Diabetes-induced increased oxidative stress in cardiomyocytes

28 We previously reported that acute inhibition of the RhoA/Rho kinase (ROCK) pathway 29 normalized contractile function of diabetic rat hearts, but the underlying mechanism is unclear. 30 PKCβ2 has been proposed to play a major role in diabetic cardiomyopathy at least in part by 31 increasing oxidative stress. Further evidence suggests that PKC positively regulates RhoA 32 expression through induction of iNOS in diabetes. However, in preliminary studies we found that 33 inhibition of ROCK itself reduced RhoA expression in diabetic hearts. We hypothesized that 34 there is an interaction between RhoA/ROCK and PKCβ2 in the form of a positive feedback loop 35 that sustains their activation and the production of reactive oxygen species (ROS). This was 36 investigated in cardiomyocytes isolated from diabetic and control rat hearts, incubated with or 37 without cytochalasin D or inhibitors of ROCK, RhoA, PKCβ2 or iNOS. Inhibition of RhoA and 38 ROCK markedly attenuated the diabetes-induced increases in PKCβ2 activity and iNOS and 39 RhoA expression in diabetic cardiomyocytes, while having no effect in control cells. Inhibition 40 of PKCβ2 and iNOS also normalized RhoA expression and ROCK overactivation, while iNOS 41 inhibition reversed the increase in PKCβ2 activity. Each of these treatments also normalized the 42 diabetes-induced increase in production of ROS. Actin cytoskeleton disruption attenuated the 43 increased expression and/or activity of all of these targets in diabetic cardiomyocytes. These data 44 suggest that, in the diabetic heart, the RhoA/ROCK pathway contributes to contractile 45 dysfunction at least in part by sustaining PKCβ2 activation and ROS production via a positive 46 feedback loop that requires an intact cytoskeleton. 47