Y27632, a RHO kinase inhibitor, limits myocardial reperfusion injury through a nitric oxide-dependent mechanism

Shabaz A Hamid, Hugo SF Bower, Gary F Baxter, Royal Veterinary College, London NW1 0TU

Rho kinases (ROCK) are signalling proteins mediating a diverse range of physiological responses. Cytoprotective properties of ROCK inhibitors in myocardial ischaemia and reperfusion have been reported. In experimental myocardial infarction, pre-ischaemic treatment with the ROCK inhibitor, Y27632, reduced infarct size through augmentation of the PI3K/Akt/NO pathway (Wolfrum et al., 2004). Recent studies have highlighted the salvage role of PI3K/Akt/NO activation specifically in reperfusion (e.g. Hamid and Baxter, 2005). Therefore, the aim of the present study was to investigate whether Y27632 given only during early reperfusion protected against reperfusion injury on mechanism involving the PI3K/Akt/NO pathway.

Male Sprague-Dawley rats (300-400g) were anaesthetised with pentobarbital sodium (50mg/kg). Hearts were excised and Langendorff perfused at 11.3 kPa with Krebs-Henseleit buffer. A 3-0 suture was placed around the left main coronary artery to permit reversible regional ischaemia. After 35 min coronary occlusion and 120 min reperfusion, infarct size was determined by Evans’ blue and triphenylterazolium staining and expressed as % risk zone volume (I/R %). Y27632 5μM was administered from 30 min ischaemia until 10 min after reperfusion. To examine the involvment of PI3K/Akt/NO activation, further groups were assigned to receive either the NO synthase inhibitor L-NAME 100μM or the PI3K inhibitor wortmannin 10 nM, co-perfused with Y27632.

Table1 Baseline cardiac function and risk zone data for treatment groups.

Data are mean±SEM. *p<0.05 vs Control, ψ not significant vs Y27632 (1-way ANOVA with Tukey’s post-hoc analysis.)

Y-27632 in early reperfusion significantly limited infarct size, suggesting a role of ROCK in the development of reperfusion injury. L-NAME abolished the cardioprotective effect of Y27632 while wortmanin attenuated, but did not abolish, the protective effect of Y27632. These studies provide evidence for a NO-dependent cytoprotective effect of Y27632 at early reperfusion and suggest that ROCK inhibition leads to NO generation independently of PI3K/Akt activation.

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