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

Abstract

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 μM fasudil, 18.2 ± 3.1%; 0.3 μM Y-27632, 19.4 ± 4.4%; 5 μM Y-27632, 9.2 ± 2.9%). When 5 μM 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 Nω-nitro-l-arginine methyl ester (30.4 ± 5.7%), confirming a protective mechanism involving PI3K/Akt/NO. 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.