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Investigation of mechanisms underlying altered alpha-adrenergic receptor-induced contractile responses… Guo, Tianhai 2005

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INVESTIGATION OF MECHANISMS UNDERLYING ALTERED ALPHA-ADRENERGIC  RECEPTOR-INDUCED  CONTRACTILE RESPONSES IN THE STREPTOZOTOCIN-DIABETIC RAT HEART by  TIANHAI GUO B. Med., Sun Yat-sen University of Medical Sciences (Zhongshan University), China, 2002  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR THE DEGREE OF  M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES ,  ^ P H A R M A C E U T I C A L SCIENCES  T H E UNIVERSITY OF BRITISH C O L U M B I A February 2005  © T I A N H A I G U O , 2005  Abstract  Diabetic cardiomyopathy is one o f the major chronic complications i n diabetes mellitus.  Alterations i n the ai-adrenergic receptor (at-AR)-induced positive inotropic  effect (PIE) have been shown i n the diabetic heart, but the results have not been consistent.  The molecular signaling mechanisms underlying the oci-AR-induced P I E i n  the heart, though still under debate, have been suggested to be related to two protein kinases, protein kinase C ( P K C ) and Rho kinase, both o f which consist o f several isoforms. Thus it was hypothesized that specific P K C and/or Rho Kinase isoforms play a role i n the altered a i - A R - i n d u c e d P I E i n the diabetic heart.  The effects o f chronic  streptozotocin-induced diabetes on the basal contractile function and the a i - A R - i n d u c e d PIE, as well as the associated changes i n four P K C isoforms (a, 182, 5 and e) and two Rho kinase isoforms ( R O C K 1 and R O C K 2) i n the rat heart were investigated. Three cardiac contractile parameters, left ventricular developed pressure ( L V D P ) , maximal rate o f contraction  (+dP/dt) and  relaxation (-dP/dt),  were  measured  using the  isolated  Langendorff-perfused isovolumic heart model. In the absence o f adrenergic stimulation, all three contractile parameters were attenuated i n hearts from 6-7 week and 12-15 week diabetic rats.  The selective a i - A R agonist, phenylephrine (PE), produced greater  maximal increase (Rmax) values for L V D P and -dP/dt i n both 6-7 week and 12-15 week diabetic hearts compared to age-matched controls.  It also produced greater pD2 (-log  [ED50]) values for +dP/dt i n both 6-7 week and 12-15 week diabetic hearts, and greater PD2 values for L V D P and -dP/dt in 12-15 week diabetic hearts compared to age-matched controls. In the presence o f the non-isoform-selective P K C inhibitor, chelerythrine ( C E ) , the increase i n all three contractile parameters in response to P E was partially suppressed  ii  in both diabetic and control hearts, and the increase to P E i n L V D P and -dP/dt was not different i n diabetic and control hearts. The non-isoform-selective Rho kinase inhibitors, Y-27632 and H I 152, had no effect on the oii-AR-induced P I E i n either diabetic or control hearts. Western immunoblotting showed that i n the absence o f adrenergic stimulation, the basal levels o f P K C 5 and P K C s i n the particulate fraction o f 12~15 week diabetic hearts were increased compared to control, without any change i n the soluble fraction. There was no change i n the subcellular distribution o f P K C a , P K C / 3 , R O C K 1 or R O C K 2  2 in diabetic hearts compared to control. P E produced a significant increase in levels o f P K C 8 and P K C e i n the particulate fraction o f both 12-15 week diabetic and control hearts, but without a corresponding decrease i n the soluble fraction.  The increase i n  particulate P K C S over its own basal levels i n diabetic hearts was significantly greater than control, whereas the increase i n particulate P K C c over its own basal levels i n diabetic and control hearts was not different.  In the presence o f C E , the PE-induced  increase i n levels o f P K C S and P K C s i n the particulate fraction o f both diabetic and control hearts was completely suppressed.  P E had no effect  on the subcellular  distribution o f P K C a , P K C / 3 , R O C K 1 or R O C K 2 i n either diabetic or control hearts. 2  Activation o f the renin-angiotensin contribute  to  diabetic  cardiomyopathy.  system It  has  ( R A S ) has been been  suggested  to  shown that i n isolated  cardiomyocytes from diabetic rats, P K C s translocated from the soluble to the particulate fraction, while treatment with the angiotensin II type 1 receptor antagonist, L - l 5 8 , 8 0 9 , normalized the alteration i n P K C s .  Thus it was hypothesized that treatment with this  antagonist would improve the attenuated basal contractile function and normalize the enhanced ai-AR-induced P I E , as well as the associated changes in P K C isoforms i n the  in  diabetic heart. The results showed that treatment with L-158,809 significantly improved the basal contractile function o f 12-week diabetic hearts. However, it did not normalize the enhanced ai-AR-induced P I E . This antagonist also had no effect on the basal levels o f P K C 5 and P K C s in the particulate fraction o f diabetic hearts, nor did it affect the P E induced changes i n these two P K C isozymes i n either diabetic or control hearts. The results from the present study suggest a role for P K C S and/or P K C s i n the P I E to a i - A R stimulation i n the heart, and that P K C 5 may contribute to the enhanced aiAR-induced P I E in the diabetic heart. These two P K C isoforms appear to be activated under basal conditions in the diabetic heart. The present study does not support a role for Rho kinase in the ai-AR-induced P I E i n the heart or i n diabetic cardiomyopathy. activation o f R A S contributes to cardiac contractile dysfunction i n diabetes.  The  However,  this study does not support an involvement o f P K C i n this process.  iv  Table of contents Abstract  ii  Table of contents  v  List of figures  x  List of tables  xii  List of abbreviations  xiii  Acknowledgements  xv  1. INTRODUCTION  1  1.1.  Excitation-contraction coupling  1  1.2.  Overview o f regulation o f cardiac contractile function by the autonomic nervous system  1.3.  1.4.  3  Signaling mechanisms underlying p-AR-induced positive inotropic, lusitropic and chronotropic effects  3  Signaling mechanisms underlying the a i - A R - i n d u c e d P I E  5  1.4.1.  Possible role o f protein kinase C ( P K C ) i n the a i - A R - i n d u c e d P I E  1.4.2.  Possible role o f Rho kinase i n the a i - A R - i n d u c e d P I E  10  1.4.3.  Other possible mechanisms underlying the a i - A R - i n d u c e d P I E  12  1.4.4.  Summary o f the signaling mechanisms underlying the a i - A R - i n d u c e d P I E in  1.5.  6  adult rat hearts  13  Diabetes mellitus  14  1.5.1.  Definition o f diabetes mellitus  15  1.5.2.  Diagnostic criteria for diabetes mellitus  16  1.5.3.  Etiological classification o f diabetes mellitus  17  1.5.4.  The two major types - Type 1 and Type 2 diabetes mellitus  18  1.6.  Diabetic cardiomyopathy  19  1.7.  Streptozotocin-diabetic animal models  22  1.8.  Effects o f STZ-diabetes on the ai-AR-induced P I E and on the components o f the signaling pathways coupled to a i - A R  23  v  1.9.  Contribution o f the  activated  renin-angiotensin  system  ( R A S ) to  diabetic  cardiomyopathy  25  1.10. Experimental rationales and hypotheses  28  2. MATERIALS AND METHODS  30  2.1.  30  Chemicals and materials  2.1.1.  Langendorff heart studies  30  2.1.2.  Enhanced chemiluminescence Western blot studies  30  2.2.  Experimental protocols  31  2.2.1.  Animals and blood samples  31  2.2.2.  Measurement o f plasma glucose and insulin  32  2.2.3.  Langendorff heart studies  32  2.2.3.1.  Preliminary experiment to determine the coronary perfusion pressure (CPP) at which the heart developed optimal basal contractile performance and c t i AR-induced P I E  33  2.2.3.2.  Preliminary experiment to confirm that P E selectively activates a - A R s  34  2.2.3.3.  Cardiac Function Study #1: Investigation o f the effects o f diabetes on the c t i AR-mediated P I E  2.2.3.4.  34  Cardiac Function Study #2: Investigation o f the role for P K C i n the oti-ARinduced P I E  35  2.2.3.4.1.  Choice o f P K C inhibitor  35  2.2.3.4.2.  Effect o f chelerythrine on the P I E induced b y /3-AR stimulation i n normal hearts  2.2.3.4.3.  36  Effect o f chelerythrine on basal contractile performance and the a i - A R induced P I E i n hearts from 12-15 week diabetic and age-matched control rats  2.2.3.5.  37  Cardiac Function Study #3: Investigation o f the role for Rho kinase in the a\AR-induced P I E  2.2.3.5.1.  Effect o f Y-27632 on basal contractile performance induced P I E i n normal hearts  38 and the  ai-AR38  vi  2.2.3.5.2.  Effect o f H I 152 on basal contractile performance and the a i - A R - i n d u c e d P I E i n hearts from normal and 12-week diabetic rats  2.2.3.6.  38  Cardiac Function Study #4: Investigation o f effects o f A T i receptor blockade on basal contractile performance and the a i - A R - i n d u c e d P I E i n hearts from 12-week diabetic and age-matched control rats  2.2.4.  Enhanced chemiluminescence Western blot studies  2.2.4.1.  41  Effect o f diabetes, a i - A R stimulation and P K C inhibition on the subcellular distribution o f P K C and Rho kinase isoforms  2.2.4.3.  40  Preliminary experiment to determine the appropriate amount o f protein that should be loaded  2.2.4.2.  39  42  Effect o f L-158,809 treatment on the levels o f P K C 8 , P K C s , R O C K 1 and R O C K 2 i n the particulate fraction i n unstimulated and PE-stimulated hearts from 12-week diabetic and age-matched control rats  2.3.  Statistical analyses  42 42  3. RESULTS  44  3.1.  44  Langendorff heart studies  3.1.1.  Preliminary experiment to determine the C P P at which the heart developed optimal basal contractile performance and a i - A R - i n d u c e d P I E  44  3.1.2.  Preliminary experiment to confirm that P E selectively activates a - A R s  46  3.1.3.  B o d y weight, plasma glucose level and plasma insulin level o f 6-7 week and 12-15 week diabetic and age-matched control rats  3.1.4.  Cardiac Function Study #1: Investigation o f the effects o f diabetes on the ociAR-mediated P I E  3.1.4.1.  3.1.4.3. 3.1.5.  48  Heart weight, coronary perfusion flow rate and ratio o f heart weight / flow rate from 12-15 week diabetic and age-matched control hearts  3.1.4.2.  48  48  Basal contractile performance o f hearts from 6-7 week and 12-15 week diabetic and age-matched control rats  51  Effect o f chronic diabetes on the ai-AR-induced P I E  51  Cardiac Function Study #2: Investigation o f the role for P K C i n the oti-ARinduced P I E  56  vn  3.1.5.1.  Choice of P K C inhibitor  3.1.5.2.  Effect of chelerythrine on the PIE induced by /3-AR stimulation in normal hearts  3.1.5.3.  56  58  Effect of chelerythrine on basal contractile performance and the oii-ARinduced PIE in hearts from 12-15 week diabetic and age-matched control rats  3.1.6.  Cardiac Function Study #3: Investigation of the role for Rho kinase in the aiAR-induced PIE  3.1.6.1.  3.1.6.2.  62  Effect of Y-27632 on basal contractile performance and the ai-AR-induced PIE in normal hearts  62  Effect of H1152 on basal contractile performance and the ai-AR-induced PIE in hearts from normal and 12-week diabetic rats  3.1.7.  60  ....65  Cardiac Function Study #4: Investigation of effects of A T ] receptor blockade on basal contractile performance and the ai-AR-induced PIE in hearts from 12week diabetic and age-matched control rats  3.2. 3.2.1.  Enhanced chemiluminescence Western blot studies  71  Effect of diabetes, a i - A R stimulation and P K C inhibition on the subcellular distribution of P K C and Rho kinase isoforms  3.2.4.  69  Effect of diabetes on the protein levels o f actin in the soluble and the particulate fractions  3.2.3.  69  Preliminary experiment to determine the appropriate amount of protein that should be loaded  3.2.2.  67  72  Effect of L-158,809 treatment on the levels of P K C 5 , P K C s , R O C K 1 and R O C K 2 in the particulate fraction in unstimulated and PE-stimulated hearts from 12-week diabetic and age-matched control rats  80  4. DISCUSSION  86  4.1.  Summary of results  86  4.2.  Choice of cardiac preparation  87  4.3.  Setting of experimental conditions  88  vm  4.4.  Basal contractile function and the ai-AR-mediated P I E are two  independent  processes  89  4.5.  Changes i n the subcellular distribution o f P K C isoforms  90  4.6.  Effect o f diabetes on the subcellular distribution o f cardiac P K C isoforms  92  4.7.  P K C isozymes and diabetic cardiomyopathy  93  4.8.  Diabetes may not have effects on the subcellular distribution o f Rho kinase i n the  4.9.  heart  96  Contribution o f P K C isozymes to the ai-AR-mediated P I E  97  4.10. Contribution o f P K C to the enhanced  a i - A R - m e d i a t e d P I E i n the  heart  diabetic 98  4.11. Activation o f P K C : good or bad?  100  4.12. Role o f Rho kinase i n the ai-AR-mediated P I E  101  4.13. P K C may not be involved i n the improvement o f the impaired basal contractile function o f the diabetic heart b y the inhibition o f R A S  102  4.14. The attenuated basal contractile performance and the enhanced ai-AR-mediated P I E may be two relatively independent alterations i n the diabetic heart  104  4.15. Summary and future directions  105  5. BIBLIOGRAPHY  108  ix  List of figures Figure 1 Possible signaling pathways underlying the ai-AR-mediated P I E  14  Figure 2 Basal L V D P , +dP/dt, -dP/dt and the increase i n these parameters i n response to P E in the C P P 5 0 group and the C P P 7 0 group  45  Figure 3 Increase i n L V D P , +dP/dt and -dP/dt produced b y cumulative addition o f P E i n phentolamine-treated hearts and control hearts  47  Figure 4 B o d y weight, plasma glucose level and plasma insulin level o f the 6-7 week and 12-15 week diabetic and age-matched control rats  49  Figure 5 Heart weight, coronary perfusion flow rate and ratio o f heart weight / flow rate from the 12-15 week diabetic and age-matched control hearts  50  Figure 6 Basal L V D P , +dP/dt and -dP/dt o f 6-7 week and 12-15 week diabetic and agematched control hearts  53  Figure 7 L V D P , +dP/dt and -dP/dt produced b y cumulative addition o f P E i n 6-7 week and 12-15 week diabetic and age-matched control hearts  54  Figure 8 Increase i n L V D P , +dP/dt and -dP/dt produced b y cumulative addition o f P E i n 6-7 week and 12-15 week diabetic and age-matched control hearts  .....55  Figure 9 Basal L V D P , +dP/dt, -dP/dt and the increase i n these parameters i n response to P E in the control, B I M I-treated, RO-treated and CE-treated normal hearts  57  Figure 10 Basal L V D P , +dP/dt, -dP/dt and the increase in these parameters in response to isoproterenol i n the control and CE-treated normal hearts  59  Figure 11 Basal L V D P , +dP/dt, -dP/dt and the increase i n these parameters i n response to P E i n 12-15 week diabetic and control hearts, in the absence or presence o f C E  61  Figure 12 L V D P , +dP/dt, -dP/dt and the increase in these parameters produced by cumulative addition o f P E in control and Y-27632-treated normal hearts  63  Figure 13 Basal L V D P , +dP/dt, -dP/dt and the increase in these parameters in response to P E in control and H I 152-treated hearts from normal and 12-week diabetic rats  66  Figure 14 Basal L V D P , +dP/dt, -dP/dt and the increase in these parameters i n response to P E in hearts from 12-week untreated control, untreated diabetic, L-158,809-treated control and L-158,809-treated diabetic rats  68  x  Figure 15 Densitometric reading vs. amount o f soluble and particulate protein loaded, for incubation with antibodies to P K C 5, P K C a , R O C K 1 or R O C K 2  70  Figure 16 Representative blot o f soluble and particulate actin i n unstimulated and P E stimulated hearts from 12-15 week diabetic and age-matched control rats  71  Figure 17 Relative protein levels and a representative blot o f P K C a in the soluble and particulate fractions o f basal, PE-treated and C E plus PE-treated hearts from 12-15 week diabetic and age-matched control rats  74  Figure 18 Relative protein levels and a representative blot o f P K C p i n the soluble and 2  particulate fractions o f basal, PE-treated and C E plus PE-treated hearts from 12-15 week diabetic and age-matched control rats  75  Figure 19 Relative protein levels and a representative blot o f P K C 8 i n the soluble and particulate fractions o f basal, PE-treated and C E plus PE-treated hearts from 12-15 week diabetic and age-matched control rats  76  Figure 20 Relative protein levels and a representative blot o f P K C e i n the soluble and particulate fractions o f basal, PE-treated and C E plus PE-treated hearts from 12-15 week diabetic and age-matched control rats  77  Figure 21 Relative protein levels and a representative blot of R O C K 1 i n the soluble and particulate fractions o f basal, PE-treated and C E plus PE-treated hearts from 12-15 week diabetic and age-matched control rats  78  Figure 22 Relative protein levels and a representative blot o f R O C K 2 i n the soluble and particulate fractions o f basal, PE-treated and C E plus PE-treated hearts from 12-15 week diabetic and age-matched control rats  79  Figure 23 Relative protein levels and a representative blot o f P K C S i n the particulate fraction o f basal and PE-treated hearts from 12-week diabetic and age-matched control rats with or without L-158,809 treatment  82  Figure 24 Relative protein levels and a representative blot o f P K C s i n the particulate fraction of basal and PE-treated hearts from 12-week diabetic and age-matched control rats with or without L - l 5 8 , 8 0 9 treatment  83  Figure 25 Relative protein levels and a representative blot o f R O C K 1 i n the particulate fraction o f basal and PE-treated hearts from 12-week diabetic and age-matched control rats with or without L-158,809 treatment  84  xi  Figure 26 Relative protein levels and a representative blot o f R O C K 2 i n the particulate fraction o f basal and PE-treated hearts from 12-week diabetic and age-matched control rats with or without L-158,809 treatment  85  List of tables Table 1 Rmax and p D values for the PE-induced P I E i n 6~7 week and 12-15 week 2  diabetic and age-matched control hearts  56  Table 2 Rmax and p D values for the PE-induced P I E i n the control and Y-27632-treated 2  normal hearts  64  xn  List of abbreviations  +dP/dt  M a x i m a l rate o f contraction  -dP/dt  M a x i m a l rate o f relaxation  ACE  Angiotensin-converting enzyme  AEBSF  4-(2-aminoethyl)benzenesulfonylfluoride  ANP  Atrial natriuretic peptide  aPKC  Atypical protein kinase C  AR  Adrenergic receptor, adrenoceptor  A T i receptor  Angiotensin II type 1 receptor  AT2 receptor  Angiotensin II type 2 receptor  BIM  Bisindolylmaleimide  /3-MHC  /3-myosin heavy chain  CE  Chelerythrine  cPKC  Conventional protein kinase C  CPP  Coronary perfusion pressure  CRC  Concentration-response curve  DAG  Diacylglycerol  EDTA  Ethylenediaminetetraacetic acid  EGTA  Ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid  ERK  Extracellular-regulated kinase  GAP  GTPase-activating protein  GEF  Guanine nucleotide exchange factor  GLUT  Glucose transporter  G-protein  Guanine nucleotide-binding protein  G  Stimulatory G-protein  s  HLA-D  Class-II major histocompatability complex  If channel  Hyperpolarization-activated cyclic nucleotide-gated cation channel  n>3  Inositol 1,4,5-triphosphate  JAK  Janus kinase  INK  c-Jun N-terminal kinase  K - H buffer  Krebs-Henseleit buffer  LVDP  Left ventricular developed pressure  MAPK  Mitogen-activated protein kinase  MHC  M y o s i n heavy chain  Xlll  MLC  M y o s i n light chain  MLC1  Essential myosin light chain  MLC2  Regulatory myosin light chain  nPKC  N o v e l protein kinase C  pD  -log[EC ]  2  50  PE  Phenylephrine  PIE  Positive inotropic effect  PIP2  Phosphatidylinositol-4,5-biphosphate  pK  Apparent affinity constant  B  PKC  Protein kinase C  PLC  Phospholipase C  PMA  Phorbol 12-myristate 13-acetate  PS  Phosphatidylserine  PTX  Pertussis toxin  RACK  Receptor for activated C-kinase  RAS  Renin-angiotensin system  Rho kinase  Rho-associated kinase  Rmax  M a x i m a l response  RO  Ro318220, bisindolylmaleimide I X  ROCK  Rho kinase  SDS  Sodium dodecyl sulfate  SR  Sarcoplasmic reticulum  STAT  Signal transducer and activator o f transcription  STZ  Streptozotocin  T  Triiodothyronine  3  TBS  Tris buffer saline  Tn  Troponin  TnC  Ca  Tnl  ATPase inhibitory subunit o f troponin  TnT  Tropomyosin binding subunit o f troponin  Tris  Tris[hydroxymethyl]aminomethane  Tween 20  Polyoxyethylenesorbitan monolaurate  WHO  W o r l d Health Organization  2 +  binding subunit o f troponin  Acknowledgements  W i t h deep pleasure and satisfaction, I would like to express m y greatest appreciation to m y research supervisor, Dr. Kathleen M a c L e o d , for all her patience, guidance and encouragement throughout the course o f m y study.  