Measuring local gradients of intramitochondrial [Ca2+] in cardiac myocytes during sarcoplasmic reticulum Ca2+ release

Abstract

RATIONALE:: Mitochondrial [Ca2+] ([Ca2+] mito) regulates mitochondrial energy production, provides transient Ca 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 Ca 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 Ca 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 release sites, because the mitochondrial Ca 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 Ca signaling. © 2012 American Heart Association, Inc.