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INOS mediates increased RhoA expression and altered cell signaling in diabetic cardiomyopathy Craig, Graham Peter 2006

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INOS M E D I A T E S I N C R E A S E D R H O A E X P R E S S I O N A N D A L T E R E D C E L L S I G N A L I N G IN D I A B E T I C C A R D I O M Y O P A T H Y . by G R A H A M P E T E R C R A I G B . S c , The University of York, 2004 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Pharmaceut ica l Sc iences ) T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A December 2006 © G r a h a m Peter Cra ig , 2006 ABSTRACT The R h o A / R O C K pathway is a well establ ished signal ing pathway which regulates cellular contractility. Prev ious studies, from this lab have shown that acute inhibition of R O C K improves heart function in 12 week STZ-d iabet ic rats. Here we wished to determine, 1) whether the R h o A / R O C K pathway w a s upregulated in the diabetic rat heart, 2) what factors may. be contributing to altered activity of this pathway during diabetes, and 3) determine the consequences of the altered R h o A / R O C K signal ing might be. R h o A express ion was significantly elevated in hearts and card iomyocytes from rats with STZ- induced diabetes. Our preliminary studies a lso showed elevated i N O S express ion in hearts from rats with STZ- induced diabetes. W e hypothesized that N O from i N O S mediates elevated R h o A express ion. Th is hypothesis is supported by a number of our observat ions. Elevated i N O S expression and activity was observed to be concomitant with increased R h o A express ion in diabetic rat hearts. In cultured card iomyocytes exposure to the N O donor, S N P , and the i N O S inducer, L P S , c a u s e d elevated R h o A express ion. The i N O S inhibitor, L-NIL, b locked increased R h o A express ion in L P S treated card iomyocytes and in hearts from diabetic animals. Increases in R h o A express ion correlated strongly with increased levels of active R h o A in diabetic card iomyocytes. W e also observed increased phosphorylated L IMK, a marker for activation of the R h o A / R O C K pathway. L IMK phosphorylat ion was reduced to levels similar to control after chronic treatment of diabetic rats with L-NIL, suggest ing that i N O S a lso contributes to increased activity of this pathway in the STZ-d iabet ic rat heart. The actin cytoskeleton is a well establ ished downstream target of the R h o A / R O C K pathway. The level of polymer ized actin in diabetic rat card iomyocytes was found to be significantly e levated, but was normal ized after acute R O C K inhibition. G iven this observat ion, we suggest that normalization of actin polymerizat ion may contribute to R O C K inhibitor-mediated improvement of contracti le function of hearts from diabetic rats. The f indings presented in this thesis indicate a central role for i N O S in the upregulation of the R h o A / R O C K pathway, which is bel ieved to contribute to impaired contractility in the diabetic heart. Our f indings a lso support the suggest ion that R O C K is an excel lent therapeutic target in the treatment of diabetic cardiomyopathy. T A B L E OF CONTENTS P a g A B S T R A C T ii T A B L E O F C O N T E N T S iv LIST OF FIGURES vii LIST O F ABBREVIATIONS viii A C K N O W L E D G E M E N T S x 1.0 INTRODUCTION 1 1.1 Diabetes Mell i tus. 2 1.1.1 Classi f icat ion of Diabetes. 3 1.1.2 The Streptozotocin- induced diabetic rat model . .4 1.1.3 Diabet ic cardiomyopathy. .6 1.1.3.1 Biochemica l and pathological changes in the heart during diabetes. 7 1.1.3.2 Metabol ic changes in the heart during d iabetes. 8 1.2 The R h o G T P a s e s . 10 1.2.1 The cellular functions of RhoA . 13 1.2.1.1 R h o A and the actin cytoskeleton. 13 1.2.2 A pathological role for the R h o A / R O C K pathway? 18 1.3 Nitric oxide and the heart. 24 1.3.1 Ev idence for an interaction between N O and R h o A . 26 1.4 Hypothesis and rationale for proposed experiments. 27 1.5 Spec i f ic objectives. 28 2.0 MATERIALS AND METHODS 30 2.1 C h e m i c a l s and materials. 30 2.2 An ima ls . 32 2.2.1 Induction of d iabetes. 32 2.2.2 Chron ic inhibition of i N O S in control and S T Z diabetic rats. 33 2.3 Preparat ion of rat cardiac t issue. 33 2.3.1 Preparat ion of isolated rat ventricular myocytes. 33 2.3.1.1 Pr imary culture of isolated ventricular myocytes. 34 2.3.1.2 Induction of i N O S by L P S in cultured ventricular myocytes. 35 2.3.2 Extraction of total protein from cultured myocytes. 35 2.3.3 Extraction of total protein from rat ventricular t issue. 37 2.4 Protein concentrat ion determination. 37 2.5 SDS-Po l yac ry l am ide gel electrophoresis and immunoblott ing of proteins from rat ventricular t issue and rat ventricular myocytes. 38 2.6 i N O S activity assay . 39 2.7 G-LISA™ R h o A Activation A s s a y . .40 2.8 G-act in / F-actin A s s a y . .40 2.9 Immunof luorescence labeling and Confoca l Microscopy. 41 2.10 Statist ical Ana lys is . .41 3.0 RESULTS 42 3.1 Express ion of R h o A in control and diabetic card iomyocytes. .42 3.2 Express ion of i N O S in control and diabetic card iomyocytes. .44 3.3 Effect of S N P on the express ion of R h o A in cultured card iomyocytes. .46 3.4 Effect of i N O S induction on the express ion of R h o A in cultured card iomyocytes. 48 v 3.5 Effect of chronic i N O S inhibition on express ion of R h o A in control and diabet ic rat hearts. 51 3.6 Effect of chronic i N O S inhibition on activity of the R h o A / R O C K signal ing pathway. 54 3.7 State of activation of the R h o A / R O C K pathway in control and diabetic card iomyocytes. 56 3.8 Effect of R O C K inhibition on the actin cytoskeleton in control and diabet ic diabetic cardiomyocytes. 59 4.0 DISCUSSION 64 4.1 Increased R h o A and i N O S express ion in S T Z diabetic rat card iomyocytes. 64 4.2 N O and induction of i N O S elevate R h o A express ion in vitro. 65 4.3 Chron ic i N O S inhibition results in decreased i N O S express ion in diabet ic rat hearts. ." 68 4.4 Chron ic inhibition of i N O S normal izes express ion of R h o A and activity of the R h o A / R O C K pathway in heart t issue from rats with STZ - i nduced d iabetes. 69 4 .5 Increased activity of R h o A in diabetic rat card iomyocytes. 70 4.6 Effects of acute R h o A / R O C K inhibition on the actin cytoskeleton in diabet ic cardiomyocytes. 72 4.7 Further Studies. 75 5.0 SUMMARY AND CONCLUSIONS .78 BIBLIOGRAPHY 80 vi LIST O F FIGURES P a g 1. R h o G T P a s e regulation. 12 2. M o d e of action of R h o / R O C K in cytoskeletal reorganization and cellular contractility. 17 3. Effect of Y -27632 on the function of isolated working hearts from control and diabetic rats. 21 4. Effect of H-1152 on percent fractional shortening (% FS) of control and diabetic left ventricle. 23 5. Induction of i N O S by L P S . 36 6. Relat ive express ion of R h o A in control and diabetic card iomyocytes, as a s s e s s e d by western blotting. 43 7. Relat ive express ion of i N O S in control and diabetic card iomyocytes, as a s s e s s e d by western blotting. 45 8. Effect of 10 u M sodium nitroprusside ( S N P ) treatment for 18hrs on R h o A express ion . 47 9A. Effect of L P S treatment on R h o A and i N O S express ion. 49 9B . Effect of L P S treatment (50 pg/ml for 18hrs) on R h o A express ion . 50 10. i N O S express ion and activity in control and diabetic card iac ventricular t issue following chronic i N O S inhibition. 52 11. Relat ive R h o A express ion levels in control and diabetic card iac ventricular t issue following chronic i N O S inhibition. 53 12. Relat ive levels of L I M K - P in card iac ventricular t issue. 55 13. Relat ive amount of active R h o A in Control and 12 week S T Z diabetic rat ventricular myocytes. 57 14. L IMK-P/To ta l L IMK in Contro l and 12 week S T Z diabet ic rat ventricular myocytes. 58 15. Compar i son o f the F-actin to G-act in ratio in freshly isolated myocytes. 61 16. Con foca l microscopy images of f luorescently labeled card iomyocytes. 63 17. G P C R mediated activation of R h o G T P a s e s . 71 LIST OF ABBREVIATIONS °C degrees Ce ls ius +dP/dT maximal rate in rise of deve loped pressure -dP /dT maximal rate of decl ine in deve loped pressure u micro (1 x 10"6) pm micron (1 x 10" 6 metre) A N O V A analys is of var iance A T P adenos ine tr iphosphate B S A bovine serum albumin C a C b calc ium chloride c G M P guanos ine 3':5'-cycl ic monophosphate [Ca 2 + / ] intracellular calc ium concentrat ion C 0 2 carbon dioxide D T T dithiothreitol E G T A ethylene glycol bis(3-aminoethyl ether) N,N'-tetraacetic ac id g relative centrifugal force g gram HCI hydrogen chloride H E P E S /V-2-hydroxyethylpiperazine-/V-2-ethanesulfonic ac id hrs hours i N O S inducible nitric oxide synthase i.p. intraperitoneal kDa kilo Dalton kg kilogram Kj inhibition constant, concentrat ion for half maximal inhibition L litre L-NIL L-N6-(1-iminoethyl)lysine L V D P left ventricular deve loped pressure m milli (1 x 10" 3), if used as a prefix min minute/s M molar N a C l sodium chloride viii N a O H sodium hydroxide N O nitric oxide N O S nitric oxide synthase P A G E polyacrylamide gel electrophoresis S D S sodium dodecyl sulfate S E M standard error of the mean S N P sodium nitroprusside T E M E D N, N, N', N'-tetramethylethylenediam ine Y -27632 (R)-(+)-trans-4-(1-Aminoethyl)-N-(4-Pyridyl) -cyc lohexanecarboxamide dihydrochloride ACKNOWLEDGEMENTS Firstly, I would like to express my greatest thanks to my supervisor Dr. Kath leen M a c L e o d , for her pat ience, her k indness, her constantly open door and her thoughts and guidance throughout the course of my studies at U B C . I express my appreciat ion to my supervisory committee. Drs. Roger Brownsey, G lenn Tibbits, W a y n e Riggs and Brian Rodr igues. Thank you for your time, your input, your constructive crit icism and directions. Thanks a lso go to the members of the Tibbits lab with whom we attempted preliminary exper iments to measure intracellular calc ium concentrat ions. J ingbo Huang 's help and guidance in the isolation of card iomyocytes was greatly appreciated. Our chronic i N O S inhibition studies would not have been possib le without the kind help Prabhakar Nagareddy, who generously provided us with heart t issue from his L-NIL treated animals. Prabhakar a lso conducted the i N O S activity assays . I would a lso like to thank my fellow lab co l leagues Rui Zhang , Lili Zhang , B a o h u a W a n g , Dr. Guorong Lin and Mar ia C h a n for their technical help, advice and fr iendship. Finally, thanks go to the Canad ian Insitute of Health Resea rch for providing funding for this research, and to Dr. John McNei l l for f inancial support in the form of an academic scholarsh ip. Thanks to my parents, for support ing my move to C a n a d a and for all your advice and love. x 1.0 INTRODUCTION The last decade has seen growing public awareness of the prevalence of d iabetes mellitus in the western world. A number of startling statistics and projections are in part responsible for this growing concern . Recent est imates suggest that 6 .5% of the U .S . population have diabetes (Amer ican Diabetes Assoc ia t ion , 2005). The number of people worldwide with diabetes, which was est imated to be around 135 million people in 1995, is projected to increase to 300 million in 2025 (King etal., 1998). C o m p a r e d to many other d i seases , the treatment of d iabetes consumes a disproportionate share of health care resources (Winer and Sowers , 2004). This is because diabetic patients show heightened risk of development of a number of different medical compl icat ions, including retinopathy, nephropathy, neuropathy and cardiovascular d i sease . Of these compl icat ions cardiovascular d i sease (CVD) is the biggest cause of mortality in patients with d iabetes. It is bel ieved that a typical diabetic patient has an increased risk of development of cardiovascular d i sease by a factor of up to four t imes that of their healthy counterpart (Haffner, 2000). Card iovascu lar d i seases such as atherosclerosis, coronary artery d i sease and card iac hypertrophy can ultimately lead to heart failure. In fact, heart failure is cited as the cause of death in 6 5 % of people with d iabetes (Ge iss et al., 1995). Therefore there is t remendous interest in research that may help us to understand the factors contributing to the development of C V D s , and why diabetic patients in particular show increased risk of development of card iovascular compl icat ions which can ultimately lead to heart failure. 1 1.1 Diabetes Mellitus. Diabetes mellitus is a group of d isorders character ized by persistent aberrat ions in carbohydrate, protein and lipid metabol ism, as a result of impaired insulin action and/or insufficient insulin production. Th is is a metabolic disorder that requires medical d iagnosis , treatment and lifestyle changes . It has been suggested that the history of d iabetes can be traced to as far back as 1552 B . C . when the 3rd Dynasty Egypt ian physic ian H e s y - R a documented on papyrus a number of symptoms of d iabetes (Canad ian Diabetes Assoc ia t ion, 2006). Diabetes w a s first specif ical ly descr ibed by the ancient G reek physician Aretaeus, who in around 70 A . D . named the d i sease 'Diabetes' . Th is name derived from an ancient Greek term meaning 'pass ing through' or 's iphon' , a reference to one of d iabetes ' major symptoms: polyuria (excess ive urine production). In 1670 Thomas Wil l is, physic ian to King Char les , added 'mellitus', from the Latin word for honey, after he noted that a person with d iabetes had urine and blood that had a sweet taste and aroma. The d iscovery of the role of the pancreas in d iabetes is general ly ascr ibed to J o s e p h von Mer ing and Oska r Minkowski , who showed that pancreatectomized dogs deve loped hyperglycemia, and died shortly after surgery (Von Mehr ing and Minkowsk i , 1890). The Canad ian researchers Sir Freder ick Grant Bant ing and John J a m e s Richard Mac leod expanded upon the work of V o n Mering and Minkowski , when they demonstrated that they could reverse pancreactomy- induced diabetes in dogs by giving them extracts from healthy pancreat ic islets of Langerhans (Banting et al., 1922). Bant ing and M a c L e o d and their co l leagues, went on to isolate the hormone insulin from bovine pancreata. In recognition of these findings Banting and M a c L e o d were awarded 2 the Nobe l prize for medicine in 1923. The increased world-wide availabil ity of insulin for the treatment of diabetes led to the distinction of insulin dependent and non insulin dependent forms of diabetes (Himsworth, 1936). The main forms of d iabetes are now well identified and dist inguished and are known as type 1 and type 2 diabetes. 