In the heart, reperfusion following an ischaemic episode results in a marked increase in intracellular Ca²⁺ ([Ca²⁺]_i) and causes cellular dysfunction and death. Although Ca²⁺ influx via reverse-mode Na⁺/Ca²⁺ exchange has been implicated (Eigel & Hadley, 2001), all of the ionic mechanisms that are responsible have not yet been identified (Blaustein & Lederer, 1999). The hypothesis that the diastolic membrane potential influences Na⁺/Ca²⁺ exchange during chemically induced hypoxia/ reoxygenation in ventricular myocytes has been tested. Adult rats were humanely killed with pentobarbital according to the local and Canadian Guidelines for Laboratory Animal Care. Right ventricular myocytes were then obtained by enzymatic dissociation (Shimoni et al. 1998).

Superfusion with extracellular K⁺ ([K⁺]_o) of 2.5, 5, 7, 10, 15 and 0.5 mM gave the following resting membrane potentials: -102.2 ± 1.89, -86.5 ± 1.03, -80.1 ± 1.25, -73.6 ± 1.02, -66.4 ± 1.03 and -27.6 ± 1.63 mV, respectively (n = 7-9 cells, means ± S.E.M.). In parallel, myocytes were subjected to chemically induced hypoxia (4 mM NaCN and 5 mM 2-deoxy-glucose), followed by reoxygenation at these same [K⁺]_o, while [Ca²⁺]_i was monitored using the calcium-sensitive dye fura-2 AM. After chemically induced hypoxia/reoxygenation had caused an increase in [Ca²⁺]_i, hyperpolarization of myocytes with 2.5 mM [K⁺]_o significantly reduced [Ca²⁺]_i levels (7.5 ± 0.32 vs. 16.9 ± 0.55 %, n = 6 experiments, P < 0.05, Student’s paired t test); 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, n = 6 experiments, P < 0.05). Myocyte hypercontracture and mortality increased in parallel with Ca²⁺ overload at depolarized membrane potentials. The involvement of Na⁺/Ca²⁺ exchanger in Ca²⁺ overload was evaluated using the Na⁺/Ca²⁺ exchanger inhibitor KB-R7943. During reoxygenation KB-R7943 (5 µM) almost completely prevented the increase in [Ca²⁺]_i in both control conditions (in 5 mM [K⁺]_o: 2.2 ± 0.40 vs. 10.8 ± 0.14 %, n = 6 experiments) and in depolarized myocytes (in 15 mM [K⁺]_o: -2.1 ± 0.51 vs. 11.3 ± 0.05 %, n = 6 experiments).

These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca²⁺]_i during hypoxia/reoxygenation, and suggest that this is due to an effect of diastolic membrane potentials on the Na⁺/Ca²⁺ exchanger, since at depolarized potential this exchanger mechanism works in reverse mode, causing Ca²⁺ influx.

This work was supported by the Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research.