Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes.

Cardiac mitochondria can take up Ca(2+), competing with Ca(2+) transporters like the sarcoplasmic reticulum (SR) Ca(2+)-ATPase. Rapid mitochondrial [Ca(2+)] transients have been reported to be synchronized with normal cytosolic Ca(2+) transients. However, most intra-mitochondrial free [Ca(2+)] (Ca(2+)) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated Ca(2+) in single rat myocytes using the ratiometric Ca(2+) indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state Ca(2+) dependence on Ca(2+) (with 5 mM EGTA) was sigmoid with Ca(2+)<Ca(2+) for Ca(2+) below 475 nM. With low [EGTA] (50 microM) and 150 nM Ca(2+) (+/-15 mM Na(+)) cyclical spontaneous SR Ca(2+) release occurred (5-15/min). Changes in Ca(2+) during individual Ca(2+) transients were small ( approximately 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca(2+) handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in Ca(2+) and spontaneous Ca(2+) transients (confirming that SR Ca(2+) releases caused the Ca(2+) rise). Confocal imaging of local Ca(2+) (using rhod-2) showed that Ca(2+) rose rapidly with a delay after SR Ca(2+) release (with amplitude up to 10 nM), but declined much more slowly than Ca(2+) (time constant 2.8+/-0.7 s vs. 0.19+/-0.06 s). Total Ca(2+) uptake for larger Ca(2+) transients was approximately 0.5 micromol/L cytosol (assuming 100:1 mitochondrial Ca(2+) buffering), consistent with prior indirect estimates from Ca(2+) measurements, and corresponds to approximately 1% of the SR Ca(2+) uptake during a normal Ca(2+) transient. Thus small phasic Ca(2+) transients and gradually integrating Ca(2+) signals occur during repeating Ca(2+) transients.