Effect of metabolic inhibition on couplon behavior in rabbit ventricular myocytes.

We investigated the effect of combined inhibition of oxidative and glycolytic metabolism on L-type Ca(2+) channels (LCCs) and Ca(2+) spikes in isolated patch-clamped rabbit ventricular myocytes. Metabolic inhibition (MI) reduced LCC open probability, increased null probability, increased first latency, and decreased open time but left conductance unchanged. These results explain the reduction in macroscopic Ca(2+) current observed during MI. MI also produced a gradual reduction in action potential duration at 90% repolarization (APD(90)), a clear decline in spike probability, and an increase in spike latency and variance. These effects are consistent with the changes we observed in LCC activity. MI had no effect on the amplitude or time to peak of Ca(2+) spikes until APD(90) reached 10% of control, suggesting preserved sarcoplasmic reticulum Ca(2+) stores and ryanodine receptor (RyR) conductance in those couplons that remained functioning. Ca(2+) spikes disappeared completely when APD(90) reached <2% of control, although in two cells, spikes were reactivated in a highly synchronized fashion by very short action potentials. This reactivation is probably due to the increased driving force for Ca(2+) entry through a reduced number of LCCs that remain open during early repolarization. The enlarged single channel flux produced by rapid repolarization is apparently sufficient to trigger RyRs whose Ca(2+) sensitivity is likely reduced by MI. We suggest that loss of coupling fidelity during MI is explained by loss of LCC activity (possibly mediated by Ca(2+)-calmodulin kinase II activity). In addition, the results are consistent with loss of RyR activity, which can be mitigated under conditions likely to enlarge the trigger.

Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1.

The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.