Trigger Ca²⁺ is assumed to be the Ca²⁺ influx through the L-type Ca²⁺ channel that causes Ca²⁺ release from the sarcoplasmic reticulum (SR). Although Na⁺ Ca²⁺ exchange (NCX) is essential for Ca²⁺ extrusion, its participation in excitation-contraction coupling is controversial. We investigated action potential (AP)-evoked Ca²⁺ release in cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. These mice live into adulthood and exhibit reduced L-type Ca²⁺ current due to high subsarcolemmal Ca²⁺ ([Ca²⁺]sm). Myocytes were loaded with Fluo-3 and 0 or 3 mM EGTA via the patch pipette to image Ca²⁺ sparks using high-speed confocal microscopy in linescan mode (0.28 ms/line).

In WT-myocytes, a single population of Ca²⁺ sparks was evoked in a synchronous fashion, in presence and absence of EGTA. However, KO-myocytes displayed two Ca²⁺ spark populations in both buffering conditions: the first population had a latency of ~12 ms (~11 ms for WT), the second population appeared more than 35 ms after the stimulus. In absence of EGTA, spark probability was not affected in KO-mice. Interestingly, when 3 mM EGTA was present, Ca²⁺ spark probability was reduced by 60% in KO-mice. Differences found among concentrations of EGTA tested suggest that, in absence of EGTA, high [Ca²⁺]sm may help to prime the dyadic cleft. Nevertheless, when EGTA is present, [Ca²⁺]sm may be reduced and the effect of lacking NCX is unmasked, suggesting that NCX may participate in triggering of AP-evoked Ca²⁺ release. Supported by NIH HL70828 and HL48509. PÑ is a Fulbright Scholar.