1.1.1 Classification of Diabetes. The clinical symptoms of d iabetes are the primary factors used in the classif icat ion of this d isease . Type 1, formerly known as insulin dependent d iabetes mellitus (IDDM), makes up 5 -10% of all d iagnosed c a s e s and is assoc ia ted with insulin def iciency. It has been shown that Type 1 d iabetes is the outcome of select ive ablation of the insulin-producing B-cells of the pancreas (Foul is and Stewart, 1984). It is widely accepted that a T-cel l mediated, immunological response is involved in the pathogenesis of type 1 diabetes. Th is is supported by histological examinat ion of the pancreata from children at the onset of the d i sease and from a number of animal models (Bertera et al., 1999). However, despite this ev idence, the precise mechan ism triggering this immunologic B-cell loss is yet to be establ ished and remains subject to debate. Studies have suggested that viral infection can act as an initiator of type 1 d iabetes (Yoon etal., 1979), and infection of pr imates with Coxsack i e v i ruses has been shown to induce abnormali t ies in g lucose homeostas is (Yoon etal., 1986). Despi te lack of a specif ic mechan ism descr ib ing the initiation of type 1 diabetes, ev idence strongly supports the autoimmune nature of the pathogenesis of type 1 diabetes. Serum from patients with type 1 diabetes tests positive for ant ibodies raised specif ical ly against pancreact ic islet cel ls (Pietropaolo et al., 1998), temporary immunosuppress ion can delay the onset of type 1 diabetes (Bach, 1993), and there is 3 ev idence that type 1 d iabetes can be transmitted to non-diabetic patients via bone marrow transplants (Lampeter et al., 1993). Type 2 d iabetes represents 9 0 - 9 5 % of all d iagnosed c a s e s . It is widely bel ieved that type 2 d iabetes develops in response to over-nutrition and an assoc ia ted insulin resistance (see review by Prentki and Nolan, (2006)). The normal 6-cell response to over-nutrition and obesi ty-associated insulin resistance is compensatory insulin hypersecret ion in order to maintain normoglycemia. Insulin hypersecret ion leads to hyper insul inaemia. Over time, the compensatory B-cell hypersecret ion fails, resulting in a progressive decl ine in B-cell function. A s a consequence , subjects progress to hyperglycemia and establ ished type 2 diabetes. A number of different factors have been assoc ia ted with a heightened incidence of type 2 d iabetes within certain populat ions. It has been proposed that genetic predisposit ion, environmental factors, lifestyle cho ices and ethnicity may influence an individuals risk of acquir ing type 2 d iabetes (Pimenta et al., 1995). A comprehens ive explanat ion of the pathology of type 1 and type 2 d iabetes is yet to be establ ished. In order to further our understanding of the mechan isms and compl icat ions of d iabetes mellitus further experimentat ion using appropriate animal models of d iabetes are required. 1.1.2 The streptozotocin-induced diabetic rat model. A s descr ibed above, the historical exper iments of von Mer ing demonstrated that surgical removal of the pancreas from an animal served as a useful model of d iabetes mellitus. S ince then non-surgical methods of inducing hyperglycemia by damaging the pancreas have been deve loped. Th is includes the administration of toxins such as 4 streptozotocin (STZ). S T Z was initially indicated as broad spectrum antibiotic isolated from the organism Streptomyces achromogenes (Vavra et al., 1959). Subsequent ly it was found that S T Z was a lso a potent and select ive toxin caus ing necros is of pancreat ic B-cells. Th is highlighted the potential use of the drug in animal models of d iabetes (Mansford and Opie , 1968) and a s a medical treatment for pancreat ic carc inomas (Murray-Lyon etal., 1968). The chemica l structure of S T Z consis ts of a g lucose moiety conjugated to a nitrosourea side chain. The uptake of S T Z by pancreat ic B-cells is mediated by the g lucose transporter G L U T 2 . Reduced express ion of G L U T 2 has been found to prevent the diabetogenic action of S T Z (Schnedl etal., 1994, Thu lesen etal., 1997). O n c e taken up by the pancreas, the highly reactive nitrosourea side chain of S T Z is bel ieved to mediate its cytotoxic effect. However, the exact mechan ism by which S T Z exerts its specif ic and select ive toxic effect on B-cells remains a content ious topic. Stud ies have suggested that STZ- induced B-cell death is primarily as a result of D N A damage (Delaney et a l . 1995; E isner et a l . 2000). It has been demonstrated that S T Z is a powerful alkylating agent which can induce multiple D N A strand breaks (Bolzan and Bianchi , 2002). In support of this proposal previous studies have shown increased purine methylation in pancreat ic t issue from STZ-d iabet ic rats (Bennett and P e g g , 1981). It has also been proposed that nitric oxide production (Kwon etal., 1994; Haluzik and Nedv idkova, 2000) and free radical generat ion (Oberley, 1988) a lso contribute to B-cell death after S T Z administration. W h e n administered to rats, S T Z ' s action in B-cells is accompan ied by characterist ic alterations in blood insulin and g lucose concentrat ions. Two hours after injection, hyperglycemia is observed with a concomitant drop in blood insulin. About six 5 hours later, hypoglycemia occurs with high levels of blood insulin. Within 24-48 hours, hyperglycemia deve lops and blood insulin levels dec rease (Junod et al., 1969). Th is hyperglycemic and hypoinsul inemic effect is assoc ia ted with the eventual onset of hyperl ipidemia (Smith and Novotny, 1980). Thus this diabetic rat model demonstrates three of the c lass ic hal lmarks of untreated type 1 diabetes. The feasibility of using this model in chronic studies w a s a s s e s s e d by Ar 'Ra jab and Ahren (1993). In a three month study it was shown that administration of S T Z at doses exceed ing 40 mg/kg resulted in a long-term, stable hyperglycemia. At these d o s e s g lucose- induced insulin release remained undetectable for the duration of the study. The S T Z rat has been widely adopted as an experimental model for chronic, untreated, type 1 d iabetes, and its use has been well reviewed by Rodr igues etal., (1999). 1.1.3 Diabetic cardiomyopathy. Although the d iscovery of insulin el iminated many acute compl icat ions of d iabetes and prolonged the life of many patients with d iabetes, it unfortunately highlighted a number of chronic compl icat ions in these patients. Card iomyopathy progressing to heart failure is one of the most common chronic compl icat ions observed in diabetic patients (Mahgoub, 1998). The first descript ion of diabetic card iomyopathy was reported over thirty years ago (Rubier et al., 1972). This compl icat ion is character ized by impaired card iac function and can be detected in the absence of hypertension and ischemic heart d i sease . Subsequen t to the initial descr ipt ion of diabetic cardiomyopathy numerous epidemiological , animal , and cl inical studies have supported the existence of diabetic cardiomyopathy as a distinct compl icat ion of d iabetes (Hamby et al., 1974; R e g a n e r a / . , 1977; Mizush ige etal., 2000). Diabetic card iomyopathy is assoc ia ted with a ser ies of morphological , b iochemical and functional 6 abnormal i t ies which are seen in both human patients and animal models of both type I and type II d iabetes (Young et al., 2002; Taegtmeyer et al., 2002). Card iac abnormali t ies, including diastol ic dysfunct ion, manifested by dec reased compl iance, prolonged myocardial relaxation and impaired left ventricular filling, as well as an increased number of supraventr icular and ventricular premature beats can be detected early in the course of d iabetes in both humans and animal models (Dent et al., 2001 ; Mihm et a l . , 2001 ; Schanwel l etal., 2002). Direct ev idence of impaired card iac function has been obtained both in vivo and in vitro. Echocard iography studies have shown that hearts from diabetic rats have impaired fractional shortening (an index of contractility) and defects in systol ic and diastol ic function (Mihm etal., 2001 ; Dent etal., 2001 ; Lin et al., unpubl ished data). Impaired contractility has also been demonstrated in the isolated whole heart and in isolated card iomyocytes (Horackova e ra / . , 1988; Fein e ra / . , 1990), indicating that altered contracti le function is due to changes occurr ing at the cellular and molecular level. 1.1.3.1 Biochemical and pathological changes in the heart during diabetes. A ser ies of b iochemical and physiological abnormal i t ies at the level of the heart, occurr ing during diabetes, have been establ ished. T h e s e changes may contribute to the assoc ia ted impaired card iac performance. For example , alterations in ion homeostas is have been demonstrated in the diabetic heart, particularly alterations in calc ium handl ing have been observed. T h e s e alterations include: impaired S R calc ium release (Cannel l et al., 1995; Cho i et al., 2002), dec reased express ion of ca lc ium release channe ls (Guner et al., 2004) and dec reased express ion of the S R C a 2 + - A T P a s e or C a 2 + pump ( S E R C A 2 ) (Zhong et al., 2001). Any alterations in the regulation of the 7 intracellular calc ium concentrat ion would have the potential to impact dramatical ly on card iac contractility. N a + - K + - A T P a s e activity has a lso been shown to be depressed and the depress ion appears to correlate with depressed atrial contractility (Tahil iani and McNei l l , 1986). There is a lso ev idence of altered regulation of contractile proteins in the diabetic heart. Severa l studies have demonstrated that myosin isoform express ion shifts from VT to V 3 in diabetic rat hearts (Malhotra etal., 1981; Di l lmann etal., 1982; Malhotra et al., 1985). These myosin isoforms differ in their A T P a s e activity, velocity of contraction and energy requirements (d'Albis etal., 1979; Schwar tz etal., 1985) . It has been suggested that very smal l myosin isoform shifts could result in marked changes in myofibrillar activity and shortening velocit ies of card iac muscle (Malhotra and Sangh i , 1997). 1.1.3.2 Metabolic changes in the heart during diabetes. Insulin insufficiency or insulin resistance results in compromised g lucose utilization during diabetes. Th is change, along with an increase in circulating free fatty ac ids (Denton and Randle , 1964), results in a switch in card iac energy metabol ism in the heart during diabetes. Increases in fatty ac id oxidation accompan ied by dec reases in g lucose uptake and metabol ism during diabetes are well establ ished (Stanley et al., 1997; Lopaschuk, 2002) . In fact, in the diabetic state energy supply to the heart is almost exclusively via fatty ac ids (Rodr igues et al., 1997). For s o m e time it has been bel ieved that this alteration in metabol ism contributes to the impaired cardiac performance observed in the diabetic myocard ium. Elevated concentrat ions of free fatty ac ids have been shown to impair card iac function in control rat hearts (Henderson etal., 1970). Fatty ac ids are suppl ied to the heart v ia the hydrolysis of l ipoproteins. In the S T Z diabetic rat heart, increased lipoprotein hydrolysis and lipoprotein l ipase activity have 8 been demonstrated (Sambandam etal., 1999; Pulinilkunnil etal., 2005). Increased fatty acid metabol ism is a lso assoc ia ted with impaired card iac function in chronic d iabetes (Borradaile et a l . , 2005). The involvement of abnormal card iac metabol ism in impaired card iac function is further supported by studies in transgenic mice. Card iac-spec i f ic overexpress ion of cardiac P P A R - a increases F A uptake and oxidation (Finck et al., 2002). Hearts from these transgenic mice, exhibited a metabolic phenotype similar to diabetic hearts (Finck et al., 2002). This was concomitant with systol ic dysfunction and ventricular hypertrophy, suggest ing altered cardiac metabol ism can induce card iac contracti le dysfunction (Finck et al., 2002). A number of explanat ions have been proposed to explain how both increased F A metabol ism and F A uptake impacts upon card iac performance during d iabetes, however, the exact mechan isms and the relative contribution of these two changes to impaired card iac function remains unclear. One possibil ity is that F A metabol ism dec reases the energy eff iciency of the heart compared to g lucose metabol ism. Oxidat ion of F A consumes more oxygen than g lucose (2.58 vs . 2.33 A T P / o x y g e n atom). Ca rd iac efficiency, the ratio of cardiac work to myocardia l oxygen consumpt ion, is therefore reduced when F A oxidation is increased. A n other effect of increased free fatty ac id levels is lipotoxicity. It is bel ieved that lipotoxicity is mediated via the accumulat ion of fatty ac ids, which can , either by themselves or v ia production of second messengers such as ceramides, provoke cell death (Listenberger and Schaffer, 2002). Lipotoxicity has been proposed to play an important role in the development of diabetic cardiomyopathy (Finck etal., 2003). The mechan isms by which lipotoxicity might mediate impaired card iac function are not clear. It is possib le that cell death reduces the number of viable card iomyocytes in the heart, thus reducing the potential for the heart to generate force. 