Resting Membrane Potential Regulates Na+–Ca2+ Exchange‐Mediated Ca2+ Overload during Hypoxia–Reoxygenation in Rat Ventricular Myocytes

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Abstract
In the heart, reperfusion following an ischaemic episode can result in a marked increase in [Ca2+]i and cause myocyte dysfunction and death. Although the Na+–Ca2+ exchanger has been implicated in this response, the ionic mechanisms that are responsible have not been identified. In this study, the hypothesis that the diastolic membrane potential can influence Na+–Ca2+ exchange and Ca2+ homeostasis during chemically induced hypoxia–reoxygenation has been tested using right ventricular myocytes isolated from adult rat hearts. Superfusion with selected [K+]o of 0.5, 2.5, 5, 7, 10 and 15 mm yielded the following resting membrane potentials: −27.6 ± 1.63 mV, −102.2 ± 1.89, −86.5 ± 1.03, −80.1 ± 1.25, −73.6 ± 1.02 and −66.4 ± 1.03, respectively. In a second set of experiments myocytes were subjected to chemically induced hypoxia–reoxygenation at these different [K+]o, while [Ca2+]i was monitored using fura‐2. These results demonstrated that after chemically induced hypoxia–reoxygenation had caused a marked increase in [Ca2+]i, hyperpolarization of myocytes with 2.5 mm[K+]o significantly reduced [Ca2+]i (7.5 ± 0.32 vs. 16.9 ± 0.55 %); while depolarization (with either 0.5 or 15 mm[K+]o) significantly increased [Ca2+]i (31.8 ± 3.21 and 20.8 ± 0.36 vs. 16.9 ± 0.55 %, respectively). As expected, at depolarized membrane potentials myocyte hypercontracture and death increased in parallel with Ca2+ overload. The involvement of the Na+–Ca2+ exchanger in Ca2+ homeostasis was evaluated using the Na+–Ca2+ exchanger inhibitor KB‐R7943. During reoxygenation KB‐R7943 (5 μm) almost completely prevented the increase in [Ca2+]i both in control conditions (in 5 mm[K+]o: 2.2 ± 0.40 vs. 10.8 ± 0.14 %) and in depolarized myocytes (in 15 mm[K+]o: −2.1 ± 0.51 vs. 11.3 ± 0.05 %). These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca2+]i during hypoxia–reoxygenation. This appears to be due mainly to an effect of diastolic membrane potential on the Na+–Ca2+ exchanger, since at depolarized potentials this exchanger mechanism operates in the reverse mode, causing a significant Ca2+ influx.