Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium

Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium. Am J Physiol Heart Circ Physiol 292: H2598-H2606 2007. First published January 12, 2007; doi: 10.1152/ajpheart.01393.2006.-Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here we investigate the role of Rho kinase (ROCK) activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide (NO) synthase (eNOS). Rat isolated hearts underwent 35 min of left coronary artery Occlusion and 120 min of reperfusion. Phosphorylation of the ROCK substrate protein complex ezrin-radixin-moesin, assessed by immunoblotting and immunofluorescence, was used as a marker of ROCK activation. Infarct size was determined by tetrazolium staining, and terminal dUTP nick-end labeling (TUNEL) positivity was used as an index of apoptosis. The ROCK inhibitors fasudil or Y-27632 given 10 min before ischemia until 10 min after reperfusion reduced infarct size (control, 34.1 +/- 3.8%; 5 mu M fasudil, 18.2 +/- 3.1%; 0.3 mu M Y-27632, 19.4 +/- 4.4%; 5 mu M Y-27632, 9.2 +/- 2.9%). When 5 mu M Y-27632 was targeted specifically during early reperfusion, robust infarct limitation was observed (14.2 +/- 2.6% vs. control 33.4 +/- 4.4%, P < 0.01). The protective action of Y-27632 given at reperfusion was attenuated by wortmannin (29.2 +/- 6.1%) and N-omega-nitro-L-arginine methyl ester (30.4 +/- 5.7%), confirming a protective mechanism involving PI3K/Akt/N Ezrin-radixin-moesin phosphorylation in risk zone myocardium confirmed early and sustained ROCK activation during reperfusion and its inhibition by Y-27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL-positive nuclei in the infarcted region. In conclusion, ROCK activation occurs specifically during early reperfusion. Inhibition of ROCK at reperfusion onset limits infarct size through an Akt/eNOS-dependent mechanism, Suggesting that ROCK activation at reperfusion may be deleterious through Suppression of the RISK pathway.