C A M K : Autocamtide-2 K K A L R R Q E T V D A L ; bold indicates phosphorylated residue). Prior to A M P K assay , samp les were purified by immunoprecipitat ion with ant ibodies speci f ic for the a1 or a2 A M P K subunits, or with an antibody speci f ic for both a-1 and a-2 A M P K subunits (a-pan). Prior to P K A and C A M K assays , samp les were purified by immunoprecipi tat ion with ant ibodies speci f ic for P K A or C A M K > II. The immunoprecipitat ion protocol is descr ibed in sect ion IVd. 45 (b) CPT-1 Assay C P T - 1 activity was est imated by measur ing the production of 1 4 C -palmitoylcarnit ine by the reaction: Palmitoyl C o A + 1 4 C-Carn i t i ne => 1 4 C - Palmitoylcarnit ine + C o A - S H Under the condit ions of this assay , the equil ibrium favours production of 1 4 C - palmitoylcarnit ine by both C P T - 1 and C P T - 2 . However, only C P T - 1 is inhibited by malonyl C o A . C P T - 1 activity was defined as the activity which is inhibited by 2 0 0 u M malonyl C o A . T h e heart t issue was homogen ised in 15mM KCI / 0 .5mM Tris, pH 7.2 by two 10s bursts using a Polytron. Total protein concentration was determined by the B i o R a d Protein A s s a y . A s s a y buffer composit ion was as fol lows: 1 0 5 m M Tris-CI (pH 7.2), 50uM palmitoyl C o A , 500uM carnitine, 0 .25uCi /ml [ 1 4 C]-carnit ine, 1% B S A , 4 m M A T P , 0 .25mM glutathione, 40ug/ml rotenone and 4 m M K C N . The reaction was started by the addition of 10Oul homogenate and al lowed to proceed for 5 minutes at 30°C. The reaction was terminated by the addition of 500ul concentrated HCI. 500ul of 1-butanol was added and the samp les vortexed for 1 minute prior to centrifugation at 3000g for 8 minutes. 300ul of the butanol layer was be taken, added to 1ml butanol-saturated water, vortexed for 30 seconds and centrifuged in a microcentrifuge for 2 minutes. 10Oul of the washed butanol layer was taken and counted in a scintillation counter. C P T - 1 activity was calculated as C P T (total) - C P T (activity in the presence of 2 0 0 u M malonyl C o A ) . Data were expressed as nmol / min/ mg protein. To measure the sensitivity of C P T - 1 to malonyl C o A , C P T - 1 activity w a s assayed in the presence of 0, 10, 20, 50, 100, 150 uM malonyl C o A . The dose -response data were subjected to non-l inear regression analys is and converged 46 to s igmoidal dose- response curves using G r a p h P a d Pr ism 5.0 curve fitting software. The IC50 va lue was calculated. To determine whether metoprolol is a pharmacological inhibitor of C P T - 1 , C P T - 1 activity was assayed in whole heart homogenates from 5 control hearts in the p resence of increasing concentrat ions of metoprolol (ranging from 2-50 ag/ ml) and 0, 50 or 100 uM malonyl C o A . (c) Acyl CoA Dehydrogenase Assay Acy l C o A Dehydrogenase activity was measured by following the reaction: A c y l C o A + [Fc] + => Trans- A 2 -Enoy l C o A + [Fc] In this assay , the ferricenium ion ([Fc] +) replaced F A D + as the electron acceptor, and the reaction was fol lowed by measur ing the rate of reduction of [Fc] + . Reduct ion of- [Fc] + to [Fc] is assoc ia ted with a dec rease in absorpt ion at 300 nm. Octanoyl C o A was used a s the substrate a s it can p a s s freely through the mitochondrial membrane (215). Ferr icenium hexaf luorophosphate (Fc + PFe) is not avai lable commercia l ly and was synthes ized in our laboratory as previously descr ibed (215). Heart t issue was homogenised in 100mM 4-(2-hydroxyethyl)-1-piperazineethanesul fonic acid ( H E P E S ) , pH 7.6 by two 10s bursts using a Polytron. Total protein concentrat ion was determined by the B i o R a d Protein A s s a y . The a s s a y buffer composit ion was as fol lows: 2 0 0 u M F c + P F 6 , 100mM H E P E S , 190ml homogenate. The reaction was started by the addit ion of 25ul 5 m M octanoyl C o A , and the absorpt ion at 300nm was measured every 5 seconds for 5 minutes. Linear rates of reaction were obtained by this protocol. The data were col lected and a zero-order rate constant calculated using E n z y m e 47 Kinet ics Pro ( S y n e x C h e m , Fairf ield, California). Data were expressed as iamol/min/mg protein. (d) Citrate Synthase Assay Citrate synthase activity was measured by following the reaction: Acety l C o A + Oxaloacetate + H 2 0 => C o A - S H + Citrate + H + + H 2 0 The reaction was fol lowed for one minute by measur ing the rate of appearance of C o A - S H spectrophotometrical ly using dithio-bis (2-nitrobenzoic acid) (DTNB) . The thiol (SH) group of C o A - S H re leases T N B from D T N B , caus ing an increase in absorption a t 4 1 2 n m (216). Heart t issue was homogen ised in 0 .1M tris (hydroxymethyl) aminomethane hydrochloride (tris-CI), pH 8.1 by two 10s bursts using a Polytron. Total protein concentrat ion was determined by the B ioRad Protein A s s a y . The a s s a y buffer composi t ion was as follows: 0 .2mM D T N B , 0 .3mM acetyl C o A , 0.1 m M Tris-CI, 0.05 ml homogenate. The reaction was started by the addition of 0.05ml 10mM oxaloacetate, and the absorption at 412nm was measured every 5 seconds for 1 minute. Linear rates of reaction were obtained by this protocol. The data were col lected and a zero-order rate constant calculated using the E n z y m e Kinet ics Pro software (SynexChem, Fairf ield, California). Data were expressed as pmol/min/mg protein. 48 IV: Immunoprecipitation and Measurement of Protein Expression by Western Blotting (a) Overview of Experimental Design Measurements were only carried out on samp les that had been perfused with insulin. Wheneve r possib le, phosphorylat ion of speci f ic res idues on the protein of interest was measured using phospho-speci f ic ant ibodies. The blots were str ipped and re-blotted for total express ion of the protein of interest. However, if phospho-speci f ic ant ibodies were not avai lable to probe the protein of interest, we measured the co-immunoprecipi tat ion (co-IP) of the protein with pan-speci f ic phosphoser ine and phosphothreonine antibodies, either individually or in combinat ion, (Upstate Biotechnology/ Mill ipore, Bi l ler ica, Massachusse ts ) as an index of the total phosphorylat ion state. To obtain a measure of A C C and M C D activities, we measured the express ion of A C C (Upstate Biotechnology/ Mill ipore, Bi l ler ica, Massachusse ts ) and M C D (a generous gift from Dr. G .D Lopaschuk and Dr. J . Dyck, University of Alberta), A M P K - m e d i a t e d phosphorylat ion of Ser ine 79 on A C C and the total ser ine phosphorylat ion state of A C C . S E R C A - 2 express ion was measured as an index of ca lc ium handling and fetal gene program effects. To investigate changes in C P T - 1 express ion, we measured total C P T - 1 express ion with a pan-speci f ic C P T - 1 antibody, as well as with ant ibodies speci f ic for C P T - 1 A and C P T - 1 B (Santa-Cruz Biotechnology, San ta -Cruz , Cali fornia). W e measured the total protein express ion of P G C 1 a , U S F - 1 and U S F - 2 (all f rom Upstate Biotechnology/ Mil l ipore, Bi l ler ica, Massachusse ts ) , and P P A R - a and P D K - 4 (both from San ta -Cruz Biotechnology, San ta Cruz , Cal i fornia), a-myosin heavy chain (a-MHC) express ion (San ta -Cruz Biotechnology, San ta Cruz , California) was measured as an index of U S F 49 activity, and P D K - 4 express ion (Upstate Biotechnology/ Mil l ipore, Bi l ler ica, Massachusse ts ) was measured as an index of P P A R - a activity. P P A R - a was purified by IP prior to Western blotting. The co-IP of P G C 1 a with M E F 2 A (Santa-C r u z Biotechnology, San ta -Cruz , California) and P P A R - a and to its repressor U S F - 2 was measured as an index of the associat ion between these proteins. Finally, to determine whether P P A R - a binds the P G C 1 a . M E F 2 A functional complex, we measured the co-IP of P P A R - a with M E F 2 A and U S F - 2 . The express ion of (31, P2 and p3 adrenoceptors (all from San ta -Cruz Biotechnology, San ta -Cruz , California) was measured . p2 associat ion with G s or G i was measured by co-IP. P K A and C A M K II activities were measured using radioisotopic a s s a y s as descr ibed in sect ion Ilia. PI3K/ Akt activation was determined by measur ing the phosphorylat ion of Akt. Efforts to measure N O S activity in our laboratory were unsuccessfu l . W e therefore measured the express ion and P K A / PI3K-mediated phosphorylat ion of e N O S , and the express ion of i N O S . T issue nitrate/ nitrite levels were measured as an indirect index of N O levels. Total protein glutathiolation and tyrosine nitration were measured by dot blotting as biomarkers of reactive nitrogen spec ies . The total phosphorylat ion states of C P T - 1 and A K A P 1 4 9 (Santa-Cruz Biotechnology, San ta -Cruz , California) were measured using phosphoser ine and phosphothreonine ant ibodies in combinat ion! The nitrosylation, glutathiolation and nitration of C P T - 1 were determined by measur ing the co-immunoprecipitat ion of C P T - 1 with pan-speci f ic anti-nitrotyrosine ant ibodies (Upstate Biotechnology/ Mil l ipore, Bi l ler ica, Massachusse ts ) , pan-speci f ic anti-glutathione ant ibodies (Santa-Cruz Biotechnology, San ta -Cruz , California) and pan-speci f ic anti- nitrosocysteine antibodies ( A G Scientif ic, S a n Diego, Cali fornia). To determine the relationship between A K A P 149 binding and P K A binding to C P T - 1 , co-immunoprecipitat ion of P K A with C P T - 1 was measured . The blots were then stripped and reblotted for A K A P 1 4 9 . Co- immunoprecip i tat ion of CAMK- I I with C P T - 1 was also measured . 50 (b) Sample Preparation 10-30mg of heart t issue was homogenized in total protein extraction buffer containing 20 m M H E P E S , 1mM ethylenediamine tetraacetic acid (EDTA) , 250 m M sucrose , 100 m M sodium pyrophosphate, 10 m M sodium orthovanadate, 100 m M sodium fluoride and 4pJ/ ml protease inhibitor cocktai l (S igma-Ald ich, Saint -Louis , Missouri) at pH 7.4. The total extraction buffer was found to interfere with the B i o R a d protein assay . Protein concentration was therefore determined using the P ie rce bjcinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, Illinois). To prepare samples for sod ium dodecyl sulphate polyacry lamide gel electrophoresis ( S D S - P A G E ) , 50 jag of protein was diluted with water and a reducing' buffer (6% S D S , 185mM Tris pH 6.8, 3 0 % glycerol , 14% mercaptoethanol , 0 .7% bromophenol blue) to a total vo lume of 20 ut. The samp les were boiled for 5 minutes and stored on ice prior to loading. Dot blotting samp les were boiled for 5 minutes, but, to ensure adherence of the protein to the nitrocellulose membrane, an equivalent vo lume of water rather than reducing buffer was added . For immunoprecipitates, 15 pi of the immunoprecipitate was diluted with 5 u,l sample buffer and boiled for 5 minutes. (c) SDS-PAGE, Western Blotting and Dot Blotting S a m p l e s were loaded onto an acry lamide/ bis acry lamide gel of the appropriate percentage (5, 7.5 or 10%) and subjected to S D S - P A G E as previously descr ibed (210). The protein bands were transferred to a nitrocellulose membrane and stained with P o n c e a u Red to measure total protein. The membrane was blocked in 5 % bovine serum albumin in Tris-buffered sal ine containing 0 . 1 % polyoxyethylenesorbitan monopalmitate ( T W E E N ) . The membrane was subsequent ly incubated with primary antibody overnight at 4°C, washed three t imes with tris-buffered sal ine and incubated in secondary antibody at room temperature for 1 hour. Fol lowing three further w a s h e s with tris-buffered 51 sal ine, the blots were visual ised using chemi luminescent detect ion. Due to the low abundance of s o m e proteins of interest, blots were deve loped using the Supe r S igna l ® Wes t Femto Max imum Sensitivity substrate (Pierce Biotechnology, Rockford, Illinois). The blots were imaged using a Chemigen iusQ Image Ana lyse r (Genef low, A lexandr ia , Virginia) so the images obtained are inverted. Band intensity was quantif ied using ImageJ software avai lable from the National Institutes of Health. For most ana lyses , 3 or 4 samp les from each group were run on a single gel. However, blots were repeated to ensure that the observed patterns were consistent for all samples . (d) Immunoprecipitation Protocol 500 uq protein was diluted into 500 ul total protein extraction buffer to create a 1 uq/ ul protein solution. 2 ul of antibody was added and the samp les incubated on a rotator overnight at 4°C. For combined phosphoser ine and phosphothreonine IP, 1 ul of each antibody was added . 20 pi of protein A sepharose slurry was added and the samples incubated on the rotator at 4°C for a further hour. S a m p l e s were centrifuged to pellet the immunoprecipitate and the supernatant was d iscarded. The pellet was washed three t imes in total protein extraction buffer. Finally, the pellet w a s resuspended in suspens ion buffer (0.1 M Tris base , 1 m M E D T A , 1 m M E G T A , 50 m M sodium fluoride, 5 m M sodium pyrophosphate, 10% glycerol, 0.02% sodium az ide, pH 7.5). (e) Dot Blotting 5 ug of protein was loaded directly onto a dry nitrocellulose membrane. Total protein was determined by P o n c e a u Red staining. The membrane was then blotted according to the Western blotting protocol. Pan-spec i f ic anti-nitrotyrosine or glutathione ant ibodies were used as the primary antibody. 52 V: Functional Effects of CPT-1 Covalent Modifications in Isolated Mitochondria (a) Isolation of Mitochondria Left ventricular t issue from control hearts was homogen ized in 10ml of mitochondrial homogenisat ion buffer containing 20 m M 3-(N-Morphol ino)-propanesul fonic acid ( M O P S ) , 250 m M sucrose, 2 m M E D T A , 2 m M E G T A , 2.5 m M reduced glutathione and 3 % B S A at pH 7.2. The samp les were centrifuged at 1000g for 5 minutes to remove the nuclei and incompletely disrupted t issue, and the supernatant was centrifuged at 10,000g for 10 minutes. The supernatant was d iscarded and the pellet resuspended in mitochondrial homogenisat ion buffer without B S A prior to further centrifugation at 10,000g for 10 minutes. The final pellet was resuspended in 0.6 ml of mitochondrial homogenisat ion buffer without B S A . A 50ul aliquot of the mitochondrial fraction was taken for protein quantif ication using the B ioRad assay . The mitochondrial preparat ions were used on the day of isolation. (b) Phosphorylation of Proteins in Isolated Mitochondria Purif ied preparations of active P K A , C A M K II and Akt were purchased from Upstate Biotechnology/ Mil l ipore. Phosphorylat ion of isolated mitchondria was ach ieved by incubating 100 JLXI (0.4 - 0.7 mg protein) of the mitochondrial isolate with the active k inase. For the control reaction, 100 uJ of the s a m e mitochondrial isolate was incubated without the active k inase. The reaction condit ions were the s a m e as those used to measure k inase activity: 20 m M M O P S , 25 m M p-glycerophosphate, 5 m M E G T A , 1mM sodium orthovanadate, 1 m M dithiothreitol, 12.5 m M magnes ium chloride, 83 ulvl A T P , 1.7 uM c A M P (for P K A react ions only), 1 m M calc ium chloride (for C A M K reactions only) and 0.83 jag/ ml act ive k inase at pH 7.2. The final reaction vo lume was 600 u.l. The 53 samp les were incubated at 30°C for 10 minutes and the reaction terminated by f lash freezing in liquid nitrogen. Separate reactions were carried out to generate samp les for C P T - 1 assay or measurement of total C P T - 1 phosphorylat ion and co-immunoprecipi tat ion of C P T - 1 with the k inase. (c) Peroxynitrite Dose Response in Isolated Mitochondria Peroxynitr i te has a half life of less than 5 seconds at physiological p H ; 8 0 % of the peroxynitrite loaded at pH 7.4 is degraded by protonation within 12 seconds (217). However, this t imeframe is sufficient for peroxynitrite to produce measurab le covalent modif ications of target proteins (218). In order to examine the effects of tyrosine nitration, glutathiolation and cysteine nitrosylation on C P T -1, we incubated mitochondrial isolates with increasing concentrat ions of peroxynitrite. Peroxynitrite is stable at high pH as the equil ibrium does not favour protonation. Peroxynitrite stock solutions were made in 0 .3M N a O H . Mitochondrial isolates from four control hearts were pooled. 100 pi (0.4 -0.7 mg protein) of the pooled mitochondrial isolate was incubated with 0, 0.1, 1, 10, 100, 500 and 1000 p M peroxynitrite in mitochondrial homogenisat ion buffer without B S A . The final reaction vo lume was 600 pi. The samp les were incubated at room temperature for 5 minutes. The addition of the 0.3M N a O H vehic le raised the pH of the buffer from 7.2 to 8. Separate reactions were carried out to generate samp les for C P T - 1 assay or measurement of total C P T - 1 phosphorylat ion and co-immunoprecipitat ion of C P T - 1 with the k inase. Immunoprecipitation was started immediately following the incubation with peroxynitrite. C P T - 1 activity samp les were frozen in liquid nitrogen and stored at -70°C until the day of assay . 54 (d) Measurement of CPT-1 Activity, Phosphorylation and Kinase Binding C P T - 1 activity and sensitivity to malonyl C o A was measured as descr ibed in sect ion III (b) with two modif ications: 50 pi of the sample was added and the reaction was al lowed to proceed for 10 minutes rather than 5: Addit ion of the pH 8 mitochondrial samp les had no effect on the final pH of the C P T - 1 reaction mixture. VI: Identification of CPT-1 Phosphorylation Sites by LC MS/ MS The rat C P T - 1 B primary sequence was searched for consensus sites of the following k inases: P K A , P K C , C A M K I/ II, A M P K and Akt. Putative phosphorylat ion sites for P K A and C A M K I/ II were identified. To maximize our chances of finding phosphorylat ion, C P T - 1 B was purified by IP and phosphorylat ion enrichment was performed on the tryptic digests of the C P T - 1 bands. C P T - 1 in whole cell homogenates was purified by IP with speci f ic C P T - 1 B ant ibodies. The immunoprecipi tates were subjected to S D S - P A G E and stained with C o u m a s s i e Blue. The bands corresponding to C P T - 1 (88kDa) were exc ised and stored at -70°C until the day of assay . Tryptic digests of the C P T - 1 bands were subjected to titanium TopTip phosphopept ide enrichment at the U B C M S L / L M B Proteomics Co re Facility. The digests were appl ied to titanium tips for 30 minutes to al low binding of phosphopept ides. The tips were then washed with binding solution (0 .1% trif luoroacetic ac id , 10% acetonitrile) to remove unphosphorylated pept ides. Phosphory la ted peptides were eluted from the tips by a ser ies of 50 pi w a s h e s with increasing concentrat ions of ammonium bicarbonate (10, 20, 50 and 100 mM), which re lease low to moderately phosphorylated peptides, and a final 50 pi wash with ammon ium hydroxide, which re leases highly phosphorylated pept ides. 55 The ammon ium bicarbonate eluents for each sample were pooled, and the ammon ium bicarbonate and ammonium hydroxide eluents for each sample were subjected to L C / M S / M S separately. Phosphory lat ion detection was carried out using Liquid Chromatography/ M a s s Spec t roscopy / M a s s Spec t roscopy ( L C / M S / M S ) on an A P I Q S T A R P U L S A R i Hybrid L C / M S / M S at the U B C M S L / L M B Proteomics Co re Facil ity. Tryptic digests of C P T - 1 bands underwent reversed-phase H P L C . The co lumn eluent w a s subjected to M S / M S as it eluted from the co lumn. In this procedure, atmospher ic pressure ionization (API) was applied to generate a spectrum of mass- to-charge ratio (m/z) peaks . A s each amino acid produces a character ist ic m/z peak, the sequence of the eluted peptide can be determined and searched in the M A S C O T protein database to confirm the identity of the protein. Ions with a change in m/z suggest ing that phosphorylat ion had occurred (indicated by an increase of 80 Da) were selected as parent ions for M S / M S to confirm that the phosphorylat ion event was present and identify the affected residue. Vll: Data Analysis Data are expressed as mean ± standard error of the mean ( S E M ) . For statistical analys is , data were ana lysed using Number Cruncher Statistical Software ( N C S S , Kaysvi l le, Utah). Starl ing curves were ana lysed using G L M A N O V A with Neumann-Keu ls post hoc test. A l l other data were ana lysed using O n e - W a y A N O V A with Neumann-Keu ls post hoc test. Compar i sons between two groups were carried out using an unpaired- student's t-test. Acu te perfusion and chronic treatment data are presented separately, 1 although the control and diabetic groups for each are the same , and the data were subjected to composi te analys is . 