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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 CARDIAC CARNITINE  PALMITOYLTRANSFERASE-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  DIABETIC  HEARTS by VIJAY S H A R M A  B S c (Hon), University of E d i n b u r g h , 1998  M B C h B , University of E d i n b u r g h , 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 THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES (Pharmaceutical Sciences)  T H E UNIVERSITY O F BRITISH C O L U M B I A October 2007 © Vijay S h a r m a , 2 0 0 7 .  11  ABSTRACT  Diabetic c a r d i o m y o p a t h y m a y be initiated or c o m p o u n d e d by the h e a v y reliance of the heart on fatty a c i d s and ketones a s m e t a b o l i c fuels, p-blockers h a v e b e e n p r o p o s e d to inhibit fatty acid oxidation by d e c r e a s i n g the activity of the enzyme  carnitine  palmitoyltransferase-1  (CPT-1).  By  inhibiting  fatty  acid  oxidation, p-blockers could improve myocardial efficiency a n d ameliorate the c y t o p l a s m i c accumulation of toxic fatty acid a n d g l u c o s e intermediates.  In this  study, w e  investigated whether  metoprolol  improves c a r d i a c  function a n d inhibits fatty a c i d oxidation in the streptozotocin ( S T Z ) diabetic rat, a m o d e l of poorly controlled type 1 diabetes. T h e a n i m a l s w e r e injected with 6 0 m g / kg S T Z a n d w e r e e u t h a n i z e d six w e e k s following the induction of d i a b e t e s . W e investigated the effects of c h r o n i c metoprolol treatment (75 m g / k g / day), a n d a c u t e metoprolol perfusion on c a r d i a c function, substrate utilization a n d three major s y s t e m s of C P T - 1 regulation: malonyl C o A levels, C P T - 1 transcription a n d covalent modifications (phosphorylation, nitrosylation, glutathiolation, nitration).  C h r o n i c metoprolol treatment improved c a r d i a c function in the diabetic heart. W h e r e a s , chronic metoprolol treatment i n c r e a s e d fatty acid oxidation in control hearts but d e c r e a s e d it in diabetic hearts, acute metoprolol perfusion a l w a y s inhibited fatty acid oxidation. Metoprolol lowered m a l o n y l C o A levels in control hearts, a n d both acute metoprolol perfusion a n d chronic metoprolol treatment  led to d e c r e a s e d C P T - 1 m a x i m u m activity a n d d e c r e a s e d C P T - 1  m a l o n y l C o A sensitivity. C P T - 1 sensitivity w a s i n c r e a s e d by c a l c i u m / c a l m o d u l i n d e p e n d e n t protein k i n a s e phosphorylation a n d d e c r e a s e d by protein k i n a s e A d e p e n d e n t phosphorylation in vitro. C P T - 1 activity w a s inhibited by nitrosylation a n d glutathiolation, and stimulated treatment  by nitration  in  vitro.  C h r o n i c metoprolol  d e c r e a s e d the binding a n d coactivation of peroxisome-proliferator  receptor-y coactivator  1-a  (PGC-1a)  a n d peroxisome-proliferator  receptor-a  Ill  ( P P A R - a ) , a n d a l s o i n c r e a s e d the binding of the repressor protein u p s t r e a m stimulatory factor-2 (USF-2).  In c o n c l u s i o n , 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 C P T - 1 itself. T h e effects of metoprolol on C P T - 1 w e r e mediated acutely by covalent modifications and chronically by inhibition of the transcriptional c o m p l e x that induces CPT-1 e x p r e s s i o n .  TABLE OF CONTENTS Page Abstract  »  T a b l e of C o n t e n t s  iv  List of T a b l e s  vii  List of F i g u r e s  ."  viii  List of S c h e m e s  x  List of A b b r e v i a t i o n s  xi  Acknowledgments  xiv  Dedication  xvi  INTRODUCTION  1  I: Diabetic C a r d i o m y o p a t h y  1  II: C a r d i a c M e t a b o l i s m  8  III: Modulation of C a r d i a c M e t a b o l i s m a s a T h e r a p e u t i c Strategy  20  IV: T h e Benefits of p-Adrenergic B l o c k a d e  22  V : p - A d r e n o c e p t o r Signalling  24  VI: Potential Links B e t w e e n p-Adrenoceptors a n d C a r d i a c M e t a b o l i s m .  30  V l l : S p e c i f i c R e s e a r c h P r o b l e m and R e s e a r c h Strategy  33  Vlll: Working Hypotheses  35  IX: O b j e c t i v e s . .  36  MATERIALS AND METHODS  39  I: M e a s u r e m e n t of e x vivo Left Ventricular Function  39  (a) A n i m a l T r e a t m e n t s  39  (b) M e a s u r e m e n t of P l a s m a P a r a m e t e r s  39  (c) Direct M e a s u r e m e n t of Left Ventricular P r e s s u r e  40  II: M e a s u r e m e n t of ex wVo C a r d i a c M e t a b o l i s m  40  (a) A n i m a l T r e a t m e n t s a n d M e a s u r e m e n t of P l a s m a P a r a m e t e r s  40  (b) M e a s u r e m e n t of C a r d i a c M e t a b o l i s m  42  (c) M e a s u r e m e n t of T i s s u e G l y c o g e n a n d Triglyceride L e v e l s  43  (d) M e a s u r e m e n t of T i s s u e Malonyl C o A a n d A d e n i n e Nucleotide Levels  44  (e) M e a s u r e m e n t of T i s s u e Nitrate/ Nitrite L e v e l s  44  III: M e a s u r e m e n t of K i n a s e a n d B i o c h e m i c a l E n z y m e Activities  44  (a) A M P K , P K A a n d C A M K Activities  44  (b) C P T - 1 A s s a y  45  (c) A c y l C o A D e h y d r o g e n a s e A s s a y  46  (d) Citrate S y n t h a s e A s s a y  47  IV: Immunoprecipitation and M e a s u r e m e n t of Protein E x p r e s s i o n by W e s t e r n Blotting  48  (a) O v e r v i e w of Experimental D e s i g n  48  (b) S a m p l e Preparation  50  (c) S D S P A G E , W e s t e r n Blotting a n d Dot Blotting  50  (d) Immunoprecipitation Protocol  51  (e) Dot Blotting  51  V : Functional Effects of C P T - 1 C o v a l e n t Modifications in Isolated Mitochondria  52  (a) Isolation of Mitochondria  52  (b) K i n a s e P h o s p h o r y l a t i o n of Isolated Mitochondria  53  (c) Peroxynitrite D o s e - R e s p o n s e C u r v e in Isolated Mitochondria  54  (d) M e a s u r e m e n t of C'PT-1 Activity, P h o s p h o r y l a t i o n and K i n a s e Binding  54  VI: Identification of C P T - 1 Phosphorylation S i t e s by L C M S / M S  54  VII: D a t a A n a l y s i s  55  RESULTS  56  I: G e n e r a l C h a r a c t e r i s t i c s .  .'.  II: F u n c t i o n a l a n d Metabolic Effects of C h r o n i c Metoprolol Treatment  56 56  VI  III: M a l o n y l C o A L e v e l s  78  IV: C P T - 1 Activity and Malonyl C o A Sensitivity  78  V : Regulation of C P T - 1 E x p r e s s i o n  79  VI: p-Adrenoceptor Signalling  100  VII: C P T - 1 C o v a l e n t Modifications  117  DISCUSSION  137  I: Effects of Metoprolol on C a r d i a c Function and M e t a b o l i s m  137  II: C P T - 1 Activity a n d Regulation by Malonyl C o A  142  III: Regulation of C P T - 1 E x p r e s s i o n  145  IV: p-Adrenoceptor Signalling P a t h w a y s : Modulation of K i n a s e s andeNOS  149  V : ' N O / R N S - Induced C o v a l e n t Modifications of C P T - 1  152  VI: P h o s p h o r y l a t i o n of C P T - 1  157  VII: S i g n i f i c a n c e of the P r e s e n t S t u d i e s  160  VIII: C o n c l u s i o n s  164  BIBLIOGRAPHY  168  vii  LIST OF TABLES Table  Page  1. G e n e r a l characteristics a n d p l a s m a parameters at termination  57  2. Lactate production a n d tissue g l y c o g e n , triglyceride a n d malonyl C o A levels following chronic metoprolol treatment  64  3. M y o c a r d i a l e n e r g e t i c s a n d A M P K activity following c h r o n i c metoprolol treatment  65  4. G l y c o l y s i s a n d fatty acid oxidation e n z y m e activities following chronic metoprolol treatment  67  5. Lactate production a n d tissue g l y c o g e n , triglyceride a n d malonyl C o A levels following acute metoprolol perfusion  70  6. M y o c a r d i a l energetics a n d A M P K activity following a c u t e metoprolol perfusion 7. T i s s u e activities of P K A a n d C A M K  71 109  viii  LIST OF FIGURES  Figure  Page  1. M e c h a n i c a l p e r f o r m a n c e of isolated perfused hearts: left ventricular p r e s s u r e m e a s u r e m e n t s  58  2. M e c h a n i c a l performance of isolated perfused hearts: flow a n d rate-pressure product m e a s u r e m e n t s  60  3. Effects of chronic in vivo metoprolol treatment on m e t a b o l i s m of isolated perfused hearts  62  4. A c u t e effects of metoprolol on metabolism of isolated perfused hearts  68  5. A C C a n d M C D e x p r e s s i o n a n d phosphorylation  72  6. C P T - 1 activity a n d malonyl C o A sensitivity  74  7. P h a r m a c o l o g i c a l effects of metoprolol on C P T - 1 activity  76  8. Total C P T - 1 e x p r e s s i o n  80  9. C P T - 1 B ( M u s c l e Isoform) e x p r e s s i o n  82  10. C P T - 1 A (Liver Isoform) e x p r e s s i o n  84  11. P P A R - a , P G C 1 a , P D K - 4 e x p r e s s i o n following c h r o n i c treatment with metoprolol  86  12. Densitometric a n a l y s i s of P P A R - a , P G C 1 a , P D K - 4 e x p r e s s i o n following chronic treatment with metoprolol  88  13. U S F - 1 , U S F - 2 , M H C a n d S E R C A - 2 e x p r e s s i o n following chronic treatment with metoprolol  90  14. Densitometric a n a l y s i s of U S F - 1 and U S F - 2 e x p r e s s i o n following chronic treatment with metoprolol  92  15. Densitometric analysis of M H C a n d S E R C A e x p r e s s i o n following c h r o n i c 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 , a n d of M E F - 2 A a n d U S F - 2 to P P A R - a m e a s u r e d by immunoprecipitation <  96  ix  17. Densitometric a n a l y s i s of P G C 1 a binding  98  18. E x p r e s s i o n of p-Adrenoceptor s u b t y p e s  101  19. Densitometric a n a l y s i s of e x p r e s s i o n of p-Adrenoceptor s u b t y p e s following chronic metoprolol treatment  103  2 0 . Binding of G s a n d G i to p2-adrenoceptors  105  2 1 . Akt P h o s p h o r y l a t i o n  107  2 2 . E x p r e s s i o n of e N O S a n d i N O S  111  2 3 . P h o s p h o r y l a t i o n of e N O S a t S e r 1177 a n d T h r 4 9 5  113  24. B i o m a r k e r s of N O a n d R N S  115  2 5 . C o v a l e n t modifications of C P T - 1 m e a s u r e d by immunoprecipitation 26. Densitometric a n a l y s i s of C P T - 1 covalent modifications  118 120  27. Binding of P K A a n d A K A P - 1 4 9 to C P T - 1 , a n d phosphorylation state of A K A P - 1 4 9  122  28. Densitometric a n a l y s i s of the binding of P K A and A K A P - 1 4 9 to C P T - 1 , a n d phosphorylation state of A K A P - 1 4 9  124  2 9 . Binding of C A M K - I I to C P T - 1  126  30. P h o s p h o r y l a t i o n of C P T - 1 by P K A in isolated mitochondria  128  3 1 . P h o s p h o r y l a t i o n of C P T - 1 by C A M K in isolated mitochondria  130  32. P h o s p h o r y l a t i o n of C P T - 1 by A k t in isolated mitochondria  132  33. Incubation of isolated mitochondria with peroxynitrite  134  X  LIST OF SCHEMES  Scheme  Page  1. M e c h a n i s m s involved in the p a t h o g e n e s i s of diabetic c a r d i o m y o p a t h y  10  2. S u m m a r y of fatty acid and g l u c o s e metabolism  14  3. R e g u l a t i o n of malonyl C o A levels.  18  4. p-adrenergic signaling pathways  26  5. P r o p o s e d m e c h a n i s m of action of metoprolol  147  6. N O a n d R N S - m e d i a t e d modifications of thiol r e s i d u e s  154  7. S u m m a r y of the acute effects of metoprolol on malonyl C o A levels, C P T - 1 malonyl C o A sensitivity a n d C P T - 1 activity  166  LIST OF ABBREVIATIONS  +dP/ dt  M a x i m u m R a t e of Contraction  -dP/dt  M a x i m u m R a t e of Relaxation  ACC  Acetyl C o A Carboxylase  Acyl C o A  Acyl CoEnzyme A  ADP  A d e n o s i n e diphosphate  AGE  A d v a n c e d G l y c o s y l a t i o n Endproduct  Akt  Protein K i n a s e B  AMP  Adenosine monophosphate  cAMP  Cyclic A M P  ANOVA  A n a l y s i s of V a r i a n c e  AMPK  A M P - a c t i v a t e d protein k i n a s e  ANP  Atrial Natriuretic Peptide  API  A t m o s p h e r i c P r e s s u r e Ionization  ATP  A d e n o s i n e triphosphate  BH  4  Tetrahydrobiopterin  BSA  Bovine Serum Albumin  CAMK  C a l c i u m / calmodulin d e p e n d e n t protein k i n a s e  CAPRICORN  C a r v e d i l o l Post-Infarction Survival Control in Left Ventricular Dysfunction Trial  CAT  Carnitine A c y l t r a n s f e r a s e  CD36  Fatty acid t r a n s l o c a s e  CPT  Carnitine Palmitoyltransferase  DCA  Dichloroacetate  DIGAMI  D i a b e t e s Insulin G l u c o s e in A c u t e M y o c a r d i a l Infarction Trial  EDTA  E t h y l e n e d i a m i n e Tetraacetic A c i d  EGTA  -  Ethylene-glycol-bis(p-aminoethyl ether)tetraacetic A c i d  ERGO-1  Etomoxir for the R e c o v e r y of G l u c o s e Oxidation Trial  ERR  Estrogen-Related Receptor  FACS  Fatty A c y l C o A S y n t h a s e  FADH  2  Flavine A d e n i d e Dinucleotide  FABP  Fatty A c i d Binding Protein  LPL  Lipoprotein L i p a s e  GAPDH  Glyceraldehyde-3-phosphate Dehydrogenase  GLM-ANOVA  G e n e r a l linear m o d e l A N O V A  GLP-1  G l u c a g o n - l i k e Peptide 1  cGMP  Cyclic guanosine monophosphate  HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic A c i d  MHC  Myosin Heavy Chain  HMG CoA  p-hydroxy-p-methylglutaryl-CoA  HPLC  High-performance Liquid C h r o m a t o g r a p h y  IP  Intraperitoneal  IV  Intravenous  3-KAT  3-Ketoacyl T r a n s f e r a s e  LC M S M S  Liquid C h r o m a t o g r a p h y T a n d e m M a s s S p e c t r o s c o p y  LVDP  Left Ventricular D e v e l o p e d P r e s s u r e  LVEDP  Left Ventricular E n d - D i a s t o l i c P r e s s u r e  MCD  Malonyl C o A Decarboxylase  MCT  M o n o c a r b o x y l a t e Transporter  MEF-2A  M y o c y t e e n h a n c e r factor-2A  MERIT-HF  Metoprolol C R / X L R a n d o m i s e d Intervention Trial in C o n g e s t i v e Heart Failure Trial  MOPS  3-(N-Morpholino)-propanesulfonic A c i d  m/z  M a s s to C h a r g e Ratio  NADH  Nicotinamide a d e n i d e dinucleotide  NO  Nitric O x i d e  eNOS  Endothelial Nitric O x i d e S y n t h a s e  iNOS  Inducible Nitric O x i d e S y n t h a s e  mtNOS  Mitochondrial Nitric O x i d e S y n t h a s e  PDE  Phosphodiesterase  PDH  Pyruvate Dehydrogenase  PDK  P D H kinase  PFK  Phosphofructokinase  PGC-1  P P A R - y coactivator protein-1  PI3K  P h o s p h a t i d y l inositol-3 kinase,  PKA  Protein K i n a s e A  PKC  Protein K i n a s e C  PKG  Protein K i n a s e G  PARP  Poly (ADP-ribose) Polymerase  PPAR  P e r o x i s o m e proliferator activated  RAGE  R e c e p t o r for A G E  RNA  Ribonucleic Acid  RNS  R e a c t i v e Nitrogen S p e c i e s  ROS  Reactive Oxygen Species  RXR  Retinoic A c i d R e c e p t o r  SEM  S t a n d a r d Error of the M e a n  SERCA  S a r c o p l a s m i c Reticulum C a l c i u m A T P a s e  STZ  Streptozotocin  T C A cycle  Tricarboxylic A c i d C y c l e  USF  U p s t r e a m Stimulatory F a c t o r  receptor  xiv  ACKNOWLEDGMENTS First a n d foremost, I want to set down here my d e e p gratitude to Dr. J o h n H. M c N e i l l , my supervisor, tutor a n d mentor for providing m e with the opportunity to work with him over the past four y e a r s a n d for his unfailing e n c o u r a g e m e n t , inspiration, thoughtfulness a n d c a r e . I a l s o wish to thank my s u p e r v i s o r Dr. M i c h a e l A l l a r d for his a d v i c e , constant support and for his thoughtfulness a n d meticulous attention to my work carried out in his laboratory. I a l s o o w e thanks to all the m e m b e r s of my r e s e a r c h committee, Dr. R o g e r B r o w n s e y , Dr. Katherine M a c L e o d , Dr. Brian R o d r i g u e s and Dr. W a y n e R i g g s for their invaluable input a n d constructive a d v i c e throughout the c o u r s e of my P h D program. I a m a l s o indebted to Dr. R o g e r B r o w n s e y for m a n y stimulating a n d invaluable d i s c u s s i o n s , a n d to his technician, J e r z y K u l p a , for carrying out the m e a s u r e m e n t s of a d e n i n e dinucleotides a n d C o A esters. S p a c e forbids m e from a p e r s o n a l mention of m a n y wonderful p e o p l e both in the F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s a n d at the iCapture center, but I must m a k e an e x c e p t i o n of M s . Violet Y u e n a n d thank her for her friendship and tireless support over the y e a r s ; I found  her technical expertise to be truly  r e m a r k a b l e . I a m a l s o indebted to Mr. R i c h a r d W a m b o l t a n d M r s . H a n n a h P a r s o n s , two outstanding t e c h n i c i a n s , for all the time they d e v o t e d to training a n d supporting m e a s well a s for their friendship. I would a l s o like to thank P a v a n Dhillon, a p h a r m a c y student, w h o h a s w o r k e d diligently with m e a s my right-hand m a n a n d friend for three y e a r s . I have had the good fortune to work with m a n y outstanding work study a n d s u m m e r students, S h a h i l e e n R e m t u l l a , L i z a T o n g , D a l e Dhillon, K a r e n W i n a n d Sherry W u , w h o s e help a n d devotion to work I gratefully a c k n o w l e d g e . M y t h a n k s are d u e to M s . M o i r a G r e a v e n for her expert secretarial a s s i s t a n c e , a n d a l s o to our former laboratory m a n a g e r , M s . M a r y Battel!, for her  XV  help a n d support. T h e support of the personnel in the animal c a r e and p u r c h a s e / ordering facilities at U B C and St. P a u l ' s Hospital is greatly a p p r e c i a t e d . I a m grateful to Dr. H e l e n Burt and Mr. J o h n J a c k s o n for allowing m e to u s e their spectrophotometer to do e n z y m e kinetics, to Dr. Katherine T h o m p s o n and Dr. C h r i s Orvig for the use of the infra-red spectrophotometer, and to Dr. J a s o n D y c k and Dr. G a r y L o p a s c h u k at the University of Alberta for the g e n e r o u s gift of M C D antibodies. I a l s o thank Dr. S u z a n n e Perry and Dr. S h o u m i n g H e at the U B C P r o t e o m i c s C o r e 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 financial help and I wish to a c k n o w l e d g e the support  I received from the C a n a d i a n Institutes of  Health  R e s e a r c h ( C I H R ) , the R x & D Health Foundation and the C a n a d i a n D i a b e t e s A s s o c i a t i o n with s c h o l a r s h i p s over the c o u r s e of my studies. I would like to mention my c l o s e friends Dr. S h o u m a Dutta, Dr. H e l e n E n g d a r a n d M s B a r b a r a B u c k i n x for their m a n y y e a r s of loyal friendship. Finally, I wish to thank my wonderful parents for their e n d l e s s love and e n c o u r a g e m e n t ; they h a v e b e e n the s o u r c e of my inspiration and I love them dearly.  xvi  DEDICATION  D e d i c a t e d to my prescient parents w h o s e timely nudge led m e to the study of m e d i c i n e a n d s c i e n c e s , a n d o p e n e d up for m e a n e w a n d exciting world to explore life's s e c r e t s in all their w o n d e r a n d beauty. I pray:  " T e a c h m e , my G o d a n d K i n g , In all things thee to s e e , A n d what I d o in any thing, T o d o it for thee."  G e o r g e Herbert (1593-1662): T h e Elixir'  A n d I know:  " . . . e x p e r i e n c e is an arch wherethrough G l e a m s that untraveled world, w h o s e margin f a d e s For ever and ever when I move  T o strive, to s e e k , to find a n d not to yield."  Alfred Lord T e n n y s o n (1809-1892), ' U l y s s e s '  1  INTRODUCTION I. Diabetic Cardiomyopathy C a r d i o v a s c u l a r d i s e a s e is t h e leading c a u s e of death a m o n g diabetic patients, a c c o u n t i n g for 8 0 % of all deaths in this group (1). Indeed, d i a b e t e s is an independent  risk  factor  for c a r d i o v a s c u l a r  death,  a n d mortality  following  m y o c a r d i a l infarction is i n c r e a s e d in diabetic patients (2-5). T h e most c o m m o n c a u s e of this c a r d i o v a s c u l a r mortality is heart failure. T h e F r a m i n g h a m Heart study r e v e a l e d that heart failure is twice a s c o m m o n in diabetic m a l e s a n d five times a s c o m m o n in diabetic f e m a l e s a g e d 4 5 - 7 4 y e a r s w h e n c o m p a r e d to n o n diabetic a g e - m a t c h e d controls (6). Furthermore, according to the results of the U K prospective diabetes study, for e a c h 1% i n c r e a s e in the H b A  1 C l  the risk of  heart failure i n c r e a s e s by 1 5 % (7). T a k e n together, t h e s e data clearly e s t a b l i s h that there is a link between diabetes a n d heart failure. T h e p r o g n o s i s of heart failure in the context of diabetes is very poor. In the Diabetes Insulin G l u c o s e in Acute  Myocardial  Infarction  m y o c a r d i a l infarctions following  myocardial  (DIGAMI)  study,  a prospective  study  of first  in diabetic patients, 6 6 % of d e a t h s in t h e first y e a r infarction  were  caused  by heart  failure  (8). S y s t o l i c  dysfunction in diabetic patients carries a n a n n u a l mortality of 1 5 - 2 0 % . T h r e e pathophysiological myocardial  p r o c e s s e s a c c o u n t for d i a b e t e s - a s s o c i a t e d heart  ischemia,  hypertension,  a n d diabetic  cardiomyopathy  failure: (the  "cardiotoxic triad") (9). T h e first two c o m p o n e n t s of this triad are not unique to diabetes, but are important pathophysiological p r o c e s s e s which d i a b e t e s c a n trigger, sustain a n d e x a c e r b a t e .  Diabetic cardiomyopathy is a d i s e a s e p r o c e s s in w h i c h d i a b e t e s p r o d u c e s a direct a n d continuous myocardial insult e v e n in the a b s e n c e of i s c h e m i c , hypertensive or valvular d i s e a s e ( S c h e m e 1). It c a n a c t synergistically with hypertension o r i s c h e m i a to d a m a g e heart m u s c l e , but c a n a l s o c a u s e heart failure in its o w n right. Diabetic cardiomyopathy w a s first d e s c r i b e d by R u b i e r et  2  al w h o reported four c a s e s of heart failure in normotensive diabetic patients with no e v i d e n c e of coronary artery d i s e a s e , valvular pathology or congenital heart d i s e a s e (10). E x p e r i m e n t a l e v i d e n c e for the e x i s t e n c e of this entity b e g a n to a p p e a r shortly thereafter, a n d epidemiological e v i d e n c e a l s o s u g g e s t e d that a n additional c a r d i a c insult w a s present in diabetic patients. H o w e v e r , the direct clinical demonstration of diabetic cardiomyopathy proved challenging until the 1990's,  when  a  series  of  studies  echocardiography documented  using  echocardiography  e v i d e n c e of left ventricular  and  Doppler  hypertrophy  and  diastolic dysfunction in both type 1 a n d type 2 diabetic patients independent of other risk factors (see (11) for review). S t u d i e s using Doppler ultrasound have revealed that the p r e v a l e n c e of diabetic cardiomyopathy is alarmingly  high.  P r e v a l e n c e rates of >40% in young normotensive type 1 diabetic patients a n d 5 0 6 0 % in well-controlled type 2 diabetic patients h a v e b e e n reported (12-15). In a recent c a s e - c o n t r o l study, Bertoni et al reported that diabetes is independently a s s o c i a t e d with the d e v e l o p m e n t of idiopathic cardiomyopathy (16).  T h e clinical c o u r s e of diabetic cardiomyopathy is long, a n d c a n be divided into three s t a g e s (17). In the early s t a g e , the cardiomyopathy p r e s e n t s with mild asymptomatic  diastolic dysfunction  which  is a s s o c i a t e d with  ultrastructural  c h a n g e s in t i s s u e architecture, impaired c a l c i u m handling, oxidative s t r e s s a n d c h a n g e s in c a r d i a c metabolism. A s the d i s e a s e p r o g r e s s e s , e v i d e n c e of left ventricular hypertrophy a p p e a r s which is a s s o c i a t e d with more s e v e r e diastolic dysfunction  and  mild  systolic  dysfunction.  Cardiomyocyte  apoptosis  and  n e c r o s i s , m y o c a r d i a l fibrosis, mild autonomic neuropathy a n d activation of the renin-angiotensin s y s t e m a p p e a r at this stage. Finally, c o m b i n e d systolic a n d diastolic dysfunction o c c u r which are a s s o c i a t e d with c a r d i a c m i c r o v a s c u l a r disease,  severe autonomic  neuropathy  and  systemic sympathetic  nervous  s y s t e m activation. T h i s late s t a g e is frequently a s s o c i a t e d with hypertension a n d the onset of i s c h e m i a (17). T h e m e c h a n i s m s underlying the p r o c e s s are poorly understood, but a n overall picture is e m e r g i n g . T h e s u s t a i n e d diabetic c a r d i a c insult a p p e a r s to be p r o d u c e d by two major factors: h y p e r g l y c e m i a , a major  3 mediator of m a n y diabetic complications, and a shift in energy substrate selection by the  heart  (18).  T h i s d i s e a s e p r o c e s s impairs  both  passive and  active  m e c h a n i c a l properties of the m y o c a r d i u m ; the c o m p l i a n c e of the heart  wall  d e c r e a s e s (due to i n c r e a s e d cross-linking of c o l l a g e n , c a r d i a c hypertrophy a n d fibrosis (18; 19)), a n d contractility a l s o d e c r e a s e s .  C a r d i a c m e t a b o l i s m in the diabetic heart differs from that in the n o n diabetic hypertrophied a n d failing heart. It has b e e n s h o w n that, in the a d v a n c e d s t a g e s of d i s e a s e , the hypertrophied and failing heart i n c r e a s e s its reliance o n glycolysis a n d g l u c o s e oxidation, although fatty acid oxidation predominant Transport  of  fuel  (20-22).  glucose  The  into the  reverse is true in diabetic cardiomyocyte  is d e p e n d e n t  remains  the  cardiomyopathy. on  the  Glut-4  transporter w h o s e translocation, translation a n d transcription are all d e c r e a s e d by d i a b e t e s , a n d the constitutive Glut-1 transporter (18). T h e diabetic heart is therefore l e s s a b l e to u s e g l u c o s e a s a n energy s o u r c e , a n d relies m o r e heavily on alternative substrates s u c h a s fatty a c i d s a n d ketones. P l a s m a fatty acid levels are i n c r e a s e d . Fatty acid delivery to the heart from the coronary l u m e n by the e n z y m e lipoprotein lipase ( L P L ) , a n d its s u b s e q u e n t uptake by fatty a c i d transporters, is a l s o i n c r e a s e d (23).  T h e heavy reliance of the diabetic heart on fatty a c i d s is harmful for two r e a s o n s : it d e c r e a s e s myocardial efficiency, a n d it i n d u c e s 'lipotoxicity'. Despite the fact that fatty acid oxidation i n c r e a s e s dramatically in the diabetic heart, the delivery of free fatty a c i d s into the cardiomyocyte m e t a b o l i s e a n d store them. Intermediate  e x c e e d s its capacity  to  products of fatty acid m e t a b o l i s m ,  particularly long-chain a c y l - c o e n z y m e A ' s (Acyl C o A ' s ) , therefore a c c u m u l a t e in the c y t o p l a s m (23). This effect could be e x a c e r b a t e d if ketone body utilisation is i n c r e a s e d , a s ketone b o d i e s are competing substrates with fatty a c i d s . L o n g chain a c y l - C o A ' s are converted into c e r a m i d e s , toxic s u b s t a n c e s w h i c h induce reactive o x y g e n s p e c i e s ( R O S ) a n d cardiomyocyte a p o p t o s i s (24).  Interventions  w h i c h selectively i n c r e a s e cytoplasmic fatty acid influx ( o v e r e x p r e s s i o n of fatty  4  acid transport protein-1 or L P L ) or which selectively i n c r e a s e long-chain a c y l C o A s y n t h e s i s ( o v e r e x p r e s s i o n of long chain acyl C o A synthetase) p r o d u c e a c a r d i a c p h e n o t y p e w h i c h is similar to diabetic c a r d i o m y o p a t h y (23). T a k e n together, t h e s e d a t a provide convincing e v i d e n c e that lipotoxicity is a significant d i s e a s e inducing m y o c a r d i a l injury.  Fatty a c i d s bind and activate p e r o x i s o m e proliferator-activated (PPAR's)  of w h i c h P P A R - a  receptors  isoform in the heart. P P A R - a  is a major  is a  transcription factor which acts a s a 'lipostat', inducing g e n e s involved in e v e r y step of fatty a c i d m e t a b o l i s m (25). P P A R - a upregulates e n z y m e s at e v e r y step of the  fatty  acid  oxidation  pathway,  but  it  is  the  transcriptional  control  of  mitochondrial long-chain acyl C o A uptake w h i c h h a s the greatest impact on the overall fatty phenotype  a c i d oxidation similar  to  that  rate  (26).  When  in  diabetic  seen  PPAR-a  is o v e r e x p r e s s e d , a  cardiomyopathy  is  induced.  Furthermore, w h e n P P A R - a is o v e r e x p r e s s e d in diabetic hearts, the p h e n o t y p e p r o d u c e d by d i a b e t e s is w o r s e n e d (23). C o n v e r s e l y , deletion of P P A R - a sufficient  to  protect  cardiomyopathy  (23;  diabetic 27).  hearts  PPAR-a  against  activation  p a t h o g e n e s i s of diabetic cardiomyopathy.  the  development  is therefore  of  diabetic  e s s e n t i a l to  It is not clear, however,  is  the  whether  P P A R - a activation is a c o n s e q u e n c e or a c a u s e of i n c r e a s e d fatty acid oxidation. O v e r a l l , the reliance of the diabetic heart on fatty acid oxidation is harmful for three  reasons: decreased  myocardial  efficiency,  lipotoxicity  and  PPAR-a-  activation.  Protein m e t a b o l i s m is a l s o affected by d i a b e t e s , a n a s p e c t w h i c h is rarely c o n s i d e r e d . R i b o n u c l e i c acid ( R N A ) levels fall. D i a b e t e s p r o d u c e s a m a r k e d a r t e r o - v e n o u s difference in b r a n c h e d chain a m i n o a c i d s a c r o s s the m y o c a r d i u m , s u g g e s t i n g that protein  c a t a b o l i s m is i n c r e a s e d . T h i s w o u l d  d e c r e a s e the  availability of a m i n o a c i d s for translation. T h e combination of i n c r e a s e d protein b r e a k d o w n a n d d e c r e a s e d protein s y n t h e s i s would be e x p e c t e d to qualitative a n d quantitative d e c r e a s e s in myocardial proteins (18).  