9 Interestingly treatment of diabetic rat hearts with agents that promote a shift from fatty ac id metabol ism to g lucose oxidation (Tan e r a / . , 1984; Nicholl etal., 1991) have been shown to acutely improve card iac function in vitro. This ev idence strongly suggests that alterations in metabol ism contribute significantly to impaired card iac function in the diabetic heart. Ultimately, maladaptat ions in the diabetic heart result in cardiomyopathy and can lead to heart failure (Fein and Sonnenbl ick, 1985; Sander and Gi les , 2003). Al though a number of factors have been identified, the biochemical bas is of diabetic cardiomyopathy remains incompletely understood! At the cellular level a group of proteins cal led R h o - G T P a s e s have be studied for a number of years, and are well establ ished as regulators of cell morphology and cellular contractility. T h e s e proteins have been implicated in the development of a number of card iovascular compl icat ions including hypertension, atherosclerosis, and hypertrophic heart failure (Ren etal., 2005; Brown etal., 2006), and may therefore play a role in the pathology of diabetic cardiomyopathy. 1.2 The Rho-GTPases. R h o G T P a s e s are members of the R a s superfamily of monomer ic 20-30 k D A GTP-b ind ing proteins (Bishop and Hal l , 2000). The first R h o family G-proteins were identified in the s e a slug Ap lys ia (Madaule, 1988). Twenty two R h o family members have so far been identified (Wherlock and Mellor, 2002), which can further be subdiv ided into 8 subc lasses . However, most research work has focused on the three members , R a d , R h o A and C d c 4 2 . Th is is most likely due to the fact that they were the first G-proteins to be descr ibed as playing an important role in the morphological responses of cel ls to extracellular stimuli around 14 years ago (Ridley and Hal l , 1992). 10 The R h o G T P a s e s are molecular swi tches that function to control complex cel lular p rocesses . Rho cyc les between two conformational states (figure 1). In its active state guanosine-tr iphosphate (GTP) is bound. In its inactive state, guanos ine-diphosphate ( G D P ) is bound. O n c e active, Rho G-proteins have the ability to bind target molecu les and modulate the activity of these targets. Rho G-proteins p o s s e s s intrinsic G T P a s e activity and will generate a response until G T P hydrolysis returns the G-protein to its inactive state (with G D P bound). The activity of Rho G T P a s e s is highly modulated by severa l accessory proteins (figure 1). The binding and intrinsic hydrolysis of G T P is regulated by guanine-nucleot ide-exchange factors ( G E F s ) and GTPase-ac t i va t ing proteins ( G A P s ) , respectively. In addition Rho can a lso be bound by guanine-nucleot ide-dissociat ion inhibitors (GDIs), which alter activity by preventing the exchange of G D P for G T P , keeping R h o in an inactive state (Aspenstrom etal, 2004). The general mechan ism of activation of R h o is well establ ished. The inactive form of Rho ( R h o . G D P ) is present in the cytosol bound to a GDI . Receptor- induced activation of Rho can be mediated by G a q and/or Ga13-coup led receptors via var ious R h o G E F s , which promote the exchange of G D P for G T P (Sahai and.Marshal l , 2002). R h o A in particular has received much research attention. A vast number of studies have shown the importance of R h o A in regulating signal ing c a s c a d e s which affect cel lular contractility (Hall , 1998; Nobes and Hal l , 1999; Fukata and Kaibuch i , 2001 ; Et ienne-Mannevi l le and Hal l , 2002; Somlyo and Somlyo , 2003; Aspenst rom etal., 2004). For some time, the literature has been unclear when d iscuss ing the role of the Rho proteins. Three subc lasses of Rho G-proteins: RhoA, R h o B and R h o C have now been identified (Wheeler and Ridley, 2004). T h e s e three G T P a s e s share 8 5 % amino acid sequence identity, but despite their similarity, 11 Receptor Stimulus :'RhoA-GDP) * ( R h o - G D I / " Sequestration •* |f?hoA-GDP) Inactive '^SSi*-^^ Active RhoA-GTPJ ROCK Cytoskeletal Regulation F i g u r e 1: R h o G T P a s e regu la t i on . G E F s promote the exchange of G D P for G T P ; GDIs interact with G D P - b o u n d Rho and inhibit exchange of G D P for G T P ; G A P s increase the intrinsic G T P a s e activity of Rho G T P a s e s . [(adapted from: Nature Rev iews C a n c e r 2; 133-142 [2002]). 12 regulators and effectors show preferential interaction with e a c h of the three spec ies , R h o A , B, or C . The three proteins exhibit distinct functions in cel ls (Wheeler and Ridley, 2004; Aspens t rom era / . , 2004). 1.2.1 The cellular functions of RhoA. The wel l -establ ished functions of R h o A highlight this protein as an important target for research when investigating the molecular bas is of compl icat ions such as cardiomyopathy, where cardiomyocyte contractility is significantly impaired. It has been shown that R h o A plays an important role in the regulation of a number of dynamic cellular p rocesses including: fatty acid metabol ism (Pulinilkunnil et al., 2005), hypertrophic responses to stimuli (Torsoni e r a / . , 2005), regulation of intracellular C a 2 + concentrat ion (Suematsu et a l . , 2002; Yatani et al., 2005), C a 2 + sensit izat ion (Somlyo, 2002) and regulation of the actin cytoskeleton (Ridley and Hal l , 1992b; N o b e s and Hal l . 1995). 1.2.1.1 RhoA and the actin cytoskeleton. Actin is a structural protein that is a vital component of the cardiomyocyte cytoskeleton. The individual subunits of actin are known as globular actin (G-actin). G -actin subunits polymerise to form a f i lamentous polymer (F-actin), a lso known as a microfi lament. These microfi laments form the cytoskeleton, a three d imensional network found inside eukaryotic cel ls. The cardiomyocyte cytoskeleton provides structural support and compartmental isat ion of intracellular components (Watson, 1991) as well as regulating intracellular vesicular transport and protein synthesis (Rogers and Ge l fand , 2000). In both neonatal and adult cultured myocytes, distinct sarcomer ic and costamer ic (cytoskeletal) actin have been identified (Sadosh ima etal., 1992; Messer l i and Perr iard 1995; Larsen et al., 1999 and Larsen et al., 2000). Here we will focus 13 solely upon cytoskeletal act in, the functions it p lays in the cardiomyocyte and regulation of the actin cytoskeleton by RhoA . The ability of the actin cytoskeleton to modulate both electrical and mechanica l activity of the cardiomyocyte has been well reviewed (Ca laghan et al., 2004). The microfi laments have been implicated in regulating the concentrat ion of intracellular C a 2 + (Lange and Brandt, 1996), a key regulator of cardiomyocyte contractility (Bers and G u o , 2005). Lange and Brandt (1996) suggest that actin f i laments can bind C a 2 + with high affinity and that actin polymerisat ion renders bound C a 2 + less avai lable, effectively creating a C a 2 + store. Th is is supported by their ev idence showing that the F-actin stabil izing agent, phalloidin, strongly inhibited A T P -dependent C a 2 + uptake and reduced the IP3-sensit ive C a 2 + pool by 7 0 % . Converse ly , when actin f i laments were depolymer ised, for example following treatment with Cytocha las in-D, free C a 2 + was elevated from 50 nM to 500 n M (Lange and Brandt, 1996). Ev idence is emerging on the role played by the actin cytoskeleton in the regulation of p lasma membrane C a 2 + - p e r m e a b l e channe ls as well as the intracellular calc ium concentrat ion. Disruption of the cytoskeleton by actin depolymeriz ing agents inhibits hormone- induced C a 2 + re lease from l n s P 3 - and ryanodine-sensit ive stores in NIH 3T3 cel ls (Ribiero et a l . , 1997), A R 4 - 2 J cel ls (Bozem et a l . , 2000), and hepatocytes (Wang et a l . , 2002). Moreover, actin depolymerizat ion has been reported to reduce C a 2 + influx through voltage-gated C a 2 + channe ls (Ruecksch loss et a l . , .2001) , while store-operated C a 2 + influx through transient receptor potential (TRP) ion channe ls is affected by both actin polymerization and depolymerizat ion (Rosado and S a g e , 2001 ; W a n g et al., 2002). In cardiomyocytes increased actin polymerizat ion has been assoc ia ted with 14 decreased contractility due to disruption of intracellular C a release (Davani et a l . , 2004). There is a lso ev idence to suggest that in card iomyocytes the actin cytoskeleton can regulate the metabol ism of fatty ac ids (Pulinilkunnil et al., 2005), the preferred energy substrate of the heart (Randle et al., 1963; Rodr igues et al., 1995). Elevated levels of polymer ised actin were shown to be assoc ia ted with increased lipoprotein l ipase activity in rat card iomyocytes. Th is effect was blocked by actin depolymer is ing agents (Pulinilkunnil etal., 2005), strongly suggest ing a role for the actin cytoskeleton in the regulation of fatty ac id metabol ism. G iven the number of observat ions linking the actin cytoskeleton with factors known to influence myocardial contractility, it is not surprising that a number of studies have implicated the involvement of cytoskeletal abnormali t ies in condit ions of impaired cardiac performance such as cardiomyopathy and heart failure (Hein et al., 2000; Kost in et al., 2000; Py le , 2004). R h o A and it's downstream targets are known to be of great importance in maintaining the state of cel lular actin polymerization (figure 2). Rho-k inase ( R O C K ) , a wel l -known downstream target, which is directly activated by R h o A , inc ludes two isoforms, R O C K I (also known as p 1 6 0 R O C K or R O K b ) and ROCKI I ( R O K a ) (Nakagawa et al., 1996). R O C K activity can be select ively b locked by the competit ive inhibitor Y -27632 , as well as the isoquinoline sulfonyl derivative H A 1 0 7 7 and its more potent and select ive derivative, H1152 (Uehata et al., 1997; S a s a k i et al., 2002; Brei tenlechner et al., 2003). R O C K is a serine/threonine kinase which phosphorylates and act ivates its downstream target, LIM k inase, which in turn directly phosphorylates cofilin on the N-terminal Se r3 residue (Arber et a l . , 1998; Bamburg , 1999). Cofi l in is an actin regulatory protein that plays a critical role in regulating actin fi lament dynamics v ia 15 its actin sever ing and depolymer iz ing activity (Huang et a l . , 2006). The phosphorylated form of cofilin is unable to bind and depolymer ise F-actin (Agnew et a l . , 1995). In this way R O C K , v ia LIM k inase and Cofi l in, inhibits actin depolymerisat ion, thus stabil izing f i lamentous actin. A functional R h o A / R O C K / L I M - k i n a s e pathway has been shown to be required for actin polymerizat ion in lymphocytes (Lou et a l . , 2001) and in rat card iomyocytes (Zeidan et a l . , 2006). Activation of RhoA , has been shown to result in significantly increased levels of polymer ized actin in rat card iomyocytes (Pulinilkunnil et a l . , 2005 ; Ze idan et a l . , 2006). 16 (mDja) • (Profiling (ROCK) Myosin phosphatase Actin polymerization (Myosin light chain Xg) (Cofi l in)-® Actomyosin contractility Inhibition of actin depolymerization cell contractility Figure 2: Mode of action of Rho/ROCK in cytoskeletal reorganization and cellular contractility. [Taken from: Taka i et a l . , (2001)] 17 1.2.2 A pathological role for the RhoA/ROCK Pathway? The R h o A / R O C K pathway is an important regulator of physiological function (Narumiya, 1996; Burridge and Wnnerberg , 2004), and is bel ieved to play a role in the pathophysiology of many d i seases , including hypertension (Chitaley and Weber , 2001), myocardial hypertrophy (Kobayash i et al., 2001) and heart failure (Hu and Lee, 2003). The development of specif ic R O C K inhibitors has al lowed extensive studies a imed at determining potential pathological roles for this signall ing pathway. R O C K activity can be select ively b locked by the competit ive inhibitor Y -27632 , a s well a s the isoquinol ine sulfonyl derivative HA1077 , and its' more potent and select ive derivative, H1152 (Sasak i et al., 2002; Brei tenlechner et al., 2003). Treatment with R O C K inhibitors has been shown to effectively reduce blood pressure in rats with spontaneous or angiotensin II -mediated hypertension (Uehata etal., 1997; Mukai etal., 2001), and in hypertensive patients in a cl inical trial (Masumoto et al., 2001). The importance of Rho-media ted signall ing in card iac contractility w a s illustrated by Kobayash i et a l . , (2002). In this study Dahl salt-sensit ive hypertensive rats were chronical ly treated with the specif ic R O C K inhibitor Y -27632 . Inhibition of this downstream signal ing target of R h o A resulted in signif icant improvement in card iac function and helped prevent progression to heart failure. A role for the R h o A / R O C K pathway in card iac hypertrophy is supported by a number of studies. Mutational deletion of R O C K was shown to prevent pressure over load- induced hypertrophy in a mouse knock-out model (Zhang et al., 2006). Furthermore activation of R h o A by L P A has been shown to induce hypertrophy in cultured rat card iomyocytes (Hi la l -Dandan e ra / . , 2004). Th is effect was b locked by the R h o A inhibitor C3 -exoenzyme (Hi la l -Dandan etal., 2004; W e i , 2004). 18 Increased activation of the R h o A / R O C K pathway has been demonstrated in vascu lar (Tang etal., 2006) and erecti le t issue from animal models of type 1 and type 2 d iabetes (Chang et al., 2003), but whether this a lso occurs in the heart and contributes to contractile dysfunction is not yet known. However, previous work in this lab has demonstrated that acute inhibition of R O C K with the inhibitors Y -27632 and H-1121 significantly improves card iac function in 12 week S T Z diabetic rats (unpubl ished data). Th is has been demonstrated both in vitro using isolated, perfused working hearts (figure 3) and in vivo using echo cardiography (figure 4). 