I would also like to  thank m y supervisory committee, Dr. Stelvio Bandiera, D r . John M c N e i l l , D r . M i k e Allard, D r . Roger Brownsey and D r . Brian Rodrigues, for their valuable suggestions, constructive criticisms and thought-provoking questions.  M y big thanks to m y lab  colleagues Irem Mueed, L i l i Zhang, R u i Zhang, Graham Craig and Guorong L i n for their help and support. Great appreciation to all who have helped me and made m y study an unforgettable experience. Finally I need to express m y deepest gratitude to m y family, who are always there for me.  xv  1  1.1  INTRODUCTION  Excitation-contraction coupling  Cardiomyocytes have the ability to contract and relax, which is the fundamental basis for the heartbeat. The heartbeat is initiated by the generation o f electrical impulses in specialized cells i n the sinoatrial node within the right atrium. The electrical impulse is then transmitted along a conduction system, which is made up o f specialized cardiac cells, to individual cardiomyocytes. The cell membrane o f cardiomyocytes, namely the sarcolemma, contains receptors, ion channels, ion pumps and transporters embedded in its lipid bilayer. This structure allows the cardiomyocyte to communicate with adjacent cardiomyocytes and the extracellular environment. The intracellular mechanisms underlying each contraction-relaxation cycle o f the cardiomyocyte are called excitation-contraction coupling (Korzick 2003). In response to the electrical impulse, voltage-dependent  Na  +  channels i n the sarcolemma open for  several milliseconds and allow N a entry. The sarcolemma is then depolarized, resulting +  in the opening o f the L-type voltage-dependent C a extracellular C a intracellular C a  channels. This permits the entry o f  down a concentration gradient, resulting i n a small increase o f  2 +  2 +  2 +  concentration. This small elevation o f intracellular C a  the binding o f C a  2 +  2 +  levels allows  to the sarcoplasmic reticulum (SR), which is an intracellular C a  pool, resulting in the release o f the stored C a (a process named Ca -induced C a 2+  increase to a much greater extent.  2 +  2 +  release). Ca  via the C a  2 +  2 +  releasing channels on the S R  A s a result, the intracellular C a  2 +  levels  ions subsequently interact with the troponin-  1  tropomyosin complex, the contraction and relaxation unit o f the cardiomyocyte.  The  complex is composed o f tropomyosin and three troponin (Tn) subunits: the C a - b i n d i n g 2+  subunit (TnC), the ATPase inhibitory subunit (Tnl) and the tropomyosin-binding subunit (TnT).  In the relaxed state, the troponin-tropomyosin complex lies between actin and  myosin, preventing the interaction o f the two. When the intracellular C a in response to the electrical impulse, C a  2 +  2 +  levels increase  ions bind to T n C , shifting away the troponin-  tropomyosin complex, allowing the actin molecules to interact with myosin crossbridges. The myosin-bound A T P is then hydrolyzed, providing energy for the persistent interaction o f actin and myosin. A s a result, the myosin cross-bridges make a "rowing" movement along the actin chain and the cardiomyocyte shortens.  The magnitude o f  contractile force generated is dependent on the number o f myosin interacting with actin.  The more intracellular C a  2 +  cross-bridges  ions available to the interaction,  and/or the higher affinity o f the contractile proteins (i.e. troponin-tropomyosin complex, actin and myosin) for C a  2 +  ions, the greater the contractile force can be obtained.  Positive inotropic agents exert their effects either by elevating intracellular C a  2 +  levels or  sensitizing the contractile proteins to C a . During the relaxation o f the cardiomyocyte, 2 +  Ca  2 +  ions dissociate from the troponin-tropomyosin complex as a result o f the decrease i n  intracellular C a  2 +  concentration.  The complex shifts back to its original location,  preventing the interaction between myosin and actin. The decrease in intracellular C a levels is the result o f three mechanisms: C a the SR, the C a  2 +  2 +  ions being taken up by the C a  pumps i n the sarcolemma, and the N a - C a +  2 +  2 +  2 +  pumps on  exchangers in the  sarcolemma.  2  1.2  Overview of regulation of cardiac contractile function by the autonomic nervous system  Cardiac function is mainly regulated by the autonomic nervous systems (i.e. sympathetic and parasympathetic nervous systems), which act v i a adrenergic receptors (adrenoceptors, A R s ) and muscarinic acetylcholine receptors, respectively. Activation o f the sympathetic system results i n elevation i n intracellular cyclic A M P and intracellular Ca  2 +  levels or increase i n myofibrillar C a  2 +  sensitivity, leading to increased cardiac  contractile performance; activation o f the parasympathetic system decreases intracellular cyclic A M P and intracellular C a  2 +  levels, thus attenuates contractile force.  endogenous neurotransmitter o f the sympathetic nervous system is noradrenaline. least nine adrenoceptor subtypes have been identified in mammalian tissues: « I D , ot2A,  ot2B,  P ' P  '  1  tt2C  2 a n c  CCIA,  The At oti , B  * P (Brodde et al. 1999). In the heart, noradrenaline acts on 3  ocr, P i - and p - A R s to produce positive inotropic and chronotropic effects, increasing 2  contractile force and heart rate, resulting i n increased cardiac output (Brodde et al. 1999).  1.3  Signaling mechanisms underlying p-AR-induced positive inotropic, lusitropic and chronotropic effects  There is ample evidence showing that P - A R s are the predominant A R s through which noradrenaline exerts its actions in the heart (Leone et al. 2002). P i - A R s are more prominent than p - A R s , i n terms o f the number o f receptors, and their positive inotropic 2  and positive chronotropic effects (Lohse et al. 2003). Both P i - and p - A R s couple to 2  3  stimulatory small guanine nucleotide-binding proteins ( G proteins). s  kinds o f receptors leads to the activation o f G  s  Activation o f both  and subsequently the activation o f  adenylyl cyclase, resulting i n increased intracellular levels o f cyclic A M P . This leads to the activation o f protein kinase A , which regulates the phosphorylation o f several cellular structures, including the L-type voltage-dependent C a Ca  2 +  2 +  channels i n the sarcolemma, the  releasing channels on the S R , T n l and phospholamban (Leone et al. 2002; Korzick  2003; Lohse et al. 2003).  Phosphorylation o f L-type C a  opening time, allowing more C a induced C a more C a  2 +  2 +  2 +  channels increases their  to enter the cardiomyocyte, and enhancing C a 2 +  release. Phosphorylation of the C a  ions to be released from the SR.  mechanisms is an increase i n intracellular C a  2 +  2 +  releasing channels on the S R allows The overall outcome o f these two  levels, allowing more intracellular C a  2 +  ions available for the interaction between myosin and actin, thus enhancing contractile force (positive inotropic effect). decreased C a  2 +  Phosphorylation o f T n l by protein kinase A results i n  sensitivity of contractile proteins, facilitating the dissociation o f C a  the troponin-tropomyosin complex. the C a  2 +  2 +  from  When unphosphorylated, phospholamban inhibits  pumps on the S R and prevents intracellular C a  2 +  uptake. Once phosphorylated  by protein kinase A , phospholamban is inhibited, resulting i n increased C a  2 +  uptake into  the SR. Phosphorylation o f T n l and phospholamban accelerates the relaxation of the cardiomyocyte and shortens the diastolic phase (positive lusitropic effect). O n the other hand, elevation o f intracellular cyclic A M P levels by the activation o f P r and P2-ARS contributes to positive chronotropic effects.  The heart rate is controlled by specialized  cardiomyocytes that generate electrical impulses i n the sinoatrial node.  In the  sarcolemma, the hyperpolarization-activated cyclic nucleotide-gated cation channels (If  4  channels),  which  are permeable  to both K  and N a , determine  the  speed  of  depolarization, thus controlling heart rate. U p o n the activation o f P i - and p2-ARs, cyclic A M P binds to If channels and accelerates their activation kinetics (Biel et al. 2002), resulting i n increased heart rate (positive chronotropic effect).  1.4  Signaling mechanisms underlying the ai-AR-induced PIE  A s early as the 1960's, a group o f researchers reported an a-AR-mediated positive inotropic effect (PEE) i n rat ventricular strips (Wenzel et al. 1966). Subsequently, similar observations have been confirmed i n a number o f investigations using different cardiac preparations from a variety o f species ( L i et al. 1997), even from humans (Schumann et al. 1978; Bruckner et al. 1984). T w o types o f a - A R s - ct\ and a - have been found so 2  far, but only a i - A R mediates the P I E in heart, because selective a - A R agonists cause no 2  positive inotropy (Williamson et al. 1987; Housmans 1990). Three subtypes o f « i - A R have been identified pharmacologically and through molecular cloning: « I A , a m and a.\n (Hieble et al. 1995). Both (X\A and aie have been proposed to mediate P I E i n adult rat hearts (Williamson et al. 1994a; Williamson et al. 1994b; Deng et al. 1996a). However, a m seems to play little role i n the ai-AR-mediated P I E (Deng et al. 1996b). The mechanisms underlying the ai-AR-mediated P I E in heart have been the target o f intensive investigations during the last decade. several signaling pathways have been proposed.  Though not completely elucidated, a i - A R s couple to their signal  transduction machinery mainly v i a pertussis toxin ( P T X ) - insensitive G-proteins o f the Gq/n family (Graham et al. 1996).  U p o n stimulation, a i - A R s subsequently activate  5  phospholipase C (PLC) and this results in the formation o f inositol 1,4,5-triphosphate and diacylglycerol  (IP3)  ( D A G ) after  biphosphate (PIP2) (Divecha et al. 1995).  the cleavage  o f phosphatidylinositol-4,5-  Both IP3 and D A G are important second  messengers upon ai-AR activation. IP3 binds to specific IP receptors located on the SR, 3  resulting in the release o f stored C a  2 +  into cytosol and elevation o f intracellular C a  2 +  (Divecha et al. 1995). However, in studies using saponin-skinned cardiomyocytes and isolated SR, there was no evidence  showing rP -induced 3  (Movsesian et al. 1985). Whether IP3 can release C a  2 +  Ca  2 +  release from SR  from S R in cardiomyocytes, and  even i f IP3 does modulate the mobilization of intracellular C a , whether this is associated 2 +  with the ai-AR-mediated PEE, is still controversial (Terzic et al. 1993).  1.4.1  Possible role of protein kinase C (PKC) in the ai-AR-induced PIE  D A G activates protein kinase C (PKC), which consists o f a family o f serinethreonine kinases that play a critical role in signal transduction by phosphorylating a variety o f substrates (Terzic et al. 1993). P K C isozymes are classified into three groups based on their structures and the co factors bound upon activation (Mackay et al. 2001): •  The conventional P K C (cPKC) isozymes, comprising a, ft, 182 and 7, which are activated by C a , D A G and phosphatidylserine (PS); 2 +  •  The novel P K C (nPKC) isozymes, comprising 8, e, rj and 6, which do not respond to Ca  •  2 +  but are activated by D A G , PS and unsaturated fatty acids;  The atypical P K C (aPKC) isozymes, comprising £* and T/X, which are unresponsive to Ca  and phorbol esters but can also be activated by PS and unsaturated fatty acids.  6  The presence o f P K C isozymes is species-, tissue- and developmental stagedependent. P K C a , 8 and e are consistently found i n adult rat hearts (Puceat et al. 1994; Mackay et al. 2001; Das 2003). However, P K C f o has not been consistently detected i n adult rat hearts (Rybin et al. 1994; M a c k a y et al. 2001). For the last two decades, translocation o f P K C isoforms has been considered as a hallmark o f their activation.  In their inactive state, P K C isozymes are mainly i n the  soluble fraction o f cells (Nishizuka 1992). U p o n activation, catalytically competent P K C isozymes translocate from the soluble (cytosolic) to the particulate (membrane) fraction of cells where they are thought to bind to "receptors for activated C-kinase" ( R A C K s ) and subsequently interact with their subcellular targets (Kraft et al. 1983; Mochly-Rosen etal. 1990; Mochly-Rosen et al. 1991). The translocation is rapidly followed by a return of P K C back to the soluble fraction (Feng et al. 1998b), a process referred to as reverse translocation and thought to require autophosphorylation (Feng et al. 1998a; Feng et al. 2000).  Based on this attribute, and the available access to isoform-selective P K C  antibodies, the activation o f P K C isozymes can be detected and measured by immunoblot (Western blot) analysis, i n which the protein levels i n soluble and particulate fractions are quantified. Although total P K C activity (i.e. the activity o f all isoforms) can be directly measured using radioactive or non-radioactive methods, it is difficult to measure the activity o f individual isoforms due to the lack o f selective substrates. The measurement of total P K C activity cannot be adopted i n cases when the activity o f a single isoform or several isoforms needs to be determined.  Immunofluorescent studies showed that each  P K C isozyme localizes to unique subcellular sites o f cardiomyocytes upon stimulation (Disatnik et al. 1994; Johnson et al. 1996): PKC/?2 is mainly found i n fibrillar structures  7  in the unstimulated state and is translocated to the perinucleus and cell periphery after stimulation; on the other hand, P K C e translocates from the nucleus and perinucleus to cross-striated structures and cell-cell contacts upon stimulation. In addition, although still under debate, the involvement o f different P K C isoforms has been proposed i n specific physiological and pathological processes i n the heart; moreover, even i f several P K C isozymes participate i n the progression o f the same disease, each isoform may have its own contribution (Puceat et al. 1996; K o y a et al. 1998; Mackay et al. 2001; Das 2003; Sabri et al. 2003). A l l these studies suggest different P K C isozymes may have unique functions, thus the measurement o f the activation o f individual isozymes is necessary. A s a result, i n current investigations, Western blot with isoform-selective antibodies to detect translocation from the soluble to the particulate fraction is still widely used as a measure of P K C isoform activation. It has been well established that a i - A R agonists increase myofibrillar sensitivity to C a  2 +  (Endoh et al. 1988; Puceat et al. 1990). PKC-dependent regulation o f contractile  proteins (mainly the regulatory myosin light chain) may play a role i n myofibrillar C a sensitization.  2 +  Studies using pig (Morano et al. 1985; Morano et al. 1990) and human  (Morano et al. 1988) cardiac preparations suggested that the phosphorylation o f myosin light chain ( M L C ) increases the C a  2 +  sensitivity o f atrial or ventricular strips. T w o types  of M L C have been found in the heart: essential M L C ( M L C 1 ) and regulatory M L C ( M L C 2 ) (Morano 1999). M L C 1 may act as a myosin heavy chain ( M H C ) / actin tether, imposing a load on the myosin cross-bridge. Relieving or weakening o f this tether has been suggested to decrease this load, accelerate cross-bridge cycling and enhance the tension output per cross-bridge, thus increasing contractility (Morano et al. 1995). The  8  elimination o f M L C 2 has been shown to increase the attachment rate constant, leading to an increased number o f force-generating cross-bridges at a given C a and consequently to increased C a  2 +  activation level  sensitivity o f myosin (Brenner 1988).  suggested i n another study that the elimination o f M L C 2 increases C a  2 +  It has been sensitivity o f  isometric tension generation (Hofmann et al. 1990). In all, M L C may act as an inhibitory factor i n the unphosphorylated state; once phosphorylated, its inhibitory effect is relieved, resulting i n increased C a force (Morano 1999).  2 +  sensitivity o f the contractile proteins and cardiac contractile  There is evidence showing the phosphorylation o f M L C 2 is  regulated by P K C . A group o f researchers (Venema et al. 1993a; Venema et al. 1993b) showed that P K C incorporated phosphate stoichiometrically into M L C 2 i n cardiac myofibrils i n vitro; direct activation o f P K C by phorbol 12-myristate 13-acetate ( P M A , a non-isoform-selective P K C activator) induced the phosphorylation o f M L C 2 i n isolated cardiomyocytes.  Besides these direct effects o f P K C on M L C 2 , P K C has also been  proposed to increase the phosphorylation o f M C L 2 v i a an action on M L C kinase. P K C was suggested to enhance the effect o f M L C kinase on force development and ATPase activity (Clement et al. 1992).  M L C 2 phosphorylation by cardiac M L C kinase or by  P K C has been suggested to increase actin-stimulated myosin M g A T P a s e activity (Noland et al. 1993a). These investigations suggest P K C may play a role i n the phosphorylation of M L C 2 , resulting i n increased C a  2 +  sensitivity o f myofibrils (Morano et al. 1985) and  the P I E . It should be noted that some o f these experiments were performed in cell-free systems.  The exact role for P K C in regulating the phosphorylation o f M L C 2 upon the  stimulation o f a i - A R s i n whole cardiomyocytes or i n vivo is still not clear (Puceat et al. 1996).  There are other contractile proteins that have been proposed to be possible  9  substrates for P K C in the heart, including C-protein, T n l and T n T . C-protein has been reported to be phosphorylated by P K C both in vitro (Lim et al.  1985) and in vivo  (Venema et al. 1993a). Since the function o f C-protein in the contractile process is not clear, the significance o f its phosphorylation by P K C remains unresolved. There is ample evidence showing T n l and T n T can be phosphorylated by P K C , resulting in decreased Ca  2 +  sensitivity o f myofilaments and attenuated contractile force (Katoh et al. 1983;  Noland et al.  1991; Clement et al.  1992; Noland et al.  1993b).  In summary, the  regulation o f cardiac contractile proteins by P K C is a complicated process, and the influences on contractile force may be opposite to each other.  However, compared to  other contractile proteins phosphorylated by P K C , the phosphorylation o f M L C 2 may be most prominent in the ai-AR-mediated PIE (Puceat et al. 1996). In studies using intact cardiomyocytes, among the three P K C isoforms ( P K C a , e and 5) that are consistently detected in adult rat hearts, only P K C e and 8 translocate from the soluble fraction to the particulate fraction o f cardiomyocytes upon the activation of a i - A R by phenylephrine (PE, selective a i - A R agonist) (Puceat et al. 1994; Wang et al. 2003).  A s already mentioned, P K C f o has not been consistently detected in adult rat  hearts (Mackay et al. 2001). The effect o f a p A R activation on this isoform is still not clear.  1.4.2  Possible role of Rho kinase in the ai-AR-induced PIE  a i - A R s not only couple to the G / n family, but also couple to the G12/13 family o f q  G-proteins (Katoh et al. 1998). The activation o f a i - A R leads to the activation o f R h o A ,  10  which is a member o f the small G-protein subfamily, Rho. L i k e other small G-proteins, inactive R h o A localizes i n the cytosol and once activated, it translocates from the soluble fraction to the particulate fraction o f cells (Bokoch et al. 1994).  