The RISK of ROCK.

SINCE MYOCARDIAL INFARCT SIZE is the major contributor to the development of heart failure, interventions aiming to limit irreversible damage of ischemic myocardium are essential. In the clinical setting, reduction of myocardial oxygen demand and restoration of blood flow to the ischemic myocardium remain the primary targets to accomplish this goal, despite experimental evidence that reperfusion by itself might elicit irreversible injury. In the laboratory, phenomena such as ischemic myocardial preand postconditioning and ischemic preconditioning of remote organs have all been shown to limit infarct size, independent of the employed animal species. Several groups of investigators have expressed that pharmacological mimicking of these phenomena might also benefit the patient. Especially ischemic postconditioning (a form of graded reperfusion) is clinically attractive because this does not require application before the onset of the ischemic insult. Interestingly, preand postconditioning have been proposed to act via similar signaling pathways and target similar end points. Thus both have been shown to involve activation of the reperfusion injury salvage kinase (RISK) pathway and ultimately prevent opening of the mitochondrial permeability transition pore (7). Despite advances in our understanding, the exact mechanisms involved in reperfusion damage and the protection by preand postconditioning remain incompletely understood. In this issue of American Journal of PhysiologyHeart and Circulatory Physiology, Hamid et al. (6) provide evidence that Rho-associated kinases (ROCKs) contribute to irreversible myocardial damage and that blockade of ROCK during early reperfusion can limit infarct size. These findings suggest that the Rho/ROCK pathway is also an active part in reperfusion injury (Fig. 1). The ROCKs consist of two isoforms, ROCK-1 and ROCK-2. ROCK-1 is known as ROK, whereas ROCK-2 is known as ROKor Rho kinase (14, 15, 18). The small guanosine triphosphate-binding protein Rho activates ROCKs, which, in turn, phosphorylate various downstream targets. Both Rho and Rho kinase have been shown to play a central role in diverse cellular functions such as smooth muscle contraction, stress fiber formation, cell migration, and cell proliferation (20). Y-27632 and fasudil are selective ROCK inhibitors that target their ATP-dependent kinase domains and therefore are equipotent in terms of inhibiting both ROCK-1 and ROCK-2 (14). ROCK-1 is highly expressed in kidney, liver, lung, spleen, and testis, whereas ROCK-2 is preferentially expressed in cardiac and brain tissues (14). ROCK may also play a role in a number of cardiovascular diseases because increased activity of ROCKs has been demonstrated in cerebral ischemia, coronary vasospasm, hypertension, vascular inflammation, atherosclerosis, erectile dysfunction, and cardiac hypertrophy (14). Recent evidence indicates that pharmacological inhibition of ROCK is protective against ischemia-reperfusion injury in liver and kidney (8, 21). These findings suggest that ROCK activation contributes to ischemia-reperfusion-induced cell death, possibly via upregulation of Bax and induction of apoptosis (3). Sanada et al. (17) were the first to link Rho kinase to irreversible myocardial cell death when they reported that, in an in vivo dog model, intracoronary infusion of Y-27632 for 30 min after 90 min of ischemia limited infarct size, although they did not investigate or speculate about the possible mechanisms involved. Capitalizing on these observations, Hamid et al. (6) studied the role of ROCK in the development of infarct size in isolated rat hearts that were subjected to 35 min of regional myocardial ischemia. The results confirm earlier reports of a cardioprotective effect of ROCK inhibition before ischemia (1, 5), which involves phosphatidylinositol 3-kinase (PI3K)/Akt and nitric oxide (NO) synthase (NOS) activation (23), linking the ROCK to the RISK pathway. In addition, Hamid et al. (6) reported that ROCK activity increases during early reperfusion and, most importantly, that inhibition of ROCK, selectively during the reperfusion phase, limits infarct size. These observations suggest that patients undergoing interventions to restore myocardial blood flow might benefit from ROCK inhibition. However, the study by Hamid et al. (6) raises several questions that need to be addressed before ROCK inhibition can be advocated as treatment for limitation of reperfusion damage. A concern with any pharmacological study is the selectivity of the drugs employed. Hamid et al. (6) used the ROCK inhibitors fasudil and Y-27632, which are known to inhibit both ROCK-1 and ROCK-2. Although it remains to be determined whether ROCK-1 and/or ROCK-2 are involved in the effects of fasudil and Y-27632, ROCK-2 is the most likely candidate because it is the principal isoform present in the heart. Another concern is that fasudil has been reported to be able to inhibit protein kinase A and protein kinase C, whereas Y-27632 at higher concentrations may also inhibit protein kinase C-related kinase protein kinase N and citron kinase C (16). Nevertheless, both antagonists display a high degree of selectivity for ROCK inhibition (14). Furthermore, Hamid et al. (6) showed similar infarct size limitation with fasudil and Y-27632, and showed that Y-27632 inhibited ROCK activation. Taken together, these observations suggest that infarct limitation was the result of ROCK inhibition. Hamid et al. (6) employed a single duration of index ischemia, and, as a result, it remains unclear whether ROCK activation occurred as a specific result of reperfusion or that it was, at least in part, a time-dependent response. Thus the authors used an ischemic episode of 35 min and determined ROCK activity at the end of ischemia and after 10 min of reperfusion. They interpreted the observation that ROCK acAddress for reprint requests and other correspondence: D. J. Duncker, Experimental Cardiology, Thoraxcenter, Erasmus MC, Univ. Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands (e-mail: [email protected]). Am J Physiol Heart Circ Physiol 292: H2563–H2565, 2007; doi:10.1152/ajpheart.00179.2007.