19 Figure 3: Effect of Y-27632 on the function of isolated working hearts from control and diabetic rats. Control (black squares) , control treated with inhibitor (black triangles), diabetic (white squares) , and diabetic treated with inhibitor (white triangles). Hearts were perfused with 10" 6 M Y -27632 (n=7 in each group), # significantly different from the diabetic treated with the inhibitor. *, P< 0.05 significantly different from all other groups. @ Three of seven diabetic hearts failed at this L A F P . [Taken from Lin et al., unpubl ished data]. 20 Figure 4: Effect of H-1152 on percent fractional shortening (% FS) of control and diabetic left ventricle. % F S determined by transthoracic echocard iogram in vivo. Control before (C), control following treatment (C+H), diabetic before (D), and diabetic fol lowing treatment (D+H). #, significantly different (P<0.05) from control (C); and @ , significantly different (P<0.05) from diabetic untreated (D) by repeated measure A N O V A fol lowed by Newman- Keu ls test (n=6 in each group). [Taken from Lin et al., unpubl ished data]. 22 1.3 Nitric oxide and the heart. Nitric oxide (NO) is a gaseous product generated from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS) . Three forms of N O S have so far been identified; endothelial nitric oxide synthase (eNOS) , inducible nitric oxide synthase ( iNOS) and neuronal nitric oxide synthase (nNOS) . n N O S and e N O S are constitutively active isoforms with calmodul in binding (and, consequent ly, enzyme activity) regulated by physiological [Ca 2 + ] , (Kelly et al., 1996). Unlike the other isoforms, i N O S activity is independent of [Ca 2 + ] , and is regulated primarily at the transcriptional level. O n c e express ion is induced i N O S cont inuously produces N O until the enzyme is degraded (MacMick ing et al., 1997). For some time N O has been regarded as an important bioregulatory molecule and its roles in the nervous, immune and card iovascular sys tems have been well reviewed (Anggard, 1994; B la ise et al., 2005). It is well establ ished that N O plays a regulatory role in the modulat ion of coronary vesse l tone, thrombogenicity, and proliferative and inflammatory properties as well as the cellular cross-talk support ing angiogenes is . Numerous studies have suggested that N O is an important, indirect regulator of card iac function (see review by Kelly et al., 1996). The recognition that all three isoforms of N O S are expressed in card iac myocytes (Bal l ingand etal., 1994; Feron etal., 1996; Elfering etal., 2002) has raised severa l intriguing quest ions regarding the role of N O in the heart, and whether direct effects on myocardial contractility might exist. Stud ies in the past decade have provided extensive ev idence to suggest that N O can in fact directly modulate myocardial contractility (Brady etal., 1992; Bal l igand etal., 1993; Mass ion etal., 2003 ; Be ige etal., 2005). The exact mechan isms of these direct effects on myocardial contractility remain unclear. A number of studies have been content ious in that they suggest that N O S or N O can play both 24 cardio-protective as well as detrimental roles, though this appears to depend on the source of N O and the level of N O output (Cotton et al., 2005). Genet ic knock-out models have helped our understanding of the role of the N O S and N O in pathological condit ions such as heart failure (see review by Mungrue et al., 2002b). In e N O S knockout mice S R calc ium handling is altered and cardiomyocyte contractility reduced (Sears et al., 2003). i N O S knock-out mice show improved cardiac performance and remodell ing after myocardial infarction (Sam et al., 2001 ; Liu et al., 2005). It has a lso been shown that i N O S knockout mice have better survival rates after card iac allograft surgery (Koglin et al., 1999). n N O S knockout mice have been shown to have significantly dec reased l i fe-span and card iac performance (Li et al., 2004). It is important to appreciate that these genetic knock-out models may not truly reflect the cellular functions of the targeted protein or its product. Attributing physiological re levance to observat ions from knock-out models is difficult. Other experimental techniques have been adopted to a s s e s s the roles of the N O S isoforms. Us ing an i N O S inhibitor, S-methyl isothiourea, Wildhirt et al., (1996), were able to improve left ventricular performance and survival rates after acute myocardial infarction in New Zea land rabbits. Us ing a different experimental approach, Mungrue e r a / . , (2002), have deve loped a transgenic mouse model condit ionally targeting the express ion of human i N O S c D N A to the myocardium. T h e s e mice have been shown to display card iac f ibrosis, hypertrophy, and dilatation as well as a high incidence of sudden card iac death due to bradyarrhythmia (Mungrue e ra / . , 2002a). Stud ies have a lso looked for ev idence of altered N O S express ion under pathological condit ions. It has been shown that i N O S express ion is significantly elevated in biopsy samp les taken from the myocardium of patients with heart failure (Haywood et al., 1996; Habib e r a / . , 1996). In 12-week S T Z 25 diabetic rats, i N O S express ion is significantly e levated (Nagareddy etal., 2005), at time point when cardiomyopathy is well establ ished in this model . The ev idence reviewed above suggests that i N O S plays a pathological role in c a s e s of impaired card iac function. However, the mechanist ic and functional consequences of altered N O S express ion and N O bioactivity in the failing human heart remain unclear. 1.3.1 Evidence for an interaction between NO and RhoA. A s descr ibed above (1.2, figure 1), the regulation of activation of R h o A and its role as a 'molecular switch' controll ing cel l s ignal ing pathways has been well character ized. O n the other hand, very few studies have addressed which factors regulate the express ion of RhoA . However, recent ev idence points towards an interaction between N O and RhoA . It has been shown that express ion of R h o A in rat aortic smooth musc le cel ls is highly inducible by nitric oxide (Sauzeau et a l . , 2003). In this study, exposure of cultured vascular smooth musc le cel ls to the N O donor molecule, S N P , for 18 hours resulted in a significant increase in R h o A express ion. Interestingly it has been shown that in S T Z diabetic rat hearts, express ion of i N O S is significantly elevated (Nagareddy et a l . , 2005). Th is is concomitant with significantly increased R h o A express ion in the s a m e model (preliminary data, not shown). Taken alone these are solely assoc ia ted observat ions, nevertheless they provide us with the foundation for an interesting hypothesis to be tested using the S T Z diabetic rat model . 26 1.4 Hypothesis and rationale for proposed experiments. 'During diabetes, the elevated expression of iNOS in rat hearts results in increased RhoA expression and impaired cardiomyocyte contractility via alterations in actin cytoskeleton dynamics'. A s d i scussed above, our lab and others have shown that heightened i N O S express ion is concomitant with increased R h o A express ion (preliminary data, not shown), in the 12 week S T Z diabetic rat model . O n c e induced, i N O S generates large amounts of N O until the enzyme is degraded (MacMick ing et al., 1997). Ev idence from vascular smooth muscle cel ls suggests that N O can induce increased levels of R h o A express ion (Sauzeau et a l . , 2003). W e therefore wish to investigate whether increased i N O S express ion is responsib le for the increased R h o A express ion observed in the heart during diabetes. Work conducted in this lab has shown that acute inhibition of the R h o A / R O C K signal ing pathway c a u s e s rapidly improved card iac performance in hearts from chronic diabetic rats (figures 3 and 4). Th is ev idence suggests that the R h o A / R O C K signal ing pathway may be upregulated in hearts from 12 week S T Z diabetic rats. W e therefore wished to a s s e s s whether increased express ion of R h o A in the diabetic rat heart is assoc ia ted with increased activity of the R h o A / R O C K pathway. It is a lso important to determine what consequences any change in activity of the R h o A / R O C K pathway might have in the diabetic rat heart. G iven the well establ ished role of R h o A in the regulation of the actin cytoskeleton in eukaryotic cel ls , we proposed to investigate whether any alterations in actin cytoskeletal dynamics could be detected in card iomyocytes from diabetic hearts, and a s s e s s the effects of acute R O C K inhibition on the actin cytoskeleton in these cel ls. 27 1.5 Specific objectives. 1) Conf i rm that express ion of R h o A and i N O S is up-regulated in diabetic card iomyocytes. 2) Determine whether N O induces elevated express ion of R h o A in isolated card iomyocytes. 3) Determine if i N O S induction results in increased R h o A express ion in isolated card iomyocytes. 4) A s s e s s any alterations in the activity of the R h o A / R O C K pathway in diabetic card iomyocytes. 5) Determine if chronic inhibition of i N O S restores basa l levels of express ion of R h o A and activity of the R h o A / R O C K pathway in diabetic hearts. 6) A s s e s s any changes in the dynamics of the actin cytoskeleton in diabetic card iomyocytes, and the influence of R O C K inhibition on these changes . The molecular mechan isms that underlie pathogenic changes in the heart during chronic d iabetes remain poorly understood. In this study we will attempt to determine the cause of altered R h o A / R O C K signal ing during d iabetes and illustrate the pathological consequences of these changes . This investigation should provide insight 28 into the mechan isms contributing to the development of diabetic cardiomyopathy, and may potentially highlight new pharmacological targets for the treatment of this chronic compl icat ion of d iabetes. 29 2.0 M A T E R I A L S A N D M E T H O D S 2.1 C h e m i c a l s a n d ma te r i a l s Chemica l s and materials were purchased from or provided by the following sources : Amersham International E C L Western blotting detecting reagent, P o n c e a u S stain. B i o R a d Laborator ies 2-mercaptoethanol , Acry lamide (99.9%), glycine, polyvinyl idene fluoride ( P V D F ) , Prec is ion P lus Protein™ Standards, tris base, 1.5M tris pH 8.8, 0 .5M tris pH 6.8, sodium dodecyl sulfate (SDS) , T E M E D . B D B iosc iences M o u s e ant i - iNOS primary antibody. Boehr inqer Manhe im G m b H Bovine serum albumin (BSA) . Ca lb iochem C o r p DNAasel-AlexaFluor®594, Phal loidin-AlexFluor®488. C a y m a n Chemica l i N O S activity a s s a y kit. Ce l l S ignal ing Technology Phospho-L IMK1 Antibody. Cytoskeleton Inc. G-act in/F-act in In Vivo A s s a y Kit, G-LISA™ R h o A Activation A s s a y B iochem K i t™. 30 Fisher Scienti f ic C o Ethanol , Methanol . G I B C O Life Techno log ies Steri le Phosphate Buffered Sal ine ( P B S ) . Med iaas Ca rbogen (5% Carbon Dioxide, 9 5 % Oxygen) . M T C Pharmaceut ica ls Somnito l® (Sodium Pentobarbital). New England Bio labs Inc. L-N6-(1-iminoethyl)- lysine (L-NIL), Y -27632 . Organon Techn ika Inc Hepar in Sulfate. Roche Appl ied Sc ience Laminin. San ta C ruz Biotechnology Inc G A P D H antibody, R-Actin antibody, R h o A antibody, R O C K I antibody, all secondary H R P conjugated ant ibodies. Sa feway Stores Sk im milk powder. S i g m a Chemica l C o . Ammon ium persulfate, aprotinin, ca lc ium chloride, E D T A , E G T A , g lucose (99.9%), halothane, H E P E S , l-carnitine, leupeptin, lysopolysacchar ides, magnes ium sulfate, medium 199, penicil l in, phenylmethylsulfonyl fluoride ( P M S F ) , phosphatase inhibitor cocktai l I, phosphatase inhibitor cocktai l II, potassium chloride, potassium phosphate, 31 sodium chlor ide, sodium fluoride, sod ium nitroprusside, streptomycin, Streptozotocin, taurine, Triton X -100 , trypan blue solution. Worthington B iochemica l Corp Co l l agenase (Type II, C L S II) 2.2 Animals. Male Wistar rats were obtained from the U B C Animal Ca re Facility. An ima ls were housed in pairs. Water and standard rat chow was avai lable ad libitum. An ima l care was given in accordance with the principles and guidel ines of the Canad ian Counc i l on An imal C a r e and the U B C Animal C a r e Commit tee. 2.2.1 Induction of diabetes. Male Wistar rats weighing 180-200 g were al lowed to accl imat ize to the local vivarium for at least 3 days prior to induction of d iabetes. Rats were al located into groups randomly and made diabetic v ia a single tail vein injection of S T Z d isso lved in citrate buffer (55 mg/kg body weight). Control rats were administered equivalent vo lumes of citrate buffer v ia single tail vein injection. B lood g lucose concentrat ion was measured 72 hours after S T Z administration. Induction of d iabetes was conf i rmed by the presence of hyperglycemia (blood g lucose £18 mmol/L). For most exper iments animals were kept for 12-15 weeks prior to sacri f ice and col lection of heart t issue and card iomyocytes for analys is. At time of sacri f ice blood samp les were taken, and blood g lucose levels were a s s e s s e d . Al l diabetic an imals exhibited blood g lucose levels of >18 mmol/L. The S T Z diabetic rat model is well establ ished in our laboratory and all an imals exhibited the typical character ist ics and symptoms which we have previously 32 observed in this lab, this includes significant dec reases in body weight, polyuria, polydipsia and diarrhea. 2.2.2 Chronic inhibition of iNOS in control and STZ diabetic rats. Age-matched male Wistar rats were separated into two groups at random. O n e group was made diabetic using S T Z , while the other was treated with vehicle as descr ibed above (2.2.1). O n e week after injection of S T Z , control and diabetic rats were sub-div ided into two groups; treated and untreated. Treated animals received the select ive i N O S inhibitor, L-NIL at a dose of 3mg/kg/day by oral gavage (Ferrini et a l . , 2004; Lee et a l . , 2005). Untreated control and untreated diabetic an imals received equivalent vo lumes of vehicle by oral gavage. Eight weeks later an imals were sacr i f iced. Ventr icular card iac t issue was prepared as desr ibed below (2.3.2) 2.3 Preparation of rat cardiac tissue. 