The activation and  inactivation o f R h o A are regulated b y guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively. Inactive R h o A binds to G D P and is activated by exchange o f G D P for G T P . This process is enhanced and regulated b y G E F . The innate GTPase activity o f small G-proteins hydrolyzes bound G T P to G D P , returning them to the inactive state. This GTPase activity is stimulated by G A P (Kaibuchi et al. 1999). The activation o f R h o A results i n the activation o f downstream Rho kinase. Translocation o f Rho kinase isoforms has also been considered as a hallmark o f their activation.  In their inactive state, Rho kinase isozymes mainly localize i n the soluble  fraction o f the cell (Leung et al. 1995); once activated by R h o A , they translocate to the particulate fraction (Sin et al. 1998). Unlike P K C , there are no direct methods for the measurement o f Rho kinase activity. However, there are isoform-selective Rho kinase antibodies commercially available.  A s a result, the activation o f this kinase can be  detected b y Western blot analysis. Two Rho kinase isoforms have been identified: ROK/3 ( R O C K 1) (Leung et al. 1996) and R O K a ( R O C K 2) (Leung et al. 1995). The kinase domains o f R O C K 1 and R O C K 2 are 92% identical and so far there is no evidence that they phosphorylate different substrates or have different functions (Riento et al. 2003). Rho kinase has also been suggested to regulate M L C . proposed  to  promote  the  phosphorylation  Rho kinase has been  o f M L C (Amano et  al.  1996)  by  11  phosphorylating  the  inhibitory subunit  o f myosin phosphatase, resulting i n  the  inactivation o f the latter (Kimura et al. 1996) and increased M L C phosphorylation. Y 27632 (a non-isoform-selective Rho kinase inhibitor) was reported to reduce the P E induced P I E i n rat left ventricular papillary muscles (Andersen et al. 2002) and it also blocked the PE-induced C a 2001).  2 +  sensitization i n isolated cardiomyocytes (Suematsu et al.  It was also suggested that in failing hearts, a i - A R - G - R h o A signaling is upq  regulated, resulting i n increased levels o f activated Rho kinase, increased myofibrillar Ca  2 +  sensitivity and elevated contractility, which might be a compensatory mechanism i n  heart failure (Suematsu et al. 2001).  A l l these investigations suggest a role for Rho  kinase i n the ai-AR-mediated P I E .  1.4.3  Other possible mechanisms underlying the ai-AR-induced PIE  It has been suggested that a i - A R agonists increase intracellular p H by activating the N a - H +  +  exchanger on the cell membrane (Terzic et al. 1993) possibly v i a aiA-AR  (Yokoyama et al. 1998).  There is a correlation between the magnitude o f the a i - A R -  mediated P I E and the degree o f intracellular alkalinization (Vaughan-Jones et al. 1987; Gambassi et al. 1992; Terzic et al. 1992). N a - H e x c h a n g e r blockers inhibit the increase +  +  in PE-induced contractile force i n cardiac myocytes (Otani et al. 1990; Gambassi et al. 1992). Intracellular alkalinization has been suggested to increase the affinity o f T n C for C a , sensitize the actomyosin ATPase to C a 2 +  contractility (Fabiato  et  al.  1978).  2 +  P K C has  and thus result in an increase i n been  proposed  to modulate  the  12  phosphorylation o f N a - H +  +  exchangers in the heart, resulting in a P I E (Wallert et al.  1992; Puceat et al. 1993). a i - A R agonists inhibit voltage-dependent  K  +  current in isolated rat ventricular  cardiomyocytes (Apkon et al. 1988). This results i n the prolongation o f action potential duration, leading to increased C a  2 +  influx (Terzic et al. 1993) and P I E . However, this  mechanism is still controversial because there is evidence suggesting that the a i - A R mediated P I E is associated with a decrease i n action potential duration i n ventricular papillary muscles (Arreola et al. 1994).  1.4.4  Summary of the signaling mechanisms underlying the ori-AR-induced PIE in adult rat hearts  The mechanisms underlying the ai-AR-mediated P I E i n adult rat hearts may have the following components (Figure 1): •  A n increase i n intracellular C a  •  Ca  2 +  2 +  due to the release o f C a  2 +  from S R b y IP3  sensitization o f contractile proteins, which may be related to P K C and/or Rho  kinase activation •  Activation o f the N a - H +  +  exchanger and intracellular alkalization, which may be  related to P K C activation •  Prolongation  of  action  potential  duration  that  increases  Ca  2 +  influx  into  cardiomyocytes.  13  Figure 1  1.5  Possible signaling pathways underlying the ai-AR-mediated PIE.  Diabetes mellitus  The earliest known record o f diabetes mellitus is from 1552 B.C..  The 3rd  Dynasty Egyptian physician Hesy-Ra mentioned frequent urination as a symptom o f the disease in his papyrus.  The first description is usually credited to Arataeus of  Cappadocia in Asia Minor in the first century A D , who gave the disease its name, diabetes (the Greek word for siphon) mellitus (meaning honey).  This was because the  disease was thought to be like sweet water passing through a siphon (Medvei 1993). The discovery o f insulin b y Sir Frederick Banting and Dr. Charles Best and subsequently the  14  application o f insulin i n clinical treatment i n the early 1920s were great medical triumphs of the last century (Ionescu-Tirgoviste 1996). The prevalence o f diabetes is increasing rapidly. In 2000, the W o r l d Health Organization ( W H O ) estimated that over 177 million people have diabetes.  This figure w i l l go up to 300 million (5.4% o f the world  population) by 2025. In Canada, more than two million Canadians have diabetes. B y the end o f the decade, this number is expected to rise to three million (Leiter et al. 2001). Diabetic complications such as heart disease,  stroke, kidney disease, blindness,  amputation and erectile dysfunction are great threats to patients' health. Life expectancy for people with diabetes may be shortened by 5—15 years.  O n the other hand, the  financial burden o f diabetes and its complications on people with the disease and on the Canadian healthcare system is enormous.  A person with diabetes incurs medical costs  that are 2-3 times higher than that o f a person without diabetes (data from the Canadian Diabetes Association, www.diabetes.ca).  1.5.1  Definition of diabetes mellitus  The term diabetes mellitus describes a metabolic disorder o f multiple etiologies characterized by chronic hyperglycemia with disturbances o f carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both ( W H O 1999).  Diabetes may have characteristic symptoms such as unusual thirst  (polydipsia), frequent urination (polyuria), unexplained weight loss, extreme fatigue or lack o f energy, blurred vision and slow healing. In more severe situation, ketoacidosis or a non-ketotic hyperosmolar state may occur, which lead to stupor, coma, and even death.  15  The long-term effects o f diabetes include the progressive development o f cardiovascular, peripheral vascular and cerebrovascular complications, resulting i n cardiomyopathy, atherosclerosis, heart failure, nephropathy, skin ulcers, retinopathy and stroke, etc. The destruction o f pancreatic P-cells with consequent insulin deficiency and tissue resistance to insulin action are the two pathogenetic processes o f diabetes.  The abnormalities o f  carbohydrate, fat and insulin metabolism are the consequence o f lack o f insulin and/or insensitivity o f target tissues to insulin.  1.5.2  Diagnostic criteria for diabetes mellitus  In  1997,  The  International  Expert  Committee  On  The  Diagnosis A n d  Classification O f Diabetes Mellitus revised the diagnostic criteria o f diabetes, which were based  on  the  1979  publication  of  the  National  Diabetes  Data  Group  (NationalDiabetesDataGroup 1979) and subsequent W H O study group ( W H O 1985). The following diagnostic criteria are recommended by W H O for the diagnosis o f diabetes: •  Fasting plasma glucose > 7.0 mM/1  •  Two-hour postprandial plasma glucose > 11.1 mM/1 during an oral glucose tolerance test with an oral glucose load o f 75g  •  For clinical purposes, the diagnosis o f diabetes mellitus should be confirmed by repeating the test on another day  16  1.5.3  Etiological classification of diabetes mellitus  W H O has also revised the etiological classification o f diabetes mellitus. The new classification ( W H O 1999) contains four categories: •  Type 1 diabetes mellitus (P-cell destruction, usually leading to absolute insulin deficiency) o  Autoimmune (major type, with identified autoimmune disorders that lead to P-cell destruction)  o •  Idiopathic (rarely seen, without evidence o f autoimmune disorders)  Type 2 diabetes mellitus (may range from predominantly insulin resistance with relative insulin deficiency to a predominantly secretory defect with or without insulin resistance)  •  •  Other specific types o f diabetes mellitus o  Genetic defects o f beta-cell function  o  Genetic defects i n insulin action  o  Diseases o f the exocrine pancreas  o  Endocrinopathies  o  Drug- or chemical-induced  o  Infections  o  Uncommon forms o f immune-mediated diabetes  o  Other genetic syndromes sometimes associated diabetes  Gestational diabetes (glucose intolerance during pregnancy; i n most cases, this disorder is normalized after labor)  17  1.5.4  The two major types - Type 1 and Type 2 diabetes mellitus  Type 1 diabetes mellitus accounts for about 10% o f all diabetic cases.  It can  occur at any age, but mostly starts to develop i n youth. Three mechanisms have been proposed i n the pathogenesis o f Type 1 diabetes: genetic susceptibility, autoimmunity and environmental factors (Krolewski et al. 1987; Rossini et al. 1988).  The genetic  susceptibility, mainly a defect i n the allele o f the class-II major histocompatability complex ( H L A - D ) , predisposes the individual to dysfunctioning o f the antigen specific cytotoxic T-lymphocytes, resulting i n a slow and progressive immunological attack on pancreatic (3-cells. This process is augmented by cytokine release from macrophages and N K - c e l l s . The autoimmunity can occur spontaneously, or can be triggered by a variety o f environmental factors, such as viruses and chemicals. These three mechanisms interact with each other, leading to progressive destruction o f P-cells.  Symptomatic diabetes  mellitus and insulin dependence occur only when the P-cell mass is reduced to 10% o f normal.  Exogenous insulin injection is the only effective treatment for patients with  Type 1 diabetes mellitus, which was identified as "insulin-dependent diabetes mellitus" in an earlier classification. Type 2 diabetes accounts for nearly 90% o f all cases, and usually occurs over the age o f 35. Obesity is prominent among 5 0 - 9 0 % o f all Type 2 diabetic patients (Valle 1997). The pathogenesis o f Type 2 diabetes mellitus (in the earlier classification, noninsulin-dependent diabetes mellitus) is poorly understood. The primary defect is hepatic and peripheral insulin resistance.  Subsequently, a compensatory hyperinsulinemia  18  occurs. W i t h time, pancreatic P-cells fail to secret sufficient insulin and to overcome the insulin resistance (Valle 1997). A s a result, hyperglycemia and Type 2 diabetes occur. Patients  with this  type  o f diabetes have  a  strong  genetic  predisposition,  and  environmental factors, such as imbalance o f nutrition and lack o f exercise, also contribute to the onset o f the disease.  Several cellular mechanisms have been proposed i n insulin  receptors and intracellular signaling pathways that may contribute to insulin resistance (Valle 1997).  Multiple treatment choices, including changing life style and diet,  medications and exogenous insulin supplementation, can be applied to patients with Type 2 diabetes. The exact treatment plan may vary from individual to individual. However, in the final stages o f Type 2 diabetes, most patients require insulin injection, since endogenous insulin decreases to a very low level due to the dysfunction o f pancreatic Pcells.  1.6  Diabetic cardiomyopathy  Diabetes mellitus can result i n a host o f acute and chronic complications. The most dangerous acute complication is ketoacidosis, a state o f absolute or relative insulin deficiency aggravated  by ensuing hyperglycemia (plasma glucose > 300 mg/dL),  dehydration, and acidosis (plasma p H < 7.30). Ketoacidosis often occurs i n severe Type 1 diabetic cases.  The chronic complications are mainly macro- and microvascular  diseases. Macrovascular diseases may occur in peripheral vessels leading to gangrene; in cerebral vessels leading to intracerebral bleeding and stroke; i n cardiac vessels leading to coronary artery diseases, atherosclerosis and myocardial infarction (Uccella et al. 1991).  19  Microvascular diseases result i n retinopathy and nephropathy.  Autonomic neuropathy,  another kind o f chronic diabetic complication, may contribute to diabetic hypertension. The diabetes-induced cardiac muscle disease, diabetic cardiomyopathy, is one o f the chronic diabetic complications.  Cardiovascular complications are responsible for  about 80% o f deaths among diabetic patients (Kannel et al. 1979; Valle 1997), most o f which has been attributed to coronary artery disease. However, diabetic cardiomyopathy has gained intensive focus since the 1970's. It was first recognized i n a study on diabetic patients with heart failure, but without evidence o f vascular diseases, valvular heart diseases or congenital heart diseases (Rubier et al. 1972).  Diabetic cardiomyopathy  refers to a disease process that affects the myocardium in diabetic patients, causing a wide range o f structural abnormalities, eventually leading to left ventricular hypertrophy and diastolic and systolic dysfunction or a combination o f both. This disease can occur and be detected without the presence o f any vascular diseases (Feuvray 2004). The cellular mechanisms underlying diabetic cardiomyopathy, though not yet completely elucidated, have been investigated i n a host o f studies. Hyperglycemia leads to the excess formation o f advanced glycation end-products and mitochondrial reactive oxygen species, resulting in myocardial collagen deposition and fibrosis (Singh et al. 2001). Hyperglycemia also leads to advanced glycation o f the C a  2 +  pumps on the SR,  resulting i n the inactivation o f the latter and prolongation o f cardiac relaxation (Bidasee et al. 2004). accumulation  Hyperlipidaemia results i n increased P-oxidation and mitochondrial o f long-chain acyl  carnitines,  phosphorylation (Stanley et al. 1997).  leading to  uncoupling o f oxidative  Hypoinsulinemia decreases the utilization o f  glucose i n cardiomyocytes, resulting i n enhanced  utilization o f fatty  acids and  20  perturbation o f myocardial bioenegetics (Rodrigues et al. 1998).  These lead to the  dysfunction o f contraction / relaxation coupling and apoptosis o f cardiomyocytes (Zhou et al. 2000). In patients with hyperglycemia, aldosterone has been suggested to mediate cardiac fibrosis through the stimulation o f myofibroblast growth (Neumann et al. 2002). In diabetes, the renin-angiotensin system is activated (Fein et al. 1985), leading to cardiac hypertrophy and apoptosis (Leri et al. 1999; Fiordaliso et al. 2000; Kajstura et al. 2001). The cardiac expression o f vascular endothelial growth factor and its receptors is decreased i n diabetes, resulting i n inadequate angiogenic response to ischemia and poor collateral formation, thus the patients may have an increased propensity to infarction due to a reduced reparative response (Chou et al. 2002). m R N A levels o f the N a - K  In diabetic animals, depressed  ATPase and increased m R N A levels o f the N a / C a  2 +  exchanger have been found i n cardiomyocytes, which may result i n intracellular C a  2 +  +  +  +  overload and contractile deficiency (Golfman et al. 1998).  The m R N A levels and  sarcolemmal protein density o f the K channels (Kv2.1, K v 4 . 2 and Kv4.3) i n ventricular +  myocytes from diabetic animals are decreased, which may lead to cardiac arrhythmia (Qin et al. 2001). cardiomyopathy.  Cardiac autonomic neuropathy has been suggested in diabetic  Patients with Type 1 diabetes exhibit cardiac autonomic neuropathy  and abnormal diastolic filling (Kahn et al. 1986).  Sympathetic dysfunction has been  related to both systolic and diastolic dysfunction in Type 2 diabetes (Annonu et al. 2001). In all, a range o f molecular changes may underlie the development  o f diabetic  cardiomyopathy.  21  1.7  Streptozotocin-diabetic animal models  Diabetic animal models are widely used as they provide a means to understand and explore the etiology, pathogenesis and treatment strategies i n human diabetes.  A  variety o f Type 1 and Type 2 diabetic animal models have been developed (Rodrigues et al. 1999b). Genetic Type 1 diabetic models include the diabetic biobreeding ( B B ) rats and the  non-obese  diabetic  ( N O D ) mouse.  In these models,  diabetes  spontaneously, and the animals depend on exogenous insulin for survival.  occurs  Chemically  induced Type 1 models include alloxan-induced and streptozotocin (STZ)-induced diabetic rats. Both o f the chemicals specifically destroy pancreatic p-cells. Since these models closely reproduce the lesions i n human Type 1 diabetes, and they produce permanent and stable diabetes, they are o f specific interest in diabetic research. S T Z has replaced alloxan as the principal chemical to induce experimental diabetes because o f its greater selectivity for p-cells, lower mortality rate and longer half-life (Rodrigues et al. 1999b). Genetic Type 2 diabetic models include db/db mice, fa/fa diabetic Zucker rats, etc. These models demonstrate some manifestations o f human Type 2 diabetes, such as hyperglycemia, hyperinsulinemia and obesity. There are also some chemically induced Type 2 diabetic models. Although genetic models provide the possibility to investigate the genetic predisposition o f diabetes and the influence o f environmental factors on the pathogenesis o f the disease, the use o f these models is limited due to their high cost. Chemically induced models are less expensive. Moreover, their duration o f diabetes and the severity o f the disease can be better controlled. A s a result, these models, especially S T Z models, have been used widely in diabetic research.  22  STZ-diabetic rats exhibit characteristic symptoms similar to human Type 1 diabetes, such as ploydipsia, polyphagia (increased food intake) and weight loss. Hyperglycemia, hypoinsulinemia and increased levels of plasma lipids also occur in these models (Junod et al. 1969). The etiology of diabetic cardiomyopathy in STZ-diabetic rats appears to be similar to that in human Type 1 diabetes. The subcellular changes in the sarcolemma, the mitochondria, the SR and the contractile proteins are found in STZdiabetic hearts (Rodrigues et al. 1999a).  These eventually lead to left ventricular  hypertrophy and diastolic and systolic dysfunction (Tahiliani et al. 1983; Mihm et al. 2001). Depressed responses to noradrenaline, which subsequently result in attenuated cardiac contractile reserve, is one major type of cardiac dysfunction in STZ-diabetic animals (Gotzsche 1983a; Gotzsche 1983b; Smith et al. 1984). As mentioned previously, (3-AR is the predominant adrenergic receptor through which noradrenaline exerts its actions on cardiac muscle (Leone et al. 2002).  As a result, the depressed cardiac  response to noradrenaline in these animals is possibly due to defects in the /3-ARmediated signaling cascade.  Several studies have been reported in support of this  hypothesis (Gotzsche 1983a; Gotzsche 1983b; Smith et al. 1984). On the other hand, alterations in the ai-AR-mediated PIE in the STZ-diabetic heart have also been wellrecognized.  1.8  Effects of STZ-diabetes on the <Xi-AR-induced PIE and on the components of the signaling pathways coupled to ai-AR  23  The ai-AR-mediated P I E in right ventricular strips (Wald et al. 1988; Y u et al. 1991) and working hearts (Heijnis et al. 1992) isolated from STZ-induced diabetic rats was enhanced.  Similar findings were shown i n atria (Canga et al. 1986; Jackson et al.  1986; Durante et al. 1989; B r o w n et al. 1994) and left ventricular papillary muscles (Brown et al. 1994).  These findings are intriguing because when jS-AR-mediated  responses in diabetic heart are diminished, the augmented a.