produce  5  H y p e r g l y c e m i a , c o u p l e d with the m a r k e d d e c r e a s e in g l u c o s e oxidation, increases  the  formation  of  reactive  oxygen  species  (ROS)  by  several  m e c h a n i s m s (28). P r o l o n g e d e x p o s u r e of proteins to h y p e r g l y c e m i a i n d u c e s a s e r i e s of c h e m i c a l reactions which eventually lead to the irreversible formation of a d v a n c e d - g l y c a t i o n end-products ( A G E ' s ) which act on their own  receptors  ( R A G E ) to i n c r e a s e the synthesis of diacylglycerol (29; 30). Increased de novo s y n t h e s i s of diacylglycerol, either by i n c r e a s e d flux of g l u c o s e through the a l d o s e r e d u c t a s e / polyol pathway or by R A G E activation, l e a d s to activation of protein k i n a s e C ( P K C ) isoforms a n d stress signaling p a t h w a y s (31; 32). Activation of t h e s e p a t h w a y s i n c r e a s e s mitochondrial R O S production, stimulates a p o p t o s i s a n d i n c r e a s e s the transcription of pro-inflammatory a n d pro-fibrotic g e n e s (28). In addition, flux of g l u c o s e through the h e x o s a m i n e biosynthetic pathway l e a d s to O - l i n k e d - N - a c e t y l g l u c o s a m i n a t i o n a n d activation of transcription factors w h i c h also  regulate  pro-inflammatory  and  pro-fibrotic  genes  (33).  Diabetic  c a r d i o m y o p a t h y is therefore a s s o c i a t e d with w i d e s p r e a d deleterious c h a n g e s in fatty a c i d , protein a n d g l u c o s e m e t a b o l i s m . H o w e v e r , b e c a u s e g l u c o s e utilisation is d e c r e a s e d in the diabetic heart, fatty acid oxidation m a y be a m o r e important regulator of oxidative stress in this setting.  In addition to the stress a n d P K C pathways activated by g l u c o s e , G protein-mediated signaling pathways (particularly Gj a n d G ) activated by cellq  s u r f a c e receptors a l s o contribute to the induction of pro-hypertrophic a n d profibrotic g e n e s (34). R h o A is a small molecular weight m o n o m e r i c G-protein which activates R h o K i n a s e to induce c a r d i a c hypertrophy a n d fibrosis. T h e R h o - A / R h o - k i n a s e pathway is known to be activated by ai a d r e n o c e p t o r stimulation, a s well  as  endothelium-derived  vasoconstrictors  such  as  endothelin-1  and  thromboxane A . 2  A t the level of the cardiomyocyte, e l e c t r o m e c h a n i c a l coupling is impaired. M y o c y t e shortening and lengthening is s l o w b e c a u s e the action potential is  6  prolonged  and  c a l c i u m efflux  is too  s l o w (35).  C a r d i a c contractile  protein  a d e n o s i n e triphosphatase ( A T P a s e ) is crucial for the generation of c a r d i a c force a n d is markedly  d e p r e s s e d . In animal m o d e l s , the s o - c a l l e d 'fetal g e n e program',  which c o n s i s t s of re-expression of atrial natriuretic peptide ( A N P ) , p-myosin a n d a-actin, a n d d e c r e a s e d e x p r e s s i o n of a - m y o s i n a n d s a r c o p l a s m i c reticulum c a l c i u m A T P a s e - 2 ( S E R C A - 2 ) in the ventricle, is i n d u c e d . T h i s shift in contractile protein e x p r e s s i o n from a c a r d i a c pattern to a skeletal m u s c l e pattern is a l s o o b s e r v a b l e w h e n the pattern is a s s e s s e d functionally. T h e s e e n in the normal heart gives w a y to a slower V  3  i s o m y o s i n pattern  pattern in the diabetic heart  (18). Total contractile protein levels m a y a l s o be d e c r e a s e d b e c a u s e of the effects  of  diabetes  cardiomyopathy  on  protein  synthesis  and  p r o g r e s s e s to s y m p t o m a t i c  breakdown.  As  diabetic  heart failure, downregulation  of  m y o c y t e - e n h a n c e r factor-2 ( M E F - 2 ) a n d its target g e n e s o c c u r s , a n d a more s e v e r e contractile dysfunction e n s u e s (36).  T h e d e c r e a s e in S E R C A - 2 e x p r e s s i o n s e e n a s part of the fetal g e n e p r o g r a m , c o m b i n e d with a concomitant d e c r e a s e in S E R C A - 2 function a n d N a / +  Ca  2 +  exchanger  expression  and  function,  disturbs  cardiomyocyte  calcium  handling. In the diabetic heart, the uptake, s a r c o l e m m a l binding a n d myofibrillar calcium-ATPase-mediated sensitivity activation,  of  intake  myofilaments  p-adrenergic  activity  are  all d e c r e a s e d .  b e c o m e s abnormally  responsiveness  high  decreases  as a (28)  The  calcium  result  of P K C  and  autonomic  neuropathy d e c r e a s e s sympathetic nervous s y s t e m input (18); a s a result, the cardiomyocyte  is cut  off  from  s y s t e m i c regulation  of  excitation-contraction  coupling.  A s the heart fails, the tissue renin-angiotensin s y s t e m , the sympathetic n e r v o u s s y s t e m a n d other neuro-hormonal regulatory s y s t e m s are stimulated in a n effort to maintain s y s t e m i c perfusion. Activation of t h e s e s y s t e m s i n c r e a s e s a p o p t o s i s a n d necrosis (via p i - a d r e n o c e p t o r s ) (37-41), a n d fibrosis (via adrenoceptors)  and  R O S generation  (through  the  tissue  ctr  renin-angiotensin  7 system) (28; 42-44). This time-course is reflected by pathological findings at autopsy a n d e c h o c a r d i o g r a p h i c findings. Left ventricular m a s s , t h i c k n e s s a n d s i z e are found to i n c r e a s e progressively a s diabetic cardiomyopathy p r o g r e s s e s from a s y m p t o m a t i c diastolic dysfunction to symptomatic systolic dysfunction (17).  The  architecture  of the  myocardium  is a l s o progressively disturbed.  Fibrosis is the most prominent histopathological finding in the diabetic heart a n d is diffuse. P e r i v a s c u l a r a n d interstitial fibrosis a p p e a r during the  intermediary  s t a g e s of the d i s e a s e p r o c e s s . Interstitial fibrosis d e c r e a s e s c a r d i a c c o m p l i a n c e , but p e r i v a s c u l a r fibrosis has additional functional c o n s e q u e n c e s b e c a u s e it b r e a k s the c o n n e c t i o n between the endothelium and the c a r d i o m y o c y t e s , thereby impairing relaxation (45). P r o g r e s s i o n to systolic dysfunction is a s s o c i a t e d with m y o c y t e cell death which i n d u c e s replacement fibrosis (17). Both a p o p t o s i s a n d n e c r o s i s are s e e n , but only necrotic cell death stimulates fibrosis (17).  Focal  microangiopathy is a l s o present in the intermediary a n d a d v a n c e d s t a g e s of the d i s e a s e a n d m a y contribute to the o b s e r v e d fibrosis; it is unlikely to be the s o l e c a u s e of fibrosis b e c a u s e , in diabetic cardiomyopathy, fibrosis is diffuse, not focal (17). T h e ultrastructure of the cardiomyocyte is profoundly d a m a g e d ; c y t o p l a s m i c a r e a is i n c r e a s e d , with an i n c r e a s e in c y t o p l a s m i c lipid content (consistent with the p r e s e n c e of lipotoxicity) a n d i n c r e a s e d collagen-fibre c r o s s - s e c t i o n a l a r e a (consistent  with  ultrastructure  increased  disrupts  collagen  mitochondria,  cross-linking). impairing  Disturbance  mitochondrial  of  the  function  and  inducing oxidative stress (18).  T o s u m m a r i z e , diabetic cardiomyopathy is initiated by h y p e r g l y c e m i a a n d a shift in substrate selection in favour of fatty a c i d s . Both p r o c e s s e s i n c r e a s e oxidative stress a n d stimulate pro-apoptotic, pro-fibrotic a n d pathways.  Necrosis  also  occurs,  contraction  coupling  is impaired  stimulating  by disordered  further  pro-inflammatory  fibrosis.  c a l c i u m handling,  Excitationimpaired  A T P a s e activity a n d a shift in contractile protein e x p r e s s i o n from a c a r d i a c to a skeletal m u s c l e pattern. Excitation-contraction coupling is d i s c o n n e c t e d from  8  a u t o n o m i c regulation a s a result of impaired (3-adrenergic r e s p o n s i v e n e s s a n d a u t o n o m i c neuropathy. T h e s e functional c h a n g e s are a c c o m p a n i e d by e x t e n s i v e d a m a g e to the architecture of the m y o c a r d i u m a n d the ultrastructure  of the  c a r d i o m y o c y t e . T h e result is a d i s e a s e p r o c e s s which c a n be initiated y e a r s before the a p p e a r a n c e of hypertensive or i s c h e m i c d i s e a s e . Initially presenting as a s y m p t o m a t i c diastolic dysfunction, the d i s e a s e p r o g r e s s e s to  combined  s y m p t o m a t i c systolic a n d diastolic dysfunction, a n d the m y o c a r d i u m is rendered m o r e s u s c e p t i b l e to d a m a g e from the other c o m p o n e n t s of the cardiotoxic triad.  D e s p i t e recent a d v a n c e s in drug therapy for heart failure, this condition still carries a w o r s e prognosis than significant  morbidity.  Current  most c a n c e r s , a n d is a s s o c i a t e d with  therapies  are  directed  at  symptomatic  relief  (diuretics, (3-adrenergic agonists, p h o s p h o d i e s t e r a s e inhibitors) a n d attenuation of left ventricular  remodelling  (angiotensin  converting  e n z y m e inhibitors,  p-  blockers, aldosterone antagonists). A g e n t s which restore the normal b a l a n c e of c a r d i a c m e t a b o l i s m could improve the m e c h a n i c a l efficiency of the m y o c a r d i u m , a n d prevent the harmful s e q u e l a e of shifts in energy substrate s e l e c t i o n . T h i s m e c h a n i s m h a s b e e n p r o p o s e d a s a useful a v e n u e to pursue in the identification of new drug targets for heart failure and m a y be particularly useful in diabetic c a r d i o m y o p a t h y (46-48).  II. Cardiac Metabolism  T h e heart requires a constant supply of a d e n o s i n e triphosphate ( A T P ) for m u s c u l a r contraction a n d the m a i n t e n a n c e of ionic h o m e o s t a s i s (49). U n d e r a e r o b i c conditions most of this A T P (>95%) is g e n e r a t e d by  mitochondrial  oxidative phosphorylation. Oxidation of energy substrates is c o u p l e d to  the  reduction of nicotinamide a d e n i n e dinucleotide ( N A D H ) a n d flavoproteins. N A D H and flavoproteins are then re-oxidised by o x y g e n , a n d the reducing equivalents they r e c e i v e d are p a s s e d on to the electron transport chain w h i c h p u m p s protons out of the mitochondria; the result is the creation of an e l e c t r o c h e m i c a l gradient  9  consisting of a t r a n s m e m b r a n e p H gradient a n d a m e m b r a n e potential. T h e F i F o A T P a s e located on the inner mitochondrial m e m b r a n e allows protons to reenter the mitochondrial matrix d o w n their concentration gradient, h a r n e s s i n g the electrical potential energy of the gradient to generate A T P from A D P a n d Pj (50). T h e o x y g e n c o n s u m e d is converted to carbon dioxide, a n d the reducing equivalents are eventually transferred to hydrogen and o x y g e n , forming water. A T P is exported in e x c h a n g e for A D P by the a d e n i n e nucleotide transporter, and a continuous supply of mitochondria  are  Pi is maintained  by the  p h o s p h a t e translocator. T h e  precisely fixed between two T-tubules, a n d both A T P a n d its  metabolic intermediates are continuously c h a n n e l e d b e t w e e n the mitochondria, the myofibrils, the s a r c o p l a s m i c reticulum a n d the s a r c o l e m m a . T h i s exquisite coupling s y s t e m b e t w e e n oxidative phosphorylation a n d myofibril  contraction  allows A T P d e m a n d to be precisely a n d instantly met over a w i d e range of w o r k l o a d s ( s e e (51) for review).  Although  functional  phosphorylation  matches  communication  coupling ATP  between  production  the to  myofibrils  ATP  and  consumption,  oxidative further  is required to e n s u r e that the s u p p l y of e n e r g y s u b s t r a t e s  r e s p o n d s to c h a n g e s in A T P d e m a n d . T h i s role is fulfilled by A M P activated protein k i n a s e ( A M P K ) , which is activated by an i n c r e a s e in the A M P / A T P ratio (a signal of A T P depletion) a n d acts to deactivate A T P - c o n s u m i n g p a t h w a y s (protein,  triglyceride  and  glycogen  synthesis)  and  activate  ATP-producing  p a t h w a y s (protein, g l y c o g e n and triglyceride c a t a b o l i s m , g l u c o s e a n d fatty acid uptake a n d oxidation) (52).  T h e heart is an omnivorous organ which h a s the ability to u s e a n y e n e r g y substrate provided to it (lipids, c a r b o h y d r a t e s , ketone b o d i e s , a m i n o a c i d s ) ; however, the normal heart derives most of its A T P from the m e t a b o l i s m of fatty a c i d s a n d c a r b o h y d r a t e s (53). Although fatty acid oxidation p r o d u c e s m o r e A T P , g l u c o s e oxidation is more efficient in terms of o x y g e n c o n s u m p t i o n . S e v e r a l  10  SCHEME 1 M e c h a n i s m s involved in the p a t h o g e n e s i s of diabetic cardiomyopathy. T h e c a r d i o m y o p a t h y a r i s e s a s a result of a d e c r e a s e in heart m u s c l e c o m p l i a n c e , p r o d u c e d by fibrosis and i n c r e a s e d collagen cross-linking, a n d a d e c r e a s e in contractility, p r o d u c e d a s a result of w i d e s p r e a d c h a n g e s in contractile signaling p a t h w a y s , c a r d i a c metabolism and oxidative stress.  Myocyte Altered Cardiac Apoptosis Gene Expression  ft R O S  \  Remodelling/ Hypertrophy  /  Alterations in Gproteins, ^ " " RhoA^ Rho Kinase, PKC o  t  S  ,  R  h  •DECREASED CONTRACTILITY Disordered calcium handling  Metabolic 'Switch'  DIABETIC CARDIOMYOPATHY  DECREASED COMPLIANCE 1T Collagen Cross Linking  Myocardial Fibrosis  12  factors a c c o u n t for this. Firstly, to maintain a fixed A T P production rate, fatty acid oxidation requires greater o x y g e n c o n s u m p t i o n . T h e A T P p r o d u c e d per unit o x y g e n is theoretically predicted to be 3.17 for g l u c o s e but only 2.80 for palmitate (48).  S e c o n d l y , fatty a c i d s allow protons to  membrane, Indeed,  uncoupling oxidative  fatty  acids  have  leak a c r o s s the  phosphorylation  recently  been  a n d wasting  shown  to  activate  mitochondrial oxygen  (54).  mitochondrial  uncoupling in m o d e l s of type 2 diabetes (55; 56). Thirdly, fatty a c i d s activate s a r c b l e m m a l c a l c i u m c h a n n e l s . C a l c i u m p u m p s must i n c r e a s e their activity, a n d therefore their A T P utilization, to c o m p e n s a t e for the resulting i n c r e a s e in c a l c i u m influx (57). F o r optimal c a r d i a c efficiency, the normal heart maintains a b a l a n c e of 6 0 - 8 0 % fatty acid oxidation a n d 2 0 - 4 0 % pyruvate oxidation; the heart d e r i v e s approximately e q u a l a m o u n t s of pyruvate from glycolysis a n d lactate m e t a b o l i s m (58; 59). K e t o n e utilization is concentration dependent; in situations w h e r e blood ketone levels rise (starvation, diabetes), ketones b e c o m e a major c a r d i a c fuel (60; 61).  T h e m e t a b o l i s m of g l u c o s e a n d fatty a c i d s by the heart is s u m m a r i z e d in s c h e m e 2.  G l u c o s e - 6 - p h o s p h a t e , the glycolytic substrate, is obtained from  e n d o g e n o u s g l y c o g e n stores and from e x o g e n o u s g l u c o s e taken up by the Glut1 a n d Glut-4 g l u c o s e transporters. G l u c o s e uptake is regulated by Glut-4 v e s i c l e translocation, d o c k i n g a n d fusion to the s a r c o l e m m a (stimulated by insulin a n d A M P - a c t i v a t e d protein kinase) a n d is responsible for the majority of g l u c o s e uptake (59; 6 2 ; 63). G l y c o l y s i s c o m p r i s e s a s e r i e s of reactions w h i c h convert g l u c o s e - 6 - p h o s p h a t e to pyruvate. In the normal aerobic heart, the major fate of pyruvate  is  decarboxylation  to  acetyl  CoA,  catalysed  by  the  pyruvate  d e h y d r o g e n a s e c o m p l e x ( P D H ) in the mitochondria (64). Lactate is m e t a b o l i s e d to  pyruvate,  but  pyruvate  is  also  metabolized  to  lactate.  Under  aerobic  conditions, the normal heart is a net c o n s u m e r of lactate; the heart only b e c o m e s a net p r o d u c e r of lactate w h e n the glycolytic flux e x c e e d s the rate of oxidation  (59).  A  small  proportion  of  pyruvate  is  metabolized  pyruvate to  either  13  o x a l o a c e t a t e or malate in order to replenish tricarboxylic acid c y c l e ( T C A cycle) intermediates, a p r o c e s s known a s a n a p l e r o s i s (65).  Fatty a c i d s are highly hydrophobic a n d are therefore transported  as  triglycerides in c h y l o m i c r o n s or very-low-density lipoproteins ( V L D L ) , although a s m a l l proportion are also transported bound to s e r u m albumin. Fatty a c i d s are r e l e a s e d from c h y l o m i c r o n s a n d V L D L by lipoprotein lipase ( L P L ) on the luminal s u r f a c e a n d are then taken up into the cardiomyocyte by fatty acid transporters s u c h a s C D 3 6 (66). U p o n entry to the c y t o p l a s m , fatty a c i d s are bound by fatty acid binding protein ( F A B P ) until they are esterified to fatty acyl C o A by fatty a c y l C o A s y n t h e t a s e ( F A C S ) . Fatty acyl C o A h a s two major fates: it c a n enter the mitochondria to be o x i d i z e d , or it c a n be esterified to triglyceride a n d stored (24; 67). A small proportion of palmitoyl C o A ' s are a l s o converted to c e r a m i d e s (24).  W h e r e a s short a n d m e d i u m - c h a i n acyl C o A ' s c a n p a s s freely into the mitochondria  to  be  oxidized,  long-chain  acyl  CoA's  cannot  cross  the  mitochondrial m e m b r a n e and must be transported. T h i s function is carried out by a  carnitine-dependent  shuttle  system.  In  the  first  step,  carnitine  palmitoyltransferase 1 ( C P T - 1 ) converts acyl C o A to acyl-carnitine. T h i s is a major control step of overall fatty acid oxidation. Acyl-carnitine is then transported to the mitochondrial matrix by carnitine acyltransferase ( C A T ) in e x c h a n g e for free carnitine. Finally, carnitine palmitoyltransferase 2 ( C P T - 2 ) r e v e r s e s the initial reaction a n d regenerates acyl C o A (68). A c y l C o A enters the p-oxidation spiral, a s e r i e s of four reactions which c l e a v e s two c a r b o n s (one acetyl C o A molecule) from the acyl C o A m o l e c u l e per c y c l e a n d g e n e r a t e s N A D H a n d F A D H 2 . T h e r e are specific e n z y m e s for long-; m e d i u m - a n d short-chain acyl C o A ' s (69). A c e t y l C o A g e n e r a t e d by carbohydrate or fatty acid oxidation (as well a s from other pathways) enters the T C A cycle to generate additional reducing equivalents which drive oxidative phosphorylation.  14  SCHEME 2  S u m m a r y of fatty acid a n d g l u c o s e m e t a b o l i s m . G l u c o s e is taken up by Glut-1 and Glut-4 transporters a n d is converted by glycolysis to pyruvate. P y r u v a t e then enters the mitochondria to be o x i d i z e d , producing acetyl C o A . Fatty a c i d s are liberated from lipoproteins by L P L , a n d are taken up by C D 3 6 a n d 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 carnitine shuttle s y s t e m to the mitochondria. T h e fatty a c y l C o A u n d e r g o e s p-oxidation, removing two c a r b o n s per turn of the c y c l e a n d generating acetyl C o A . A c e t y l C o A , g e n e r a t e d by either pathway, enters the T C A cycle to generate reducing equivalents ( N A D H ) . T h e s e p a s s electrons to the electron transport c h a i n w h i c h creates  an  electrochemical  proton  gradient  to  drive  A T P synthesis. A T P  s y n t h e s i s is exquisitely c o u p l e d to the s y s t e m s which create the A T P d e m a n d (abbreviations: L P L = lipoprotein lipase, C D 3 6 = fatty acid t r a n s l o c a s e , F A B P = fatty acid binding protein, F A C S = fatty acyl C o A s y n t h a s e , C P T  = carnitine  palmitoyltransferase, C A T = carnitine acyl transferase, C o A = c o e n y m e A , T C A cycle = tricarboxylic acid cycle, A G E = a d v a n c e d glycosylation endproduct, P D H =  pyruvate  dehydrogenase,  MCT =  monocarboxylate  transporter,  PDH  =  pyruvate d e h y d r o g e n a s e , N A D H = r e d u c e d nicotinamide a d e n i d e dinucleotide, A T P = a d e n o s i n e triphosphate, A D P = a d e n o s i n e m o n o p h o s p h a t e ),  Glucose [• Oxidation  Lactate  The Rail die Cycle  - Fatty Acid ^ Oxidation  16 T h e rate of fatty acid oxidation is determined by the p l a s m a concentration of fatty a c i d s , the work performed by the heart, the entry of fatty a c i d s into the c y t o p l a s m , the entry of fatty a c i d s into the mitochondrion, and the activity of the e n z y m e s involved in the p-oxidation spiral (62; 70). High rates of fatty acid oxidation i n c r e a s e the ratios of N A D H / N A D + a n d acetyl G o A / free C o A , both of which f e e d b a c k and inhibit g l u c o s e oxidation by d e c r e a s i n g flux through P D H (62).  High rates of fatty acid oxidation a l s o i n c r e a s e citrate production. Citrate  inhibits glycolysis by inhibiting the key glycolytic e n z y m e p h o s p h o f r u c t o k i n a s e ( P F K ) . T h i s is known a s the R a n d l e cycle. C o n v e r s e l y , high rates of glycolysis a n d g l u c o s e oxidation c a n feed back to inhibit fatty acid oxidation.  A t the level of C P T - 1 , fatty acid oxidation is controlled by malonyl C o A , a potent inhibitor w h i c h binds C P T - 1 on the cytosolic side (68); by altering malonyl C o A levels in the cytosolic microdomain adjacent to the site of fatty a c y l - C o A uptake, fatty acid oxidation c a n be controlled; tonic inhibition of C P T - 1 by malonyl C o A is a l w a y s present, but a rise in malonyl C o A levels inhibits fatty acid oxidation while a fall relieves inhibition (71; 72). T w o isoforms of C P T - 1 are e x p r e s s e d in the heart: C P T - 1 A (the isoform which p r e d o m i n a t e s in the liver) a n d C P T - 1 B (the isoform w h i c h predominates in the heart). T h e sensitivity of C P T - 1 B to malonyl C o A is 30 times greater than that of C P T - 1 A (71; 72). T h e turnover of malonyl C o A in the heart is rapid. M a l o n y l C o A is s y n t h e s i z e d from acetyl C o A by acetyl C o A c a r b o x y l a s e ( A C C ) a n d is broken d o w n to acetyl C o A by malonyl C o A decarboxylase  (MCD)  (73;  74).  The  activity  of  ACC  is  inhibited  by  phosphorylation; the major k i n a s e responsible is A M P K , although P K A m e d i a t e s p-adrenoceptor induced A C C phosphorylation (73) . T h e m e c h a n i s m s by w h i c h M C D is regulated are still u n k n o w n ; it is p o s s i b l e that M C D is activated by A M P K (75). M a l o n y l C o A levels c a n be i n c r e a s e d either by stimulating A C C or by inhibiting M C D ( S c h e m e 3). M C D is a transcriptional target of the p e r o x i s o m e proliferator activated receptor-a, w h o s e actions are d i s c u s s e d below.  17  In the long term, regulation of the e x p r e s s i o n of the e n z y m e s , transporters a n d k i n a s e s that c o m p r i s e the metabolic machinery e n a b l e s the heart to adapt more permanently w h e n the stimulus to do s o is s u s t a i n e d . T h e mitochondria contain D N A which c o d e s for 13 electron transport chain subunits (for c o m p l e x e s 1, III, IV, a n d V ) , but all other c o m p o n e n t s of the metabolic m a c h i n e r y are c o d e d for in the nuclear D N A (76).  T h e r e are s e v e r a l important inter-related nuclear  transcriptional regulators of metabolism g e n e s . T h e first are the p e r o x i s o m e 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 c o m p l e x requires binding of 9-c/s-retinoic acid to the R X R and binding of long chain fatty a c i d s or an  exogenous  PPAR  ligand to the  PPAR.  U p o n activation, the  complex  translocates to the n u c l e u s and binds to P P A R r e s p o n s e e l e m e n t s  (PPREs)  within the promoter regions of its target g e n e s . In the heart, the major isoform is PPAR-a,  and  its target  g e n e s e n c o m p a s s the  full pathway  of fatty  acid  m e t a b o l i s m from fatty acid uptake to the p-oxidation spiral, a s well a s pyruvate d e h y d r o g e n a s e kinase-4 (PDK-4), the major inhibitory k i n a s e of P D H  (77).  T h e s e c o n d 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 s o that, w h e n A T P d e m a n d i n c r e a s e s , the reserve of the metabolic m a c h i n e r y c a n meet the d e m a n d  79).  (78;  T o this e n d , the major role of PGC-1a in c a r d i a c m u s c l e is to i n c r e a s e  mitochondrial b i o g e n e s i s and the capacity of e a c h mitochondrion for oxidative phosphorylation. PGC-1a binds and e n h a n c e s the action of other transcription factors including P P A R - a (fatty acid oxidation control), m y o c y t e - e n h a n c e r factor 2A  (MEF-2A,  contractile  estrogen-related  machinery  receptor a  (ERR-a,  control)  and  carbohydrate  orphan  nuclear  and fatty acid  receptor oxidation  control, contractile machinery control, oxidative phosphorylation g e n e s ) (80;  81).  18  SCHEME 3  Regulation of malonyl C o A levels. Malonyl C o A is synthesised from cytosolic acetyl C o A by acetyl C o A carboxylase (ACC), and is broken down to acetyl C o A by malonyl C o A decarboxylase (MCD). M C D 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 C o A 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 =  monophosphate-activated protein kinase).  protein  kinase A, A M P K  = adenosine  +  n  Malonyl CoA Decarboxylase  Acetyl CoA  I  Malonyl CoA  Acetyl CoA Carboxylase  PKA  AMPK  ^CPT-1  f  FATTY ACID OXIDATION  20 111: Modulation of Cardiac Metabolism as a Therapeutic Strategy  Inhibition of fatty acid uptake a n d oxidation, or stimulation of g l u c o s e oxidation, w o u l d be e x p e c t e d to be beneficial to the diabetic heart for three r e a s o n s : improvement in myocardial efficiency, amelioration of lipotoxicity a n d amelioration of glucotoxicity. M o s t experimental a n d clinical data demonstrating the effectiveness of this therapeutic strategy pertain to non-diabetic heart failure, although a s m a l l body of data also exists for diabetic animal m o d e l s a n d patients. Intravenous  infusion of dichloroacetate ( D C A ) ,  a drug w h i c h  inhibits P D K ,  p r o d u c e s a rapid improvement in left ventricular p e r f o r m a n c e in patients with heart failure (82). T h i s s u g g e s t s that d e c r e a s e d flux through P D H contributes significantly to the c a r d i a c dysfunction of heart failure. W e h a v e previously s h o w n that the administration of D C A also p r o d u c e s a m a r k e d improvement in left ventricular function in the diabetic heart (83). Increasing p l a s m a insulin levels c a n a l s o stimulate g l u c o s e oxidation both directly by increasing g l u c o s e uptake, a n d indirectly by d e c r e a s i n g fatty acid delivery to the heart, thereby relieving R a n d l e c y c l e - m e d i a t e d inhibition of g l u c o s e oxidation (84). Infusion either of insulin in heart failure patients (85) or the insulinotropic peptide glucagon-like peptide 1 (GLP-1)  in d o g s with p a c i n g - i n d u c e d heart failure improved  function  (86).  In  the  Diabetes  Mellitus  Insulin-Glucose  left  Infusion  ventricular in  Acute  M y o c a r d i a l Infarction (DIGAMI) study, stimulation of g l u c o s e oxidation r e d u c e d mortality following myocardial infarction in diabetic patients (87). T a k e n together, t h e s e data s u g g e s t that acute stimulation of g l u c o s e oxidation m a y be beneficial to the failing heart, but more definitive clinical studies are required to confirm this. Furthermore, there are, at present, no p h a r m a c o l o g i c a l a g e n t s w h i c h act a s chronic g l u c o s e oxidation stimulators.  A  number  of  agents  have  been developed which  inhibit  fatty  acid  oxidation, thereby stimulating g l u c o s e oxidation indirectly through the R a n d l e  21  cycle. Fatty acid oxidation inhibitors have b e e n classified a c c o r d i n g to w h e t h e r they p r o d u c e reversible (partial fatty acid oxidation inhibitors) or irreversible inhibition. Etomoxir is a n irreversible C P T - 1 inhibitor.  It h a s b e e n s h o w n to  improve c a r d i a c function in rats with left ventricular hypertrophy (88; 89) a n d diabetic c a r d i o m y o p a t h y (90-92). In experimental studies, etomoxir  treatment  improved S E R C A - 2 e x p r e s s i o n a n d c a l c i u m handling, indicating that C P T - 1 inhibition p r o d u c e s improvements in c a l c i u m handling (89; 93). Inhibition of C P T 1 without concomitant inhibition of fatty acid delivery a n d uptake results in a n a c c u m u l a t i o n of long-chain fatty acyl C o A ' s in the cytosol a n d activation of the P P A R - a / R X R c o m p l e x . R u p p et al s p e c u l a t e d that the i n c r e a s e d S E R C A - 2 expression  induced  by etomoxir  is mediated  by  PPAR-a,  noting  that  the  regulatory region of the S E R C A - 2 g e n e contains a s e q u e n c e similar to the P P R E (89).  A  small  preliminary  trial  of  etomoxir  in  patients  demonstrated  an  improvement in left ventricular function (94). H o w e v e r , etomoxir p r o d u c e d a n u n a c c e p t a b l e rate of side-effects in the 'etomoxir for the recovery of g l u c o s e oxidation' ( E R G O - 1 ) P h a s e II clinical trial, a n d the trial w a s terminated early (95). Etomoxir h a s a l s o b e e n s h o w n to induce mild myocardial hypertrophy w h i c h w a s prevented by feeding rats a m e d i u m - c h a i n fatty acid diet, s u g g e s t i n g that the effect is attributable to C P T - 1 inhibition (96; 97). Etomoxir h a s a l s o b e e n s h o w n to c a u s e oxidative stress in H e p G 2 cells (98). T h e fact that etomoxir is a potent irreversible inhibitor of C P T - 1 m a y explain its t e n d e n c y to p r o d u c e a d v e r s e effects. It is not clear whether e t o m o x i r - a s s o c i a t e d hypertrophy is related to e x c e s s i v e P P A R - a activation, a mild lipotoxicity or the fact that chronic C P T - 1 inhibition m i m i c s a growth-promoting a n a b o l i c state.  