2.3.1 Preparation of isolated rat ventricular myocytes. The isolation procedure was modif ied from the protocol of Huang etal., (2005). Rats were anaesthet ized via a single intraperitoneal injection of sod ium pentobarbital (60 mg/kg body weight), co-administered with heparin (1000 units/kg body weight). The hearts were rapidly exc ised into ice-cold, pre-oxygenated C a 2 + - f r e e Tyrode 's solution (containing 100 m M N a C l , 10mM K C ! , 1.2 m M K H 2 P 0 4 , 5 m M M g S 0 4 , 5 0 m M taurine, 20 m M g lucose, 10 m M H E P E S ) . A n y extraneous t issue was quickly removed from the heart. Hearts were then mounted on Langendorff perfusion apparatus and perfused retrogradely at a constant flow of 8 ml/min with oxygenated C a 2 + - f r e e Tyrode 's solution at 37 °C. After the coronary perfusate had c leared of blood, perfusion was cont inued for 4 min with C a 2 + - f r e e Tyrode 's solut ion. Subsequent ly the hearts were perfused for 16 33 min with Tyrode 's solution containing 0.05 m M C a , 0.8 mg/ml co l lagenase and 0 . 1 % B S A . After this time, the ventricles were exc ised from the heart, minced and gently shaken in the col lagenase-conta in ing isolation solution. Th is cell suspens ion was filtered through a 200 micron nylon mesh into a 50 ml culture tube. The solution was then centrifuged for 45 seconds at approximately 60g in a cl inical centrifuge. The resulting supernatant was decanted and the cell pellet was resuspended in Tyrodes solution containing 0.2 m M C a 2 + . Ce l l s were al lowed to settle for 10 minutes. Th is washing step was repeated twice with Tyrode 's solution containing 0 .5mM and then 1 m M C a 2 + . Card iomyocyte counts were taken using a haemocytometer and viability was determined by assess ing the percentage of cel ls that exc luded trypan blue dye. Ce l l viability was greater than 6 5 % in all groups. Card iomyocytes were then used in exper iments as descr ibed below or snap frozen in liquid nitrogen for future western blotting experiments. 2.3.1.1 Primary culture of isolated ventricular myocytes. Card iomyocytes were isolated and made calc ium tolerant as descr ibed above. Myocytes were then resuspended in medium 199 supplemented with 1% B S A , 100 units/ml penicil l in, 100 pg/ml streptomycin, 1.2 m M L-carnitine and 2.5 M H E P E S (pH 7.4). Ce l l s prepared from a single rat heart were plated on laminin coated culture-d ishes. Ce l l s were plated at a density of 0.75 x 1 0 6 cel ls per 100 mm dish. Culture d ishes were p laced in a 37 °C incubator with a maintained atmospher ic C 0 2 level of 5%. V iab le cel ls were given 3hrs to recover from the isolation p rocess and adhere to the laminin-coated culture d ish. After this incubation period the media and unattached cel ls were aspirated off. Fresh supplemented media 199 was then added to the culture d ish . At this time any drugs were a lso added to the cel ls if required. 34 2.3.1.2 Induction of iNOS by LPS in cultured ventricular myocytes. Card iomyocytes were isolated and plated on laminin coated culture d ishes as descr ibed above. After attachment, med ia and unattached cel ls were aspirated off. Fresh supplemented med ia 199 was then added to the culture d ish. Ce l l s were then exposed to 50 ug/ml L P S for 18hrs. Prel iminary studies had shown that a concentrat ion of at least 40 ug/ml L P S was required for i N O S induction in this culture preparation (figure 5). After 18hrs cel ls were inspected to ensure that.they had maintained viability and retained a rod shaped phenotype. At this time media was aspirated away and cell lysates prepared as desr ibed below (2.3.2) 2.3.2 Extraction of total protein from cultured myocytes. After complet ion of the required incubation per iod, cultured myocytes were washed twice with ice-cold P B S . After being well dra ined, the cel ls, kept on ice, were treated with 200 pL of lysis buffer (100 p M Tr i s -HCl pH 7.5, 5 0 m M N a F , 5 m M Sod ium Pyrophosphate, 0.5 m M E D T A , 2 m M E G T A , 1% (v/v) G lycero l , 2 % Sod ium Az ide , 1% N P 4 0 , 0 . 1 % S D S , 2pg/ml aprotinin, 25ug/ml leupeptin, 1% (v/v) phosphatase inhibitor cocktail). Ce l l s were incubated with the lysis buffer on ice for 10 minutes. Ce l l s were then scraped off the dish surface and the cell lysates were col lected in Eppendorf tubes. Ce l l s were homogen ized via fine needle tituration and brief sonicat ion on ice. To remove unbroken cel ls and nuclei , lysates were spun at 700g for 5 minutes in a B e c k m a n Al legra® 21R centrifuge. At this point samp les were taken for protein a s s a y (see 2.4). 35 iNOS G A P D H 20 40 (Mg/mL LPS) B 20 ug/mL LPS F i g u r e 5: Induc t ion of i N O S b y L P S . (A) Representat ive western blot showing effect of varying concentrat ions of L P S on i N O S express ion . (B) Band O .D . va lues were normal ized by their corresponding G A P D H band value and then expressed relative to the mean control value. Resul ts shown are means (± S E M ) . *, P< 0.05 significantly different from control group. Statistical analys is was performed using a one-way A N O V A . 36 2.3.3 Extraction of total protein from rat ventricular tissue. Rats were anaesthet ized a s descr ibed above. Hearts were removed and rinsed in ice-cold P B S to remove e x c e s s b lood. Ventr icular t issue was cut away from the heart and snap frozen in liquid nitrogen. Frozen ventricular t issue was subsequent ly c rushed in a liquid nitrogen cooled mortar and pestle. Heart powders were col lected in cryovials and stored at -70 °C for future exper iments. For homogenizat ion, approximately 150 mg of the heart powder was col lected in ice-cold polyethylene homogenizat ion tubes. The tubes were kept on ice and 1.5 ml homogenizat ion buffer w a s added to each tube. The composi t ion of the homogenizat ion buffer was as fol lows: 20 m M Tr i s -HCl (pH 7.5), 50 m M 3-mercaptoethanol , 5 m M E G T A , 2 m M E D T A , 10 m M N a F , 1mM P M S F , 25 pg/ml leupeptin, 2 pg/ml aprotinin, 0 . 1 % N P 4 0 , 0 . 1 % S D S , 0 . 1 % deoxychol ic ac id , 1% phosphatase inhibitor cocktai l . Heart powder was homogen ized using a K inemat ica Polytron homogeniser ( 3 x 1 0 second bursts, probe at setting 7, with the samp les kept on ice). To remove unsolubi l ized protein and cel ls, homogen ized samp les were centrifuged at 700g for 5 minutes in a B e c k m a n Model J2-21® centrifuge. Supernatants from this centrifugation were transferred to Eppendorf tubes and kept as 'total protein samples ' . Protein concentrat ions in these samp les was then determined (see 2.4). 2.4 Protein concentration determination. After harvesting protein samp les from cel ls or t issues the concentrat ion of protein in these samp les was determined using the B io -Rad Protein Assay™. This a s s a y is based on the Bradford dye-binding procedure (Bradford, 1976) which measures the colour change of C o o m a s s i e Brilliant B lue G-250 dye when it binds to protein through basic and aromatic amino acid residues. To determine protein concentrat ion the 37 manufacturers instructions were carefully fol lowed. For each sample absorbance was read at 595 nm for compar ison with a B S A standard curve (0.0625 mg/ml - 1 mg/ml). 2.5 SDS-Polyacrylamide gel electrophoresis and immunoblotting of proteins from rat ventricular tissue and rat ventricular myocytes. Prior to electrophoresis all samp les were made up to a final volume of 500 pL at a concentrat ion of 2 mg/ml in sample buffer ( S D S reducing buffer). The composi t ion of the sample buffer w a s as fol lows: 2 % (w/v) S D S , 120 m M tris-HCI pH 6.8, 1 0 % (v/v) glycerol , 5 % (v/v) B-Mercaptoethanol , 0 .004% (w/v) bromophenol blue. The samp les were then heated to 100 °C for 5 minutes. S D S - P A G E gels were cast following the method of Laemml i (1970). The resolving gel contained 1 0 % (w/v) total acry lamide, 375 m M tris-HCI, 0 . 1 % S D S (w/v), 0 .08% (w/v) ammonium persulfate and 0 .03% (w/v) T E M E D . The stacking gel contained 4 % (w/v) total acry lamide, 125 m M tris-HCI (pH 6.8), 0 . 1 % S D S (w/v), 0 .08% (w/v) ammonium persulfate and 0 .05% (w/v) T E M E D . The upper and lower tank was filled with running buffer (25 m M tris, 192 m M glycine, 0 . 1 % (w/v) S D S , pH 6.8). Un less otherwise stated 25 uL of each sample or 15 pL of Prec is ion P lus Protein™ standards were loaded into their appropriate wells in the stacking gel . Proteins were electrophoretical ly separated through the gel under a constant voltage of 125 volts for 75 minutes using a B io -Rad Protein II e lectrophoresis unit. After electrophoresis a B io -Rad Cri ter ion™ Blotter was used to transfer the resolved proteins onto a P V D F membrane. Transfer was conducted at a constant current of 250 mA for 1 hour in ice-cold transfer buffer. The transfer buffer composi t ion was : 25 m M tris, 192 m M glycine, 2 0 % (v/v) methanol , pH 8.3. After transfer, membranes were briefly stained 38 with P o n c e a u S stain to confirm successfu l protein transfer. E x c e s s stain was subsequent ly removed by wash ing the membrane for 10 minutes in T T B S . The P V D F membrane was treated for 2 hours at room temperature in blocking buffer (3% (v/w) sk im milk powder in T T B S ) , to reduce non-specif ic antibody binding. The membranes were subsequent ly washed with T T B S (3 x 10 minutes). If more than one protein was being detected membranes were cut using a sterile surgical b lade to isolate molecular weight regions containing the protein of interest. The membrane was then incubated overnight at 4 °C with the appropriate primary antibody. Pr imary ant ibodies were diluted in 5 % (w/v) B S A in T T B S . The membrane was then washed (3 x 10 minutes) in T T B S and then incubated for 1 hour at room temperature with an appropriate secondary antibody. Secondary ant ibodies were diluted 1:10,000 in 5 % milk powder in T T B S . Membranes were washed again with T T B S ( 3 x 1 0 minutes). Pr imary antibody binding was detected using the E C L technique. Equa l amounts of reagent A (0.45 mg/ml Luminal in 0.1 M tris, pH 8.5) and reagent B (0.2% H 2 0 2 in 0.1 M tris, pH 8.5) were mixed thoroughly. The mixed reagents were incubated on the blots for 2 minutes before exposing the drained blots to the x-ray film. 2.6 iNOS Activity Assay. A commercia l ly avai lable i N O S activity a s s a y was used to determine i N O S activity in whole heart homogenates (Cayman Chemica l Ml) . Extraction of protein from ventricular card iac t issue was carr ied out following the manufacturers protocol. Al l samp les were measured in triplicate. 39 2.7 G-LISA™ RhoA Activation Assay. A commercia l ly avai lable R h o A G - L I S A kit (Cytoskeleton Inc, C O ) was used to determine the amount of active R h o A present in protein samp les from ventricular myocytes. Myocytes were isolated as descr ibed previously (2.3.1). Ce l l lysates from freshly isolated myocytes were col lected as descr ibed in the manufacturers protocol. Samp le absorbances were read at 490 nm using a microplate spectrophotometer. Samp le wells containing lysis buffer only were designated a s the assay blank. A constitutively active Rho control protein was used as the assay positive control. Al l samp les were measured in triplicate. 2.8 G-actin / F-actin Assay. 0.5 x 1 0 6 freshly isolated, viable cel ls were resuspended in 0.5 ml lysis and F-actin stabil ization buffer (LAS) . The L A S composi t ion was as fol lows: 50 m M P I P E S (pH 6.9), 50 m M KCI, 5 m M M g C I 2 , 5 m M E G T A , 5 % (v/v) Glycero l , 0 . 1 % N P 4 0 , 0 . 1 % Triton X -100 , 0 . 1 % Tween 20, 0 . 1 % G-mercaptoethanol, 0 . 001% Anti foam C . Ce l l s were gently homogen ized v ia fine needle tituration. Lysates were then spun at 2000 rpm for 5 minutes to pellet unbroken cel ls. The supernatant from this spin was spun at 100,000g for 1 hour at 4 °C in a B e c k m a n L8-60M® ultracentrifuge. The supernatants from this spin were col lected in labeled eppendorf tubes and kept as the G-act in containing fraction. Pel lets were resuspended to the s a m e volume as the G-act in fraction using L A S . Th is was kept as the F-actin containing fraction. 40 pL of S D S sample reducing buffer was added to 160 pL of each of the F-actin and G-act in fractions. S a m p l e s were then heated to 100 °C for 5 minutes. S a m p l e s were then loaded onto a 1 0 % S D S -polyacrylamide gel and electrophoretical ly separated following the protocol descr ibed previously (2.5). G e l s were blotted onto P V D F membranes and actin immunoblots were 40 carr ied out as decr ibed previously (2.5). The optical density of each band was determined using a scann ing densitometer. The ratio of G-act in to F-actin was calculated by dividing the optical density value of the appropriate F-actin band by its corresponding G-act in band. 2.9 Immunofluorescence labeling and Confocal Microscopy. Myocytes were isolated from hearts as descr ibed previously (2.3.1). Ce l l s were then plated on poly-L- lysine coated cover s l ips and rinsed with P B S . Myocytes were fixed for 10 min with 4 % paraformaldehyde in P B S , permeabi l ized with 0 . 1 % Triton X -100 in P B S for 3 min, treated with P B S containing 1% B S A for 20 minutes, and finally r insed with P B S . Ce l l s were double stained with DNAasel-AlexaFluor®594 and Phalloidin-AIexFluor®488 to colocal ize monomer ic globular actin (red, G-actin), and polymerized f i lamentous actin (green, F-actin) (Pullinikunil et al., 2005). The unbound f luorescent probe was rinsed with P B S buffer and s l ides were v isual ized using a Bio-Rad Radiance-600® Confoca l M ic roscope at 1260X magnif ication. 2.10 Statistical Analysis. Un less otherwise stated, all va lues are expressed as means ± S E M ; n denotes the number of an imals in each group. For all results the level of s igni f icance was set at P < 0.05. For multiple compar isons of more than two experimental groups a 2-way A N O V A fol lowed by a Newman-Keu l s post-hoc test was conducted using using N C S S statistical analys is sys tem ( N C S S ) . W h e n compar ing only two groups, for instance, control and diabetic samp les , a one-way A N O V A was employed using G r a p h P a d Pr ism® (GraphPad Software). 