Calcium Signaling in Transgenic Mice Overexpressing Cardiac Na(+)-Ca(2+) Exchanger

We have produced transgenic mice which overexpress cardiac Na(+)-Ca(2+) exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes. In whole cell clamped myocytes dialyzed with 0.1–0.2 mM Fura-2, the magnitude of I(Ca )and Ca(2+) (i) -transient triggered by I(Ca )or caffeine were not significantly different in transgenic vs. control myocytes. In transgenic myocytes, activation of I(Ca), however, was followed by a large slowly inactivating transient inward current representing I(Na-Ca). This current depended on Ca(2+) release as it was abolished when sarcoplasmic reticulum (SR) Ca(2+) was depleted using thapsigargin. Ca(i)-transients triggered by rapid application of 5 mM caffeine, even though equivalent in control and transgenic myocytes, activated larger I(Na-Ca )(∼5 pA/pF at −90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca(2+) (i) -transient and I(Na-Ca )was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni(2+) was equally effective in blocking I(Na-Ca )in control or transgenic myocytes. In 9 out of 26 transgenic myocytes, but none of the controls, Ca(2+) influx via the exchanger measured at +80 mV caused a slow rise in [Ca(2+)](i )triggering rapid release of Ca(2+) from the SR. SR Ca(2+) release triggered by the exchanger at such potentials was accompanied by activation of transient current in the inward direction. In 2 mM Fura-2–dialyzed transgenic myocytes caffeine-triggered Ca(i)-transients failed to activate I(Na-Ca), even though the kinetics of inactivation of I(Ca )slowed significantly in caffeine-treated myocytes. In 0.1 mM Fura-2–dialyzed transgenic myocytes 100 μM Cd(2+) effectively blocked I(Ca )and suppressed Ca(i)-transients at −10 or +50 mV. Our data suggests that in myocytes overexpressing the exchanger, the content of intracellular Ca(2+) pools and the signaling of its release by the Ca(2+) channel vis-à-vis the Na(+)-Ca(2+) exchanger were not significantly altered despite an up to ninefold increase in the exchanger activity. We conclude that the exchanger remains functionally excluded from the Ca(2+) microdomains surrounding the DHP/ryanodine receptor complex.