Effects of [Ca²⁺]_i, SR Ca²⁺ load, and rest on Ca²⁺ spark frequency in ventricular myocytes

In heart, spontaneous local increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) called "Ca²⁺ sparks" may be fundamental events underlying both excitation-contraction coupling and resting Ca²⁺ leak from the sarcoplasmic reticulum (SR). In this study, resting Ca²⁺ sparks were analyzed in rabbit and rat ventricular myocytes with laser scanning confocal microscopy and the fluorescent Ca²⁺ indicator fluo 3. During the first 20 s of rest after regular electrical stimulation, both the frequency of Ca²⁺ sparks and SR Ca²⁺ content gradually decreased in rabbit. When rabbit SR Ca²⁺ content was decreased by reduction of stimulation rate, the initial resting spark frequency was also decreased, even though resting [Ca²⁺]_i was unchanged. The rest-dependent decrease in spark frequency in rabbit cells was prevented by inhibition of Na⁺/Ca²⁺ exchange (which also prevents SR Ca²⁺ depletion during rest). These results suggest that elevation of SR Ca²⁺ content can increase Ca²⁺ spark frequency. In contrast to rabbit cells, 20 s of rest produced a gradual increase in spark frequency in rat cells, although SR Ca²⁺ content was constant and Ca²⁺ influx was completely prevented. This indicates that there is a time-dependent increase in spark probability during rest that is independent of [Ca²⁺]_i or SR Ca²⁺. This effect was also apparent in rabbit cells when SR Ca²⁺ depletion was prevented by blocking Na⁺/Ca²⁺ exchange. Stimulation of Ca²⁺ extrusion via Na⁺/Ca²⁺ exchange in the rat (by Ca²⁺-free superfusion, which slowly depletes SR Ca²⁺ content) converted the normal rest-dependent increase in spark frequency to a decrease. The amplitude of individual Ca²⁺ sparks increased with increasing SR Ca²⁺ content. In the Ca²⁺-overloaded state, fusion of sparks or long-lasting localized increases of [Ca²⁺]_i were observed with increased spark frequency. We conclude that the resting frequency of Ca²⁺ sparks can be independently affected by changes in SR Ca²⁺ content, [Ca²⁺]_i, or rest period. The latter may reflect recovery of the SR Ca²⁺ release channels from inactivation or adaptation.