| In human end-stage heart failure patients, the activation of Rho kinase 1 (ROCK1) is observed in the heart. ROCK1 activation promotes the activation of caspase 3, which results in a feed-forward loop contributing to myocyte apoptosis. Rnd3, a small GTPase, is an endogenous inhibitor of ROCK1. I observed a decrease in Rnd3 protein levels in human failing hearts, suggesting a pathological significance of Rnd3 alteration in the transition to heart failure. However, the biological function of Rnd3 in the heart remains unexplored. To investigate the functional role of Rnd3 in the heart and the underlying mechanism, the Rnd3 knockout mouse line was generated to mimic the down-regulation of Rnd3 in the diseased human heart. Rnd3 deficiency (Rnd3 -/-) resulted in embryoniclethality. Severe cardiac apoptosis along with elevated ROCK1 activity were observed in the Rnd3-/- mice at the E10.5 stage. The haploinsufficient (Rnd3+/-) mice were viable,however, were predisposed to heart failure after transverse aortic constriction (TAC) induced hemodynamic stress. Remarkable cardiomyocyte apoptosis, increased caspase 3 activity, and activation of ROCK1 were detected in the Rnd3+/- heart compared to the wild-type (WT) control. Pharmacological inhibition of ROCK1 by Fasudil administration partially improved cardiac function and attenuated apoptosis in the Rnd3+/- mice after TAC. To determine whether ROCK1 contributed to the Rnd3 deficiency-mediated heart failure and cardiac apoptosis, I generated a double knockout (DKO) mouse line with Rnd3+/- ROCK1-/- background. Again, the genetic deletion of ROCK1 partially rescued the phenotype. My data suggest that Rnd3 is a novel anti- apoptotic factor that protects the heart from cardiac apoptosis partially through the inhibition of the ROCK1 pathway.;Determining the ROCK1 independent mechanisms involved in the Rnd3 deficiency-mediated cardiac remodeling is the goal for further investigation. Compensatory angiogenesis is necessary in the cardiac response to the ischemic stimuli. Compared to the wild-type mice, the post-TAC Rnd3+/- hearts showed significantly smaller capillaryareas, fewer capillary numbers, a 20.8% reduction of coronary flow reserve, and a 5.9-fold increase in the hypoxic myocardial areas. Key pro-angiogenic factors were down-regulated, including hypoxia-inducible factor 1alpha (HIF1alpha) and vascular endothelial growth factor A (VEGFA), suggesting a stress-responsive angiogenic defect. Using loss- and gain-of-function approaches, I revealed that Rnd3 physically interacted with and stabilized HIF1alpha. The deficiency of Rnd3 facilitated the HIF1alpha protein degradation, reduced the VEGFA expression, and compromised the endothelial cell tube formation. Finally, the angiogenesis defect and the heart failure phenotype in the Rnd3 +/- mice werepartially rescued by cobalt chloride treatment, a HIF1alpha stabilizer. My data suggests that Rnd3 is a pro-angiogenic factor involved in the responsive angiogenesis in the heart through HIF1alpha-VEGF signaling promotion. Animals with Rnd3 haploinsufficiency showed cardiac dysfunction with impaired angiogenesis in response to the pressure overload. The down-regulation of Rnd3 observed in the heart failure patients may explain the insufficiency in the compensatory angiogenesis, which contributes to the transition to heart failure. |
