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 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+]mitoare limited, and spatial [Ca2+]mitogradients have not been directly measured.Objective:To directly measure local [Ca2+]mitoduring normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial [Ca2+]mitogradients.Methods and Results:Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured [Ca2+]mitoduring SR Ca2+release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Δ[Ca2+]mitoamplitude 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+]mitonear SR Ca2+release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37±4 versus 26±4 nmol/L, respectivelyP<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+]mitogradients.Conclusions:We developed methods to measure spatiotemporal [Ca2+]mitogradients quantitatively during excitation–contraction coupling. The amplitude and kinetics of [Ca2+]mitotransients 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.