\ responses have been proposed to help compensate to maintain cardiac performance (Corr et al. 1981; M i l l i g a n et al. 1994; Beaulieu et al. 1997; Skomedal et al. 1997). However, not all studies are i n agreement with these findings, as i n some investigations the ai-AR-mediated P I E was reported to be attenuated i n myocardial preparations from STZ-diabetic rats (Heyliger et al. 1982; Williams et al. 1983; Sunagawa et al. 1987). These discrepancies may be due to differences i n the duration o f diabetes or i n experimental conditions between studies. Besides the above functional studies, a number o f investigations have suggested diabetes-induced changes i n the components o f the signaling pathways coupled to a i - A R in the heart. Binding studies have consistently found that in diabetic cardiomyocytes, the number o f oii-AR-binding sites is reduced (Heyliger et al. 1982; W a l d et al. 1988). This is associated with no change (Heyliger et al. 1982; Tanaka et al. 1992) or an increase (Wald et al. 1988) i n their affinity constants.  The enhanced ai-AR-mediated P I E in the  diabetic heart was associated with increased IP3 production (Xiang et al. suggesting that ai-AR-mediated stimulation o f P L C is enhanced.  1991),  However, opposite  evidence suggested that I P production in response to « i - A R stimulation was decreased in 3  diabetic cardiomyocytes (Tanaka et al. 1992; Tanaka et al. 1993). This discrepancy may be due to the different rat strains and different experimental protocols.  A number o f  24  investigations have indicated that STZ-diabetes affects P K C activity or levels o f P K C isoforms i n the particulate fractions i n rat hearts, but the results are far from consistent. For instance, a high basal P K C activity was found i n diabetic rat hearts, associated with a decrease in cell surface a i - A R density and reduced IP3 production i n response to a p A R stimulation (Tanaka et al. 1992).  Increased activity o f particulate P K C along with  elevated particulate levels o f PKCp2, and increased intracellular levels o f D A G were found i n diabetic hearts, without any change i n particulate levels o f P K C a (Inoguchi et al. 1992). In another study, particulate P K C s was increased i n diabetic cardiomyocytes while no change was found i n PKC8, however P K C (5 was not even detected (Malhotra et al. 1997).  O n the other hand, L i u et al. (1999) reported that the total (soluble plus  particulate) levels o f P K C a , P and s were increased i n STZ-diabetic rat hearts, but the particulate levels o f these isoforms were not changed ( L i u et al. 1999). In another study, the total level o f P K C a was increased, accompanied by reduced total levels o f P K C s and no change i n P K C P i , P2 and 5 (Kang et al. 1999). These varied results may be due to the difference in strains o f rats, duration o f diabetes, cardiac tissues and experimental conditions. Though there are discrepancies i n these studies, they still suggest there may be changes i n P K C activity and particulate levels o f P K C isoforms in diabetic hearts. However, whether diabetes affects ai-AR-induced changes i n P K C i n rat hearts has not been established.  1.9  Contribution of the activated renin-angiotensin system ( R A S ) to diabetic cardiomyopathy  25  The R A S is classically viewed as an enzymatic protein cascade (Volpe et al. 2002). The first component o f the system is angiotensinogen, which forms angiotensin I in the presence o f renin, an enzyme synthesized and released from kidney. Angiotensin I is subsequently transformed to angiotensin II by the action o f angiotensin-converting enzyme  (ACE).  Angiotensin II is the terminal biologic effector o f the system. Under  physiological conditions, angiotensin II plays an important role i n regulating the cardiovascular system to maintain homeostasis.  It also participates in the regulation o f  salt and water balance and cellular growth (Volpe et al. 2002). Angiotensin II, which is a biologically active peptide, can bind to two receptor subtypes, type 1 ( A T i ) and type 2 (AT2) receptors (Bumpus et al. 1991). The principal actions o f R A S in heart, vessels, kidney, brain, and other tissues and organs are mainly mediated b y A T j receptors (Volpe et al. 2002). A number o f investigations suggest that the R A S is altered i n STZ-diabetic animal models. Plasma renin concentration (Ubeda et al. 1988) and activity (Funakawa et al. 1983) are reduced, which may be due to hyalinization o f the renin secreting structure i n the kidney (Nakamura et al. 1978) and a reduction i n renal prostaglandin production (Funakawa et al. 1983). Circulating levels o f A C E are increased i n S T Z models, but the mechanisms for this phenomenon are not clear (Valentovic et al. 1987; Hartmann et al. 1988). Alterations i n the other components o f R A S are less obvious as compared to renin and angiotensin-converting enzyme. Plasma angiotensinogen levels have been found to be unchanged (Cassis 1992) or reduced (Brown et al. 1997). Circulating angiotensin II levels have also been found normal (Vallon et al. 1995) or reduced (Kigoshi et al. 1986). However, the existence o f a local cardiac R A S comprising all components has been  26  shown (Dostal et al. 1992a; Dostal et al. 1992b; Silvestre et al. 1998).  Although the  circulating levels o f angiotensin II may not increase i n diabetes, left ventricles from S T Z diabetic rats have been reported to have higher A C E levels than normal (Goyal et al. 1998), and an up-regulation o f the local R A S has been suggested in the diabetic heart (Fein et al. 1985; Rosen et al. 1995; Hayat et al. 2004). Though there are uncertainties i n the diabetes-induced changes i n the R A S , the contribution o f this system to diabetic cardiomyopathy, mainly through A T i receptors (Dzau 2001), has been suggested by several i n vitro, i n vivo, and even clinical studies. ATi  receptor blockers prevent  the attenuated contractile performance  o f isolated  cardiomyocytes exposed to hyperglycemic medium (Privratsky et al. 2003). Incubation o f diabetic cardiomyocytes with A C E inhibitors or A T i receptor blockers restores their depressed electrical properties (Shimoni 2001). Treatment o f STZ-diabetic rats with A T i receptor blockers has been shown to prevent the decline o f glucose transporters ( G L U T 4 s ) (Hoenack et al. 1996) and to improve glucose uptake in the heart (Raimondi et al. 2004). Treatment o f diabetic rats with A C E inhibitors has been reported to improve cardiac function (Goyal et al. 1998; Al-Shafei et al. 2002).  A C E inhibitors have also  been shown to improve the outcome o f heart failure i n diabetic patients (Shekelle et al. 2003). A T i receptor blockers have been suggested to be a novel therapeutic approach for the treatment o f diabetic cardiomyopathy and the prevention o f sudden cardiac death (Taegtmeyer et al. 2002). The  molecular  mechanisms  underlying  the  role  for  R A S in  diabetic  cardiomyopathy, though still not clear, have been related to increased expression o f A T i receptors (Sechi et al. 1994) and the up-regulation o f the downstream signaling pathways,  27  which are very similar to those of aj-ARs. A T i receptors also couple to G /n proteins. q  Activation of the receptor also leads to the activation of P L C and subsequently the generation of D A G and IP3 (Mattiazzi 1997).  Thus PKC-related pathways may  participate in this process. Malhotra et al. (1997) have found that PKCe translocated from the soluble to the particulate fraction in isolated cardiomyocytes from 4-week STZdiabetic rats, accompanied by increased Tnl phosphorylation. Treatment of the diabetic rats with the selective A T i antagonist, L-l58,809, completely prevented both the change in the subcellular distribution of PKCs and the elevated phosphorylation of Tnl. Since phosphorylation of Tnl by P K C in vitro is associated with inhibition of the C a 2+  stimulated MgATPase activity both of myofibrils and of reconstituted actomyosin complexes (Noland et al. 1993b; Venema et al. 1993a), and abnormalities in the regulatory proteins in myofibrils and myocytes from diabetic animals are associated with diminished C a  2+  sensitivity and impaired contractile performance (Hofmann et al. 1995;  Malhotra et al. 1995), this PKC-dependent pathway is a possible mechanism that is involved in A T i receptor-mediated diabetic cardiomyopathy.  1.10 Experimental rationales and hypotheses  Although it has been shown that experimental diabetes affects the ai-AR-induced PIE, results from previous studies have not been consistent.  Because P K C and Rho  kinase are downstream of ai-ARs, and have been shown to contribute to the ai-ARinduced PIE, in this study, it was hypothesized that P K C and/or Rho kinase play a role in the altered ai-AR-induced PIE in diabetes.  28  Previous studies indicated that diabetes is associated with changes in P K C activity and the subcellular distribution of P K C isozymes; on the other hand, activation of the R A S contributes to diabetic cardiomypathy, in which a PKC-dependent pathway may be involved. In the present study, it was hypothesized that blockade of A T i receptors would improve the impaired cardiac contractile function, prevent the enhanced ai-AR-induced PEE in diabetes and normalize the associated changes in P K C . To test the first hypothesis, the isolated Langendorff-perfused isovolumic heart model was used to determine the effect of STZ-diabetes on the ai-AR-mediated PIE. P K C and Rho kinase inhibitors were also used to clarify their role in diabetes-induced changes in the ai-AR-mediated PEE. Moreover, Western blot was performed to measure the associated changes in the subcellular distribution of the isozymes of these two kinases, as an index of their activation. To test the second hypothesis, chronic treatment with A T i receptor antagonists was performed in long-term STZ-diabetic rats. Basal cardiac contractile function and the PEE to Q!i-AR stimulation were subsequently measured, and Western blot was used to determine the associated changes in P K C isoforms.  29  2  MATERIALS AND METHODS  2.1  2.1.1  Chemicals and materials  Langendorff heart studies  The following chemicals were purchased from Sigma Chemical C o . (St. Louis, M O ) : Streptozotocin, sodium chloride, calcium chloride, potassium chloride, potassium phosphate monobasic, magnesium sulfate,  sodium bicarbonate,  ethylenediaminetetraacetic  timolol,  acid  (EDTA),  glucose, pyruvate,  phenylephrine,  isoproterenol,  chelerythrine chloride ( C E ) , bisindolylmaleimide I (BDvl I), bisindolylmaleimide DC (Ro318220,RO). Sodium pentobarbital was purchased from M T C Pharmaceuticals (Cambridge, O N ) . H I 152 and Y-27632 were purchased from Calbiochem C o . (Mississauga, O N ) . L 158,809 was purchased from Merck (Rahway, N J ) .  2.1.2  Enhanced chemiluminescence Western blot studies  The following chemicals and materials were purchased from Bio-Rad (Hercules, C A ) : Tris[hydroxymethyl]aminomethane (Tris), 2-mercaptoethanol,  sodium dodecyl  sulfate (SDS), glycine, protein assay dye reagent, skim milk powder, nitrocellulose membrane.  30  The following chemicals were purchased from Sigma Chemicals C o . (St. Louis, MO):  polyoxyethylenesorbitan  aminoethyl  monolaurate  ether)-N,N,N',N'-tetraacetic  acid  (Tween  20),  (EGTA),  ethylene  EDTA,  glycol-bis(P-  sodium  fluoride,  leupeptin, aprotinin, deoxycholic acid, N P 4 0 . 4-(2-aminoethyl)benzenesulfonylfluoride  (AEBSF)  was  purchased  from  CalBiochem C o . (Mississauga, O N ) . Enhanced chemiluminescence detection kit was purchased from Amersham Biosciences (United Kingdom). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, C A ) : P K C a , p , 5 and s (rabbit polyclonal), R O C K 1 and R O C K 2 (rabbit 2  polyclonal), actin (goat polyclonal), goat anti-rabbit I g G - H R P , donkey anti-goat IgGHRP.  2.2  2.2.1  Experimental protocols  Animals and blood samples  Male Wistar rats weighing 180-200g were obtained from the University o f British Columbia A n i m a l Care Unit and were housed and treated i n accordance with the guidelines o f the Canadian Council o f A n i m a l Care. S T Z was dissolved i n citrate buffer (pH 4.5) and diabetes was induced by injection o f 60 mg/kg S T Z into the lateral tail vein o f rats lightly anesthetized with halothane. Control rats received the citrate buffer vehicle. STZ-treated rats with blood glucose levels o f 13 m m o l / L or greater, measured with an Ames glucometer one week after injection, were considered diabetic and were kept for  31  experiments.  The diabetic state o f the animals was confirmed at the time o f the  experiments, by measurement o f plasma insulin and glucose levels.  Six to seven or  twelve to fifteen weeks later, animals were weighed and given an overdose o f sodium pentobarbital. After the rat was deeply anesthetized, the heart was excised. B l o o d was collected from the chest cavity (in the presence o f heparin) and spun i n a centrifuge for 20 minutes at 17,000 x g for 15 minutes for the separation o f plasma.  The plasma  samples were stored i n -20°C for the later measurement o f glucose and insulin levels.  2.2.2  Measurement of plasma glucose and insulin  Plasma glucose was determined using an assay kit from Roche (Laval, P Q ) . Plasma insulin was measured with a radioimmunoassay kit from Cedarlane (St. Charles, MO).  2.2.3  Langendorff heart studies  The excised heart was placed i n ice-cold Krebs-Henseleit ( K - H ) bicarbonate buffer (95% 0  2  - 5% C 0 ) containing (in m M ) 1.75 C a C l , 117.4 N a C l , 4.7 K C 1 , 1.2 2  2  M g S 0 , 1.3 K H P 0 , 24.7 N a H C 0 , 11.0 glucose, 5.0 pyruvate and 0.5 E D T A . 4  2  4  3  The  non-selective /3-AR anatagonist, timolol ( l u M ) , was present in K - H buffer in all the following experiments (unless specified) to minimize P E effects through /3-ARs.  The  heart was quickly cannulated via the aorta and perfused with the above K - H buffer (37°C). A pressure transducer was connected to the aortic cannula to monitor coronary  32  perfusion pressure (CPP). A balloon was inserted into the left ventricle v i a a cut i n the left atria, and a second pressure transducer was connected to the balloon for the measurement o f left ventricular developed pressure ( L V D P ) , from which the maximal rate o f contraction (+dP/dt) and relaxation (-dP/dt) were calculated using custom-written computer software. The balloon volume was adjusted to yield an end-diastolic pressure of 5 m m H g , which was then maintained constant throughout the experiment.  The  coronary flow rate was adjusted to give a C P P o f 80 m m H g , which was determined in preliminary experiments to result i n optimal basal contractile performance and P I E to a i A R stimulation, and then remained constant throughout the experiment. The heart was then paced at 300 beats/min, and was allowed to stabilize and equilibrate before the administration o f adrenergic agonists.  2.2.3.1 Preliminary experiment to determine the coronary perfusion pressure (CPP) at which the heart developed optimal basal contractile performance and <Xi-AR-induced PIE  Two groups o f normal hearts (four i n each group) were used, one o f which was perfused with a C P P o f 50 m m H g (CPP50 group) and the other with a C P P o f 70 m m H g (CPP70 group).  After a brief equilibration, the basal L V D P , +dP/dt and -dP/dt were  measured. Subsequently a single dose o f P E (10" M ) was added to the perfusion buffer, 5  and the increases in these contractile parameters i n response to P E were measured.  33  2.2.3.2 Preliminary experiment to confirm that PE selectively activates a-ARs  Although P E is a selective a i - A R agonist, it could have potential effects through other A R s i n the heart, such as (3-ARs.  T o ensure that the PEE was produced b y the  stimulation o f a - A R alone, not b y the stimulation o f (3-ARs, the following experiment was performed. Two groups o f normal hearts (three i n each group) were used. Phentolamine (an a - A R antagonist; 10" M ) was added to the K - H buffer i n one group, while the other 5  group was perfused with normal K - H buffer.  Timolol (10~ M ) was added to the K - H 6  buffer i n both groups. After 20 minutes o f equilibration, cumulative P E concentrationresponse curves (PE C R C s ) were performed i n both groups. Subsequently an apparent affinity constant (PKB value) for phentolamine was calculated using the equation p K s = log (concentration ratio -  1) - log [molar antagonist concentration], where the  concentration ratio = EC50 o f P E i n the phentolamine group / E C 5 0 o f P E i n the control group (Arunlakshana et al. 1959; Bowman et al. 1980; Kenakin 1987). The p K e value was then compared to the pKj values o f phentolamine for a i - A R obtained from previous binding studies.  2.2.3.3 Cardiac Function Study #1: Investigation of the effects of diabetes on the o^-AR-mediated PIE  Hearts from two sets o f diabetic rats (6-7 weeks and 12-15 weeks, respectively) and age-matched control rats were used (n = 9-10 hearts i n each group). After a brief 34  equilibration period, the basal LVDP, +dP/dt and -dP/dt were measured. Subsequently PE concentration-response curves (PE CRCs, 10" M ~ 10~ M) were determined in each 9  4  group. The maximal response (Rmax) values and -log[ECso] (PD2) values of the PE CRCs were calculated using GraphPad Prism 4 computer software. In some hearts from the second set of rats (12-15 week diabetic and control), the heart weight and coronary perfusion flow rate (at a CPP of 80 mmHg, in the absence of PE) were measured, and the ratios of flow rate / heart weight were calculated.  2.2.3.4 Cardiac Function Study #2: Investigation of the role for PKC in the ai-ARinduced PIE  To clarify the role for PKC in the PIE to the stimulation of ai-ARs, the effects of a PKC inhibitor on PE-induced PIE were determined.  2.2.3.4.1 Choice of PKC inhibitor  Three non-isoform selective PKC inhibitors were used: bisindolylmaleimide I (BEvI I), bisindolylmaleimide IX (Ro318220, RO) and chelerythrine (CE). Eight normal hearts were divided into four groups (two in each group): no PKC inhibitor was present in the control group, while BIM I (3*10~ M), RO (2*10~ M) and CE (10~ M) were 6  6  5  present in the other three groups, respectively. The hearts were perfused with K-H buffer or K-H buffer containing the PKC inhibitor for 20 minutes, at the end of which the  35  LVDP, +dP/dt and -dP/dt were determined. Subsequently a single dose of PE (10~ M) 5  was added to the perfusion buffer. At the end of a 2-minute perfusion with PE, the increases in these parameters were measured.  2.2.3.4.2 Effect of chelerythrine on the PIE induced by /8-AR stimulation in normal hearts  To investigate whether CE had any nonspecific effects on other signaling cascades, the effect of this PKC inhibitor on the PEE induced by the /3-adrenoceptor agonist, isoproterenol, was determined. Nine normal hearts were divided into two groups, a control group (n = 5) and a CE-treated group (10~ M CE was present in the perfusion buffer, n = 4). The hearts were 5  perfused with K-H buffer or K-H buffer containing CE for 20 minutes, at the end of which the LVDP, +dP/dt and -dP/dt were determined. Subsequently a single dose of isoproternol (10~ M) was added to the perfusion buffer. At the end of a 1-minute 6  perfusion with isoproternol, the increases in these parameters were measured. It should be noted that in this experiment, all the hearts were perfused at a basal CPP of 70 mmHg and paced at 350 beats/min before the administration of isoproterenol. As a result, a lower basal contractile performance as compared to previous experiments was obtained (section 3.1.5.2).  36  2.2.3.4.3 Effect of chelerythrine on basal contractile performance and the ai-ARinduced PIE in hearts from 12~15 week diabetic and age-matched control rats  Hearts from 12-15 week diabetic and age-matched control rats were divided into groups and treated as follows: •  Basal group (B) - hearts were perfused with K - H buffer for 22 minutes;  •  C E group (CE) - hearts were perfused with K - H buffer containing C E (10 M ) for 22 5  minutes; •  PE group (PE) - hearts were perfused with K - H buffer for 20 minutes, followed by treatment of a single dose of PE (10~ M) for 2 minutes; 5  •  CE + PE group (CE+PE) - hearts were perfused with K - H buffer containing C E (10~  5  M) for 20 minutes, followed by treatment with a single dose of PE (10~ M ; in the 5  presence of 10" M CE) for 2 minutes. 