Measuring Local Gradients of Intramitochondrial [Ca2+] in Cardiac Myocytes During Sarcoplasmic Reticulum Ca2+ Release

Rationale: Mitochondrial [Ca2+] ([Ca2+](mito)) regulates mitochondrial energy production, provides transient Ca2+ buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca2+](mito) are limited, and spatial [Ca2+](mito) gradients have not been directly measured. Objective: To directly measure local [Ca2+](mito) during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca2+](mito) gradients. Methods and Results: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca2+](mito) during SR Ca2+ release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Delta[Ca2+](mito) amplitude was 29 +/- 3 nmol/L, rising rapidly (similar to cytosolic free [Ca2+]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca2+](mito) near SR Ca2+ release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37 +/- 4 versus 26 +/- 4 nmol/L, respectively P<0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca2+ release sites, because the mitochondrial Ca2+ uniporter was homogeneously distributed, and elevated [Ca2+] applied laterally did not produce longitudinal [Ca2+](mito) gradients. Conclusions: We developed methods to measure spatiotemporal [Ca2+](mito) gradients quantitatively during excitation-contraction coupling. The amplitude and kinetics of [Ca2+](mito) transients differ significantly from those in the cytosol and are respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca2+ signaling. (Circ Res. 2013;112:424-431.)