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

Lu, X., Ginsburg, K.S., Kettlewell, S., Bossuyt, J., Smith, G.L. and Bers, D.M. (2013) Measuring local gradients of intramitochondrial [Ca2+] in cardiac myocytes during sarcoplasmic reticulum Ca2+ release. Circulation Research, 112(3), pp. 424-431. (doi:10.1161/CIRCRESAHA.111.300501)

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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+]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 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.

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 Ca2+ signaling.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Professor Godfrey and Kettlewell, Dr Sarah
Authors: Lu, X., Ginsburg, K.S., Kettlewell, S., Bossuyt, J., Smith, G.L., and Bers, D.M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Circulation Research
Publisher:American Heart Association
ISSN:0009-7330
ISSN (Online):1524-4571
Published Online:14 December 2012

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