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UBC Theses and Dissertations

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

Abstract

The RhoA/ROCK pathway is a well established signaling pathway which regulates cellular contractility. Previous studies, from this lab have shown that acute inhibition of ROCK improves heart function in 12 week STZ-diabetic rats. Here we wished to determine, 1) whether the RhoA/ROCK pathway was 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 RhoA/ROCK signaling might be. RhoA expression was significantly elevated in hearts and cardiomyocytes from rats with STZ-induced diabetes. Our preliminary studies also showed elevated iNOS expression in hearts from rats with STZ-induced diabetes. We hypothesized that NO from iNOS mediates elevated RhoA expression. This hypothesis is supported by a number of our observations. Elevated iNOS expression and activity was observed to be concomitant with increased RhoA expression in diabetic rat hearts. In cultured cardiomyocytes exposure to the NO donor, SNP, and the iNOS inducer, LPS , caused elevated RhoA expression. The iNOS inhibitor, L-NIL, blocked increased RhoA expression in LPS treated cardiomyocytes and in hearts from diabetic animals. Increases in RhoA expression correlated strongly with increased levels of active RhoA in diabetic cardiomyocytes. We also observed increased phosphorylated LIMK, a marker for activation of the RhoA/ROCK pathway. LIMK phosphorylation was reduced to levels similar to control after chronic treatment of diabetic rats with L-NIL, suggesting that iNOS also contributes to increased activity of this pathway in the STZ-diabetic rat heart. The actin cytoskeleton is a well established downstream target of the RhoA/ROCK pathway. The level of polymerized actin in diabetic rat cardiomyocytes was found to be significantly elevated, but was normalized after acute ROCK inhibition. Given this observation, we suggest that normalization of actin polymerization may contribute to ROCK inhibitor-mediated improvement of contractile function of hearts from diabetic rats. The findings presented in this thesis indicate a central role for iNOS in the upregulation of the RhoA/ROCK pathway, which is believed to contribute to impaired contractility in the diabetic heart. Our findings also support the suggestion that ROCK is an excellent therapeutic target in the treatment of diabetic cardiomyopathy.

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