Rapid Changes in Mitochondrial Ca2+-Concentration in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin Null Mice

Abstract

Mitochondrial Ca2+-uptake is important for the regulation of aerobic ATP production and is involved in apoptosis. Muscle fibers contract in response to transient elevation in intracellular calcium concentration. At rest, the Ca2+ stored inside the sarcoplasmic reticulum (SR) is predominantly bound to calsequestrin (CASQ). Since muscle fibers lacking calsequestrin (CASQ) have a reduced SR Ca2+ content and present alterations in mitochondrial morphology that may affect function, we investigated mitochondrial Ca2+-handling using a ratiometric FRET-based calcium indicator (mtD3cpv Cameleon) targeted to the mitochondrial matrix. Experiments were conducted in enzymatically dissociated FDB muscle fibers from wild type (WT) and CASQ-null mice, lacking both CASQ isoforms either in the presence (1 mM) or absence of extracellular calcium. Small but significant differences in free mitochondrial Ca2+-concentration ([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 10% - 90% rise time of 18.4±0.4 ms. The decline in [Ca2+]mito during and after stimulation trains was governed by 3 temporally distinct processes with rate constants of approximately 40 s−1, 1.6 s−1 and 0.2 s−1 (at 26 oC). During the 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 increase in free [Ca2+]mito was less pronounced, and in the absence of extracellular Ca2+, the increase in free [Ca2+]mito was virtually absent. These results provide direct evidence for rapid Ca2+ uptake by the mitochondria and suggest that mitochondrial Ca2+ uptake is sensitive to the amount of Ca2+ available inside the sarcoplasmic reticulum as well as in the extracellular spaces.