Perhexiline a n d oxfenicine are C P T - 1 inhibitors which are c o n s i d e r e d to be partial fatty acid oxidation inhibitors. N o c a s e s of c a r d i a c hypertrophy arising from either the experimental or clinical u s e of t h e s e a g e n t s h a v e b e e n reported. Perhexiline fell into disfavor w h e n it w a s found to induce hepatotoxicity  and  neuropathy in a s u b s e t of patients; this toxicity is now known to be d u e to s l o w c y t o c h r o m e P 4 5 0 metabolism of the drug a n d c a n be a v o i d e d by appropriate  22  d o s e titration (99). In a small randomized-control trial, perhexiline w a s s h o w n to improve  ejection  fraction,  myocardial  efficiency  and  symptoms  (100).  Trimetazidine is a partial fatty acid oxidation inhibitor which m a y act by inhibiting 3-ketoacyl C o A thiolase ( 3 - K A T ) , the final step of the p-oxidation spiral (101). S e v e r a l s m a l l clinical trials, including o n e r a n d o m i z e d control trial, h a v e s h o w n that trimetazidine improves ejection fraction a n d s y m p t o m s in patients receiving optimal treatment for their heart failure (102). Trimetazidine w a s tested in diabetic patients with i s c h e m i c heart d i s e a s e a n d w a s found to significantly improve heart function (103-105).  T a k e n together, t h e s e d a t a indicate that inhibition of fatty  acid oxidation is beneficial to the failing heart a n d is a m e c h a n i s m w h i c h c a n p r o d u c e meaningful improvements in function.  IV: The Benefits of P-Adrenergic Blockade Heart failure is a s s o c i a t e d with activation of the sympathetic  nervous  s y s t e m . T h e sympathetic drive to a failing resting heart is equivalent to the m a x i m u m drive a normal heart is subjected to during s e v e r e e x e r c i s e ; spillover of c a t e c h o l a m i n e s i n c r e a s e s a s m u c h a s 50-fold, producing m a r k e d elevation of c a r d i a c a n d s y s t e m i c c a t e c h o l a m i n e levels  (106-109).  T h i s large r e s p o n s e is  initiated in an effort to maintain s y s t e m i c perfusion, but sympathetic activation is harmful to the failing heart, regardless of the c a u s e of the failure, a n d correlates inversely with survival (110).  C o n v e r s e l y , the  p-blocking  a g e n t s bisoprolol,  carvedilol a n d metoprolol have b e e n s h o w n in large-scale r a n d o m i z e d controlled trials to r e d u c e heart failure mortality by a third or more (111). p-blocking drugs produce  negative  chronotropic  and  inotropic  responses when  administered  acutely. F o r this r e a s o n , they were contraindicated in heart failure for m a n y years.  H o w e v e r , in the  1970's, p-blockers w e r e p i o n e e r e d a s heart  failure  treatments (112), and they are now a m o n g the a g e n t s of c h o i c e for the treatment of heart failure (111).  23  T h e r e h a v e b e e n no clinical or experimental studies e x a m i n i n g whether pblocking a g e n t s are beneficial in diabetic cardiomyopathy. H o w e v e r , a n u m b e r of clinical studies have e x a m i n e d the effects of t h e s e agents in the context of i s c h e m i a . In patients taking p-blocker therapy post-myocardial infarction,  the  improvement in survival p r o d u c e d by treatment is greater in diabetic patients than  non-diabetic  patients  patients  (113).  The  DIGAMI  study  receiving a p-blocker had a 5 0 % reduction  s h o w e d that in mortality  diabetic  (87),  and  carvedilol p r o d u c e d a 2 9 % reduction in mortaility in the Carvedilol Post-Infarction Survival Control in Left Ventricular Dysfunction ( C A P R I C O R N ) trial (114). Similar improvements  in survival are also s e e n in the  p r e s e n c e of  asymptomatic  coronary artery d i s e a s e (115).  L o o k i n g at heart failure patients a s a whole, more than 2 0 clinical trials h a v e b e e n published covering more than 10,000 patients w h o s e  symptoms  ranged from mild to s e v e r e ; t h e s e studies consistently found that p-blocker therapy r e d u c e s all c a u s e mortality by more than a third, a similar improvement to that s e e n following p-blocker treatment of myocardial infarction or i s c h e m i a (116-120).  H o w e v e r , the d e c r e a s e in mortality is greater for s u d d e n d e a t h s than  other c a u s e s . W h e n d i a b e t e s - a s s o c i a t e d i s c h e m i c heart failure is a s s e s s e d , pblocker therapy p r o d u c e s a large improvement in heart function and survival. In the Metoprolol C R / X L R a n d o m i s e d Intervention Trial in C o n g e s t i v e Heart Failure ( M E R I T - H F ) trial, metoprolol produced a greater functional improvement in heart failure patients without diabetes than t h o s e with diabetes (119). In the C a r v e d i l o l P r o s p e c t i v e R a n d o m i s e d Cumulative Survival Trial, survival w a s improved by more than a third in both diabetic and non-diabetic patients with s e v e r e heart failure (116). T a k e n together, t h e s e 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 following myocardial infarction is greater in the diabetic heart than the non-diabetic heart. T h i s raises the possibility that p-blockers could h a v e a m e c h a n i s m of action that is particularly beneficial to the diabetic c a r d i o m y o p a t h y  24  c o m p o n e n t of the cardiotoxic triad in terms of protecting against a c u t e injury. In the context of i s c h e m i c heart failure, however, this s p e c i a l benefit is lost. It s h o u l d be noted that, in all heart failure clinical trials to date, (3-blocker therapy had b e e n instigated following the onset of systolic failure in a c c o r d a n c e with existing e v i d e n c e a n d guidelines, a n d the diabetic patients could therefore h a v e had m o r e s e v e r e heart failure to begin with. T h e crucial difference may be the timing of the intervention. It is not known whether 3-blocker therapy c a n reverse the diabetic cardiomyopathy c o m p o n e n t at a m u c h earlier s t a g e b e c a u s e this possibility h a s not b e e n investigated either experimentally or clinically.  Putative  mechanisms  for  the  chronic  effect  of  p-blockers  include  antiarrhythmic effects, amelioration of cardiomyocyte hypertrophy, n e c r o s i s a n d apoptosis,  reversal of the fetal  gene  program  (thereby  improving  calcium  handling a n d force of contraction), i n c r e a s e s in c a r d i a c receptor density (for some  p-blockers  including  metoprolol),  anti-inflammatory  effects  (p-blockers  lower s e r u m C - r e a c t i v e protein levels) a n d partial restoration of c a r d i a c g l u c o s e oxidation (37; 121). Metoprolol (122), carvedilol (123) a n d bucindolol (124) h a v e all b e e n s h o w n to induce a switch from fatty acid to g l u c o s e oxidation in n o n diabetic patients with heart failure.  Furthermore, metoprolol was  shown  to  i n c r e a s e lactate uptake in heart failure patients, an effect w h i c h is consistent with an  increase  in  carbohydrate  oxidation  (125).  A  study  in  dogs  with  m i c r o e m b o l i s m - i n d u c e d heart failure revealed a potential m e c h a n i s m for this effect:  CPT-1 was  inhibited  by  chronic  treatment  with  metoprolol  (126).  C o n s i d e r i n g the hypothesis that the switch from g l u c o s e to fatty acid oxidation plays a n important role in diabetic cardiomyopathy, the ability of p-blockers to partially reverse this switch could be especially beneficial to the diabetic heart.  V: P-Adrenoceptor Signalling  In 1948, Ahlquist first demonstrated the e x i s t e n c e of two broad s u b t y p e s of a d r e n o c e p t o r s : a - a d r e n o c e p t o r s a n d p-adrenoceptors (127). T w o s u b t y p e s of  25  pi  and  while a third,  p3,  p-adrenoceptors,  (128),  p2, w e r e  identified a n d characterized in the late  w a s isolated a n d c l o n e d in  1989 (129).  1960's  A l l three  s u b t y p e s are e x p r e s s e d in the heart, but the major s u b t y p e s are p1 a n d p2, the  p1: p2  ratio of expression  being approximately  (130). T h e effects  60-70%: 40-30  of the putative  %, with very low  p4 adrenoceptor  p3  are n o w believed  to be mediated by a low-affinity state of the pi adrenoceptor (131;  132).  The  receptor reserve is low b e c a u s e the absolute e x p r e s s i o n levels are in the femtomolar range  (50-70  fmol/ m g protein for the  pi  adrenoceptor)  affinities of t h e s e receptors for their ligands differ: pi noradrenaline = 4  u M , isoproterenol  noradrenaline = 26  uM, isoproterenol = 0.5  noradrenaline =  4 uM, isoproterenol  =  0.2  u M ) , p2 uM),  (37). T h e  (adrenaline = 4  (adrenaline  uM,  = 0.7 u M ,  p3 (adrenaline = 130  uM,  2 uM) (133).  P-adrenoceptor signaling pathways are s u m m a r i z e d in s c h e m e 4.  p-  a d r e n o c e p t o r s are G-protein coupled receptors. In the c l a s s i c a l p-adrenoceptor pathway, pi  a n d p2 adrenoceptors, acting via G s , p r o d u c e a n acute positive  inotopic r e s p o n s e mediated by i n c r e a s e d c A M P levels a n d stimulation of protein k i n a s e A ( P K A ) . P K A then phosphorylates s e v e r a l key proteins involved in c a l c i u m handling a n d c a l c i u m sensitivity of myofilaments. Phosphorylation a n d activation of L-type c a l c i u m c h a n n e l s a n d ryanodine receptors i n c r e a s e s c a l c i u m uptake a n d r e l e a s e , while phosphorylation of p h o s p h o l a m b a n relieves inhibition of S E R C A , thereby increasing s a r c o p l a s m i c reticulum c a l c i u m uptake Finally,  P K A modulates  calcium  sensitivity  of  myofilaments  phosphorylation of troponin I a n d myosin binding protein B (137;  138).  (134-136). through P K A also  activates protein p h o s p h a t a s e inhibitor-1, sustaining its effects by preventing dephosphorylation of its targets  (139).  Recently, a major paradigm shift h a s occurred in a d r e n o c e p t o r biology. T h e p-adrenoceptors are now known to form c o m p l e x ' s i g n a l o m e s ' w h i c h are temporally a n d spatially o r g a n i z e d . A s i g n a l o m e c a n be defined a s all g e n e s ,  26  SCHEME 4 p-adrenergic signaling pathways, p i - a d r e n e r g i c receptors activate P K A , which regulates c a l c i u m sensitivity a n d c a l c i u m handling. P r o l o n g e d activation of this receptor  activates a harmful  C A M K - I I pathway  which  is pro-apoptotic  and  i n d u c e s pathological remodeling. p2-adrenergic receptors also activate P K A , but prolonged activation c a u s e s a switch to G i signaling which activates  PDE4,  inhibiting c A M P formation, a n d activates the cardioprotective P I 3 K / A k t pathway. D e s e n s i t i z a t i o n of p2-adrenergic receptors by p-arrestin c a n recruit p 3 8 a n d E R K , w h i c h protect the cell from apoptosis. p3-adrenergic receptors p r o d u c e a negative inotopic effect which is mediated by N O p r o d u c e d via the P I 3 K / Akt pathway.  P2 Gs  Gi  cAMPh-PDE4 PI3K CAMK-II  PKA  Akt  ERK + p38  Calcium Handling Calcium Sensitivity Pathological Remodeling Apoptosis  28  phosphorylation, mediated by (3-arrestins acting together with G proteincoupled  receptor  k i n a s e s or  P K A itself  (140-142).  In  addition  to  receptor  d e s e n s i t i z a t i o n , proteins a n d ligands w h i c h are involved in the transduction a n d r e s p o n s e to a biological s i g n a l .  With  regard  to temporal  organization,  it is  w e l l - e s t a b l i s h e d that p-adrenoceptors, a n d most particularly the p 2 - a d r e n o c e p t o r , d e s e n s i t i z e by uncoupling from their G-proteins. T h i s dissociation is stimulated by receptor p-adrenoceptors  c h a n g e their coupling to d o w n s t r e a m  signaling  p a t h w a y s . P r o l o n g e d activation of p i a d r e n o c e p t o r s c a u s e s a switch from P K A to c a l c i u m / calmodulin  dependent  protein  kinase-ll ( C A M K  II) -  dependent  signaling, leading to C A M K - I I mediated a p o p t o s i s a n d pathological  hypertrophy  (143). In contrast, prolonged activation of p 2 - a d r e n o c e p t o r s s w i t c h e s G-protein coupling from G s to G i , which is cardioprotective (144).  W h e r e a s p i adrenoceptor signaling is widely d i s s e m i n a t e d throughout the cell, p2 a d r e n o c e p t o r signaling is c o m p a r t m e n t a l i z e d , a n d the positive inotopic effect  elicited  adrenoceptor  by  p2/  Gs  signaling  compartmentalization  is is  therefore partly  smaller  (145;  a c h i e v e d . by  the  146).  p2  selective  e n r i c h m e n t of p2 a d r e n o c e p t o r s in c a v e o l a e (147; 148). It h a s b e e n s u g g e s t e d that translocation  of p2 a d r e n o c e p t o r s  out  of c a v e o l a e following  sustained  stimulation c a u s e s the switch from G s to G i a s s o c i a t i o n (149). p2 a d r e n o c e p t o r G i signaling activates the phosphoinositol-3 k i n a s e (PI3K) - protein k i n a s e B (Akt) pathway a n d p h o s p h o d i e s t e r a s e 4 (145). P h o s p h o d i e s t e r a s e 4 i n c r e a s e s the b r e a k d o w n of c A M P g e n e r a t e d by p 1 - a d r e n o c e p t o r - G s stimulation, e n a b l i n g the p 2 - a d r e n o c e p t o r - G i pathway to functionally a n t a g o n i z e the p i - a d r e n o c e p t o r - G s pathway. T h e P I 3 K - A k t pathway protects the c a r d i o m y o c y t e against a p o p t o s i s (145). R e c e n t l y , a role for the extracellular-signal-regulated k i n a s e ( E R K ) 1/2 in mediating p 2 - a d r e n o c e p t o r - G i cardioprotection h a s b e e n s u g g e s t e d (150). T a k e n together,  these  downstream  data  signaling  indicate pathways  that  the  coupling  of  is c o m p a r t m e n t a l i z e d  pand  adrenoceptors  to  time-dependent.  29  Sustained  activation  of  (31  adrenoceptors  is  harmful,  whereas  sustained  activation of p2 a d r e n o c e p t o r s could b e cardioprotective.  A n o t h e r c o n s e q u e n c e of P I 3 K / Akt activation is stimulation of nitric oxide (NO) production. N O is s y n t h e s i z e d from the terminal guanidine nitrogen atom of the a m i n o a c i d L-arginine a n d m o l e c u l a r o x y g e n by nitric oxide s y n t h a s e ( N O S ) . T h i s p r o c e s s requires tetrahydrobiopterin ( B H ) a s a cofactor; without B K , e N O S 4  becomes  'uncoupled',  peroxynitrite,  and  instead of  produces  4  reactive  oxygen  N O . Endothelial nitric  species,  including  oxide s y n t h a s e ( e N O S ) is  constitutively e x p r e s s e d in adult c a r d i o m y o c y t e s , producing p h y s i o l o g i c a l N O signaling in the n a n o m o l a r range. Inducible  nitric oxide s y n t h a s e ( i N O S ) is  e x p r e s s e d in r e s p o n s e to inflammatory stimuli (151; 152) a n d p r o d u c e s higher levels of N O , mediating pathophysiological effects (153; 154) . N O a n d related reactive nitrogen s p e c i e s (e.g. peroxynitrite) covalently modify target proteins in o n e of three w a y s : nitrosylation, oxidation or nitration. Binding of N O to a protein, termed nitrosylation, is a reversible reaction a n d the modification p r o d u c e d is labile. Oxidation (e.g. glutathiolation of cysteine residues) or nitration of a protein (on  tyrosine  residues) p r o d u c e s m o r e  stable covalent modifications  T y r o s i n e nitration, nitrosylation a n d oxidation c a n be stimulatory or  (155).  inhibitory  d e p e n d i n g on the target protein and residue affected. Nitrosylation of the h e m e moiety of s o l u b l e guanylyl c y c l a s e by N O activates the e n z y m e , stimulating the production of c y c l i c 3', 5 ' g u a n o s i n e m o n o p h o s p h a t e ( c G M P ) f r o m g u a n o s i n e triphosphate (156). Just a s c A M P activates P K A , c G M P activates protein k i n a s e G ( P K G ) isoforms. T h e N O / c G M P signaling pathway i n d u c e s a negative inotropic effect in the heart (151). p3-adrenoceptors a l w a y s c o u p l e to G i , activating the P I 3 K / A k t pathway. p 3 - a d r e n o c e p t o r s produce a negative inotropic effect w h i c h is mediated by N O . Therefore, p2 a d r e n o c e p t o r - G i signaling a n d p3 a d r e n o c e p t o r G i signaling both stimulate N O production (157; 158).  T h e effects of d i a b e t e s o n c a r d i a c p-adrenergic r e s p o n s i v e n e s s h a v e b e e n studied for m a n y y e a r s , but the results obtained h a v e b e e n conflicting.  30 V a d l a m u d i a n d M c N e i l l (159) s h o w e d a d e c r e a s e in the c a r d i a c relaxant effects without a n effect on heart rate or contractility. Z o l a et al (160) s h o w e d a d e c r e a s e in the chronotropic  r e s p o n s e in rabbit heart in vivo.  Foy and Lucas  (161)  d e m o n s t r a t e d a n i n c r e a s e d chronotropic r e s p o n s e a n d a d e c r e a s e d inotropic response  in  atria.  Most  recent  studies  report  d e c r e a s e d sensitivity  to  p-  a d r e n e r g i c stimulation in c a r d i a c t i s s u e s (162; 163). T h e effects of diabetes on preceptor e x p r e s s i o n a n d d o w n s t r e a m signalling are a l s o controversial (163-167). 14 w e e k s but not 8 w e e k s of diabetes blunted the chronotropic r e s p o n s e to noradrenaline,  but the  r e s p o n s e to fenoterol,  a selective p2 agonist,  p r e s e r v e d (168). T h i s s u g g e s t s that p i - m e d i a t e d  was  r e s p o n s e s are selectively  blunted in the diabetic heart. T h e e x p r e s s i o n of p i is markedly d e c r e a s e d a n d that of p2 a d r e n o c e p t o r s modestly d e c r e a s e d in the diabetic heart, w h e r e a s the e x p r e s s i o n of p3 a d r e n o c e p t o r s is i n c r e a s e d twofold (163). A similar i n c r e a s e in P3 a d r e n o c e p t o r e x p r e s s i o n h a s b e e n reported in failing h u m a n hearts (169). T h e significance of this shift in receptor s u b t y p e s towards p3 a d r e n o c e p t o r s r e m a i n s to be determined; it is p o s s i b l e that this shift contributes to c a r d i a c dysfunction by promoting  a negative inotropic effect; on the other h a n d , a  cardioprotective effect m a y a l s o result if p3 adrenoceptor-mediated activation of the P I 3 K / A k t pathway a l s o prevents a p o p t o s i s .  VI: Potential Links Between B-Adrenoceptors and Cardiac Metabolism  M e c h a n i s m s linking p-adrenergic signalling with c a r d i a c m e t a b o l i s m have not b e e n investigated in great detail. W e therefore e m p l o y e d a c o m b i n a t i o n of 'bottom-up'  (known  rate-limiting  enzymes)  and  'top-down'  (known  p-  a d r e n o c e c e p t o r pathways) a p p r o a c h e s to unravel the p a t h w a y s involved. A s discussed  a b o v e , a previous study  in m i c r o e m b o l i s m - i n d u c e d heart  failure  d e m o n s t r a t e d that chronic metoprolol treatment d e c r e a s e d the activity of C P T - 1 (126). In the heart, the major m e c h a n i s m by which C P T - 1 is regulated is through modulation of malonyl C o A levels. Isoproterenol has previously b e e n s h o w n to  31  lower malonyl C o A levels by increasing P K A - m e d i a t e d phosphorylation of A C C (73). It is therefore p o s s i b l e that p-adrenergic b l o c k a d e could h a v e the o p p o s i t e effect,  preventing A C C phosphorylation  and increasing malonyl C o A levels.  Recently, a study in isolated c a r d i o m y o c y t e s using activators a n d inhibitors of cAMP  revealed that stimulation of fatty acid oxidation by contraction is P K A -  d e p e n d e n t (170).  C h r o n i c p-adrenergic b l o c k a d e could a l s o d e c r e a s e the e x p r e s s i o n of C P T - 1 . T h e e x p r e s s i o n of C P T - 1 is controlled by P P A R - a , but the P P A R - a / R X R c o m p l e x p r o d u c e s only m o d e s t induction of C P T - 1 w h e n acting a l o n e (26; 171; 172). P G C 1 a greatly e n h a n c e s C P T - 1 induction by P P A R - a , but c a n a l s o 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 e x p r e s s i o n of C P T - 1 h a s b e e n s h o w n to be r e p r e s s e d in isolated c a r d i o m y o c y t e s upstream  stimulatory  factor  (USF)-2.  Upstream  stimulatory  factors  by are  transcription factors of the b a s i c helix-loop-helix leucine z i p p e r family w h i c h bind to the E-box c o n s e n s u s s e q u e n c e C A N N T G  (174). In the heart, U S F ' s  are  involved in excitation-transcription coupling, responding to s u s t a i n e d i n c r e a s e s in electrical stimulation by increasing the e x p r e s s i o n of s a r c o m e r i c g e n e s s u c h a s s a r c o m e r i c mitochondrial creatine k i n a s e a n d M H C (175; 176).  p-blockers, by  improving function a n d thereby indirectly increasing electrical stimulation, could activate U S F ' s with the s e c o n d a r y effect that U S F - 2 r e p r e s s e s P G C 1 a - m e d i a t e d CPT-1  e x p r e s s i o n . Alternatively,  if p-blockers are acute fatty acid  oxidation  inhibitors, the activation of P P A R - a a n d its binding to coactivators could be altered a s a result of c h a n g e s in c y t o p l a s m i c long chain fatty acid levels.  In the study by P a n c h a l et al (126), a d e c r e a s e in C P T - 1 activity w a s detected using the in vitro a s s a y ; allosteric effects are typically lost during s a m p l e preparation, s o the o b s e r v e d d e c r e a s e could be d u e to d e c r e a s e d C P T - 1 e x p r e s s i o n , or alternatively to a covalent modification of C P T - 1 itself. F e w studies have e x a m i n e d whether covalent modifications of C P T - 1 occur. P h o s p h o r y l a t i o n of C P T - 1 A h a s b e e n demonstrated in vitro (177), and the stimulation of C P T - 1 by  32  o k a d a i c acid in hepatocytes w a s prevented by a specific inhibitor of C A M K indicating that C A M K  II is involved in stimulation of C P T - 1 A activity  II,  (178).  P h o s p h o r y l a t i o n of C P T - 1 B in the heart has never b e e n d e m o n s t r a t e d . H o w e v e r , activation of the sympathetic nervous s y s t e m centrally by cerulinin w a s found to stimulate C P T - 1 B activity in s o l e u s m u s c l e within 3 hours (179).  T h i s effect  must h a v e b e e n mediated by an as-yet unidentified covalent modification of C P T 1B; the modification in question could c o n c e i v a b l y be phosphorylation. It is p o s s i b l e that phosphorylation of C P T - 1 requires the p r e s e n c e of other proteins present  on  or  recruited  to  the  outer  mitochondrial  membrane.  C o m p a r t m e n t a l i z a t i o n of P K A signaling in the cardiomyocyte is a c h i e v e d in part by the action of A - k i n a s e anchoring proteins ( A K A P s ) , a group of proteins w h i c h bind to P K A targets in order to regulate P K A - d e p e n d e n t phosphorylation of t h o s e targets (180). T h r e e mitochondrial A K A P ' s have b e e n identified - A K A P 1 2 1 , DA K A P - 1 a n d A K A P 1 4 9 - but functional studies of their role in the heart are awaited (180). It is p o s s i b l e that mitochondrial A K A P ' s mediate, a n d are e v e n essential  for,  CPT-1  phosphorylation.  This  possibility  has  never  been  T y r o s i n e nitration of C P T - 1 by peroxynitrite has b e e n s h o w n to  inhibit  investigated.  C P T - 1 activity following e n d o t o x e m i a (181). Furthermore, s u p e r o x i d e , N O a n d peroxynitrite w e r e all s h o w n to inhibit C P T - 1 activity in vitro w h e n C P T - 1 w a s c o incubated with s y s t e m s which continuously generated these reactive o x y g e n and nitrogen s p e c i e s (182). This s u g g e s t s that C P T - 1 c a n be regulated by covalent modifications mediated by nitrogen s p e c i e s . It is likely that, in addition to tyrosine nitration, c y s t e i n e nitrosylation and glutathiolation a l s o occur. Indeed, c y s t e i n e s c a n n i n g m u t a g e n e s i s of the m u s c l e isoform of C P T - 1 revealed that c y s t e i n e 3 0 5 w a s important for catalysis; nitrosylation or glutathiolation of this residue could c o n c e i v a b l y i n c r e a s e or d e c r e a s e the catalytic activity of the e n z y m e .  p-blockers could c o n c e i v a b l y modulate C P T - 1 activity through  nitrogen  s p e c i e s g e n e r a t e d a s a result of p2 a d r e n o c e p t o r - G i or p3 a d r e n o c e p t o r - G i  33  signaling. H o w e v e r , N O is known to h a v e more extensive effects o n c a r d i a c m e t a b o l i s m which must be c o n s i d e r e d . Firstly, N O is well-known to inhibit overall o x y g e n utilization by irreversibly inhibiting mitochondrial respiration (183; 184). S e c o n d l y , N O c a n alter myocardial substrate selection. N O inhibits glycolysis by nitrosylating  and  inhibiting  the  enzyme  glyceraldehyde-3-phosphate  d e h y d r o g e n a s e ( G A P D H ) (185-187). In isolated hearts perfused without fatty acids, N O w a s found to inhibit Glut-4-mediated g l u c o s e uptake a n d glyolytic flux (188). In the s a m e study, 8 B r - c G M P , a stable a n a l o g u e of c G M P , w a s found to, d o u b l e the activity of A C C (188).  Treatment of d o g s with N O S inhibitors  stimulated g l u c o s e u s e and reduced fatty acid oxidation, and administration of a long-acting N O donor switched metabolism back to fatty acid u s e (189). T h e s e data s u g g e s t that N O is an inhibitor of g l u c o s e use acting primarily to inhibit g l u c o s e uptake and glycolytic flux. Modulation of N O levels by p-blockers could therefore affect g l u c o s e oxidation directly.  VII: Specific Research P r o b l e m and Research Strategy  T h e r e is no specific treatment strategy for diabetic cardiomyopathy despite its e m e r g i n g importance a s a c a u s e of heart failure in diabetic patients  and  alarmingly high p r e v a l e n c e , p-blockers have b e e n s h o w n to improve function and survival  in  experimental  diabetic  patients  with  ischemic  or clinical studies have  heart  disease.  investigated whether  However, p-blockers  no also  improve function in diabetic cardiomyopathy. C o n s i d e r i n g the hypothesis that the reliance of the diabetic heart on fatty acid oxidation is a c a u s a t i v e injury of diabetic cardiomyopathy, the putative ability of p-blockers to inhibit fatty acid oxidation could be especially beneficial to the diabetic heart. Data obtained using fatty acid oxidation inhibitors support the idea that this is a m e c h a n i s m which c a n p r o d u c e meaningful improvements in c a r d i a c function, p-blockers are p r o p o s e d to inhibit fatty acid oxidation by inhibiting long chain acyl C o A uptake into the mitochondria, c a t a l y s e d by C P T - 1 . T h e y could do this by s e v e r a l m e c h a n i s m s : increasing malonyl C o A levels, d e c r e a s i n g C P T - 1 e x p r e s s i o n or d e c r e a s i n g  34  CPT-1  activity through  covalent modifications (phosphorylation,  nitrosylation,  glutathiolation, nitration). W e therefore undertook the present study to determine whether p-blocker treatment ameliorates diabetic cardiomyopathy by inhibiting fatty acid oxidation, and to determine the m e c h a n i s m of the effect.  Three  p-blockers  have  been  shown  to  exert  effects  on  cardiac  m e t a b o l i s m : metoprolol, carvedilol a n d bucindolol. Carvedilol a n d metoprolol are u s e d clinically in the treatment of heart failure. Carvedilol is a non-selective pblocker w h i c h a l s o b l o c k s the a 1 - a d r e n e r g i c receptor a n d , at high d o s e s , c a l c i u m c h a n n e l s . Metoprolol is a s e c o n d generation p-blocker which is selective for the pi  receptor a n d is an inverse agonist at this receptor. W e c h o s e to  use  metoprolol in the present study a s it d o e s not p r o d u c e a 1 - a d r e n e r g i c , c a l c i u m c h a n n e l blocking or antioxidant effects which would complicate interpretation of the results. Although metoprolol is classically regarded a s being highly  pi-  selective, its selectivity in intact cells is less than w a s previously s u p p o s e d ; the selectivity ratio of metoprolol for p i and p2 adrenoceptors w a s recently s h o w n to be 2.3 (190; 191). Metoprolol will therefore block both pi and p2 a d r e n o c e p t o r s at clinical d o s e s . T h e d o s e of metoprolol u s e d in our studies w a s 7 5 m g / k g / d a y by  intraperitoneal  injection. T h i s d o s e is equivalent  to  a human  dose  of  approximately 100 mg per day after correction for inter-species differences in s u r f a c e a r e a (the surface a r e a of 150 mg rat is 0.025 m a r e a of a 6 0 kg h u m a n is 1.6 m  2  (192) ).  2  w h e r e a s the surface  Furthermore, this d o s e w a s well  tolerated by the rats in our preliminary studies, and p r o d u c e d a d e m o n s t r a b l e improvement in c a r d i a c function (see results).  