I ntracellular ATP is required for mitochondrial apoptotic pathways in isolated hypoxic rat cardiac myocytes

Objectives: The present study examined the possibility that intracellular ATP levels dictate whether hypoxic cardiac myocytes die by apoptosis or necrosis. Background: Although apoptosis and necrosis may appear to be distinct forms of cell death, recent studies suggest that the two may represent different outcomes of a common pathway. In ischemic myocardium, apoptosis appears early, while energy stores are presumably still available, followed only later by necrosis. Methods: Neonatal rat cardiac myocytes were exposed to continuous hypoxia, during which the intracellular ATP concentration was modulated by varying the glucose content in the medium. The form of cell death was determined at the end of the hypoxic exposure. Results: Under total glucose deprivation, ATP dropped precipitously and cell death occurred exclusively by necrosis as determined by nuclear staining with ethidium homodimer-1 and smearing on DNA agarose gels. However, with increasing glucose concentrations (10, 20, 50, 100 mg/dl) cellular ATP increased correspondingly, and apoptosis progressively replaced necrosis until it became the sole form of cell death, as determined by nuclear morphology, DNA fragmentation on agarose gels, and caspase-3 activation. The data showed a signiﬁcantly positive correlation between myocyte ATP content and the percentage of apoptotic cells. Hypoxia resulted in lactate production and cellular acidiﬁcation which stimulates apoptosis. However, acidiﬁcation-induced apoptosis was also increased in an ATP-dependent fashion. Loss of mitochondrial membrane potential and cytochrome c release from the mitochondria was observed in both the apoptotic and necrotic cells. Furthermore, translocation of Bax from cytosol into mitochondria preceded these events associated with mitochondrial permeability transition. Increased lactate production and a lack of effect by the mitochondrial inhibitor oligomycin indicated that ATP was generated exclusively through glycolysis. Conclusions: We demonstrate that ATP, generated through glycolysis, is a critical determinant of the form of cell death in hypoxic myocytes, independently of cellular acidiﬁcation. Our data suggest that necrosis and apoptosis represent different outcomes of the same pathway. In the absence of ATP, necrosis prevails. However, the presence of ATP favors and promotes apoptosis. The clinical ramiﬁcations of a common mechanism for apoptosis and necrosis are likely to be profound. Our present data suggest that the apoptotic machinery in these cells was arrested during the ischemia-associated depletion of ATP, and that the subsequent replenishing of the cellular energy during reperfusion re-initiated the interrupted apo-sis ptotic process. Moreover, our data suggest that ‘Reperfu-[7] sion Injury’, the postulated ability of reperfusion-associ-or ated phenomena to lethally injure myocytes at the onset of reperfusion, may in fact be an ATP-induced acceleration of apoptosis. On the other hand, several previous reports have indeed shown that ischemia results in a signiﬁcant increase in TUNEL-positive myocytes in vivo. However, it is very