56 RESULTS I: General Characteristics W e measured p lasma g lucose and insulin levels to confirm success fu l induction of d iabetes. S T Z successfu l ly induced marked and susta ined hyperglycemia which was assoc ia ted with a decrease in insulin levels and a mild elevation of p lasma lipids. A s expected, metoprolol treatment had no effect on either g lucose or insulin (Table 1). Metoprolol had no effect on p lasma lipids, but amel iorated the increase in ketone levels in the diabetic group (Table 1). Body weights were lower in the diabetic groups. Metoprolol had no significant effect on body weight. However, metoprolol significantly lowered heart weight in both control and diabetic rats (Table 1). II: Functional and Metabolic Effects of Chronic Metoprolol Treatment To establ ish whether chronic metoprolol treatment improves function in the S T Z - m o d e l , cardiac function was measured ex vivo by direct left ventricular pressure measurements in paced isolated working hearts following chronic treatment with metoprolol. Metoprolol treatment (75mg/kg/day IP) significantly improved contracti le function in diabetic cardiomyopathy as measured ex vivo by L V D P , +dP/ dt and - d P / dt from the left ventricle in paced hearts (Figure 1). During subsequent studies for the measurement of card iac metabol ism, in which the hearts were not paced and fatty acid was present in the perfusate, metoprolol a lso amel iorated the depress ion in rate-pressure product, card iac output and hydraul ic power in diabetic hearts (Figure 2). Chron ic treatment with metoprolol increased palmitate oxidation and dec reased g lucose oxidation in control hearts (Figure 3). Lactate production was unchanged, but g lycogen levels were decreased (Table 2). In the diabet ic hearts, there w a s a marked increase in palmitate oxidation relative to controls and 57 TABLE 1 GENERAL CHARACTERISTICS AND PLASMA PARAMETERS AT TERMINATION C CT D DT Body Weight (g) 486.4 ±31.3 480.3 ± 41.0 387.8 ± 37.5* 351.6 + 53.7* Heart Weight (S) 1.82 ±0.10 1.47 ± 0.07 + 1.51 ±0.10* 1.38 ± 0.07+ Plasma Glucose (mmol/1) 7.25 ± 0.26 7.35 ± 0.40 27.99 ±1 .1* 24.09 ± 5.41* Plasma Insulin (ng/ml) 1.59 ± 0.41 1.78 ± 0.78 0.49 ± 0.3* 0.37 + 0.12* Plasma Triglycerides (mmol/ 1) 0.19 ±0.01 0.16 ±0.01 0.25 ± 0.05* 0.26 ± 0.05* Plasma Cholesterol (mmol/ 1) 1.89±0.05 1.86±0.10 2.05±0.18* 2.10 ±0.15* Plasma Free Fatty Acids (mmol/1) 0.19 ± 0.02 0.23 ± 0.01 0.20 ± 0.02 0.19 ± 0.03 Plasma Ketones (mmol/1) 0.76 ± 0.05 0.50 ± 0.04 2.43 ± 0.57* 1.36 ± 0.28+ Anima ls were fasted for 5 hours prior to blood col lection. Data represent means ± S E M . Data were ana lysed using one-way A N O V A with Neumann-Keu ls post-hoc test. * = significantly different from C, + = significantly different from untreated group (p<0.05) (C=control, n=8; CT=control treated with metoprolol, n=8; D=diabetic, n=8; DT=diabetic treated with metoprolol, n=8). 58 FIGURE 1 Mechan ica l performance of Isolated Per fused Hearts: Left Ventr icular P ressu re Measurements . Hearts were exc ised and perfused in working heart mode with Chenoweth -Keu ls buffer containing 5.5 m M g lucose but no palmitate. The hearts were paced at 300 bpm and direct left ventricular pressure measurements were taken. A - C : Left ventricular developed pressure (LVDP) , max imum rate of contraction (+dP/dt) and maximum rate of relaxation (-dP/ dt) from direct left ventricular pressure measurements . Data represent means ± S E M and were ana lysed using G L M A N O V A with Neumann Keuls post-hoc test. * = significantly different from C , C T , DT at the same filling pressure, # = significantly different from C and D at the s a m e filling pressure (C=control, n=12; CT=control treated, n=12; D= diabetic, n=12; DT= diabetic treated, n=12). A. LEFT ATRIAL FILLING PRESSURE (mmHg) 60 FIGURE 2 Mechan ica l performance of Isolated Per fused Hearts: F low and Ra te -P ressu re Product Measurements . Card iac function measurements obtained by aortic pressure and heart rate measurements , and pulmonary and aortic flow measurements . Unpaced hearts were perfused with Krebs-Hensele i t buffer containing 5.5 m M g lucose and 0.8 mmol palmitate at constant preload (11.5 mm Hg) and afterload (80 mm Hg). Heart rate, peak systol ic pressure, rate-pressure product, card iac output and hydraulic power. Data represent means ± S E M and were ana lysed using G L M A N O V A with Neumann Keu ls post-hoc test. * = signif icantly different from D and DT at s a m e timepoint, # = significantly different from C , C T , D at s a m e timepoint, + = significantly different from C , C T and DT at s a m e timepoint (p<0.05), (C=control, n=5; CT=control treated, n=5; D= diabetic, n=5; DT= diabet ic treated, n=5). 61 10 20 30 40 50 60 ' 10 20 30 40 50 60 TIME (MINUTES) TIME (MINUTES) 62 FIGURE 3 Effects of Chron ic In Vivo Metoprolol Treatment on Metabol ism of Isolated Per fused Hearts. A - B G lucose and palmitate oxidation during a 60 minute aerobic perfusion. Perfus ions were carried out in the presence (left panel) or absence (right panel) of insulin (100 uUnits/ml) as indicated. Data represent means ± S E M . Data were ana lysed using O n e - W a y A N O V A with Neumann Keu ls post-hoc test, * = significantly different from C of metoprolol , + = significantly different from corresponding untreated group (p<0.05), CD PALMITATE OXIDATION GLUCOSE OXIDATION 100 UU7 ml Insulin Present 1 0 0 p U / m | | n s u M n P r e s e n t (nmol/ mm/ g dry weight) ( n m 0 l / m i n / g d r y w e j g n t ) PALMITATE OXIDATION Insulin Absent (nmol/ min/ g dry weight) S GLUCOSE OXIDATION Insulin Absent (nmol/ min/ g dry weight) 64 TABLE 2 LACTATE PRODUCTION AND TISSUE GLYCOGEN, TRIGYLCERIDE AND MALONYL CoA LEVELS FOLLOWING CHRONIC IN VIVO METOPROLOL TREATMENT AND EX VIVO PERFUSION IN THE PRESENCE OF INSULIN C CT D DT Lactate Production (nmol/ min/ g dry weight) 10.2 ± 2.2 13.9 ± 4.1 10.6 ±3.6 10.8 ± 4.0 Tissue Glycogen Levels (u.mol/ g dry weight) 180.4±31.2 95.9 ± 18.3 + 285.0± 32.0 * 286.6± 35.2 * Tissue Triglyceride Levels (umol/ g dry weight) 41.1 ±2.6 31.2 ± 1.5 + 59.1 ±3.9* 44.6± 2.7 + Malonyl CoA Levels (u.mol/ g wet weight) 17.1 ±2 .4 7.7 ± 1.4 + 20.7 ± 5.2 17.4 ±3.8 Data represent means ± S E M . Data were analysed using one-way A N O V A with Neumann-Keu l s post-hoc test. * = significantly different from corresponding unperfused group, + = significantly different from corresponding untreated group, (p<0.05) (C=control, n=5; CT=control treated, n=5; D=diabetic, n=5; DT=diabetic treated, n=5). 65 TABLE 3 MYOCARDIAL ENERGETICS AND AMPK ACTIVITY FOLLOWING CHRONIC IN VIVO METOPROLOL TREATMENT AND EX VIVO PERFUSION IN THE PRESENCE OF INSULIN C CT D DT ATP (umol/g wet weight) 6.3 ± 0.3 7.2 +1.7 6.0 ± 0.5 5.1 ± 1.2 ADP (umol/g wet weight) 2.3 ± 0.2 1.6 + 0.3 2.5 ± 0.3 1.6 ± 0.5 + AMP (umol/g wet weight) 0.74 ± 0.09 1.03 ± 0.08 0.83 ± 0.08 0.77 ± 0.03 ATP/ADP Ratio 2.9 ± 0.3 6.0 ± 1.4+ 2.5 ± 0.2 3.0 ± 1.0 a-1 AMPK Activity (pmol ATP incorporated/ min/ mg protein) 2.7 ± 0.3 3.5 ± 0.6 3 .1±0 .4 3.6 ± 0.5 a-2 AMPK Activity (pmol ATP incorporated/ min/ mg protein) 3.6 ± 0.4 3.6 ± 0.3 3.4 ± 0.6 3.4 ± 0.4 a-pan AMPK Activity (pmol ATP incorporated/ min/ mg protein) 4.0 ± 0.9 3.8 ± 0.3 3.6 ± 0.5 3.7 ± 0.4 Data represent means ± S E M . Data were analysed using one-way A N O V A with Neumann-Keu l s post-hoc test. * = significantly different from corresponding unperfused group, + = significantly different from corresponding untreated group (p<0.05) (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). 66 g lucose oxidation was negligible; in these hearts, chronic metoprolol treatment dec reased palmitate oxidation and increased g lucose oxidation (Figure 3). G l ycogen levels were elevated in diabetic hearts as compared with controls; metoprolol treatment had no effect on either g lycogen levels or lactate production (Table 2). T i ssue triglyceride levels were significantly elevated in the diabetic group. In both control and diabetic hearts, metoprolol treatment significantly lowered t issue triglyceride levels (Table 2). Myocard ia l energet ics, as determined by t issue levels of A T P , A D P and A M P , and A M P K activity, were not altered either by metoprolol or by d iabetes (Table 3). W h e n perfusions were repeated in the a b s e n c e of insulin, the effect of metoprolol on g lucose oxidation was obli terated, but the effect on palmitate oxidation was preserved (Figure 3). In a preliminary study, chronic metoprolol treatment had no effect on glycolysis in either control or diabetic heart (Table 4), but improved coupl ing between glycolysis and g lucose oxidation in diabetic hearts by increasing g lucose oxidat ion. W h e n control or diabetic hearts were perfused for 30 minutes with metoprolol , palmitate oxidation was inhibited and g lucose oxidation markedly st imulated (Figure 4) producing an increase in t issue A T P levels and a dec rease in A M P levels (Table 5). Stimulation of g lucose oxidation was assoc ia ted with a fall in lactate production without any change in g lycogen levels (Table 5). In both control and diabetic hearts, acute metoprolol perfusion lowered t issue triglyceride levels (Table 5). In the absence of insulin, the pattern of changes observed for g lucose oxidation was obliterated in diabetic, but not control, hearts, whereas the pattern of changes observed for palmitate oxidation was preserved (Figure 4). 67 TABLE 4 GLYCOLYSIS AND FATTY ACID OXIDATION ENZYME ACTIVITIES FOLLOWING IN VIVO METOPROLOL TREATMENT AND EX VIVO PERFUSION IN THE PRESENCE OF INSULIN C CT D DT Glycolysis (nmol/ min/ g dry weight) 3900 ± 539 4484 + 1116 1335 ± 358 # 1729 ± 313 # Glucose Oxidation (nmol/ min/ g dry weight) 2336± 564 3749 ±1378 123 ± 52 # 854 ±312* % Coupling of Glycolysis to Glucose Oxidation 72 ± 2 0 82±19 11 ± 6 # 40 ± 18* Acyl CoA Dehydrogenase Activity (mmol/ min/ mg protein) 5.0 ±0.4 5.5 ± 0.4 4.6 ± 0.3 4.2 ± 0.4 Citrate Synthase Activity (mmol/ min/ mg protein) 1.3 ± 0.1 1.2 ±0.1 1.3 ±0.2 1.4 ±0 .2 G l u c o s e and fatty acid metabol ism measurements from a preliminary study. Data represent means ± S E M . Data were ana lysed using one-way A N O V A with Neumann-Keu l s post-hoc test. # = significantly different from C and C T , = significantly different from D (p<0.05) (Glycolysis and g lucose oxidation, C=control, n=4; CT=control treated with metoprolol, n=4; D=diabetic, n=4; DT=diabetic treated with metoprolol, n=4; Acy l C o A dehydrogenase and citrate synthase activit ies, C=control, n=8; CT=control treated with metoprolol , n=8; D=diabetic, n=8; DT=diabetic treated with metoprolol, n=8). 68 FIGURE 4 Acute Effects of Metoprolol on Metabol ism of Isolated Per fused Hearts. A - B : G l u c o s e and palmitate oxidation during a 60 minute aerobic perfusion. Per fus ions were carried out in the presence (left panel) or absence (right panel) of insulin (100 uJvl/ml) as indicated. Data represent means ± S E M . Data were ana lysed using O n e - W a y A N O V A with Neumann Keu ls post-hoc test, * = significantly different from C of metoprolol, + = significantly different from corresponding untreated group (p<0.05), DO PALMITATE OXIDATION 100 nil/ ml Insulin Present (nmol/ min/ g dry weight) GLUCOSE OXIDATION 100 nU/ ml Insulin Present (nmol/ min/ g dry weight) PALMITATE OXIDATION Insulin Absent (nmol/ min/ g dry weight) GLUCOSE OXIDATION Insulin Absent (nmol/ min/ g dry weight) O 3 O o 70 TABLE 5 LACTATE PRODUCTION AND TISSUE GLYCOGEN, TRIGYLCERIDE AND MALONYL CoA LEVELS FOLLOWING ACUTE METOPROLOL PERFUSION EX-VIVO IN THE PRESENCE OF INSULIN C CP D DP Lactate Production (nmol/ min/ g dry weight) 10.2 ± 2.2 6.7 ± 1.3 + 10.6 + 3.6 2.4 ± 0.8 + Tissue Glycogen Levels (u,mol/ g dry weight) 180.4± 31.2 160.6 +18.1 285.0 ±32.0 A 237.3±35.7 + Tissue Triglyceride Levels (umol/ g dry weight) 41.1+2.6 35.3± 3.0 + 59.1 ±3.9 * 31.5± 0.8 + Malonyl CoA Levels (umol/ g wet weight) 17.1 ±2.4 8.1 +1.1 + 20.7 ± 5.2 22.1 ± 4.7 Data represent means + S E M . Data were analysed using one-way A N O V A with Neumann-Keu l s post-hoc test. * = significantly different from corresponding unperfused group, + = significantly different from corresponding untreated group, (p<0.05) (C=control, n=5; C P = control perfused, n=5; D=diabetic, n=5; D P = diabetic perfused, n=5). 71 TABLE 6 MYOCARDIAL ENERGETICS AND AMPK ACTIVITY FOLLOWING ACUTE METOPROLOL PERFUSION EX-VIVO IN THE PRESENCE OF INSULIN C CP D DP ATP (umol/g wet weight) 6.3 ± 0.3 7.3 ± 0.4 + 6.0 ± 0.5 9.7± 0.9 + ADP (umol/g wet weight) 2.3 ± 0.2 2.3 ±0.1 2.5 ± 0.3 3.3 ± 0.4+ AMP (umol/g wet weight) 0.74 ± 0.03 0.22 ± 0.03+ 0.83 ± 0.08 0.39 ± 0.10+ ATP/ADP Ratio 2.9 ± 0.3 3.2 ±0.14 2.5 ± 0.2 2.3 ± 0.4 a-1 AMPK Activity (pmol [P] incorporated/ min/ mg protein) 2.7 ± 0.3 3 . 8 ± 0 . 1 + 3.1 ±0 .4 3.7 ± 0.4 a-2 AMPK Activity (pmol [P] incorporated/ min/ mg protein) 3.6 ± 0.4 3.0 ±0.5 3.4 ± 0.6 3.8 ±0.7 a-pan AMPK Activity (pmol [P] incorporated/ min/ mg protein) 4.0 ± 0.9 4.0 ± 0.9 3.6 ± 0.5 3.3 ± 0.4 Myocard ia l Energet ics and A M P K Activity. Data represent means ± S E M . Data were ana lysed using one-way A N O V A with Neumann-Keu ls post-hoc test. + = significantly different from corresponding untreated group (p<0.05) (C=control, n=5; C P = control perfused with metoprolol, n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol). 72 FIGURE 5 A C C and M C D Express ion and Phosphorylat ion. A : Express ion of A C C and M C D measured by Western blotting. (C=c.ontrol; CT=control treated; D=diabetic; DT=diabetic treated). B: Phosphorylat ion of A C C measured by Weste rn blotting. Band intensity was quantified using ImageJ software. *= significantly different, p<0.05. (C=control; CT=control treated; D=diabetic; DT=diabetic treated). 74 FIGURE 6 C P T - 1 Activity and Malonyl C o A Sensitivity. A : C P T - 1 Activity in whole t issue homogenates . Data represent means ± S E M . Data were ana lysed using one-way A N O V A with Neumann-Keu ls post-hoc test. + = significantly different from * corresponding untreated group, = significantly different from all other groups (p<0.05) (C=control, n=5; C P = control perfused, n=5 (Cone of metoprolol?); CT=control treated, n=5; D=diabetic, n=5; D P = diabetic per fused, n=5; DT=diabetic treated, n=5) B. Malonyl C o A I C 5 0 va lues calculated following curve-fitting analys is of C P T - 1 dose- response curves. Data represent means ± S E M . Data were ana lysed using one-way A N O V A with Neumann-Keu ls post-hoc test. * = significantly different from control, + = significantly different from corresponding untreated group, p<0.05. (C=control, n=5; C P = control perfused, n=5; CT=control treated, n=5; D=diabetic, n=5; D P = diabetic perfused, n=5; DT=diabetic treated, n=5). 76 FIGURE 7 Pharmaco log ica l Effects of Metoprolol on C P T - 1 Activity. C P T - 1 Activity fol lowing incubation of control t issue homogenates with increasing concentrat ions of metoprolol and in the presence of 0, 50 or 100u.M malonyl C o A . Data represent means ± S E M (n=5). 77 4.0 -i 3.5 S- B 3.0 E 2 > Q . 2.5 i= °> ^ » ^ c |I E 1.5 CL =S O o £ 1.0 H c 0.5 0.0 - •— 0 (xm Malonyl CoA -o— 50 nmol Malonyl CoA 100|imol Malonyl CoA ' 7 / -10000 50000 0 2000 4000 6000 8000 METOPROLOL CONCENTRATION (ng/ ml) 78 III: Malonyl CoA Levels In vivo treatment with metoprolol lowered malonyl C o A levels in control hearts, as did acute treatment of control hearts with metoprolol during perfusion (Tables 2 and 5). Diabetes had no effect on malonyl C o A levels, and malonyl C o A levels in the diabetic heart remained unchanged by chronic metoprolol treatment and acute metoprolol perfusion. A C C and M C D express ion in control and diabet ic hearts was unchanged by chronic metoprolol treatment (Figure 5), and A M P K - m e d i a t e d phosphorylat ion of A C C , a s s e s s e d by phosphorylat ion of S e r 79 on A C C , was also unchanged either by metoprolol perfusion or chronic in vivo metoprolol treatment (Figure 5). Furthermore, metoprolol had no effect on the total phosphorylat ion state of A C C , a s s e s s e d by reactivity with pan-speci f ic ant i -phosphoser ine and ant i-phosphothreonine antibodies. Overal l , malonyl C o A levels did not correlate with the observed changes in the rate of fatty acid oxidat ion, and the observed dec rease in malonyl C o A levels in control hearts could not be expla ined by changes in A C C or M C D . IV: CPT-1 Activity and Malonyl CoA Sensitivity In a preliminary study, we measured the activities of C P T - 1 (entry of fatty acyl C o A to mitochondria), a c y l - C o A dehydrogenase (a p-oxidation enzyme) and citrate synthase (a T C A cycle enzyme) . Chron ic metoprolol treatment dec reased C P T - 1 activity in both control and diabetic hearts but had no effect on the other enzymes (Table 4 and Figure 6). Acu te metoprolol perfusion also dec reased C P T - 1 activity. To investigate whether metoprolol altered C P T - 1 sensitivity, we assayed C P T - 1 activity in the presence of increasing concentrat ions of malonyl C o A to obtain dose- response curves for each of the groups. The IC 5 o of malonyl C o A w a s calculated (Figure 6). Chron ic metoprolol treatment dec reased the sensitivity of C P T - 1 to malonyl C o A in diabetic, but not control, hearts. Acute metoprolol perfusion decreased the sensitivity in both control and diabet ic hearts. 79 The rightward shift in the dose- response curve was preserved when absolute activity data were plotted (data not shown). To test whether metoprolol is a direct pharmacological inhibitor of C P T - 1 , or whether it can directly interfere with malonyl C o A inhibition of C P T - 1 , we incubated C P T - 1 with increasing concentrat ions of metoprolol in the p resence of 0, 50 and 100pM malonyl C o A . Metoprolol did not inhibit C P T - 1 activity directly, and did not dec rease or enhance inhibition of C P T - 1 by malonyl C o A (Figure 7). V: Regulation of CPT-1 Expression Chron ic metoprolol treatment decreased total C P T - 1 express ion in control and diabetic hearts (Figure 8). Metoprolol dec reased C P T - 1 B express ion , but did not alter C P T - 1 A express ion, which was present at low levels (Figures 9 and 10). C h a n g e s in C P T - 1 sensitivity cannot, therefore, be attributed to a shift in C P T - 1 isoform express ion . Metoprolol did not alter the total express ion of P P A R - a , P G C 1 a or P D K - 4 in either control or diabetic hearts (Figures 11 and 12). In control hearts, metoprolol increased the express ion of U S F - 1 but the express ion of a -MHC was not significantly changed. The express ion of both U S F - 1 and U S F - 2 was dec reased in the diabetic heart, as was a -MHC. Metoprolol increased the express ion of U S F - 2 in control and diabetic hearts, but this w a s only assoc ia ted with an increase in M H C express ion in diabetic hearts; indeed, although the a-M H C band in control treated hearts was larger and more diffuse, the band was less intense in densitometric analys is, indicating that a -MHC express ion may actually have dec reased (Figures 13-15). S E R C A express ion was markedly dep ressed in diabetic hearts, and metoprolol restored S E R C A express ion . In control hearts, metoprolol dec reased the associat ion of the coactivator P G C 1 a with the transcription factors P P A R - a and M E F - 2 A without changing the 80 FIGURE 8 Total C P T - 1 express ion . Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). 18 82 FIGURE 9 C P T - 1 B (muscle isoform) express ion. Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). CPT-1 MUSCLE ISOFORM EXPRESSION (Normalised to Total Protein) o -fc §» w 4k CPT-1 MUSCLE ISOFORM EXPRESSION (Normalised to Total Protein) O O O O O - i — — b k > j > . a > b b k j j > . D CPT-1 MUSCLE ISOFORM EXPRESSION (Normalised to Total Protein) © o © © o -* b j» b b fo 70 m > - i a JO O c 13 84 FIGURE 10 C P T - 1 A (liver isoform) express ion. Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 ( O c o n t r o l , n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). CPT-1 LIVER ISOFORM EXPRESSION (Normalised to Total Protein) CPT-1 LIVER ISOFORM EXPRESSION (Normalised to Total Protein) o o o © o -* e M ^ o» OD © CPT-1 LIVER ISOFORM EXPRESSION (Normalised to Total Protein) © © 0 0 0 - » - f c - » - » - » K > b r o * b > c o b » o * c » c » b H 86 FIGURE 11 P P A R - a , P G C 1 a and P D K - 4 , express ion following chronic treatment with metoprolol . Densitometr ic ana lyses of these data are presented in Figure 12. (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). CT PPAR-a PGC1a PDK-4 Ponceau PPAR-a PGC1a PDK-4 Ponceau DT * PPAR-a PGC1a D PDK-4 Ponceau 88 FIGURE 12 Densitometr ic analys is of P P A R - a , P G C 1 a and PDK -4 , express ion following chronic treatment with metoprolol. Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). PDK-4 EXPRESSION (Normalised to Total Protein) PGC-1a EXPRESSION (Normalised to Total Protein) PPAR-a EXPRESSION (Normalised to Total Protein) PDK-4 EXPRESSION (Normalised to Total Protein) PGC-1a EXPRESSION (Normalised to Total Protein) PPAR-a EXPRESSION (Normalised to Total Protein) 68 90 FIGURE 13 U S F - 1 , U S F - 2 , M H C and S E R C A - 2 express ion following chronic treatment with metoprolol . Densitometr ic ana lyses of these data are presented in F igures 14 and 15. (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). 91 CT USF-1 USF-2 MHC SERCA Ponceau DT 92 FIGURE 14 Densitometr ic analys is of U S F - 1 and U S F - 2 express ion following chronic treatment with metoprolol. Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). USF-2 EXPRESSION (Normalised to Total Protein) USF-1 EXPRESSION (Normalised to Total Protein) 7> m > H s m USF-2 EXPRESSION (Normalised to Total Protein) USF-1 EXPRESSION (Normalised to Total Protein) P p 7* ^ NJ NJ W b "ui b ui b ui b > s m z JO m > — i m z USF-2 EXPRESSION (Normalised to Total Protein) O U l O Ul O U l USF-1 EXPRESSION (Normalised to Total Protein) p o -» -k NJ ro o i n b i n o i n b 94 FIGURE 15 Densitometr ic analys is of M H C and S E R C A express ion following chronic treatment with metoprolol. Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). SERCA EXPRESSION (Normalised to Total Protein) o in o in b MYOSIN HEAVY CHAIN EXPRESSION (Normalised to Total Protein) p p ^ -» M ro p eft o en b in JO m > H 3 m z SERCA EXPRESSION (Normalised to Total Protein) MYOSIN HEAVY CHAIN EXPRESSION (Normalised to Total Protein) m w > -i 3 m z H CD JO 8 3 •o m w > -i _ m z — i o JO 8 5 •o SERCA EXPRESSION (Normalised to Total Protein) O O -k -» K> JO b in b in p in MYOSIN HEAVY CHAIN EXPRESSION (Normalised to Total Protein) JO m > -m z H G) JO m > -i 3 m z 96 FIGURE 16 Binding of P P A R - a , M E F - 2 A and U S F - 2 to P G C 1 a , and of M E F - 2 A and U S F - 2 to P P A R - a measured by immunoprecipitat ion. S a m p l e s underwent immunoprecipitat ion with anti- P G C 1 a ant ibodies as indicated, and were subsequent ly subjected to immunoblotting for P P A R - a , M E F - 2 A or U S F - 2 . S a m p l e s a lso underwent immunoprecipitation with an t i -PPAR - a ant ibodies and immunoblott ing with anti- M E F - 2 A and U S F - 2 ant ibodies as indicated. Densi tometr ic ana lyses of these data are presented in Figure 17. (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). 97 C CT D DT IP: PGC1a Blot: PPAR-a IP: PGC1a Blot: MEF -2A IP: PGC1a f * • fc • » 4 M I ! • § I N I i Jlt__> j——^ _ ^ _•»_•. - ja» :\Z Blot: USF-2 IP 1 » wm^^f WBK^f • • • p*t"W PJMmf H i W pjVlPJPlHP**1^ C CT D DT IP: PPAR-a Blot: MEF -2A 9 8 FIGURE 17 Densitometr ic analys is of P G C 1 a binding. Data represent means ± S E M . Data were ana lyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. * = significantly different from untreated group, # = significantly different from all groups, + = significantly different from C and D, (p<0.05). (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol , n=5). 