Measuring Local Gradients of Intramitochondrial [Ca 2+ ] in Cardiac Myocytes During Sarcoplasmic Reticulum Ca 2+ Release

Abstract

Rationale: Mitochondrial [Ca 2+ ] ([Ca 2+ ] mito ) regulates mitochondrial energy production, provides transient Ca 2+ 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 [Ca 2+ ] mito are limited, and spatial [Ca 2+ ] mito gradients have not been directly measured. Objective: To directly measure local [Ca 2+ ] mito during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca 2+ ] mito gradients. Methods and Results: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca 2+ ] mito during SR Ca 2+ release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Δ[Ca 2+ ] mito amplitude was 29±3 nmol/L, rising rapidly (similar to cytosolic free [Ca 2+ ]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that [Ca 2+ ] mito near SR Ca 2+ 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 Ca 2+ release sites, because the mitochondrial Ca 2+ uniporter was homogeneously distributed, and elevated [Ca 2+ ] applied laterally did not produce longitudinal [Ca 2+ ] mito gradients. Conclusions: We developed methods to measure spatiotemporal [Ca 2+ ] mito gradients quantitatively during excitation–contraction coupling. The amplitude and kinetics of [Ca 2+ ] 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 Ca 2+ signaling.