T h e streptozotocin (STZ) diabetic rat is a model of poorly controlled type 1 d i a b e t e s which is a s s o c i a t e d with a marked d e c r e a s e in insulin levels. S T Z is a n antibiotic  s y n t h e s i s e d by the bacterium S t r e p t o m y c e s a c h r o m o g e n e s w h i c h  selectively targets and destroys the insulin-secreting p-cells of the p a n c r e a s (193; 194). T h e m e c h a n i s m of this cytotoxicity is incompletely u n d e r s t o o d . It is  35  believed that S T Z induces D N A strand breaks through the generation of o x y g e n free radicals or c a r b o n i u m ions (195; 196). A s part of the repair p r o c e s s , poly ( A D P - r i b o s e ) p o l y m e r a s e ( P A R P ) is activated. P A R P u s e s large a m o u n t s of A T P a n d N A D , a n d the resulting depletion of t h e s e m o l e c u l e s impairs the s y n t h e s i s +  a n d secretion of insulin a n d , if sufficiently s e v e r e , triggers cell death (197-199). T h e induction of oxidative stress m a y be partly related to the ability of N O to inhibit oxidative phosphorylation; A D P is shunted into degradation  pathways  w h i c h p r o d u c e xanthine, which via xanthine o x i d a s e g e n e r a t e s o x y g e n free radicals a n d uric acid (200).  Intravenous  (IV)  or intraperitoneal  (IP)  injection of S T Z (>40mg/  kg)  p r o d u c e s stable insulin-deficient diabetes, but, at low d o s e s , sufficient insulin secretion is p r e s e r v e d to e n a b l e the animals to survive without e x o g e n o u s insulin. 2-4 hours following injection of S T Z , insulin secretion is inhibited a n d g l u c o s e levels rise. O v e r the next 2-8 hours, h y p o g l y c e m i a e n s u e s , finally giving w a y to s u s t a i n e d h y p e r g l y c e m i a 24 hours following the injection (201). In our laboratory, w e routinely u s e a d o s e of 60 m g / kg. S T Z diabetic rats d e v e l o p both the s y m p t o m s (hyperphagia, polyuria, polydipsia, weight loss), a n d c o m p l i c a t i o n s (diabetic retinopathy, nephropathy a n d cardiomyopathy) of clinical d i a b e t e s (202204).  T h e diabetic cardiomyopathy of the S T Z rat closely r e s e m b l e s that w h i c h  is s e e n clinically, a n d a p p e a r s 6 w e e k s following S T Z injection (202-204). S T Z rats d o not d e v e l o p a t h e r o s c l e r o s i s or hypertension, thereby e n a b l i n g diabetic c a r d i o m y o p a t h y to be studied in the a b s e n c e of i s c h e m i c or hypertensive heart disease.  VIII: Working Hypotheses  It w a s h y p o t h e s i z e d that:  36  1.  C h r o n i c treatment with metoprolol would improve c a r d i a c function  in the diabetic heart by directly inhibiting fatty a c i d oxidation a n d indirectly stimulating g l u c o s e oxidation through the R a n d l e C y c l e . 2.  A c u t e perfusion with metoprolol directly inhibits fatty acid oxidation  and indirectly stimulates g l u c o s e oxidation through the R a n d l e C y c l e . 3.  A c u t e perfusion a n d c h r o n i c treatment with metoprolol d e c r e a s e  phosphorylation of A C C , leading to a n i n c r e a s e in malonyl C o A levels. 4.  A c u t e perfusion a n d chronic treatment with metoprolol d e c r e a s e the  activity of C P T - 1 without affecting the sensitivity of C P T - 1 to malonyl C o A . 5.  C h r o n i c treatment with metoprolol d e c r e a s e s the e x p r e s s i o n of  C P T - 1 by increasing the e x p r e s s i o n , activity a n d P G C 1 a - b i n d i n g of U S F - 2 . 6.  C h r o n i c treatment with metoprolol i n c r e a s e s the e x p r e s s i o n of all  three p-adrenoceptor s u b t y p e s without affecting G-protein a s s o c i a t i o n . A c u t e perfusion or c h r o n i c treatment with metoprolol d e c r e a s e p i - a n d p 2 - a d r e n o c e p t o r signaling, but a c c e n t u a t e p 3 - a d r e n o c e p t o r signaling. 7.  P h o s p h o r y l a t i o n of C P T - 1 by P K A and C A M K II is d e m o n s t r a b l e in  isolated mitochondria a n d modulated by acute metoprolol perfusion in w h o l e hearts. 8.  P K A , A K A P 149 a n d C A M K II bind to a n d co-immunoprecipitate  with C P T - 1 . A K A P 149 m e d i a t e s the binding of P K A to C P T - 1 . 9.  Nitrosylation, glutathiolation a n d nitration of C P T - 1 is d e m o n s t r a b l e  in isolated mitochondria a n d m o d u l a t e d by a c u t e metoprolol perfusion in w h o l e hearts.  IX: Objectives  T h e first objective w a s to determine whether metoprolol i m p r o v e s c a r d i a c function  in the  diabetic heart  a n d whether  the functional  improvement  is  a s s o c i a t e d with direct inhibition of fatty acid oxidation. T h e s e c o n d objective w a s to d e t e r m i n e w h e t h e r metoprolol directly inhibits fatty a c i d oxidation during shortterm perfusion. In a s e r i e s of studies, g l u c o s e oxidation a n d fatty acid oxidation  37  w e r e m e a s u r e d in the p r e s e n c e or a b s e n c e of insulin. T o c h a r a c t e r i s e the metabolic effects more fully, m e a s u r e s of g l u c o s e a n d fatty acid d i s p o s a l (lactate production, g l y c o g e n levels, triglyceride levels) a n d m y o c a r d i a l e n e r g e t i c s (tissue a d e n i n e nucleotide levels, A M P K activity) w e r e obtained. T h e third objective w a s to determine the effect  of short term  metoprolol  perfusion a n d long  term  metoprolol treatment on C P T - 1 activity a n d regulation by malonyl C o A , a s well a s on  the  activities  dehydrogenase,  of  other  citrate  key  fatty  synthase).  We  acid  oxidation  measured  enzymes  CPT-1  (acyl  activity,  CoA  CPT-1  sensitivity to m a l o n y l C o A a n d t i s s u e malonyl C o A levels. T o investigate the regulation of malonyl C o A levels, w e m e a s u r e d M C D a n d A C C e x p r e s s i o n , a s well a s A C C phosphorylation by A M P K a n d P K A .  The  fourth objective w a s to determine whether  metoprolol  treatment  controls C P T - 1 e x p r e s s i o n by increasing the e x p r e s s i o n , activation a n d P G C 1 a binding of U S F - 2 .  W e a l s o investigated whether metoprolol d e c r e a s e s the  binding of P G C 1 a to its coactivators. T h e fifth objective w a s to c h a r a c t e r i s e the acute a n d chronic effects of metoprolol on the e x p r e s s i o n a n d G-protein coupling of (3-adrenoceptors a n d the activation of d o w n s t r e a m s e c o n d m e s s e n g e r s ( P K A , C A M K II, P I 3 K / A k t , N O )  The  sixth objective w a s to determine whether short-term  metoprolol  perfusion regulates C P T - 1 activity a n d malonyl C o A sensitivity through direct covalent modifications of C P T - 1 .  W e investigated whether P K A a n d C A M K I I  directly bind to C P T - 1 , whether A K A P 1 4 9 binds to C P T - 1 a n d m e d i a t e s P K A binding a n d whether C P T - 1 is p h o s p h o r y l a t e d . W e a l s o m e a s u r e d the c y s t e i n e nitrosylation, glutathiolation a n d tyrosine nitration of C P T - 1 . In order to determine w h e t h e r t h e s e c h a n g e s w e r e s u s t a i n e d , the m e a s u r e m e n t s w e r e a l s o carried out following c h r o n i c metoprolol treatment. T h e s e v e n t h objective w a s to m e a s u r e the effects of k i n a s e phosphorylation a n d peroxynitrite-mediated nitrosylation, glutathiolation a n d nitration on C P T - 1 activity a n d C P T - 1 sensitivity. T h e final  38  objective w a s to u s e a m a s s s p e c t r o s c o p y a p p r o a c h to identify phosphorylation sites on C P T - 1 .  39  MATERIALS AND METHODS  I: Measurement of ex vivo Left Ventricular Function  (a)  Animal Treatments  A n i m a l s w e r e c a r e d for in a c c o r d a n c e with the guidelines of the C a n a d i a n C o u n c i l o n A n i m a l C a r e . M a l e W i s t a r Rats (weight m a t c h e d 2 0 0 - 2 2 0 g ) w e r e p u r c h a s e d from C h a r l e s R i v e r Laboratories a n d allowed to a c c l i m a t i z e for 1 w e e k prior to t h e beginning of the study. R a t s w e r e allowed ad libitum  a c c e s s to  standard rat c h o w and water for the duration of the study. R a t s w e r e randomly divided into four groups: control (C), control treated (CT), diabetic (D) a n d diabetic treated  (DT).  D i a b e t e s w a s induced by t h e injection  of 6 0 m g / k g  streptozotocin ( S T Z ) into the c a u d a l vein. O n e w e e k following t h e induction of d i a b e t e s , treatment w a s c o m m e n c e d . T h e treated g r o u p s received 7 5 m g / k g / d a y metoprolol b y intraperitoneal injection while untreated g r o u p s r e c e i v e d a n equivalent v o l u m e of vehicle (normal saline). S i x w e e k s following the induction of d i a b e t e s , the a n i m a l s w e r e e u t h a n i z e d . 5-hour fasting blood s a m p l e s w e r e t a k e n o n e w e e k following STZ-injection a n d immediately prior to termination. F o r perfused g r o u p s , metoprolol w a s a d d e d to the perfusate in the isolated working heart preparation a s d e s c r i b e d below.  (b) Measurement of Plasma Parameters  F i v e - h o u r fasting p l a s m a s a m p l e s w e r e collected o n e w e e k following S T Z injection a n d immediately prior to termination. P l a s m a g l u c o s e concentration w a s determined measured  using  the B e c k m a n n  Glucose  analyser.  Plasma  using t h e r a d i o i m m u n o a s s a y kit available from  insulin w a s  Millipore/  LINCO  (Billerica, M a s s a c h u s s e t s ) . P l a s m a free fatty a c i d s , cholesterol a n d triglycerides were  determined  by colorimetric  assay  kits  available from  Roche  (Basel,  40  Switzerland).  Plasma  ketone  levels w e r e  measured  using the C a r d i o C h e k  A n a l y z e r from P o l y m e r T e c h n o l o g y S y s t e m s , Inc (Indianapolis, Indiana).  (c)  Direct Measurement of Left Ventricular Pressure  Six  weeks  intraperitoneal  after  STZ  injection,  the  rats  were  euthanized  by  an  injection of 60 mg/kg s o d i u m pentobarbital. T h e hearts w e r e  r e m o v e d , a n d mounted on the working heart apparatus by cannulation of the aorta. T h e hearts w e r e first perfused in Langendorff m o d e with w a r m o x y g e n a t e d ( 9 5 % 0 , 5 % C 0 ) C h e n o w e t h - K o e l l e buffer (composition: 120 m M N a C I ; 5.6 2  2  m M K C I , 2.18 m M C a C I , 2.1 m M M g C I , 19.2 m M N a H C 0 , 10 m M g l u c o s e , 2  2  3  T e m p 37°C). Following cannulation of the pulmonary vein, the a p p a r a t u s w a s switched to working heart m o d e s o that the heart w a s being p e r f u s e d via the pulmonary v e i n . T h e afterload w a s set by c o l u m n of H 0 (height=19cm). 2  The  heart w a s p a c e d at 300 beats per minute. A 2 0 - g a u g e n e e d l e w a s inserted into the left ventricle to m e a s u r e left ventricular p r e s s u r e via a S t r a t h a m p r e s s u r e t r a n s d u c e r . Following equilibration for 10 minutes, the hearts w e r e s u b j e c t e d to atrial filling p r e s s u r e s from 3 to 11 m m H g . T h e left ventricular d e v e l o p e d pressure  (LVDP),  left ventricular  end  diastolic p r e s s u r e ( L V E D P ) ,  rate  of  contraction (+dP/dT) a n d rate of relaxation (-dP/dT) w e r e calculated for e a c h atrial filling p r e s s u r e by a microcomputer a s it collected the data.  II: Measurement of ex vivo Cardiac Metabolism  (a)  Animal Treatments and Measurement of Plasma Parameters  F o r preliminary studies in which g l u c o s e oxidation a n d glycolysis w e r e m e a s u r e d , rats w e r e divided into four g r o u p s ( C , C T , D, DT). F o r the m a i n studies in w h i c h g l u c o s e a n d fatty acid oxidation w e r e m e a s u r e d ,  rats w e r e  randomly divided into six groups: control (C), control + acute metoprolol perfusion  41  ( C P ) , control + chronic metoprolol treatment (CT), diabetic (D), diabetic + acute metoprolol perfusion (DP) a n d diabetic + chronic metoprolol treatment (DT). T h e treatment protocol w a s the s a m e a s d e s c r i b e d in section I (a). T h e treated g r o u p s r e c e i v e d 7 5 m g / k g / d a y metoprolol by intraperitoneal injection, while the remaining g r o u p s received a n equivalent v o l u m e of vehicle (normal saline). In the a c u t e metoprolol perfusion groups, isolated hearts w e r e perfused with metoprolol ex vivo a s d e s c r i b e d below. P l a s m a parameters w e r e m e a s u r e d a s d e s c r i b e d in s e c t i o n I (b) for all g r o u p s . A n i m a l s . w e r e terminated six w e e k s following S T Z injection. T i s s u e a n a l y s e s w e r e only undertaken in s a m p l e s that w e r e perfused with insulin.  T o improve clarity, the acute and chronic effects of metoprolol on function a n d m e t a b o l i s m are presented separately. H o w e v e r , acute a n d chronic effects w e r e a l w a y s investigated together in the s a m e experiment a n d 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 l u c o s e oxidation a n d glycolysis w e r e m e a s u r e d to confirm w h e t h e r metoprolol i m p r o v e s g l u c o s e u s e by the heart. Activities of key e n z y m e s involved in fatty acid  oxidation  (CPT-1,  acyl  C o A dehydrogenase,  citrate  synthase)  were  m e a s u r e d . F o r the m a i n studies, the effects of a c u t e metoprolol perfusion a n d chronic metoprolol treatment on g l u c o s e oxidation a n d palmitate oxidation w e r e m e a s u r e d . In a s e r i e s of studies, perfusions w e r e carried out in the p r e s e n c e or a b s e n c e of insulin to determine w h e t h e r inhibition of fatty a c i d oxidation by metoprolol is direct or mediated by the R a n d l e cycle in r e s p o n s e to  direct  stimulation of g l u c o s e oxidation. B a s e d on the findings of the preliminary study, w e did not m e a s u r e a c y l C o A d e h y d r o g e n a s e or citrate s y n t h a s e activities in the m a i n studies.  42  Measurement  of  c a r d i a c metabolism  was  carried  out  as  previously  d e s c r i b e d (20; 2 0 5 - 2 0 9 ) . T h e hearts w e r e perfused in working heart m o d e with K r e b s - H e n s e l e i t buffer  (composition:  118 m M  NaCI, 4.7 m M  K C I , 1.2  mM  K H P 0 , 1.2 m M M g S 0 , 2 m M C a C I , 5.5 m M o l g l u c o s e , 0.5 m M o l lactate, 100 2  4  4  2  or 0 uunits insulin, 0.8 m M o l palmitate bound to 3 % B S A ) in a n a e r o b i c perfusion for 6 0 minutes. T h e preload w a s set at 11.5 m m H g a n d the afterload set at 80 m m H g . T h e palmitate concentration w a s s e l e c t e d b a s e d on the p l a s m a lipid profile. A physiological g l u c o s e concentration w a s s e l e c t e d . F o r s i m u l t a n e o u s m e a s u r e m e n t of g l u c o s e a n d palmitate oxidation, the production of 3  H 0 from  1 4  2  C glucose and  3  H palmitate w a s m e a s u r e d .  H 0 from  1 4  2  C0  2  and  For simultaneous  m e a s u r e m e n t of g l u c o s e oxidation a n d glycolysis, the production of 3  1 4  1 4  C0  and  2  C g l u c o s e a n d H g l u c o s e w a s m e a s u r e d . C a r d i a c output, aortic a n d 3  pulmonary flow w e r e m e a s u r e d by p r o b e s positioned upstream of the pulmonary c a n n u l a a n d d o w n s t r e a m of the aortic c a n n u l a . P r e s s u r e w a s m e a s u r e d by a p r e s s u r e t r a n s d u c e r positioned d o w n s t r e a m of the aortic c a n n u l a . F o r p e r f u s e d g r o u p s ( C P , D P ) , 2 0 0 0 n g / ml (4.8 uM) metoprolol w a s a d d e d to the perfusate after 30 minutes. F o r other g r o u p s (C, C T , D, DT), an equivalent v o l u m e of v e h i c l e w a s a d d e d . Lactate production w a s m e a s u r e d in the perfusate u s i n g the colorimetric  lactate  a s s a y kit from  B i o V i s i o n . Following completion  of  the  perfusion, t i s s u e s w e r e immediately flash-frozen in liquid nitrogen, w e i g h e d a n d stored at -70°C for further a s s a y .  (c) Measurement of Tissue Glycogen and Triglyceride Levels  G l y c o g e n levels w e r e determined by m e a s u r e m e n t of g l y c o g e n - d e r i v e d g l u c o s e following extraction of g l y c o g e n in K O H a n d hydrolysis in H S 0 2  previously  described  (210).  T i s s u e triglyceride  levels  were  measured  4  as by  performing a chloroform-methanol extraction a n d redissolving the lipid pellet in phosphate-buffered saline ( P B S ) containing 1% Triton X . Triglyceride levels in the P B S Triton X solution w e r e 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  R a t heart s a m p l e s that had b e e n s n a p frozen in liquid nitrogen following the isolated working heart perfusions w e r e extracted in 0 . 4 M perchloric acid containing 0 . 5 m M ethylene-glycol-bis(B-aminoethyl ether)tetraacetic a c i d ( E G T A ) in a ratio of 100 m g / ml. H P L C a s s a y s for malonyl C o A a n d a d e n i n e nucleotides w e r e carried out on the perchoric acid extracts.  T i s s u e a d e n i n e nucleotide levels w e r e m e a s u r e d by gradient ion pair r e v e r s e d - p h a s e H P L C a s a m e a s u r e of c a r d i a c energetics. T h e H P L C p r o c e d u r e has  b e e n d e s c r i b e d previously (212)  a n d c a n be s u m m a r i z e d a s follows.  S a m p l e s of a c i d extracts w e r e a p p l i e d to a C 1 8 r e v e r s e - p h a s e c o l u m n via a p r e c o l u m n cartridge at a flow rate of 0.5ml/min. Buffer A ( 2 5 m M K H P 0 , 6 m M 2  4  tetrabutylammonium hydrogensulphate, p H 6.0, 1 2 5 m M E D T A ) a n d buffer B (1:1 v/v mixture of buffer A a n d H P L C - g r a d e acetonitrile), w e r e filtered through a 0.2 um m e m b r a n e filter a n d helium d e - g a s s e d . After 10 minutes of isocratic elution with 9 8 % A a n d 2 % B, W a t e r s curvilinear program no 3 w a s u s e d , e n d i n g with a gradient of 4 5 % A and 5 5 % B after 10 minutes. T h i s gradient w a s be maintained at a flow rate of 1.5 ml/min for a further 5 minutes. T h e c o l u m n w a s reequilibrated with 9 8 % A a n d 2 % B.  T h e H P L C procedure for m e a s u r e m e n t of malonyl C o A levels w a s a s follows (213). S a m p l e s of acid extracts w e r e be applied to a C 1 8 r e v e r s e - p h a s e c o l u m n via a p r e c o l u m n cartridge at a flow rate of 0.5ml/min. T h e applied gradient w a s a s follows: Buffer A , 0 . 2 M N a H P 0 , Buffer B, 0 . 2 5 M N a H P 0 2  4  2  4  and  acetonitrile ( 2 0 % v/v). A t the time of s a m p l e application, the buffer c o m p o s i t i o n w a s 2 0 % B. T h e gradient rose linearly to 5 7 % at 16.7 minutes, r e m a i n e d at 5 7 % until 18 minutes, rose to 9 0 % B by 22 minutes, a n d fell b a c k to 2 0 % B by 30 minutes. C o A a n d C o A ester elution w e r e detected by a flow through a monitor set at 2 5 4 n m .  44  (e) Measurement of Tissue Nitrate/ Nitrite Levels N O is rapidly s c a v e n g e d and h a s a half life of 4 s e c o n d s in biological fluids. It is c o n v e r t e d , by c h e m i c a l reactions, to nitrate and nitrite. T h e s u m of nitrate a n d nitrite levels provides a n indirect index of total N O production. T i s s u e nitrate a n d nitrite levels were m e a s u r e d using the colorimetric a s s a y available from C a y m a n c h e m i c a l s (Ann Arbor, Michigan). In the first step of the a s s a y , nitrate r e d u c t a s e is a d d e d to convert nitrate to nitrite. In the s e c o n d step, the addition of the G r i e s s reagents converts nitrite to a purple a z o product w h o s e a b s o r b a n c e is read at 540  nm.  Ill: Measurement of Kinase and Biochemical Enzyme Activities  (a)  AMPK, PKA and CAMK Activities  Protein k i n a s e and e n z y m e activities w e r e only m e a s u r e d in s a m p l e s that had b e e n perfused with insulin.  T h e activities of A M P K , P K A and C A M K w e r e a s s a y e d using kits available from  Upstate Biotechnology/ Millipore (Billerica, M a s s a c h u s s e t s )  a s s a y s are b a s e d on the rate of incorporation of  3 2  (214).  The  P from [A, P] A T P into 32  synthetic peptides containing specific c o n s e n s u s s e q u e n c e s for the k i n a s e of interest. ( A M P K : A M A R A  peptide, s e q u e n c e = A M A R A A S A A A L A R R R ; P K A :  kemptide, s e q u e n c e = L R R A S L G ; C A M K : Autocamtide-2  KKALRRQETVDAL;  bold indicates phosphorylated residue). Prior to A M P K a s s a y , s a m p l e s w e r e purified by immunoprecipitation with antibodies specific for the a1 subunits, or with a n antibody specific for both a-1 pan).  Prior  to  PKA  immunoprecipitation  and  with  CAMK  assays,  antibodies  specific  and a-2 samples  for  PKA  immunoprecipitation protocol is d e s c r i b e d in section IVd.  or a2  AMPK  A M P K subunits  (a-  were  by  or  purified  C A M K > II.  The  45  (b) CPT-1 Assay CPT-1  activity  was  estimated  by  m e a s u r i n g the  production  of  1 4  C-  palmitoylcarnitine by the reaction:  Palmitoyl C o A + C - C a r n i t i n e => 14  1 4  C - Palmitoylcarnitine + C o A - S H  U n d e r the conditions of this a s s a y , the equilibrium f a v o u r s production of 1 4  C-  palmitoylcarnitine  by both C P T - 1 a n d C P T - 2 .  H o w e v e r , only C P T - 1 is  inhibited by malonyl C o A . C P T - 1 activity w a s defined a s the activity w h i c h is inhibited by 2 0 0 u M malonyl C o A .  T h e heart tissue w a s h o m o g e n i s e d in 1 5 m M K C I / 0 . 5 m M Tris, p H 7.2 by two 10s bursts using a Polytron. Total protein concentration w a s determined by the B i o R a d Protein A s s a y . A s s a y buffer composition w a s a s follows: 1 0 5 m M Tris-CI  (pH  7.2),  50uM  palmitoyl  C o A , 5 0 0 u M carnitine,  0.25uCi/ml  [ C]14  carnitine, 1% B S A , 4 m M A T P , 0 . 2 5 m M glutathione, 4 0 u g / m l rotenone a n d 4 m M K C N . T h e reaction w a s started by the addition of 10Oul h o m o g e n a t e a n d allowed to p r o c e e d for 5 minutes at 30°C. T h e reaction w a s terminated by the addition of 500ul c o n c e n t r a t e d HCI. 500ul of 1-butanol w a s a d d e d a n d the s a m p l e s vortexed for 1 minute prior to centrifugation at 3 0 0 0 g for 8 minutes. 300ul of the butanol layer w a s be t a k e n , a d d e d to 1ml butanol-saturated water, vortexed for 30 s e c o n d s a n d centrifuged in a microcentrifuge for 2 minutes. 10Oul of the w a s h e d butanol layer w a s taken a n d counted in a scintillation counter. C P T - 1 activity w a s calculated a s C P T (total) - C P T (activity in the p r e s e n c e of 2 0 0 u M malonyl C o A ) . D a t a w e r e e x p r e s s e d a s n m o l / min/ mg protein.  T o m e a s u r e the sensitivity of C P T - 1 to malonyl C o A , C P T - 1 activity w a s a s s a y e d in the p r e s e n c e of 0, 10, 20, 50, 100, 150 u M malonyl C o A . T h e d o s e r e s p o n s e d a t a w e r e subjected to non-linear regression a n a l y s i s a n d c o n v e r g e d  46  to s i g m o i d a l d o s e - r e s p o n s e c u r v e s using G r a p h P a d software. T h e  IC50  P r i s m 5.0 curve fitting  value was calculated.  T o determine whether metoprolol is a p h a r m a c o l o g i c a l inhibitor of C P T - 1 , C P T - 1 activity w a s a s s a y e d in w h o l e heart h o m o g e n a t e s from 5 control hearts in the p r e s e n c e of increasing concentrations of metoprolol (ranging from 2-50 a g / ml) a n d 0, 50 or 100 u M malonyl C o A .  (c) Acyl CoA Dehydrogenase Assay  A c y l C o A D e h y d r o g e n a s e activity w a s m e a s u r e d by following the reaction:  A c y l C o A + [ F c ] => T r a n s - A - E n o y l C o A + [Fc] +  2  In this a s s a y , the ferricenium ion  ([Fc] ) r e p l a c e d F A D +  +  a s the electron  acceptor, a n d the reaction w a s followed by m e a s u r i n g the rate of reduction of [Fc] . R e d u c t i o n of- [Fc] to [Fc] is a s s o c i a t e d with a d e c r e a s e in absorption at +  +  3 0 0 n m . O c t a n o y l C o A w a s u s e d a s the substrate a s it c a n p a s s freely through the mitochondrial m e m b r a n e (215).  F e r r i c e n i u m hexafluorophosphate ( F c P F e ) is not available c o m m e r c i a l l y +  a n d w a s s y n t h e s i z e d in our laboratory a s previously d e s c r i b e d (215). tissue  was  homogenised  in  p i p e r a z i n e e t h a n e s u l f o n i c acid ( H E P E S ) ,  Heart  100mM  4-(2-hydroxyethyl)-1-  p H 7.6 by two  10s bursts using a  Polytron. Total protein concentration w a s determined by the B i o R a d Protein A s s a y . T h e a s s a y buffer composition w a s a s follows: 2 0 0 u M F c P F , +  6  100mM  H E P E S , 190ml h o m o g e n a t e . T h e reaction w a s started by the addition of 25ul 5 m M octanoyl C o A , a n d the absorption at 3 0 0 n m w a s m e a s u r e d e v e r y 5 s e c o n d s for 5 minutes.  Linear rates of reaction w e r e obtained by this protocol.  T h e d a t a w e r e collected a n d a z e r o - o r d e r rate constant calculated using E n z y m e  47  Kinetics  Pro  ( S y n e x C h e m , Fairfield,  California).  Data  were  expressed  as  iamol/min/mg protein.  (d) Citrate Synthase Assay Citrate s y n t h a s e activity w a s m e a s u r e d by following the reaction:  A c e t y l C o A + O x a l o a c e t a t e + H 0 => C o A - S H + Citrate + H + H 0 +  2  2  T h e reaction w a s followed for o n e minute by m e a s u r i n g the rate a p p e a r a n c e of C o A - S H spectrophotometrically  using dithio-bis  acid) ( D T N B ) . T h e thiol (SH) group of C o A - S H  of  (2-nitrobenzoic  r e l e a s e s T N B from  DTNB,  c a u s i n g a n i n c r e a s e in absorption a t 4 1 2 n m (216).  Heart  tissue  was  homogenised  in  0.1M  tris  (hydroxymethyl)  a m i n o m e t h a n e hydrochloride (tris-CI), p H 8.1 by two 10s bursts using a Polytron. Total protein concentration w a s determined by the B i o R a d Protein A s s a y . T h e a s s a y buffer composition w a s a s follows: 0 . 2 m M D T N B , 0 . 3 m M acetyl C o A , 0.1 m M Tris-CI, 0.05 ml h o m o g e n a t e . T h e reaction w a s started by the addition of 0.05ml 1 0 m M oxaloacetate, a n d the absorption at 4 1 2 n m w a s m e a s u r e d every 5 s e c o n d s for 1 minute. Linear rates of reaction w e r e obtained by this protocol. T h e d a t a w e r e collected and a zero-order rate constant calculated using the E n z y m e Kinetics P r o software ( S y n e x C h e m , Fairfield, California). D a t a w e r e e x p r e s s e d a s p m o l / m i n / m g protein.  48  IV:  Immunoprecipitation and  Measurement of  Protein Expression  by  Western Blotting  (a)  Overview of Experimental Design  M e a s u r e m e n t s were only carried out on s a m p l e s that had b e e n perfused with insulin. W h e n e v e r possible, phosphorylation  of specific residues on the  protein of interest w a s m e a s u r e d using p h o s p h o - s p e c i f i c antibodies. T h e blots w e r e stripped  a n d re-blotted  for total e x p r e s s i o n of the protein  of  interest.  H o w e v e r , if p h o s p h o - s p e c i f i c antibodies w e r e not available to probe the protein of interest, w e m e a s u r e d the co-immunoprecipitation (co-IP) of the protein with pan-specific p h o s p h o s e r i n e and phosphothreonine antibodies, either individually or in combination, (Upstate Biotechnology/ Millipore, Billerica, M a s s a c h u s s e t s ) a s an index of the total phosphorylation state.  T o obtain a m e a s u r e of A C C and M C D activities, w e m e a s u r e d  the  e x p r e s s i o n of A C C (Upstate Biotechnology/ Millipore, Billerica, M a s s a c h u s s e t s ) a n d M C D (a g e n e r o u s gift from Dr. G . D L o p a s c h u k and Dr. J . Dyck, University of Alberta), A M P K - m e d i a t e d phosphorylation of S e r i n e 79 on A C C a n d the total s e r i n e phosphorylation state of A C C . S E R C A - 2 e x p r e s s i o n w a s m e a s u r e d a s an index of c a l c i u m handling and fetal g e n e program effects.  T o investigate c h a n g e s in C P T - 1 e x p r e s s i o n , w e m e a s u r e d total C P T - 1 e x p r e s s i o n with a pan-specific C P T - 1 antibody,  a s well a s with  specific for  Biotechnology,  CPT-1A  and  CPT-1 B  (Santa-Cruz  antibodies  Santa-Cruz,  California). W e m e a s u r e d the total protein e x p r e s s i o n of P G C 1 a , U S F - 1 a n d U S F - 2 (all from Upstate Biotechnology/ Millipore, Billerica, M a s s a c h u s s e t s ) , and PPAR-a  and  California),  PDK-4 a-myosin  Biotechnology,  (both  from  heavy  Santa-Cruz  chain  Biotechnology,  (a-MHC)  expression  Santa  Cruz,  (Santa-Cruz  S a n t a C r u z , California) w a s m e a s u r e d a s an index of U S F  49  activity,  and  PDK-4  expression  (Upstate  Biotechnology/  Millipore,  Billerica,  M a s s a c h u s s e t s ) w a s m e a s u r e d a s an index of P P A R - a activity. P P A R - a w a s purified by IP prior to W e s t e r n blotting. T h e co-IP of P G C 1 a with M E F 2 A ( S a n t a C r u z Biotechnology, S a n t a - C r u z , California) and P P A R - a a n d to its r e p r e s s o r U S F - 2 w a s m e a s u r e d a s an index of the a s s o c i a t i o n between t h e s e proteins. Finally, to determine whether  P P A R - a binds the P G C 1 a .  MEF2A  functional  c o m p l e x , w e m e a s u r e d the co-IP of P P A R - a with M E F 2 A a n d U S F - 2 .  T h e e x p r e s s i o n of (31, P2 and p3 adrenoceptors  (all from S a n t a - C r u z  Biotechnology, S a n t a - C r u z , California) w a s m e a s u r e d . p2 a s s o c i a t i o n with G s or G i w a s m e a s u r e d by co-IP. P K A and C A M K II activities w e r e m e a s u r e d using radioisotopic  a s s a y s as described  in section  Ilia.  P I 3 K / A k t activation  was  determined by m e a s u r i n g the phosphorylation of Akt. Efforts to m e a s u r e N O S activity  in  expression  our  laboratory  and  were  unsuccessful. W e  P K A / PI3K-mediated  therefore  phosphorylation  of  measured  eNOS,  and  the the  e x p r e s s i o n of i N O S . T i s s u e nitrate/ nitrite levels were m e a s u r e d a s a n indirect index of N O levels. Total protein  glutathiolation  a n d tyrosine  nitration w e r e  m e a s u r e d by dot blotting a s biomarkers of reactive nitrogen s p e c i e s .  