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.

To directly measure local [Ca²⁺]_mito during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca²⁺]_mito gradients.

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, Δ[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.

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.