41 3.0 RESULTS 3.1 Expression of RhoA in control and diabetic cardiomyocytes. Prev ious work in this laboratory had shown that acute R O C K inhibition improved card iac function in diabetic rats. G iven this finding a major objective of this thesis was to determine any upregulation of the R h o A / R O C K pathway in hearts from 12 week S T Z diabetic rats. Accordingly we isolated card iomyocytes from 12 weeks S T Z diabetic male rats and their age-matched controls, and determined the relative express ion of R h o A in control and diabetic card iomyocytes using western blotting. Pr imary R h o A antibody (mouse monoclonal) was used at a concentrat ion of 1:250. A s shown in figure 6, when compared to control, express ion of R h o A in freshly isolated diabetic card iomyocytes was significantly e levated. 42 RhoA Actin Control Diabetic Figure 6: Relative expression of RhoA in control and diabetic cardiomyocytes, as assessed by western blotting. (A) A representative blot showing RhoA and Actin expression in freshly isolated control and diabetic cardiomyocytes. (B) RhoA band O.D. values were corrected by their equivalent actin band O.D. value and expressed relative to mean control value. Means are shown (± SEM), n = 8. *, P< 0.05 significantly different from control group. Statistical analysis was performed using a one-way ANOVA. 43 3.2 Expression of iNOS in control and diabetic cardiomyocytes. A n important objective of this thesis was to confirm the f indings of Nagareddy et al., (2005), who had shown that i N O S express ion was significantly elevated in whole hearts from 12 week S T Z diabetic rats. W e wished to confirm if this was a lso the c a s e in isolated cardiomyocytes. The relative levels of i N O S express ion in control and diabetic card iomyocytes was determined using western blotting. Primary i N O S antibody (mouse monoclonal) was used at a concentrat ion of 1:200. It was noticeable that i N O S was expressed , albeit weakly, in freshly isolated control card iomyocytes (figure 7B) . There was a strong increase in i N O S express ion in diabetic card iomyocytes. Dens i tomet ry data (figure 7A) , conf irmed that the express ion of i N O S in diabetic card iomyocytes was significantly e levated compared to control. 44 .„V. ,,. . iNOS Act in Control Diabetic Figure 7: Relative expression of iNOS in control and diabetic cardiomyocytes, as assessed by western blotting. (A) A representative blot showing iNOS and actin expression in freshly isolated control and diabetic cardiomyocytes. (B) iNOS band O.D. values were corrected by their equivalent actin band O.D. value and expressed relative to mean control value. Means are shown (± SEM), n = 8. *, P< 0.05 significantly different from control group. Statistical analysis was performed using a one-way ANOVA. 45 3.3 Effect of SNP on the expression of RhoA in cultured cardiomyocytes. S a u z e a u et al., (2003) demonstrated that when exposed to S N P , cultured vascular smooth muscle cel ls exhibited a significant increase in the express ion of RhoA . W e wished to determine whether the s a m e effect occurred in cultured primary rat card iomyocytes. Isolated card iomyocytes were cultured for 18 hours in the presence or absence of 10 p M S N P . After this time lysates were col lected and express ion of R h o A was compared in control and treated cel ls using western blotting. Pr imary R h o A antibody (mouse monoclonal) was used at a concentrat ion of 1:250. A s shown in figure 8, there was a significant increase in R h o A express ion in S N P treated cel ls compared to control. 46 SNP + Figure 8: Effect of 10 uM sodium nitroprusside (SNP) treatment for 18hrs on RhoA expression. 0.75 x 1 0 6 viable myocytes were cultured per plate. Express ion levels were a s s e s s e d using western blotting. Band O .D . va lues were normal ised to the mean control va lue. Resul ts shown are normal ised means (± S E M ) . *, P< 0.05 significantly different from control group. Statistical analys is w a s performed using a one-way A N O V A . 47 3.4, Effect of iNOS induction on the expression of RhoA in cultured cardiomyocytes. W h e n induced, i N O S cata lyzes the production of large amounts of N O from I-arginine and molecular oxygen (Palmer et a l . , 1988). Ce l l s were exposed to 50 pg/ml L P S for 18hrs. Myocytes were a lso co-treated with 50 pg/ml L P S and 100 p M N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL), a select ive i N O S inhibitor. Previously, this concentrat ion of L-NIL had been shown effectively inhibit i N O S in vitro (Moore et a l . , 1994). A control group treated with 100 p M L-NIL alone was also implemented. After this incubation period western blotting was performed to confirm induction of i N O S and to ana lyze the level of express ion of R h o A in these cel ls. Pr imary ant ibodies were used at the concentrat ions descr ibed previously (see 3.1 and 3.2). A s shown in figure 9A, induction of i N O S by L P S resulted in a significant increase in R h o A express ion. Co-treatment of cel ls with L P S and L-NIL resulted in the induction of i N O S , however, R h o A express ion in these cel ls was reduced to levels not different from control (figure 9B.) . Treatment with L-NIL alone had no effect on R h o A express ion. There was no ev idence of i N O S induction in cel ls treated with L-NIL alone (figure 9A). 48 F i g u r e 9A: E f fec t of L P S t rea tment o n R h o A a n d i N O S e x p r e s s i o n . A representative blot showing i N O S , R h o A and G A P D H express ion in control (1), L P S treated (2), L P S and LNIL treated (3), and LNIL treated card iomyocytes (4). 49 Control LPS LPS + LNIL LNIL Figure 9B: Effect of LPS treatment (50 pg/ml for 18hrs) on RhoA expression. Representat ive Band O .D . va lues were normal ised by their cor responding G A P D H band value and then expressed relative to the mean control value. Resu l ts shown are means (± S E M ) . *, P< 0.05 significantly different from control group. Statistical analys is was performed using a one-way A N O V A . 50 3.5 Effect of chronic iNOS inhibition on expression of RhoA in control and diabetic rat hearts. Control and diabetic male Wistar rats were chronical ly administered L-NIL as descr ibed (2.2.2). It was important to determine whether treatment with the chosen dose of L-NIL had been effective in inhibiting i N O S activity. i N O S activity was found to be significantly increased in hearts from untreated diabetic rats (figure 10B). Treatment of diabetic rats with L-NIL at 3mg/kg/day for nine weeks was effective at reducing i N O S activity to levels not significantly different from control. E levated i N O S express ion in untreated diabetic hearts was conf i rmed using western blotting (figure 10A). Chron ic treatment with L-NIL appeared to normal ise i N O S express ion in diabetic rat hearts (figure 10A). Concomi tant with elevated i N O S express ion, R h o A express ion in hearts from 9 week diabetic rats was significantly e levated. Hearts from diabetic rats treated with L-NIL will showed normal ized R h o A express ion (figure 11). L-NIL had no effect on R h o A express ion in hearts from control rats. 51 Figure 10: iNOS expression and activity in control and diabetic cardiac ventricular tissue following chronic iNOS inhibition. (A) A representative blot showing i N O S and G A P D H express ion in ventricular t issue. Treatment was with L-NIL at 3mg/kg/day for 9 weeks via oral gavage . (B) Graph displaying i N O S activity va lues (counts per min per 80pg heart t issue). Activity va lues are expressed relative to the mean control value. Data shown are m e a n s ±SEM. Statistical analys is w a s performed using a one-way A N O V A fol lowed by a Newman-Keu l s post hoc test. *, P< 0.05 significantly different from control group (n = 6). C = Control , D = Diabetic, C T = Treated Contro l , DT = Treated Diabet ic. 5 2 Figure 11: Relative RhoA expression levels in control and diabetic cardiac ventricular tissue following chronic iNOS inhibition. (A) A representative blot showing R h o A and G A P D H express ion in ventricular t issue. Treatment was with L-NIL at 3mg/kg/day for 9 weeks v ia oral gavage. (B) Band O .D . va lues were corrected by their loading control band O.D. value and expressed relative to the mean control. Data shown are means ±SEM. Statistical analysis was performed using a one-way A N O V A fol lowed by a Newman-Keu l s post hoc test. *, P< 0.05 significantly different from control group (n = 6). C = Contro l , D = Diabetic, C T = Treated Contro l , DT = Treated Diabetic. 53 3.6 Effect of chronic iNOS inhibition on activity of the RhoA/ROCK signaling pathway. To date no commercia l ly avai lable R O C K a s s a y has been establ ished. However, it is well establ ished that, once activated by RhoA , R O C K phosphorylates and act ivates its downstream target, L IMK (Bamburg, 1999). Therefore, phosphorylated L IMK (LIMK-P) was used as a marker for activity of R h o A / R O C K in this experiment. Protein samp les from ventricular t issue (from 3.4) were prepared for western blotting. Weste rn blotting was performed using an antibody specif ic for L I M K - P . This rabbit polyclonal primary antibody was used at a concentrat ion of 1:1000. W e observed ev idence for increased activation of the R h o A / R O C K pathway in diabetic rat hearts (figure 12). There was a significant elevation in the levels of L I M K - P in diabetic samp les , chronic treatment with L-NIL reduced L I M K - P to levels that were not significantly different from control. 54 LIMK-P GAPDH C CT D DT Figure 12: Relative levels of LIMK-P in cardiac ventricular tissue. (A) A representative blot showing LIMK and GAPDH expression in ventricular tissue. C = Control, D = Diabetic, CT = Treated Control, DT = Treated Diabetic. Treatment was with L-NIL at 3mg/kg/day for 9 weeks via oral gavage. (B) Band O.D. values were normalized by their corresponding GAPDH band value and expressed relative to the mean control value. Data shown are means ±SEM. Statistical analysis was performed using a one-way ANOVA followed by a Newman-Keuls post hoc test. *, P< 0.05 significantly different from control group (n = 6). 55 3.7 State of activation of the RhoA/ROCK pathway in control and diabetic cardiomyocytes. It was important that a s well as determining an increase in express ion of R h o A we also confirm whether the activity of R h o A is altered in diabetic card iomyocytes. To this end , we measured levels of active R h o A in freshly isolated myocytes from control and 12-week S T Z diabetic rat hearts. To a s s e s s activation of R O C K , we used western blotting to measure the levels of phosphorylated L IMK relative to total L IMK in cel ls lysates from control and diabetic card iomyocytes. Compared to control card iomyocytes there was a significantly increased amount of active R h o A in diabetic card iomyocytes (figure 13). Concomitant ly, there was a significant increase in the levels of phosphorylated L IMK in diabetic card iomyocytes compared to control (figure 14). 56 2.0n Control Diabetic Figure 13: Relative amount of active RhoA in Control and 12 week STZ diabetic rat ventricular myocytes. 0.75 x 1 0 6 cel ls per sample , n = 12 in both groups. Al l data shown are means ± S E M . *Signif icantly different from control sample (P<0.05), as determined by a one-way A N O V A . 57 C C D D B Figure 14: LIMK-P/Total LIMK in Control and 12 week STZ diabetic rat ventricular myocytes. (A) A representative blot showing L IMK and L I M K - P in ventricular t issue. (B) L I M K - P band O .D . va lues were normal ized by their cor responding L IMK band value and expressed relative to the mean control value. Data shown are m e a n s ±SEM. Statistical analys is was performed using a one-way A N O V A fol lowed by a Newman-Keu l s post hoc test. *, P< 0.05 significantly different from control group (n = 6). 58 3.8 Effect of ROCK inhibition on the actin cytoskeleton in control and diabetic cardiomyocytes. The actin cytoskeleton is a wel l -establ ished down stream target of R h o A / R O C K in eukaryotic cel ls. It was therefore of interest in this study to determine the state of actin cytoskeleton dynamics in control and diabetic card iomyocytes, and to determine the effect of acute R O C K inhibition on cytoskeletal actin in these cel ls. A commercia l ly avai lable a s s a y kit w a s used to isolate and determine the free globular actin (G-actin) and f i lamentous actin (F-actin) content from freshly isolated control and diabetic card iac myocytes. Ce l l s from control and diabetic an imals were a lso treated with the R O C K inhibitor, Y -27632 (1 u M , 15 minutes). For each sample , actin was extracted from 0.5 x 1 0 6 viable cel ls. W e also used a qualitative f luorescence confocal microscopy approach to a s s e s s the levels of f i lamentous actin in control and diabetic cel ls in the absence and presence of Y -27632 . It was found that there was a consistent, significant elevation in the F-act in /G-actin ratios in diabetic card iomyocytes (figure 15). Acute treatment with Y -27632 reduced the F-act in/G-act in ratios in diabetic cel ls to levels which were not significantly different from control. Acute treatment of control cel ls with Y -27632 had no effect on the F-act in/G-act in ratio. Us ing f luorescence microscopy it was found that f luorescence from the labeled F-actin (green, Phalloidin-AlexFluor®488) appeared to be much more intense in diabetic cel ls than in controls (figure 16). The levels of F-actin f luorescence (green) in Y -27632 treated diabetic cel ls was not noticeably different from that in control and control-treated cel ls. 59 Figure 15: Comparison of the F-actin to G-actin ratio in freshly isolated myocytes. Myocytes were isolated from control (C), 12 week S T Z diabetic rats (Q). The effect of treatment with Y - 2 7 6 2 3 (1uM, 15 mins) on control (CT) and treated diabetic (DT) myocytes was a lso a s s e s s e d . (A) The western blot shown is representative of all samp les a s s a y e d . Weste rn blot bands are labeled as follow: (1) = Control F-Act in, (2) = Control G-Act in , (3) = Diabetic F-Act in, (4) = Diabetic G-Act in , (5) = Treated control F-Act in, (6) = Treated control G-Act in , (7) = Treated diabetic F-Act in, (8) = Treated diabetic G-Act in . (B) Resul ts are mean F/G-act in ratios ± S E M (n=6 in each group), *, P<0.05 different from all other groups. Statist ics were performed by performing a one-way A N O V A fol lowed by a Newman-Keu l s post-hoc test. 60 Figure 16: Confocal microscopy images of fluorescently labeled cardiomyocytes. Freshly isolated myocytes from control and 12-week S T Z diabetic rats were untreated or treated with 1uM Y-27632 for 15 mins. Myocytes were f ixed, permeabi l ized and double stained with AlexaFluor®594-DNAsel and AlexaFluor®488-Phal loidin to colocal ize G-act in (red) and F-actin (green). S l ides were v isual ized using a B i o - R a d Rad iance confocal microscope at 1260x magnif ication. Control (A), Diabetic (B), Control-Treated (C), Diabet ic-Treated (D). 