T h e total phosphorylation  states of C P T - 1 and A K A P 1 4 9  (Santa-Cruz  Biotechnology, S a n t a - C r u z , California) w e r e m e a s u r e d using p h o s p h o s e r i n e a n d phosphothreonine and  nitration  antibodies in combination! T h e nitrosylation, of  immunoprecipitation  CPT-1 of  were  determined  by  C P T - 1 with pan-specific  glutathiolation  measuring  anti-nitrotyrosine  the  co-  antibodies  (Upstate Biotechnology/ Millipore, Billerica, M a s s a c h u s s e t s ) , p a n - s p e c i f i c antiglutathione pan-specific  antibodies ( S a n t a - C r u z Biotechnology, S a n t a - C r u z , California) and anti-  nitrosocysteine  antibodies  (AG  Scientific,  San  Diego,  California). T o determine the relationship between A K A P 149 binding a n d P K A binding to C P T - 1 , co-immunoprecipitation  of P K A with C P T - 1 w a s m e a s u r e d .  T h e blots w e r e then stripped and reblotted for A K A P 1 4 9 . of C A M K - I I with C P T - 1 w a s a l s o m e a s u r e d .  Co-immunoprecipitation  50  (b)  Sample Preparation  1 0 - 3 0 m g of heart tissue w a s h o m o g e n i z e d in total protein extraction buffer containing 2 0 m M H E P E S , 1 m M ethylenediamine tetraacetic acid ( E D T A ) , 2 5 0 m M s u c r o s e , 100 m M s o d i u m pyrophosphate, 10 m M s o d i u m  orthovanadate,  100 m M s o d i u m fluoride and 4pJ/ ml protease inhibitor cocktail ( S i g m a - A l d i c h , S a i n t - L o u i s , Missouri) at p H 7.4. T h e total extraction buffer w a s found to interfere with the B i o R a d protein a s s a y . Protein concentration w a s therefore  determined  using the P i e r c e bjcinchoninic acid ( B C A ) protein a s s a y (Pierce Biotechnology, Rockford,  Illinois).  To  prepare  samples  for  sodium  dodecyl  sulphate  p o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) , 50 jag of protein w a s diluted with water a n d a reducing' buffer (6% S D S , 1 8 5 m M Tris p H 6.8, 3 0 % glycerol, 1 4 % m e r c a p t o e t h a n o l , 0 . 7 % b r o m o p h e n o l blue) to a total v o l u m e of 2 0 ut. T h e s a m p l e s w e r e boiled for 5 minutes a n d stored on ice prior to loading. Dot blotting s a m p l e s w e r e boiled for 5 minutes, but, to e n s u r e a d h e r e n c e of the protein to the nitrocellulose m e m b r a n e , an equivalent v o l u m e of water rather than  reducing  buffer w a s a d d e d . For immunoprecipitates, 15 pi of the immunoprecipitate w a s diluted with 5 u,l s a m p l e buffer and boiled for 5 minutes.  (c)  SDS-PAGE, Western Blotting and Dot Blotting  S a m p l e s w e r e loaded onto an a c r y l a m i d e / bis a c r y l a m i d e gel of the appropriate  percentage  (5, 7.5  or  10%)  and  subjected  to  SDS-PAGE  as  previously d e s c r i b e d (210). T h e protein b a n d s w e r e transferred to a nitrocellulose membrane  and  stained with  Ponceau  R e d to  m e a s u r e total protein.  m e m b r a n e w a s blocked in 5 % bovine s e r u m albumin in Tris-buffered containing  0.1%  polyoxyethylenesorbitan  monopalmitate  (TWEEN).  The saline The  m e m b r a n e w a s s u b s e q u e n t l y incubated with primary antibody overnight at 4 ° C , w a s h e d three times with tris-buffered saline and incubated in s e c o n d a r y antibody at room temperature for 1 hour. Following three further w a s h e s with tris-buffered  51  saline, the blots w e r e visualised using c h e m i l u m i n e s c e n t detection. D u e to the low a b u n d a n c e of s o m e proteins of interest, blots w e r e d e v e l o p e d using the Super  Signal  ®  West  Femto  Maximum  Sensitivity  substrate  (Pierce  Biotechnology, R o c k f o r d , Illinois). T h e blots w e r e i m a g e d using a C h e m i g e n i u s Q Image A n a l y s e r (Geneflow, A l e x a n d r i a , Virginia) s o the i m a g e s obtained are inverted. B a n d intensity w a s quantified using ImageJ software available from the National Institutes of Health. For most a n a l y s e s , 3 or 4 s a m p l e s from e a c h group w e r e run on a single gel. However, blots w e r e repeated to e n s u r e that the o b s e r v e d patterns w e r e consistent for all s a m p l e s .  (d)  Immunoprecipitation Protocol  500  uq protein w a s diluted into 500  ul total protein extraction buffer to  create a 1 uq/ ul protein solution. 2 ul of antibody w a s a d d e d a n d the s a m p l e s incubated on a rotator  overnight  at 4°C.  For combined phosphoserine  p h o s p h o t h r e o n i n e IP, 1 ul of e a c h antibody w a s a d d e d . 20  pi of protein  and A  s e p h a r o s e slurry w a s a d d e d a n d the s a m p l e s incubated on the rotator at 4°C for a further hour. S a m p l e s w e r e centrifuged to pellet the immunoprecipitate a n d the supernatant w a s d i s c a r d e d . T h e pellet w a s w a s h e d three times in total protein extraction buffer. Finally, the pellet w a s r e s u s p e n d e d in s u s p e n s i o n buffer (0.1  M  Tris b a s e , 1 m M E D T A , 1 m M E G T A , 50 m M s o d i u m fluoride, 5 m M s o d i u m p y r o p h o s p h a t e , 10% glycerol, 0.02%  (e)  sodium azide, pH  7.5).  Dot Blotting  5 ug of protein w a s loaded directly onto a dry nitrocellulose m e m b r a n e . Total protein w a s determined by P o n c e a u R e d staining. T h e m e m b r a n e w a s then blotted a c c o r d i n g to the W e s t e r n blotting protocol. P a n - s p e c i f i c anti-nitrotyrosine or glutathione antibodies w e r e u s e d a s the primary antibody.  52  V:  Functional  Effects  of  CPT-1  Covalent  Modifications  in  Isolated  Mitochondria  (a)  Isolation of Mitochondria  Left ventricular tissue from control hearts w a s h o m o g e n i z e d in 10ml of mitochondrial  homogenisation  buffer  containing  20  mM  3-(N-Morpholino)-  p r o p a n e s u l f o n i c acid ( M O P S ) , 2 5 0 m M s u c r o s e , 2 m M E D T A , 2 m M E G T A , 2.5 m M r e d u c e d glutathione a n d 3 % B S A at p H 7.2. T h e s a m p l e s w e r e centrifuged at 1000g for 5 minutes to r e m o v e the nuclei a n d incompletely disrupted tissue, a n d the supernatant w a s centrifuged at 10,000g for 10 minutes. T h e supernatant w a s d i s c a r d e d a n d the pellet r e s u s p e n d e d in mitochondrial  homogenisation  buffer without B S A prior to further centrifugation at 10,000g for 10 minutes. T h e final pellet w a s r e s u s p e n d e d in 0.6 ml of mitochondrial h o m o g e n i s a t i o n buffer without B S A . A 50ul aliquot of the mitochondrial fraction w a s t a k e n for protein quantification using the B i o R a d a s s a y . T h e mitochondrial preparations w e r e u s e d on the d a y of isolation.  (b)  Phosphorylation of Proteins in Isolated Mitochondria  Purified preparations of active P K A , C A M K II a n d A k t w e r e p u r c h a s e d from U p s t a t e B i o t e c h n o l o g y / Millipore. P h o s p h o r y l a t i o n of isolated mitchondria w a s a c h i e v e d by incubating 100 JLXI (0.4 - 0.7 m g protein) of the mitochondrial isolate with the active k i n a s e . F o r the control reaction,  100 uJ  of the s a m e  mitochondrial isolate w a s incubated without the active k i n a s e . T h e  reaction  conditions w e r e the s a m e a s those u s e d to m e a s u r e k i n a s e activity: 2 0 m M M O P S , 2 5 m M p-glycerophosphate, 5 m M E G T A , 1 m M s o d i u m orthovanadate, 1 m M dithiothreitol, 12.5 m M m a g n e s i u m chloride, 83 ulvl A T P , 1.7 uM c A M P (for P K A reactions only), 1 m M c a l c i u m chloride (for C A M K reactions only) a n d 0.83 jag/ ml active k i n a s e at p H 7.2. T h e final reaction v o l u m e w a s 6 0 0 u.l. T h e  53  s a m p l e s w e r e incubated at 30°C for 10 minutes a n d the reaction terminated by flash freezing in liquid nitrogen. S e p a r a t e reactions w e r e carried out to g e n e r a t e s a m p l e s for C P T - 1 a s s a y or m e a s u r e m e n t of total C P T - 1 phosphorylation a n d co-immunoprecipitation of C P T - 1 with the k i n a s e .  (c)  Peroxynitrite Dose Response in Isolated Mitochondria  Peroxynitrite h a s a half life of less than 5 s e c o n d s at physiological p H ; 8 0 % of the peroxynitrite loaded at p H 7.4 is d e g r a d e d by protonation within 12 s e c o n d s (217). H o w e v e r , this timeframe is sufficient for peroxynitrite to p r o d u c e m e a s u r a b l e covalent modifications of target proteins (218). In order to e x a m i n e the effects of tyrosine nitration, glutathiolation a n d cysteine nitrosylation on C P T 1,  we  incubated  mitochondrial  isolates  with  increasing  concentrations  of  peroxynitrite. Peroxynitrite is stable at high p H a s the equilibrium d o e s not favour protonation. Peroxynitrite stock solutions w e r e m a d e in 0 . 3 M N a O H .  Mitochondrial isolates from four control hearts w e r e p o o l e d . 100 pi (0.4 0.7 m g protein) of the pooled mitochondrial isolate w a s incubated with 0, 0.1, 1, 10, 100, 5 0 0 a n d 1000 p M peroxynitrite in mitochondrial h o m o g e n i s a t i o n buffer without B S A . T h e final reaction v o l u m e w a s 6 0 0 pi. T h e s a m p l e s w e r e incubated at room temperature for 5 minutes. T h e addition of the 0 . 3 M N a O H v e h i c l e raised the p H of the buffer from 7.2 to 8. generate  samples  phosphorylation  and  Immunoprecipitation  for  CPT-1  S e p a r a t e reactions w e r e carried out to  assay  or  co-immunoprecipitation was  started  immediately  measurement of  CPT-1  following  of  total  CPT-1  with  the  kinase.  the  incubation  with  peroxynitrite. C P T - 1 activity s a m p l e s w e r e frozen in liquid nitrogen a n d stored at -70°C until the d a y of a s s a y .  54  (d)  Measurement of CPT-1 Activity, Phosphorylation and Kinase Binding  C P T - 1 activity a n d sensitivity to malonyl C o A w a s m e a s u r e d a s d e s c r i b e d in s e c t i o n III (b) with two modifications: 50 pi of the s a m p l e w a s a d d e d a n d the reaction w a s allowed to p r o c e e d for 10 minutes rather than 5: Addition of the p H 8 mitochondrial s a m p l e s had no effect on the final p H of the C P T - 1 reaction mixture.  VI: Identification of CPT-1 Phosphorylation Sites by LC MS/ MS  T h e rat C P T - 1 B primary s e q u e n c e w a s s e a r c h e d for c o n s e n s u s sites of the  following  kinases: P K A , P K C , C A M K  I/  II,  AMPK  and  Akt.  Putative  phosphorylation sites for P K A a n d C A M K I/ II w e r e identified. T o m a x i m i z e our chances  of  finding  phosphorylation,  CPT-1 B  was  purified  by  IP  and  phosphorylation enrichment w a s performed on the tryptic digests of the C P T - 1 bands.  C P T - 1 in w h o l e cell h o m o g e n a t e s w a s purified by IP with s p e c i f i c C P T - 1 B antibodies. T h e immunoprecipitates w e r e subjected to S D S - P A G E a n d stained with C o u m a s s i e Blue. T h e b a n d s corresponding to C P T - 1 (88kDa) w e r e e x c i s e d a n d stored at -70°C until the d a y of a s s a y .  Tryptic digests of the C P T - 1 b a n d s were subjected to titanium T o p T i p p h o s p h o p e p t i d e enrichment at the U B C M S L / L M B P r o t e o m i c s C o r e Facility. T h e digests w e r e  applied to titanium  tips for  p h o s p h o p e p t i d e s . T h e tips w e r e then trifluoroacetic  acid,  1 0 % acetonitrile)  30  minutes  to  allow  w a s h e d with binding  binding  solution  to r e m o v e unphosphorylated  of  (0.1%  peptides.  P h o s p h o r y l a t e d peptides w e r e eluted from the tips by a s e r i e s of 50 pi w a s h e s with i n c r e a s i n g concentrations of a m m o n i u m bicarbonate (10, 2 0 , 50 a n d 100 m M ) , w h i c h r e l e a s e low to moderately phosphorylated peptides, a n d a final 50 pi w a s h with a m m o n i u m hydroxide, which r e l e a s e s highly p h o s p h o r y l a t e d peptides.  55  The ammonium  bicarbonate eluents for e a c h s a m p l e w e r e p o o l e d , a n d  the  a m m o n i u m bicarbonate a n d a m m o n i u m hydroxide eluents for e a c h s a m p l e w e r e subjected to L C / M S / M S separately.  P h o s p h o r y l a t i o n detection w a s carried out using Liquid C h r o m a t o g r a p h y / M a s s Spectroscopy/ Mass Spectroscopy (LC/ M S / M S ) on an API Q  STAR  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 P r o t e o m i c s C o r e Facility. Tryptic digests of C P T - 1 b a n d s underwent r e v e r s e d - p h a s e H P L C . T h e c o l u m n eluent w a s subjected to M S / M S a s it eluted from the c o l u m n . In this p r o c e d u r e , a t m o s p h e r i c p r e s s u r e ionization (API) w a s applied to generate a s p e c t r u m of m a s s - t o - c h a r g e ratio (m/z) p e a k s . A s e a c h a m i n o acid p r o d u c e s a characteristic m/z peak, the s e q u e n c e of the eluted peptide c a n be determined a n d s e a r c h e d in the M A S C O T protein d a t a b a s e to confirm the identity of the protein. Ions with a c h a n g e in m/z s u g g e s t i n g that phosphorylation had occurred (indicated by a n i n c r e a s e of 80 Da) w e r e s e l e c t e d a s parent ions for M S / M S to confirm that the phosphorylation event w a s present a n d identify the affected residue.  Vll: Data Analysis  D a t a are e x p r e s s e d a s m e a n ± standard error of the m e a n ( S E M ) . F o r statistical  a n a l y s i s , data w e r e  a n a l y s e d using  Number  Cruncher  Statistical  Software ( N C S S , Kaysville, Utah). Starling c u r v e s w e r e a n a l y s e d using G L M A N O V A with N e u m a n n - K e u l s post h o c test. A l l other d a t a w e r e a n a l y s e d u s i n g O n e - W a y A N O V A with N e u m a n n - K e u l s post h o c test. C o m p a r i s o n s b e t w e e n two g r o u p s w e r e carried out using a n unpaired- student's t-test. A c u t e perfusion a n d chronic treatment data are presented separately,  1  although  the control  and  diabetic g r o u p s for e a c h are the s a m e , a n d the data w e r e subjected to c o m p o s i t e analysis.  56  RESULTS I: General Characteristics  W e m e a s u r e d p l a s m a g l u c o s e a n d insulin levels to confirm s u c c e s s f u l induction  of  diabetes.  STZ  successfully  induced  marked  and  sustained  h y p e r g l y c e m i a which w a s a s s o c i a t e d with a d e c r e a s e in insulin levels a n d a mild elevation of p l a s m a lipids. A s e x p e c t e d , metoprolol treatment had no effect on either g l u c o s e or insulin (Table 1). Metoprolol had no effect on p l a s m a lipids, but ameliorated the i n c r e a s e in ketone levels in the diabetic group (Table 1). B o d y weights w e r e lower in the diabetic groups. Metoprolol had no significant effect on body weight.  H o w e v e r , metoprolol significantly lowered heart weight  in  both  control a n d diabetic rats (Table 1).  II: Functional and Metabolic Effects of Chronic Metoprolol Treatment  T o establish whether chronic metoprolol treatment improves function in the S T Z - m o d e l , c a r d i a c function w a s m e a s u r e d ex vivo by direct left ventricular p r e s s u r e m e a s u r e m e n t s in p a c e d isolated working treatment with metoprolol. Metoprolol treatment  hearts following  (75mg/kg/day IP)  chronic  significantly  improved contractile function in diabetic cardiomyopathy a s m e a s u r e d ex vivo by L V D P , +dP/ dt a n d - d P / dt from the left ventricle in p a c e d hearts (Figure 1). During s u b s e q u e n t studies for the m e a s u r e m e n t of c a r d i a c m e t a b o l i s m , in w h i c h the hearts w e r e not p a c e d and fatty acid w a s present in the perfusate, metoprolol also ameliorated the d e p r e s s i o n in rate-pressure product, c a r d i a c output a n d hydraulic power in diabetic hearts (Figure 2).  C h r o n i c treatment  with metoprolol  i n c r e a s e d palmitate  oxidation  and  d e c r e a s e d g l u c o s e oxidation in control hearts (Figure 3). Lactate production w a s u n c h a n g e d , but g l y c o g e n levels w e r e d e c r e a s e d (Table 2). In the diabetic hearts, there w a s a m a r k e d i n c r e a s e in palmitate oxidation relative to controls a n d  57  TABLE 1 GENERAL CHARACTERISTICS AND PLASMA PARAMETERS AT TERMINATION  Body Weight (g) Heart Weight (S) Plasma Glucose (mmol/1) Plasma Insulin (ng/ml) Plasma Triglycerides (mmol/ 1) Plasma Cholesterol (mmol/ 1) Plasma Free Fatty Acids (mmol/1) Plasma Ketones (mmol/1)  C 486.4 ±31.3  CT 480.3 ± 41.0  DT D 387.8 ± 37.5* 351.6 + 53.7*  +  1.51 ±0.10*  1.38 ± 0.07  7.25 ± 0.26  7.35 ± 0.40  27.99 ± 1 . 1 *  24.09 ± 5.41*  1.59 ± 0.41  1.78 ± 0.78  0.49 ± 0.3*  0.37 + 0.12*  0.19 ±0.01  0.16 ±0.01  0.25 ± 0.05*  0.26 ± 0.05*  1.89±0.05  1.86±0.10  2.05±0.18*  2.10 ± 0 . 1 5 *  0.19 ± 0.02  0.23 ± 0.01  0.20 ± 0.02  0.19 ± 0.03  0.76 ± 0.05  0.50 ± 0.04  2.43 ± 0.57*  1.36 ± 0.28  1.82 ±0.10  1.47 ± 0.07  +  +  A n i m a l s w e r e fasted for 5 hours prior to blood collection. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using o n e - w a y A N O V A with N e u m a n n - K e u l s p o s t - h o c test. * = significantly different from C , group  (p<0.05)  (C=control,  +  = significantly different from untreated  n=8; CT=control  treated  with  D=diabetic, n=8; DT=diabetic treated with metoprolol, n=8).  metoprolol, n=8;  58  FIGURE 1  M e c h a n i c a l p e r f o r m a n c e of Isolated P e r f u s e d Hearts: Left Ventricular P r e s s u r e M e a s u r e m e n t s . Hearts w e r e e x c i s e d a n d perfused in working heart m o d e with C h e n o w e t h - K e u l s buffer containing 5.5 m M g l u c o s e but no palmitate. T h e hearts w e r e p a c e d at 3 0 0 bpm and direct left ventricular p r e s s u r e m e a s u r e m e n t s w e r e t a k e n . A - C : Left ventricular d e v e l o p e d p r e s s u r e ( L V D P ) ,  maximum  rate  of  contraction (+dP/dt) a n d m a x i m u m rate of relaxation (-dP/ dt) from direct left ventricular p r e s s u r e m e a s u r e m e n t s . D a t a represent m e a n s ± S E M a n d w e r e a n a l y s e d using G L M A N O V A with N e u m a n n K e u l s post-hoc test. * = significantly different from C , C T , D T at the s a m e filling p r e s s u r e , #  = significantly different  from C a n d D at the s a m e filling p r e s s u r e (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  M e c h a n i c a l p e r f o r m a n c e of Isolated P e r f u s e d Hearts: F l o w a n d R a t e - P r e s s u r e Product Measurements. pressure  and  measurements.  heart  rate  C a r d i a c function m e a s u r e m e n t s obtained by aortic measurements,  U n p a c e d hearts w e r e  and  pulmonary  and  aortic  flow  perfused with K r e b s - H e n s e l e i t buffer  containing 5.5 m M g l u c o s e a n d 0.8 m m o l palmitate at constant preload (11.5 m m Hg) a n d afterload (80 m m Hg). Heart rate, peak systolic p r e s s u r e , rate-pressure product, c a r d i a c output a n d hydraulic power. D a t a represent m e a n s ± S E M a n d w e r e a n a l y s e d using G L M A N O V A with N e u m a n n K e u l s p o s t - h o c test. * = significantly different from D a n d D T 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 a n d D T at s a m e timepoint (p<0.05), (C=control, n=5; CT=control treated, n=5; D= diabetic, n=5; DT= diabetic treated, n=5).  61  10  20  30  40  TIME (MINUTES)  50  60  '  10  20  30  40  TIME (MINUTES)  50  60  62  FIGURE 3  Effects of C h r o n i c In Vivo P e r f u s e d Hearts.  Metoprolol Treatment  on M e t a b o l i s m of  A - B G l u c o s e a n d palmitate oxidation during a 6 0  Isolated minute  a e r o b i c perfusion. P e r f u s i o n s w e r e carried out in the p r e s e n c e (left panel) or a b s e n c e (right panel) of insulin (100 uUnits/ml) a s indicated. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using O n e - W a y A N O V A with N e u m a n n Keuls  p o s t - h o c test,  * = significantly  different  from  C  of  metoprolol,  significantly different from c o r r e s p o n d i n g untreated group (p<0.05),  +  =  CD PALMITATE OXIDATION 100 U7 ml Insulin Present (nmol/ mm/ g dry weight) U  PALMITATE OXIDATION Insulin Absent (nmol/ min/ g dry weight)  S  GLUCOSE OXIDATION | | l/ j )  1 0 0 p U / m ( n m 0  n s u M n  m i n / g  d r y  P r e s e n t  w e  g n t  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  Lactate Production (nmol/ min/ g dry weight) Tissue Glycogen Levels (u.mol/ g dry weight) Tissue Triglyceride Levels (umol/ g dry weight) Malonyl CoA Levels (u.mol/ g wet weight)  C 10.2 ± 2.2  CT 13.9 ± 4.1  D 10.6 ± 3 . 6  DT 10.8 ± 4.0  180.4±31.2  95.9 ± 18.3  285.0± 32.0  286.6± 35.2  59.1 ± 3 . 9 *  44.6± 2.7  +  *  41.1 ± 2 . 6  31.2 ± 1.5  17.1 ± 2 . 4  7.7 ± 1.4  +  +  20.7 ± 5.2  *  +  17.4 ± 3 . 8  D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using o n e - w a y A N O V A with N e u m a n n - K e u l s post-hoc test. * = significantly different u n p e r f u s e d group,  +  from  corresponding  = significantly different from c o r r e s p o n d i n g 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  ATP (umol/g wet weight) ADP (umol/g wet weight) AMP (umol/g wet weight) ATP/ADP Ratio a-1 A M P K Activity (pmol ATP incorporated/ min/ mg protein) a-2 A M P K Activity (pmol ATP incorporated/ min/ mg protein) a-pan A M P K Activity (pmol ATP incorporated/ min/ mg protein)  DT 5.1 ± 1.2  C 6.3 ± 0.3  CT 7.2 +1.7  D 6.0 ± 0.5  2.3 ± 0.2  1.6 + 0.3  2.5 ± 0.3  0.74 ± 0.09  1.03 ± 0.08  0.83 ± 0.08  0.77 ± 0.03  2.9 ± 0.3  6.0 ± 1.4  2.5 ± 0.2  3.0 ± 1.0  2.7 ± 0.3  3.5 ± 0.6  3.1±0.4  3.6 ± 0.5  3.6 ± 0.4  3.6 ± 0.3  3.4 ± 0.6  3.4 ± 0.4  4.0 ± 0.9  3.8 ± 0.3  3.6 ± 0.5  3.7 ± 0.4  +  1.6 ± 0.5  +  D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using o n e - w a y A N O V A with N e u m a n n - K e u l s post-hoc test. * = significantly different u n p e r f u s e d group,  +  from  corresponding  = significantly different from c o r r e s p o n d i n g 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 l u c o s e oxidation w a s negligible; in t h e s e hearts, chronic metoprolol treatment decreased  palmitate  oxidation  and  i n c r e a s e d g l u c o s e oxidation  (Figure  3).  G l y c o g e n levels w e r e e l e v a t e d in diabetic hearts a s c o m p a r e d with controls; metoprolol treatment had no effect on either g l y c o g e n levels or lactate production (Table 2). T i s s u e triglyceride levels w e r e significantly elevated in the diabetic group. In both control a n d diabetic hearts, metoprolol treatment significantly lowered t i s s u e triglyceride levels (Table 2). M y o c a r d i a l e n e r g e t i c s , a s d e t e r m i n e d by t i s s u e levels of A T P , A D P a n d A M P , and A M P K activity, w e r e not altered either by metoprolol or by d i a b e t e s (Table 3). W h e n perfusions w e r e repeated in the a b s e n c e of insulin, the effect  of metoprolol on g l u c o s e oxidation  was  obliterated, but the effect o n palmitate oxidation w a s p r e s e r v e d (Figure 3). In a preliminary study, chronic metoprolol treatment had no effect on glycolysis in either  control  glycolysis  or diabetic heart  and  g l u c o s e oxidation  (Table 4), but in diabetic  improved  hearts  by  coupling  between  increasing g l u c o s e  oxidation.  When  control or diabetic hearts w e r e  perfused for 30 minutes  with  metoprolol, palmitate oxidation w a s inhibited and g l u c o s e oxidation m a r k e d l y stimulated (Figure 4) producing a n i n c r e a s e in t i s s u e A T P levels a n d a d e c r e a s e in A M P levels (Table 5). Stimulation of g l u c o s e oxidation w a s a s s o c i a t e d with a fall in lactate production without any c h a n g e in g l y c o g e n levels (Table 5). In both control a n d diabetic hearts, acute metoprolol perfusion lowered t i s s u e triglyceride levels ( T a b l e 5). In the a b s e n c e of insulin, the pattern of c h a n g e s o b s e r v e d for g l u c o s e oxidation w a s obliterated in diabetic, but not control, hearts, w h e r e a s the pattern of c h a n g e s o b s e r v e d for palmitate oxidation w a s p r e s e r v e d (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  Glycolysis (nmol/ min/ g dry weight) Glucose Oxidation (nmol/ min/ g dry weight) % Coupling of Glycolysis to Glucose Oxidation Acyl CoA Dehydrogenase Activity (mmol/ min/ mg protein) Citrate Synthase Activity (mmol/ min/ mg protein)  DT  D  CT  C  1729 ± 313  3900 ± 539  4484 + 1116 1335 ± 358  2336± 564  3749 ± 1 3 7 8  123 ± 52  72 ± 2 0  82±19  11 ± 6  5.0 ± 0 . 4  5.5 ± 0.4  4.6 ± 0.3  4.2 ± 0.4  1.3 ± 0.1  1.2 ± 0 . 1  1.3 ± 0 . 2  1.4 ± 0 . 2  #  #  #  #  854 ± 3 1 2 * 40 ± 18*  G l u c o s e a n d fatty acid metabolism m e a s u r e m e n t s from a preliminary study. D a t a represent m e a n s ± S E M . Data w e r e a n a l y s e d using o n e - w a y A N O V A  with  N e u m a n n - K e u l s post-hoc test. # = significantly different from C a n d C T , = significantly C=control,  different  from  n=4; CT=control  D (p<0.05) treated  (Glycolysis a n d glucose  with metoprolol,  DT=diabetic treated with metoprolol, n=4;  oxidation,  n=4; D=diabetic, n=4;  A c y l C o A d e h y d r o g e n a s e a n d citrate  s y n t h a s e activities, 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  A c u t e Effects of Metoprolol on M e t a b o l i s m of Isolated P e r f u s e d Hearts. Glucose  and  palmitate  oxidation  during  a  60  minute  aerobic  A-B:  perfusion.  P e r f u s i o n s w e r e carried out in the p r e s e n c e (left panel) or a b s e n c e (right panel) of insulin (100 uJvl/ml) a s indicated. D a t a represent m e a n s ± S E M . D a t a w e r e analysed  using O n e - W a y A N O V A  significantly  different  from  with N e u m a n n K e u l s p o s t - h o c test,  C of metoprolol,  c o r r e s p o n d i n g untreated group (p<0.05),  + = significantly  different  * = from  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  Lactate Production (nmol/ min/ g dry weight) Tissue Glycogen Levels (u,mol/ g dry weight) Tissue Triglyceride Levels (umol/ g dry weight) Malonyl CoA Levels (umol/ g wet weight)  C 10.2 ± 2.2 180.4± 31.2  CP 6.7 ± 1.3  +  160.6 +18.1  D 10.6 + 3.6 285.0 ±32.0  DP 2.4 ± 0.8  +  237.3±35.7  +  A  41.1+2.6  35.3± 3.0  17.1 ± 2 . 4  8.1 +1.1  +  +  59.1 ± 3 . 9 *  20.7 ± 5.2  31.5± 0.8  +  22.1 ± 4.7  D a t a represent m e a n s + S E M . Data w e r e a n a l y s e d using o n e - w a y A N O V A with N e u m a n n - K e u l s post-hoc test. * = significantly different unperfused group,  +  from  corresponding  = significantly different from c o r r e s p o n d i n g untreated group,  (p<0.05) (C=control, n=5; C P = control perfused, n=5; D=diabetic, n=5; D P = diabetic p e r f u s e d , n=5).  71  TABLE 6 MYOCARDIAL ENERGETICS AND AMPK ACTIVITY FOLLOWING ACUTE METOPROLOL PERFUSION EX-VIVO IN THE PRESENCE OF INSULIN  ATP (umol/g wet weight) ADP (umol/g wet weight) AMP (umol/g wet weight) ATP/ADP Ratio a-1 A M P K Activity (pmol [P] incorporated/ min/ mg protein) a-2 A M P K Activity (pmol [P] incorporated/ min/ mg protein) a-pan A M P K Activity (pmol [P] incorporated/ min/ mg protein)  D 6.0 ± 0.5  DP 9.7± 0.9  2.5 ± 0.3  3.3 ± 0.4  0.22 ± 0.03  0.83 ± 0.08  0.39 ± 0.10  2.9 ± 0.3  3.2 ±0.14  2.5 ± 0.2  2.3 ± 0.4  2.7 ± 0.3  3.8±0.1  3.1 ± 0 . 4  3.7 ± 0.4  3.6 ± 0.4  3.0 ± 0 . 5  3.4 ± 0.6  3.8 ± 0 . 7  4.0 ± 0.9  4.0 ± 0.9  3.6 ± 0.5  3.3 ± 0.4  C 6.3 ± 0.3  CP 7.3 ± 0.4  2.3 ± 0.2  2.3 ± 0 . 1  0.74 ± 0.03  +  +  +  +  +  +  M y o c a r d i a l E n e r g e t i c s a n d A M P K Activity. Data represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using o n e - w a y A N O V A with N e u m a n n - K e u l s p o s t - h o c test.  +  =  significantly different from c o r r e s p o n d i n g 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 a n d M C D E x p r e s s i o n and P h o s p h o r y l a t i o n . A : E x p r e s s i o n of A C C a n d M C D measured  by W e s t e r n blotting.  (C=c.ontrol;  CT=control treated;  D=diabetic;  DT=diabetic treated). B: P h o s p h o r y l a t i o n of A C C m e a s u r e d by W e s t e r n blotting. B a n d intensity w a s 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 a n d M a l o n y l C o A Sensitivity. A : C P T - 1 Activity in w h o l e t i s s u e h o m o g e n a t e s . D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using o n e - w a y ANOVA  with N e u m a n n - K e u l s post-hoc test.  + = significantly  different  from  *  c o r r e s p o n d i n g untreated group,  = significantly different from all other g r o u p s  (p<0.05) (C=control, n=5; C P = control perfused, n=5 ( C o n e of metoprolol?); CT=control  treated,  n=5;  D=diabetic,  n=5;  DT=diabetic treated, n=5) B. M a l o n y l C o A I C  5 0  DP  =  diabetic  perfused,  n=5;  v a l u e s calculated following curve-  fitting a n a l y s i s of C P T - 1 d o s e - r e s p o n s e curves. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using o n e - w a y A N O V A with N e u m a n n - K e u l s p o s t - h o c test. * = significantly different from control, untreated CT=control  group,  p<0.05.  treated,  n=5;  DT=diabetic treated, n=5).  +  = significantly different from c o r r e s p o n d i n g  (C=control,  n=5;  CP  D=diabetic,  n=5;  DP  = =  control  perfused,  n=5;  diabetic  perfused,  n=5;  76  FIGURE 7 P h a r m a c o l o g i c a l Effects of Metoprolol on C P T - 1 Activity. C P T - 1 Activity following incubation  of control tissue h o m o g e n a t e s with increasing concentrations  of  metoprolol a n d in the p r e s e n c e of 0, 50 or 100u.M malonyl C o A . Data represent m e a n s ± S E M (n=5).  77  4.0 -i  -•—  0 (xm Malonyl CoA  -o— 50 nmol Malonyl CoA  3.5  100|imol Malonyl CoA S-  B  3.0  E  2  >  Q . 