Mitochondrial Ca2+ Dynamics in the Heart

Abstract

Compelling reports suggest that during an intracellular [Ca2+]i transient a large influx of Ca2+ enters the mitochondrial matrix and that this influx is followed by an equally enormous Ca2+ efflux. These Ca2+ fluxes underlie a dramatic increase and then decrease in matrix free Ca2+, [Ca2+]mito Yet, other equally compelling investigations suggest that small fluxes occur with little or no measureable changes. Here we take advantage of the high temporal and spatial resolution of confocal microscopy imaging to examine this question in primary cultures of rat and rabbit ventricular cardiomyocytes (48 hours). Using a mitochondrially targeted Ca2+-sensitive fluorescent protein “Mitycam”, we examined the time course of the changes in [Ca2+]mito in isolated ventricular myocytes.Additionally, we have examined isolated Mitycam-containing mitochondria to investigate the rapidity of the kinetics of the changes in [Ca2+]mito as measured by Mitycam in response to changes in extra-mitochondrial [Ca2+] (0 to 100uM). The measured changes in [Ca2+]mito occurred within milliseconds. We therefore conclude that Mitycam (which co-localizes with mitochondrial-specific markers such as MitoTracker red), is not rate-limiting when it reports changes in [Ca2+]mito of ∼1 s ) in intact (i.e. non-permeabilized) cardiac ventricular myocytes where a large change in cytosolic [Ca2+]i (from ∼100 nM to ∼10 uM) following caffeine (10 mM) application. However, in these same cells, physiologic [Ca2+]i transients produced no significant measureable increase in [Ca2+]mito in a beat-to-beat manner. They do, however, report modest time-averaged changes in [Ca2+]mito following changes in heart rate. These observations suggest that [Ca2+]mito responds to changes in [Ca2+]i like a low-pass-filter.We conclude that the cardiac [Ca2+]i transient does not significantly change [Ca2+]mito in cardiac myocytes in a beat-to-beat manner but slowly influences the time-averaged [Ca2+]mito signal.