99 100 binding of U S F - 2 . In the diabetic heart, the associat ion, of P G C 1 a with P P A R - a w a s enhanced but its assoc ia t ions with M E F - 2 A and U S F - 2 were abo l ished. Metoprolol dec reased the associat ion between P G C 1 a and P P A R - a in the diabetic heart but the associat ion between P G C 1 a and M E F - 2 A increased. However, in the diabetic heart, metoprolol markedly increased the binding of U S F - 2 to P G C 1 a (Figures 16 and 17). VI: 3-Adrenoceptor Signalling Chron ic treatment with metoprolol increased 31 receptor express ion in control hearts. In the diabetic heart, 31 receptor levels were dec reased , and chronic metoprolol treatment increased 31 receptor levels (Figures 18 and 19). 32 receptor levels were increased in both control and diabetic hearts, but d iabetes itself did not alter 32 receptor express ion (Figures 18 and 19). Both metoprolol treatment and diabetes induced marked increases in the express ion of the 33 receptor (Figure 14). Overal l , metoprolol caused a shift in 3-receptor express ion towards p2 and p3 in control hearts, and increased the express ion of all three receptor subtypes in the diabetic heart. There was no clear shift in the assoc iat ion of the p2 adrenoceptor with G s or G i in any of the groups (Figure 20). The activity of P K A , as expected, was decreased by metoprolol (Table 7). By contrast, the activity of the PI3K/ Akt pathway was increased by metoprolol Figure 21), whereas C A M K activity was not significantly altered (Table 7). At tempts to measure t issue peroxynitrite levels and e N O S and i N O S 101 FIGURE 18 Express ion of (5-Adrenoceptor subtypes following chronic metoprolol treatment. Densi tometr ic ana lyses of these data are presented in Figure 19. (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). 102 C CT P1 AR P2 AR P3 AR Ponceau DT P1 AR p2 AR p3 AR Ponceau P1 AR P2 AR P3 AR Ponceau 103 FIGURE 19 Densitometr ic analys is of express ion of p-Adrenoceptor subtypes following chronic metoprolol treatment. Data represent means ± S E M . Data were ana lysed using an unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol, n=5). B3 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) B2 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) B1 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) 2 o B3 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) B2 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) B1 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) o o B3 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) B2 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) B1 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein) 71 o 105 FIGURE 20 Binding of G s and G i to p2 -Adrenoceptors. Densitometr ic analys is was carried out and data ana lyzed using an one-factor A N O V A with Neumann-Keu ls post hoc test, (data not shown). There were no significant dif ferences between groups. (C=control, n=5; C P = control perfused with metoprolol, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol , n=5; DT=diabetic treated with metoprolol, n=5). C T D T IP: P2 AR Blot: Gs IP: P2 AR Blot: Gi IP: p2 AR Blot: P2 AR tn ^ • w • if » < P » ^ « o 3> 107 FIGURE 21 A . Akt phosphorylat ion. B. Densitometr ic analys is of Akt phosphorylat ion. Data represent means ± S E M . Data were ana lyzed using one-factor A N O V A with Neumann-Keu l s post hoc test. * = significantly different from C and C P , p<0.05. (C=control, n=5; C P = control perfused with metoprolol, n=5; CT=control treated with metoprolol , n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5). 108 109 TABLE 7 TISSUE ACTIVITIES OF PKA AND CAMK C C P C T D DP D T P K A Activity 828 + 52 ± 214 ± 792 ± 276 ± 454 ± (pmol A T P incorporated/ 279 8* 42* 281 36 + 143 min/ mg protein) C A M K Activity 611 ± 563 ± 714 ± 426 ± 388 ± 325 ± (pmol A T P incorporated/ 90 94 90* 118 102 96 min/ mg protein) P K A and CAMK- I I were purified by immunoprecipitat ion prior to assay . Data represent m e a n s + S E M . Data were ana lysed using O n e - W a y A N O V A with Neumann Keu ls post-hoc test, * = significantly different from C, + = significantly different f rom D (p<0.05). (C=control, n=5; C P = control per fused with metoprolol , n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; D P = diabet ic perfused with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5). 110 activity were unsuccessfu l . W e therefore used a combinat ion of western blotting, b iomarkers and indirect measures of N O S activity to determine how metoprolol inf luences the generat ion of peroxynitrite (Figures 22-24). Metoprolol dec reased the express ion of e N O S in control hearts (Figure 22). Acu te metoprolol perfusion decreased Akt- and PKA-med ia ted phosphorylat ion of e N O S in control hearts at Se r 1177 and Thr 495 respectively, but the dec rease was not sustained by chronic treatment (Figure 23). Functional ly, N O production, as indicated by nitrate/ nitrite levels, was increased by metoprolol despi te the dec rease in e N O S express ion (Figure 24). In diabetic hearts, e N O S express ion was very low but i N O S was induced. Metoprolol prevented the induction of i N O S without restoring e N O S (Figure 22). Akt-mediated phosphorylat ion of e N O S was not detected in diabetic hearts. PKA-med ia ted phosphorylat ion of e N O S was high, and was dec reased only by chronic metoprolol treatment (Figure 23). N O production, as indicated by nitrate/ nitrite levels, was low in the diabetic heart and was not altered by metoprolol (Figure 24). Total protein glutathiolation, a biomarker of N O and reactive nitrogen spec ies , was increased by acute and chronic metoprolol treatment in control hearts. In diabetic hearts, total protein glutathiolation was low, and was dec reased further by chronic metoprolol treatment. Total protein tyrosine nitration, a biomarker of peroxynitrite, was unchanged by metoprolol in control hearts, and was unchanged in diabetic hearts as compared to control.. In diabetic hearts, chronic metoprolol treatment produced a marked dec rease in tyrosine nitration (Figure 24). I l l FIGURE 22 A . Express ion of e N O S and i N O S . B. Densitometr ic analys is of e N O S express ion. Data represent means ± S E M . Data were ana lyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. + = significantly different from C , D and DT, * = significantly different from C and C T , (p<0.05). (C=control, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; DT=diabetic treated with metoprolol , n=5). 112 A. C CT D DT eNOS iNOS * #»••"» Ponceau i i mm* img m ti> M i to* HI mm 'm^m^. m^mf mm B. C CT D DT TREATMENT GROUP 113 FIGURE 23 Phosphory lat ion of e N O S at S e r 1177 and Thr 495 . Data represent m e a n s ± S E M . S e r 1177 phosphorylat ion was not detected in diabetic hearts (data not shown). Data were ana lyzed using one-factor A N O V A with Neumann -Keu l s post hoc test. * = significantly different from other groups, (p<0.05). (C=control, n=5; C P = control per fused with metoprolol, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5). C CP CT C CP CT TREATMENT GROUP C CP CT C CP ( TREATMENT GROUP 115 FIGURE 24 Biomarkers of N O and R N S . A : T i ssue nitrate and nitrite levels. Data represent means ± S E M . Data were analyzed using one-factor A N O V A with Neumann -Keu ls post hoc test. * = significantly different from C groups, # = significantly different from C , (p<0.05). B: Total protein glutathiolation measured by Dot Blotting. Data were analyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. * = significantly different from all groups, # = significantly different from C , (p<0.05). C : Total protein tyrosine nitration measured by Dot Blotting. Data were ana lyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. * = significantly different from all groups, (p<0.05). (C=control, n=5; C P = control per fused with metoprolol, n=5; CT=control treated with metoprolol , n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5). o CO TOTAL PROTEIN TYROSINE NITRATION (Normalised to Total Protein) 73 m > m z H O o 73 O TOTAL PROTEIN GLUTATHIOLATION (Normalised to Total Protein) NITRATE + NITRITE LEVELS (nmol/ mg protein) K» 117 VII: CPT-1 Covalent Modifications Phosphory lat ion of C P T - 1 was detected by immunoprecipitat ion, and the total phosphorylat ion state of C P T - 1 w a s increased by acute metoprolol perfusion in control and diabetic hearts. Cyste ine nitrosylation, glutathiolation and tyrosine nitration were all detected. Acute metoprolol perfusion increased nitrosylation and glutathiolation but abol ished tyrosine nitration (Figures 25 and 26). Having conf irmed that phosphorylat ion of C P T - 1 is detectable by immunoprecipi tat ion, we next investigated whether k inases are physical ly assoc ia ted with C P T - 1 . Both P K A and C A M K were found to co-immunoprecipi tate with C P T - 1 (Figures 27-29). Increased phosphorylat ion of A K A P - 1 4 9 was assoc ia ted with increased binding of P K A to C P T - 1 and a corresponding dec rease in A K A P - 1 4 9 binding to C P T - 1 . Converse ly , as A K A P -149 phosphorylat ion dec reased , P K A binding to C P T - 1 dec reased and the assoc ia t ion between C P T - 1 and A K A P - 1 4 9 increased. In control hearts, acute metoprolol perfusion increased the phosphorylat ion of A K A P - 1 4 9 and the binding of P K A to C P T - 1 , while A K A P - 1 4 9 binding to C P T - 1 was decreased . These changes persisted with chronic treatment. C A M K binding to C P T - 1 was not decreased acutely, but chronic treatment with metoprolol did produce a modest dec rease in C A M K binding to C P T - 1 . In diabetic hearts, a different pattern is observed. A K A P - 1 4 9 binding to C P T - 1 was low in diabetic hearts, and metoprolol increased its binding. Metoprolol modest ly dec reased C A M K binding acutely in diabetic hearts. Th is dec rease persisted and became more marked following chronic metoprolol treatment. W h e n P K A and C A M K were incubated with isolated mitochondria, both k inases bound to and phosphorylated C P T - 1 . By contrast, Akt neither bound nor 118 FIGURE 25 Covalent modif ications of C P T - 1 measured by immunoprecipitat ion. S a m p l e s underwent immunoprecipitat ion with combined pan-speci f ic ant i -phosphoser ine and ant i -phosphothreonine antibodies, anti-glutathione ant ibodies or anti-ni trosocysteine antibodies, fol lowed by immunoblotting with pan-speci f ic C P T - 1 ant ibodies. Densitometr ic ana lyses of these data are presented in Figure 26. (C=control, n=5; C P = control perfused with metoprolol, n=5; CT=control treated with metoprolol , n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5). IP: Phosphoserine/ threonine Blot: CPT-1 IP: Glutathione Blot: CPT-1 IP: Nitrosocysteine Blot: CPT-1 IP: Nitrotyrosine Blot: CPT-1 IP: Phosphoserine/threonine Blot: CPT-1 IP: Glutathione Blot: CPT-1 IP: Nitrosocysteine Blot: CPT-1 IP: Nitrotyrosine Blot: CPT-1 120 FIGURE 26 Densitometr ic analys is of C P T - 1 covalent modif ications. Data represent means ± S E M . Data were ana lyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. * = significantly different from C or D (p<0.05). (C=control, n=5; C P = control perfused with metoprolol, n=5; CT=control treated with metoprolol , n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5). 121 122 FIGURE 27 Co-immunoprecip i tat ion of P K A and A K A P - 1 4 9 with C P T - 1 , and phosphorylat ion state of A K A P - 1 4 9 . (C=control, n=5; C P = control perfused with metoprolol, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; D P = diabetic perfused with metoprolol , n=5; DT=diabetic treated with metoprolol, n=5). IP: CPT-1 Blot: AKAP 149 IP: Phosphoserine/threonine Blot: AKAP 149 IP: CPT-1 Blot: PKA IP: CPT-1 Blot: AKAP 149 IP: Phosphoserine/threonine Blot: AKAP 149 IP: CPT-1 Blot: PKA 124 FIGURE 28 Densitometr ic analys is of the binding of P K A and A K A P - 1 4 9 to C P T - 1 , and phosphorylat ion state of A K A P - 1 4 9 . Data represent means ± S E M . Data were ana lyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. * = significantly different from C or D, # = significantly different from C and C P or D and D P (p<0.05). 126 FIGURE 29 Binding of CAMK- I I to C P T - 1 . Data represent means ± S E M . Data were ana lyzed using one-factor A N O V A with Neumann-Keu ls post hoc test. * = significantly different from C or D, # = significantly different from C and C P or D and D P (p<0.05). 127 128 FIGURE 30 Phosphory lat ion of C P T - 1 by P K A in isolated mitochondria. Data represent means + S E M . Phosphorylat ion, k inase binding, densitometr ic analys is of phosphorylat ion, malonyl C o A dose response and catalytic activity following incubation of isolated mitochondria with P K A . Data were ana lyzed using student 's t-test. * = significantly different (p<0.05). n=4 for each intervention. 130 FIGURE 31 Phosphory lat ion of C P T - 1 by C A M K in isolated mitochondria. Data represent means ± S E M . Phosphorylat ion, k inase binding, densitometr ic analys is of phosphorylat ion, malonyl C o A dose response and catalytic activity following incubation of isolated mitochondria with C A M K . Data were ana lyzed using student 's t-test. * = significantly different (p<0.05). n=4 for each intervention. 131 CONTROL + CAMK IP: Phosphoserine/ threonine I Blot: CPT-11 Control +CAMK TREATMENT GROUP 132 FIGURE 32 Phosphory lat ion of C P T - 1 by Akt in isolated mitochondria. Data represent means + S E M . Phosphorylat ion, k inase binding, densitometr ic analys is of phosphorylat ion, malonyl C o A dose response and catalytic activity following incubation of isolated mitochondria with C A M K . Data were ana lyzed using student 's t-test. * = significantly different (p<0.05). n=4 for each intervention. CONTROL + Akt IP: Phosphoserine/ threonine Blot: CPT-1 IP: CPT-1 Blot: Akt 200 -I 180 -Z 160 -O _ 0 20 40 60 80 100 120 140 160 180 200 MALONYL CoA CONCENTRATION (uM) 1.8 1.6 Control +Akt TREATMENT GROUP 134 FIGURE 33 Incubation of isolated mitochondria with peroxynitrite. C P T - 1 nitrosylation, glutathiolation and tyrosine nitration, and C P T - 1 activity following exposure to increasing concentrat ions of peroxynitrite. Three separate exper iments were run using the s a m e pooled mitochondrial isolate; the n-number is therefore 1. Data represent means . PEROXYNITRITE CONCENTRATION (uM) 0 0.1 1 10 100 500 1000 IP: Glutathione mmm Blot: CPT-1 IP: Nitrosocysteine Blot: CPT-1 IP: Nitrotyrosine Blot: CPT-1 -0.0 0.1 1.0 10.0 100.0 500.0 1000.0 PEROXYNITRITE CONCENTRATION (LIM) 136 phosphorylated C P T - 1 , and had no effect on C P T - 1 activity or sensitivity. P K A -mediated phosphorylat ion of C P T - 1 decreased the sensitivity of C P T - 1 to malonyl C o A without affecting activity. C A M K - m e d i a t e d phosphorylat ion of C P T -1 increased the sensitivity of C P T - 1 to malonyl C o A without affecting activity (Figures 30-32). Having confirmed that functionally-significant phosphorylat ion of C P T - 1 does occur, we undertook a study to search for the speci f ic phosphorylat ion sites. C P T - 1 was eluted in the ammonium hydroxide fraction. C P T - 1 was definitively identified by peptide mass fingerprinting and Masco t search . However, coverage of the C P T - 1 B sequence was poor (< 10%) and only one phosphorylat ion site of interest was covered by the search . Phosphory lat ion of that site was not detected. Incubation of isolated mitochondria from control hearts with increasing concentrat ions of peroxynitrite revealed that basal levels of C P T - 1 nitration and glutathiolation were very low (Figure 33). W h e n 0.1-1 u,M peroxynitrite was added , a marked increase in glutathiolation occurred which was assoc ia ted with very modest inhibition of C P T - 1 activity. In the range 10-500 uJvl, glutathiolation was susta ined and then decreased while tyrosine nitration increased, and a dose-dependent increase in C P T - 1 activity was observed. By contrast, basa l levels of cysteine nitrosylation were high, and peroxynitrite produced a dose -dependent dec rease in cysteine nitrosylation. Loss of cysteine nitrosylation w a s assoc ia ted with an increase in C P T - 1 activity. 1mM peroxynitrite was toxic to the enzyme. Acu te metoprolol perfusion increased glutathiolation in control and diabetic hearts, and increased S-nitrosylation in diabet ic hearts only. Acu te metoprolol perfusion a lso decreased tyrosine nitration of C P T - 1 . With the except ion of tyrosine nitration in control hearts, all changes produced by acute metoprolol perfusion were sustained with chronic treatment (Figure 25). 137 DISCUSSION I: Effects of Metoprolol on Cardiac Function and Metabolism Our current knowledge of the benefits of p-blockers in diabetic heart failure is limited to patients with concurrent d iabetes and ischemia. W e have demonstrated, for the first time, that metoprolol a lso amel iorates the card iac dysfunction produced by diabetic cardiomyopathy. This improvement was evident both from Starl ing curves generated by direct left ventricular pressure measurements and from measurements of cardiac output and hydraulic power taken over the course of an hour-long perfusion at constant preload and afterload. Having confirmed that metoprolol improves card iac function, we investigated whether inhibition of fatty acid oxidation could account for this effect. W e began by investigating the effects of metoprolol on known card iac fuels. A s expected, metoprolol had no effects on p lasma g lucose levels. To account for the putative ability of p-blockers to suppress lipolysis, we measured circulating p lasma lipids. Metoprolol had no effect on p lasma free fatty ac ids, tr iglycerides or cholesterol . Surprisingly, however, metoprolol attenuated the observed increase in p lasma ketone bodies in the diabetic rats. It is not c lear why metoprolol would produce such an effect. The rate of ketogenesis is determined by the rate of fatty acid delivery to the liver, the rate of fatty acid oxidation in the liver, and the activity of mitochondrial p-hydroxy-p-methylglutaryl-CoA synthase ( H M G C o A synthase), which cata lyses the first step in ketogenesis. Stimulation of H M G - C o A synthase is the underlying mechan ism by which low insulin levels, starvation and low-carbohydrate/ low protein/ high fat diets increase ketogenesis (219). p-adrenergic stimulation is known to increase lipolysis in adipocytes. The effect is mediated partly by hormone-sensi t ive l ipase and partly by a poorly understood alternative 138 pathway (220). p-blockers, acting on the same pathway, may dec rease l ipolysis, thereby decreas ing the delivery of fatty ac ids to the heart. The fact that metoprolol treatment had no effect on circulating p lasma lipids argues against a major effect on lipolysis. However, there are two components to l ipolysis: a pulsati le re lease under the control of the sympathet ic nervous system and a basa l re lease which is independent of the sympathet ic nervous system (221); attenuation of the pulsatile re lease could be sufficient to reduce ketogenesis without affecting cholesterol synthesis by the l iver . Alternatively, the effect could be mediated by inhibition of C P T - 1 in the liver. It is a lso possib le that metoprolol increases the peripheral utilization of ketones. However, present ev idence suggests that ketone utilization by peripheral t issues is largely unregulated, being determined solely by ketone body supply (222). It is therefore difficult to postulate a mechan ism by which metoprolol could directly increase the peripheral utilization of ketones. Further studies are needed to investigate the mechan ism of this intriguing effect of metoprolol on p lasma ketones. W e next measured ex vivo fatty acid and g lucose metabol ism in the heart to explore the acute and chronic effects of metoprolol. By studying the effects of chronic metoprolol treatment, we hoped to gain insights into the susta ined changes in card iac metabol ism that accompany the improvement in card iac function. By studying the rapid effects of acute metoprolol perfusion, we hoped to gain insights into positive events that could occur in vivo immediately following the commencement of treatment, preceding the improvements in function. The pattern of changes we observed in our studies was complex and depended on the d i sease state and the duration of metoprolol exposure. The heavy rel iance of the diabetic heart on fatty acid oxidation observed in pur studies was expected and agrees with previous exper imental f indings in isolated perfused hearts; palmitate oxidation was markedly increased, glycolysis was dec reased by 5 0 % and g lucose oxidation was negl igable (27; 47; 60; 223-226). Surprisingly, however, myocardial energet ics, as indicated by t issue 139 adenine nucleot ide levels, were not altered. Chron ic diabetes is known to be assoc ia ted with a fall in card iac A T P production (227). It is possib le that six weeks of d iabetes is too soon to observe a fall in A T P levels as this is the timepoint at which cardiac dysfunction first appears. Furthermore, we did not observe any activation of A M P K in the diabetic heart. This is consistent with previous reports (74; 228), and a recent study suggested that A M P K activation is prevented by high circulating and t issue lipids (228). However, circulating fatty ac ids were only mildly elevated in our studies. Contrary to expectat ions, we observed that chronic metoprolol treatment increased palmitate oxidation and decreased g lucose oxidation in control hearts. However, in diabetic hearts, chronic metoprolol treatment had the expected effect of lowering fatty acid oxidation and increasing g lucose oxidation. Before attempting to resolve this apparent paradox, it was important to establ ish whether the main target of metoprolol was in the fatty acid or the g lucose oxidation pathway. In a preliminary