2.5  i= °> ^ » ^ c |I E 1.5 CL =S  O o £  c  1.0 H  0.5 0.0  0  2000  4000  6000  8000  '7/10000  50000  METOPROLOL CONCENTRATION (ng/ ml)  78 III: Malonyl CoA Levels  In vivo treatment with metoprolol lowered malonyl C o A levels in control hearts, a s did a c u t e treatment of control hearts with metoprolol during perfusion (Tables 2 a n d 5). D i a b e t e s had no effect on malonyl C o A levels, a n d malonyl C o A levels in the diabetic heart remained u n c h a n g e d by chronic  metoprolol  treatment a n d a c u t e metoprolol perfusion. A C C a n d M C D e x p r e s s i o n in control a n d diabetic hearts w a s u n c h a n g e d by chronic metoprolol treatment (Figure 5), a n d A M P K - m e d i a t e d phosphorylation of A C C , a s s e s s e d by phosphorylation of S e r 79 on A C C , w a s a l s o u n c h a n g e d either by metoprolol perfusion or chronic in vivo metoprolol treatment  (Figure 5). Furthermore, metoprolol had no effect on  the total phosphorylation state of A C C , a s s e s s e d by reactivity with p a n - s p e c i f i c a n t i - p h o s p h o s e r i n e and anti-phosphothreonine antibodies. O v e r a l l , malonyl C o A levels did not correlate with the o b s e r v e d c h a n g e s in the rate of fatty acid oxidation, a n d the o b s e r v e d d e c r e a s e in malonyl C o A levels in control hearts could not be e x p l a i n e d by c h a n g e s in A C C or M C D .  IV: CPT-1 Activity and Malonyl CoA Sensitivity  In a preliminary study, w e m e a s u r e d the activities of C P T - 1 (entry of fatty acyl C o A to mitochondria), a c y l - C o A d e h y d r o g e n a s e (a p-oxidation e n z y m e ) a n d citrate s y n t h a s e (a T C A c y c l e e n z y m e ) . C h r o n i c metoprolol treatment d e c r e a s e d C P T - 1 activity in both control a n d diabetic hearts but had no effect on the other e n z y m e s (Table 4 a n d Figure 6). A c u t e metoprolol perfusion a l s o d e c r e a s e d C P T - 1 activity. T o investigate whether metoprolol altered C P T - 1 sensitivity, w e a s s a y e d C P T - 1 activity in the p r e s e n c e of increasing concentrations of malonyl C o A to obtain d o s e - r e s p o n s e c u r v e s for e a c h of the groups. T h e IC o of malonyl 5  C o A w a s c a l c u l a t e d (Figure 6). C h r o n i c metoprolol treatment d e c r e a s e d the sensitivity of C P T - 1 to malonyl C o A in diabetic, but not control, hearts. A c u t e metoprolol perfusion d e c r e a s e d the sensitivity in both control a n d diabetic hearts.  79 T h e rightward shift in the d o s e - r e s p o n s e curve w a s p r e s e r v e d w h e n absolute activity data w e r e plotted (data not shown).  T o test whether metoprolol is a direct p h a r m a c o l o g i c a l inhibitor of C P T - 1 , or whether it c a n directly interfere with malonyl C o A inhibition of C P T - 1 , w e incubated C P T - 1 with increasing concentrations of metoprolol in the p r e s e n c e of 0, 50 a n d 1 0 0 p M malonyl C o A . Metoprolol did not inhibit C P T - 1 activity directly, a n d did not d e c r e a s e or e n h a n c e inhibition of C P T - 1 by malonyl C o A (Figure 7).  V: Regulation of CPT-1 Expression  C h r o n i c metoprolol treatment d e c r e a s e d total C P T - 1 e x p r e s s i o n in control a n d diabetic hearts (Figure 8). Metoprolol d e c r e a s e d C P T - 1 B e x p r e s s i o n , but did not alter C P T - 1 A e x p r e s s i o n , which w a s present at low levels (Figures 9 a n d 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 e x p r e s s i o n .  Metoprolol did not alter the total e x p r e s s i o n 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 i n c r e a s e d the e x p r e s s i o n of U S F - 1 but the e x p r e s s i o n of a - M H C w a s not significantly  c h a n g e d . T h e e x p r e s s i o n of both  d e c r e a s e d in the diabetic heart, a s w a s a - M H C .  U S F - 1 and  USF-2  was  Metoprolol i n c r e a s e d the  e x p r e s s i o n of U S F - 2 in control and diabetic hearts, but this w a s only a s s o c i a t e d with a n i n c r e a s e in M H C e x p r e s s i o n in diabetic hearts; i n d e e d , although the  a-  M H C b a n d in control treated hearts w a s larger and more diffuse, the b a n d w a s less intense in densitometric a n a l y s i s , indicating that a - M H C e x p r e s s i o n m a y actually h a v e d e c r e a s e d (Figures 13-15). S E R C A e x p r e s s i o n w a s  markedly  d e p r e s s e d in diabetic hearts, and metoprolol restored S E R C A e x p r e s s i o n .  In control hearts, metoprolol d e c r e a s e d the association of the coactivator P G C 1 a with the transcription factors P P A R - a and M E F - 2 A without c h a n g i n g the  80  FIGURE 8 Total C P T - 1 e x p r e s s i o n . D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using a n unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5;  CT=control treated with metoprolol, n=5;  treated with metoprolol, n=5).  D=diabetic,  n=5;  DT=diabetic  18  82  FIGURE 9  C P T - 1 B ( m u s c l e isoform) e x p r e s s i o n . D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using an unpaired student's t-test. * = significantly different, (C=control,  n=5;  CT=control treated with metoprolol, n=5;  DT=diabetic treated with metoprolol, n=5).  p<0.05  D=diabetic,  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  >  — .  70 D m > -i  a  JO O c 13  CPT-1 MUSCLE ISOFORM EXPRESSION (Normalised to Total Protein) © b  o  © j»  ©  o b  -* b  fo  84  FIGURE 10  C P T - 1 A (liver isoform) e x p r e s s i o n . D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using a n unpaired student's t-test. * = significantly different, (Ocontrol,  n=5;  CT=control treated with  DT=diabetic treated with metoprolol, n=5).  metoprolol, n=5;  p<0.05  D=diabetic,  n=5;  CPT-1 LIVER ISOFORM EXPRESSION (Normalised to Total Protein)  CPT-1 LIVER ISOFORM EXPRESSION (Normalised to Total Protein) o  e  o M  o ^  © o»  o  -*  OD  ©  CPT-1 LIVER ISOFORM EXPRESSION (Normalised to Total Protein) © © 0 0 0 - » - f c - » b r o * b > c o b » o *  H  » c  - » K » c »  > b  86  FIGURE 11  PPAR-a,  PGC1a  and  PDK-4,  e x p r e s s i o n following  chronic treatment  with  metoprolol. Densitometric a n a l y s e s of t h e s e data are presented in Figure 12. (C=control,  n=5;  CT=control  treated  with metoprolol,  DT=diabetic treated with metoprolol, n=5).  n=5;  D=diabetic,  n=5;  CT PPAR-a  PGC1a PDK-4 Ponceau DT PPAR-a  *  PGC1a PDK-4 Ponceau D PPAR-a  PGC1a PDK-4 Ponceau  88  FIGURE 12  Densitometric a n a l y s i s of P P A R - a , P G C 1 a and P D K - 4 , e x p r e s s i o n following chronic treatment with metoprolol. Data represent m e a n s ± S E M . D a t a were a n a l y s e d using a n unpaired student's t-test. * = significantly different, (C=control,  n=5;  CT=control  treated  with metoprolol,  DT=diabetic treated with metoprolol, n=5).  n=5;  p<0.05  D=diabetic,  n=5;  PDK-4 EXPRESSION (Normalised to Total Protein)  PDK-4 EXPRESSION (Normalised to Total Protein)  68  PGC-1a EXPRESSION (Normalised to Total Protein)  PGC-1a EXPRESSION (Normalised to Total Protein)  PPAR-a EXPRESSION (Normalised to Total Protein)  PPAR-a EXPRESSION (Normalised to Total Protein)  90  FIGURE 13  U S F - 1 , U S F - 2 , M H C a n d S E R C A - 2 e x p r e s s i o n following chronic treatment with metoprolol. Densitometric a n a l y s e s of t h e s e data are presented in F i g u r e s 14 a n d 15. (C=control, n=5; CT=control treated with metoprolol, n=5; n=5; DT=diabetic treated with metoprolol, n=5).  D=diabetic,  91  CT USF-1 USF-2 MHC SERCA Ponceau DT  92  FIGURE 14  Densitometric  a n a l y s i s of  U S F - 1 and  USF-2  e x p r e s s i o n following  chronic  treatment with metoprolol. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using a n unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5;  CT=control treated with metoprolol,  treated with metoprolol, n=5).  n=5;  D=diabetic,  n=5;  DT=diabetic  USF-2 EXPRESSION (Normalised to Total Protein)  USF-1 EXPRESSION (Normalised to Total Protein)  7>  m > H  s m  USF-1 EXPRESSION (Normalised to Total Protein)  USF-2 EXPRESSION (Normalised to Total Protein)  P b  p  "ui  7* b  ^  ui  NJ b  NJ  ui  W b  JO m > —i m z  > s m z  USF-1 EXPRESSION (Normalised to Total Protein)  USF-2 EXPRESSION (Normalised to Total Protein) O  Ul  O  Ul  O  Ul  p o  o in  -» b  -k in  NJ  o  ro in  b  94  FIGURE 15  Densitometric  a n a l y s i s of  M H C and  SERCA  e x p r e s s i o n following  chronic  treatment with metoprolol. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using a n unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5;  CT=control treated with metoprolol,  treated with metoprolol, n=5).  n=5;  D=diabetic,  n=5;  DT=diabetic  MYOSIN HEAVY CHAIN EXPRESSION (Normalised to Total Protein)  SERCA EXPRESSION (Normalised to Total Protein)  o  o  in  in  b  p p  p eft  ^ o  -» en  M b  JO  m > H  3 m z  MYOSIN HEAVY CHAIN EXPRESSION (Normalised to Total Protein)  SERCA EXPRESSION (Normalised to Total Protein)  m  >  m  w  -i 3 m z  w  > -i  _  H CD JO  m z  8 3  o  —i  JO  •o  8 5 •o  MYOSIN HEAVY CHAIN EXPRESSION (Normalised to Total Protein)  SERCA EXPRESSION (Normalised to Total Protein) O b  JO  m >  -  m z  H  G)  O in  -k b  -» in  K> p  JO in  JO  m > -i 3 m z  ro in  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 a n d U S F - 2 to PPAR-a  measured  immunoprecipitation  by  with  immunoprecipitation.  anti-  PGC1a  antibodies  s u b s e q u e n t l y subjected to immunoblotting  for  Samples as  PPAR-a,  underwent  indicated, MEF-2A  and or  were USF-2.  S a m p l e s a l s o underwent immunoprecipitation with a n t i - P P A R - a antibodies and immunoblotting  with  anti-  MEF-2A  and  USF-2  antibodies  as  indicated.  Densitometric a n a l y s e s of t h e s e data are presented in Figure 17. (C=control, n=5;  CT=control treated with metoprolol, n=5;  treated with metoprolol, n=5).  D=diabetic,  n=5;  DT=diabetic  97  C  IP: PGC1a  CT  D  f * •fc• »  Blot: PPAR-a  IP: PGC1a  Blot: M E F - 2 A  IP: PGC1a  Blot: USF-2  4MI ! • § INI  J l t _ _ > j——^ IP  i  _ »  1  C IP: PPAR-a Blot: M E F - 2 A  DT  wm^^f WBK^f  ^ •  •  _•»_•. - j a »  :\Z  • p*t"W PJMmf H i W pjVlPJPlHP** ^  CT  1  D  DT  98  FIGURE 17  Densitometric a n a l y s i s of P G C 1 a binding. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. * = significantly different from untreated group, # = significantly different from all g r o u p s , + = significantly different from C a n d 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 association, of P G C 1 a with P P A R - a was  e n h a n c e d but its a s s o c i a t i o n s with M E F - 2 A a n d U S F - 2 w e r e a b o l i s h e d .  Metoprolol d e c r e a s e d the a s s o c i a t i o n between  PGC1a  diabetic heart but the a s s o c i a t i o n between P G C 1 a  a n d P P A R - a in the  and M E F - 2 A  increased.  H o w e v e r , in the diabetic heart, metoprolol markedly i n c r e a s e d the binding of U S F - 2 to P G C 1 a (Figures 16 a n d 17).  VI: 3-Adrenoceptor Signalling  C h r o n i c treatment with metoprolol i n c r e a s e d 31 receptor e x p r e s s i o n in control hearts. In the diabetic heart, 31 receptor levels w e r e d e c r e a s e d , a n d chronic metoprolol treatment i n c r e a s e d 31 receptor levels (Figures 18 a n d 19). 32 receptor levels w e r e i n c r e a s e d in both control a n d diabetic hearts,  but  d i a b e t e s itself did not alter 32 receptor e x p r e s s i o n (Figures 18 a n d 19). Both metoprolol treatment a n d diabetes induced marked i n c r e a s e s in the e x p r e s s i o n of the 33 receptor (Figure 14). Overall, metoprolol c a u s e d a shift in 3-receptor e x p r e s s i o n towards p2 a n d p3 in control hearts, a n d i n c r e a s e d the e x p r e s s i o n of all three receptor s u b t y p e s in the diabetic heart. T h e r e w a s no clear shift in the a s s o c i a t i o n of the p2 adrenoceptor with G s or G i in any of the groups (Figure 20).  T h e activity of P K A , a s e x p e c t e d , w a s d e c r e a s e d by metoprolol (Table 7). B y contrast, the activity of the P I 3 K / A k t pathway w a s i n c r e a s e d by metoprolol Figure 21), w h e r e a s C A M K activity w a s not significantly altered (Table 7).  A t t e m p t s to m e a s u r e tissue peroxynitrite levels a n d e N O S a n d i N O S  101  FIGURE 18  E x p r e s s i o n of (5-Adrenoceptor s u b t y p e s following chronic metoprolol  treatment.  Densitometric a n a l y s e s of t h e s e data are presented in Figure 19. (C=control, n=5;  CT=control treated  with metoprolol,  treated with metoprolol, n=5).  n=5;  D=diabetic,  n=5;  DT=diabetic  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  Densitometric  a n a l y s i s of e x p r e s s i o n of  p-Adrenoceptor s u b t y p e s  following  chronic metoprolol treatment. Data represent m e a n s ± S E M . D a t a w e r e a n a l y s e d using a n unpaired student's t-test. * = significantly different, p<0.05 (C=control, n=5; CT=control treated with metoprolol, n=5; treated with metoprolol, n=5).  D=diabetic, n=5;  DT=diabetic  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)  B2 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein)  B1 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein)  o o  B3 ADRENOCEPTOR EXPRESSION (Normalised to Total Protein)  71 o  105  FIGURE 20  Binding of G s a n d G i to p 2 - A d r e n o c e p t o r s . Densitometric a n a l y s i s w a s carried out a n d d a t a a n a l y z e d using a n one-factor A N O V A with N e u m a n n - K e u l s post h o c test,  (data not shown). T h e r e w e r e  no significant differences  between  g r o u p s . (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).  CT IP: P2 AR Blot: Gs  tn ^ •  w • if  DT »  <P»  ^  «  IP: P2 AR Blot: Gi IP: p2 AR Blot: P2 AR  o 3>  107  FIGURE 21  A . A k t phosphorylation. B. Densitometric a n a l y s i s of Akt phosphorylation. D a t a represent m e a n s ± S E M . D a t a w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. * = significantly different from C a n d 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  CP  CT  D  DP  DT  P K A Activity (pmol A T P incorporated/ min/ mg protein)  828 + 279  52 ± 8*  214 ± 42*  792 ± 281  276 ± 36  454 ± 143  C A M K Activity (pmol A T P incorporated/ min/ mg protein)  611 ± 90  563 ± 94  714 ± 90*  426 ± 118  388 ± 102  325 ± 96  +  P K A a n d C A M K - I I w e r e purified by immunoprecipitation prior to a s s a y . Data represent m e a n s + S E M . D a t a w e r e a n a l y s e d using O n e - W a y A N O V A N e u m a n n K e u l s p o s t - h o c test, * = significantly different from C , different  from  D (p<0.05).  (C=control,  n=5; C P = control  +  with  = significantly perfused  with  metoprolol, n=5; CT=control treated with metoprolol, n=5; D=diabetic, n=5; D P = diabetic p e r f u s e d with metoprolol, n=5; DT=diabetic treated with metoprolol, n=5).  110  activity w e r e u n s u c c e s s f u l . W e therefore u s e d a combination of w e s t e r n blotting, biomarkers a n d indirect m e a s u r e s of N O S activity to determine how metoprolol influences the generation of peroxynitrite (Figures 22-24).  M e t o p r o l o l d e c r e a s e d the e x p r e s s i o n of e N O S in control hearts (Figure 22).  Acute  metoprolol  perfusion  decreased  Akt-  and  PKA-mediated  phosphorylation of e N O S in control hearts at S e r 1177 a n d T h r 4 9 5 respectively, but  the  decrease was  not  sustained  by  chronic  treatment  (Figure  23).  Functionally, N O production, a s indicated by nitrate/ nitrite levels, w a s i n c r e a s e d by metoprolol d e s p i t e the d e c r e a s e in e N O S e x p r e s s i o n (Figure 24). In diabetic hearts, e N O S  e x p r e s s i o n w a s very low but i N O S w a s i n d u c e d . Metoprolol  prevented the induction of i N O S without restoring e N O S (Figure 22).  Akt-mediated  phosphorylation  of e N O S w a s not detected  in diabetic  hearts. P K A - m e d i a t e d phosphorylation of e N O S w a s high, a n d w a s d e c r e a s e d only by chronic metoprolol treatment (Figure 23). N O production, a s indicated by nitrate/ nitrite levels, w a s low in the diabetic heart a n d w a s not altered by metoprolol (Figure 24).  T o t a l protein glutathiolation, a biomarker of N O a n d reactive  nitrogen  s p e c i e s , w a s i n c r e a s e d by a c u t e a n d chronic metoprolol treatment  in control  hearts.  and  In  decreased  diabetic further  hearts, total protein by  chronic  metoprolol  glutathiolation treatment.  was  Total  low, protein  was  tyrosine  nitration, a biomarker of peroxynitrite, w a s u n c h a n g e d by metoprolol in control hearts, a n d w a s u n c h a n g e d in diabetic hearts a s c o m p a r e d to control.. In diabetic hearts, chronic metoprolol treatment p r o d u c e d a m a r k e d d e c r e a s e in tyrosine nitration (Figure 24).  Ill  FIGURE 22  A.  E x p r e s s i o n of  eNOS  and  iNOS.  B.  Densitometric  a n a l y s i s of  eNOS  e x p r e s s i o n . D a t a represent m e a n s ± S E M . Data w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. + = significantly different from C , D and D T , * = significantly different from C a n d 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  D  CT  DT  eNOS iNOS Ponceau  *  ii  mm*  img mti> M  i  to*  HI  mm  #»••"»  'm^m^.  m^mf  B.  C  CT  D  TREATMENT GROUP  DT  mm  113  FIGURE 23  P h o s p h o r y l a t i o n of e N O S at S e r 1177 a n d T h r 4 9 5 . D a t a represent m e a n s ± SEM.  S e r 1177 phosphorylation w a s not detected in diabetic hearts (data not  s h o w n ) . D a t a w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. * = significantly different from other groups, (p<0.05). (C=control, n=5; C P = control p e r f u s e d 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  C  CP  CT  CP  CT  TREATMENT GROUP C  C  CP  CT  CP  (  TREATMENT GROUP  115  FIGURE 24  B i o m a r k e r s of N O a n d R N S . A : T i s s u e nitrate a n d nitrite levels. D a t a represent m e a n s ± S E M . Data w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n K e u l s post h o c test. * = significantly different from C groups, # = significantly different from C , (p<0.05). B: Total protein glutathiolation  m e a s u r e d by Dot  Blotting. D a t a w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. * = significantly different from all groups, # = significantly different from C , (p<0.05). C : Total protein tyrosine nitration m e a s u r e d by Dot Blotting. D a t a w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. * = significantly different from all groups, (p<0.05). (C=control, n=5; C P = control perfused  with  D=diabetic,  metoprolol,  n=5;  n=5;  CT=control  treated  with  metoprolol,  D P = diabetic perfused with metoprolol, n=5;  treated with metoprolol, n=5).  n=5;  DT=diabetic  o  TOTAL PROTEIN TYROSINE NITRATION (Normalised to Total Protein)  73  m > m  z  H O 73  O  o  CO  TOTAL PROTEIN GLUTATHIOLATION (Normalised to Total Protein)  NITRATE + NITRITE LEVELS (nmol/ mg protein) K»  117  VII: CPT-1 Covalent Modifications  P h o s p h o r y l a t i o n of C P T - 1 w a s detected by immunoprecipitation, a n d the total  phosphorylation  state  of  CPT-1 was  i n c r e a s e d by  acute  metoprolol  perfusion in control a n d diabetic hearts. C y s t e i n e nitrosylation, glutathiolation a n d tyrosine  nitration  were  all detected. A c u t e  metoprolol  perfusion  increased  nitrosylation a n d glutathiolation but a b o l i s h e d tyrosine nitration (Figures 2 5 a n d 26).  Having  confirmed  immunoprecipitation, associated  with  we  that  phosphorylation  next  investigated  CPT-1.  Both  PKA  of  CPT-1  whether  and  CAMK  is  detectable  kinases  are  were  found  by  physically to  co-  immunoprecipitate with C P T - 1 (Figures 27-29). Increased phosphorylation  of  A K A P - 1 4 9 w a s a s s o c i a t e d with i n c r e a s e d binding of P K A to C P T - 1 a n d a c o r r e s p o n d i n g d e c r e a s e in A K A P - 1 4 9 binding to C P T - 1 . C o n v e r s e l y , a s A K A P 149 phosphorylation d e c r e a s e d , P K A binding to C P T - 1 d e c r e a s e d a n d  the  association between C P T - 1 and A K A P - 1 4 9 increased.  In  control  hearts,  acute  metoprolol  perfusion  increased  the  phosphorylation of A K A P - 1 4 9 a n d the binding of P K A to C P T - 1 , while A K A P - 1 4 9 binding  to  CPT-1 was  decreased.  T h e s e c h a n g e s persisted with  chronic  treatment. C A M K binding to C P T - 1 w a s not d e c r e a s e d acutely, but chronic treatment with metoprolol did produce a modest d e c r e a s e in C A M K binding to C P T - 1 . In diabetic hearts, a different pattern is o b s e r v e d . A K A P - 1 4 9 binding to CPT-1  was  low  in  diabetic  hearts,  and  metoprolol  i n c r e a s e d its  binding.  M e t o p r o l o l modestly d e c r e a s e d C A M K binding acutely in diabetic hearts. T h i s d e c r e a s e persisted and b e c a m e more m a r k e d following  chronic  metoprolol  treatment.  W h e n P K A a n d C A M K w e r e incubated with isolated mitochondria, both k i n a s e s b o u n d to and phosphorylated C P T - 1 . By contrast, A k t neither b o u n d nor  118  FIGURE 25  C o v a l e n t modifications of C P T - 1 m e a s u r e d by immunoprecipitation.  Samples  underwent immunoprecipitation with c o m b i n e d pan-specific a n t i - p h o s p h o s e r i n e and  anti-phosphothreonine  antibodies,  anti-glutathione  antibodies  or  anti-  nitrosocysteine antibodies, followed by immunoblotting with p a n - s p e c i f i c C P T - 1 antibodies. Densitometric a n a l y s e s of t h e s e data are presented in Figure 2 6 . (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  Densitometric a n a l y s i s of C P T - 1 covalent modifications. D a t a represent m e a n s ± SEM.  D a t a w e r e a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post  h o c 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,  treated with metoprolol, n=5).  n=5;  DT=diabetic  121  122  FIGURE 27  C o - i m m u n o p r e c i p i t a t i o n of P K A a n d A K A P - 1 4 9 with C P T - 1 , a n d phosphorylation 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: A K A P 149 IP: Phosphoserine/threonine Blot: A K A P 149 IP: CPT-1 Blot: P K A  IP: CPT-1 Blot: A K A P 149 IP: Phosphoserine/threonine Blot: A K A P 149 IP: CPT-1 Blot: P K A  124  FIGURE 28  Densitometric a n a l y s i s of the binding of P K A a n d A K A P - 1 4 9 to C P T - 1 , phosphorylation state of A K A P - 1 4 9 .  and  D a t a represent m e a n s ± S E M . D a t a w e r e  a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post h o c test. * = significantly different from C or D, # = significantly different from C a n d C P or D and D P (p<0.05).  126  FIGURE 29  Binding of C A M K - I I to C P T - 1 .  Data represent  means ± S E M .  Data  were  a n a l y z e d using one-factor A N O V A with N e u m a n n - K e u l s post hoc test. * = significantly different from C or D, # = significantly different from C a n d C P or D a n d D P (p<0.05).  127  128  FIGURE 30  Phosphorylation means + S E M . phosphorylation, incubation  of  of C P T - 1 by P K A in isolated mitochondria.  Data  Phosphorylation, k i n a s e binding, densitometric  analysis  malonyl C o A d o s e r e s p o n s e and catalytic activity  isolated  mitochondria  with  P K A . Data  were  represent of  following  analyzed  student's t-test. * = significantly different (p<0.05). n=4 for e a c h intervention.  using  130  FIGURE 31  P h o s p h o r y l a t i o n of C P T - 1 by C A M K in isolated mitochondria. D a t a means ± S E M . phosphorylation, incubation  Phosphorylation, k i n a s e binding, densitometric  analysis  malonyl C o A d o s e r e s p o n s e and catalytic activity  of isolated  mitochondria  with C A M K .  Data w e r e  represent of  following  analyzed  student's t-test. * = significantly different (p<0.05). n=4 for e a c h intervention.  using  131 CONTROL  + CAMK  IP: Phosphoserine/ threonine I Blot: CPT-11  Control  +CAMK  TREATMENT GROUP  132  FIGURE 32  P h o s p h o r y l a t i o n of C P T - 1 by Akt in isolated mitochondria. D a t a represent m e a n s +  SEM.  Phosphorylation,  phosphorylation, incubation  kinase  binding,  densitometric  analysis  malonyl C o A d o s e r e s p o n s e and catalytic activity  of isolated  mitochondria  with C A M K .  Data w e r e  of  following  analyzed  student's t-test. * = significantly different (p<0.05). n=4 for e a c h intervention.  using  CONTROL  + Akt  IP: Phosphoserine/ threonine Blot: CPT-1 IP: CPT-1 Blot: Akt  200  -I  180 Z  O _  160 -  0  20  40  60  80  100  120  140  160  180  MALONYL CoA CONCENTRATION (uM) 1.8 1.6  Control  +Akt  TREATMENT GROUP  200  134  FIGURE 33  Incubation  of  isolated  mitochondria  with  peroxynitrite.  CPT-1  nitrosylation,  glutathiolation a n d tyrosine nitration, a n d C P T - 1 activity following e x p o s u r e to increasing concentrations of peroxynitrite. T h r e e s e p a r a t e e x p e r i m e n t s w e r e run using the s a m e pooled mitochondrial isolate; the n-number is therefore 1. D a t a represent m e a n s .  PEROXYNITRITE CONCENTRATION (uM) 0  0.1  1  10  100  500 1000  mmm  IP: Glutathione 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  p h o s p h o r y l a t e d C P T - 1 , a n d had no effect on C P T - 1 activity or sensitivity. P K A mediated  phosphorylation  of C P T - 1 d e c r e a s e d 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 phosphorylation of C P T 1 i n c r e a s e d the sensitivity of C P T - 1 to malonyl C o A without affecting  activity  (Figures 30-32).  H a v i n g confirmed that functionally-significant phosphorylation of C P T - 1 d o e s occur, w e undertook a study to s e a r c h for the specific phosphorylation sites. C P T - 1 w a s eluted  in the a m m o n i u m  definitively  peptide  identified  by  mass  hydroxide fraction. C P T - 1 w a s  fingerprinting  and  Mascot search.  H o w e v e r , c o v e r a g e of the C P T - 1 B s e q u e n c e w a s poor (< 10%) a n d only o n e phosphorylation site of interest w a s c o v e r e d by the s e a r c h . P h o s p h o r y l a t i o n of that site w a s not detected.  Incubation of isolated mitochondria from control hearts with increasing concentrations of peroxynitrite revealed that b a s a l levels of C P T - 1 nitration a n d glutathiolation  w e r e very low (Figure 33). W h e n 0.1-1  u,M peroxynitrite  was  a d d e d , a m a r k e d i n c r e a s e in glutathiolation occurred which w a s a s s o c i a t e d with very m o d e s t inhibition of C P T - 1 activity. In the range 10-500 uJvl, glutathiolation was  s u s t a i n e d a n d then d e c r e a s e d while tyrosine nitration i n c r e a s e d , a n d a  d o s e - d e p e n d e n t i n c r e a s e in C P T - 1 activity w a s o b s e r v e d . B y contrast, b a s a l levels of c y s t e i n e nitrosylation w e r e high, and peroxynitrite p r o d u c e d a d o s e d e p e n d e n t d e c r e a s e in cysteine nitrosylation. L o s s of cysteine nitrosylation w a s a s s o c i a t e d with an i n c r e a s e in C P T - 1 activity. 1 m M peroxynitrite w a s toxic to the enzyme.  A c u t e metoprolol perfusion i n c r e a s e d glutathiolation in control a n d diabetic hearts, a n d i n c r e a s e d S-nitrosylation in diabetic hearts only. A c u t e metoprolol perfusion a l s o d e c r e a s e d tyrosine nitration of C P T - 1 . With the e x c e p t i o n of tyrosine nitration in control hearts, all c h a n g e s p r o d u c e d by acute metoprolol perfusion w e r e s u s t a i n e d with chronic treatment (Figure 25).  137  DISCUSSION I: Effects of Metoprolol on Cardiac Function and Metabolism  O u r current knowledge of the benefits of p-blockers in diabetic  heart  failure is limited to patients with concurrent diabetes and i s c h e m i a . W e h a v e d e m o n s t r a t e d , for the first time, that metoprolol a l s o ameliorates the c a r d i a c dysfunction p r o d u c e d by diabetic cardiomyopathy. This improvement w a s evident both  from  Starling  curves  generated  by  direct  left  ventricular  pressure  m e a s u r e m e n t s and from m e a s u r e m e n t s of cardiac output and hydraulic p o w e r taken over the c o u r s e of an hour-long  perfusion at constant preload  and  afterload.  Having  confirmed  that  metoprolol  improves  cardiac  function,  we  investigated whether inhibition of fatty acid oxidation could a c c o u n t for this effect. W e b e g a n by investigating the effects of metoprolol on known c a r d i a c fuels. A s e x p e c t e d , metoprolol had no effects on p l a s m a g l u c o s e levels. T o a c c o u n t for the putative ability of p-blockers to s u p p r e s s lipolysis, w e m e a s u r e d circulating p l a s m a lipids. Metoprolol had no effect on p l a s m a free fatty acids, triglycerides or cholesterol. Surprisingly, however, metoprolol attenuated the o b s e r v e d i n c r e a s e in p l a s m a ketone bodies in the diabetic rats. It is not clear why metoprolol would p r o d u c e s u c h a n effect.  T h e rate of k e t o g e n e s i s 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 s y n t h a s e ( H M G C o A s y n t h a s e ) , which c a t a l y s e s the first step in k e t o g e n e s i s . Stimulation of H M G - C o A s y n t h a s e is the underlying m e c h a n i s m by which low insulin levels, starvation and lowc a r b o h y d r a t e / low protein/ high fat diets increase k e t o g e n e s i s (219). p-adrenergic stimulation is known to i n c r e a s e lipolysis in adipocytes. T h e effect is m e d i a t e d partly by hormone-sensitive lipase and partly by a poorly understood alternative  138 pathway (220). p-blockers, acting on the s a m e pathway, m a y d e c r e a s e lipolysis, thereby  d e c r e a s i n g the delivery  of fatty a c i d s to the heart. T h e fact  that  metoprolol treatment had no effect on circulating p l a s m a lipids a r g u e s against a major effect o n lipolysis. However, there are two c o m p o n e n t s to lipolysis: a pulsatile r e l e a s e under the control of the sympathetic nervous s y s t e m a n d a b a s a l r e l e a s e w h i c h is independent of the sympathetic nervous s y s t e m (221); attenuation of the pulsatile r e l e a s e could be sufficient to r e d u c e k e t o g e n e s i s without affecting cholesterol s y n t h e s i s by the l i v e r . Alternatively, the effect c o u l d be mediated by inhibition of C P T - 1 in the liver. It is a l s o p o s s i b l e that metoprolol i n c r e a s e s the peripheral  utilization  of ketones.  