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

Abstract

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.

Excitation-contraction coupling and mitochondrial energetics

“…Several studies indicate that by buffering [Ca 2+ ] c , rapid mitochondrial Ca 2+ uptake directly affects EC coupling [61,100,117,119,124,147,148,171]. When blocking mitochondrial Ca 2+ uptake, an increase of cytosolic Ca 2+ transients was observed ( Fig.…”

“…6D; [12,117]). Accordingly, localized Ca 2+ sparks induced transient focal depolarizations of Δψ m by ∼10-15 mV [61]. The propagation of caffeine-induced Ca 2+ sparks was suppressed by mitochondria, since inhibition of the mCU (with Ru360) resulted in regenerative Ca 2+ waves across the whole cell [171].…”

“…In the following years, a number of studies attempted to measure mitochondrial Ca 2+ uptake during EC coupling more directly, with quite controversial results [12,26,33,[59][60][61]68,[72][73][74]100,117,119,136,137,140,141,144,147,148,161,170,171,176,191,200,201,213] [26,117,119,148,176,191,200,201] or fluo-3 [33,144,170]) penetrate sarcolemmal and mitochondrial membranes and accumulate (to different degrees) in the mitochondrial matrix. To avoid contamination of the fluorescence signal by cytosolic Ca 2+ , the groups of Hansford, Silverman [59,60,74,136,137] and Bers [26,222] applied MnCl 2 in the extracellular solution.…”

Widespread depolarization during expiration: A source of respiratory drive?

“…The researchers proposed that these partial, transient depolarizations may reflect the mitochondria alternating between active and inactive oxidative phosphorylation [63]. Other studies have also shown mitochondrial membrane potential changes in isolated mitochondria [64,65], and in mitochondria within different cell types, such as cardiomyocytes [66], vascular endothelium [65], and pancreatic B-cells [67]. Buckman and Reynolds found that oligomycin, which inhibits ATP-synthase, decreased the number of fluctuations, suggesting that the fluctuations are related to oxidative phosphorylation [63].…”

Cardiac cell modelling: Observations from the heart of the cardiac physiome project

“…This omission generally has been justified by findings that the role of mitochondria as a calcium sink or source in beat-to-beat regulation of the cytosolic calcium concentration is rather small (Bassani et al 1992). However, recent results from various groups suggest that mitochondrial calcium uptake and release can have a significant effect on cellular calcium homeostasis (Duchen et al 1998, Trollinger et al 2000, see Dedkova and Blatter (2008) for a review. Thus, it appears that the addition of a mitochondrial calcium compartment should be addressed in future modelling studies.…”