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

Cardiac mitochondria can take up Ca2+ competing with Ca2+ transporters like the sarcoplasmic reticulum (SR) Ca2+-ATPase. Rapid mitochondrial [Ca2+] transients have been reported to be synchronized with normal cytosolic [Ca2+], transients. However, most intra-mitochondrial free [Ca2+] ([Ca2+](mito)) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca2+](mito) in single rat myocytes using the ratiometric Ca2+ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca2+](mito) dependence on [Ca2+](i) (with 5 mM EGTA) was sigmoid with [Ca2+](mito) < [Ca2+](i) for [Ca2+](i) below 475 nM. With low [EGTA] (50 mu M) and 150 nM [Ca2+](i) (+/- 15 mM Na+) cyclical spontaneous SR Ca2+ release occurred (5-15/min). Changes in [Ca2+](mito) during individual [Ca2+](i) transients were small (similar to 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SIR Ca2+ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca2+]mito and spontaneous Ca2+ transients (confirming that SR Call releases caused the [Ca2+](mito) rise). Confocal imaging of local [Ca2+](mito) (using rhod-2) showed that [Ca2+](mito) rose rapidly with a delay after SR Ca2+ release (with amplitude up to 10 nM), but declined much more slowly than [Ca2+], (time constant 2.8 +/- 0.7 s vs. 0.19 +/- 0.06 s). Total Call uptake for larger [Ca2+](mito) transients was similar to 0.5 mu mol/L cytosol (assuming 100:1 mitochondrial Ca2+ buffering), consistent with prior indirect estimates from [Ca2+](i) measurements, and corresponds to similar to 1% of the SR Ca2+ uptake during a normal Ca2+ transient. Thus small phasic [Ca2+](mito) transients and gradually integrating [Ca2+](mito) signals occur during repeating [Ca2+], transients. (C) 2009 Elsevier Inc. All rights reserved.