However,  present  evidence  s u g g e s t s that ketone utilization by peripheral t i s s u e s is largely unregulated, being determined solely by ketone body supply (222). It is therefore difficult to postulate a  mechanism  by w h i c h  metoprolol  could  directly  i n c r e a s e the  peripheral  utilization of ketones. Further studies a r e n e e d e d to investigate the m e c h a n i s m of this intriguing effect of metoprolol o n p l a s m a ketones.  W e next m e a s u r e d ex vivo fatty acid a n d g l u c o s e m e t a b o l i s m in the heart to explore the acute a n d chronic effects of metoprolol. B y studying the effects of c h r o n i c metoprolol treatment, w e h o p e d to gain insights into the s u s t a i n e d c h a n g e s in c a r d i a c metabolism that a c c o m p a n y the improvement  in c a r d i a c  function. B y studying the rapid effects of acute metoprolol perfusion, w e h o p e d to gain insights into positive events that could o c c u r in vivo immediately following the c o m m e n c e m e n t of treatment, preceding the improvements in function. T h e pattern of c h a n g e s w e o b s e r v e d in our studies w a s c o m p l e x a n d d e p e n d e d on the d i s e a s e state a n d the duration of metoprolol e x p o s u r e .  T h e h e a v y reliance of the diabetic heart on fatty acid oxidation o b s e r v e d in pur studies w a s e x p e c t e d a n d a g r e e s with previous experimental findings in isolated perfused hearts; palmitate oxidation w a s markedly i n c r e a s e d , glycolysis w a s d e c r e a s e d by 5 0 % a n d g l u c o s e oxidation w a s negligable (27; 4 7 ; 6 0 ; 2 2 3 226).  Surprisingly, however, myocardial energetics, a s indicated  by t i s s u e  139  a d e n i n e nucleotide levels, w e r e not altered. C h r o n i c diabetes is k n o w n to be a s s o c i a t e d with a fall in c a r d i a c A T P production (227). It is p o s s i b l e that six w e e k s of d i a b e t e s is too s o o n to o b s e r v e a fall in A T P levels a s this is the timepoint at w h i c h c a r d i a c dysfunction first a p p e a r s . Furthermore, w e did not o b s e r v e any activation of A M P K in the diabetic heart. T h i s is consistent with previous reports (74; 228), and a recent study s u g g e s t e d that A M P K activation is prevented by high circulating a n d tissue lipids (228). H o w e v e r , circulating fatty a c i d s w e r e only mildly elevated in our studies.  C o n t r a r y to expectations, w e o b s e r v e d that chronic metoprolol treatment i n c r e a s e d palmitate oxidation a n d d e c r e a s e d g l u c o s e oxidation in control hearts. H o w e v e r , in diabetic hearts, chronic metoprolol treatment had the e x p e c t e d effect of  lowering  fatty  acid  oxidation  and  increasing g l u c o s e oxidation.  Before  attempting to resolve this apparent paradox, it w a s important to establish w h e t h e r the m a i n target of metoprolol w a s in the fatty acid or the g l u c o s e oxidation pathway. In a preliminary study, w e found that chronic metoprolol treatment had no effect on glycolysis, but i n c r e a s e d coupling between g l u c o s e oxidation a n d glycolysis in the diabetic heart by increasing g l u c o s e oxidation. T o determine whether the o b s e r v e d c h a n g e s in g l u c o s e oxidation w e r e direct, or mediated through the R a n d l e C y c l e by direct c h a n g e s in fatty acid oxidation, w e repeated the perfusions in the a b s e n c e of insulin to reduce g l u c o s e uptake a n d utilization to low levels. W h e n this w a s d o n e , the effect of metoprolol on g l u c o s e oxidation w a s a b o l i s h e d while the effect on palmitate  oxidation w a s p r e s e r v e d . This  strongly s u g g e s t s that fatty acid oxidation is the direct target of metoprolol. It a l s o indicates that the effect of metoprolol is independent of insulin.  Short term perfusion with metoprolol inhibited fatty acid oxidation and p r o d u c e d m a r k e d stimulation of g l u c o s e oxidation in both control a n d diabetic hearts w h i c h w a s a s s o c i a t e d with a d e c r e a s e in lactate production, reflecting a m a r k e d improvement in glycolytic/ g l u c o s e oxidation coupling, a n d an i n c r e a s e in t i s s u e A T P levels. W h e n the perfusions w e r e repeated in the a b s e n c e of insulin,  140  the effect of metoprolol on g l u c o s e oxidation w a s attenuated in control hearts and a b o l i s h e d in diabetic hearts. H o w e v e r , the effect on palmitate oxidation w a s p r e s e r v e d . O n c e a g a i n , this s u g g e s t s that fatty acid oxidation is.the direct target of metoprolol, a n d inhibition of fatty acid oxidation o c c u r s immediately following e x p o s u r e to the drug.  Intriguingly,  we  found  that  acute  metoprolol  perfusion  and  chronic  metoprolol treatment lowered tissue triglyceride levels regardless of w h e t h e r fatty acid oxidation w a s i n c r e a s e d or d e c r e a s e d . T h i s effect cannot be e x p l a i n e d on the b a s i s of fatty acid oxidation c h a n g e s alone. Indeed, inhibition of C P T - 1 by metoprolol in d o g s p r o d u c e d an i n c r e a s e in tissue triglycerides (126); treatment of rats with C P T - 1 inhibitors a l s o i n c r e a s e d tissue triglyceride levels (229). H o w e v e r , our tissue triglyceride m e a s u r e m e n t s w e r e carried out in hearts that had b e e n perfused ex vivo. T h e heart is known to utilize its e n d o g e n o u s triglyceride pool over the c o u r s e of an ex vivo perfusion, w h i c h m a y partly a c c o u n t for the difference. N e v e r t h e l e s s , inhibition of C P T - 1 would be e x p e c t e d to d e c r e a s e the utilization of fatty a c i d s from all s o u r c e s , s o the d e c r e a s e in tissue  triglyceride  levels following  metoprolol  treatment  is  unlikely  to  be  attributable to i n c r e a s e d triglyceride utilization. It is p o s s i b l e that metoprolol d e c r e a s e s the uptake of fatty a c i d s into the c y t o p l a s m . Uptake of long c h a i n fatty a c i d s into the c y t o p l a s m is known to be stimulated by contraction, but the effect is likely to be mediated by A M P K (which w a s unaffected in our studies) a n d P K C isoforms  (230). Triglyceride  levels c a n  be d e c r e a s e d by the  secretion  of  lipoproteins by the heart itself. Indeed, o v e r e x p r e s s i o n of apolipoprotein B (apo B) prevents triglyceride accumulation in'the diabetic heart (231; 232). H o w e v e r , there is presently no e v i d e n c e to s u g g e s t that p-adrenoceptors regulate this process.  T h e p-blocker propanolol w a s reported to induce an i n c r e a s e in C P T - 1 activity  in normal S p r a g u e - D a w l e y rats (233).  Metoprolol, by contrast, w a s  reported to d e c r e a s e C P T - 1 activity in c o n s c i o u s d o g s with m i c r o - e m b o l i s m -  141  induced heart failure (126). In d o g s with p a c i n g - i n d u c e d heart failure, g l u c o s e uptake w a s improved by carvedilol but not metoprolol (234). H o w e v e r , in clinical studies, metoprolol, carvedilol a n d bucindolol (122-124)  have all b e e n s h o w n to  inhibit fatty acid oxidation. T h e wide variation in r e s p o n s e s reported  in the  literature reflects the complexity o b s e r v e d in our own studies in w h i c h the effect of metoprolol on fatty acid oxidation varied according to the length of e x p o s u r e to the drug a n d the d i s e a s e state.  An  increase  in  diastolic  filling  i n c r e a s e s c a r d i a c work  and  oxygen  c o n s u m p t i o n in direct proportion via the Frank-Starling m e c h a n i s m . H o w e v e r , in the n o r m a l heart, A T P supply is maintained at a s t e a d y level r e g a r d l e s s of c a r d i a c work or o x y g e n c o n s u m p t i o n . T h i s m e a n s that c a r d i a c m e t a b o l i s m is driven by c a r d i a c function (51). W h a t is less clear, however, is how c a r d i a c function influences c a r d i a c energy substrate selection.  It is p o s s i b l e that s o m e of  the beneficial effects of metoprolol on c a r d i a c metabolism m a y be attributable  to,  rather than r e s p o n s i b l e for, its effects on cardiac function. W h e n palmitate a n d g l u c o s e oxidation  rates w e r e normalized to c a r d i a c function, the pattern  of  c h a n g e s o b s e r v e d w a s p r e s e r v e d , a n d , in the c a s e of palmitate oxidation, e v e n a c c e n t u a t e d . However, to fully a c c o u n t for effects of function on m e t a b o l i s m , future studies are n e e d e d to investigate whether the effect of metoprolol  is  p r e s e r v e d in isolated c a r d i o m y o c y t e s , in w h i c h the effects of c a r d i a c function a n d the Frank-Starling m e c h a n i s m do not apply.  T o identify the s e c o n d - m e s s e n g e r signalling pathways involved in this response,  we  employed  both  'top  down'  (known  B-adrenoceptor  signalling  pathways) a n d 'bottom up' (known regulatory e n z y m e s of c a r d i a c metabolism) a p p r o a c h e s . W e had established that metoprolol  acts directly on fatty, acid  oxidation. In a preliminary study, w e found that neither diabetes nor chronic metoprolol treatment had any effect on the activities of a c y l - C o A d e h y d r o g e n a s e or citrate s y n t h a s e . B a s e d on t h e s e findings, and previous reports of the effects  142  of p-blockers  on C P T - 1 ,  we  investigated whether the  o b s e r v e d 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 h y p o t h e s i z e d that metoprolol would i n c r e a s e malonyl C o A levels by d e c r e a s i n g the phosphorylation of A C C . However, malonyl C o A levels w e r e d e c r e a s e d by metoprolol in control hearts a n d w e r e u n c h a n g e d in diabetic hearts. T h e m e c h a n i s m of this effect  is unclear, b e c a u s e A C C a n d M C D  e x p r e s s i o n w e r e u n c h a n g e d , a n d w e found no e v i d e n c e of c h a n g e s in A M P K or P K A - m e d i a t e d phosphorylation of A C C .  Dobutamine, a non-selective p-agonist,  w a s previously found to d e c r e a s e 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 a n d M C D , malonyl C o A levels are a l s o d e p e n d e n t on the cytosolic supply of acetyl C o A (236). M o s t of the acetyl C o A in the c a r d i o m y o c y t e is present in the mitochondria (237), and cytosolic  acetyl  C o A is derived  from  peroxisomal  p-oxidation,  citrate  and  acetylcarnitine (238). Intriguingly, a c u t e inhibition of C P T - 1 h a s b e e n s h o w n to p r o d u c e a fall in malonyl C o A levels independent of A C C a n d M C D (238). T h e fall in malonyl C o A levels o b s e r v e d in control hearts c o u l d , therefore, h a v e b e e n secondary  to the inhibition of C P T - 1 . It is unclear w h y s u c h a m e c h a n i s m would  only lower malonyl C o A levels in control hearts. O n e possibility is that fatty acid o x i d a t i o n , rates, a n d therefore the acetyl C o A / C o A ratio, are higher in the diabetic heart, a n d the fall in cytosolic acetyl C o A levels p r o d u c e d by C P T - 1 inhibition in this context m a y not be sufficient to d e c r e a s e malonyl C o A levels. Metoprolol t e n d e d to d e c r e a s e tissue acetyl C o A levels in our studies, but m e a s u r e m e n t s of the cytosolic a n d mitochondrial acetyl C o A pools would be required to confirm t h e s e speculations. O v e r a l l , however, malonyl C o A levels did not correlate with the o b s e r v e d c h a n g e s in fatty acid oxidation. T h e action of metoprolol, therefore, could not be explained solely on the b a s i s of malonyl C o A regulation.  143  W e next investigated the effects of metoprolol on C P T - 1 itself. Metoprolol d e c r e a s e d the m a x i m u m capacity of C P T - 1 activity a s m e a s u r e d in vitro. T h i s effect w a s o b s e r v e d following both short-term  perfusion with metoprolol a n d  chronic metoprolol treatment, and w a s s e e n in both control and diabetic hearts. S i n c e allosteric effects are lost during s a m p l e preparation, t h e s e effects could only be e x p l a i n e d by a d e c r e a s e in C P T - 1 e x p r e s s i o n or a covalent modification. Surprisingly, metoprolol a l s o d e c r e a s e d the sensitivity of C P T - 1 to malonyl C o A . T o our k n o w l e d g e , this is the first study to demonstrate that regulation of C P T - 1 sensitivity o c c u r s in the heart. Long-term c h a n g e s in C P T - 1 catalytic activity a n d malonyl C o A sensitivity w e r e previously believed to o c c u r only in the liver (239; 240).  T a k e n together, the time a n d d i s e a s e - d e p e n d e n t c h a n g e s in fatty acid oxidation c a n be d e s c r i b e d a s follows.  In control  hearts, acute  metoprolol  perfusion c a u s e s malonyl C o A levels to fall. T h e sensitivity of C P T - 1 to malonyl C o A d e c r e a s e s , a n d the activity of C P T - 1 is markedly d e c r e a s e d . 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 a n d the inhibition of C P T - 1 activity is less m a r k e d . Fatty acid oxidation is therefore inhibited following acute e x p o s u r e to the drug, but this effect is lost with time. In diabetic hearts, acute metoprolol perfusion markedly r e d u c e s C P T - 1 activity. With chronic treatment, this reduction is s u s t a i n e d a n d p r o d u c e s inhibition of fatty acid oxidation despite a concomitant d e c r e a s e in malonyl C o A sensitivity. T h e major determinants of the fatty acid oxidation rate are C P T - 1 activity a n d malonyl C o A levels. Using metabolic control a n a l y s i s , it h a s b e e n s h o w n that C P T - 1 only b e c o m e s rate-limiting  w h e n its activity  is  inhibited by approximately 5 0 % (241). C o n s i s t e n t with this o b s e r v a t i o n , in our studies, fatty acid oxidation w a s a l w a y s inhibited if C P T - 1 activity w a s inhibited by approximately 5 0 % . T h e o b s e r v e d c h a n g e s in C P T - 1 sensitivity w o u l d be e x p e c t e d to i n c r e a s e flux through C P T - 1 ; however, they m a y represent a fine tuning m e c h a n i s m of the s y s t e m s i n c e at no point do they hold s w a y 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). T h e net IC50  of malonyl C o A in the heart is intermediate between the high sensitivity of  C P T - 1 B a n d 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 . Catalytic activity a n d malonyl C o A sensitivity could c h a n g e for s e v e r a l r e a s o n s . Firstly, total C P T - 1 e x p r e s s i o n could be altered. S e c o n d l y , isoform switching b e t w e e n C P T - 1 A a n d C P T - 1 B could alter sensitivity; the fetal heart e x p r e s s e s C P T - 1 A , a n d C P T - 1 B e x p r e s s i o n is a s s e r t e d during d e v e l o p m e n t , eventually b e c o m i n g the major isoform (243). H o w e v e r , C P T - 1 isoform switches in the adult heart have not b e e n reported. Finally, two splicing variants of C P T - 1 have b e e n identified in the heart which are predicted to be malonyl C o A - i n s e n s i t i v e (244; 245).  T h e N - a n d C - termini of C P T - 1 both f a c e the cytosol, s e p a r a t e d by a loop region inserted into the outer mitochondrial membrane  m e m b r a n e which contains  s p a n n i n g d o m a i n s . T h e C-terminus  is the  catalytic region,  two and  r e s i d u e s w h i c h regulate malonyl C o A sensitivity h a v e b e e n found within the C terminus, the N-terminus a n d 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, a n d h a s b e e n attributed to regulation by cytoskeletal e l e m e n t s (250), c h a n g e s in the membrane  environment  (251)  and  direct  phosphorylation  of  CPT-1  (177).  Peroxynitrite-mediated nitration of C P T - 1 B has b e e n s h o w n to d e c r e a s e C P T - 1 B catalytic activity following e n d o t o x e m i a in the heart (181). H o w e v e r , no other covalent modifications of C P T - 1 B have b e e n identified. W e therefore p u r s u e d two lines of enquiry. Firstly, w e investigated the effects of chronic metoprolol treatment on C P T - 1 A and C P T - 1 B e x p r e s s i o n . S e c o n d l y , w e investigated the effects of short-term metoprolol perfusion and chronic metoprolol treatment on C P T - 1 B covalent modifications.  145  III: Regulation of CPT-1 Expression  C h r o n i c metoprolol treatment d e c r e a s e d total C P T - 1 e x p r e s s i o n in the heart, a n d this w a s attributable to a d e c r e a s e in C P T - 1 B e x p r e s s i o n . T h e d e c r e a s e w a s only s e e n in diabetic hearts. C P T - 1 A w a s detected at low levels, but its e x p r e s s i o n w a s not altered either by diabetes or by metoprolol. T h e protein e x p r e s s i o n of P P A R - a a n d P G C 1 a r e m a i n e d u n c h a n g e d , a s did the e x p r e s s i o n of the P P A R - a target, P D K - 4 . H o w e v e r , w h e n w e investigated the binding of P G C 1 a to the transcription factors it coactivates a n d to U S F - 2 , w e u n c o v e r e d s o m e intriguing a s s o c i a t i v e c h a n g e s w h i c h s u g g e s t an explanation for the c h a n g e s in C P T - 1 B e x p r e s s i o n .  T h e data obtained to date only establish a s s o c i a t i v e effects. B a s e d on t h e s e d a t a , w e p r o p o s e the following m o d e l . In control hearts, U S F - 2 maintains a constant level of tonic repression of C P T - 1 e x p r e s s i o n , and C P T - 1 e x p r e s s i o n is modulated through the activation of P G C 1 a and P P A R - a . T h i s p r o d u c e s m o d e s t changes increased  in C P T - 1 e x p r e s s i o n . E v e n though by  metoprolol  in control  U S F - 1 a n d 2 e x p r e s s i o n are  hearts, M H C e x p r e s s i o n is  unaffected,  indicating that U S F activity is u n c h a n g e d . In the diabetic heart, U S F e x p r e s s i o n , and U S F activity a s indicated by M H C e x p r e s s i o n , are both d e c r e a s e d in the diabetic heart, a n d metoprolol i n c r e a s e s U S F e x p r e s s i o n a n d activity. T h e result is that tonic r e p r e s s i o n 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 p r o d u c e s m a r k e d c h a n g e s in C P T - 1 e x p r e s s i o n . T h e s e effects are s u m m a r i z e d in s c h e m e 5. T h e role of U S F - 2 could be tested in t r a n s g e n i c m i c e with U S F - 2 knockout targeted to the heart, or alternatively in a conditional knockout m o d e l . Alternatively, U S F - 2 could be s i l e n c e d using a n interfering R N A a p p r o a c h . If U S F - 2 mediates repression of C P T - 1 by metoprolol, the effect w o u l d be attenuated or lost following U S F - 2 knockout a n d m i m i c k e d by U S F - 2 o v e r e x p r e s s i o n . Furthermore, w e would e x p e c t U S F - 2 knockout to be a s s o c i a t e d with a n i n c r e a s e in C P T - 1 e x p r e s s i o n . W e w e r e able to d e m o n s t r a t e  146  that both U S F - 2 a n d M E F - 2 A co-immunoprecipitate with P P A R - a , s u g g e s t i n g that PGC1a, P P A R - a , M E F - 2 A a n d U S F - 2 could form a single transcriptional complex.  In a recent study, M o o r e et al demonstrated that P G C 1a/ dependent  induction  of  CPT-1 was  repressed  by  USF-2  in  MEF2Aisolated  c a r d i o m y o c y t e s (173). W e have d e m o n s t r a t e d that binding of U S F - 2 to PGC1a o c c u r s in the heart with the native proteins, a n d that this is a s s o c i a t e d with functionally significant r e p r e s s i o n of C P T - 1 e x p r e s s i o n : O u r results s u g g e s t that binding of U S F - 2 c a n be induced by the transcriptional activation of U S F - 2 itself, s i n c e U S F - 2 binding to PGC1a a l w a y s c h a n g e d in the s a m e direction a s U S F activity.  U S F is activated  by i n c r e a s e s in electrical stimulation  (176).  It is  therefore likely that activation of U S F by metoprolol is mediated by the i n c r e a s e in electrical stimulation that a c c o m p a n i e s the improvement  in function;  this  e x p l a i n s w h y the effect is only s e e n in the diabetic heart. Function did not c h a n g e in  control  hearts.  However,  the  more  global  regulation  of the  PGC1a  transcriptional c o m p l e x o b s e r v e d in our studies is not explicable solely o n the b a s i s of U S F binding. T h e d e c r e a s e in PGC1a a s s o c i a t i o n with P P A R - a a n d M E F 2 A c o u l d b e a n indirect effect of the acute c h a n g e s in fatty a c i d m e t a b o l i s m . H o w e v e r , it is more likely that active regulation of the c o m p l e x is occurring. P h o s p h o r y l a t i o n of p 3 8 mitogen-activated protein k i n a s e ( M A P K ) i n c r e a s e s both P G C W P P A R - a coactivation a n d d o w n s t r e a m signaling to PGC1a a n d P P A R - a targets (252-255). It h a s b e e n s u g g e s t e d that phosphorylation by p 3 8 M A P K m a y s e r v e to integrate a n d coordinate contractile a n d metabolic g e n e e x p r e s s i o n (173).  Activation of (32-adrenoceptors in the heart h a s b e e n s h o w n to i n c r e a s e signaling through the p 3 8 M A P K pathway (256). It is therefore p o s s i b l e that metoprolol  decreases  p 3 8 phosphorylation  by  blocking  p2-adrenoceptors,  leading to a d e c r e a s e in the a s s o c i a t i o n of PGC1a with its coactivators. Further  147  SCHEME 5  Proposed  m e c h a n i s m of action of metoprolol: chronic metoprolol  treatment  d e c r e a s e s the activation of P G C I o c , possibly by preventing p38 activation, a n d increases  the  repression  of  PGC1a  by  USF-2  (abbreviations:  PPAR-a:  p e r o x i s o m e proliferator activated receptor - a ; P G C 1 a : P P A R - y coactivator 1 a ; M E F = m y o c y t e e n h a n c e r factor; U S F = upstream stimulatory factor, C P T - 1 = carnitine palmitoyltransferase-1.)  METOPROLOL  ft Function  P2 Adrenoceptor  i  1  USF-2  p38  PGGla  MEF-2A  PGCla  I CPT-1  PGCla  PPAR-a  4oo  149 studies are required to investigate the role of s t r e s s - k i n a s e signaling in the regulation of the P G C l a transcriptional complex.  a - M H C e x p r e s s i o n is d e c r e a s e d a s part of the fetal g e n e  program  induction. In the diabetic heart, a fall in both a - M H C and S E R C A e x p r e s s i o n w a s o b s e r v e d , both of which were improved by metoprolol. T h i s improvement in fetal g e n e program e x p r e s s i o n is consistent with what is known about the m e c h a n i s m of action of p-blockers. a - M H C is regulated by U S F ' s , while S E R C A h a s b e e n s h o w n to be induced by M E F - 2 A (257) and is p r o p o s e d to be induced by P P A R <x (89). It is therefore possible that the P G C l a / P P A R a / M E F 2 A / U S F c o m p l e x m a y be able to prevent or reverse the induction of at least s o m e c o m p o n e n t s of the fetal g e n e program. In other words, improvement of g e n e e x p r e s s i o n and modulation  of  cardiac metabolism  could  occur  in parallel  as  a  result  of  modulation by the s a m e transcriptional complex. It is not clear whether D N A binding of P P A R - a or M E F - 2 A  w a s altered by metoprolol treatment;  further  e x p e r i m e n t s are required to m e a s u r e o c c u p a n c y of P P R E and M E F - 2 A  binding  sites.  IV: B-Adrenoceptor Signalling Pathways: Modulation of Kinases and eNOS  C o n s i s t e n t with previous reports, diabetes p r o d u c e d a d e c r e a s e in p i a d r e n o c e p t o r e x p r e s s i o n and a marked increase in p 3 - a d r e n o c e p t o r - e x p r e s s i o n . Metoprolol i n c r e a s e d the e x p r e s s i o n of all 3 adrenoceptor subtypes. P K A activity was  d e c r e a s e d by both acute metoprolol  treatment,  perfusion and chronic  metoprolol  w h e r e a s P I 3 K activity, a s indicated by Akt phosphorylation,  was  i n c r e a s e d by metoprolol only following chronic treatment. C A M K activity w a s not significantly affected by metoprolol. T h e r e w a s no clear shift in p2-adrenoceptor a s s o c i a t i o n with G s or G i ; association with both G-proteins w a s detected. T h e s e results indicate that, in the whole heart, the major acute effect of metoprolol is to d e c r e a s e c l a s s i c a l c A M P / P K A signaling. C h r o n i c treatment with metoprolol, in  150  addition, i n c r e a s e s P I 3 K / Akt signaling, a n d w e s p e c u l a t e that this is primarily d u e to the m a r k e d i n c r e a s e in (33-adrenoceptor-expression.  e N O S is regulated by two main m e c h a n i s m s ; phosphorylation of S e r 1177, mediated by the P I 3 K / Akt pathway, w a s s h o w n to i n c r e a s e e N O S activity in transfected C O S cells (258; 259); phosphorylation of T h r 4 9 5 , mediated by P K A , partially b l o c k s the phosphorylation of S e r 1177 in bovine aortic endothelial cells (260). C a l c i u m - d e p e n d e n t translocation of e N O S from c a v e o l a e in the p l a s m a m e m b r a n e to calmodulin in the cytosol is a l s o a s s o c i a t e d with a n i n c r e a s e in e N O S activity (261; 262). Activation of e N O S by p 3 - a d r e n o c e p t o r s has b e e n s h o w n to be d u e both to an i n c r e a s e in S e r 1177 phosphorylation a n d to translocation from c a v e o l a e , but the importance of t h e s e m e c h a n i s m s is region-specific; in atria, translocation is the predominant m e c h a n i s m w h e r e a s , in the  left  ventricle,  phosphorylation  is  the  predominant  mechanism  (263).  Intriguingly, p 3 - a d r e n o c e p t o r stimulation has a l s o b e e n s h o w n to u n c o u p l e e N O S a n d i n c r e a s e o x y g e n free radical formation (263).  O u r efforts to m e a s u r e N O S activity in the heart w e r e u n s u c c e s s f u l . W e therefore  m e a s u r e d nitrate/  nitrite levels and total protein glutathiolation  as  b i o m a r k e r s of N O a n d physiological reactive nitrogen s p e c i e s ( R N S ) production respectively, a n d correlated t h e s e with c h a n g e s in the phosphorylation  and  e x p r e s s i o n of N O S isoforms. T h e pattern of c h a n g e s p r o d u c e d for both markers w a s the s a m e , with the exception of the D T group, a n d c a n be interpreted a s follows.  In control  hearts, acute  metoprolol  perfusion  increased N O / R N S  production by d e c r e a s i n g the inhibitory phosphorylation of T h r 4 8 6 , a P K A - s i t e . Stimulatory phosphorylation of S e r 1177 by Akt w a s a l s o d e c r e a s e d , but w e s p e c u l a t e that the d e c r e a s e in P K A - m e d i a t e d phosphorylation exerted a greater effect on activity. Following chronic treatment with metoprolol in control hearts, N Q production remained high despite a surprising d e c r e a s e in e N O S e x p r e s s i o n a n d a l o s s of a n y effect on e N O S phosphorylation. W e s p e c u l a t e that this  151  i n c r e a s e in activity could be d u e to i n c r e a s e d e N O S translocation from c a v e o l a e to the cytosol.  In the diabetic heart, N O production is reduced a n d is d e p e n d e n t on i N O S rather than e N O S . i N O S is not regulated by p-adrenoceptors acutely, s o a c u t e perfusion with metoprolol has no effect on N O production.  C h r o n i c metoprolol  treatment prevented the induction of i N O S without restoring e N O S e x p r e s s i o n . T h e net result w a s that chronic treatment with metoprolol had no effect on N O production. H o w e v e r , a s indicated by the fall in glutathiolation, prevention of i N O S induction by chronic metoprolol treatment did d e c r e a s e R N S production. T h e c h a n g e s p r o d u c e d by metoprolol on the phosphorylation of e N O S by Akt did not correlate with the c h a n g e s in Akt-phosphorylation p r o d u c e d by the s a m e treatment; this s u g g e s t s that the elevated Akt signal w a s c o m p a r t m e n t a l i z e d , and that e N O S w a s not its primary target. It is important to note that s e v e r a l cell types would  have  been  cardiomyocytes  and  present  in  endothelial  the cells;  whole we  heart did  homogenate,  including  differentiate  between  not  endothelial a n d cardiomyocyte N O signaling in our studies.  W h e n tyrosine nitration, a marker of peroxynitrite, w a s m e a s u r e d , levels of total protein tyrosine nitration remained constant a s long a s either e N O S or i N O S w e r e present. W h e n e N O S e x p r e s s i o n w a s . l o w and  i N O S w a s absent, total  protein tyrosine nitration fell. T h e s e data indicate that nitrosative stress w a s not significantly i n c r e a s e d in diabetic hearts, although e N O S e x p r e s s i o n w a s low a n d N O levels h a d fallen. T h e fact that prevention of i N O S induction had a m a r k e d effect on R N S production but no effect on N O production s u g g e s t s that i N O S w a s producing predominantly R N S . A s d i s c u s s e d a b o v e , N O h a s b e e n reported to inhibit g l u c o s e utilisation predominantly through inhibition of glycolysis (264). H o w e v e r , chronic metoprolol treatment  had no effect on glycolysis in control  hearts d e s p i t e the fact that it i n c r e a s e d N O production.  