Quantification of Mitochondrial Calcium Dynamic Changes During Voltage-Induced Calcium Release in Mammalian Skeletal Muscle

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Mitochondrial Ca2+ uptake regulates mitochondrial metabolism and synthesis of ATP to meet demands of muscle contraction. In a recent study, we found that mitochondrial Ca2+ uptake also plays a critical role in modifying rapid Ca2+ transients in skeletal muscle of amyotrophic lateral sclerosis transgenic mice (Zhou 2010). To better understand how mitochondria are involved in the control of Ca2+ transients in healthy and diseased conditions, we need to trace dynamic changes of Ca2+ inside mitochondria during contractile activation. Rudolf (2004) first demonstrated mitochondrial Ca2+ uptake in skeletal muscle during contraction using the cameleon YC2. The maximal ratio change, however, was not more than 0.4, and Ca2+ uptake was not quantified. We have now used the improved biosensor YC3.6 (Nagai 2004) with a dynamic range close to 6 (in in-situ calibration) to monitor changes of free [Ca2+] inside mitochondria during voltage clamp-induced Ca2+ release (VICR). We targeted YC3.6 to mitochondria by adding a mitochondrial signal sequence at 5’ of the cDNA to obtain mt11-YC3.6. One week after FDB muscles of adult mice were transfected by electroporation, enzyme-isolated single FDB fibers expressing mt11-YC3.6 were patch-clamped. Following a depolarizing pulse, the cytosolic Ca2+ transient (monitored by x-rhod-1) and the change of Ca2+ inside mitochondria were simultaneously recorded in a confocal microscope. We found that the maximal ratio change of mt11-YC3.6 reached 3.25 following a membrane depolarization. The free [Ca2+] inside mitochondria during VICR was calculated using parameters from an in-situ calibration. [Ca2+]mito increased to 200 nM during a 10 ms pulse and reached 3 μM during a 800 ms pulse. These results and similar measurements in progress will allow us to evaluate the uptake of Ca2+ by mitochondria during single twitches and tetanic contraction. Supported by MDA/NIAMS.