5  The basal L V D P , +dP/dt, -dP/dt and the increase in these parameters in response to PE (at the end of the 2-minute perfusion) were measured. Following the treatment period the hearts were quickly removed from the cannula and the aorta, atrium and right ventricles were removed and discarded. Left ventricles (including the left ventricular walls and the septa) were snap frozen in liquid nitrogen and stored at -70°C for Western blot analysis.  37  2.2.3.5 Cardiac Function Study #3: Investigation of the role for Rho kinase in the ai-AR-induced PIE  To clarify the role for Rho kinase in the PEE to ai-AR stimulation, the effects of two non-isoform-selective Rho kinase inhibitors, Y-27632 and HI 152, on the PE-induced PEE were determined.  2.2.3.5.1 Effect of Y-27632 on basal contractile performance and the ai-ARinduced PIE in normal hearts  Six normal hearts were divided into two groups, a control group (n = 3) and a Y27632-treated group (10~ M Y-27632 was present in the perfusion buffer, n = 3). The 6  hearts were perfused with K-H buffer or K-H buffer containing Y-27632 for 20 minutes, at the end of which the LVDP, +dP/dt and -dP/dt were determined. Subsequently PE CRCs(10"°M~ \QTM) were performed and the Rmax and pD values were calculated. 2  2.2.3.5.2 Effect of HI 152 on basal contractile performance and the ai-AR-induced PIE in hearts from normal and 12-week diabetic rats  HI 152, which is also a non-isoform-selective Rho kinase inhibitor, is more potent than Y-27632. Nine hearts obtained from normal rats weighing 400~450 g were divided into two groups, a control group (n = 4) and an H1152-treated group (10" MH1152 was 6  38  present in the perfusion buffer, n = 5). The hearts were perfused with K - H buffer or K - H buffer containing H I 152 for 20 minutes, at the end of which the L V D P , +dP/dt and dP/dt were determined. Subsequently a single dose of PE (10~ M ) was added to the 5  perfusion buffer. At the end of a 2-minute perfusion with PE, the increases in these parameters were measured. In order to investigate whether Rho kinase also plays a role in the PIE to «i-AR stimulation in diabetic state, nine hearts obtained from 12-week diabetic rats were divided into two groups, a control group (n = 4) and an H I 152-treated group (n = 5). The treatment protocol was the same as above.  2.2.3.6 Cardiac Function Study #4: Investigation of effects of ATj receptor blockade on basal contractile performance and the ai-AR-induced PIE in hearts from 12-week diabetic and age-matched control rats  A n A T i receptor antagonist, L-158,809, was used. One week after STZ or vehicle injection, diabetic and control rats were divided into two groups. One group of diabetic or control rats was treated with L-l58,809 (1 mg/kg/day) orally in their drinking water for eleven weeks. The other group of diabetic or control rats remained untreated. At the time of termination, the rat hearts were further separated into 2 subgroups: •  Basal group (B) - hearts were perfused with K - H buffer for 22 minutes;  •  PE group (PE) - hearts were perfused with K - H buffer for 20 minutes, followed by treatment with a single dose of PE (10~ M ) for 2 minutes. 5  39  The basal LVDP, +dP/dt, -dP/dt and the increase in these parameters at the end of PE perfusion were measured. Following the treatment period the hearts were quickly removed from the cannula and the aorta, atrium and right ventricles were removed and discarded. The left ventricles (including the septum) were snap frozen in liquid nitrogen and stored at -70°C for Western blot analysis.  2.2.4  Enhanced chemiluminescence Western blot studies  The frozen left ventricle preparations were powdered, homogenized and sonicated in EGTA buffer containing Tris-HCl (20 mM), 2-mercaptoethanol (50 mM), EGTA (5 mM), EDTA (2 mM), NaF (1 mM), AEBSF (1 mM), leupeptin (25 fig/ml) and aprotinin (2 |J.g/ml). The homogenized samples were spun in a centrifuge at 600 x g for 3 minutes to precipitate unbroken cardiomyocytes and organelles and the supernatant was then centrifuged at 100,000 x g for 1 hour. The resulting supernatant was retained as the soluble fraction, and the pellets were re-suspended in EGTA buffer containing (v/v) 1% NP40, 0.1% SDS and 0.5% deoxycholic acid. Following centrifugation at 100,000 x g for another 1 hour, the supernatant was collected and used as the particulate fraction. The protein content of each fraction was determined using the Bradford protein assay. Equal amounts of protein (50 ug) from each fraction were subjected to SDS-PAGE on 11% polyacrylamide gels. This amount of protein was shown to fall within the linear range of densitometric detection in preliminary experiments (section 3.2.1). The resolved proteins were electrophoretically transferred to a nitrocellulose membrane.  Membranes were  blocked with 5% skim milk in 0.05% (v/v) Tween-20/TBS (tris buffer saline containing  40  20 mM tris and 250 mM NaCI) solution and incubated with the appropriate PKC isoformspecific primary antibodies [a, p , 8 and e; rabbit polyclonal, 1:500 (v/v)] or Rho kinase 2  isoform-specific primary antibody [ROCK 1 and ROCK 2; rabbit polyclonal, 1:180 (v/v)] overnight at 4°C. Actin was used as an internal control and membranes were incubated with actin primary antibody [goat polyclonal, 1:200 (v/v)] in the manner described above. Lmmune complexes were detected following incubation of membranes with horseradish peroxidase conjugated anti-rabbit or anti-goat secondary antibody [1:20,000 (v/v); in 3% skim milk] for two hours at room temperature using an enhanced chemiluminescence detection kit. Band intensity was analyzed by densitometry and normalized for actin on the same membrane using a method similar to that described by a study (Ping et al. 1997). In brief, the average density of actin bands for each fraction (control soluble, control particulate, diabetic soluble and diabetic particulate) was calculated. The density of actin in each lane was divided by the corresponding average density, generating a correction value. The density of PKC or Rho kinase isoform band in each lane was then adjusted by dividing it by the corresponding correction value.  2.2.4.1 Preliminary experiment to determine the appropriate amount of protein that should be loaded  This preliminary experiment was done to determine the appropriate amount of protein that should be loaded into each lane to ensure that this amount of protein falls within the linear range of densitometric detection. Increasing amounts of cardiac protein (20, 30, 40, 50, 60 ug) were loaded onto the same polyacrylamide gel and the  41  densitometric values of soluble and particulate PKC5, PKCa, ROCK 1 and ROCK 2 were obtained. The densitometric reading was then plotted against the amount of protein and subjected to linear regression analysis, from which the R values were calculated.  2.2.4.2 Effect of diabetes, cii-AR stimulation and PKC inhibition on the subcellular distribution of PKC and Rho kinase isoforms  In this experiment, the left ventricle preparations were from the previous experiment (section 2.2.3.4.3). The soluble and particulate protein levels of four PKC isoforms (a, p2, 8 and s) and two Rho kinase isoforms (ROCK 1 and ROCK 2) were measured.  2.2.4.3 Effect of L-158,809 treatment on the levels of PKC8, PKCe, ROCK 1 and ROCK 2 in the particulate fraction in unstimulated and PE-stimulated hearts from 12-week diabetic and age-matched control rats  In this experiment, the left ventricle preparations were from the previous experiment (section 2.2.3.6). The particulate protein levels of two PKC isoforms (8 and s) and two Rho kinase isoforms (ROCK 1 and ROCK 2) were measured.  2.3  Statistical analyses  42  All data were presented as mean ± standard error of mean, unless specified. PE CRCs were analyzed by non-linear regression for calculation of pD (-log[ECso]) values 2  and maximum responses (Rmax) values. Statistical significance was evaluated by oneway or two-way ANOVA followed by Newman-Keuls post-hoc test for multiple comparisons in NCSS 2000 computer software.  A P-value < 0.05 was considered  statistically significant.  43  3  RESULTS  3.1  3.1.1  Langendorff heart studies  Preliminary experiment to determine the CPP at which the heart developed optimal basal contractile performance and a^-AR-induced PIE  As shown in Figure 2, the basal LVDP, +dP/dt, -dP/dt and the increase in these parameters in response to PE in hearts perfused at a CPP of 70 mmHg (CPP70) were all significantly higher than those in hearts perfused at a CPP of 50 mmHg (CPP50). This suggests that a higher CPP produces a better basal cardiac performance and a greater response to PE. However, there is a limit to the increase in CPP, because the load on the heart increases with the increase in CPP, eventually resulting in heart failure. In order to determine the optimal CPP at which the best basal cardiac performance and the greatest PE response could be obtained, several normal hearts were perfused with a CPP of 90 mmHg. Unfortunately all these hearts failed quickly (i.e. the diastolic pressure was not steady and increased gradually). However, at a CPP of 80 mmHg the hearts did not fail (section 3.1.2). Therefore, in all the following experiments, the hearts were perfused at a basal CPP of 80 mmHg, unless specified.  44  Basal LVDP  Increase in LVDP  Basal +dP/dt  Increase in +dP/dt  CPP50  CPP70 c  CPP50  CPP70 f  Figure 2 Basal LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) in response to PE (10~ M) in the CPP50 group (in which the hearts were perfused at 5  a basal CPP of 50 mmHg) and the CPP70 group (in which the hearts were perfused at a basal CPP of 70 mmHg). n = 4 hearts in each group. * Significantly different from corresponding CPP50 groups. P<0.05  45  3.1.2  Preliminary experiment to confirm that PE selectively activates a-ARs  As shown in Figure 3, 10" M phentolamine produced a significant rightward shift 5  of the PE CRC. The calculated pKe values of phentolamine were 7 . 7 7 , 7 . 6 6 and 7 . 6 6 for the increase in LVDP, +dP/dt and -dP/dt, respectively.  The mean pKj values of  phentolamine for cloned human Q!IA-,Q;IB- andai -ARs were reported to be 8 . 1 7 , 7 . 2 0 and D  7.48,  respectively (Yan et al.  2001);  while the mean pKj value of phentolamine for rat  cardiac ai-ARs was found to be 8 . 2 5 (Asano et al.  1990).  Therefore, the results from the  current study were within the range of the values reported for phentolamine acting at a.\ARs in other studies.  It should be noted that only one concentration (10~ M) of 5  phentolamine was used in the experiment and no Schild graph was plotted. However, the observation that the pKe values were close to the pKj values in the literature, suggests that in the presence of 10" M timolol, the PIE to PE was produced by the stimulation of 6  a-AR alone. In this preliminary experiment, the concentration range of the PE CRCs in the control group was lO^-lO" M (Figure 3 ) . In all three contractile parameters, 10" M PE 4  9  produced a small PIE, while the PIE produced by 10" M PE was nearly maximal. This 5  suggests that  10" ~10" 9  4  M is the appropriate concentration range of PE CRCs. In the  following experiments with PE CRCs, this concentration range was used, unless specified.  46  -B-C  -»"P Increase in LVDP  PE(-logM)  Increase in +dP/dt  tn  E  "3) x E E  7  6  4  5  PE(-logM)  Increase in -dP/dt 0.8n 0.6 SOA-  E E  0.20.0 PE(-logM)  Figure 3 Increase in LVDP (top), +dP/dt (middle) and -dP/dt (bottom) produced by cumulative addition of PE in phentolamine (10" M) treated hearts (P) and control hearts 5  (C). n = 3 hearts in each group.  47  3.1.3  Body weight, plasma glucose level and plasma insulin level of 6-7 week and 12-15 week diabetic and age-matched control rats  The body weights, plasma glucose levels and plasma insulin levels of the 6-7 week and 12-15 week diabetic and control animals are presented in Figure 4. In both cases the diabetic animals weighed significantly less than the age-matched controls. The 12-15 week control animals weighed significantly more than the 6-7 week controls while there was no difference in body weight between the two diabetic groups. The plasma glucose levels were significantly higher in 6-7 week and 12-15 week diabetic animals compared to control, while the plasma insulin levels were significantly lower in both groups of diabetic rats.  3.1.4  Cardiac Function Study #1: Investigation of the effects of diabetes on the a.iAR-mediated PIE  3.1.4.1 Heart weight, coronary perfusion flow rate and ratio of heart weight / flow rate from 12-15 week diabetic and age-matched control hearts  As shown in Figure 5, the diabetic hearts weighed significantly less than control. When perfused at the same basal CPP of 80 mmHg, the coronary perfusion flow rate in the diabetic hearts was also significantly lower than the control. However, when normalized for heart weight, the ratio of flow rate / heart weight in the diabetic hearts was not different from the control.  48  Body Weight 600n  A  III II  400-  1  Con 6-7  Dia  week  Con Dia 12-15 week  Plasma Glucose 30  20-I  -  10H  Con  Dia  6-7 week  Con  Dia  12-15 week  Plasma Insulin  X c  Con  Dia  6-7 week  Con  Dia  12-15 week  Figure 4 Body weight (top), plasma glucose level (middle) and plasma insulin level (bottom) of the 6-7 week and 12-15 week diabetic (Dia) and age-matched control (Con) rats, n = 9-10 animals in each group. * Significantly different from age-matched controls. P<0.05 A  Significantly different from 6-7 week controls. P<0.05  49  Heart Weight  Con  Dia Flow Rate  Con  Dia  Flow Rate / Heart Weight 9n  Con  Dia  Figure 5 Heart weight (top), coronary perfusion flow rate (middle) and ratio of heart weight / flow rate (bottom) from the 12-15 week diabetic (Dia) and age-matched control (Con) hearts, n = 6 hearts in each group. * Significantly different from age-matched controls. P<0.05  50  3.1.4.2 Basal contractile performance of hearts from 6-7 week and 12-15 week diabetic and age-matched control rats  In the absence of PE, the basal LVDP, +dP/dt and -dP/dt in both the 6-7 week and 12-15 week diabetic hearts were all significantly attenuated compared to the agematched controls (Figure 6).  3.1.4.3 Effect of chronic diabetes on the ai-AR-induced PIE  PE CRCs (10" ~ 10" M) were obtained in 6-7 week and 12-15 week diabetic and 9  4  age-matched control hearts (Figure 7). The increase in LVDP, +dP/dt and -dP/dt produced by the cumulative addition of PE is shown in Figure 8. The corresponding Rmax (maximal response) and pD (-logfECso]) values for the PE-induced PEE are shown 2  in Table 1. Ln 6-7 week diabetic hearts, the maximal increases in the PE CRCs and Rmax values for both LVDP and -dP/dt in response to PE were significantly greater than control, although there was no significant change in the PE pD values (Figure 8 a, c; 2  Table 1). In contrast, while Rmax value for +dP/dt produced by PE was similar in control and diabetic hearts, the PE CRC for this parameter was shifted to the left, resulting in a significant increase in the PE pD value in the diabetic hearts (Figure 8 b; 2  Table 1). Ln 12-15 week diabetic hearts, similar changes in response to PE were seen. The Rmax values for LVDP and -dP/dt but not +dP/dt produced by PE were significantly  51  increased, but in addition, the PE pD values for all three parameters were significantly 2  greater at this time (Figure 8 d, e, f; Table 1). Therefore, in hearts from 12-15 week diabetic rats, there was not only an increase in maximal response to PE, but also an increase in sensitivity to this agonist. Despite the impairement in the basal contractile performance of the 6-7 week diabetic hearts, the maximal LVDP, +dP/dt and -dP/dt that these hearts attained in the presence of PE were not different from control (Figure 7 a, b, c). However, since the basal contractile performance of the 12-15 week diabetic hearts was far lower than control, the maximal LVDP, +dP/dt and -dP/dt that these hearts attained in the presence of PE remained below those in control despite the greater PIE to PE (Figure 7 d, e, f).  52  Basal LVDP 10(H  75 E  *  50 25'  Con  Dia  6-7 week  Con  Dia  12-15 week  Basal +dP/dt 2i  O)  I  * 11  Con  Dia  6-7 week  Con  Dia  12-15 week  * n Jl Basal -dP/dt  2n  6-7 week  Con  Dia  12-15 week  Con  Dia  Figure 6 Basal L V D P (top), +dP/dt (middle) and -dP/dt (bottom) o f 6-7 week and 12-15 week diabetic (Dia) and age-matched control (Con) hearts,  n = 9-10 hearts in each  group. * Significantly different from age-matched controls. P<0.05  53  -©- Control  - • - Diabetic  LVDP  LVDP  140-.  140-1  D) 105X  O) 105X  35-  35  E E  70-  E E  —i  1  1  1  r •i  1—  70-  B 9 8 7 6 5 4 PE (-logM) d  B 9 8 7 6 5 4 PE (-logM) a  +dP/dt  +dP/dt in E o>  (A E X  X  E E  E E  —I  I  1  1  I  l  1—  1 1 1 1 1 1  1—  B 9 8 7 6 5 4 PE (-logM) b  B 9 8 7 6 5 4 PE (-logM) e  -dP/dt  -dP/dt in E  ID  E  O) X  ~S>  x E E  E E  —I  I  I  1  1  1 1—  B 9 8 7 6 5 4 PE (-logM) c  —I  1  l  l  i  1 I  B 9 8 7 6 5 4 PE (-logM) f  Figure 7 LVDP, +dP/dt and -dP/dt produced by cumulative addition of PE (10-10 M) in 6-7 week (a, b, c) and 12-15 week (d, e, f) diabetic and age-matched control hearts. "B" in the x-axes stands for basal contractile performance before the addition of PE. n = 9-10 hearts in each group.  54  - Control  Diabetic  Increase in LVDP  Increase in LVDP  9 8 7 6 5 4 PE (-logM)  9 8 7 6 5 4 PE (-logM)  •  i  i  i  i  d  Increase in +dP/dt 1.5 | 1.2 *S>0.9-| E 0.6  Increase in +dP/dt 1.5  n  0.3]  E  0.0  (A  8 7 6 5 PE (-logM) b  8 7 6 5 PE (-logM)  Increase in -dP/dt  Increase in  9 8 7 6 5 4 PE (-logM) c  9 8 7 6 5 4 PE (-logM) f  -dP/dt  0.9  -I.0-6 O)  x  | 0.3H 0.0  Figure 8 Increase in LVDP, +dP/dt and -dP/dt produced by cumulative addition of PE (10~ ~10~ M) in 6-7 week (a, b, c) and 12-15 week (d, e, f) diabetic and age-matched 9  4  control hearts, n = 9-10 hearts in each group.  55  6-7 week 12-15 week  Rmax L PD2 LVDP +dP/dt -dP/dt LVDP +dP/dt -dP/dt (mmHg) (mmHg/ms) (mmHg/ms) (-logM) (-logM) (-logM) Control 34.6±4.3 0.53±0.08 6.72±0.21 6.46±0.08 6.73±0.24 1.13±0.13 Diabetic 51.6±3.4* 1.26±0.11 0.79±0.05* 7.02±0.19 6.94±0.18* 7.09±0.21 Control 29.9±2.6 0.99±0.07 0.44±0.05 6.61±0.09 6.42±0.09 6.61±0.13 Diabetic 46.7-5.3* 1.00±0.12 0.72±0.10* 6.95±0.07* 6.87±0.09* 7.02±0.09*  Table 1 Rmax (maximal response) and pD (-log[EC o]) values for the PE-induced PIE 2  5  in 6-7 week and 12-15 week diabetic and age-matched control hearts, n = 9-10 hearts in each group. * Significantly different from age-matched controls. P<0.05  3.1.5  Cardiac Function Study #2: Investigation of the role for PKC in the <Xi-ARinduced PIE  3.1.5.1 Choice of PKC inhibitor  Figure 9 shows the basal LVDP, +dP/dt, -dP/dt and the increase in these parameters in response to PE in control hearts, BEVI I-treated hearts, RO-treated hearts and CE-treated hearts. The basal contractile performance was slightly attenuated in BIM I- and RO-treated hearts, suggesting non-specific inhibitory effects of these two inhibitors, but was not changed in CE-treated hearts compared to control. The increase in the three contractile parameters in response to PE was not changed in BEVI I- and ROtreated hearts.  However, the increase in these parameters in response to PE was  attenuated in CE-treated hearts. Higher concentrations of BEVI I (5*10~ M), RO (5*10" 6  6  M)andCE (5*10 M) were tried in a few normal hearts, but all of them failed before the 5  administration of PE. Therefore, 10" M CE was chosen for further investigations. 5  56  Basal LVDP  Increase in LVDP  120  30  90 at E  201  60  ioH  30  Con  BIMI  RO  CE  Con  BIM I  Basal +dP/dt  RO  CE  Increase in +dP/dt  2.5-1  0.75-1  2.0H CD  |  1.5-  1.0-1  1  0.50-1  I  0.25H  0.5' 0.0  Con  0.00  CE  BIM I  Con  BIM I  RO  CE  e  Basal -dP/dt  Increase in -dP/dt  2.5  0.75  2.0H E E E  1.5 1.0-1  1  0.50-1  E E  0.25-  0.5  0.0  Con  BIM I  RO  0.00  CE  Con  BIM I  c  RO  CE  f  Figure 9 Basal LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) in response to PE (10" M ; 5  RO-treated  (2*10"  6  M;  20min)  2min)  in the control, BLM I-treated (3* 10"° M ;  and CE-treated  (10°  M;  20min)  20min)  normal hearts, n =  2  hearts in each group. All values are presented as means.  