Measuring Local Gradients of Intra-Mitochondrial [Ca] in Cardiac Myocytes During SR Ca 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: We used in-situ calibration of the mitochondrially targeted inverse pericam indicator Mitycam and 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 &[plusmn] 3 nM, rising rapidly (similar to cytosolic [Ca2+]i) 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-lines) vs. mid sarcomere (M-line) reached a higher peak ampli¬tude (37 &[plusmn] 4 vs. 26 &[plusmn] 4 nM, 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 quanti¬tatively during excitation-contraction coupling. The amplitude and kinetics of [Ca2+]mito transients differ significantly from those in the cytosol and are higher and faster near the Z- vs. M-line. This approach will help clarify SR-mitochondrial Ca2+ signaling.
- Received November 12, 2012.
- Revision received December 5, 2012.
- Accepted December 12, 2012.
- Copyright © 2012, Circulation Research