| Diabetes mellitus is a common disease that seriously threats human health. According to statistics, diabetes mellitus affects more than 380 million people worldwide, and the number is predicted to reach 600 million by the year 2035. Diabetic cardiomyopathy (DCM) is one of the most common cardiovascular complications of diabetes, which is independent of valvular heart disease, hypertension, and coronary artery disease. Early stage DCM is characterized by diastolic dysfunction, and systolic dysfunction later develops leading to advanced heart failure. Unfortunately, at present, there are no specific drugs available for treatment of DCM.The pathogenesis of DCM is complicated. Among these hypothesis, impaired calcium homeostasis and abnormal sarcomere structure are important causes accounting for diabetes-induced cardiac dysfunction. On the one hand, in diabetic heart, the expression and function of the sarcoplasmic reticulum Ca2+-ATPase (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) and sodium calcium exchanger (Na+-Ca2+ exchanger, NCX) decreased, which further lead to delayed cytoplasmic calcium clearance, decreased sarcoplasmic reticulum calcium uptake and depressed amplitude of calcium transient. On the other hand, the structure and function of sarcomere, including abnormal cross-bridge attachment and detachment, actin disarrangement and conformational change, were also impaired in diabetic heart. These changes, mentioned before, eventually lead to abnormal excitation-contraction coupling and thus result in cardiac dysfunction.RhoA, a small GTP-binding protein, and its downstream effector, Rho kinase (ROCK), regulate a variety of cellular functions. Recently, RhoA/ROCK pathway has been demonstrated to associate with various cardiovascular diseases, such as ischemia-reperfusion injury, atherosclerosis, and cardiac hypertrophy. In addition, a growing body of evidence suggested that RhoA/ROCK activation was implicated in the development of DCM, while the mechanism was not completely addressed. Recently, we have reported that inhibition of RhoA/ROCK pathway restored the decreased amplitude of intracellular Ca2+([Ca2+]i) transient in anoxia-induced H9C2 cells, indicating that RhoA/ROCK pathway may participate in the regulation of Ca2+handling. However, whether RhoA/ROCK activation affects the [Ca2+]i cycle in diabetic cardiomyocytes is still unclear. In addition, RhoA/ROCK pathway has been demonstrated to participate in rearrangement of actin cytoskeleton, but whether inhibition RhoA/ROCK activity could improve actin organization and dynamics in diabetic heart is still uncertain.Thus, the aim of this study is to determine whether the ROCK inhibitor, fasudil, improves diabetes-induced cardiac dysfunction by regulating Ca2+ handling and actin remodeling. Furthermore, the underlying molecular mechanism and associated signaling pathway are also discussed in present study.Part 1:The effect of Rho kinase inhibitor, fasudil, on cardiac dysfunction and abnormal calcium homeostasis of diabetic heartObjectiveTo investigate the effect of fasudil on cardiac dysfunction and abnormal calcium homeostasis in streptozotocin-induced diabetic heart.MethodsRats were given a single intraperitoneal injection of streptozotocin or citrate buffer alone. One week after STZ administration, rats with blood glucose>16.7mmoI/L were considered diabetic. Then, the diabetic rats with similar degrees of hyperglycemia and body weight were randomly divided into untreated diabetes group and fasudil-treated group. The fasudil-treated rats received fasudil by intraperitoneal injection (lOmg/kg/day). The cardiac function was assessed by transthoracic echocardiography and hemodynamics. Single LV cardiomyocyte was isolated and the change of function was recorded. The intracellular Ca2+ transient was detected by an IonOptix dual-excitation fluorescence photomultiplier system with Fura-2 as indicator. Caffeine-induced Ca2+ transient was used to assess the function of Na+/Ca2+exchanger and as an index of SR Ca2+ store. Western blotting was used to analyze the expression of protein responsible for Ca2+ handling.ResultsCompared with control group, the RhoA/ROCK activity significantly elevated in diabetic heart. In addition, the diabetic rats displayed obvious cardiac dysfunction, decreased amplitude of Ca2+ transient, delayed Ca2+ removal, reduced SR Ca2+stores and depressed NCX function. Corresponding with these functional changes, the expression of Ca2+ regulating proteins including SERCA2、NCX、p-PLB、p-Akt was decreased while the expression of PLB and p-PKCβ2 was increased in the diabetic heart. Importantly, we demonstrate that long-term administration of the ROCK inhibitor, fasudil, can ameliorate the cardiac dysfunction induced by type 1 diabetes both at the cellular and the organ level. Along with improved mechanical performance, the impaired Ca2+ removal and altered expression of SERCA2、NCX、p-PLB、PLB、p-Akt and p-PKCβ2 were also normalized by inhibiting RhoA/ROCK activity with fasudil. However, fasudil failed to attenuate the depressed amplitude of Ca2+ transient and reduced SR Ca2+ stores in diabetic cardiomyocyte.ConclusionsLong-term administration of fasudil can attenuate diabetes-induced cardiac dysfunction both at the cellular and the organ level by inhibiting of RhoA/ROCK activity. The mechanism underlying the improved cardiac diastolic dysfunction with fasudil treatment might be associated with its beneficial effect on Ca2+ clearance and expression of protein responsible for Ca2+ handling.Part 2:The effect of Rho kinase inhibitor, fasudil, on actin remodeling of diabetic heartObjectiveTo investigate the effect of fasudil on actin remodeling of diabetic heart.MethodsRats were given a single intraperitoneal injection of streptozotocin or citrate buffer alone. One week after STZ administration, rats with blood glucose>16.7mmol/L were considered diabetic. Then, the diabetic rats with similar degrees of hyperglycemia and body weight were randomly divided into untreated diabetes group and fasudil-treated group. The fasudil-treated rats received fasudil by intraperitoneal injection (10mg/kg/day). The arrangement of F-actin was observed with phalloidin staining. Ultracentrifugation method was used to separate F-actin from G-actin. Cardiac interstitial fibrosis was assessed with Masson staining. Western blotting was used to analyze the expression of G-actin, F-actin and a-SMA.ResultsCompared with control, the diabetic hearts exhibited disorganized F-actin arrangements, reduced G/F-actin ratio, and elevated expression of a-SMA and collagen deposition. Fasudil treatment attenuated F-actin disorganization, restored the ratio of G/F-actin, and reduced the amount of α-SMA expression and cardiac interstitial collagen deposition in diabetic hearts.ConclusionThe protective effects of fasudil on diabetes-induced cardiac dysfunction occur via its beneficial effect on F-actin arrangement, actin remodeling and cardiac interstitial fibrosis. |
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