57  3.1.5.2 Effect of chelerythrine on the PIE induced by  jS-AR stimulation  in normal  hearts  In this experiment, because the hearts were perfused at a lower basal CPP and paced at a faster rate as compared to previous experiments (section 2.2.3.4.2), a lower basal contractile performance was attained (Figure 10 a, b, c). As was found previously (Figure 9 a, b, c), the basal LVDP, +dP/dt and -dP/dt values were not affected by CE. Similarity, the PIE induced by isoproterenol was not changed in the CE-treated hearts compared to control (Figure 10 d, e, f). In summary, incubation of the hearts with 10" M 5  CE for 20 minutes did not affect basal contractile function, nor did it inhibit the /3-ARinduced PEE. In the next experiment, the effect of CE (10" M ; 20min) on the PIE to PE 5  in diabetic and age-matched control hearts was determined.  58  Basal LVDP  Increase in LVDP  70-  70-  60-  6050-  50-  01 X 40E E 3020-  X 40| 302010-  100-  Con  0-  CE  Con  a  CE d  Basal +dP/dt  Increase in +dP/dt  1.5n  E  "S> x E E  1*  1-0-|  "3)  x E E  0.5H  0.0  1-  J  Con  CE  Con  CE  b  e  Basal -dP/dt  Increase in -dP/dt  1.5n  i  1-0H  E  I  0.5H  E E  0.0  J  Con  CE  Con  c  Figure 10 Basal LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) in response to isoproterenol (10~ M ; lmin) in the control and CE-treated (10" M ; 6  5  20min) normal hearts, n = 4-5 hearts in each group.  59  3.1.5.3 Effect of chelerythrine on basal contractile performance and the ai-ARinduced PIE in hearts from 12~15 week diabetic and age-matched control rats  Figure 11 shows the basal LVDP, +dP/dt, -dP/dt and the PEE to PE in 12-15 week diabetic and age-matched control hearts, in the absence or presence of CE. As was found previously in normal hearts (Figure 9 a, b, c; Figure 10 a, b, c), the basal LVDP, +dP/dt and -dP/dt were not affected by CE. Consistent with the results obtained from the PE CRCs (Figure 8 d, e, f; Table 1), 10" M PE produced a significantly greater increase in 5  LVDP and -dP/dt, but no difference in +dP/dt in diabetic hearts compared to control. CE significantly attenuated the PE-induced increase in all three parameters in both diabetic and control hearts (Figure 11 d, e, f). Moreover, in the presence of CE, the increases in LVDP and -dP/dt to PE in diabetic hearts were no longer significantly greater than control (Figure 11 d, f).  60  C3 Con PE  HUH Con CE+PE  IDiaPE  Basal L V D P  100-i co  75H  Increase in L V D P  E  25-1  Basal +dP/dt  1.5  in 0.75H  E | 0.50 E 0.25  O)  LOH  |  E  Increase in +dP/dt  1.00-1  2.0-1  E  0.5 0.0-  0.00' Basal -dP/dt  2.0in  Dia CE+PE  50-| 40' 2E 3020100'  § 50-1  in  ^  Increase in -dP/dt  1.00 m 0.75 E o> X 0.50-I E 0.25-f 0.00  1.5H  E | 1.«H E 0.5 0.0-  Figure 11 Basal LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) in response to PE (10" M ; 2min) in 12-15 week diabetic (Dia) and control (Con) 5  hearts, in the absence (PE) or presence (CE+PE) of CE (10" M ; 20min). n = 5 hearts in 5  each group. * Significantly different from Con PE and Con CE+PE groups. P<0.05 @ Significantly different from all other three groups. P<0.05 $ Significantly different from Con PE and Dia PE groups. P<0.05  61  3.1.6  Cardiac Function Study #3: Investigation of the role for Rho kinase in the «iAR-induced PIE  3.1.6.1 Effect of Y-27632 on basal contractile performance and the ai-AR-induced PIE in normal hearts  PE CRCs (lO^-lO" M) and the increase in LVDP, +dP/dt and -dP/dt produced by 4  cumulative addition of PE in the absence or presence of Y-27632, are shown in Figure 12. Table 2 shows the Rmax and pD values calculated from the PE CRCs. The basal 2  LVDP, +dP/dt and -dP/dt were not affected by Y-27632 (Figure 12 a, b, c). In all three contractile parameters, there was no change in the maximal increase or rightward shift in the PE CRCs of the Y-27632-treated hearts compared to control (Figure 12 d, e, f). Correspondingly, none of the Rmax and pD values were changed in the Y-27632-treated 2  hearts compared to control (Table 2). Therefore, treatment with Y-27632 (10~ M ; 6  20min) did not affect the basal contractile performance or the PE-induced PIE in normal hearts.  62  -e-Con  — Y LVDP  Increase in L V D P  120n O)  X  E E  50 40  90-  o  60-  |  30H  30-  E  20 10  0-  B  8 7 6 5 PE (-logM)  0  4  +dP/dt  8  1  1  1  7 6 5 PE (-logM) d  p-  4  Increase in +dP/dt 1.2-,  2.5-. |2.0-  E0.9i  o>1.5-  X0.6-I  E 1.0E  -1  E 0.3  0.50.0-  B  0.0  8 7 6 5 P E (-logM) b  8  -dP/dt  7 6 5 PE (-logM)  4  Increase in -dP/dt  2.5-,  1.2-.  §2.0-1 "5)1.5 E 1.0E  0.50.0  B  8 7 6 5 P E (-logM) c  4  8  7 6 5 P E (-logM) f  4  Figure 12 LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) produced by cumulative addition of PE (10~ ~10" M) in control (Con) and Y-276328  4  treated (Y; 10" M ; 20min) normal hearts. "B" in the x-axes (a, b, c) stands for basal 6  contractile performance before the addition of PE. n = 3 hearts in each group.  63  Control Y-27632  Rmax +dP/dt -dP/dt LVDP (mmHg/ms) (mmHg/ms) (-logM) 0.91±0.10 0.52±0.08 6.52-0.26 1.04-0.15 0.59±0.15 6.61±0.18  LVDP (mmHg) 30.2±2.2 36.9-6.1  pD +dP/dt (-logM) 6.28-0.24 6.51±0.16 2  -dP/dt (-logM) 6.10-0.66 6.64±0.29  Table 2 Rmax and pD values for the PE-induced PEE in the control and Y-27632-treated 2  normal hearts, n = 3 hearts in each group.  64  3.1.6.2 Effect of H1152 on basal contractile performance and the aj-AR-induced PIE in hearts from normal and 12-week diabetic rats  Due to the ineffectiveness of 10" M Y-27632, and the expense associated with the 6  perfusion with a higher concentration of this inhibitor, HI 152, which is a more potent non-isoform-selective Rho kinase inhibitor than Y-27632, was used in this experiment. Unpublished data from our lab showed that 10" M HI 152 significantly attenuated 7  the contraction induced by PE in vascular smooth muscles.  HI 152 at the same  concentration also improved the function of isolated working hearts from diabetic rats, while having no effect on the control heart function. HI 152 at 5*10" M produced a 7  further improvement in the function of isolated working hearts from diabetic rats; however, this concentration slightly attenuated the function of control hearts.  In  Langendorff-perfused hearts, perfusion with 5*10" M HI 152 for 20 minutes before 7  treatment with a single dose of 10" M PE for 2 minutes had no effect on the PE-induced 5  PIE. As a result, a higher concentration (10" M) of HI 152 was used in the current 6  experiment. Before the addition of PE, 10" M HI 152 did not significantly affect the basal 6  contractile performance of either normal or 12-week diabetic hearts (Figure 13 a, b, c). On the other hand, the PE-induced PIE was not significantly affected by HI 152 in either normal or 12-week diabetic hearts (Figure 13 d, e, f). The concentration of HI 152 was not further increased, because the cost of the inhibitor is high, and the basal contractile function of normal hearts was already slightly (though not significantly) impaired at the concentration of 10" M (Figure 13 a, b, c). 6  65  Basal LVDP  Increase in LVDP  90  45-  60H  30H  30  15'  Con  H  Con  H  Con  12-week Diabetic  Normal  H  Con  H  12-week Diabetic  Normal d  Basal +dP/dt 2.0  Increase in +dP/dt 1.2  n  1.5H  <o 0.9H E  1.0H  £ E  0.5H 0.0-  Con  0.3' 0.0  Con  Normal  0.6H  Con  H  Con  Normal  12-week Diabetic  H  12-week Diabetic e  Basal -dP/dt  Increase in -dP/dt 0.75  1-5i  |  1.0-  £  X  I  n  0.500.50  x 0.5-  0.0  |  Con  H  Con  H  12-week Diabetic  Normal  0.25H  coo-Li  Con  L _ J ^ H  Con  Normal f  c  12-week Diabetic  Figure 13 Basal LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) in response to PE (10" M ; 2min) in the control hearts and H1152-treated (10" M ; 5  6  20min) hearts from normal and 12-week diabetic rats, n = 4-5 hearts in each group.  66  3.1.7  Cardiac Function Study #4: Investigation of effects of ATi receptor blockade on basal contractile performance and the aj-AR-induced PIE in hearts from 12-week diabetic and age-matched control rats  Figure 14 shows the basal LVDP, +dP/dt, -dP/dt, and the PIE to PE in hearts from 12-week diabetic and age-matched control rats, with or without L-158,809 treatment. Consistent with previous results, hearts from untreated diabetic rats exhibited attenuated basal contractile performance compared to untreated controls. cardiac function of hearts from L-158,809-treated  However, the basal  diabetic rats was  significantly  improved compared to that of hearts from untreated diabetic rats, and was no longer significantly different than control. Treatment of control rats with L-l58,809 had no effect on the basal cardiac function. Consistent with results obtained from the PE CRCs (Figure 8 d, e, f, Table 1), and the results from the chelerythrine study (Figure 11 d, e, f), 10" M PE produced a significant greater increase in L V D P and -dP/dt, but no difference 5  in +dP/dt in untreated diabetic hearts compared to untreated controls.  However, L -  158,809 treatment had no significant influence on the PE-induced PIE in either diabetic or control hearts, and the PE-induced increase in L V D P and -dP/dt in L-158,809-treated diabetic hearts remained significantly elevated compared to control (Figure 14 d, f).  67  Basal LVDP  Increase in LVDP  Figure 14 Basal LVDP (a), +dP/dt (b), -dP/dt (c) and the increase in these parameters (d, e, f) in response to PE (10~ M ; 2min) in hearts from 12-week untreated control (C), 5  untreated diabetic (D), L-158,809-treated control (CT) and L-158,809-treated diabetic (DT) rats, n = 7-13 hearts in each group. @ Significantly different from all other three groups. P<0.05 # Significantly different from C and CT groups. P<0.05  68  3.2  3.2.1  Enhanced chemiluminescence Western blot studies  Preliminary experiment to determine the appropriate amount of protein that should be loaded  The plots of "Densitometric reading vs. Amount of protein" are shown in Figure 15. The points were subjected to linear regression analysis. No points deviated from the linear regression trend lines, and all the R values were higher than 0.99. These data 2  suggest that 50 fag protein of PKC or Rho kinase in the soluble or particulate fration fell within the linear range of densitomertric detection. As a result, in all the following Western blot experiments, 50 p.g of soluble or particulate protein was loaded into lanes of polyacrylamide gels.  69  PKCAIplia (Idubls)  PKC Alprra'(paritajlate)  1  .10  c*  !  i.  :'--P. fi  zi  ta  2  SD  •  10  Am ountbf prutoln Cuo) PKC,Delta(sdLt]le) ID  f 3 n E n." S n•  a  5 •a n  B  o  6  E  JS  b  I *  •I  •, 0 10 ZD 3D 4D SC . SO 70  a ?••  • ID '20  D  Am ou nt of q m tj In Cu o).  ROCK 1 (lOluDU)  —i E  •£ B'  1 Su iz. 10 .  m tD 50 SD . 'AmbuntnTprafaIn Cub)  • R O C K 1 (parltcul'ate)  [ZD  E 7  a.  .* I s  •'•  j  W  20  40  ZD tD SD Am auntof protpln-Cug)  SO  Am • un to Tp in InC . up )  ROCK 2  E  7. or6  I  n  e s  fi 10  I-  • £'. E  *  J ^ * 2  •  R O C K 2(p articulate ]  (JOlUble)  16 U i iz  fi  70  9, s  R/-D9S  E; 6; s 2.  •P-  so 7D  PKC Delta (particiiae)  10  a  2 0 ' 3i * a SJ Amauntpfpnjfoln (ug)  2d 40 '60 - Amn unto f prvbln Cu b)  1 •  20 tD SO Am nuntpf protein (up)  SO  Figure 15 Densitometric reading vs. amount of soluble and particulate protein loaded, for incubation with antibodies to PKC5, PKCa, ROCK 1 or ROCK 2. Trend lines for linear 2  regression and R values are also shown in the figure.  70  3.2.2  Effect of diabetes on the protein levels of actin in the soluble and  the  particulate fractions  Actin is used as an internal control to minimize the errors that could occur during protein loading and/or transfer in Western blot assays (see section 2.2.4 for detailed correction methods).  A representative blot of soluble and particulate actin in  unstimulated and PE-stimulated hearts from 12-15 week diabetic and age-matched control rats is shown in Figure 16. Diabetes did not have distinguishable influence on the protein levels of actin in either the soluble or the particulate fractions.  Control  Basal  Diabetic  PE  Basal  PE  Soluble  Particulate  Figure 16  Representative blot of soluble and particulate actin in unstimulated and PE-  stimulated hearts from 12-15 week diabetic and age-matched control rats.  71  3.2.3  Effect of diabetes, ai-AR stimulation and PKC inhibition on the subcellular distribution of PKC and Rho kinase isoforms  In this set of experiments (Figure 17-22), all the soluble and particulate values were expressed relative to the mean value in the soluble fraction of the Control Basal group, which was set at 1. Four isoforms of PKC (a, p , 5 and s) were investigated. No significant changes 2  in the levels of PKCa and p in the soluble or particulate fractions were detected in 2  untreated 12-15 week diabetic hearts, and PE produced no significant change in the soluble or particulate levels of either isoform in either control or diabetic hearts (Figure 17; Figure 18). In unstimulated diabetic hearts, levels of both PKC5 and PKCe in the particulate fraction were significantly increased, but no significant change in their levels in the soluble fraction was detected (Figure 19; Figure 20). Exposure of both control and diabetic hearts to PE for 2 minutes, the time required for the peak PIE to this agonist, resulted in a significant increase in the particulate levels of PKC8, but without a corresponding decrease in the soluble levels of this isoform (Figure 19). The increase in PKCS over its own basal levels in diabetic hearts (0.74±0.07) was significantly greater than the PE-induced increase in levels of this isoform in control hearts (0.45±0.10). PE also produced a significant increase in the levels of PKCe in the particulate fraction of both control and diabetic hearts, again without a corresponding decrease in levels in the soluble fraction (Figure 20). Although in the presence of PE, total levels of PKCe in the particulate fraction of diabetic hearts were significantly greater than control, the  72  magnitude of the PE-induced increase in PKCs over the corresponding basal levels was not significantly different in diabetic (0.7210.17) and control (0.63±0.08) hearts. Although CE was without effect on basal levels of PKCa, (3 , 8 and e (data not shown), it 2  completely prevented the PE-induced increases in the particulate levels of PKCS and € in both control and diabetic hearts (Figure 19; Figure 20). No significant changes in the levels of ROCK 1 and ROCK 2 in the soluble or particulate fractions were detected in unstimulated 12-15 week diabetic hearts, and PE produced no significant change in the soluble or particulate levels of either isoform in either control or diabetic hearts (Figure 21; Figure 22).  73  PKCa Control B  PE  CE+PE  Diabetic B  PE  CE+PE  Soluble Particulate 1.5 > _l  .E  m o  1.0  i_  DL  >  I3  PE CE+PE  Control • Soluble  PE CE+PE Diabetic Particulate  Figure 17 Relative protein levels and a representative blot o f P K C a i n the soluble and particulate fractions o f basal (B), PE-treated (PE) and C E plus PE-treated (CE+PE) hearts from 12-15 week diabetic and age-matched control rats. A l l the soluble and particulate values are expressed relative to the mean value i n the soluble fraction o f the Control B group, which is set at 1. n = 10 i n B groups; n = 5 in P E and C E + P E groups.  74  PKC p Control  B c  ..  l  l  h  i.  -  PE CE+PE  2  Diabetic B PE CE+PE  .. .. Mtl  Particulate 1.25n 0)  >  1.00-  X  X  c jE 0.75u  > | 0.25H  III  o.oo II BB IPE • CE+PE I B Control a Soluble  •  m  LB  PE CE+PE  Diabetic Particulate  Figure 18 Relative protein levels and a representative blot of PKCP2 in the soluble and particulate fractions of basal (B), PE-treated (PE) and CE plus PE-treated (CE+PE) hearts from 12-15 week diabetic and age-matched control rats. All the soluble and particulate values are expressed relative to the mean value in the soluble fraction of the Control B group, which is set at 1. n = 10 in B groups; n = 5 in PE and CE+PE groups.  75  PKC 6 Control B  B  PE CE+PE  PE  CE+PE  Control  a  Diabetic  Soluble  B  B  PE CE+PE  PE  CE+PE  Diabetic  • • Particulate  Figure 19 Relative protein levels and a representative blot of PKC5 in the soluble and particulate fractions of basal (B), PE-treated (PE) and CE plus PE-treated (CE+PE) hearts from 12-15 week diabetic and age-matched control rats. All the soluble and particulate values are expressed relative to the mean value in the soluble fraction of the Control B group, which is set at 1. n = 10 in B groups; n = 5 in PE and CE+PE groups. # Significantly different from all control groups and other diabetic groups. P<0.05 & Significantly different from control B, control CE+PE and diabetic PE groups. P<0.05  76  PKC e Control  B  Diabetic  PE CE+PE  B  PE CE+PE  Soluble  mm mm  Particulate 2.5n  mm mm mm  a>  % 2.0H  B  PE CE+PE Control  C Z 3 Soluble  B  PE CE+PE Diabetic  •  Particulate  Figure 20 Relative protein levels and a representative blot of PKCs in the soluble and particulate fractions of basal (B), PE-treated (PE) and C E plus PE-treated (CE+PE) hearts from 12-15 week diabetic and age-matched control rats. A l l the soluble and particulate values are expressed relative to the mean value in the soluble fraction of the Control B group, which is set at 1. n = 10 in B groups; n = 5 in PE and CE+PE groups. # Significantly different from all control groups and other diabetic groups. P<0.05 & Significantly different from control B, control CE+PE and diabetic PE groups. P<0.05 % Significantly different from control B, control CE+PE, diabetic CE+PE and diabetic PE groups. P<0.05  77  ROCK 1 Control soluble  Diabetic  B  PE  CE+PE  mm  mm  mm  Particulate  «•«•*  mam  B  PE  CE+PE  mam  1.25-1  S I.OOH c *  o u  0.75-1  S" 0.50-1  > |  0.25-  o.oo  II •  IM Im  PE CE+PE  Control  •Soluble  B  PE CE+PE  Diabetic  • • Particulate  Figure 21 Relative protein levels and a representative blot of ROCK 1 in the soluble and particulate fractions of basal (B), PE-treated (PE) and CE plus PE-treated (CE+PE) hearts from 12-15 week diabetic and age-matched control rats. A l l the soluble and particulate values are expressed relative to the mean value in the soluble fraction of the Control B group, which is set at 1. n = 10 in B groups; n = 5 in PE and CE+PE groups.  78  ROCK 2 Control PE  B  Diabetic  CE+PE  B  PE  CE+PE  Soluble Particulate 1.5  > £  1.0  o a. m  liiL o,o  B I I I  PE CE+PE I I  I  Control  insoluble  I  L  PE CE+PE Diabetic  •  Particulate  Figure 22 Relative protein levels and a representative blot of ROCK 2 in the soluble and particulate fractions of basal (B), PE-treated (PE) and CE plus PE-treated (CE+PE) hearts from 12-15 week diabetic and age-matched control rats. All the soluble and particulate values are expressed relative to the mean value in the soluble fraction of the Control B group, which is set at 1. n = 10 in B groups; n = 5 in PE and CE+PE groups.  79  3.2.4  Effect of L-158,809 treatment on the levels of PKC8, PKCe, ROCK 1 and ROCK 2 in the particulate fraction in unstimulated and PE-stimulated hearts from 12-week diabetic and age-matched control rats  In this set of experiments (Figure 23—26), only changes in the particulate levels of PKCS, PKCs, ROCK 1 and ROCK 2 were investigated, as no changes in the soluble fractions or other PKC isoforms were detected in previous experiments. All particulate values were expressed relative to the mean value of the Control Basal group, which was set at 1. In the absence of PE treatment, the particulate levels of both PKC5 and PKCs were significantly increased in diabetic hearts. L-158,809 treatment did not significantly change the particulate levels of either isoform in either diabetic or control hearts. Exposure of both control and diabetic hearts to PE for 2 minutes resulted in a significant increase in the particulate levels of PKC5 (Figure 23). As was found in the previous experiment, the increase in PKCS over its own basal levels in diabetic hearts (0.86±0.11) was also significantly greater than the PE-induced increase of this isoform in control hearts (0.32±0.13) this time. PE also produced a significant increase in the levels of PKCe in the particulate fraction of both control and diabetic hearts (Figure 24). Similar to what had been found in the previous experiment, in the presence of PE, total levels of PKCe in the particulate fraction of diabetic hearts were significantly greater than control, while the magnitude of the PE-induced increase in PKCs over its corresponding basal level was also not significantly different in diabetic (0.49±0.07) and control (0.47+0.14)  80  hearts this time. L-158,809 treatment did not significantly change the particulate levels of either isoform in either PE-treated diabetic or PE-treated control hearts. However, in hearts from L-158,809-treated rats, particulate levels of PKCs in the presence of PE were not significantly different from those in the absence of PE. No significant changes in the particulate levels of ROCK 1 and ROCK 2 were detected in diabetic hearts. Neither L-158,809 nor PE treatment had any significant effect on the particulate levels of either isoform in either control or diabetic hearts (Figure 25; Figure 26).  