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© The Rockefeller University Press, 0021-9525/1998//975 $5.00
The Journal of Cell Biology, Volume 142, Number 4, , 1998 975-988

Articles

Transient Mitochondrial Depolarizations Reflect Focal Sarcoplasmic Reticular Calcium Release in Single Rat Cardiomyocytes



Michael R. Duchen*, Anne Leyssens*, and Martin Crompton{ddagger}

* Department of Physiology, {ddagger} Department of Biochemistry, University College London, London WC1E 6BT, United Kingdom

Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call "flicker." These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K+, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 µM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.

Key Words: mitochondria • intracellular calcium • mitochondrial potential • sarcoplasmic reticulum • cardiomyocyte

Abbreviations used in this paper: DAPPAC, diaminopentane pentammine cobalt; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; PTP, permeability transition pore; SR, sarcoplasmic reticulum; TMRE, tetra-methyl rhodamine-ethyl ester.

We would like to dedicate this paper to the late Dr. Louis Bohm, whose generosity helped to make much of this work possible.

Some animated sequences of images that have been used to provide data for this paper can be viewed on the web at http://www.geribolsover.physiol. ucl.ac.uk/Duchen/jcb.html

Address all correspondence to Michael R. Duchen, Department of Physiology, University College London, Gower St., London WC1E 6BT, UK. Tel.: 44 171 419 3207. Fax: 44 171 916 3239. E-mail: m.duchen{at}ucl.ac.uk


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