Mitochondrial Ca2+ (Ca2+m) plays a fundamental role in the cardiac excitation-contraction process. Cytosolic Ca2+ (Ca2+c) enters mitochondria through the mitochondrial uniporter and is extruded by the mitochondrial Na+-Ca2+ exchanger. Mitochondria buffer Ca2+c during normal and pathological conditions. For example an increase in ventricular cardiomyocyte workload elevates the cytosolic Ca2+ concentration (Ca2+c), which effects Ca2+m concentration (Ca2+m), permitting Ca2+-dependent activation of mitochondrial enzymes to match ATP production to the increase in ATP demand. Under pathological conditions Ca2+c overload may cause Ca2+m overload, promoting cell injury. In contrast, Ca2+m buffering, up to a point, counteracts cardiac glycoside toxicity derived from Ca2+c overload. Normal automaticity in sinoatrial node cells (SANC) involves Ca2+c cycling within a coupled-clock system: periodic local, subsarcolemmal Ca2+ releases (LCRs) from sarcoplasmic reticulum (Ca2+ clock) activate an inward Na+-Ca2+ exchange current that accelerates the diastolic depolarization prompting the ensemble of surface membrane ion channels (membrane clock) to generate the next action potential (AP). We hypothesized that dynamic Ca2+m cycling impacts on SANC normal automaticity. Specific inhibitors of Ca2+ influx into (Ru360) and Ca2+ efflux from (CGP-37157) mitochondria in intact single isolated SANC were used. Ca2+m was indexed by selectively quenching the cytosolic fluorescence Ca2+ probe, Indo-1 by Mn2+. Ru360 decreased Ca2+m over 10-min and increased SANC spontaneous AP firing rate. Conversely, CGP-37157 increased Ca2+m over 10-min and reduced AP firing rate. Inhibition of Ca2+m influx significantly increased the SR Ca2+ load, LCR size, duration and amplitude (imaged via confocal linescan), and shortened the LCR period. Conversely, inhibition of Ca2+ efflux from mitochondria decreased the SR Ca2+ load, LCR characteristics, and lengthened the LCR period. Changes in total Ca2+ released by the LCR ensemble were highly correlated with the change in the SR Ca2+ load (r2=0.97). Changes in LCR period induced by changes in Ca2+m flux predicted (r2=0.84) concurrent changes in the spontaneous SANC AP cycle length. Our novel numerical model simulations reproduce our experimental findings and validate our interpretation of these findings: changes in mitochondrial Ca2+ flux affect the spontaneous AP firing rate of SANC via changes in SR Ca2+ loading and release. Furthermore, while our experimental method to measure Ca2+m does not permit evaluation of systolic and diastolic Ca2+m, our model predicts that under basal conditions both diastolic and systolic Ca2+m are lower than the diastolic and systolic Ca2+c, respectively. However, the systolic Ca2+m is higher than diastolic Ca2+c. In summary, a change in SANC Ca2+m flux translates into a change in the AP firing rate by effecting changes in Ca2+c and SR Ca2+ loading, which affects the characteristics of spontaneous SR Ca2+ release.