81  PKCtf Control L PE L+PE  B  B  Diabetic PE L+PE  mm mm mm mm  # #  2.5« a 2.0c 2 1.5H >  &  &  &  &  B  L  i.oH  ro  o 0.50.0'  B  L  PE L+PE  Control  PE L+PE  Diabetic  Figure 23 Relative protein levels and a representative blot of PKC8 in the particulate fraction of basal (B) and PE-treated (PE) hearts from 12-week diabetic and age-matched control rats with (L) or without L-158,809 treatment. All particulate values are expressed relative to the mean value of the Control B group, which is set at 1. n = 5 hearts in each group. # Significantly different from all control groups and diabetic B, diabetic L group. P<0.05 & Significantly different from control B, control L, diabetic PE and diabetic L+PE group. PO.05  82  PKC  e  Control  B  L  PE L+PE  Diabetic  B  jjMftfe* ^ ^ ^ ^ ^ ^^^^^  L ^jgg^  PE L+PE ^HHlk ^ttl^k  2.(h 0) >  1.5H C  '5  o 1.0H >  ^ 0.5H o  0.0-  B  L  PE L+PE  Control  Figure 24  B  L  PE L+PE  Diabetic  Relative protein levels and a representative blot of PKCs in the particulate  fraction of basal (B) and PE-treated (PE) hearts from 12-week diabetic and age-matched control rats with (L) or without L-158,809 treatment. All particulate values are expressed relative to the mean value of the Control B group, which is set at 1. n = 5 hearts in each group. # Significantly different from all control groups and diabetic B, diabetic L group. P<0.05 $ Significantly different from control B, control L, control PE, and diabetic B group. PO.05 A  Significantly different from control B, diabetic PE and diabetic L+PE group. PO.05  & Significantly different from control B and diabetic PE group. PO.05  83  ROCK 1 Control B  L  PE  Diabetic L+PE  B  L  L+PE  mm* mmm . mm*  4 H H H I flHttifr'  iMMiM^  PE  1.5q  B  L  P E L+PE  Control  B  L  P E L+PE  Diabetic  Figure 25 Relative protein levels and a representative blot of ROCK 1 in the particulate fraction of basal (B) and PE-treated (PE) hearts from 12-week diabetic and age-matched control rats with (L) or without L-158,809 treatment. All particulate values are expressed relative to the mean value of the Control B group, which is set at 1. n = 5 hearts in each group.  S4  ROCK 2 Control B  L  PE  Diabetic L+PE  Control  B  L  PE  L+PE  Diabetic  Figure 26 Relative protein levels and a representative blot of ROCK 2 in the particulate fraction of basal (B) and PE-treated (PE) hearts from 12-week diabetic and age-matched control rats with (L) or without L-158,809 treatment. All particulate values are expressed relative to the mean value of the Control B group, which is set at 1. n = 5 hearts in each group.  85  4 DISCUSSION 4.1  Summary of results The present study showed that in the absence of adrenergic stimulation, the basal  contractile performance was attenuated in diabetic hearts, whereas the PIE in response to PE was enhanced compared to control. In the presence of CE, the PE-induced PIE in both diabetic and control hearts was suppressed, and the PIE in diabetic hearts was no longer significantly different than control. Under basal conditions, the subcellular distribution of PKCa, PKC/3 , ROCK 1 and ROCK 2 was not altered in diabetic hearts. 2  However, the levels of PKCS and s in the particulate fraction of diabetic hearts were increased, without a corresponding decrease in the soluble fraction. PE produced a significant increase in the levels of PKCS and s in the particulate fraction of hearts from both diabetic and control rats, again without a corresponding decrease in the soluble fraction. The increase in particulate PKCS over its own basal levels in diabetic hearts was significantly greater than control, whereas the increase in particulate PKCe over its own basal levels in diabetic and control hearts was not different. In the presence of CE, the PE-induced increase in the levels of PKCS and s in the particulate fraction of both diabetic and control hearts was completely blocked. PE had no detectable effect on the subcellular distribution of PKCa, PKC/3 , ROCK 1 or ROCK 2. Treatment with the A T i 2  receptor antagonist, L-158,809, significantly improved the basal contractile function of diabetic hearts. However, it did not normalize the enhanced ai-AR-induced PIE. L158,809 had no effect on the basal levels of PKCS, PKCs, ROCK 1 or ROCK 2 in the  86  particulate fraction in either diabetic or control hearts, nor did it affect the PE-induced changes in these two PKC isozymes.  4.2  Choice of cardiac preparation  One purpose of the present study was to investigate the effect of long-term STZdiabetes on the ai-AR-induced contractile responses in the heart. Several functional studies have reported the ai-AR-mediated PIE is enhanced in diabetic rat hearts. Most of these investigations used isolated cardiac muscle strip preparations, such as intact atria or atrial strips (Canga et al. 1986; Jackson et al. 1986; Durante et al. 1989; Brown et al. 1994), right ventricular strips (Wald et al. 1988; Yu et al. 1991) or left ventricular papillary muscles (Brown et al. 1994). Since it is the left ventricle that determines cardiac output and drives the systemic circulation, the studies using atrial or right ventricular preparations may not reflect the effects of diabetes on cardiac function. As a result, left ventricular preparations were used in the study. The left ventricular papillary muscle preparation is a relatively simple and easy technique compared to whole heart perfusion.  However, since multiple biochemical assays had to be performed after  functional measurements, left ventricular papillary muscles were not able to provide enough protein for Western blot assays. A second disadvantage of the left ventricular papillary muscle preparation is that due to its thickness, it is highly susceptible to hypoxia. The perfused whole heart preparations, in which the coronary vessels are perfused and the oxygenated buffer rapidly gains access to all cardiomyocytes, are less likely to be subject to ischemia. Whole heart preparations such as the working heart or  87  the Langendorff iosvolumic heart are ideal choices because the function of the whole left ventricle can be measured, and the amount of protein available for subsequent biochemical assays is relatively high. Heijnis et al. (1992), using the working heart model, showed the ai-AR-mediated PEE was elevated in hearts from STZ-diabetic rats. So far there has been no investigation using the Langendorff isovolumic heart for the measurement of the ai-AR-mediated PIE. Compared to working heart techniques, the Langendorff-perfused heart is simpler and less demanding in terms of equipment and the operator's skill.  Secondly, in the working heart model, the coronary perfusion is  dependent on left ventricular function, while in the Langendorff heart, the coronary perfusion is independent of ventricular function, leading to the suggestion that the Langendorff heart is a better model to study the concentration-dependent effects of inotropic agents on cardiac contractile function (Fawzi 1997). As a result, in the study, the Langendorff isovolumic heart model was chosen.  4.3  Setting of experimental conditions  One important issue of this study was to treat the diabetic and age-matched control hearts with the same basal conditions. Contractile parameters such as LVDP, +dP/dt and -dP/dt of the Langendorff heart are affected by CPP, as well as by other factors, such as the temperature, balloon size and heart rate (Fawzi 1997).  In the  Langendorff setup of this study, the temperature, balloon size and heart rate were controlled and maintained the same in diabetic and control hearts. In addition, a pressure transducer was connected to the aortic cannula to monitor CPP, and adjusted the  88  perfusion flow rate to give the same CPP (80 mmHg) in both diabetic and control hearts. Because diabetic hearts were smaller than age-matched control hearts, the coronary vessels were also smaller in diabetic hearts. When perfused at the same CPP, the coronary perfusion flow rate in diabetic hearts was lower than that in control hearts. However, when normalized for heart weight, the ratio of flow rate / heart weight in diabetic hearts was not different from that in control hearts (Figure 5).  4.4  Basal contractile function and the ai-AR-mediated PIE are two independent processes  The present study demonstrated an attenuated basal contractile performance as well as an elevated ai-AR-mediated PIE in diabetic hearts compared to control. While it might be argued that the greater maximal increase (though not sensitivity) to PE is simply the result of the lower basal contractile performance in the diabetic hearts, two observations from the study argue against this possibility. First of all, in preliminary experiments, two groups of normal hearts were perfused with a basal CPP of 50 mmHg and 70 rnrriHg, respectively. The group perfused at a CPP of 70 rnjnHg exhibited a higher basal contractile performance. If the above argument is true, this group would be expected to be associated with a smaller maximal increase to PE. In fact, this group showed a greater response to PE. Moreover, in Cardiac Function Study #4, the basal contractile function of hearts from L-158,809-treated diabetic rats was significantly improved compared to that of hearts from untreated diabetic rats, and was no longer significantly different than control. However, the hearts from L-158,809-treated diabetic  89  rats still exhibited a significantly greater maximal increase to PE compared to control. Based on these two findings, it could be ruled out that a lower basal contractile performance mechanically allowed the heart to produce a higher contractile response to PE. There is no mechanical correlation between the basal cardiac function and the oci.AR-mediated PIE.  4.5  Changes in the subcellular distribution of PKC isoforms  The present study showed that both diabetes and «i-AR stimulation elevated the particulate levels of PKCS and e, without a corresponding decrease in the soluble levels of these two isozymes. Similar observations have also been found in other investigations with diabetic animal models. For example, Inoguchi et al. (1992) showed a significant increase in particulate PKC02 in hearts from 2-week STZ-diabetic rats compared to control, but the soluble levels of this isoform remained unchanged. Kang et al. (1999) found that the particulate levels of PKCa and 8 were increased in renal tissues from 4week STZ-diabetic rats, but the percentage of the soluble versus the particulate levels of these two isoforms was unaltered, hi addition, some investigations also showed that PE as well as other G-protein-coupled receptor agonists induced a significant elevation in particulate PKC isozymes, but without a corresponding decrease in the soluble levels. For instance, Puceat et al. (1994) exposed isolated cardiomyocytes from adult rats to PE and produced 2-3 fold increases in particulate PKCs, but without a decrease in the soluble levels. Wang et al. (2003) also found an elevation in particulate PKCs produced by PE, associated with a much smaller decrease in the soluble levels of this isoform. In  90  another study (Henry et al. 1996), adenosine receptor agonists produced 2-3 fold increases in particulate PKC8 in isolated cardiomyocytes from adult rats, without an apparent concomitant decrease in the soluble fraction. Two arguments may help to explain the above observations. Firstly, although the translocation of PKC isozymes from the soluble fraction to the particulate fraction has been recognized as a hallmark of their activation, some studies have suggested that additional changes in PKC isoforms may occur upon their activation. Recent studies have shown that stimulation of PKCS and e by neurohormones or phorbol 12-myristate 13-acetate (PMA, a non-isoform-selective PKC activator) not only produces a translocation of the isozymes from the soluble fraction to the particulate fraction, but also induces phosphorylation as well as conformational changes in the isoforms (Rybin et al. 2003; Rybin et al. 2004). Thus, the binding of the PKC isoform molecules with their corresponding antibodies may be affected, resulting in changes in their immunoreactivity. Therefore, in the present study, the changes in PKCS and e in diabetes or in response to PE stimulation could have resulted not only from a physical translocation, but also from a structural modification of the isozymes leading to altered immunoreactivity. Secondly, the increase in levels of PKC5 and PKCs in the particulate fraction without a corresponding decrease in the soluble fraction could be due to the uneven distribution of the isoforms between the two fractions. This theory was uttered by Henry et al. (1996), who also detected a significant increase in particulate PKC8 in isolated rat cardiomyocytes after the treatment with an adenosine receptor agonist, but without a corresponding decrease in the soluble fraction. In the present study, if we take PKCs as an example, examination of Figure 20 suggests that there are approximately equal  91  amounts of P K C E in 50 jag of soluble and 50 p,g of particulate protein. However, on fractionation of left ventricular preparations, approximately 80% of the total protein remains in the soluble fraction, while 20% is found in the particulate fraction. Therefore, there is about 4 times more PKCs in the soluble than that in the particulate fraction. Following stimulation with PE, there is an approximately 50% increase in PKCs in the particulate fraction, but this would correspond to only a 12.5% decrease in the soluble fraction, an amount that would be difficult to detect given the variability between hearts and assays.  4.6  Effect of diabetes on the subcellular distribution of cardiac PKC isoforms  A number of studies have suggested that in hearts from STZ-diabetic rats, the membrane-associated (particulate) or total PKC activity is increased (Inoguchi et al. 1992; Tanaka et al. 1992; Xiang et al. 1992; Liu et al. 1999). Although the mechanisms are not fully understood, hyperglycemia, which increases the D A G content in rat myocardium, has been suggested to play a causal role in the activation of PKC (Okumura et al. 1988; Inoguchi et al. 1992; Porte et al. 1996). On the other hand, changes in the subcellular distribution of PKC isoforms in STZ-diabetic hearts have been shown in some studies, but the results are far from consistent. Inoguchi et al. (1992) reported that in whole heart preparations from 2-week diabetic male Sprague-Dawley rats, there was an increase in particulate P K C P 2 with no change in the soluble fraction, and the subcellular distribution of PKCa was not altered. Malhotra et al. (1997) showed that in isolated cardiomyocytes from 3~4 week diabetic female Wistar rats, PKCs translocated from the  92  soluble fraction to the particulate fraction, while no change in PKC5 was detected. Liu et al. (1999) demonstrated that in ventricular preparations from 8-week diabetic male Sprague-Dawley rats, levels of PKCa, (3 and s were increased in the total protein and in the soluble fraction, but there was no change in the particulate fraction. Kang et al. (1999) showed that in whole heart preparations from 4-week diabetic male SpragueDawley rats, the total protein levels of PKCa were increased, with no changes in PKCp2 and 8, and a decrease in PKCs. In the present study, a significant increase in the levels of PKC5 and s in the particulate fraction was detected, without a corresponding decrease in the soluble fraction, and the subcellular distribution of PKCa and p was not altered. 2  Note that in the present study, the duration of STZ-diabetes was much longer (12-15 week), only left ventricular preparations (left ventricular walls and ventricular septa) were used, and the hearts were perfused with K - H buffer for some time (22 minutes) before being frozen for Western blot assays. Therefore, the discrepancies between the present study and the investigations mentioned above could be due to difference in the types of rats, the duration of diabetes, the cardiac preparations and/or the experimental conditions.  4.7  PKC isozymes and diabetic cardiomyopathy  PKC has been implicated in the pathological progress of myocardial diseases, including diabetic cardiomyopathy and other disease-induced cardiac dysfunctions. As mentioned above, diabetes appears to induce the activation of specific PKC isozymes in  93  the heart.  The involvement of PKC5, 8 and P2 in the pathogenesis of diabetic  cardiomyopathy has been suggested by a variety of studies. Activation of PKCS and s has been suggested to contribute to the over-expression of (3-myosin heavy chain (P-MHC) and to the increased secretion of atrial natriuretic peptide (ANP) and angiotensin-converting enzyme (ACE) in the heart; on the other hand, the levels of these proteins in the hypertrophic heart have been shown to be increased (Zarich et al. 1989; Uusitupa et al. 1990). Thus the activation of PKC5 and s, and the subsequent elevation in p-MHC, ANP and ACE, may be an important component in the development of diabetic cardiomyopathy (Steinberg et al. 1995). More recent studies have suggested PKC8 and s might have distinct effects.  Over-expression of a  constitutively-active PKCe mutant in cardiac culture has been reported to induce cellular remodeling and elongation (Strait et al. 2001), which are fundamental processes for cardiac hypertrophy. Similarly, transgenic over-expression of PKCe has been shown to cause concentric cardiac hypertrophy (Takeishi et al. 2000). On the other hand, PKC5 over-expression has been shown to result in cell detachment and cardiomyocyte apoptosis, which are different outcomes than the hypertrophic effect of PKCe (Heidkamp et al. 2001). Though the exact signaling mechanisms underlying the apoptotic effect of PKC5 and the hypertrophic effect of PKCe are not fully clear, some reports have suggested that the activation of the two PKC isoforms leads to the selective activation of specific terminal kinases of the mitogen-activated protein kinase (MAPK) cascade, a signaling cascade for cardiac hypertrophic and apoptotic gene expression (Bueno et al. 2002). PKC5 preferentially activates two terminal kinases, c-Jun N-terminal kinase (INK) and p38-MAPK, resulting in apoptosis, while PKCe selectively activates  94  extracellular-regulated kinase (ERK), a terminal kinase of the MAPK cascade generally implicated in growth responses and hypertrophy (Heidkamp et al. 2001). Malhotra et al. (1997) showed a tranlocation of PKCe and an increased phosphorylation of Tnl in cardiomyocytes from diabetic rats, and they also suggested this isoform might participate in diabetic cardiomyopathy. In all, both PKC8 and s may be activated in the diabetic heart and could contribute to the development of diabetic cardiomyopathy. PKCP2 has been suggested to participate in several chronic pathological processes in the heart, such as cardiac hypertrophy, heart failure and diabetic cardiomyopathy (Koya et al. 1998; Sabri et al. 2003). In short-term (2-week) STZ-diabetic rat hearts, particulate levels of PKC & were increased (Inoguchi et al. 1992). In pressure-overload cardiac hypertrophic rats, an increase in particulate levels of PKCfo was also observed (Gu et al. 1994). Ventricles from patients with end-stage heart failure showed increased expression of PKC/32 (Bowling et al. 1999). Transgenic mice over-expressing PKC/3  2  specifically in myocardium developed cardiac hypertrophy, cardiomyocyte injuries and fibrosis at 8-12 weeks of life. Later, cardiac atrophy and severe fibrosis were observed. Treatment with a selective PKC/3 inhibitor, LY333531, prevented most of the functional and pathological changes in hearts from these transgenic mice (Wakasaki et al. 1997). This study, along with two other investigations (Bowman et al. 1997; Takeishi et al. 1998), in which similar findings were observed, has implicated the role for this PKC isoform in cardiomyopathy and cardiac contractile dysfunction. Moreover, in a number of phase 2 clinical trials, LY333531 has been shown to be efficacious in diabetes-induced cardiac dysfunction (Hayat et al. 2004). Transforming growth factor p and connective tissue growth factor can induce production of collagen and fibronectin in cardiac  95  fibroblasts and cardiomyocytes, resulting in myocardial stiffness and attenuated contractility (Ohnishi et al. 1998; Chen et al. 2000). A correlation between the activation of PKC/32 and the expression of these two growth factors in hearts from STZ-diabetic mice has been suggested (Way et al. 2002). In all, P K C ^ has been implicated in the development of diabetic cardiomyopathy. In the present study, no significant change was detected in the subcellular distribution of P K C ^ in the diabetic heart. However, there are a number of differences in the animal models, duration of diabetes and experimental conditions between the present study and the ones mentioned above. Thus at this point, it cannot be ruled out that this PKC isozyme is involved in diabetic cardiomyopathy.  