62 63 4.0 DISCUSSION 4.1 Increased RhoA and iNOS expression in STZ diabetic rat cardiomyocytes. The compel l ing finding that acute inhibition of R O C K improves heart function in 12 week S T Z diabetic rats (figures 3 and 4) was a major inspiration for conduct ing this research project. O n the bas is of these observat ions we wished to determine what factors may be contributing to any change in activity of the R h o A / R O C K pathway during chronic S T Z diabetes and determine what the cellular consequences of altered R h o A / R O C K signal ing might be. In this study we found that express ion of R h o A was upregulated in hearts from 12 week S T Z diabetic rats (figure 6). Th is observat ion is at odds with a recent report that R h o A m R N A and protein express ion are dec reased in hearts from diabetic rats (Tang et al., 2005). The reason for this difference is not clear, but it may be due to dif ferences in the duration of d iabetes between the two studies. The study by Tang et al., (2005) was conducted in hearts from 3 week S T Z diabetic rats, a time which is prior to the development of overt impaired contractile function in most investigations. It should be noted that in further studies conducted in this laboratory we were not able to detect altered express ion of R h o A in whole heart t issue from 3 week S T Z diabetic rats, and in 5-6 week S T Z diabetic rats we have observed increased R h o A express ion (data not shown). It is interesting that the increase in express ion of R h o A in the heart correlates well with the onset of diabetic card iac dysfunct ion. Taken together with ev idence that card iac specif ic over-expression of R h o A leads to diminished ventricular contractility (Sah et al., 1999), these findings suggest a strong associat ion between increased R h o A expression and impaired myocardial function in the chronic S T Z diabetic rat. 64 The observat ion that i N O S express ion is elevated in diabetic card iomyocytes is in agreement with Nagareddy e ra / . , (2005), who showed increased express ion of i N O S in whole heart t issue from 12 week S T Z diabetic rats. In this thesis we have shown that i N O S and R h o A express ion are concomitantly elevated in card iomyocytes and heart t issue from S T Z diabetic rats (figure 6, figure 7). Interestingly, this is not the first study which has shown concomitant increases in R h o A and i N O S express ion in a pathological model of impaired cardiac function. Dong et al., (2005), investigated the effects of iron-def iciency on card iac ultrastructure and found that iron-deficient rats exhibited significantly elevated R h o A and i N O S express ion. Unfortunately Dong et al., (2005) did not a s s e s s card iac function in these animals. However, a previous investigation using the same model has shown that impaired myocardial function deve lops in iron-deficient rats (Goldstein et al., 1996). Interestingly Goldste in et al., observed impaired L-type C a 2 + currents and depressed rates of ventricular contraction and relaxation. T h e s e pathological observat ions are similar to those observed in hearts from animals with chronic type 1 diabetes. These findings strongly support an associat ion between elevated i N O S and R h o A express ion the pathology of impaired cardiac function. 4.2 NO and induction of iNOS elevates RhoA expression in vitro. Given the associat ion of elevated i N O S and R h o A express ion in whole heart t issue and cardiomyocytes from 12 week S T Z diabetic rats, we wished to determine if an interaction existed between these two proteins. S a u z e a u etal., (2003), demonstrated that the N O donor, S N P , caused significant increases in R h o A express ion in cultured vascu lar smooth muscle cel ls. In agreement with this work, we have shown that using the s a m e concentrat ion of S N P , a significant increase in R h o A express ion can be induced in cultured card iomyocytes over an 18 hour period. 65 The mechan ism by which N O might be elevating R h o A express ion is still not fully understood. The elevated levels of R h o A that we have observed could be due to transcriptional and translational upregulation of the R h o A gene or dec reased degradat ion of the R h o A protein. There is very limited publ ished data on the regulation of stability or express ion of Rho G T P a s e s . S a u z e a u etal., (2002) suggest that that N O activates P K G , which phosphorylates R h o A on the C-terminal domain, thus protecting the protein from degradat ion and resulting in R h o A accumulat ion in the cel l . S a u z e a u et al., have shown elevated P K G activity and R h o A phosphorylat ion in cel ls exposed to S N P . They a lso show that co-treatment of cel ls with S N P and the P K G inhibitor (Rp)-8-B r - P E T - c G M P - S blocks the S N P - i n d u c e d elevation of R h o A , support ing the author's hypothesis. It should be appreciated that with other proteins, cel l - type-specif ic express ional regulation mechan isms have been establ ished (Park et al., 2003). It is therefore possib le that the mechan isms proposed in f ibroblasts and vascular smooth musc le cel ls by S a u z e a u et al., (2003) may not necessar i ly be mirrored in cardiomyocytes. The activity of i N O S is primarily regulated at the level of protein express ion. Unlike e N O S and n N O S , regulation of protein activity is uncommon for i N O S (Kleinert et al., 2003). After express ional induction, i N O S cont inuously produces N O until the enzyme is degraded (MacMick ing et al., 1997). In cultured card iomyocytes we were able to induce i N O S express ion using L P S . This observat ion is supported by a number of reports which have indicated that cytokines including L P S induce i N O S and N O production in card iomyocytes (see review by Kel ly etal,, 1996). Prev ious studies in rats have used neonatal card iomyocytes and have demonstrated a significant induction of i N O S after L P S exposure (Kinugawa etal., 1997a; K inugawa etal., 1997b; Ke i ra etal., 66 2002). A difference between our studies and previous ones is that in the previous studies a lower L P S concentrat ion (10 pg/ml) was required for i N O S induction. In our exper iments a concentrat ion of at least 40 pg/ml L P S was required for induction of i N O S . T h e s e dif ferences may reflect a difference in respons iveness to L P S between cultured neonatal card iomyocytes and primary cultured adult rat card iomyocytes. It should be noted that in all exper iments descr ibed here cardiomyocyte cultures were examined carefully and appeared to be free of any contaminat ion from other cell types. Th is is an important point as other cell types, such as macrophages, are highly responsive to L P S (Fujihara et al., 2003). Al though we cannot completely rule out the possibil ity of a very low level of contaminat ion by macrophages, given the number of card iomyocytes cultured in each experiment, it is unlikely that this would impact on the total i N O S or R h o A pool. The results presented here suggest that the increased N O produced by i N O S results in increased R h o A express ion. Induction of i N O S resulted in a concomitant increase in R h o A express ion. Th is effect was blocked by co-treatment of card iomyocytes with L P S and L-NIL (figure 9). L-NIL is an L-arginine analogue which acts as a select ive, irreversible inhibitor of i N O S (Moore et al., 1994). Our results strongly support the hypothesis that induction of i N O S results in increased R h o A express ion. 67 4.3 Chronic iNOS inhibition results in decreased iNOS expression in diabetic rat hearts. The express ion and activity of i N O S in heart t issue from diabetic rats was significantly e levated compared to control. Treatment of diabetic rats with L-NIL at a dose of 3mg/kg/day for 8 weeks was effective at reducing i N O S activity in whole heart t issue to levels not different from control (figure 10B). Interestingly, in diabetic hearts, chronic treatment with L-NIL a lso appeared to reduce i N O S express ion (figure 10A). G iven that i N O S is a select ive, irreversible inhibitor of i N O S (Moore et al., 1994), it w a s not anticipated that L-NIL treatment would affect i N O S express ion. Th is data suggests that auto-regulation of i N O S express ion may be occurr ing. However our in vitro experiment using cultured card iomyocytes, there was no detectable effect on i N O S express ion after exposure to L-NIL (or L P S and L-NIL) for 18 hours (figure 9A). There are conflicting reports a s to whether treatment with i N O S inhibitors affects i N O S express ion. Us ing rat mesangia l cel ls , Muhl and Pfeilschifter (1995), showed that i N O S inhibitors acutely reduced IL-1 beta- induced i N O S express ion. In rat synovial t issue and peripheral blood leukocytes, i N O S express ion was reduced after chronic administration of L-NIL at 3 mg/kg/day (McCar tney-Franc is etal., 2001). However Beh r -Rousse l etal., (2000), did not detect a change in i N O S express ion in aortas from rats chronical ly treated with L-NIL. Taken together the results shown in this thesis and by others suggest that the dif ferences may exist in the regulation of i N O S express ion depending upon cel l and spec ies type and the duration of treatment with i N O S inhibitors. 68 4.4 Chronic inhibition of iNOS normalizes expression of RhoA and activity of the RhoA/ROCK pathway in heart tissue from rats with STZ induced diabetes. Concomitant with elevated i N O S express ion, R h o A express ion in hearts from 9 week diabetic rats was significantly e levated, while hearts from diabetic rats treated with L-NIL showed normal ized R h o A express ion (figure 11). Th is observat ion agrees with results from our in vitro experiment (figure 9) and supports the proposal that e levated i N O S express ion induces increased R h o A express ion. W e a lso wished to determine if the activity of the R h o A / R O C K pathway was affected by the observed changes in R h o A express ion in these t issues. There was a significant elevation in the levels of L I M K - P in diabetic rat hearts and chronic treatment with L-NIL reduced L I M K - P to levels that were not different from control (figure 12). L IMK is a direct downstream target of R O C K . O n c e activated R O C K phosphorylates and act ivates L IMK. These results suggest that there is increased activity of R O C K and L IMK in heart t issue from rats with diabetes. Currently there have been no identified k inases other than R O C K which phosphorylate L IMK, and other studies from this laboratory have shown that acute inhibition of R O C K normal izes L IMK phosphorylat ion in 12 week S T Z diabetic rats (Lin etal., unpubl ished data). G iven that chronic inhibition of i N O S reduced the levels of R h o A express ion and the levels of phosphorylated L IMK in heart t issue from rats with diabetes, these results suggest that i N O S induces increased express ion and activity of R h o A in the diabetic rat heart. 69 4.5 Increased activity of RhoA in diabetic rat cardiomyocytes. A s d i scussed in the introduction it was important that we a s s e s s e d the levels of active R h o A in diabetic card iomyocytes. G iven the elevated L I M K - P observed in whole heart t issue from diabetic rats, it was expected that levels of active R h o A would a lso be elevated in diabetic card iomyocytes. Our R h o A activity a s s a y showed that, compared to control, there were significantly higher levels active R h o A in card iomyocytes from diabetic hearts. There was an approximate 5 0 % increase in the levels of active R h o A in diabetic card iomyocytes compared to control. Th is correlated strongly to the relative fold-increase observed in express ion of R h o A in diabetic card iomyocytes. Us ing western blotting, e levated L I M K - P was a lso observed in diabetic card iomyocytes. Th is strongly supports the ev idence that R h o A activity is upregulated in diabetic card iomyocytes, leading to increased activity of R O C K . Elevated express ion of R h o A may be contributing to elevated levels of active R h o A in diabetic card iomyocytes, by increasing the 'total R h o A pool ' avai lable for activation. In addit ion, it has recently become apparent that G P C R signal l ing through heterotrimeric G-proteins can lead to the activation of Rho family G T P a s e s (Sah etal., 2000; Mar in issen and Gutk ind, 2001) (figure 17). Increased Ag- l l and angiotensin receptor levels have been shown in an in vivo study in streptozotocin- induced diabetic rat hearts (Fiordal iso et al., 2000). Ag- l l has been shown to activate R h o A in rat card iomyocytes (Aoki etal., 1998). 70 Figure 17: G P C R mediated activation of Rho GTPases. The binding of agonist to a G P C R promotes the exchange of G D P for G T P on the G-protein G a subunit, which leads to dissociat ion of the G a and G B y subunits. The activated proteins G a and G B Y can positively or negatively regulate various downstream effectors. G P C R s can also signal to, and activate and Rho G T P a s e s (via G a 1 2 / G a 1 3 ) . [Adapted from: Bhat tacharya etal., 2004]. 