Induction of cardioplegic arrest immediately activates the myocardial apoptosis signal pathway

Myocardial ischemia-reperfusion, including cardioplegic arrest (CA), has been associated with cardiac apoptosis induction. However, the time course of apoptosis activation and the trigger mechanisms are still unclear. Because apoptosis inhibition may represent a novel therapeutic strategy for long-term myocardial preservation, we sought to investigate the time course of apoptosis signal-pathway induction during CA. As to method, Sprague-Dawley rats (300–350 g) were anesthetized, intubated, and mechanically ventilated. CA was initiated by infusion of ice-cold crystalloid solution (Custodiol, 10 ml/kg) into the aortic root, and hearts were rapidly excised and stored for 0, 30, 60, and 120 min in 0.9% sodium chloride solution (28°C). In controls, no CA was initiated before removal and storage at 28°C. In another group, calcium-rich cardioplegia was used, and an additional group received a caspase-8 inhibitor before CA induction. Left ventricular cytosolic extracts were isolated and investigated for the activity of caspase-3 and -6 (effector caspases) and caspase-8 and -9 (involved in extrinsic and intrinsic pathways of apoptosis induction). Fluorometric activity assays were performed by using specific substrates. As a result, activities of all tested caspases were significantly increased immediately after CA induction compared with controls. Administration of the caspase-8 inhibitor significantly reduced activities of all caspases. With calcium-rich cardioplegia, caspase activities were significantly lower compared with low-calcium CA. Control hearts also showed an increase of caspase activities during cold-storage ischemia without CA but had significantly different time courses compared with hearts with CA. In conclusion, our data show rapid apoptosis signal-pathway induction immediately following CA exposure. Thus apoptosis signal-pathway inhibition as a potential strategy for improved myocardial preservation would have the greatest effect when applied before CA exposure.