Calcium (Ca2+) influx into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether mitochondrial Ca2+ transport pathways are altered in the setting of deficient mitochondrial energy synthesis, as increased matrix Ca2+ may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in cardiomyocytes. By the second postnatal week (10–15 day old mice), these mice have developed a dilated cardiomyopathy associated with impaired oxidative phosphorylation. We find evidence of increased mitochondrial Ca2+ during this period using imaging, electrophysiology, and biochemistry. The mitochondrial Ca2+ uniporter, the main portal for Ca2+ entry, displays enhanced activity, whereas the mitochondrial sodium-calcium (Na+-Ca2+) exchanger, the main portal for Ca2+ efflux, is inhibited. These changes in activity reflect changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx, the gene encoding the Na+-Ca2+ exchanger, explains diminished Na+-Ca2+ exchange, the mechanism for enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mitochondria from Tfam knockout animals to be far more sensitive to Ca2+-induced increases in respiration. In the absence of Ca2+, oxygen consumption declines to less than half of control values in these animals, but rebounds to control levels when incubated with Ca2+. Thus, we demonstrate a phenotype of enhanced mitochondrial Ca2+ in a mitochondrial cardiomyopathy model, and show that such Ca2+ accumulation is capable of rescuing deficits in energy synthesis capacity in vitro.
Ca 2þ in the mitochondrial matrix regulates several physiological processes, from ATP synthesis to cell death. Most mitochondrial Ca 2þ uptake occurs through the mitochondrial Ca 2þ uniporter, a highly-selective ion channel embedded in the inner membrane. We recently demonstrated that both Ca 2þ uptake and uniporter channel activity are increased in a mouse model of mitochondrial cardiomyopathies. In these diseases, which primarily present in infants and children, characteristic deficits in oxidative phosphorylation produce a signal that boosts mitochondrial Ca 2þ levels. Here we investigated the mechanism for such enhancement. By selective pharmacological inhibition of individual electron transport chain complexes, we found that rotenone-induced complex I dysfunction increases mitochondrial Ca 2þ uptake. Since Ca 2þ transport is governed by both uniporter activity and the transmembrane voltage gradient (DJ), we measured uniporter activity directly using whole-mitoplast patch-clamp. HEK293T cells were incubated with 0.1, 0.3 and 1 mM rotenone for 72 hr to chronically inhibit complex I. After such inhibition, we found that uniporter current density increased from À72 5 6 pA/pF for control mitoplasts (0 nM rotenone) to À90 5 6 pA/pF in 0.3 mM rotenone and À130 5 20 pA/pF in 1 mM rotenone. Such an increase was not due to acute effects of rotenone on the uniporter channel itself, as it did not affect or only mildly inhibited current densities at these concentrations. Our data indicate that chronic inhibition of complex I produces a signal that increases the activity of MCU.
Mitochondrial diseases often feature early onset of dilated cardiomyopathy, due to the large energetic demand placed by the heart. Among regulators of mitochondrial respiration, we focus on calcium, which potently stimulates ATP synthesis. We assessed the hypothesis that cells boost mitochondrial calcium to stimulate respiration as this declines in mitochondrial cardiomyopathies. To this end, we studied mice that develop a neonatal, severe cardiomyopathy caused by cardiac-specific knockout (KO) of mitochondrial transcription factor A ( Tfam ). Such deletion impairs transcription of mitochondrial DNA, which encodes multiple subunits of the electron transport chain (ETC). Tfam KO mice exhibited a dilated cardiomyopathy, with decreased fractional shortening and increased LV diameters, and had marked inhibition of multiple ETC complexes. We determined that Tfam KO mitochondria took up calcium twice as fast as mitochondria from littermate controls, while calcium efflux was approximately 70% slower. Whole-mitoplast voltage clamp revealed that the enhanced calcium uptake was due to an increase in the current carried by the uniporter. Furthermore, the larger uniporter current reflected increased amounts of uniporter subunit proteins. This occurred despite a reduction in the transcripts of genes encoding these subunits, suggesting a post-translational mechanism for the enhanced uniporter stability. Finally, we found that the rate of ADP-stimulated oxygen consumption in calcium-free solution was 50% less in the Tfam KO mitochondria compared to controls, but increased substantially more, nearly to control levels, when calcium was present (2.5-fold increase for KO versus 1.7-fold for control). In conclusion, enhanced mitochondrial calcium signaling in a mitochondrial cardiomyopathy model may serve to compensate for energetic failure.
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