study, we found that chronic metoprolol treatment had no effect on glycolysis, but increased coupling between g lucose oxidation and glycolysis in the diabetic heart by increasing g lucose oxidation. To determine whether the observed changes in g lucose oxidation were direct, or mediated through the Rand le Cyc le by direct changes in fatty acid oxidation, we repeated the perfusions in the absence of insulin to reduce g lucose uptake and utilization to low levels. W h e n this was done, the effect of metoprolol on g lucose oxidation was abol ished while the effect on palmitate oxidation was preserved. This strongly suggests that fatty acid oxidation is the direct target of metoprolol. It a lso indicates that the effect of metoprolol is independent of insulin. Short term perfusion with metoprolol inhibited fatty acid oxidation and produced marked stimulation of g lucose oxidation in both control and diabetic hearts which was assoc ia ted with a decrease in lactate production, reflecting a marked improvement in glycolytic/ g lucose oxidation coupl ing, and an increase in t issue A T P levels. W h e n the perfusions were repeated in the absence of insulin, 140 the effect of metoprolol on g lucose oxidation was attenuated in control hearts and abol ished in diabetic hearts. However, the effect on palmitate oxidation was preserved. O n c e again, this suggests that fatty acid oxidation is.the direct target of metoprolol, and inhibition of fatty acid oxidation occurs immediately following exposure to the drug. Intriguingly, we found that acute metoprolol perfusion and chronic metoprolol treatment lowered t issue triglyceride levels regardless of whether fatty acid oxidation was increased or dec reased . This effect cannot be expla ined on the basis of fatty acid oxidation changes alone. Indeed, inhibition of C P T - 1 by metoprolol in dogs produced an increase in t issue triglycerides (126); treatment of rats with C P T - 1 inhibitors a lso increased t issue triglyceride levels (229). However, our t issue triglyceride measurements were carried out in hearts that had been perfused ex vivo. The heart is known to utilize its endogenous triglyceride pool over the course of an ex vivo perfusion, which may partly account for the difference. Nevertheless, inhibition of C P T - 1 would be expected to dec rease the utilization of fatty ac ids from all sources , so the dec rease in t issue triglyceride levels following metoprolol treatment is unlikely to be attributable to increased triglyceride utilization. It is possib le that metoprolol dec reases the uptake of fatty ac ids into the cytoplasm. Uptake of long chain fatty ac ids into the cytoplasm is known to be stimulated by contraction, but the effect is likely to be mediated by A M P K (which was unaffected in our studies) and P K C isoforms (230). Triglyceride levels can be decreased by the secret ion of l ipoproteins by the heart itself. Indeed, overexpression of apol ipoprotein B (apo B) prevents triglyceride accumulat ion in' the diabetic heart (231; 232). However, there is presently no ev idence to suggest that p-adrenoceptors regulate this p rocess . The p-blocker propanolol was reported to induce an increase in C P T - 1 activity in normal Sprague-Dawley rats (233). Metoprolol, by contrast, was reported to dec rease C P T - 1 activity in consc ious dogs with micro-embol ism-141 induced heart failure (126). In dogs with pacing- induced heart failure, g lucose uptake was improved by carvedi lol but not metoprolol (234). However , in cl inical studies, metoprolol , carvedilol and bucindolol (122-124) have all been shown to inhibit fatty acid oxidation. The wide variation in responses reported in the literature reflects the complexity observed in our own studies in which the effect of metoprolol on fatty acid oxidation varied according to the length of exposure to the drug and the d isease state. A n increase in diastolic filling increases cardiac work and oxygen consumpt ion in direct proportion via the Frank-Starl ing mechan ism. However, in the normal heart, A T P supply is maintained at a s teady level regard less of card iac work or oxygen consumpt ion. This means that card iac metabol ism is driven by card iac function (51). What is less clear, however, is how card iac function inf luences cardiac energy substrate selection. It is possib le that some of the beneficial effects of metoprolol on card iac metabol ism may be attributable to, rather than responsib le for, its effects on cardiac function. W h e n palmitate and g lucose oxidation rates were normal ized to card iac function, the pattern of changes observed was preserved, and, in the case of palmitate oxidation, even accentuated. However, to fully account for effects of function on metabol ism, future studies are needed to investigate whether the effect of metoprolol is preserved in isolated cardiomyocytes, in which the effects of card iac function and the Frank-Star l ing mechan ism do not apply. To identify the second-messenger signall ing pathways involved in this response, we employed both 'top down' (known B-adrenoceptor signall ing pathways) and 'bottom up' (known regulatory enzymes of card iac metabol ism) approaches . W e had establ ished that metoprolol acts directly on fatty, acid oxidation. In a preliminary study, we found that neither diabetes nor chronic metoprolol treatment had any effect on the activities of acy l -CoA dehydrogenase or citrate synthase. Based on these f indings, and previous reports of the effects 142 of p-blockers on C P T - 1 , we investigated whether the observed effects of metoprolol on fatty acid oxidation are mediated by C P T - 1 . II: CPT-1 Activity and Regulation by Malonyl CoA W e hypothesized that metoprolol would increase malonyl C o A levels by decreas ing the phosphorylat ion of A C C . However, malonyl C o A levels were dec reased by metoprolol in control hearts and were unchanged in diabetic hearts. The mechan ism of this effect is unclear, because A C C and M C D express ion were unchanged, and we found no ev idence of changes in A M P K or PKA-med ia ted phosphorylat ion of A C C . Dobutamine, a non-select ive p-agonist, was previously found to dec rease malonyl C o A levels without an effect on A M P K , A C C or M C D (235; 236). In addition to the activities of A C C and M C D , malonyl C o A levels are a lso dependent on the cytosol ic supply of acetyl C o A (236). Most of the acetyl C o A in the cardiomyocyte is present in the mitochondria (237), and cytosol ic acetyl C o A is derived from peroxisomal p-oxidation, citrate and acetylcarnit ine (238). Intriguingly, acute inhibition of C P T - 1 has been shown to produce a fall in malonyl C o A levels independent of A C C and M C D (238). The fall in malonyl C o A levels observed in control hearts could, therefore, have been secondary to the inhibition of C P T - 1 . It is unclear why such a mechan ism would only lower malonyl C o A levels in control hearts. One possibil ity is that fatty acid ox idat ion, rates, and therefore the acetyl C o A / C o A ratio, are higher in the diabetic heart, and the fall in cytosol ic acetyl C o A levels produced by C P T - 1 inhibition in this context may not be sufficient to dec rease malonyl C o A levels. Metoprolol tended to dec rease t issue acetyl C o A levels in our studies, but measurements of the cytosol ic and mitochondrial acetyl C o A pools would be required to confirm these speculat ions. Overal l , however, malonyl C o A levels did not correlate with the observed changes in fatty acid oxidation. The action of metoprolol, therefore, could not be explained solely on the basis of malonyl C o A regulation. 143 W e next investigated the effects of metoprolol on C P T - 1 itself. Metoprolol dec reased the max imum capacity of C P T - 1 activity as measured in vitro. Th is effect w a s observed following both short-term perfusion with metoprolol and chronic metoprolol treatment, and was seen in both control and diabetic hearts. S ince al losteric effects are lost during sample preparation, these effects could only be expla ined by a decrease in C P T - 1 express ion or a covalent modif ication. Surprisingly, metoprolol a lso decreased the sensitivity of C P T - 1 to malonyl C o A . To our knowledge, this is the first study to demonstrate that regulation of C P T - 1 sensitivity occurs in the heart. Long-term changes in C P T - 1 catalytic activity and malonyl C o A sensitivity were previously bel ieved to occur only in the liver (239; 240). Taken together, the time and d isease-dependent changes in fatty acid oxidation can be descr ibed as follows. In control hearts, acute metoprolol perfusion c a u s e s malonyl C o A levels to fall. The sensitivity of C P T - 1 to malonyl C o A dec reases , and the activity of C P T - 1 is markedly dec reased . With chronic treatment, malonyl C o A levels remain low but the sensitivity of C P T - 1 to malonyl C o A is restored and the inhibition of C P T - 1 activity is less marked. Fatty acid oxidation is therefore inhibited following acute exposure to the drug, but this effect is lost with time. In diabetic hearts, acute metoprolol perfusion markedly reduces C P T - 1 activity. With chronic treatment, this reduction is susta ined and produces inhibition of fatty acid oxidation despite a concomitant dec rease in malonyl C o A sensitivity. The major determinants of the fatty acid oxidation rate are C P T - 1 activity and malonyl C o A levels. Using metabol ic control analys is, it has been shown that C P T - 1 only becomes rate-limiting when its activity is inhibited by approximately 5 0 % (241). Consistent with this observat ion, in our studies, fatty acid oxidation was a lways inhibited if C P T - 1 activity was inhibited by approximately 50%. The observed changes in C P T - 1 sensitivity would be expected to increase flux through C P T - 1 ; however, they may represent a fine tuning mechan ism of the system s ince at no point do they hold sway over the overall fatty acid oxidation rate. 144 Both C P T - 1 A and C P T - 1 B are present in the heart (242; 243). The net IC50 of malonyl C o A in the heart is intermediate between the high sensitivity of C P T - 1 B and the low sensitivity of C P T - 1 A ; in our studies, the I C 5 0 of control hearts w a s approximately 30ufVl malonyl C o A . Catalyt ic activity and malonyl C o A sensitivity could change for severa l reasons. Firstly, total C P T - 1 express ion could be altered. Second ly , isoform switching between C P T - 1 A and C P T - 1 B could alter sensitivity; the fetal heart exp resses C P T - 1 A , and C P T - 1 B express ion is asser ted during development, eventually becoming the major isoform (243). However, C P T - 1 isoform switches in the adult heart have not been reported. Finally, two spl icing variants of C P T - 1 have been identified in the heart which are predicted to be malonyl CoA- insens i t ive (244; 245). The N- and C - termini of C P T - 1 both face the cytosol, separated by a loop region inserted into the outer mitochondrial membrane which contains two membrane spanning domains. The C-terminus is the catalytic region, and res idues which regulate malonyl C o A sensitivity have been found within the C -terminus, the N-terminus and the loop region (246-249). In the liver, regulation of C P T - 1 A sensitivity is more important than regulation of malonyl C o A levels, and has been attributed to regulation by cytoskeletal e lements (250), changes in the membrane environment (251) and direct phosphorylat ion of C P T - 1 (177). Peroxynitr i te-mediated nitration of C P T - 1 B has been shown to dec rease C P T - 1 B catalytic activity following endotoxemia in the heart (181). However, no other covalent modif ications of C P T - 1 B have been identified. W e therefore pursued two l ines of enquiry. Firstly, we investigated the effects of chronic metoprolol treatment on C P T - 1 A and C P T - 1 B express ion. Secondly , we investigated the effects of short-term metoprolol perfusion and chronic metoprolol treatment on C P T - 1 B covalent modif ications. 145 III: Regulation of CPT -1 Expression Chron ic metoprolol treatment decreased total C P T - 1 express ion in the heart, and this was attributable to a decrease in C P T - 1 B express ion . The dec rease was only seen in diabetic hearts. C P T - 1 A was detected at low levels, but its express ion was not altered either by diabetes or by metoprolol. The protein express ion of P P A R - a and P G C 1 a remained unchanged, as did the express ion of the P P A R - a target, P D K - 4 . However, when we investigated the binding of P G C 1 a to the transcription factors it coact ivates and to U S F - 2 , we uncovered s o m e intriguing associat ive changeswhich suggest an explanat ion for the changes in C P T - 1 B express ion. The data obtained to date only establ ish associat ive effects. B a s e d on these data, we propose the following model . In control hearts, U S F - 2 maintains a constant level of tonic repression of C P T - 1 express ion, and C P T - 1 express ion is modulated through the activation of P G C 1 a and P P A R - a . This produces modest changes in C P T - 1 express ion. Even though U S F - 1 and 2 express ion are increased by metoprolol in control hearts, M H C express ion is unaffected, indicating that U S F activity is unchanged. In the diabetic heart, U S F express ion , and U S F activity as indicated by M H C express ion, are both dec reased in the diabetic heart, and metoprolol increases U S F express ion and activity. The result is that tonic repression of P G C 1 a by U S F - 2 is lost in the diabetic heart, and restoration of U S F - 2 repression produces marked changes in C P T - 1 express ion . T h e s e effects are summar ized in scheme 5. The role of U S F - 2 could be tested in t ransgenic mice with U S F - 2 knockout targeted to the heart, or alternatively in a condit ional knockout model . Alternatively, U S F - 2 could be s i lenced using an interfering R N A approach. If U S F - 2 mediates repression of C P T - 1 by metoprolol , the effect would be attenuated or lost following U S F - 2 knockout and mimicked by U S F - 2 overexpress ion. Furthermore, we would expect U S F - 2 knockout to be assoc ia ted with an increase in C P T - 1 express ion. W e were able to demonstrate 146 that both U S F - 2 and M E F - 2 A co-immunoprecipi tate with P P A R - a , suggest ing that PGC1a, P P A R - a , M E F - 2 A and U S F - 2 could form a single transcriptional complex. In a recent study, Moore et al demonstrated that P G C 1a/ M E F 2 A -dependent induction of C P T - 1 was repressed by U S F - 2 in isolated card iomyocytes (173). W e have demonstrated that binding of U S F - 2 to PGC1a occurs in the heart with the native proteins, and that this is assoc ia ted with functionally signif icant repression of C P T - 1 express ion: Our results suggest that binding of U S F - 2 can be induced by the transcriptional activation of U S F - 2 itself, s ince U S F - 2 binding to PGC1a a lways changed in the same direction as U S F activity. U S F is activated by increases in electrical stimulation (176). It is therefore likely that activation of U S F by metoprolol is mediated by the increase in electr ical stimulation that accompan ies the improvement in function; this expla ins why the effect is only seen in the diabetic heart. Funct ion did not change in control hearts. However, the more global regulation of the PGC1a transcript ional complex observed in our studies is not expl icable solely on the bas is of U S F binding. The decrease in PGC1a associat ion with P P A R - a and M E F 2 A could be an indirect effect of the acute changes in fatty ac id metabol ism. However, it is more likely that active regulation of the complex is occurr ing. Phosphory lat ion of p38 mitogen-activated protein k inase ( M A P K ) increases both P G C W P P A R - a coactivat ion and downstream signal ing to PGC1a and P P A R - a targets (252-255). It has been suggested that phosphorylat ion by p38 M A P K may serve to integrate and coordinate contractile and metabol ic gene express ion (173). Act ivat ion of (32-adrenoceptors in the heart has been shown to increase signal ing through the p38 M A P K pathway (256). It is therefore poss ib le that metoprolol dec reases p38 phosphorylat ion by blocking p2-adrenoceptors, leading to a dec rease in the associat ion of PGC1a with its coact ivators. Further 147 SCHEME 5 Proposed mechan ism of action of metoprolol: chronic metoprolol treatment dec reases the activation of PGCIoc , possibly by preventing p38 activation, and increases the repression of P G C 1 a by U S F - 2 (abbreviations: P P A R - a : perox isome proliferator activated receptor - a ; P G C 1a: P P A R - y coactivator 1a; M E F = myocyte enhancer factor; U S F = upstream stimulatory factor, C P T - 1 = carnit ine palmitoyltransferase-1.) METOPROLOL P2 Adrenoceptor 1 p38 ft Function i USF-2 P G C l a PGGla MEF-2A P G C l a PPAR-a I CPT-1 4-oo 149 studies are required to investigate the role of s t ress-k inase signal ing in the regulation of the P G C l a transcriptional complex. a -MHC express ion is dec reased as part of the fetal gene program induction. In the diabetic heart, a fall in both a -MHC and S E R C A express ion was observed, both of which were improved by metoprolol. This improvement in fetal gene program express ion is consistent with what is known about the mechan ism of action of p-blockers. a -MHC is regulated by U S F ' s , while S E R C A has been shown to be induced by M E F - 2 A (257) and is proposed to be induced by P P A R -<x (89). It is therefore possib le that the P G C l a / P P A R a / M E F 2 A / U S F complex may be able to prevent or reverse the induction of at least some components of the fetal gene program. In other words, improvement of gene express ion and modulat ion of cardiac metabol ism could occur in parallel as a result of modulat ion by the same transcriptional complex. It is not c lear whether D N A binding of P P A R - a or M E F - 2 A was altered by metoprolol treatment; further exper iments are required to measure occupancy of P P R E and M E F - 2 A binding si tes. IV: B-Adrenoceptor Signalling Pathways: Modulation of Kinases and eNOS Consis tent with previous reports, d iabetes produced a dec rease in p i -adrenoceptor express ion and a marked increase in p3-adrenoceptor-expression. Metoprolol increased the express ion of all 3 adrenoceptor subtypes. P K A activity was dec reased by both acute metoprolol perfusion and chronic metoprolol treatment, whereas PI3K activity, as indicated by Akt phosphorylat ion, was increased by metoprolol only following chronic treatment. C A M K activity was not significantly affected by metoprolol. There was no clear shift in p2-adrenoceptor assoc iat ion with G s or G i ; associat ion with both G-proteins was detected. T h e s e results indicate that, in the whole heart, the major acute effect of metoprolol is to dec rease c lass ica l c A M P / P K A signal ing. Chron ic treatment with metoprolol , in 150 addit ion, increases PI3K/ Akt signal ing, and we speculate that this is primarily due to the marked increase in (33-adrenoceptor-expression. e N O S is regulated by two main mechan isms; phosphorylat ion of S e r 1177, mediated by the PI3K/ Akt pathway, was shown to increase e N O S activity in transfected C O S cells (258; 259); phosphorylat ion of Thr 495 , mediated by P K A , partially b locks the phosphorylat ion of S e r 1177 in bovine aortic endothel ial cel ls (260). Ca lc ium-dependent translocation of e N O S from caveo lae in the p lasma membrane to calmodul in in the cytosol is a lso assoc ia ted with an increase in e N O S activity (261; 262). Activation of e N O S by p3-adrenoceptors has been shown to be due both to an increase in Se r 1177 phosphorylat ion and to translocat ion from caveo lae, but the importance of these mechan isms is region-specif ic; in atria, translocation is the predominant mechan ism whereas , in the left ventricle, phosphorylat ion is the predominant mechan ism (263). Intriguingly, p3-adrenoceptor stimulation has also been shown to uncouple e N O S and increase oxygen free radical formation (263). Our efforts to measure N O S activity in the heart were unsuccessfu l . W e therefore measured nitrate/ nitrite levels and total protein glutathiolation as biomarkers of N O and physiological reactive nitrogen spec ies ( R N S ) production respectively, and correlated these with changes in the phosphorylat ion and express ion of N O S isoforms. The pattern of changes produced for both markers was the same , with the except ion of the DT group, and can be interpreted as fol lows. In control hearts, acute metoprolol perfusion increased N O / R N S production by decreas ing the inhibitory phosphorylat ion of Thr 486, a PKA-s i t e . Stimulatory phosphorylat ion of S e r 1177 by Akt was also dec reased , but we speculate that the decrease in PKA-med ia ted phosphorylat ion exerted a greater effect on activity. Fol lowing chronic treatment with metoprolol in control hearts, N Q production remained high despite a surprising dec rease in e N O S express ion and a loss of any effect on e N O S phosphorylat ion. W e speculate that this 151 increase in activity could be due to increased e N O S translocation from caveo lae to the cytosol . In the diabetic heart, N O production is reduced and is dependent on i N O S rather than e N O S . i N O S is not regulated by p-adrenoceptors acutely, so acute perfusion with metoprolol has no effect on N O production. Chron ic metoprolol treatment prevented the induction of i N O S without restoring e N O S express ion . The net result was that chronic treatment with metoprolol had no effect on N O production. However, as indicated by the fall in glutathiolation, prevention of i N O S induction by chronic metoprolol treatment did dec rease R N S production. The changes produced by metoprolol on the phosphorylat ion of e N O S by Akt did not correlate with the changes in Akt-phosphorylat ion produced by the s a m e treatment; this suggests that the elevated Akt signal was compartmenta l ized, and that e N O S was not its primary target. It is important to note that severa l cell types would have been present in the whole heart homogenate, including card iomyocytes and endothelial cel ls; we did not differentiate between endothel ial and cardiomyocyte N O signaling in our studies. W h e n tyrosine nitration, a marker of peroxynitrite, was measured , levels of total protein tyrosine nitration remained constant as long as either e N O S or i N O S were present. W h e n e N O S express ion w a s . l o w and i N O S was absent, total protein tyrosine nitration fell. T h e s e data indicate that nitrosative stress was not significantly increased in diabetic hearts, although e N O S express ion was low and N O levels had fal len. The fact that prevention of i N O S induction had a marked effect on R N S production but no effect on N O production suggests that i N O S was producing predominantly R N S . A s d iscussed above, N O has been reported to inhibit g lucose utilisation predominantly through inhibition of glycolysis (264). However, chronic metoprolol treatment had no effect on glycolysis in control hearts despi te the fact that it increased N O production. 