Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown. 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 ischaemic/hypoxic stress. Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N(2) for 16 hrs in a glucose-free medium. In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone-associated-DNA-fragments, a marker of apoptosis. Addition of either apyrase, which degrades extracellular ATP, or various inhibitors of ATP release via connexin hemichannels fully abolished ischaemic/hypoxic stress-associated apoptosis. To dissect the role of specific P2 receptor subtypes, we used a combined approach: (i) non-selective and, when available, subtype-selective P2 antagonists, were added to cardiomyocytes before ischaemic/hypoxic stress; (ii) selected P2 receptors genes were silenced via specific small interfering RNAs. Both approaches indicated that the P2Y(2) and P2χ(7) receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y(4) receptor has a protective effect. Overall, these findings indicate a role for ATP and its receptors in modulating cardiomyocyte damage during ischaemic/hypoxic stress.

We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X(2)), as well as the P2Y(2,4,6,14) subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-alpha, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect 'per se', but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-alpha, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-alpha contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be 'primed' by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible 'therapeutic' window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.