152  V: NO/ RNS - Induced Covalent Modifications of CPT-1  In recent y e a r s , there h a s b e e n increasing interest in the ability of N O a n d its a s s o c i a t e d R N S to directly regulate protein function in a similar m a n n e r to phosphorylation. R e s i d u e s that are targeted by N O a n d R N S are cysteine, methionine a n d tyrosine (265; 266). T h e unique redox chemistry of protein thiol g r o u p s confers specificity a n d reversibility to thiol covalent modifications. T h e attachment of N O to thiol groups on critical cysteine r e s i d u e s within a protein, termed S-nitrosylation, is a major m e c h a n i s m by which N O acts a s a signaling m o l e c u l e . Intriguingly, there is a c o n s e n s u s s e q u e n c e , a n a l o g o u s to k i n a s e consensus  s e q u e n c e s , which  confers site specificity on  N O - m e d i a t e d thiol  modifications (267). Furthermore, S-nitrosylation is a reversible reaction, a n d a n u m b e r of e n z y m a t i c a n d n o n - e n z y m a t i c reactions have b e e n identified which can  remove  NO  from  cysteine  thiols  (268-270).  S-nitrosylation  activates  guanylate c y c l a s e , the c l a s s i c a l N O target. T h e list of targets p r o p o s e d to be regulated by S-nitrosylation is growing, a n d , in the heart, includes G A P D H and S E R C A (271).  Reversible  oxidation  or  physiological levels of N O a n d reversible (formation  modifications:  nitrosation  of  thiol  groups  R N S , a n d typically  S-nitrosylation  (addition  is  mediated  p r o d u c e s the of  NO),  by  following  glutathiolation  of mixed disulphides between the thiol group a n d glutathione)  or  oxidation from thiol to sulfenate. A n y of these modifications c a n regulate protein function,  but  glutathiolation  and  S-nitrosylation  have  been  most  frequently  implicated in the regulation of e n z y m e activity (265). Higher levels of R N S induce further oxidation of the sulfenate (one oxygen) to sulfinate (two o x y g e n s ) a n d sulfonate (three o x y g e n s ) . T h i s is toxic, c a u s i n g irreversible loss of function. Glutathiolation, by committing the thiol to an alternate reaction pathway, protects critical thiol r e s i d u e s against irreversible oxidation (265). T h e s e effects s u m m a r i z e d in s c h e m e 7.  are  153  T y r o s i n e nitration  is classically regarded a s an inhibitory  modification.  H o w e v e r , s o m e proteins are activated by tyrosine nitration including c y t o c h r o m e C , fibrinogen a n d P K C (272-275). A s with thiol-modification, tyrosine also exhibits site-specificity (276). T y r o s i n e nitration is frequently  nitration  used as a  biomarker of peroxynitrite (272), a n d w e a l s o u s e d it a s s u c h . P r e v i o u s studies h a v e d e m o n s t r a t e d that incubation of C P T - 1 with continuous peroxynitrite, N O or h y d r o g e n peroxide producing s y s t e m s p r o d u c e s a d e c r e a s e in C P T - 1 activity w h i c h is a s s o c i a t e d with tyrosine nitration  (182). Furthermore,  endotoxemia  p r o d u c e d inhibition a n d nitration of C P T - 1 in suckling rats (181).  C y s t e i n e - s c a n n i n g m u t a g e n e s i s of C P T - 1 revealed that c y s t e i n e 3 0 5 is critical for catalytic activity of the e n z y m e (277). W e therefore tested whether nitrosylation  or glutathiolation  of cysteine r e s i d u e s , or nitration  of  tyrosine  r e s i d u e s , inhibits C P T - 1 activity. T o test the effects of the modifications per s e on CPT-1  activity,  we  incubated  isolated  mitochondria  with  increasing  concentrations of peroxynitrite ranging from 100 n M to 1 m M . At neutral p H , peroxynitrite  is rapidly d e g r a d e d , but e v e n brief e x p o s u r e to peroxynitrite  is  sufficient for it to induce the full range of its target modifications. B e c a u s e of the large a m o u n t of mitochondrial isolate required for t h e s e m e a s u r e m e n t s , three duplicate e x p e r i m e n t s w e r e run on s a m p l e s taken from a single mitochondrial pool, m e a n i n g that the n-number w a s 1. Although the results w e r e consistent, further e x p e r i m e n t s are required to e n a b l e a statistical a n a l y s i s to be carried out.  Peroxynitrite induced glutathiolation at a lower concentration than tyrosine nitration, a n d c a u s e d a d e c r e a s e in S-nitrosylation. D o s e - d e p e n d e n t l o s s of snitrosylation a n d gain of tyrosine nitration w a s a s s o c i a t e d with a d o s e - d e p e n d e n t i n c r e a s e in C P T - 1 activity. T h i s s u g g e s t s that s-nitrosylation is inhibitory a n d tyrosine nitration stimulatory of C P T - 1 activity. 100 n M peroxynitrite p r o d u c e d a m a r k e d i n c r e a s e in glutathiolation but only a slight d e c r e a s e in C P T - 1 activity w h i c h did not prevent activation of the e n z y m e by higher concentrations. W e therefore s p e c u l a t e that glutathiolation of C P T - 1 s e r v e s a s a protective  154  SCHEME 6  N O a n d R N S - m e d i a t e d modifications of thiol residues. Thiol (SH) r e s i d u e s undergo a s e r i e s of reversible modifications in r e s p o n s e to c h a n g e s in the redox potential or e x p o s u r e to physiological levels of reactive nitrogen s p e c i e s or nitric oxide. Oxidation of the thiol to the corresponding sulfenide or the formation of a disulphide b o n d between the thiol a n d glutathione (glutathiolation) are reversible either by c h a n g e s in the equilibrium, or e n z y m a t i c restoration of the thiol group by thiol t r a n s f e r a s e s . Further oxidation of a glutathiolated residue is not p o s s i b l e , s o glutathiolation confers protection against oxidative d a m a g e for a s long a s it persists. H o w e v e r , e x p o s u r e of the thiol group or the sulfenide to pathological levels of reactive nitrogen or o x y g e n s p e c i e s results in the formation of sulfinate a n d then sulfonate; t h e s e are irreversible modifications which result in protein d a m a g e a n d loss of activity. Modified from Figure 2, Klatt a n d L a m a s , 2 0 0 0 (265).  Glutathiolation  R-SSG  R - S N O ^ Z Z * R-SH ^ Z Z T R-SOH S-Nitrosylation  > R-S0 H 2  Unmodified  Oxidation to  Oxidation to  Thiol Group  Sulfenide  Sulfin ate  •  R-SO3H Oxidation to Sulfonate •  Physiological RNS Levels  Pathological RNS Levels  Signal Transduction  Protein damage  Protection Against Irreversible Oxidation  Irreversible Loss of Activity  156  m e c h a n i s m against sulfonation, w h e r e a s S-nitrosylation a n d tyrosine  nitration  regulate the activity in opposite directions. E v e n a concentration of 500  uM  peroxynitrite, w h i c h c a n be toxic to s o m e e n z y m e s , stimulated C P T - 1 activity, indicating that this e n z y m e is well-protected against oxidative d a m a g e .  It w a s surprising, though nitrosylation  of  CPT-1.  As  convenient, that peroxynitrite  discussed above,  increasing  decreased S-  concentrations  of  peroxynitrite promote the glutathiolation of nitrosylated thiol g r o u p s , a n d this is the m o s t likely m e c h a n i s m of the d e c r e a s e w e o b s e r v e d . T h e possibility that tyrosine nitration of C P T - 1 could be stimulatory is surprising, c o n s i d e r i n g that nitration of C P T - 1 w a s a s s o c i a t e d with inhibition of activity following e n d o t o x e m i a (181). H o w e v e r , the authors of that study did not m e a s u r e cysteine oxidation, s o it is p o s s i b l e that sulfination  or sulfonation,  rather than  tyrosine  nitration,  p r o d u c e d inhibition of C P T - 1 activity. In our studies, tyrosine nitration of C P T - 1 a p p e a r e d following physiological d o s e s of peroxynitrite, w h e r e a s loss of activity did not a p p e a r at 1 m M . It is noteworthy that o n e or more of t h e s e covalent modifications w a s present in every treatment group, and at every peroxynitrite concentration. It is therefore p o s s i b l e that all the modifications w e r e inhibitory, but to different d e g r e e s . T h i s possibility could be tested by incubating isolated mitochondria with a reducing agent s u c h a s dithiothreitol a n d a s s a y i n g C P T - 1 activity. A l t h o u g h w e did not m e a s u r e cysteine sulfination or sulfonation, it is likely that this kind of s e v e r e oxidation, rather than tyrosine nitration, c a u s e d the loss of C P T - 1 activity o b s e r v e d at 1 m M peroxynitrite. Similarly, incubation with N O a n d peroxynitrite-producing s y s t e m s would produce continuous nitrosylation a n d , in the latter c a s e , possibly more s e v e r e oxidation which a single brief e x p o s u r e to peroxynitrite would not produce; this could explain w h y continuous N O or peroxynitrite e x p o s u r e is always inhibitory w h e r e a s a single brief e x p o s u r e to peroxynitrite  p r o d u c e s a more c o m p l e x d o s e - r e s p o n s e . T h e physiological  r e l e v a n c e of both types of r e s p o n s e would d e p e n d on the temporal regulation of C P T - 1 e x p o s u r e to N O a n d R N S .  157  We  successfully  detected  cysteine-nitrosylation,  glutathiolation  and  nitration of C P T - 1 in whole heart h o m o g e n a t e s . A c u t e metoprolol perfusion i n c r e a s e d nitrosylation and glutathiolation tyrosine  nitration  nitrosylation  was  in low  both and  control  and  in diabetic hearts, but d e c r e a s e d diabetic  glutathiolation  hearts.  i n c r e a s e d only  In  control  hearts,  following  chronic  treatment. T a k i n g into a c c o u n t the fact that nitrosylation a p p e a r s to be inhibitory, a n d nitration stimulatory, t h e s e data s u g g e s t that metoprolol acutely inhibits C P T 1 activity by increasing cysteine nitrosylation and removing tyrosine nitration of C P T - 1 . H o w e v e r , the m e c h a n i s m of t h e s e effects is not e x p l i c a b l e on the b a s i s of cell-wide c h a n g e s in N O a n d R N S production, b e c a u s e the observed"patterns in s y s t e m i c N O / R N S a n d C P T - 1 covalent modifications did not m a t c h . T h e r e is a mitochondrial isoform of N O S ( m t N O S ) , but N O a n d peroxynitrite p r o d u c e d by m t N O S affect targets within the mitochondrial matrix a n d the inner mitochondrial m e m b r a n e (278).  C P T - 1 predominantly f a c e s the cytosol, s o it is likely that  regulation of C P T - 1 by N O / R N S is mediated by e N O S a n d possibly i N O S . e N O S h a s b e e n p r o p o s e d to translocate to the mitochondria (279; 280); mitochondrial eNOS  translocation could therefore  be a major determinant  of  NO/ RNS  metoprolol  treatment  m e d i a t e d effects on C P T - 1 .  VI: Phosphorylation of CPT-1  Both  acute  metoprolol  perfusion  and  chronic  i n c r e a s e d the total phosphorylation state of C P T - 1 . H o w e v e r , w h e n P K A a n d C A M K - l l - i n t e r a c t i o n s with C P T - 1 w e r e e x a m i n e d in greater detail, a n intruiging pattern e m e r g e d . Firstly, w e found, for the first time, that P K A a n d C A M K - I I physically a s s o c i a t e with C P T - 1 . Furthermore, w e a l s o found that A K A P - 1 4 9 a l s o physically a s s o c i a t e s with C P T - 1 and a p p e a r s to mediate P K A binding.  AKAP's  bind the regulatory subunit of P K A , and activation of P K A o c c u r s following r e l e a s e of the catalytic subunit. T h e P K A antibodies w e u s e d in our studies r e c o g n i z e the catalytic subunit of P K A . Phosphorylation of A K A P - 1 4 9 w a s a l w a y s a s s o c i a t e d with a d e c r e a s e in A K A P - 1 4 9 / C P T - 1 a s s o c i a t i o n a n d an i n c r e a s e in  158  P K A / C P T - 1 binding. C o n v e r s e l y , loss of A K A P - 1 4 9 phosphorylation w a s a l w a y s a s s o c i a t e d with an i n c r e a s e A K A P - 1 4 9 / G P T - 1 binding a n d a d e c r e a s e in P K A / C P T - 1 binding. B a s e d on t h e s e findings, w e p r o p o s e the following s c h e m e . P K A is targeted to the mitochondria by A K A P - 1 4 9 . Phosphorylation 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 i s a s s o c i a t e from C P T - 1 , enabling P K A to bind a n d phosphorylate C P T - 1 . C A M K - I I binding followed a different pattern, indicating that it is regulated by another, as-yet unidentified m e c h a n i s m . T h e r e are other mitochondrial A K A P s which could mediate similar effects a s A K A P - 1 4 9 :  for  e x a m p l e , A K A P - 1 2 1 has a l s o b e e n s h o w n to target P K A to mitochondria (281). CAMK  association  proteins  (KAPs),  by  contrast,  are  not  localized  to  mitochondria, but are found only in the s a r c o p l a s m i c reticulum a n d the n u c l e u s (282). It is therefore unclear how C A M K - I I binding to C P T - 1 would be m e d i a t e d . It is c o n c e i v a b l e that C A M K - I I binds calmodulin a s s o c i a t e d with other proteins w h i c h translocate to the  mitochondria.  F o r e x a m p l e , C A M K - I I is k n o w n  to  a s s o c i a t e with e N O S (283). Alternatively, there m a y be mitochondrial C A M K - I I a s s o c i a t i o n proteins w h i c h have not b e e n identified.  P h o s p h o r y l a t i o n of C P T - 1 B has never b e e n reported, a n d w e s p e c u l a t e that this is b e c a u s e the k i n a s e s involved require other mediators s u c h a s A K A P 149 to be present in order to bind their targets. This w a s our rationale for using isolated mitochondria rather than purified e n z y m e preparations w h i c h are usually u s e d for investigating e n z y m e phosphorylation. W h e n P K A w a s incubated with isolated mitochondria, it bound and phosphorylated C P T - 1 ; the functional effect w a s a d e c r e a s e in C P T - 1 sensitivity without any effect on catalytic activity. W h e n CAMK-II  was  incubated  phosphorylated C P T - 1 ;  with  isolated  mitochondria,  however, the functional effect  it  also  bound  in this c a s e w a s  and an  i n c r e a s e in C P T - 1 sensitivity without any effect on catalytic activity. B y contrast, A k t did not bind or phosphorylate C P T - 1 , and had no effect on the activity or sensitivity of the e n z y m e .  159  The  effects of metoprolol, and the b a s a l state of the s y s t e m , differed  b e t w e e n control a n d diabetic hearts. In control hearts, acute metoprolol perfusion and  chronic metoprolol treatment i n c r e a s e d P K A binding to C P T - 1 , C h r o n i c  metoprolol treatment a l s o modestly d e c r e a s e d C A M K - I I binding to C P T - 1 . In diabetic hearts, P K A - b i n d i n g to C P T - 1 w a s d e c r e a s e d by metoprolol, but C A M K II binding w a s a l s o d e c r e a s e d , a n d , following chronic treatment, the d e c r e a s e in C A M K - I I binding w a s m a r k e d .  T a k e n together with our findings in isolated  mitochondria, t h e s e data explain the sensitivity c h a n g e s p r o d u c e d by metoprolol; Metoprolol  i n c r e a s e s P K A - m e d i a t e d desensitization  in  control  hearts,  and  d e c r e a s e s C A M K - l l - m e d i a t e d sensitization in diabetic hearts. It is noteworthy that the binding of P K A a n d C A M K - I I to C P T - 1 bears no relation to the overall activities of t h e s e k i n a s e s m e a s u r e d in the w h o l e heart. It is the translocation of the k i n a s e s to the mitochondria which is crucial. T h e m e c h a n i s m s by w h i c h ( 3 a d r e n o c e p t o r s might regulate s u c h a translocation p r o c e s s are u n k n o w n a n d require further investigation.  H a v i n g confirmed that functionally significant phosphorylation of C P T - 1 occurs,  we  attempted  to  identify specific phosphorylation  sites on  CPT-1.  C o n s e n s u s sites w e r e identified for P K A , C A M K I a n d C A M K II, s o w e p r o c e e d e d to  u s e L C M S M S to e x a m i n e phosphorylation  of t h e s e sites.  Identifying  phosphorylation events is challenging d u e to the labile nature of the modification and the o v e r w h e l m i n g n u m b e r of peptides generated by tryptic digestion. In order to m a x i m i z e our c h a n c e s of finding the phosphorylation sites, w e performed two purification steps. Firstly, w e purified C P T - 1 by immunoprecipitation. S e c o n d l y , w e performed  phosphorylation enrichment on the tryptic digest. C P T - 1 w a s  a l w a y s found in the highly phosphorylated fraction. H o w e v e r , this w a s d u e to the high concentration of a c i d i c r e s i d u e s in the peptides; peptides rich in a c i d i c r e s i d u e s are a l s o retained by the titanium tips. A l t h o u g h c o v e r a g e of C P T - 1 w a s sufficient to identify the p r e s e n c e of the protein, it w a s not sufficient to e x a m i n e the phosphorylation sites of interest; all the sites except o n e w e r e m i s s e d . S e v e r a l factors  m a y a c c o u n t for this. C P T - 1 a b u n d a n c e in the  whole-cell  160  h o m o g e n a t e m a y h a v e b e e n too low. A l s o , there w e r e relatively few trypsin cutting sites on C P T - 1 . M a n y of the C P T - 1 peptides could have b e e n too large to be eluted from the c o l u m n during the L C M S M S procedure (peptides over 2 5 0 0 D a in m a s s are retained). T h e yield of C P T - 1 could be improved by using mitochondrial fractions, and c o v e r a g e improved by the u s e of a n  alternative  digestion e n z y m e . In addition, the c h a n c e s of detecting a phosphorylation event would be greater if mitochondrial preparations which have b e e n incubated with different k i n a s e s are u s e d b e c a u s e the intensity of the signal is i n c r e a s e d a n d the time b e t w e e n the phosphorylation event and s a m p l e collection c a n be controlled more easily.  VII: Significance of the Present Studies  T h e s e studies have unraveled s e v e r a l m e c h a n i s m s by which metoprolol c a n modulate fatty acid oxidation in the heart. Metoprolol is a b l e to d e c r e a s e malonyl C o A levels in control hearts independent of A C C a n d M C D ; this effect may  be  related  to  cytosolic acetyl  C o A availability.  Acutely,  metoprolol  d e c r e a s e s C P T - 1 activity by increasing S-nitrosylation a n d d e c r e a s i n g tyrosine nitration, a n d d e c r e a s e s C P T - 1 malonyl C o A sensitivity by i n c r e a s i n g P K A mediated or d e c r e a s i n g C A M K - l l - m e d i a t e d phosphorylation. Following chronic treatment, t h e s e covalent modifications are s u s t a i n e d , but C P T - 1 e x p r e s s i o n is a l s o d e c r e a s e d . In control hearts, metoprolol d e c r e a s e s C P T - 1 e x p r e s s i o n by d e c r e a s i n g the a s s o c i a t i o n of P G C 1 a with its coactivators. In diabetic hearts, metoprolol  d e c r e a s e s C P T - 1 e x p r e s s i o n by  increasing the  binding  of  the  r e p r e s s o r U S F - 2 to P G C 1 a . T h e i n c r e a s e in U S F - 2 binding is likely to be p r o d u c e d by the i n c r e a s e in electrical stimulation p r o d u c e d by the improvement in function, w h e r e a s the d e c r e a s e in P G C I c c binding to its coactivators might be related to p 3 8 phosphorylation. W e did not investigate p38 phosphorylation in the present study s o future studies n e e d to investigate this m e c h a n i s m . W h e n combined,  these  mechanisms  result  in  a  complex  pattern  of  metabolic  161  modulation w h i c h is d e p e n d e n t on the duration of e x p o s u r e to metoprolol a n d to the d i s e a s e state.  C h a n g e s in C P T - 1 covalent modifications did not correlate with t i s s u e activity m e a s u r e m e n t s a n d levels of the s e c o n d m e s s e n g e r s w h i c h p r o d u c e d t h e m . A s e p a r a t e C P T - 1 a s s o c i a t e d m i c r o d o m a i n , consisting of A K A P - 1 4 9 , P K A , C A M K - I I a n d possibly e N O S , could exist, and covalent modifications of C P T - 1 m a y be regulated by translocation of the relevant k i n a s e s a n d e N O S into the m i c r o d o m a i n . T h i s C P T - 1 m i c r o d o m a i n is likely to contain other c o m p o n e n t s . It is well e s t a b l i s h e d that A C C a n d M C D localize c l o s e to C P T - 1 . R e c e n t studies s u g g e s t that the fatty acid transporter  C D 3 6 translocates from the p l a s m a  m e m b r a n e to the mitochondria w h e r e it a s s o c i a t e s with C P T - 1 (284). A picture is therefore e m e r g i n g of a s e c o n d a r y m e c h a n i s m for fatty acid oxidation control w h i c h c a n , a s in the c a s e of metoprolol treatment, be u n m a s k e d a n d p r o d u c e meaningful c h a n g e s in fatty acid oxidation rates.  W e c h o s e to investigate metoprolol b e c a u s e it had previously b e e n found to inhibit fatty acid oxidation, is a clinically useful drug a n d b e c a u s e its known range of actions is narrower than that of carvedilol. S e v e r a l other p-blockers h a v e b e e n reported to h a v e effects on m e t a b o l i s m . However, s e v e r a l key q u e s t i o n s remain to be a n s w e r e d . Is this effect a c l a s s effect or is it mediated only by certain p - b l o c k e r s ? A r e the effects mediated by p-adrenoceptors, a n d if s o , what is the contribution of e a c h receptor? C o m p a r a t i v e studies of a wider range of pblockers, a s well a s studies in which p-adrenoceptor e x p r e s s i o n is s i l e n c e d , must be carried out in order to a n s w e r t h e s e questions.  T h e r e h a s b e e n c o n s i d e r a b l e interest in the ability of p-blockers to act a s antioxidants. P r o p a n o l o l , pindolol, metoprolol, atenolol and sotalol w e r e all found to inhibit m e m b r a n e peroxidation, and the effect w a s attributed to c h e m i c a l rather than p h a r m a c o l o g i c a l properties (285-287). Recently, carvedilol h a s b e e n s h o w n to be a potent antioxidant (288). In a recent study, the s c a v e n g i n g activities of a  162  range of p-blockers (atenolol, labetalol, metoprolol, pindolol, propanolol, sotalol, timolol  a n d carvedilol)  compared  (289).  concentrations  In  than  against reactive o x y g e n a n d nitrogen all  cases,  effective  are a c h i e v e d clinically.  scavenging  species were  required  N o n e of the p-blockers  higher could  s c a v e n g e o x y g e n free radicals, but all could s c a v e n g e peroxynitrite. Metoprolol exhibited w e a k antioxidant  effects  in this study; the IC50 of metoprolol o n  peroxynitrite w a s greater than 5 m M . B y contrast, the beneficial effects o b s e r v e d in our study w e r e a c h i e v e d 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 w a s that the o b s e r v e d c h a n g e s in C P T - 1 a n d the factors that m a y regulate it were occurring s i m u l t a n e o u s l y under the v a r i o u s treatment conditions, a n d this m a k e s it challenging to d i s e n t a n g l e the m e c h a n i s m s w h i c h a r e of true regulatory importance. W e attempted to rationalize w h i c h c h a n g e s in C P T - 1 activity, sensitivity a n d malonyl C o A levels w e r e of greater importance b a s e d o n the m e a s u r e d rates of fatty acid oxidation. T h e patterns of activation of nitric oxide s y n t h a s e s , P K A a n d C A M K w e r e different from the patterns of phosphorylation, nitrosylation, glutathiolation a n d nitration o b s e r v e d for C P T - 1 . W e h y p o t h e s i z e d that translocation of k i n a s e s a n d e N O S to the  mitochondria  m a y be a more  important  determinant  of C P T - 1 post-  translational modifications. Further studies are required to clarify this using e N O S knockout m i c e or p h a r m a c o l o g i c a l inhibitors of the relevant k i n a s e s . It will a l s o be e s s e n t i a l to determine whether the o b s e r v e d c h a n g e s in C P T - 1 activity a n d sensitivity p r o d u c e d in vitro by P K A , C A M K a n d peroxynitrite a l s o o c c u r following metoprolol treatment in vivo. T o this e n d , it will be important to m e a s u r e the posttranslational modifications on C P T - 1 directly using m a s s s p e c t r o s c o p y .  W e h a v e s h o w n that metoprolol improves c a r d i a c function in diabetic c a r d i o m y o p a t h y , raising the question a s to whether the drug s h o u l d b e u s e d earlier in diabetic patients. However, there are a number of c o n c e r n s with the administration  of p-blockers to diabetic patients which n e e d to b e w e i g h e d  163  against the benefits of introducing the drug s o early. First and foremost are c o n c e r n s about the effects of p-blockers on g l y c e m i c control. Coadministration of a p-blocker with a thiazide h a s b e e n reported to w o r s e n g l y c e m i c control s i n c e the 1 9 8 0 ' s w h e n the effects of propanolol and hydrochlorothiazide were reported (290). R e c e n t l y , however, the use of p-blockers a s antihypertensive agents has b e e n a s s o c i a t e d with an i n c r e a s e d risk of new-onset diabetes, leading to c o n c e r n about their u s e in this context (291). Hepatic g l u c o s e output is controlled by the p2 adrenoceptor, and b l o c k a d e of this receptor, which d o e s o c c u r with the p i selective agents, d e c r e a s e s hepatic g l u c o s e output and d e l a y s recovery from h y p o g l y c e m i a (292; 293). B l o c k a d e of the s y m p t o m s of h y p o g l y c e m i a by pb l o c k a d e is no longer c o n s i d e r e d to be a problem, b e c a u s e the s y m p t o m s of sweating a n d p a r e s t h e s i a s are p r e s e r v e d , and patients c a n be e d u c a t e d to r e c o g n i z e t h e s e signs (293; 294).  A n o t h e r c o n c e r n with chronic p-blockade is the p r e s e n c e of s u s t a i n e d u n o p p o s e d a1-adrenoceptor stimulation. This is problematic in two situations. Firstly, activation of the sympathetic nervous s y s t e m by h y p o g l y c e m i a i n c r e a s e s u n o p p o s e d a1-adrenoceptor stimulation to the point w h e r e a hypertensive crisis c a n be precipitated (293). S e c o n d l y , u n o p p o s e d a1-adrenoceptor  stimulation  p r o d u c e s peripheral vasoconstriction which could w o r s e n peripheral v a s c u l a r d i s e a s e a n d , by d e c r e a s i n g m u s c l e flow, increase insulin resistance (121; 295). Indeed, u s e of p-blockers in diabetic patients i n c r e a s e s g l u c o s e levels and triglycerides and lowers high density lipoprotein cholesterol levels by d e c r e a s i n g insulin sensitivity (121). N o n e of t h e s e c o n c e r n s are c o n s i d e r e d great e n o u g h to d e n y p-blockers to patients with systolic heart failure b e c a u s e t h e s e drugs are lifesaving in this context. However, the risks and benefits of earlier p-blocker u s e will n e e d to be w e i g h e d carefully and no e v i d e n c e currently exists on which to b a s e t h e s e considerations.  164  W e studied diabetic cardiomyopathy in a model of type 1 diabetes. T h i s w a s a useful m o d e l in which to identify potential m e c h a n i s m s of action, a n d a m o d e l w h i c h d e v e l o p s the cardiomyopathy quickly w a s n e e d e d in order to w i d e n the s c o p e of the study.  H o w e v e r , future studies should e x a m i n e w h e t h e r the  beneficial effects of metoprolol are a l s o o b s e r v e d in m o d e l s of type 2 d i a b e t e s .  In c o n c l u s i o n , our studies demonstrate that metoprolol is a fatty acid oxidation  inhibitor  which  ameliorates  the  cardiac  dysfunction  of  diabetic  c a r d i o m y o p a t h y . A role for p-blocker therapy earlier in this condition m a y be c o n s i d e r e d , but careful study a n d consideration of the risks a n d benefits will be required before s u c h a r e c o m m e n d a t i o n c a n be m a d e .  VIII: CONCLUSIONS  1.  C h r o n i c metoprolol treatment improves c a r d i a c function in the diabetic  heart by inhibiting fatty acid oxidation a n d , through the R a n d l e cycle, increasing g l u c o s e oxidation.  2.  In  control  hearts,  chronic  metoprolol  treatment  i n c r e a s e s fatty  acid  oxidation a n d d e c r e a s e s g l u c o s e oxidation.  3.  C h r o n i c metoprolol treatment selectively d e c r e a s e s the e x p r e s s i o n of  CPT-1 B  by  d e c r e a s i n g the  co-activation  and  increasing  USF-2  mediated  r e p r e s s i o n of P G C 1 a .  4.  Metoprolol d e c r e a s e s malonyl C o A levels independent of A C C a n d M C D  in control hearts only.  5.  C P T - 1 u n d e r g o e s S-nitrosylation by N O and glutathiolation a n d tyrosine  nitration by  peroxynitrite.  C P T - 1 activity  is inhibited  glutathiolation, a n d stimulated by tyrosine nitration.  by S-nitrosylation  and  165  6. CPT-1  A c u t e metoprolol perfusion d e c r e a s e s the activity of C P T - 1 by increasing S-nitrosylation  and  glutathiolation,  and  decreasing  CPT-1  tyrosine  nitration. W i t h the exception of tyrosine nitration in control hearts, t h e s e c h a n g e s persist with chronic treatment.  7.  CPT-1  sensitivity,  is and  phosphorylated CAMK-II,  by  which  P K A , which increases  d e c r e a s e s malonyl CPT-1  sensitivity.  CoA PKA-  phosphorylation of C P T - 1 is mediated by A K A P - 1 4 9 .  8.  A c u t e metoprolol perfusion d e c r e a s e s the sensitivity of C P T - 1 to malonyl  C o A by increasing P K A - m e d i a t e d phosphorylation of C P T - 1 a n d d e c r e a s i n g C A M K - m e d i a t e d phosphorylation of C P T - 1 . T h e c h a n g e s persist with chronic treatment.  166  SCHEME 7  S u m m a r y of the a c u t e effects of metoprolol on malonyl C o A levels, C P T - 1 malonyl C o A sensitivity a n d C P T - 1 activity. Metoprolol lowers malonyl C o A levels in control hearts only. Metoprolol d e c r e a s e s C P T - 1 catalytic activity in both control a n d diabetic g r o u p s by increasing S-nitrosylation and glutathiolation of C P T - 1 a n d reducing tyrosine nitration of C P T - 1 . 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