152 V: NO/ RNS - Induced Covalent Modifications of CPT-1 In recent years, there has been increasing interest in the ability of N O and its assoc ia ted R N S to directly regulate protein function in a similar manner to phosphorylat ion. Res idues that are targeted by N O and R N S are cysteine, methionine and tyrosine (265; 266). The unique redox chemistry of protein thiol groups confers specificity and reversibility to thiol covalent modif icat ions. The attachment of N O to thiol groups on critical cysteine residues within a protein, termed S-nitrosylation, is a major mechan ism by which N O acts as a signal ing molecule. Intriguingly, there is a consensus sequence , ana logous to k inase consensus sequences , which confers site specificity on NO-media ted thiol modif icat ions (267). Furthermore, S-nitrosylation is a reversible reaction, and a number of enzymat ic and non-enzymat ic reactions have been identified which can remove N O from cysteine thiols (268-270). S-nitrosylation activates guanylate cyc lase , the c lass ica l N O target. The list of targets proposed to be regulated by S-nitrosylation is growing, and, in the heart, includes G A P D H and S E R C A (271). Revers ib le oxidation or nitrosation of thiol groups is mediated by physiological levels of N O and R N S , and typically produces the following reversible modif icat ions: S-nitrosylation (addition of NO) , glutathiolation (formation of mixed disulphides between the thiol group and glutathione) or oxidation from thiol to sulfenate. Any of these modif ications can regulate protein function, but glutathiolation and S-nitrosylation have been most frequently implicated in the regulation of enzyme activity (265). Higher levels of R N S induce further oxidation of the sulfenate (one oxygen) to sulfinate (two oxygens) and sulfonate (three oxygens) . This is toxic, caus ing irreversible loss of function. Glutathiolat ion, by committing the thiol to an alternate reaction pathway, protects critical thiol res idues against irreversible oxidation (265). T h e s e effects are summar ized in s c h e m e 7. 153 Tyros ine nitration is c lassical ly regarded as an inhibitory modif icat ion. However, s o m e proteins are activated by tyrosine nitration including cytochrome C , f ibrinogen and P K C (272-275). A s with thiol-modification, tyrosine nitration also exhibits site-specificity (276). Tyrosine nitration is frequently used as a biomarker of peroxynitrite (272), and we a lso used it as such . Prev ious studies have demonstrated that incubation of C P T - 1 with cont inuous peroxynitrite, N O or hydrogen peroxide producing sys tems produces a decrease in C P T - 1 activity which is assoc ia ted with tyrosine nitration (182). Furthermore, endotoxemia produced inhibition and nitration of C P T - 1 in suckl ing rats (181). Cys te ine-scann ing mutagenesis of C P T - 1 revealed that cysteine 305 is critical for catalytic activity of the enzyme (277). W e therefore tested whether nitrosylation or glutathiolation of cysteine residues, or nitration of tyrosine res idues, inhibits C P T - 1 activity. To test the effects of the modif ications per se on C P T - 1 activity, we incubated isolated mitochondria with increasing concentrat ions of peroxynitrite ranging from 100 nM to 1 m M . At neutral pH , peroxynitrite is rapidly degraded, but even brief exposure to peroxynitrite is sufficient for it to induce the full range of its target modif ications. B e c a u s e of the large amount of mitochondrial isolate required for these measurements , three dupl icate exper iments were run on samp les taken from a single mitochondrial pool , meaning that the n-number was 1. Al though the results were consistent, further exper iments are required to enable a statistical analys is to be carr ied out. Peroxynitrite induced glutathiolation at a lower concentrat ion than tyrosine nitration, and caused a decrease in S-nitrosylation. Dose-dependent loss of s-nitrosylation and gain of tyrosine nitration was assoc ia ted with a dose-dependent increase in C P T - 1 activity. This suggests that s-nitrosylation is inhibitory and tyrosine nitration stimulatory of C P T - 1 activity. 100 nM peroxynitrite produced a marked increase in glutathiolation but only a slight dec rease in C P T - 1 activity which did not prevent activation of the enzyme by higher concentrat ions. W e therefore speculate that glutathiolation of C P T - 1 serves as a protective 154 SCHEME 6 N O and RNS-med ia ted modif ications of thiol residues. Thiol (SH) res idues undergo a ser ies of reversible modif ications in response to changes in the redox potential or exposure to physiological levels of reactive nitrogen spec ies or nitric oxide. Oxidat ion of the thiol to the corresponding sulfenide or the formation of a disulphide bond between the thiol and glutathione (glutathiolation) are reversible either by changes in the equil ibrium, or enzymat ic restoration of the thiol group by thiol t ransferases. Further oxidation of a glutathiolated residue is not possib le, so glutathiolation confers protection against oxidative damage for as long as it persists. However, exposure of the thiol group or the sulfenide to pathological levels of reactive nitrogen or oxygen spec ies results in the formation of sulfinate and then sulfonate; these are irreversible modif ications which result in protein damage and loss of activity. Modif ied from Figure 2, Klatt and Lamas , 2000 (265). Glutathiolation R-SSG R - S N O ^ Z Z * R-SH ^ Z Z T R-SOH -S-Nitrosylation Unmodified Oxidation to Thiol Group Sulfenide Physiological RNS Levels Signal Transduction Protection Against Irreversible Oxidation > R - S 0 2 H • R-SO3H Oxidation to Oxidation to Sulfin ate Sulfonate • Pathological RNS Levels Protein damage Irreversible Loss of Activity 156 mechan ism against sulfonation, whereas S-nitrosylation and tyrosine nitration regulate the activity in opposite directions. Even a concentrat ion of 500 uM peroxynitrite, which can be toxic to some enzymes , stimulated C P T - 1 activity, indicating that this enzyme is well-protected against oxidative damage . It w a s surprising, though convenient, that peroxynitrite dec reased S -nitrosylation of C P T - 1 . A s d iscussed above, increasing concentrat ions of peroxynitrite promote the glutathiolation of nitrosylated thiol groups, and this is the most likely mechan ism of the dec rease we observed. The possibil i ty that tyrosine nitration of C P T - 1 could be stimulatory is surprising, consider ing that nitration of C P T - 1 was assoc ia ted with inhibition of activity following endotoxemia (181). However, the authors of that study did not measure cysteine oxidation, so it is poss ib le that sulfination or sulfonation, rather than tyrosine nitration, produced inhibition of C P T - 1 activity. In our studies, tyrosine nitration of C P T - 1 appeared following physiological doses of peroxynitrite, whereas loss of activity did not appear at 1mM. It is noteworthy that one or more of these covalent modif icat ions was present in every treatment group, and at every peroxynitrite concentrat ion. It is therefore possib le that all the modif ications were inhibitory, but to different degrees. This possibil ity could be tested by incubating isolated mitochondria with a reducing agent such as dithiothreitol and assay ing C P T - 1 activity. A l though we did not measure cysteine sulfination or sulfonation, it is likely that this kind of severe oxidation, rather than tyrosine nitration, caused the loss of C P T - 1 activity observed at 1 m M peroxynitrite. Similarly, incubation with N O and peroxynitrite-producing sys tems would produce cont inuous nitrosylation and, in the latter case , possibly more severe oxidation which a single brief exposure to peroxynitrite would not produce; this could explain why cont inuous N O or peroxynitrite exposure is always inhibitory whereas a single brief exposure to peroxynitrite produces a more complex dose- response. The physiological re levance of both types of response would depend on the temporal regulation of C P T - 1 exposure to N O and R N S . 157 W e successfu l ly detected cysteine-nitrosylation, glutathiolation and nitration of C P T - 1 in whole heart homogenates. Acu te metoprolol perfusion increased nitrosylation and glutathiolation in diabetic hearts, but dec reased tyrosine nitration in both control and diabetic hearts. In control hearts, nitrosylation was low and glutathiolation increased only following chronic treatment. Tak ing into account the fact that nitrosylation appears to be inhibitory, and nitration stimulatory, these data suggest that metoprolol acutely inhibits C P T -1 activity by increasing cysteine nitrosylation and removing tyrosine nitration of C P T - 1 . However, the mechan ism of these effects is not expl icable on the bas is of cel l-wide changes in N O and R N S production, because the observed"patterns in systemic N O / R N S and C P T - 1 covalent modif ications did not match. There is a mitochondrial isoform of N O S (mtNOS), but N O and peroxynitrite produced by m t N O S affect targets within the mitochondrial matrix and the inner mitochondrial membrane (278). C P T - 1 predominantly faces the cytosol, so it is likely that regulation of C P T - 1 by N O / R N S is mediated by e N O S and possibly i N O S . e N O S has been proposed to translocate to the mitochondria (279; 280); mitochondrial e N O S translocat ion could therefore be a major determinant of N O / R N S mediated effects on C P T - 1 . VI: Phosphorylation of CPT-1 Both acute metoprolol perfusion and chronic metoprolol treatment increased the total phosphorylat ion state of C P T - 1 . However, when P K A and CAMK- l l - in te rac t ions with C P T - 1 were examined in greater detail, an intruiging pattern emerged . Firstly, we found, for the first time, that P K A and CAMK-I I physical ly assoc ia te with C P T - 1 . Furthermore, we also found that A K A P - 1 4 9 also physical ly assoc ia tes with C P T - 1 and appears to mediate P K A binding. A K A P ' s bind the regulatory subunit of P K A , and activation of P K A occurs following re lease of the catalytic subunit. The P K A antibodies we used in our studies recognize the catalytic subunit of P K A . Phosphorylat ion of A K A P - 1 4 9 was a lways assoc ia ted with a dec rease in A K A P - 1 4 9 / C P T - 1 associat ion and an increase in 158 P K A / C P T - 1 binding. Converse ly , loss of A K A P - 1 4 9 phosphorylat ion was a lways assoc ia ted with an increase A K A P - 1 4 9 / GPT-1 binding and a dec rease in P K A / C P T - 1 binding. B a s e d on these findings, we propose the following s c h e m e . P K A is targeted to the mitochondria by A K A P - 1 4 9 . Phosphorylat ion of A K A P - 1 4 9 by P K A c a u s e s A K A P - 1 4 9 to d isassoc ia te from C P T - 1 , enabl ing P K A to bind and phosphorylate C P T - 1 . CAMK-I I binding fol lowed a different pattern, indicating that it is regulated by another, as-yet unidentified mechan ism. There are other mitochondrial A K A P s which could mediate similar effects as A K A P - 1 4 9 : for example , A K A P - 1 2 1 has also been shown to target P K A to mitochondria (281). C A M K associat ion proteins (KAPs ) , by contrast, are not local ized to mitochondria, but are found only in the sarcop lasmic reticulum and the nucleus (282). It is therefore unclear how CAMK-I I binding to C P T - 1 would be mediated. It is conce ivab le that CAMK-I I binds calmodul in assoc ia ted with other proteins which translocate to the mitochondria. For example, CAMK- I I is known to assoc ia te with e N O S (283). Alternatively, there may be mitochondrial CAMK- I I assoc iat ion proteins which have not been identified. Phosphory lat ion of C P T - 1 B has never been reported, and we speculate that this is because the k inases involved require other mediators such as A K A P -149 to be present in order to bind their targets. This was our rationale for using isolated mitochondria rather than purified enzyme preparations which are usually used for investigating enzyme phosphorylat ion. W h e n P K A was incubated with isolated mitochondria, it bound and phosphorylated C P T - 1 ; the functional effect was a dec rease in C P T - 1 sensitivity without any effect on catalytic activity. W h e n CAMK- I I w a s incubated with isolated mitochondria, it a lso bound and phosphory lated C P T - 1 ; however, the functional effect in this c a s e was an increase in C P T - 1 sensitivity without any effect on catalytic activity. By contrast, Akt did not bind or phosphorylate C P T - 1 , and had no effect on the activity or sensitivity of the enzyme. 159 The effects of metoprolol, and the basal state of the sys tem, differed between control and diabetic hearts. In control hearts, acute metoprolol perfusion and chronic metoprolol treatment increased P K A binding to C P T - 1 , Chron ic metoprolol treatment a lso modestly dec reased CAMK-I I binding to C P T - 1 . In diabetic hearts, PKA-b ind ing to C P T - 1 was decreased by metoprolol, but C A M K -II binding was also dec reased , and, following chronic treatment, the dec rease in CAMK- I I binding was marked. Taken together with our f indings in isolated mitochondria, these data explain the sensitivity changes produced by metoprolol; Metoprolol increases PKA-med ia ted desensit izat ion in control hearts, and dec reases CAMK- l l -med ia ted sensit izat ion in diabetic hearts. It is noteworthy that the binding of P K A and CAMK-I I to C P T - 1 bears no relation to the overall activities of these k inases measured in the whole heart. It is the translocation of the k inases to the mitochondria which is crucial. The mechan isms by which ( 3 -adrenoceptors might regulate such a translocation process are unknown and require further investigation. Having conf irmed that functionally significant phosphorylat ion of C P T - 1 occurs , we attempted to identify speci f ic phosphorylat ion sites on C P T - 1 . C o n s e n s u s sites were identified for P K A , C A M K I and C A M K II, so we proceeded to use L C M S M S to examine phosphorylat ion of these sites. Identifying phosphorylat ion events is chal lenging due to the labile nature of the modification and the overwhelming number of peptides generated by tryptic digest ion. In order to maximize our chances of finding the phosphorylat ion sites, we performed two purification steps. Firstly, we purified C P T - 1 by immunoprecipitat ion. Second ly , we performed phosphorylat ion enrichment on the tryptic digest. C P T - 1 was a lways found in the highly phosphorylated fraction. However, this was due to the high concentrat ion of acid ic residues in the peptides; peptides rich in acid ic res idues are a lso retained by the titanium tips. Al though coverage of C P T - 1 was sufficient to identify the presence of the protein, it was not sufficient to examine the phosphorylat ion sites of interest; all the sites except one were m issed . Severa l factors may account for this. C P T - 1 abundance in the whole-cel l 160 homogenate may have been too low. A l so , there were relatively few trypsin cutting sites on C P T - 1 . Many of the C P T - 1 peptides could have been too large to be eluted from the column during the L C M S M S procedure (peptides over 2500 Da in m a s s are retained). The yield of C P T - 1 could be improved by using mitochondrial fractions, and coverage improved by the use of an alternative digestion enzyme. In addition, the chances of detecting a phosphorylat ion event would be greater if mitochondrial preparations which have been incubated with different k inases are used because the intensity of the signal is increased and the time between the phosphorylat ion event and sample collection can be controlled more easi ly. VII: Significance of the Present Studies T h e s e studies have unraveled severa l mechan isms by which metoprolol can modulate fatty acid oxidation in the heart. Metoprolol is able to dec rease malonyl C o A levels in control hearts independent of A C C and M C D ; this effect may be related to cytosol ic acetyl C o A availability. Acutely, metoprolol dec reases C P T - 1 activity by increasing S-nitrosylation and decreas ing tyrosine nitration, and dec reases C P T - 1 malonyl C o A sensitivity by increasing P K A -mediated or decreas ing CAMK- l l -med ia ted phosphorylat ion. Fol lowing chronic treatment, these covalent modif ications are susta ined, but C P T - 1 express ion is a lso dec reased . In control hearts, metoprolol dec reases C P T - 1 express ion by decreas ing the associat ion of P G C 1 a with its coactivators. In diabetic hearts, metoprolol dec reases C P T - 1 express ion by increasing the binding of the repressor U S F - 2 to P G C 1 a . The increase in U S F - 2 binding is likely to be produced by the increase in electrical stimulation produced by the improvement in function, whereas the decrease in P G C I c c binding to its coactivators might be related to p38 phosphorylat ion. W e did not investigate p38 phosphorylat ion in the present study so future studies need to investigate this mechan ism. W h e n combined, these mechan isms result in a complex pattern of metabol ic 161 modulat ion which is dependent on the duration of exposure to metoprolol and to the d i sease state. C h a n g e s in C P T - 1 covalent modif ications did not correlate with t issue activity measurements and levels of the second messengers which produced them. A separate C P T - 1 assoc ia ted microdomain, consist ing of A K A P - 1 4 9 , P K A , CAMK- I I and possibly e N O S , could exist, and covalent modif ications of C P T - 1 may be regulated by translocation of the relevant k inases and e N O S into the microdomain. This C P T - 1 microdomain is likely to contain other components . It is well establ ished that A C C and M C D local ize c lose to C P T - 1 . Recent studies suggest that the fatty acid transporter C D 3 6 translocates from the p lasma membrane to the mitochondria where it assoc ia tes with C P T - 1 (284). A picture is therefore emerging of a secondary mechan ism for fatty acid oxidation control which can , as in the case of metoprolol treatment, be unmasked and produce meaningful changes in fatty acid oxidation rates. W e chose to investigate metoprolol because it had previously been found to inhibit fatty acid oxidation, is a clinically useful drug and because its known range of act ions is narrower than that of carvedi lol. Severa l other p-blockers have been reported to have effects on metabol ism. However, severa l key quest ions remain to be answered. Is this effect a c lass effect or is it mediated only by certain p-blockers? Are the effects mediated by p-adrenoceptors, and if so, what is the contribution of each receptor? Comparat ive studies of a wider range of p-blockers, as well as studies in which p-adrenoceptor express ion is s i lenced, must be carried out in order to answer these quest ions. There has been considerable interest in the ability of p-blockers to act as antioxidants. Propanolo l , pindolol, metoprolol, atenolol and sotalol were all found to inhibit membrane peroxidation, and the effect was attributed to chemica l rather than pharmacologica l properties (285-287). Recent ly, carvedilol has been shown to be a potent antioxidant (288). In a recent study, the scavenging activities of a 162 range of p-blockers (atenolol, labetalol, metoprolol, pindolol, propanolol , sotalol, t imolol and carvedilol) against reactive oxygen and nitrogen spec ies were compared (289). In all c a s e s , effective scavenging required higher concentrat ions than are achieved clinically. None of the p-blockers could scavenge oxygen free radicals, but all could scavenge peroxynitrite. Metoprolol exhibited weak antioxidant effects in this study; the IC50 of metoprolol on peroxynitrite was greater than 5 m M . By contrast, the beneficial effects observed in our study were ach ieved at uJVI levels of the drug. It is therefore unlikely that direct antioxidant effects of metoprolol played a significant role in our studies. A major limitation of the present studies was that the observed changes in C P T - 1 and the factors that may regulate it were occurring s imul taneously under the var ious treatment condit ions, and this makes it chal lenging to disentangle the mechan isms which are of true regulatory importance. W e attempted to rationalize which changes in C P T - 1 activity, sensitivity and malonyl C o A levels were of greater importance based on the measured rates of fatty acid oxidation. The patterns of activation of nitric oxide synthases, P K A and C A M K were different from the patterns of phosphorylat ion, nitrosylation, glutathiolation and nitration observed for C P T - 1 . W e hypothesized that translocation of k inases and e N O S to the mitochondria may be a more important determinant of C P T - 1 post-translational modif ications. Further studies are required to clarify this using e N O S knockout mice or pharmacological inhibitors of the relevant k inases. It will a lso be essent ia l to determine whether the observed changes in C