Visualization of Mitochondrial Ca(2+) Signals in Skeletal Muscle of Zebrafish Embryos with Bioluminescent Indicators

Mitochondria are believed to play an important role in shaping the intracellular Ca(2+) transients during skeletal muscle contraction. There is discussion about whether mitochondrial matrix Ca(2+) dynamics always mirror the cytoplasmic changes and whether this happens in vivo in whole organisms. In this study, we characterized cytosolic and mitochondrial Ca(2+) signals during spontaneous skeletal muscle contractions in zebrafish embryos expressing bioluminescent GFP-aequorin (GA, cytoplasm) and mitoGFP-aequorin (mitoGA, trapped in the mitochondrial matrix). The Ca(2+) transients measured with GA and mitoGA reflected contractions of the trunk observed by transmitted light. The mitochondrial uncoupler FCCP and the inhibitor of the mitochondrial calcium uniporter (MCU), DS16570511, abolished mitochondrial Ca(2+) transients whereas they increased the frequency of cytosolic Ca(2+) transients and muscle contractions, confirming the subcellular localization of mitoGA. Mitochondrial Ca(2+) dynamics were also determined with mitoGA and were found to follow closely cytoplasmic changes, with a slower decay. Cytoplasmic Ca(2+) kinetics and propagation along the trunk and tail were characterized with GA and with the genetically encoded fluorescent Ca(2+) indicator, Twitch-4. Although fluorescence provided a better spatio-temporal resolution, GA was able to resolve the same kinetic parameters while allowing continuous measurements for hours.