4.8  Diabetes may not have effects on the subcellular distribution of Rho kinase in the heart  The role for RhoA - Rho kinase in myocardial contractile function is poorly understood. There have been several investigations suggesting that RhoA - Rho kinase may be involved in myocardial hypertrophy and heart failure (Kuwahara et al. 1999; Suematsu et al. 2001; Yanazume et al. 2002). On the other hand, the mRNA levels of RhoA in hearts from transgenic diabetic mice have been shown to be elevated (Duan et al. 2003). However, there have been no studies reporting diabetes-induced changes in cardiac Rho kinase. The present study did not observe any significant effect of diabetes on the subcellular distribution of Rho kinase. Therefore it does not support a role for this kinase in diabetic cardiomyopathy.  96  4.9  Contribution of PKC isozymes to the ai-AR-mediated PIE  In the early 1990's, several groups investigated the role for PKC in the cc-ARmediated PEE (Otani et al. 1992; Talosi et al. 1992; Endoh et al. 1993). In these studies, the PKC inhibitors staurosporine and H-7 were shown to inhibit the a-AR-mediated PIE in the heart. However, both of these agents lack specificity and are equally potent inhibitors of both PKC and protein kinase A (Toullec et al. 1991). Later on, a more selective PKC inhibitor, BEM (Toullec et al. 1991), was used in a study investigating the ai-AR-mediated PIE in neonatal rat hearts (Deng et al. 1997). BIM, a non-isoformselective PKC inhibitor, significantly blocked the positive inotropic response to PE and the PE-induced increase in PKC activity in the particulate fraction. Thymeleatoxin, a selective activator of conventional PKC isoforms (Ryves et al. 1991), produced small inhibition in myocardial contraction, while Go-6976, a selective inhibitor of conventional PKC isoforms (Martiny-Baron et al. 1993), did not inhibit the PE-induced PEE. The Western blot results from this study showed that PE produced a translocation of PKCS and e from the soluble to the particulate fraction but had no effect on PKCa. Thus it was concluded in this study that the activation of novel PKCS and e by ai-AR agonists plays a key role in the ai-AR-mediated PIE in neonatal rat hearts. In studies using ventricular myocytes isolated from adult rat hearts, it was also shown that upon the stimulation with PE, the levels of PKC5 and s in the particulate fraction were elevated, but PKCa was not changed (Puceat et al. 1994; Wang et al. 2003).  In the current study, among the  conventional PKC isoforms (PKCa and P2) and the novel PKC isoforms (PKCS and e) found in the heart, only PKCS and e responded to PE stimulation and were inhibited by  97  CE. These results, along with the findings from the above studies, suggest that PKCS and/or PKCe play a role in the cci-AR-mediated PIE in both normal and diabetic hearts. However, due to a lack of selective PKCS and PKCe inhibitors, the individual role for these two isozymes in the apAR-mediated PIE could not be further clarified.  4.10 Contribution of PKC to the enhanced ai-AR-mediated PIE in the diabetic heart  Although a number of studies have shown that in the diabetic rat heart, the otiAR-mediated PIE is elevated (Canga et al. 1986; Jackson et al. 1986; Wald et al. 1988; Durante et al. 1989; Yu et al. 1991; Heijnis et al. 1992; Brown et al. 1994; Ha et al. 1999), the underlying molecular mechanisms are poorly understood. Binding studies have consistently found that in diabetic cardiomyocytes, the number of ai-AR-binding sites is reduced (Heyliger et al. 1982; Wald et al. 1988; Tanaka et al. 1992; Kamata et al. 1997). This is associated with no change (Heyliger et al. 1982; Tanaka et al. 1992; Kamata et al. 1997) or an increase (Wald et al. 1988) in their affinity constants. As a result, the enhanced ai-AR-mediated PIE may be due to post-receptor mechanisms. It has been shown that the enhanced ai-AR-mediated PIE is associated with increased D?3 production in diabetic ventricular preparations compared to control (Xiang et al. 1991), suggesting that ai-AR-mediated stimulation of PLC is enhanced in diabetes. Although there have been no studies directly measuring the effect of diabetes on the activation of PLC or the levels of DAG content upon the stimulation of ai-AR, Wald et al. (1988) obtained indirect evidence suggesting there is enhanced activation of the PLC-DAG  98  pathway upon «i-AR stimulation in hearts from acutely diabetic rats compared to control. They found an enhanced ai-AR-mediated PIE in diabetic ventricular preparations. In the presence of a PLC inhibitor, the ai-AR-mediated PEE in diabetic ventricular preparations was attenuated, reaching a level similar to that in control preparations without the presence of the PLC inhibitor. Moreover, in the presence of synthetic DAG, the ai-ARmediated PIE in control preparations was increased, reaching a level similar to that in diabetic preparations without the presence of synthetic DAG. The present study suggests a role for PKC in the enhanced ai-AR-mediated PEE in hearts from long-term diabetic rats, providing further evidence to support the hypothesis that there is enhanced activation of the PLC-DAG-PKC pathway upon apAR stimulation in the diabetic heart, and this may be one molecular mechanism underlying the enhanced ai-AR-mediated PEE in diabetes. As mentioned above, at the present stage it cannot be concluded whether PKC8, or PKCs, or both, play a role in the ai-AR-mediated PEE. However, the present study suggests a role for PKC8 in the greater PIE in response to ai-AR stimulation in diabetic hearts. With PE stimulation, the levels of PKC8 in the particulate fraction of both diabetic and control hearts were increased, and the increase in particulate levels over its own basal level in diabetic hearts was significantly greater than control. In the presence of CE, the PE-induced increase in particulate levels of this isoform in both diabetic and control hearts was completely surpressed.  Compared to PKCS, PKCe may be less  important in the enhanced ai-AR-mediated PIE in the diabetic heart, since the data from the current study showed that with PE stimulation, the increase in particulate levels of this isoform over its own basal level was not significantly different in diabetic and control  99  hearts. However, it cannot be ruled out that PKCe may also contribute to the enhanced ai-AR-mediated PIE in the diabetic heart, since there is a lack of selective PKCS and PKCe inhibitors, and the elevation in levels of PKCe the particulate fraction upon stimulation with PE is not as sustainable as that of PKCS. Puceat et al. (1994) showed that in cardiomyocytes from normal adult rats, the increase in particulate PKCs induced by PE was fast and transient. The peak elevation occurred about one minute after the addition of PE, and the levels of particulate PKCs rapidly went back to its baseline level in five minutes. However, the elevation of PKCS was much longer. Even after fifteen minutes of PE stimulation, the levels of particulate PKCS remained high compared to its baseline levels. In the present study, the time for PE treatment was two minutes (the minimal time length required to reach the maximal PIE to PE). It is possible that for PKCs, two minutes of PE treatment was too long in terms of the measurement of peak elevation, whereas, because the elevation of particulate PKCS was more sustainable, a significantly higher increase in this isoform in the diabetic heart was able to be shown. In all, the data from this study suggest a role for PKCS in the greater PIE to PE in the diabetic heart, but it cannot be excluded that PKCe may also contribute to this alteration in diabetes.  4.11 Activation of PKC: good or bad?  Due to defects in the /3-AR signaling cascade, the increase in contractile performance in response to endogenous noradrenaline is depressed in the diabetic heart (Gotzsche 1983a; Gotzsche 1983b; Smith et al. 1984). Meanwhile, the ai-AR-mediated  100  PIE is up-regulated, which has been suggested to be a compensatory mechanism to maintain cardiac contractile response to noradrenaline (Corr et al. 1981; Milligan et al. 1994; Beaulieu et al. 1997; Skomedal et al. 1997). The present study suggests a role for the novel PKC isoforms, PKCS, or both PKCS and PKCe, in the enhanced ai-ARmediated PIE in the diabetic heart. However, this may be only a transient compensatory mechanism, since long and sustained activation of these PKC isozymes may "turn on" the expression of a number of hypertrophic and apoptosis genes in the heart, including pMHC, ANP, ACE (Zarich et al. 1989; Uusitupa et al. 1990), INK, p38-MAPK and ERK (Heidkamp et al. 2001), contributing to the development of diabetic cardiomyopathy. In diabetes, DAG levels in cardiomyocytes are increased, possibly due to hyperglycemia (Okumura et al. 1988; Inoguchi et al. 1992; Porte et al. 1996). This leads to the activation of not only PKCS and PKCe, but also PKC/3, which has been implicated in many studies to contribute to the development of diabetic cardiomyopathy. In all, the overall activation of PKC in the diabetic heart may lead to the destruction and reconstitution of myocardial structure and impaired contractile performance. This may be an important component in the pathogenesis of diabetic cardiomyopathy.  4.12 Role of Rho kinase in the aj-AR-mediated PIE  A role for Rho kinase in the ai-AR-mediated PIE in the heart has been suggested in several studies using the non-isoform-selective Rho kinase inhibitor, Y-27632. This inhibitor, at the concentration of 5*10" M and 10" M , respectively, was shown to 5  5  effectively block the PE-induced PIE in rat left ventricular papillary muscles (Andersen  101  et al. 2002) and human atrium (Grimm et al. 2005). At a concentration of 10" M , it also 5  blocked the PE-induced Ca sensitization in isolated cardiomyocytes from failing hearts (Suematsu et al. 2001).  So far there has been no study showing translocation or  activation of Rho kinase in the heart or isolated cardiac preparations upon aj-AR stimulation. In the present study, at a concentration of 10" M , Y-27632 did not inhibit 6  PE-induced PIE in normal hearts. Another non-isoform-selective Rho kinase inhibitor, HI 152, which is more potent and selective for Rho kinase than Y-27632 (Sasaki 2003) was used. However, even at a concentration of 10" M , HI 152 had no effect on the PE6  induced PIE in normal or 12-week diabetic hearts. Furthermore, stimulation of a i - A R with PE did not alter the subcellular distribution of Rho kinase in either diabetic or control hearts. Therefore, a role for Rho kinase in the ai-AR-mediated PIE could not be observed in this study. The conflicting results from the above studies and from the present investigation may be due to differences in cardiac preparations. At the present stage, the existence of Rho kinase in the proposed signaling pathways underlying the ctiAR-mediated PIE is still poorly understood.  4.13 PKC may not be involved in the improvement of the impaired basal contractile function of the diabetic heart by the inhibition of RAS  There is strong evidence that activation of the RAS contributes to diabetic cardiomyopathy (Hoenack et al. 1996; Goyal et al. 1998; Al-Shafei et al. 2002; Privratsky et al. 2003; Shekelle et al. 2003), but the molecular signaling mechanisms are not clear. Malhotra et al. (1997) has suggested a PKC-dependent pathway.  These  102  investigators found a translocation of PKCe as well as an increased phosphorylation of Tnl in isolated cardiomyocytes from 4-week STZ-diabetic rats. Treatment of diabetic rats with L-158,809 completely prevented the changes in PKCs and Tnl in diabetes. However, in the present study, although treatment with L-158,809 improved the attenuated basal contractile performance in diabetic hearts, it did not normalize the increase in basal particulate PKCS and e in diabetic hearts, in contrast with the results from Malhotra's group. There are a number of discrepancies between the current study and that of Malhotra's that might account for this inconsistence. In the former, the A T i blocker was dissolved in drinking water and fed to the animals daily (1 mg/kg/day), while in Malhotra's study the rats received subcutaneous injection of the drug twice a week (10 mg/kg). The duration of diabetes (12 weeks vs 4 weeks), cardiac preparations (left ventricles vs. isolated cardiomyocytes) and experimental protocols were also different. The normalizing effect of L-158,809 on diabetic cardiac function in the absence of normalization of the subcellular distribution of PKCS and e, suggests that the A T i blocker is acting via some signaling mechanism other than PKC-dependent pathways. Some potential mechanisms have been suggested in other studies. Activation of A T i receptors leads to the activation of NADPH oxidase and consequently enhances the production of reactive oxygen species, such as superoxide anion, which can react with nitric oxide, leading to its inactivation by producing peroxynitrite. The latter can directly oxidize membrane components such as arachidonic acid, thus altering membrane integrity and cardiac function (Griendling et al. 2000; Sowers 2002). Activation of A T i receptors can also lead to the activation of Janus kinase (JAK)/signal transducer and activator of transcription  (STAT)  (Mascareno et al. 2000). This pathway was initially  103  discovered as a major signal transduction pathway of the cytokine superfamilies (Ihle 1995). The JAK/STAT proteins are involved in the production of various kinds of cytokines and growth factors that contribute to cardiac hypertrophy (Schindler et al. 1995). Although these molecular mechanisms have been proposed to contribute to cardiomyopathy, the exact role for angiotensin II, activation of ATi receptors and their downstream signaling mechanisms in the onset of cardiac dysfunction, especially during diabetes, is still poorly understood.  4.14  The attenuated basal contractile performance and the enhanced ai-ARmediated PIE may be two relatively independent alterations in the diabetic heart  Although the present study showed that treatment of diabetic rats with L-158,809 significantly improved the basal contractile function of diabetic hearts, this ATi receptor antagonist did not normalize the enhanced ai-AR-mediated PEE in diabetes, suggesting that the attenuated basal contractile function and the enhanced ai-AR-mediated PIE may be two relatively independent alterations in diabetes. This phenomenon was also shown in several studies investigating the contribution of experimental diabetes-induced hypothyroidism to the enhanced oc-AR-induced PEE and the attenuated basal cardiac function.  It has been known for a long time that STZ-diabetes is associated with  hypothyroidism, in which plasma triiodothyronine (T3) levels are significantly low (Boado et al. 1978). In animals with hypothyroidism, an increased a-AR-mediated PIE has been observed (Simpson et al. 1981). A correlation between the enhanced oci-AR-  104  mediated PEE and hypothyroidism in STZ-diabetes was suggested (Goyal et al. 1987). These investigators showed that left atrial preparations from 6-week STZ-diabetic rats exhibited greater inotropic responses to a-AR agonists, while treatment of the diabetic animals with T prevented this change. In another study (Lafci-Erol et al. 1994), it was 3  also shown that the ai-AR-mediated PIE was enhanced in atrial preparations from alloxan-induced diabetic rats. Treatment with insulin normalized this change, while thyroidectomy prevented the effect of insulin. The atrial preparations from diabetic rats with insulin treatment and thyroidectomy still exhibited enhanced inotropic responses to cti-AR stimulation. However, a study (Tahiliani et al. 1984) reported that the depression of the basal contractile performance of the diabetic heart was not normalized by T  3  treatment. In all, these studies showed that treatment of experimental diabetic rats with T normalized the greater cc-AR-mediated PIE but had no effect on the impaired basal 3  cardiac function, suggesting diabetes-induced hypothyroidism may contribute to the former but not to the latter.  Similarily, the results from the present study suggest  activation of the RAS may contribute to the impaired basal cardiac function but not to the enhanced ai-AR-mediated PIE in the diabetic heart. Although the exact molecular mechanisms are poorly understood, these two processes may be independent.  4.15  Summary and future directions  The results from the present study showed that in the absence of adrenergic stimulation, the basal contractile performance of the diabetic heart was attenuated. Since the basal levels of PKCS and e in the particulate fraction were increased in the diabetic  105  heart, activation of these two isozymes may contribute to the development of diabetic cardiomyopathy. Upon ai-AR stimulation, a PIE was seen in both diabetic and control hearts, and the diabetic heart exhibited a greater PIE compared to control. In the presence of a PKC inhibitor, the PIE was suppressed and no longer different in diabetic and control hearts. Particulate levels of PKCS and e increased in response to ai-AR stimulation and the increase in PKCS over its own basal levels in the diabetic heart was significantly greater than that in control. In the presence of a PKC inhibitor, the ai-ARinduced increase in particulate PKCS and e was totally suppressed. These results suggest that PKCS and/or PKCe may play a role in the ai-AR-induced PIE, and PKCS may contribute to the enhanced ai-AR-induced PIE in the diabetic heart. PKCa, PKCf3 , 2  ROCK 1 and ROCK 2 were detected in both diabetic and control hearts, but no significant influence of diabetes or ai-AR stimulation on these isozymes was observed. Treatment of diabetic rats with an ATi receptor antagonist improved the impaired basal cardiac performance, but it did not normalize the enhanced PIE to PE, nor did it have significant effect on the associated changes in PKCS and PKCe.  This suggests that  activation of the RAS contributes to diabetic cardiomyopathy, and PKC may not be involved in this process. The present study has suggested a role for PKCS and/or PKCe underlying the aiAR-induced PIE in the heart, and that PKCS may contribute to the enhanced ai-ARmediated PIE in the diabetic heart. However, it was not able to be excluded that PKCe may also be involved in this process. The use of selective PKCS and PKCe inhibitors would be helpful to clarify the individual role for these two PKC isozymes in the ai-ARmediated PIE, as well as in the elevated PIE in diabetes. In the present study, only the  106  Western blot assay was used for the measure of the activation of PKC isozymes. More supportive evidence for the activation of PKC could be provided. For example, since the activation of PKC5  and PKCe  by neurohormones  is associated  with their  phosphorylation, and antibodies specific for phosphorylated PKC5 and PKCs have been developed (Rybin et al. 2003; Rybin et al. 2004), investigating the levels of the phosphorylation of these isozymes would help to detect their activation. 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