DIFFERENTIAL ROLE OF DISTINCT P2 RECEPTOR SUBTYPES IN CARDIOMYOCYTE DEATH INDUCED BY ISCHEMIC/HYPOXIC STRESS

Rationale: Blood levels of extracellular nucleotides are greatly increased during heart ischemia, but, despite the presence of both P2X and P2Y receptors on cardiomyocytes, their effects on the subsequent myocardial damage are still unknown. Objective: In this study, we aimed at investigating the role of ATP and specific P2 receptors in the appearance of cell injury in a cardiac model of ischemic hypoxia (murine HL-1 cardiomyocytes). Methods and Results: Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N2 for 16 h in a glucose-free medium. In this condition, we detected a significant increase (63.6%±9.7; p< 0.005) in the release of ATP in the culture medium, and, in cultured cells, a 320.9 %±15.8 increase of cytoplasmic histone-associated-DNA-fragments, which represent a marker of apoptosis. To dissect the role of specific P2 receptors we used a combined approach by both adding different P2 antagonists to cardiomyocytes before hypoxia and by silencing selected P2 receptors genes via specific small interfering RNAs. For the latter, gene silencing was checked by real-time quantitative PCR. Both selective pharmacological inhibition and gene silencing indicated that P2Y2 and P2X7 receptors are directly involved in the induction of cell death during ischemic hypoxia, whereas the P2Y4 receptor has a protective effect. Conclusions: Averall, these findings may have important therapeutic implications for the development of novel cardioprotective agents that specifically target P2 receptors.