Regulation of Voltage-Dependent Anion Channel 2 at Glutamate 73 is Critical for its Role in Cardiac Calcium Handling

Abstract

Compelling reports suggest that intra-mitochondrial [Ca 2þ ] mito changes dramatically with intracellular [Ca 2þ ] i transients (1) while other equally compelling investigations suggest there are little to no changes (2). Here we take advantage of the high temporal and spatial resolution of confocal microscopy imaging to examine this question in rat and rabbit ventricular cardiomyocytes. Using a mitochondrially targeted Ca 2þ -sensitive fluorescent protein ''Mitycam'' (3), we examined the time course of changes in [Ca 2þ ] mito (matrix mitochondrial [Ca 2þ ]) in isolated ventricular myocytes. Examination was performed 48 hours after treatment with adenovirusdependent Mitycam expression in ventricular myocytes in primary culture. Mitycam, which co-localizes with mitochondrial-specific markers (e.g. Mito-Tracker red), responds slowly (minutes) to steep changes of [Ca 2þ ] i in saponin permeabilized myocytes ([Ca 2þ ] i << 10 to 250 nM). Mitycam responds more quickly (~1 s) in intact (i.e. non-permeabilized) myocytes where a large change in [Ca 2þ ] i (from~100 nM to~10 mM) is seen following caffeine (10 mM). However, in these cells, physiologic [Ca 2þ ] i transients produced no significant increase in [Ca 2þ ] mito in a beat-to-beat manner but can modestly affect the timeaveraged [Ca 2þ ] mito following changes in heart rate ([Ca 2þ ] mito responds to changes in [Ca 2þ ] i like a low-pass-filter). In contrast, isolated Mitycamcontaining mitochondria respond to changes in [Ca 2þ ]in milliseconds. We conclude that the cardiac [Ca 2þ ] i transient does not significantly change [Ca 2þ ] mito in cardiac myocytes in a beat-to-beat manner but slowly influences the time-averaged [Ca 2þ ] mito signal.

A Calcium Guard in the Outer Membrane: Is VDAC a Regulated Gatekeeper of Mitochondrial Calcium Uptake?

“…Nevertheless, a regulation of the channels Ca 2+ transport activity by E73 was recently confirmed in physiological experiments, where Ca 2+ transfer from lysosomes into mitochondria after lysosomal Ca 2+ release through TRPML1 was shown to depend on E73 [59]. In agreement with this, Ca 2+ transfer from the SR into mitochondria in cardiomyocytes is also regulated through E73 [60]. These findings again highlight the presence of a Ca 2+ regulated uptake of Ca 2+ through VDAC.…”