Dependence of hypoxic cellular calcium loading on Na⁺-Ca²⁺ exchange

Na⁺-Ca²⁺ exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca²⁺ loading and damage in the heart. Despite the fact that both [Na⁺]_i and [Ca²⁺]_i have been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca²⁺]_i occuring during hypoxia is linked to the rise in [Na⁺]_i via Na⁺-Ca²⁺ exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na⁺-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (Po₂<002 mm Hg), and SBFI fluorescence was monitored to index changes in [Na⁺]_i. Parallel experiments were performed with indo-1-loaded cells to index [Ca²⁺]_i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture consistent with a rise in [Na⁺]_i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na⁺]_i fell. The increase in both [Na⁺]_i and [Ca²⁺]_i, seen during hypoxia, could be markedly reduced by performing experiments in Na⁺-free buffer. These experiments suggested that hypoxic Ca²⁺ loading is linked to a rise in Na⁺_i via Na⁺-Ca²⁺ exchange. To show that Na⁺-Ca²⁺ exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na⁺ was abruptly removed after contracture occurred. The sudden removal of buffer Na⁺ would be expected to stimulate cell Ca²⁺ entry via Na⁺-Ca²⁺ exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca²⁺ loading via the exchanger. The effect of reducing hypoxic buffer [Na⁺] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na⁺-contaimng buffer (144 mM NaCl, n=38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na⁺-free buffer (144 mM choline chloride, n=17) hypercontracted at reoxygenation (p<0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na⁺ current. R 56865 blunted the rise in [Na⁺]_i typically seen after the onset of rigor, suggesting that Na⁺ entry may occur, in part, through voltage-gated Na⁺ channels. These experiments provide evidence that [Na⁺]_i rises during hypoxia and leads to cellular Ca²⁺ loading and cell destruction via Na⁺-Ca²⁺ exchange. Prevention of the rise in [Na⁺]_i during hypoxia reduces cellular injury in this model. Further studies are required to fully elucidate the mechanisms underlying the rise in [Na⁺]_i that occurs during hypoxia and ischemia.