Role of Mitochondria in Calcium Regulation of Spontaneously Contracting Cardiac Muscle Cells

Abstract

Mitochondrial involvement in the regulation of cytosolic calcium concentration ([Ca2+]i) in cardiac myocytes has been largely discounted by many authors. However, recent evidence, including the results of this study, has forced a reappraisal of this role. [Ca2+]i and Ca2+ in the mitochondria ([Ca2+]m) were measured in this study with specific fluorescent probes, fluo-3 and di-hydro-rhod-2, respectively; mitochondrial membrane potential (DeltaPsim) was monitored with JC-1. Addition of uncouplers or inhibitors of the mitochondrial respiratory chain was found to cause a twofold decrease in the rate of removal of Ca2+ from the cytosol after a spontaneously generated Ca2+ wave. These agents also caused a progressive elevation of [Ca2+]i, an increase in the number of hotspots of Ca2+ release (Ca2+ sparks), and depression of mitochondrial potential. The Ca2+-indicative fluorophore dihydro-rhod-2 has a net positive charge that contributes to selective accumulation by mitochondria, as supported by its co-localization with other mitochondrial-specific probes (MitoTracker Green). Treatment of dihydro-rhod-2-loaded cells with NaCN resulted in rapid formation of "black holes" in the otherwise uniformly banded pattern. These are likely to represent individual or small groups of mitochondria that have depressed mitochondrial potential, or have lost accumulated rhod-2 and/or Ca2+; all of these eventualities are possible upon onset of the mitochondrial permeability transition. Release of Ca2+ from the sarcoplasmic reticulum and the resultant spontaneous contractility of cardiac muscle are proposed to be triggered by the induction of the mitochondrial permeability transition and the subsequent loss of [Ca2+]m.

Examining Intracellular Organelle Function Using Fluorescent Probes

“…Although ordinary background Ca 2ϩ sparks are typically single events, the MPT-associated ones frequently occurred as clusters, as seen in Figure 1D. Although it is possible that mitochondrial Ca 2ϩ may contribute to Ca 2ϩ spark formation, 59 we were unable to exclude the possibility that such local Ca 2ϩ release from sarcoplasmic reticulum (SR) may be driven by ROS/RNS generated in and released by these adjacent mitochondria undergoing MPT induction (a "ROS/RNS-induced Ca 2ϩ release" mechanism). Regardless of whether Ca 2ϩ or ROS modulate this increased spark frequency after MPT induction, this phenomenon could induce pathological disturbances in cardiac excitation and rhythm, for example, contributing to postischemic reperfusion arrhythmias.…”

Confocal laser scanning microscopy of calcium dynamics in living cells

“…For example, calcium levels could be monitored simultaneously with organelle-specific probes that allow tracking of mitochondria (Chacon et al, 1996;Bowser et al, 1998;Dubinsky and Levi, 1998;Ohata et al, 1998;Trollinger et al, 1997), Golgi bodies (Zha et al, 1995) or the endoplasmic reticulum (Rizzuto et al, 1998;Stricker et al, 1998). Alternatively, calciumsensitive fluorophores could be used in conjunction with fluorescent dyes that monitor important molecules of signal transduction such as protein-kinase C (Moutinho et al, 1998;Olds et al, 1995;Sakai et al, 1997), cyclic AMP (Hempel et al, 1996;Tsien and Backsai, 1995), protein kinase A (Higashi et al, 1997), and calmodulin (Török et al, 1998).…”

Functional heterogeneity of mitochondria after cardiac cold ischemia and reperfusion revealed by confocal imaging

“…Using an imaging technique, the heterogeneity of the mitochondrial redox state has been established in skeletal muscle (3) and isolated cardiomyocytes (4). Heterogeneity of mitochondrial calcium has been shown in the model of simulated (chemical) ischemia of cardiac cells after cyanide treatment, with single or small groups of mitochondria that have expelled their matrix calcium (5). Heterogeneous mitochondrial damage is suggested in various abnormalities including ischemia-reperfusion injury and apoptosis, where heterogeneous release of mitochondrial cytochrome c has been demonstrated (6).…”