71 It has a lso been proposed that increased stimulation by endothel ium-derived vasoconstr ictors, such as endothelin-1 and thromboxane-A2, contribute to increased R h o A / R O C K activation in the diabetic heart (Sharma and McNei l l , 2006). In streptozotocin-diabetic rats, p lasma concentrat ions of endothelin-1 (Benrezzak et al., 2004) and thromboxane-A2 (Valentovic and Lubawy 1983) have been shown to be elevated compared to control rats. The elevated express ion of R h o A is likely to amplify the effects of increased circulating factors such as endothelin-1 and Ag- l l which are known to activate RhoA, resulting in heightened activation the R h o A / R O C K pathway the in diabetic rat heart. However, this would not account for our observat ion that R h o A activity was increased in isolated card iomyocytes where these factors would be absent. 4.6 Effects of acute RhoA/ROCK inhibition on the actin cytoskeleton in diabetic cardiomyocytes. The ev idence presented in this thesis strongly suggests increased activation of RhoA , R O C K and L IMK in diabetic rat hearts and cardiomyocytes. Prev ious f indings from this laboratory have shown that acute inhibition of R O C K significantly improved heart function in diabetic rats. T h e s e findings implicate increased activation of the R h o A / R O C K pathway in mediating impaired cardiac function in the diabetic heart. A n important aim of this thesis was to determine the consequences of R h o A / R O C K upregulation in diabetic card iomyocytes, and attempt to shed more light on the mechan ism by which acute R O C K inhibition improves card iac function in the S T Z -diabetic rat. A s descr ibed in sect ion 1.1, signal ing from R h o A to the actin cytoskeleton through R O C K and L IMK is well establ ished. M a e k a w a et al., (1999) demonstrated that 72 phosphorylat ion of L IMK by R O C K and consequent ly increased phosphorylat ion of cofilin by L IMK contribute to RhoA- induced actin polymerization in N 1 E - 1 1 5 neuroblastoma cel ls. Here we have shown that there is a significant increase in the levels of polymerized actin in card iomyocytes from 12-week S T Z diabetic rats. Acute inhibition of R O C K dec reased polymer ized actin in diabetic card iomyocytes to levels that were not different from control card iomyocytes. Ev idence from both in vitro actin polymerizat ion a s s a y s and confocal microscopy supported this finding. The ability of the actin cytoskeleton to modulate the mechanica l activity of the cardiomyocyte has been well reviewed (Ca laghan e ra / . , 2004) and a number of studies have implicated a role for the actin cytoskeleton in condit ions of impaired card iac performance (Hein et al., 2000; Kost in et al., 2000). A number of mechan isms could explain this interaction. The cardiomyocyte actin cytoskeleton has been shown to be an important regulator of the metabol ism of fatty ac ids (Pulinilkunnil et al., 2005), the preferred energy substrate of the heart (Rodr igues et al., 1995). Increased actin polymerizat ion, induced by R h o A activation, can promote the exocytosis of lipoprotein l ipase and increase fatty acid metabol ism (Pulinilkunnil etal., 2005), which is assoc ia ted with impaired card iac function and lipotoxicity in chronic d iabetes (Tan et al., 1984). Increased f i lamentous actin within the cardiomyocyte may also increase cel l rigidity, contributing to the increased myocardial stiffness which is a component of impaired card iac performance in hearts from diabetic rats (Joffe etal., 1999; Dent etal., 2001). It is possib le that normalization of actin polymerization may contribute to the R O C K inhibitor-mediated improvement in contractile function of hearts from diabetic rats, observed in previous studies conducted in this laboratory (figures 3 and 4). O n e of 73 the few speci f ic mechan isms previously known to acutely improve the contracti le function of the diabetic heart was treatment with agents that promote a shift from fatty acid metabol ism to g lucose oxidation (Tan etal., 1984; Nichol l etal., 1991). G i ven these unique previous findings, a possib le mechan ism by which R O C K inhibitors improve card iac function, is via normalization of actin polymerizat ion and reduction of fatty acid metabol ism, thereby improving the metabolic state of the heart during chronic d iabetes. However, s ince the complet ion of this research thesis, initial studies conducted in col laboration with the laboratory of Dr. Michael Al lard at the J a m e s Hogg iCapture Centre for Card iovascu lar and Pulmonary Resea rch , were unable to detect significant changes in fatty acid or g lucose metabol ism in 12 week S T Z diabetic rat hearts acutely treated with Y -27632 . It is possib le that normalization of actin polymerizat ion in diabetic card iomyocytes may not be able to acutely normal ize fatty ac id metabol ism. The actin microfi laments have also been implicated in regulating the concentrat ion of intracellular C a 2 + (Lange and Brandt, 1996), a key regulator of cardiomyocyte contractility (Bers and G u o , 2005). Increased actin polymerizat ion in card iomyocytes has been assoc ia ted with dec reased contractility due to disruption of intracellular C a 2 + re lease (Davani etal., 2004). Acute changes in intracellular [Ca 2 + ] are likely to manifest as acute changes in cardiomyocyte contractility. It would be therefore interesting to determine the effects of R O C K inhibition on intracellular [Ca 2 + ] and calc ium homeostas is in diabetic cardiomyocytes. The ev idence supports a role for increased actin polymerizat ion in impaired card iac function in the 12 week S T Z diabetic rat. G iven that acute inhibition of R O C K has been shown to normalize both actin polymerization and cardiac performance in diabetic rat hearts, we conclude that there is a strong associat ion between elevated 74 activity of the R h o A / R O C K pathway, increased actin polymerizat ion and dec reased card iac performance in the diabetic rat heart. However further studies are required to determine the mechan isms by which increased actin polymerizat ion affects card iac contractility in the diabetic rat heart. 4.7 Further Studies. The following outl ines briefly descr ibe some studies that would be interesting to pursue given the outcomes of this research thesis. To determine if inhibition of ROCK improves cardiomyocyte contractility, and if this effect is achieved via normalization of calcium transients. Previous work in this laboratory has establ ished that acute R O C K inhibition improves card iac function both in vivo and ex vivo. It would be interesting to determine if acute R O C K inhibition can improve contractility at the cellular level, and whether this is mediated via normalization of calc ium handl ing. Prev ious studies have confirmed impaired contractility and. calc ium handling in myocytes isolated from S T Z diabetic rat hearts (Choi et a l . , 2003). Ce l l shortening and intracellular [Ca 2 + ] can be measured in card iomyocytes using f luorescence microscopy as descr ibed previously (Huang et a l . , 2005). Th is investigation could provide useful information on the pathology of diabetic cardiomyopathy at the cellular level, as well as an insight into the possible mechan isms by which acute R O C K inhibition improves card iac function in the S T Z diabetic rat heart. 75 To determine whether increased RhoA expression is induced directly by NO in isolated cardiomyocytes. In this thesis we have shown that when treated with 10 u M S N P , a potent N O donor, card iomyocytes exhibit significantly e levated R h o A express ion levels. However, we have not proven causal ly that N O directly mediates this change. It would be interesting to observe if this effect could be blocked with nitric oxide scavengers . Furthermore, N O strongly interacts with with superoxide to form peroxynitrite (Stamler, 1994). Th is highly reactive nitrogen-containing radical can cause oxidative and nitrosative stress, and may lead to the activation of s t ress- response pathways (Klotz et al., 2002). Th is may ultimately affect the express ion levels of a broad range of proteins. The effect of N O (from S N P ) on the express ion of R h o A in primary culture card iomyocytes could therefore be direct or indirect. To help clarify this, cel ls could be treated with 10 p M S N P and 5 p M F e T M P y P (5,10,15,20-tetrakis(N-methyl-4*-pyridyl) porphyrinato-iron III), a peroxynitrite decomposi t ion catalyst. Ce l l s treated with F e T M P y P alone would serve as a treatment control. If F e T M P y P b locks the S N P induced increase in R h o A this would strongly suggest that the effect of N O on R h o A express ion is mediated v ia peroxynitrite. Th is experiment would be important in clarifying how N O from i N O S results in altered R h o A express ion in the diabetic cardiomyocyte. To determine if chronic iNOS inhibition improves cardiac function in STZ diabetic rats. The data provided in this thesis indicates that chronic inhibition of i N O S restores basa l levels of express ion of R h o A as well as basa l levels of phosphorylated L IMK in hearts from S T Z diabetic rats. G iven the associat ion between elevated activity of the 76 R h o A / R O C K pathway and impaired card iac function it would be interesting to determine whether chronic inhibition of i N O S improves card iac function in the S T Z diabetic rat. In this study control and diabetic rats would be chronical ly administered L-NIL and subsequent ly heart function would be measured either using echocard iography or by using the isolated work heart model . Normal izat ion of R h o A express ion and activity of the R h o A / R O C K pathway concomitant with improved card iac function would be extremely compel l ing ev idence to support a role for i N O S upregulation in altered R h o A / R O C K signal ing and impaired card iac function in the 12 week S T Z diabetic rat heart. To determine the status of the RhoA/ROCK pathway in iNOS knockout mice. To further strengthen our proposal that i N O S mediates elevated R h o A express ion and upregulation of the R h o A / R O C K pathway, we would like to examine this pathways character ist ics in the wel l -establ ished i N O S knockout mouse (MacMick ing er al., 1995). It would be interesting to investigate whether mice made diabetic, in the absence of i N O S , would still exhibit upregulation of the R h o A / R O C K pathway. Furthermore, it would be important to a s s e s s card iac function in these an imals and determine if i N O S knockout mice deve loped diabetic cardiomyopathy. If i N O S knockout mice did not exhibit diabetic cardiomyopathy, this would be compel l ing and conclus ive ev idence that i N O S mediated upregulation of the R h o A / R O C K pathway, played a central role in the development of diabetic cardiomyopathy. 77 5.0 SUMMARY AND CONCLUSIONS This thesis has provided strong ev idence that R h o A express ion, and the activity of the R h o A / R O C K pathway are significantly elevated in hearts and card iomyocytes from rats with STZ- induced diabetes. G iven that exposure to the N O donor, S N P , elevated R h o A express ion in cultured myocytes we propose that N O from i N O S is mediat ing elevated R h o A express ion in diabetic rat hearts. The conclus ion that i N O S mediates increased R h o A express ion in the STZ-d iabet ic rat heart is strongly supported by a number of other observat ions in this thesis. Western blotting and i N O S activity a s s a y s conf irmed increased express ion and activity of i N O S , concomitant with increased R h o A express ion in diabetic rat hearts. Chron ic treatment of diabetic rats with the specif ic i N O S inhibitor, L-NIL, b locked increased R h o A express ion in hearts from these animals. In cultured card iomyocytes L P S evoked increased i N O S and R h o A express ion. Co-treatment of cel ls with L P S and the specif ic i N O S inhibitor, L-NIL, b locked increased R h o A express ion, with no detectable change in express ion of i N O S . Taken together these f indings strongly support the proposal that induction of i N O S results in increased R h o A express ion in diabetic card iomyocytes. Increases in R h o A express ion correlated strongly with increased levels of active R h o A in diabetic card iomyocytes. W e a lso observed increased levels of phosphorylated LIMK, a marker for activation of the R h o A / R O C K pathway, in diabetic card iomyocytes. L IMK phosphorylat ion was reduced to levels similar to control after chronic treatment of diabetic rats with L-NIL, suggest ing that i N O S contributes to increases activity, in addition to increased express ion, of R h o A in the STZ-d iabet ic rat heart. The actin cytoskeleton is a well establ ished downstream target of the R h o A / R O C K pathway. Phosphory la ted L IMK acts as a positive regulator of actin polymerizat ion, and the levels 78 of polymer ized actin in card iomyocytes from diabetic hearts were found to be significantly e levated. The attenuation of this change via acute inhibition of R O C K implicates increased activation of the R h o A / R O C K pathway in mediat ing these changes in the diabetic heart. In conc lus ion, the present study demonstrates, for the first t ime to our knowledge, that the R h o A / R O C K pathway is upregulated in hearts, and cardiomyocytes, from rats with chronic STZ- induced diabetes. Despite the diverse b iochemical and physiological changes that occur in the diabetic heart, acute inhibition of the R h o A / R O C K pathway has been previously shown in this laboratory to improve card iac function, both in vitro and in vivo. G iven that acute R O C K inhibition was a lso shown to normal ize the levels of polymerized actin in diabetic card iomyocytes, it is possib le that normalizat ion of actin polymerization may contribute to R O C K inhibitor-mediated improvement of contracti le function of hearts from diabetic rats. Finally, the f indings presented in this thesis suggest a central role for i N O S in the upregulation of the R h o A / R O C K pathway which has been shown to contribute to impaired contractility in the diabetic heart. 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