Rho-A dependent kinase (ROCK) activation occurs during early reperfusion following prolonged ischaemia: effect of ischaemic preconditioning and postconditioning

Dwaine Burley, Bethan Rogers, Darshni Haria, Gary Baxter

Cardiff University, Cardiff, UK

Inhibition of Rho-A dependent kinase (ROCK) activation at reperfusion following myocardial ischaemia limits tissue injury (Hamid et al., 2007). Reactive oxygen species (ROS) may be potent activators of ROCK. Since ischaemic preconditioning (IPC) and postconditioning (IPost) reduce ROS generation at reperfusion, we hypothesised that ROCK activation occurs at early reperfusion via ROS generation, and that this activation is inhibited by both IPC and IPost. The aim was to investigate ROCK activity in ischaemia-reperfusion using phosphorylated ezrin/radixin/moesin (phospho-ERM) as a biomarker.

Male Sprague Dawley rats (270-350g) were anaesthetised with pentobarbital sodium (60 mg/kg i.p.) and their hearts were Langendorff perfused. After stabilisation (baseline), hearts were subjected to 35 min regional ischaemia, followed by 2 or 10 min reperfusion. IPC was induced by subjecting hearts to two 3 min periods of global ischaemia and reperfusion prior to index ischaemia. IPost was effected by six 10 sec periods of global ischaemia at the onset of reperfusion. Xanthine and xanthine oxidase (X/XO 100 μM/0.05 U/mL) a superoxide generating system, or hydrogen peroxide (H2O2 10 μM), were perfused for 10 min without ischaemia/reperfusion. Risk zone myocardium was lysed. Using immunoblotting and optical densitometry, we analysed phospho-ERM (thr-558/564/567) and total ERM signals. ROCK activity was expressed as a ratio of phospho-ERM: total ERM, shown in Figure 1.

Figure 1. **P<0.01 vs. Baseline; ##P<0.01 vs. Isch35; ¤¤P<0.01 vs. Isch35 + rep 2 min. 1-way ANOVA + Newman-Keuls post hoc; n = 4-8 per group

Transient ROCK activation occurred at early reperfusion, an effect mimicked by ROS exposure. IPost inhibited ROCK activity in reperfused myocardium but, paradoxically, IPC induced activation of ROCK. Thus, the putative deleterious role of ROCK activation at reperfusion requires further investigation.

Hamid S.A. et al. (2007). Am J Physiol Heart Circ Physiol; 292: H2598-606