P T - 1 activity and sensitivity produced in vitro by P K A , C A M K and peroxynitrite a lso occur following metoprolol treatment in vivo. To this end, it will be important to measure the post-translat ional modif ications on C P T - 1 directly using mass spectroscopy. W e have shown that metoprolol improves cardiac function in diabetic cardiomyopathy, raising the question as to whether the drug should be used earl ier in diabetic patients. However, there are a number of concerns with the administrat ion of p-blockers to diabetic patients which need to be weighed 163 against the benefits of introducing the drug so early. First and foremost are concerns about the effects of p-blockers on g lycemic control. Coadministrat ion of a p-blocker with a thiazide has been reported to worsen g lycemic control s ince the 1980's when the effects of propanolol and hydrochlorothiazide were reported (290). Recent ly , however, the use of p-blockers as antihypertensive agents has been assoc ia ted with an increased risk of new-onset d iabetes, leading to concern about their use in this context (291). Hepat ic g lucose output is controlled by the p2 adrenoceptor, and blockade of this receptor, which does occur with the p i select ive agents, dec reases hepatic g lucose output and delays recovery from hypoglycemia (292; 293). B lockade of the symptoms of hypoglycemia by p-b lockade is no longer considered to be a problem, because the symptoms of sweat ing and paresthesias are preserved, and patients can be educated to recognize these s igns (293; 294). Another concern with chronic p-blockade is the p resence of susta ined unopposed a1-adrenoceptor stimulation. This is problematic in two situations. Firstly, activation of the sympathet ic nervous system by hypoglycemia increases unopposed a1-adrenoceptor stimulation to the point where a hypertensive crisis can be precipitated (293). Secondly , unopposed a1-adrenoceptor stimulation produces peripheral vasoconstr ict ion which could worsen peripheral vascu lar d i sease and , by decreas ing musc le flow, increase insulin resistance (121; 295). Indeed, use of p-blockers in diabetic patients increases g lucose levels and tr iglycerides and lowers high density lipoprotein cholesterol levels by decreas ing insulin sensitivity (121). None of these concerns are considered great enough to deny p-blockers to patients with systol ic heart failure because these drugs are l i fesaving in this context. However, the risks and benefits of earl ier p-blocker use will need to be weighed carefully and no ev idence currently exists on which to base these considerat ions. 164 W e studied diabetic cardiomyopathy in a model of type 1 diabetes. This was a useful model in which to identify potential mechan isms of act ion, and a model which deve lops the cardiomyopathy quickly was needed in order to widen the scope of the study. However, future studies should examine whether the beneficial effects of metoprolol are a lso observed in models of type 2 d iabetes. In conc lus ion, our studies demonstrate that metoprolol is a fatty acid oxidation inhibitor which amel iorates the cardiac dysfunction of diabetic cardiomyopathy. A role for p-blocker therapy earlier in this condit ion may be cons idered, but careful study and considerat ion of the risks and benefits will be required before such a recommendat ion can be made. VIII: CONCLUSIONS 1. Chron ic metoprolol treatment improves card iac function in the diabetic heart by inhibiting fatty acid oxidation and, through the Rand le cycle, increasing g lucose oxidation. 2. In control hearts, chronic metoprolol treatment increases fatty acid oxidation and dec reases g lucose oxidation. 3. Chron ic metoprolol treatment select ively dec reases the express ion of C P T - 1 B by decreas ing the co-activation and increasing U S F - 2 mediated repression of P G C 1 a . 4. Metoprolol dec reases malonyl C o A levels independent of A C C and M C D in control hearts only. 5. C P T - 1 undergoes S-nitrosylation by N O and glutathiolation and tyrosine nitration by peroxynitrite. C P T - 1 activity is inhibited by S-nitrosylation and glutathiolation, and stimulated by tyrosine nitration. 165 6. Acu te metoprolol perfusion dec reases the activity of C P T - 1 by increasing C P T - 1 S-nitrosylation and glutathiolation, and decreas ing C P T - 1 tyrosine nitration. With the except ion of tyrosine nitration in control hearts, these changes persist with chronic treatment. 7. C P T - 1 is phosphorylated by P K A , which dec reases malonyl C o A sensitivity, and CAMK- I I , which increases C P T - 1 sensitivity. P K A -phosphorylat ion of C P T - 1 is mediated by A K A P - 1 4 9 . 8. Acu te metoprolol perfusion dec reases the sensitivity of C P T - 1 to malonyl C o A by increasing PKA-med ia ted phosphorylat ion of C P T - 1 and decreas ing C A M K - m e d i a t e d phosphorylat ion of C P T - 1 . The changes persist with chronic treatment. 166 SCHEME 7 Summary of the acute effects of metoprolol on malonyl C o A levels, C P T - 1 malonyl C o A sensitivity and C P T - 1 activity. Metoprolol lowers malonyl C o A levels in control hearts only. Metoprolol dec reases C P T - 1 catalytic activity in both control and diabetic groups by increasing S-nitrosylation and glutathiolation of C P T - 1 and reducing tyrosine nitration of C P T - 1 . Metoprolol dec reases the sensitivity of C P T - 1 to malonyl C o A by decreas ing CAMK-I I mediated phosphorylat ion of C P T - 1 and, in control hearts, by increasing P K A -mediated phosphorylat ion of C P T - 1 (abbreviations: P K A = protein k inase A , C A M K = ca lc ium/ ca lmodul in- dependent protein k inase, C P T - 1 = carnitine palmitoyltransferase-1)-METOPROLOL PKA CAMK-II Malonyl CoA CPT-1 Sensitivity T S-Nitrosylation Glutathiolation Tyrosine Nitration CPT-1 Activity 168 B I B L I O G R A P H Y 1. King H, Aubert R E , Herman W H : Globa l burden of d iabetes, 1995-2025: prevalence, numerical est imates, and projections. Diabetes Care 21 :1414-1431, 1998 2. Effects of enalapri l on mortality in severe congest ive heart failure. Resu l ts of the Cooperat ive North Scand inav ian Enalapri l Survival Study ( C O N S E N S U S ) . The C O N S E N S U S Trial Study Group. N Engl J Med 316:1429-1435, 1987 3. Effect of enalapri l on survival in patients with reduced left ventricular ejection fractions and congest ive heart failure. The S O L V D Investigators. N Engl J Med 325:293-302, 1991 4. C o h n J N , Johnson G , Z iesche S , C o b b F, Francis G , Tristani F, Smith R, Dunkman W B , Loeb H, W o n g M, et al . : A compar ison of enalapri l with hydralaz ine- isosorbide dinitrate in the treatment of chronic congest ive heart failure. N Engl J Med 325:303-310, 1991 5. P a c k e r M, Poo le -Wi lson P A , Armstrong P W , Cle land J G , Horowitz J D , M a s s i e B M , Ryden L, Thygesen K, Uretsky B F : Comparat ive effects of low and high doses of the angiotensin-convert ing enzyme inhibitor, lisinopril, on morbidity and mortality in chronic heart failure. A T L A S Study Group. Circulation 100:2312-2318, 1999 6. Kanne l W B , M c G e e DL: Diabetes and card iovascular d i sease . The Framingham study. Jama 241:2035-2038, 1979 7. Stratton IM, Ad ler A l , Neil HA, Matthews D R , Manley S E , Cul l C A , Hadden D, Turner R C , Ho lman R R : Assoc ia t ion of g lycaemia with macrovascu lar and 169 microvascular compl icat ions of type 2 diabetes ( U K P D S 35): prospect ive observat ional study. Bm/321 :405 -412 , 2000 8. Malmberg K: Prospect ive randomised study of intensive insulin treatment on long term survival after acute myocardial infarction in patients with d iabetes mellitus. DIGAMI (Diabetes Mell itus, Insulin G lucose Infusion in Acu te Myocard ia l Infarction) Study Group. Bmj 314:1512-1515, 1997 9. Bel l D S : Heart failure: the frequent, forgotten, and often fatal compl icat ion of diabetes. Diabetes Care 26 :2433-2441, 2003 10. Rubier S , Dlugash J , Yuceog lu Y Z , Kumral T, Branwood A W , Gr i shman A : N e w type of cardiomyopathy assoc ia ted with diabetic g lomerulosclerosis. Am J Cardiol 30:595-602, 1972 11. Galder is i M: Diastol ic dysfunction and diabetic cardiomyopathy: evaluat ion by Doppler echocardiography. J Am Coll Cardiol 48 :1548-1551, 2006 12. C o s s o n S , Kevork ian J P : Left ventricular diastolic dysfunction: an early sign of diabetic card iomyopathy? Diabetes Metab 29:455-466, 2003 13. Zabalgoi t ia M, Ismaeil M F , Ande rson L, Mak lady F A : Preva lence of diastol ic dysfunct ion in normotensive, asymptomat ic patients with well-control led type 2 d iabetes mellitus. Am J Cardiol 87:320-323, 2001 14. Poir ier P, Bogaty P, Garneau C , Marois L, Dumesni l J G : Diastol ic dysfunction in normotensive men with well-controlled type 2 diabetes: importance of maneuvers in echocardiographic screening for preclinical diabetic cardiomyopathy. Diabetes Care 24:5-10, 2001 170 15. Redf ield M M , J a c o b s e n S J , Burnett J C , Jr., Mahoney DW, Bai ley K R , Rodehef fer R J : Burden of systol ic and diastolic ventricular dysfunction in the community: appreciat ing the scope of the heart failure epidemic. Jama 289:194-202, 2003 16. Bertoni A G , Tsa i A , Kasper E K , Brancati FL : Diabetes and idiopathic cardiomyopathy: a nationwide case-contro l study. Diabetes Care 26:2791-2795, 2003 17. Fang Z Y , Pr ins J B , Marwick T H : Diabetic cardiomyopathy: ev idence, mechan isms, and therapeutic implications. EndocrRev 25:543-567, 2004 18. Cha tham J C , Forder J R , McNei l l J H : The Heart in Diabetes. Norwell, Massachusse t s , Kluwer A c a d e m i c Publ ishers, 1996 19. Ulr ich P, Ce ram i A : Protein glycation, diabetes, and aging. Recent Prog Horm Res 56:1-21, 2001 20. Al lard M F , S c h o n e k e s s B O , Henning S L , Engl ish DR, Lopaschuk G D : Contribution of oxidative metabol ism and glycolysis to A T P production in hypertrophied hearts. Am J Physiol 267:H742-750, 1994 21 . Zhang J , Duncker D J , Y a X , Zhang Y , Pavek T, W e i H, Merkle H, Ugurbil K, F rom A H , B a c h e R J : Effect of left ventricular hypertrophy secondary to chronic pressure over load on transmural myocardial 2-deoxyglucose uptake. A 3 1 P N M R spect roscopic study. Circulation 92:1274-1283, 1995 22. Apste in C S : Glucose- insul in-potass ium for acute myocardial infarction: remarkable results from a new prospect ive, randomized trial. Circulation 98:2223-2226, 1998 171 23. Seve rson DL: Diabetic cardiomyopathy: recent ev idence from mouse models of type 1 and type 2 diabetes. Can J Physiol Pharmacol 82:813-823, 2004 24. B ie lawska A E , Shapiro J P , J iang L, Melkonyan H S , Piot C , Wol fe C L , Tomei LD , Hannun Y A , Umansky S R : Ceramide is involved in triggering of card iomyocyte apoptosis induced by ischemia and reperfusion. Am J Pathol 151 :1257-1263 ,1997 25. Djouadi F, Brandt J M , Weinhe imer C J , Leone T C , G o n z a l e z F J , Kel ly D P : The role of the perox isome proliferator-activated receptor a lpha ( P P A R alpha) in the control of card iac lipid metabol ism. Prostaglandins Leukot Essent Fatty Acids 60:339-343, 1999 26. Brandt J M , Djouadi F, Kel ly D P : Fatty ac ids activate transcription of the musc le carnit ine palmitoyltransferase I gene in cardiac myocytes via the perox isome proliferator-activated receptor a lpha. J Biol Chem 273:23786-23792, 1998 27. Finck B N , Lehman J J , Leone T C , W e l c h M J , Bennett M J , Kovacs A , Han X , G r o s s R W , Kozak R, Lopaschuk G D , Kelly D P : The card iac phenotype induced by P P A R a l p h a overexpression mimics that caused by diabetes mellitus. J Clin //n/esf 109:121-130, 2002 28. Wo ld L E , Cey lan- ls ik A F , R e n J : Oxidat ive stress and stress signal ing: menace of diabetic cardiomyopathy. Acta Pharmacol Sin 26:908-917, 2005 29. Brownlee M: Biochemistry and molecular cell biology of diabetic compl icat ions. Nature 414:813-820, 2001 30. Brownlee M, Cerami A , V lassa ra H: Advanced glycosylat ion end products in t issue and the b iochemical basis of diabetic compl icat ions. N Engl J Med 318 :1315 -1321 ,1988 172 31. K o y a D, King G L : Protein k inase C activation and the development of diabetic compl icat ions. Diabetes 47:859-866, 1998 32. Keogh R J , Dunlop M E , Larkins R G : Effect of inhibition of a ldose reductase on g lucose flux, diacylglycerol formation, protein k inase C , and phosphol ipase A 2 activation. Metabolism 46:41-47, 1997 33. J iang T, C h e Q, Lin Y , Li H, Zhang N: A ldose reductase regulates T G F -be ta l - i nduced production of fibronectin and type IV col lagen in cultured rat mesangia l cel ls. Nephrology (Carlton) 11:105-112, 2006 34. Nishio Y , Kash iwag i A , K ida Y , K o d a m a M, A b e N, Saek i Y , Sh igeta Y : Def ic iency of card iac beta-adrenergic receptor in streptozocin- induced diabetic rats. Diabetes 37:1181-1187, 1988 35. P ie rce G N , Russe l l J C : Regulat ion of intracellular Ca2+ in the heart during d iabetes. Cardiovasc Res 34:41-47, 1997 36. Razegh i P, Young M E , Cockri l l T C , Frazier O H , Taegtmeyer H: Downregulat ion of myocardial myocyte enhancer factor 2 C and myocyte enhancer factor 2C-regulated gene express ion in diabetic patients with non ischemic heart failure. Circulation 106:407-411, 2002 37. Lohse M J , Engelhardt S , Eschenhagen T: What is the role of beta-adrenergic signal ing in heart fai lure? Circ Res 93:896-906, 2003 38. D iez J , Pan izo A , Hernandez M, V e g a F, So la I, Fortuno M A , Pardo J : Card iomyocyte apoptosis and cardiac angiotensin-convert ing enzyme in spontaneous ly hypertensive rats. Hypertension 30:1029-1034, 1997 173 39. C o m m u n a l C , Singh K, Sawyer D B , Co lucc i W S : Oppos ing effects of beta(1)-and beta(2)-adrenergic receptors on cardiac myocyte apoptosis : role of a pertussis toxin-sensit ive G protein. Circulation 100:2210-2212, 1999 40. Ches ley A , Lundberg M S , A s a i T, X iao R P , Ohtani S , Lakatta E G , C row MT: The beta(2)-adrenergic receptor delivers an antiapoptotic s ignal to card iac myocytes through G(i)-dependent coupling to phosphatidyl inositol 3 '-k inase. Circ Res 87 :1172-1179, 2000 41 . Zhu W Z , Zheng M, Koch W J , Lefkowitz R J , Kobi lka BK , X i a o R P : Dual modulat ion of cell survival and cell death by beta(2)-adrenergic signal ing in adult mouse card iac myocytes. Proc Natl Acad Sci USA 98:1607-1612, 2001 42. Y u G , L iang X , X ie X , Y a n g T, S u n M, Zhao S : Apoptos is , myocardia l f ibrosis and angiotensin II in the left ventricle of hypertensive rats treated with fosinopri l o r losar tan . Chin Med J (Engl) 115:1287-1291, 2002 43 . Frustaci A , Kajstura J , Chiment i C , Jakoniuk I, Leri A , Maser i A , Nada l -G inard B, A n v e r s a P: Myocardia l cell death in human diabetes. Circ Res 87:1123-1132, 2000 44. G o u s s e v A , Sharov V G , Sh imoyama H, Tanimura M, Lesch M, Goldste in S , S a b b a h H N : Effects of A C E inhibition on cardiomyocyte apoptosis in dogs with heart failure. Am J Physiol 275 :H626-631, 1998 45 . Tyagi S C , Rodr iguez W , Patel A M , Roberts A M , Fa lcone J C , P a s s m o r e J C , F leming JT , J o s h u a IG: Hyperhomocyste inemic diabetic cardiomyopathy: oxidative stress, remodel ing, and endothel ial-myocyte uncoupl ing. J Cardiovasc Pharmacol Ther 10:1-10, 2005 174 46. Lopaschuk G D , Rebeyka IM, Al lard M F : Metabol ic modulat ion: a means to mend a broken heart. Circulation 105:140-142, 2002 47. Lopaschuk G D : Metabol ic abnormali t ies in the diabetic heart. Heart Fail Rev 7 :149 -159 ,2002 48. Stanley W C , Chandler M P : Energy metabol ism in the normal and failing heart: potential for therapeutic interventions. Heart Fail Rev 7:115-130, 2002 49 . Brown G C : Control of respiration and A T P synthesis in mammal ian mitochondria and cel ls. Biochem J 284 ( Pt 1):1-13, 1992 50. Mitchell P: Vector ia l chemistry and the molecular mechan ics of chemiosmot ic coupl ing: power t ransmission by proticity. Biochem Soc Trans 4 :399-430, 1976 51. S a k s V A , Kuznetsov A V , Vendel in M, Guerrero K, Kay L, Seppet E K : Funct ional coupl ing as a basic mechan ism of feedback regulation of card iac energy metabol ism. Mol Cell Biochem 256-257:185-199, 2004 52. Car l ing D: The AMP-ac t i va ted protein k inase c a s c a d e - a unifying sys tem for energy control. Trends Biochem Sci 29:18-24, 2004 53. Keul J , Doll E, Keppler D, Reindel l H: [Variations of arterial substrate level under the inf luence of physical work]. Int Z Angew Physiol 22:356-385, 1966 54. Lardy HA, P r e s s m a n B C : Effect of surface active agents on the latent A T P a s e of mitochondria. Biochim Biophys Acta 21:458-466, 1956 55. Boud ina S , S e n a S , O'Neil l BT, Tathireddy P, Young M E , Abe l E D : Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupl ing impair myocardia l energet ics in obesity. Circulation 112:2686-2695, 2005 175 56. Boud ina S , S e n a S , Theobald H, Sheng X , Wright J J , Hu X X , A z i z S , Johnson J l , Bugger H, Z a h a V G , Abe l E D : Mitochondrial energet ics in the heart in obesity-related diabetes: direct ev idence for increased uncoupled respiration and activation of uncoupl ing proteins. Diabetes 56:2457-2466, 2007 57. Huang J M , X ian H, Bacaner M: Long-chain fatty ac ids activate ca lc ium channe ls in ventricular myocytes. Proc Natl Acad Sci USA 89:6452-6456, 1992 58. Lopaschuk G D : Optimizing card iac energy metabol ism: how can fatty acid and carbohydrate metabol ism be manipulated? Coron Artery Dis 12 Supp l 1:S8-11 ,2001 59. Stanley W C , Recch ia FA , Lopaschuk G D : Myocardia l substrate metabol ism in the normal and failing heart. Physiol Rev 85:1093-1129, 2005 60. Avogaro A , Nosadin i R, Doria A , Fioretto P, Ve luss i M, Vigorito C , S a c c a L, Toffolo G , Cobel l i C , Trevisan R, et al . : Myocardia l metabol ism in insulin-deficient diabetic humans without coronary artery d isease . Am J Physiol 258 :E606-618 , 1990 61 . Hal l J L , Stanley W C , Lopaschuk G D , Wisnesk i J A , Pizzurro R D , Hamilton C D , M c C o r m a c k J G : Impaired pyruvate oxidation but normal g lucose uptake in diabetic pig heart during dobutamine- induced work. Am J Physiol 271 :H2320-2329, 1996 62. Stanley W C , Lopaschuk G D , Hall J L , M c C o r m a c k J G : Regulat ion of myocardia l carbohydrate metabol ism under normal and ischaemic condit ions. Potential for pharmacological interventions. Cardiovasc Res 33:243-257, 1997 63. Y o u n g L H , C o v e n DL, Russe l l R R , 3rd: Cel lu lar and molecular regulation of card iac g lucose transport. J Nucl Cardiol 7:267-276, 2000 176 64. Rand le P J : Fuel select ion in animals. Biochem Soc Trans 14:799-806, 1986 65. G iba la M J , Young M E , Taegtmeyer H: Anapleros is of the citric acid cyc le: role in energy metabol ism of heart and skeletal musc le. Acta Physiol Scand 168:657-665, 2000 66. Augus tus A S , Kako Y , Yagyu H, Goldberg IJ: Routes of F A delivery to card iac musc le : modulation of lipoprotein lipolysis alters uptake of TG-der i ved F A . Am J Physiol Endocrinol Metab 284 :E331-339, 2003 67. Lopaschuk G D , Belke DD, G a m b l e J , Itoi T, S c h o n e k e s s B O : Regulat ion of fatty acid oxidation in the mammal ian heart in health and d i sease . Biochim BiophysActa 1213:263-276, 1994 68. Kerner J , Hoppel C : Fatty acid import into mitochondria. Biochim Biophys Acta 1486:1-17, 2000 69. Bing R J , S iege l A , Ungar I, Gilbert M: Metabol ism of the human heart. II. S tud ies on fat, ketone and amino acid metabol ism. Am J Med 16:504-515, 1954 70. Op ie L: The heart: physiology, from cell to circulation. Phi ladelphia, Lippincot-R a v e n , 1998 71. McGar ry J D , Brown N F : The mitochondrial carnitine palmitoyltransferase sys tem. From concept to molecular analysis. Eur J Biochem 244:1-14, 1997 72. McGar ry J D , Mills S E , Long C S , Foster DW: Observat ions on the affinity for carnit ine, and ma lony l -CoA sensitivity, of carnitine palmitoyltransferase I in animal and human t issues. Demonstrat ion of the presence of ma lony l -CoA in non-hepat ic t i ssues of the rat. Biochem J 214:21-28, 1983 177 73. Boone A N , Rodr igues B, Brownsey R W : Multiple-site phosphorylat ion of the 280 k D a isoform of ace ty l -CoA carboxy lase in rat card iac myocytes: ev idence that c A M P - d e p e n d e n t protein k inase mediates effects of beta-adrenergic stimulation. Biochem J 341 ( Pt 2):347-354, 1999 74. Sakamoto J , Barr R L , Kavanagh K M , Lopaschuk G D : Contribution of ma lony l -CoA decarboxy lase to the high fatty acid oxidation rates seen in the diabetic heart. Am J Physiol Heart Circ Physiol 278:H1196-1204, 2000 75. S a m b a n d a m N, Steinmetz M, C h u A , Altarejos J Y , Dyck J R , Lopaschuk G D : Ma lony l -CoA decarboxy lase (MCD) is differentially regulated in subcel lu lar compartments by 5 'AMP-act ivated protein k inase ( A M P K ) . Studies using H9c2 cel ls overexpress ing M C D and A M P K by adenoviral gene transfer technique. Eur J Biochem 271:2831-2840, 2004 76. Scarpu l la R C : Nuclear activators and coactivators in mammal ian mitochondrial b iogenesis. Biochim Biophys Acta 1576:1-14, 2002 77. Franc is G A , Annicotte J S , Auwerx J : P P A R - a l p h a effects on the heart and other vascu lar t issues. Am J Physiol Heart Circ Physiol 285:H1-9, 2003 78. Arany Z, He H, Lin J , Hoyer K, Handschin C , Toka O, A h m a d F, Matsui T, Ch in S , W u P H , Rybkin, II, Shel ton J M , Manier i M, Cinti S , S c h o e n F J , B a s s e l -Duby R, Rosenzwe ig A , Ingwall J S , Sp iege lman B M : Transcript ional coactivator P G C - 1 a lpha controls the energy state and contractile function of card iac musc le . CellMetab 1:259-271, 2005 79. Lehman J J , Barger P M , Kovacs A , Saffitz J E , Medei ros D M , Kelly D P : Perox isome proliferator-activated receptor g a m m a coactivator-1 promotes card iac mitochondrial b iogenesis. J Clin Invest 106:847-856, 2000 178 80. Huss J M , Torra IP, Stae ls B, Giguere V , Kelly D P : Estrogen-related receptor a lpha directs perox isome proliferator-activated receptor a lpha signal ing in the transcript ional control of energy metabol ism in card iac and skeletal musc le . Mol Cell Biol 24 :9079-9091, 2004 81. H u s s J M , Kel ly D P : Nuc lear receptor signal ing and card iac energet ics. Circ Res 95:568-578, 