Abstract 1525: Activation of ROCK-1 by Caspase-3 Cleavage Plays an Essential Role in Cardiac Myocyte Apoptosis
Rho-associated kinase (ROCK-1) is a direct substrate of caspase-3, and caspase-3 activation is associated with heart failure. We evaluated the contribution of ROCK-1 cleavage to cardiomyocyte apoptosis and heart failure using the gain-of-function and the loss-of-function approaches. ROCK-1 protein levels were assessed from cardiac samples taken from patients with end-stage heart failure and normal hearts. We found marked cleavage of ROCK-1, which was absent in normal hearts and in an equivalent cohort of patients with left ventricular assist devices. ROCK-1 cleavage was also demonstrated in apoptotic cardiomyocytes treated with doxorubicin and was blocked by a caspase-3 inhibitor. Similarly, an inducible caspase-3 activation system demonstrated ROCK-1 cleavage and yielded an identical 130 kDa species. In addition, a transgenic model of severe cardiomyopathy in the mouse revealed the presence of an identical 130 kDa ROCK-1 cleaved fragment. We further found that the cleaved, constitutively active ROCK-1 species expressed in cardiomyocytes led to an activation of caspase-3, suggestive of a positive feed-forward regulatory loop. To investigate the functional significance of ROCK-1 in apoptosis, loss-of-function studies were performed. In cultured cardiomyocytes, reduction in ROCK-1 expression using siRNA significantly decreased cardiac apoptosis induced by ceramide. Similarly, a genetic deletion of ROCK-1 in intact mice (ROCK-1−/ −) demonstrated reduced apoptosis in response to pressure overload. We further identified the activation of PTEN (phosphatase and tensin homolog deleted on chromosome ten) and subsequent inhibition of the Akt survival pathway as critical pro-apoptotic mechanisms downstream of ROCK-1. These data suggest an obligatory role for cleavage of ROCK-1 in the amplification of apoptotic signals, inexorably driving heart failure.