Mitochondrial Ca²⁺-uptake is important for the regulation of aerobic ATP production and is involved in signaling for apoptosis. Experiments were performed in FDB muscle fibers dissociated from wild type (WT) and CASQ-null mice. Mitochondrial free Ca²⁺ ([Ca²⁺]mito) was measured using a ratiometric FRET-based indicator (mtD3cpv Cameleon) targeted to the mitochondrial matrix, in the presence (1 mM) and absence of extracellular Ca²⁺ at 26 ^oC. Mitochondrial membrane potential was determined using TMRM. Mitochondrial density and morphology was studied by electron microscopy.

Small but significant differences in [Ca²⁺]mito were observed between quiescent WT and CASQ-null fibers. The free [Ca²⁺]mito during steady state electrical stimulation at 1 Hz showed a rapid increase with a rise time of 18.4±0.4 ms. The decline in [Ca²⁺]mito during and after stimulation trains was governed by 4 temporally distinct processes with rate constants of approximately 40 s-1, 1.6 s-1, 0.2 s-1 and 0.03 s-1. During sustained contractions in WT fibers, frequency-dependent increases in free [Ca²⁺]mito occurred, which were smaller in the absence than in the presence of external Ca²⁺. In CASQ-null fibers the increases in free [Ca²⁺]mito was less pronounced.

These results provide direct evidence for rapid Ca²⁺ uptake by the mitochondria and suggest that the mitochondrial Ca²⁺ uptake is sensitive to the amount of Ca²⁺ available in intracellular (notably the sarcoplasmic reticulum) and extracellular stores.