2004 82. Bers in R M , Wol fe C , K w a s m a n M, Lau D, Kl inski C , T a n a k a K, Khorrami P, Henderson G N , de Marco T, Chatterjee K: Improved hemodynamic function and mechan ica l eff iciency in congest ive heart failure with sodium dichloroacetate. J Am Coll Cara7o/23:1617-1624, 1994 83. Nicholl TA , Lopaschuk G D , McNei l l J H : Effects of free fatty ac ids and dichloroacetate on isolated working diabetic rat heart. Am J Physiol 261 :H1053-1059, 1991 84. Brownsey R W , Boone A N , Al lard M F : Act ions of insulin on the mammal ian heart: metabol ism, pathology and b iochemical mechan isms. Cardiovasc Res 34:3-24, 1997 85. Cottin Y , Lhuill ier I, G i lson L, Zel ler M, Bonnet C , Tou louse C , Louis P, Rochet te L, Girard C , Wol f J E : G l u c o s e insulin potass ium infusion improves systol ic function in patients with chronic ischemic cardiomyopathy. Eur J Heart Fa/7 4:181-184, 2002 86. Nikolaidis LA, Elahi D, Hentosz T, Doverspike A , Huerbin R, Zoure l ias L, Stolarski C , S h e n Y T , Shannon R P : Recombinant glucagon-l ike peptide-1 increases myocardia l g lucose uptake and improves left ventricular per formance in consc ious dogs with pacing- induced dilated cardiomyopathy. Circulation 110:955-961, 2004 179 87. Ma lmberg K, Norhammar A , W e d e l H, Ryden L: Glycometabo l ic state at admiss ion : important risk marker of mortality in conventional ly treated patients with d iabetes mellitus and acute myocardia l infarction: long-term results from the Diabetes and Insul in-Glucose Infusion in Acute Myocardia l Infarction (DIGAMI) study. Circulation 99:2626-2632, 1999 88. Turcani M, Rupp H: Etomoxir improves left ventricular per formance of pressure-over loaded rat heart. Circulation 96:3681-3686, 1997 89. R u p p H, Vetter R: Sarcop lasmic reticulum function and carnit ine palmitoyltransferase-1 inhibition during progression of heart failure. Br J Pharmacol 131:1748-1756, 2000 90. Hayash i K, Okumura K, Matsui H, Murase K, Kamiya H, Sabur i Y , Numaguch i Y , Toki Y , Hayakawa T: Involvement of 1,2-diacylglycerol in improvement of heart function by etomoxir in diabetic rats. Life Sci 68:1515-1526, 2001 91. Schmi tz F J , R o s e n P, Re inauer H: Improvement of myocardial function and metabol ism in diabet ic rats by the carnit ine palmitoyl t ransferase inhibitor Etomoxir. Horm Metab Res 27:515-522, 1995 92. Kato K, C h a p m a n D C , Rupp H, Lukas A , Dhal la N S : Alterat ions of heart function and Na+-K+-ATPase activity by etomoxir in diabetic rats. J Appl Physiol 86:812-818, 1999 93. Rupp H, E l imban V , Dhal la N S : Modif ication of subcel lu lar organel les in pressure-over loaded heart by etomoxir, a carnitine palmitoyltransferase I inhibitor. Faseb J 6 :2349-2353, 1992 180 94. Schmid t -Schweda S , Holubarsch C: First cl inical trial with etomoxir in patients with chronic congest ive heart failure. Clin Sci (Lond) 99:27-35, 2000 95. Ashra f ian H, Frenneaux M P : Metabol ic modulat ion in heart failure: the coming of age. Cardiovasc Drugs Ther 21:5-7, 2007 96. Rupp H, W a h l R, Hansen M: Influence of diet and carnitine palmitoyl transferase I inhibition on myosin and sarcop lasmic reticulum. J. Appl Physiol 72:352-360, 1992 97. Zara in-Herzberg A , Rupp H, E l imban V , Dhal la N S : Modif ication of sarcop lasmic reticulum gene express ion in pressure over load card iac hypertrophy by etomoxir. Faseb J 10:1303-1309, 1996 98. Merril l C L , Ni H, Y o o n LW, Tirmenstein M A , Narayanan P, Benav ides G R , Eas ton M J , C reech DR, Hu C X , McFar land D C , Hahn L M , Thomas H C , Morgan KT : Etomoxir - induced oxidative stress in H e p G 2 cells detected by differential gene express ion is conf irmed biochemical ly. Toxicol Sci 68 :93-101, 2002 99. Lee L, Horowitz J , Frenneaux M: Metabol ic manipulation in i schaemic heart d i sease , a novel approach to treatment. Eur Heart J 25:634-641, 2004 100. Lee L, Campbe l l R, Scheuermann-Frees tone M, Taylor R, Guna ruwan P, Wi l l iams L, Ashraf ian H, Horowitz J , Fraser A G , Clarke K, Frenneaux M: Metabol ic modulat ion with perhexil ine in chronic heart failure: a randomized, control led trial of short-term use of a novel treatment. Circulation 112:3280-3288, 2005 101. Kantor P F , Lucien A , Kozak R, Lopaschuk G D : The antianginal drug tr imetazidine shifts cardiac energy metabol ism from fatty acid oxidation to 181 g lucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl c o e n z y m e A thiolase. Circ Res 86:580-588, 2000 102. F ragasso G , Pal loshi A , Puccett i P, Sil ipigni C , Rossodiv i ta A , P a l a M, Calor i G , Alf ieri O, Margonato A : A randomized clinical trial of tr imetazidine, a partial free fatty acid oxidation inhibitor, in patients with heart failure. J Am Coll Cardiol 48:992-998, 2006 103. Thrainsdott ir IS, von Bibra H, Ma lmberg K, Ryden L: Effects of tr imetazidine on left ventricular function in patients with type 2 diabetes and heart failure. J Cardiovasc Pharmacol 44:101 -108, 2004 104. R o s a n o G M , Vitale C , Sposa to B, Mercuro G , Fini M: Tr imetazidine improves left ventricular function in diabetic patients with coronary artery d i sease : a double-bl ind placebo-control led study. Cardiovasc Diabetol 2:16, 2003 105. F ragasso G , Piatti Md P M , Monti L, Pal loshi A , Seto la E, Puccett i P, Calor i G , Lopaschuk G D , Margonato A : Short- and long-term beneficial effects of tr imetazidine in patients with diabetes and ischemic cardiomyopathy. Am Heart J 146.E18, 2003 106. Pau lson D J , Shet lar D, Light K: Catecho lamine levels in the heart, serum and adrenals of experimental diabetic rats. Federation Proceedings 39:637, 1980 107. Chr is tensen N J : P l a s m a norepinephrine and epinephrine in untreated diabet ics, during fasting and after insulin administration. Diabetes 23:1-8, 1974 108. Gangu ly P K , Beamish R E , Dhal la K S , Innes IR, Dhal la N S : Norepinephr ine storage, distribution, and release in diabetic cardiomyopathy. Am J Physiol 252 :E734-739 , 1987 182 109. Es le r M, K a y e D, Lambert G , Es ler D, Jenn ings G : Adrenerg ic nervous sys tem in. heart failure. Am J Cardiol 80 :7L-14L, 1997 110. P a c k e r M: Neurohormonal interactions and adaptat ions in congest ive heart failure. Circulation 77:721-730, 1988 111. Bristow M R : beta-adrenergic receptor b lockade in chronic heart failure. Circulation 101:558-569, 2000 112. Waags te in F, Hjalmarson A , Va rnauskas E, Wallent in I: Effect of chronic beta-adrenergic receptor b lockade in congest ive cardiomyopathy. Br Heart J 37:1022-1036, 1975 113. K jekshus J , Gi lpin E, Cal i G , B lackey A R , Henning H, R o s s J , Jr.: Diabet ic patients and beta-blockers after acute myocardial infarction. Eur Heart J 11:43-50, 1990 114. Kendal l M J , Lynch K P , Hjalmarson A , Kjekshus J : Beta-b lockers and sudden card iac death. Ann Intern Med 123:358-367, 1995 115. T s e W Y , Kendal l M : Is there a role for beta-blockers in hypertensive diabetic pat ients? Diabet Med 11:137-144, 1994 116. de Leeuw P W , Notter T, Zi l les P: Compar i son of different fixed antihypertensive combinat ion drugs: a double-bl ind, p lacebo-control led paral lel group study. J Hypertens 15:87-91, 1997 117. P a c k e r M, Bristow M R , C o h n J N , Co lucc i W S , Fowler M B , Gilbert E M , Shus te rman N H : The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedi lo l Heart Fai lure Study Group. N Engl J M e d 334:1349-1355, 1996 183 118. P a c k e r M , C o a t s A J , Fowler M B , Katus HA , Krum H, Mohacs i P, Rou leau J L , Tende ra M, Casta igne A , Roecke r E B , Schul tz M K , DeMets DL: Effect of carvedi lol on survival in severe chronic heart failure. N Engl J Med 344 :1651-1658, 2001 119. Effect of metoprolol C R / X L in chronic heart failure: Metoprolol C R / X L Randomised Intervention Trial in Congest ive Heart Fai lure (MERIT -HF) . Lancet 353:2001-2007, 1999 120. T h e Card iac Insufficiency Bisoprolol Study II (CIBIS-II): a randomised trial. Lancet 353:9-13, 1999 121. Bel l D S H : U s e of beta-blockers in the patient with d iabetes. The Endocrinologist 13:116-123, 2003 122. E ichhorn E J , Heesch C M , Barnett J H , A lvarez L G , F a s s S M , Grayburn P A , Hatfield BA , Marcoux L G , Mal loy C R : Effect of metoprolol on myocard ia l function and energet ics in patients with nonischemic dilated cardiomyopathy: a randomized, double-bl ind, placebo-control led study. J Am Coll Cardiol 24 :1310-1320, 1994 123. A l - H e s a y e n A , A z e v e d o E R , F loras J S , Hol l ingshead S , Lopaschuk G D , Parker J D : Select ive versus nonselect ive beta-adrenergic receptor b lockade in chronic heart failure: differential effects on myocardia l energy substrate utilization. Eur J Heart Fail 7:618-623, 2005 124. Eichhorn E J , Bedotto J B , Mal loy C R , Hatfield BA , Dei tchman D, Brown M, Wil lard J E , Grayburn P A : Effect of beta-adrenergic b lockade on myocard ia l function and energet ics in congest ive heart failure. Improvements in 184 hemodynamic , contractile, and diastol ic performance with bucindolol . Circulation 82 :473 -483 ,1990 125. A n d e r s s o n B, Blomstrom-Lundqvist C , Hedner T, Waags te in F: Exerc ise hemodynamics and myocardia l metabol ism during long-term beta-adrenergic b lockade in severe heart failure. J Am Coll Cardiol 18:1059-1066, 1991 126. P a n c h a l A R , Stanley W C , Kerner J , Sabbah H N : Beta-receptor b lockade d e c r e a s e s carnit ine palmitoyl t ransferase I activity in dogs with heart failure. J Card Fail 4:121 -126, 1998 127. Ahlquist R: A study of the adrenotropic receptors. American Journal of Physiology 153:586-600, 1948 128. Lands A M , Arnold A , McAuli f f J P , Luduena F P , Brown T G , Jr.: Differentiation of receptor sys tems activated by sympathomimet ic amines. Nature 214:597-598, 1967 129. Emor ine L J , Marul lo S , Br iend-Sutren M M , Patey G , Tate K, Delavier-Klutchko C , Strosberg A D : Molecular characterizat ion of the human beta 3-adrenergic receptor. Science 245:1118-1121, 1989 130. Brodde O E : Beta 1- and beta 2-adrenoceptors in the human heart: propert ies, function, and alterations in chronic heart failure. Pharmacol Rev 43:203-242, 1991 131. Kaumann A J , Preitner F, Sarsero D, Molenaar P, Revel l i J P , G iacob ino J P : ( - ) -CGP 12177 c a u s e s cardiostimulation and binds to card iac putative beta 4-adrenoceptors in both wild-type and beta 3-adrenoceptor knockout mice. Mol Pharmacol 53:670-675, 1998 185 132. Kaumann A J , Engelhardt S , Hein L, Molenaar P, Lohse M: Aboli t ion of (-)-C G P 12177-evoked cardiostimulation in double beta1/beta2-adrenoceptor knockout mice. Obligatory role of be ta l -adrenoceptors for putative beta4-adrenoceptor pharmacology. Naunyn Schmiedebergs Arch Pharmacol 363:87-93, 2001 133. Hoffmann C , Leitz M R , Oberdor f -Maass S , Lohse M J , Klotz K N : Comparat ive pharmacology of human beta-adrenergic receptor s u b t y p e s -character izat ion of stably transfected receptors in C H O cel ls. Naunyn Schmiedebergs Arch Pharmacol 369:151 -159, 2004 134. Zhao X L , Gut ierrez L M , C h a n g C F , Hosey M M : The a lpha 1-subunit of skeletal musc le L-type C a channels is the key target for regulation by A -k inase and protein phosphatase-1C. Biochem Biophys Res Commun 198:166-173, 1994 135. Gerhardste in BL , Puri T S , Ch ien A J , Hosey M M : Identification of the sites phosphory lated by cycl ic A M P - d e p e n d e n t protein k inase on the beta 2 subunit of L-type vol tage-dependent calc ium channels . Biochemistry 38 :10361-10370, 1999 136. S immerman HK, Jones L R : Phospho lamban : protein structure, mechan ism of act ion, and role in card iac function. Physiol Rev 78:921-947, 1998 137. Su lakhe P V , V o X T : Regulat ion of phospho lamban and troponin-l phosphorylat ion in the intact rat card iomyocytes by adrenergic and chol inergic stimuli: roles of cycl ic nucleot ides, ca lc ium, protein k inases and phosphatases and depolar izat ion. Mol Cell Biochem 149-150:103-126, 1995 138. Kunst G , K ress K R , Gruen M, Uttenweiler D, Gaute l M, Fink R H : Myos in binding protein C , a phosphorylat ion-dependent force regulator in musc le that controls the attachment of myosin heads by its interaction with myosin S 2 . Circ Res 86:51-58, 2000 186 139. Zhang Z Y , Zhou B, X ie L: Modulat ion of protein k inase signal ing by protein phosphatases and inhibitors. Pharmacol Ther 93:307-317, 2002 140. Lohse M J , Krase l C , Winste l R, Mayor F, Jr.: G-protein-coupled receptor k inases. Kidney Int 49 :1047-1052, 1996 141. Pi tcher J A , F reedman N J , Lefkowitz R J : G protein-coupled receptor k inases. Annu Rev Biochem 67:653-692, 1998 142. Lohse M J , Benov ic J L , Cod ina J , Caron M G , Lefkowitz R J : beta-Arrest in: a protein that regulates beta-adrenergic receptor function. Science 248:1547-1550, 1990 143. W a n g W , Zhu W , W a n g S , Y a n g D, C row MT, X iao R P , C h e n g H: Susta ined be ta l -ad renerg ic stimulation modulates card iac contractility by Ca2+/calmodul in k inase signal ing pathway. Circ Res 95:798-806, 2004 144. X i a o R P , Zhu W , Zheng M, C a o C , Zhang Y , Lakatta E G , Han Q: Subtype-speci f ic a lpha 1- and beta-adrenoceptor signal ing in the heart. Trends Pharmacol Sci 27:330-337, 2006 145. X iao R P , Zhu W , Zheng M, Chak i r K, Bond R, Lakatta E G , C h e n g H: Subtype-spec i f ic beta-adrenoceptor signal ing pathways in the heart and their potential cl inical implications. Trends Pharmacol Sci 25:358-365, 2004 146. Kusche l M, Zhou Y Y , Spurgeon HA, Bartel S , Karczewsk i P, Zhang S J , Krause E G , Lakatta E G , X iao R P : beta2-adrenergic c A M P signal ing is uncoupled from phosphorylat ion of cytoplasmic proteins in canine heart. Circulation 99:2458-2465, 1999 187 147. Rybin V O , X u X , Lisanti M P , Steinberg S F : Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyc lase to cardiomyocyte caveo lae . A mechan ism to functionally regulate the c A M P signaling pathway. J Biol Chem 275:41447-41457, 2000 148. X iang Y , Rybin V O , Steinberg S F , Kobi lka B: Caveo la r localization dictates physio logic s ignal ing of beta 2-adrenoceptors in neonatal card iac myocytes. J Biol Chem 277:34280-34286, 2002 149. Ostrom R S , Gregor ian C , Drenan R M , X iang Y , R e g a n J W , Insel P A : Receptor number and caveolar co-local izat ion determine receptor coupl ing eff iciency to adenylyl cyc lase. J Biol Chem 276:42063-42069, 2001 150. Sh izukuda Y , Buttrick P M : Subtype specif ic roles of beta-adrenergic receptors in apoptosis of adult rat ventricular myocytes. J Mol Cell Cardiol 34:823-831, 2002 151. Bal l igand J L : Regulat ion of card iac beta-adrenergic response by nitric oxide. Cardiovasc R e s 43:607-620, 1999 152. B loch W , Add icks K, Hesche le r J , F le ischmann BK : Nitric oxide synthase express ion and function in embryonic and adult cardiomyocytes. Microsc Res Tech 55:259-269, 2001 153. Pa lmer R M , M o n c a d a S : A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascu lar endothelial cel ls. Biochem Biophys Res Commun 158:348-352, 1989 154. Nathan C : Nitric oxide as a secretory product of mammal ian cel ls. Faseb J 6:3051-3064, 1992 188 155. Lane P, G r o s s S S : Ce l l signal ing by nitric oxide. Semin Nephrol 19:215-229, 1999 156. Farrel l A J , B lake DR: Nitric oxide. Ann Rheum Dis 55:7-20, 1996 157. Gauth ier C , Tavernier G , Charpent ier F, Langin D, Le Marec H: Funct ional beta3-adrenoceptor in the human heart. J Clin Invest 98:556-562, 1996 158. Gauth ier C , Leblais V , Kobzik L, Trochu J N , Khandoudi N, Bril A , Bal l igand J L , Le M a r e c H: The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle. J Clin Invest 102:1377-1384, 1998 159. Vad lamud i R V , McNei l l J H : Effect of experimental d iabetes on isolated rat heart respons iveness to isoproterenol. Can J Physiol Pharmacol 62 :124-131, 1984 160. Zo la B E , Miller B, Sti les G L , R a o P S , Sonnenbl ick E H , Fein F S : Heart rate control in diabet ic rabbits: blunted response to isoproterenol. Am J Physiol 255 :E636-641 , 1988 161. Foy J M , Lucas P D : Effect of experimental d iabetes, food deprivation and genet ic obesity on the sensitivity of pithed rats to autonomic agents. Br J Pharmacol 57:229-234, 1976 162. T a m a d a A , Hattori Y , Houzen H, Y a m a d a Y , S a k u m a I, Ki tabatake A , Kanno M : Effects of beta-adrenoceptor stimulation on contractility, [Ca2+]i, and Ca2+ current in diabetic rat cardiomyocytes. Am J Physiol 274:H1849-1857, 1998 163. Dincer U D , B idasee K R , Guner S , Tay A , Ozce l ikay A T , Al tan V M : The effect of d iabetes on express ion of b e t a l - , beta2-, and beta3-adrenoreceptors in rat hearts. Diabetes 50 :455-461,2001 189 164. Stanley W C , Dore J J , Hall J L , Hamilton C D , Pizzurro R D , Roth DA: Diabetes reduces right atrial beta-adrenergic signal ing but not agonist stimulation of heart rate in swine. Can J Physiol Pharmacol 79 :346-351,2001 165. Aust in C E , Chess-Wi l l i ams R: Transient elevation of card iac beta-adrenoceptor respons iveness and receptor number in the streptozotocin-diabetic rat. J Auton Pharmacol 12:205-214, 1992 166. Sel lers D J , Chess-Wi l l i ams R: The effect of streptozotocin- induced d iabetes on card iac beta-adrenoceptor subtypes in the rat. J Auton Pharmacol 21:15-21, 2001 167. Latifpour J , McNei l l J H : Card iac autonomic receptors: effect of long-term exper imental d iabetes. J Pharmacol Exp Ther 230:242-249, 1984 168. Dincer U D , Onay A , Ari N, Ozce l ikay A T , Al tan V M : The effects of d iabetes on beta-ad renoceptor mediated respons iveness of human and rat atria. Diabetes Res Clin Pract 40:113-122, 1998 169. Moniotte S , Kobzik L, Feron O, Trochu J N , Gauthier C , Bal l igand J L : Upregulat ion of beta(3)-adrenoceptors and altered contractile response to inotropic amines in human failing myocard ium. Circulation .103:1649-1655, 2001 170. Luiken J J , Wi l lems J , Coort S L , C o u m a n s W A , Bonen A , V a n Der V u s s e G J , G la tz J F : Effects of c A M P modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes. Biochem J 367 :881-887, 2002 190 171. M a s c a r o C , Acos ta E, Ortiz J A , Marrero P F , Hegardt F G , Haro D: Control of human muscle- type carnit ine palmitoyltransferase I gene transcription by perox isome proliferator-activated receptor. J Biol Chem 273:8560-8563, 1998 172. W a n g G L , Moore ML , McMil l in J B : A region in the first exon/intron of rat carnit ine palmitoyltransferase Ibeta is involved in enhancement of basa l transcript ion. Biochem J 362:609-618, 2002 173. Moore ML , Park E A , McMil l in J B : Upstream stimulatory factor represses the induction of carnitine palmitoyltransferase-lbeta express ion by P G C - 1 . J Biol Chem 278:17263-17268, 2003 174. S a w a d o g o M, Roeder R G : Interaction of a gene-speci f ic transcription factor with the adenovi rus major late promoter upstream of the T A T A box region. Cell 43:165-175, 1985 175. Qin W , Khuchua Z, Klein S C , Strauss A W : Elements regulating' card iomyocyte express ion of the human sarcomer ic mitochondrial creatine k inase gene in t ransgenic mice. J Biol Chem 272:25210-25216, 1997 176. O j a m a a K, Samare l A M , Klein I: Identification of a contract i le-responsive e lement in the card iac a lpha-myosin heavy chain gene. J Biol Chem 270:31276-3 1 2 8 1 , 1 9 9 5 177. Kerner J , Distler A M , Minkler P, Paf land W , Peterman S M , Hoppel C L : Phosphory lat ion of rat liver mitochondrial carnitine palmitoyltransferase-l : effect on the kinetic propert ies of the enzyme. J Biol Chem 279:41104-41113, 2004 178. V e l a s c o G , G u z m a n M, Zammit V A , G e e l e n M J : Involvement of Ca2+/calmodul in-dependent protein k inase II in the activation of carnitine 191 palmitoyltransferase I by okada ic acid in rat hepatocytes. Biochem J 321 ( Pt 1):211-216, 1997 179. J in Y J , Li S Z , Zhao Z S , A n J J , Kim R Y , K im Y M , Baik J H , Lim S K : Carni t ine palmitoyltransferase-1 (CPT-1) activity stimulation by cerulenin via sympathet ic nervous sys tem activation overr ides cerulenin's peripheral effect. Endocrinology 145:3197-3204, 2004 180. Ruehr M L , Russe l l MA , Bond M: A-k inase anchoring protein targeting of protein k inase A in the heart. J Mol Cell Cardiol 37:653-665, 2004 181. Fukumoto K, Pierro A , Zammit V A , Spi tz L, Eaton S : Tyros ine nitration of carnit ine palmitoyl t ransferase I during endotoxaemia in suckl ing rats. Biochim Biophys Acta 1683:1 -6, 2004 182. Fukumoto K, Pierro A , Spi tz L, Eaton S : Differential effects of neonatal endotoxemia on heart and kidney carnitine palmitoyl t ransferase I. J Pediatr Surg 37:723-726, 2002 183. Hibbs J B , Jr., Taintor R R , Vavr in Z, Rachl in E M : Nitric oxide: a cytotoxic act ivated macrophage effector molecule. Biochem Biophys Res Commun 157:87-94, 1988 184. Hibbs J B , Jr. , Taintor R R , Vavr in Z : Macrophage cytotoxicity: role for L-arginine de iminase and imino nitrogen oxidation to nitrite. Science 235:473-476, 1987 185. Brune B, Lapet ina E G : Activation of a cytosol ic ADP-r ibosy l t ransferase by nitric oxide-generat ing agents. J Biol Chem 264:8455-8458, 1989 186. Mol ina y V e d i a L, McDona ld B, R e e p B, Brune B, Di Silvio M, Bill iar T R , Lapet ina E G : Nitric oxide- induced S-nitrosylation of g lycera ldehyde-3-phosphate 192 dehydrogenase inhibits enzymat ic activity and increases endogenous A D P -ribosylation. J Biol Chem 267:24929-24932, 1992 187. Zhang J , Snyder S H : Nitric oxide stimulates auto-ADP-r ibosy lat ion of g lycera ldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci USA 89:9382-9385, 1992 188. Depre C , Vanoversche lde J L , Goudemant J F , Mottet I, Hue L: Protect ion against i schemic injury by nonvasoact ive concentrat ions of nitric oxide syn thase inhibitors in the perfused rabbit heart. Circulation 92:1911-1918, 1995 189. R e c c h i a FA , McConne l l PI, Loke K E , X u X , O c h o a M, Hintze T H : Nitric oxide controls card iac substrate utilization in the consc ious dog. Cardiovasc Res 44:325-332, 1999 190. Baker J G : The selectivity of beta-adrenoceptor antagonists at the human b e t a l , beta2 and beta3 adrenoceptors. BrJPharmacol 144:317-322, 2005 191. Smith C , Teitler M: Beta-blocker selectivity at c loned