CALCIUM SIGNALING IN TRANSGENIC MICE OVEREXPRESSING CARDIAC NA-CA2+ EXCHANGER()

We have produced transgenic mice which overexpress cardiac Na+-Ca2+ ex change activity. Overexpression has been assessed by Western blot, Nor thern blot, and immunofluorescence. Functional overexpression was anal yzed using membrane vesicles and isolated ventricular myocytes. In who le cell clamped myocytes dialyzed with 0.1-0.2 mM Fura-2, the magnitud e of I-Ca and Ca-i(2+)-transient triggered by I-Ca or caffeine were no t significantly different in transgenic vs. control myocytes. In trans genic myocytes, activation of I-Ca, however, was followed by a large s lowly inactivating transient inward current representing INa-Ca. This current depended on Ca2+ release as it was abolished when sarcoplasmic reticulum (SR) Ca2+ was depleted using thapsigargin. Ca-i-transients triggered by rapid application of 5 mM caffeine, even though equivalen t in control and transgenic myocytes, activated larger INa-Ca (similar to 5 pA/pF at -90 mV) in transgenic vs. control myocytes (1.5 pA/pF). The decay rate of caffeine-induced Ca-i(2+)-transient and INa-Ca was 2.5 times faster in transgenic than in control myocytes. 5 mM Ni2+ was equally effective in blocking INa-Ca in control or transgenic myocyte s. In 9 out of 26 transgenic myocytes, but none of the controls, Ca2influx via the exchanger measured at +80 mV caused a slow rise in [Ca2 +](i) triggering rapid release of Ca2+ from the SR. SR Ca2+ release tr iggered by the exchanger at such potentials was accompanied by activat ion of transient current in the inward direction. In 2 mM Fura-2-dialy zed transgenic myocytes caffeine-triggered Ca-i-transients failed to a ctivate INa-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 mu M Cd2+ effectively blocked I-Ca and suppre ssed Ca-i-transients at -10 or +50 mV. Our data suggests that in myocy tes overexpressing the exchanger, the content of intracellular Ca2+ po ols and the signaling of its release by the Ca2+ channel vis-a-vis the Na+-Ca2+ exchanger were not significantly altered despite an up to ni nefold increase in the exchanger activity. We conclude that the exchan ger remains functionally excluded from the Ca2+ microdomains surroundi ng the DHP/ryanodine receptor complex.