human beta 1- and beta 2-adrenergic receptors. Cardiovasc Drugs Ther 13:123-126, 1999 192. Freireich E J , G e h a n E A , Rai l D P , Schmidt L H , Sk ipper H E : Quantitative compar ison of toxicity of ant icancer agents in mouse, rat, hamster, dog, monkey, and man . Cancer Chemother Rep 50:219-244, 1966 193. Junod A , Lambert A E , Stauffacher W, Renold A E : Diabetogenic action of streptozotocin: relationship of dose to metabol ic response. J Clin Invest 48 :2129-2139, 1969 194. Toml inson K C , Gard iner S M , Hebden R A , Bennett T: Funct ional c o n s e q u e n c e s of streptozotocin- induced diabetes mellitus, with particular reference to the cardiovascular sys tem. Pharmacol Rev 44:103-150, 1992 193 195. T a k a s u N, Komiya I, A s a w a T, N a g a s a w a Y , Y a m a d a T: Streptozocin- and al loxan- induced H 2 0 2 generat ion and D N A fragmentation in pancreat ic islets. H 2 0 2 as mediator for D N A fragmentation. Diabetes 40:1141-1145, 1991 196. Wi l son G L , Patton N J , M c C o r d J M , Mull ins DW, M o s s m a n BT: Mechan i sms of streptozotocin- and al loxan- induced damage in rat B cel ls. Diabetologia 27:587-591, 1984 197. Y a m a m o t o H, Uchigata Y , Okamoto H: Streptozotocin and al loxan induce D N A strand breaks and poly(ADP-r ibose) synthetase in pancreat ic islets. Nature 294:284-286, 1981 198. Uchigata Y , Yamamoto H, Kawamura A , Okamoto H: Protect ion by superox ide d ismutase, cata lase, and poly(ADP-r ibose) synthetase inhibitors against a l loxan- and streptozotocin- induced islet D N A strand breaks and against the inhibition of proinsulin synthesis. J Biol Chem 257:6084-6088, 1982 199. Okamoto H: Molecular bas is of exper imental d iabetes: degenerat ion, oncogenes is and regeneration of pancreat ic beta-cel ls of islets of Langerhans. Bioessays 2 :15-21, 1985 200. Cetkovic-Cvr l je M, Sand ler S , Eizirik DL: Nicot inamide and dexamethasone inhibit interleukin-1-induced nitric oxide production by R I N m 5 F cel ls without decreas ing messenger r ibonucleic acid express ion, for nitric oxide synthase. Endocrinology 133:1739-1743, 1993 201 . Agarwa l M K : Streptozotocin: mechan isms of act ion: proceedings of a workshop held on 21 June 1980, Wash ington, D C . FEBS Lett 120:1-3, 1980 194 202. V e r m a S , Ar ikawa E, McNei l l J H : Long-term endothel in receptor b lockade improves card iovascular function in d iabetes. Am J Hypertens 14:679-687, 2001 203. X iang H, Heyl iger C E , McNei l l J H : Effect of myo-inositol and T 3 on myocard ia l l ipids and card iac function in streptozocin- induced diabet ic rats. Diabetes 37:1542-1548, 1988 204. Vad lamud i R V , Rodgers R L , McNei l l J H : The effect of chronic a l loxan- and streptozotocin- induced diabetes on isolated rat heart performance. Can J Physiol Pharmacol 60 :902-911, 1982 205 . Barr R, Lopaschuk G : Measurements of energy metabol ism in the isolated heart. In Measurement of Cardiac Function McNei l l J H , E d . B o c a Raton, F l , C R C Press , 1997 206. Wambo l t R B , Lopaschuk G D , Brownsey R W , Al lard M F : Dichloroacetate improves post ischemic function of hypertrophied rat hearts. J Am Coll Cardiol 36:1378-1385, 2000 207. Wambol t R B , Henning S L , Engl ish DR, Dyachkova Y , Lopaschuk G D , Al lard M F : G l u c o s e utilization and glycogen turnover are accelerated in hypertrophied rat hearts during severe low-flow ischemia. J Mol Cell Cardiol 31:493-502, 1999 208. Leong H S , Grist M, Pa rsons H, Wambol t R B , Lopaschuk G D , Brownsey R, Al lard M F : Acce le ra ted rates of glycolysis in the hypertrophied heart: are they a methodological artifact? Am J Physiol Endocrinol Metab 282 :E1039-1045 , 2002 209. Longnus S L , Wambol t R B , Barr R L , Lopaschuk G D , Al lard M F : Regulat ion of myocardia l fatty acid oxidation by substrate supply. Am J Physiol Heart Circ P/ i ys /o /281 :H1561-1567 , 2001 195 210. Al lard M F , Henning S L , Wambol t R B , Gran leese S R , Engl ish D R , Lopaschuk G D : G lycogen metabol ism in the aerobic hypertrophied rat heart. Circulation 96:676-682, 1997 211 . Buettner R, Newgard C B , R h o d e s C J , O'Doherty R M : Correct ion of diet-induced hyperglycemia, hyperinsul inemia, and skeletal musc le insulin res istance by moderate hyperlept inemia. Am J Physiol Endocrinol Metab 278 :E563-569 , 2000 212. Al ly A , Park G : Rap id determination of creatine, phosphocreat ine, purine b a s e s and nucleot ides ( A T P , A D P , A M P , G T P , G D P ) in heart b iops ies by gradient ion-pair reversed-phase liquid chromatography. J Chromatogr 575:19-27, 1992 213. King MT, Re iss P D , Cornel l N W : Determination of short-chain coenzyme A compounds by reversed-phase high-performance liquid chromatography. Methods Enzymol 166:70-79, 1988 214. Al lard M F , Parsons HL, Saeed i R, Wambol t R B , Brownsey R: A M P K and metabol ic adaptat ion by the heart to pressure over load. Am J Physiol Heart Circ Physiol 292 :H140-148, 2007 215. Lehman T C , Hale D E , Bha la A , Thorpe C : A n acy l - coenzyme A dehydrogenase a s s a y utilizing the ferricenium ion. Anal Biochem 186:280-284, 1990 216. Srere P A : Citrate Synthase. Methods in Enzymology 13:3-26, 1969 217. Goldste in S , Czapsk i G , Lind J , Merenyi G : Effect of * N O on the decomposi t ion of peroxynitrite: reaction of N 2 0 3 with O N O O . Chem Res Toxicol 12:132-136, 1999 196 218. T ien M, Berlett B S , Levine R L , Chock P B , Stadtman E R : Peroxynitr i te-mediated modif ication of proteins at physiological carbon dioxide concentrat ion: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation. Proc Natl Acad Sci USA 96:7809-7814, 1999 219. Rob inson A M , Wi l l iamson D H : Physio logical roles of ketone bodies as substrates and s ignals in mammal ian t issues. Physiol Rev 60:143-187, 1980 220. Fortier M, W a n g S P , Maur iege P, S e m a c h e M, Mfuma L, Li H, Levy E, R ichard D, Mitchell G A : Hormone-sensi t ive l ipase- independent adipocyte l ipolysis during beta-adrenergic stimulation, fast ing, and dietary fat loading. Am J Physiol Endocrinol Metab 287 :E282-288, 2004 221 . Huck ing K, Hami l ton-Wess ler M, El lmerer M, Bergman R N : Burst- l ike control of l ipolysis by the sympathet ic nervous system in vivo. J Clin Invest 111:257-264, 2003 222. Vanl ta l l ie T B , Nufert T H : Ketones: metabol ism's ugly duckl ing. Nutr Rev 61:327-341, 2003 223 . Lopaschuk G D : Malonyl C o A control of fatty acid oxidation in the diabetic rat heart. AdvExp Med6/'o/498:155-165, 2001 224. R a m a n a d h a m S , Brownsey R W , Cros G H , Mongold J J , McNei l l J H : Sus ta ined prevention of myocardial and metabol ic abnormali t ies in diabetic rats following withdrawal from oral vanadyl treatment. Metabolism 38:1022-1028, 1989 . 225 . Bhimji S , God in DV, McNei l l J H : B iochemical and functional changes in hearts from rabbits with diabetes. Diabetologia 28:452-457, 1985 197 226. Pulini lkunnil T, Abrahan i A , Va rghese J , C h a n N, Tang I, G h o s h S , Ku lpa J , Al lard M, Brownsey R, Rodr igues B: Ev idence for rapid "metabol ic switching" through lipoprotein l ipase occupat ion of endothelial-binding sites. J Mol Cell Cardiol 35 :1093-1103, 2003 227. Scheuermann-Frees tone M, Madsen P L , Manners D, Blamire A M , Buck ingham R E , Styles P, R a d d a G K , Neubauer S , C larke K: Abnorma l card iac and skeletal musc le energy metabol ism in patients with type 2 diabetes. Circulation 107:3040-3046, 2003 228. Kewal ramani G , A n D, K im M S , G h o s h S , Qi D, Abrahan i A , Pulini lkunnil T, S h a r m a V , Wambol t R B , Al lard M F , Innis S M , Rodr igues B: A M P K control of myocardia l fatty acid metabol ism fluctuates with the intensity of insulin-deficient d iabetes. J Mol Cell Cardiol 42:333-342, 2007 229. Okere IC, Chand ler M P , McEl f resh TA , Renn ison J H , Kung TA , Hoit B D , Ernsberger P, Young M E , Stanley W C : Carnit ine palmitoyl t ransferase- l inhibition is not assoc ia ted with card iac hypertrophy in rats fed a high-fat diet. Clin Exp Pharmacol Physiol 34:113-119, 2007 230. Koonen D P , Gla tz J F , Bonen A , Luiken J J : Long-chain fatty acid uptake and F A T / C D 3 6 translocation in heart and skeletal musc le . Biochim Biophys Acta 1736 :163 -180 ,2005 231 . N ie lsen LB : Lipoprotein production by the heart: a novel pathway of triglyceride export from cardiomyocytes. Scand J Clin Lab Invest Suppl 237:35-4 0 , 2 0 0 2 232. N ie lsen L B , Bartels E D , Bol lano E: Overexpress ion of apolipoprotein B in the heart impedes card iac triglyceride accumulat ion and development of card iac dysfunct ion in diabetic mice. J Biol Chem 277:27014-27020, 2002 198 233. J i LL , Stratman F W , Lardy HA: Effects of beta 1- and beta 1 + beta 2-antagonists on training-induced myocardial hypertrophy and enzyme adaptat ion. Biochem Pharmacol 36:3411-3417, 1987 234. Nikolaidis LA, Poorn ima I, Parikh P, Magovern M, S h e n Y T , Shannon R P : The effects of combined versus select ive adrenergic b lockade on left ventricular and sys temic hemodynamics , myocardial substrate preference, and regional perfusion in consc ious dogs with dilated cardiomyopathy. J Am Coll Cardiol 47 :1871 -1881 ,2006 235. Hall J L , Lopaschuk G D , Barr A , Br ingas J , Pizzurro R D , Stanley W C : Increased card iac fatty acid uptake with dobutamine infusion in swine is accompan ied by a dec rease in malonyl C o A levels. Cardiovasc Res 32:879-885, 1996 236. King KL , Okere IC, S h a r m a N, Dyck J R , R e s z k o A E , McEl f resh TA , Kerner J , Chand le r M P , Lopaschuk G D , Stanley W C : Regulat ion of card iac ma lony l -CoA content and fatty acid oxidation during increased card iac power. Am J Physiol Heart Circ Physiol 289 .H 1033-1037, 2005 237. Idel l-Wenger J A , Grotyohann LW, Neely J R : C o e n z y m e A and carnitine distribution in normal and ischemic hearts. J Biol Chem 253:4310-4318, 1978 238. R e s z k o A E , Kasumov T, David F, Jobb ins KA , T h o m a s K R , Hoppe l C L , Brunengraber H, Qes Ros iers C : Perox isomal fatty acid oxidation is a substantial source of the acetyl moiety of ma lony l -CoA in rat heart. J Biol Chem 279:19574-1 9 5 7 9 , 2 0 0 4 199 239. Sagge rson E D , Carpenter C A : Effects of fasting, adrenalectomy and streptozotocin-diabetes on sensitivity of hepatic carnitine acyl t ransferase to malonyl C o A . FEBS Lett 129:225-228, 1981 240. C o o k G A : Dif ferences in the sensitivity of carnitine palmitoyltransferase to inhibition by ma lony l -CoA are due to differences in Ki va lues. J Biol Chem 259:12030-12033, 1984 241 . Eaton S , Fukumoto K, Palad io Duran N, Pierro A , Spi tz L, Quant P A , Bartlett K: Carnit ine palmitoyl t ransferase I and the control of myocardia l beta-oxidation flux. Biochem Soc Trans 29:245-250, 2001 242. W e i s B C , E s s e r V , Foster DW, McGar ry J D : Rat heart exp resses two forms of mitochondrial carnitine palmitoyltransferase I. The minor component is identical to the liver enzyme. J Biol Chem 269:18712-18715, 1994 243. Brown N F , W e i s B C , Husti J E , Foster DW, McGar ry J D : Mitochondrial carnit ine palmitoyltransferase I isoform switching in the developing rat heart. J Biol Chem 270:8952-8957, 1995 244. Y u G S , Lu Y C , Gul ick T: Rat carnitine palmitoyltransferase Ibeta m R N A spl icing isoforms! Biochim Biophys Acta 1393:166-172, 1998 245. Y u G S , Lu Y C , Gul ick T: Express ion of novel isoforms of carnit ine palmitoyl transferase I (CPT-1) generated by alternative spl icing of the C P T - i b e t a gene. Biochem J 334 ( Pt 1):225-231, 1998 246. Mori l las M, Clotet J , Rubi B, Ser ra D, Ar ino J , Hegardt F G , As ins G : Inhibition by etomoxir of rat liver carnitine octanoyl transferase is produced through the co-ordinate interaction with two histidine residues. Biochem J 351 Pt 2 :495-502, 2000 200 247. J a c k s o n V N , Cameron J M , Fraser F, Zammit V A , Pr ice NT: U s e of six chimer ic proteins to investigate the role of intramolecular interactions in determining the kinetics of carnitine palmitoyltransferase I isoforms. J Biol Chem 275:19560-19566, 2000 248. J a c k s o n V N , Zammit V A , Pr ice NT: Identification of positive and negative determinants of ma lony l -CoA sensitivity and carnitine affinity within the amino termini of rat liver- and muscle- type carnit ine palmitoyltransferase I. J Biol Chem 275 :38410 -38416 ,2000 249. Da i J , Zhu H, Sh i J , Woldegiorg is G : Identification by mutagenes is of conserved arginine and tryptophan residues in rat liver carnit ine palmitoyl transferase I important for catalytic activity. J Biol Chem 275 :22020-22024, 2000 250. V e l a s c o G , G e e l e n M J , G o m e z del Pulgar T, G u z m a n M: Poss ib le involvement of cytoskeletal components in the control of hepatic carnit ine palmitoyltransferase I activity. Adv Exp Med Biol 466:43-52, 1999 251 . Zammit V A , Pr ice NT, Fraser F, Jackson V N : Structure-function relat ionships of the liver and musc le isoforms of carnitine palmitoyltransferase I. Biochem Soc Trans 29 :287-292, 2001 252. Barger P M , Brandt J M , Leone T C , Weinhe imer C J , Kelly D P : Deactivat ion of perox isome proliferator-activated receptor-alpha during card iac hypertrophic growth. J Clin Invest 105:1723-1730, 2000 253 . Oberkof ler H, Esterbauer H, L innemayr V , Strosberg A D , Krempler F, Pa tsch W : Perox isome proliferator-activated receptor ( P P A R ) g a m m a 201 coactivator-1 recruitment regulates P P A R subtype specificity. J Biol Chem 277:16750-16757, 2002 254. Barger P M , Browning A C , Garner A N , Kelly D P : p38 mitogen-act ivated protein k inase activates perox isome proliferator-activated receptor a lpha: a potential role in the cardiac metabol ic stress response. J Biol Chem 276 :44495-4 4 5 0 1 , 2 0 0 1 255. Pu igserver P, R h e e J , Lin J , W u Z, Y o o n J C , Zhang C Y , K rauss S , Mootha V K , Lowel l B B , Sp iege lman B M : Cytokine stimulation of energy expenditure through p38 M A P k inase activation of P P A R g a m m a coact ivator-1. Mol Cell 8:971-982, 2001 256. W e n z e l S , Muller C , Piper H M , Schluter K D : p38 M A P - k i n a s e in cultured adult rat ventricular cardiomyocytes: express ion and involvement in hypertrophic signal l ing. Eur J Heart Fail 7:453-460, 2005 257. Mor iscot A S , S a y e n M R , Hartong R, W u P, Dil lmann W H : Transcript ion of the rat sarcop lasmic reticulum Ca2+ adenos ine tr iphosphatase gene is increased by 3,5,3-tr i iodothyronine receptor isoform-specif ic interactions with the myocyte-speci f ic enhancer factor-2a. Endocrinology 138:26-32, 1997 258. D immeler S , F leming I, Fissl thaler B, Hermann C , B u s s e R, Ze iher A M : Activat ion of nitric oxide synthase in endothelial cel ls by Akt -dependent phosphorylat ion. Nature 399:601-605, 1999 259. Fulton D, Gratton J P , M c C a b e T J , Fontana J , Fujio Y , W a l s h K, Franke T F , Papapet ropou los A , S e s s a W C : Regulat ion of endothel ium-derived nitric oxide production by the protein k inase Akt. Nature 399:597-601, 1999 202 260. Grei f D M , Kou R, Michel T: Si te-speci f ic dephosphorylat ion of endothel ial nitric oxide synthase by protein phosphatase 2A: ev idence for crosstalk between phosphorylat ion sites. Biochemistry 41 :15845-15853, 2002 2 6 1 . Miche l J B , Feron O, S a s e K, Prabhakar P, Michel T: Caveo l in versus calmodul in. Counterbalancing allosteric modulators of endothel ial nitric oxide synthase. J Biol Chem 272:25907-25912, 1997 262. Prabhakar P, Thatte H S , Goe t z R M , C h o M R , G o l a n D E , Miche l T: Receptor-regulated translocation of endothelial nitric-oxide synthase. J Biol Chem 273:27383-27388, 1998 263 . Brixius K, B loch W , Z iskoven C , Bolck B, Napp A , Pott C , Steinritz D, J im inez M, Add i cks K, Giacob ino J P , Schwinger R H : Beta3-adrenerg ic e N O S stimulation in left ventricular murine myocardium. Can J Physiol Pharmacol 84:1051-1060, 2006 264. Recch ia F A : Ro le of nitric oxide in the regulation of substrate metabol ism in heart failure. Heart Fail Rev 7:141-148, 2002 265. Klatt P, L a m a s S : Regulat ion of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem 267:4928-4944, 2000 266. Levine R L , Moson i L, Berlett B S , Stadtman E R : Methionine res idues as endogenous antioxidants in proteins. Proc Natl Acad Sci USA 93:15036-15040, 1996 267. Pe rez -Ma to I, Cast ro C , Ru iz FA , Corra les F J , Mato J M : Methionine adenosyl t ransferase S-nitrosylation is regulated by the basic and acidic amino ac ids surrounding the target thiol. J Biol Chem 274:17075-17079, 1999 203 268. Stubauer G , Giuffre A , Sarti P: Mechan ism of S-nitrosothiol formation and degradat ion mediated by copper ions. J Biol Chem 274:28128-28133, 1999 269. Liu Z , Rudd M A , F reedman J E , Losca lzo J : S-Transnitrosat ion react ions are involved in the metabol ic fate and biological act ions of nitric oxide. J Pharmacol Exp Ther284:526-534, 1998 270. Za i A , Rudd M A , Scr ibner A W , Losca lzo J : Cel l -sur face protein disulfide i somerase cata lyzes transnitrosation and regulates intracellular transfer of nitric oxide. J Clin Invest 103:393-399, 1999 271 . Broillet M C : S-nitrosylation of proteins. Cell Mol Life Sci 55:1036-1042, 1999 272. Rad i R: Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad S c / ' L / S A 101 :4003-4008 ,2004 273. C a s s i n a A M , Hodara R, S o u z a J M , Thomson L, Cast ro L, Ischiropoulos H, F reeman B A , Rad i R: Cytochrome c nitration by peroxynitrite. J Biol Chem 275 :21409 -21415 ,2000 274. V a d s e t h C , S o u z a J M , Thomson L, Seagraves A , Nagaswami C , Sche iner T, Torbet J , Vi laire G , Bennett J S , Murc iano J C , Muzykantov V , P e n n M S , Hazen S L , W e i s e l J W , Ischiropoulos H: Pro-thrombotic state induced by post-translational modification of f ibrinogen by reactive nitrogen spec ies . J Biol Chem 279:8820-8826, 2004 275. Ba la fanova Z, Bolli R, Zhang J , Zheng Y , P a s s J M , Bhatnagar A , Tang X L , W a n g O, Cardwel l E, Ping P: Nitric oxide (NO) induces nitration of protein k inase Ceps i lon ( P K C e p s i l o n ), facilitating P K C e p s i l o n translocation via enhanced P K C e p s i l o n - R A C K 2 interactions: a novel mechan ism of no-triggered activation of P K C e p s i l o n . J Biol Chem 277:15021-15027, 2002 204 276. S o u z a J M , Daikhin E, Yudkoff M, R a m a n C S , lschiropoulos H: Factors determining the selectivity of protein tyrosine nitration. Arch Biochem Biophys 371:169-178, 1999 277. Liu H, Zheng G , Treber M, Dai J , Woldegiorgis G : Cys te ine-scann ing mutagenes is of musc le carnitine palmitoyltransferase I reveals a single cysteine res idue (Cys-305) is important for catalysis. J Biol Chem 280 :4524-4531, 2005 278. Ghafour i far P, C a d e n a s E: Mitochondrial nitric oxide synthase. Trends Pharmacol Sci 26:190-195, 2005 279. Re iner M, Bloch W , Add icks K: Functional interaction of caveolin-1 and e N O S in myocardia l capil lary endothel ium revealed by immunoelectron microscopy. J Histochem Cytochem 49:1605-1610, 2001 280. Shau l P W : Regulat ion of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol 64:749-774, 2002 281 . G insberg M D , Feliciel lo A , Jones J K , Avvedimento E V , Got tesman M E : PKA-dependen t binding of m R N A to the mitochondrial A K A P 1 2 1 protein. J Mol Biol 327:885-897, 2003 282. Nori A , Lin P J , Casset t i A , Vi l la A , Bayer K U , Vo lpe P: Targe t ing of a lpha-k inase-anchor ing protein (alpha K A P ) to sarcop lasmic reticulum and nuclei of skeletal musc le . Biochem J 370:873-880, 2003 283. F leming I, Fissl thaler B, Dimmeler S , Kemp B E , B u s s e R: Phosphory lat ion of Thr(495) regulates Ca(2+)/calmodul in-dependent endothelial nitric oxide synthase activity. Circ Res 88 :E68-75 , 2001 205 284. Campbe l l S E , Tandon N N , Woidegiorg is G , Luiken J J , Gla tz J F , Bonen A : A novel function for fatty ac id t rans locase (FAT) /CD36 : involvement in long chain fatty acid transfer into the mitochondria. J Biol Chem 279 :36235-36241, 2004 285: Mak IT, Wegl ick i W B : Protection by beta-blocking agents against free radical-mediated sarco lemmal lipid peroxidation. Circ Res 63:262-266, 1988 286. Mak IT, Ar royo C M , Wegl ick i W B : Inhibition of sarco lemmal carbon-centered free radical formation by propranolol. Circ Res 65:1151-1156, 1989 2 8 / . Wegl ick i W B , Mak IT, S im ic M G : Mechan i sms of cardiovascular drugs as antioxidants. J Mol Cell Cardiol 22:1199-1208, 1990 288. Kawa i K, Qin F, Shi te J , M a o W , Fukuoka S , Liang C S : Importance of antioxidant and antiapoptotic effects of beta-receptor b lockers in heart failure therapy. Am J Physiol Heart Circ Physiol 287:H1003-1012, 2004 289. G o m e s A , C o s t a D, L ima J L , Fernandes E: Antioxidant activity of beta-blockers: an effect mediated by scavenging reactive oxygen and nitrogen s p e c i e s ? Bioorg Med Chem 14:4568-4577, 2006 290. Popp DA, T s e T F , S h a h S D , Clutter W E , Cryer P E : Oral propranolol and metoprolol both impair g lucose recovery from insul in- induced hypoglycemia in insul in-dependent diabetes mellitus. Diabetes Care 7:243-247', 1984 2 9 1 . A k s n e s TA , Kje ldsen S E , Manc ia G : The effect of ant ihypertensive agents on new-onset d iabetes mellitus: time to amend the guidel ines? Am J Cardiovasc Drugs 6 :139-147, 2006 292. Deacon S P , Karunanayake A , Barnett D: Acebuto lo l , atenolol, and propranolol and metabol ic responses to acute hypoglycaemia in diabet ics. Br MedJ 2 :1255-1257, 1977 206 293. Kerr D, MacDona ld IA, Heller S R , Tattersall R B : Beta-adrenoceptor b lockade and hypoglycaemia. A randomised, double-bl ind, p lacebo controlled compar ison of metoprolol C R , atenolol and propranolol L A in normal subjects. Br J Clin Pharmacol 29:685-693, 1990 294. Bel l D S , Yumuk V : Frequency of severe hypoglycemia in patients with non-insul in-dependent d iabetes mellitus treated with sul fonylureas or insulin. Endocr Pract 3 :281-283, 1997 295. Toml inson B, Bompart F, G raham B R , Liu J B , Pr ichard B N : Vasodi la t ing mechan i sm and response to physiological pressor stimuli of acute d o s e s of carvedi lol compared with labetalol, propranolol and hydralazine. Drugs 36 Supp l 6:37-47, 1988 

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