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Published on: June 16, 2022
Visualization of Mitochondrial Ca2+ Signals in Skeletal Muscle of Zebrafish Embryos with Bioluminescent Indicators
Manuel Vicente1, Jussep Salgado-Almario2, Joaquim Soriano3
1Physiology and Cell Dynamics Group, Centro Regional de Investigaciones Biomédicas (CRIB) and Facultad de Medicina de Albacete, Universidad de Castilla-La Mancha, C/Almansa 14, 02006 Albacete, Spain. Manuel.Vicente@uclm.es.
Abstract:
Mitochondria are believed to play an important role in shaping the intracellular Ca2+ transients during skeletal muscle contraction. There is discussion about whether mitochondrial matrix Ca2+ dynamics always mirror the cytoplasmic changes and whether this happens in vivo in whole organisms. In this study, we characterized cytosolic and mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ transients whereas they increased the frequency of cytosolic Ca2+ transients and muscle contractions, confirming the subcellular localization of mitoGA. Mitochondrial Ca2+ dynamics were also determined with mitoGA and were found to follow closely cytoplasmic changes, with a slower decay. Cytoplasmic Ca2+ kinetics and propagation along the trunk and tail were characterized with GA and with the genetically encoded fluorescent Ca2+ 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.
Insights
Mitochondria influence skeletal muscle contraction by regulating intracellular calcium (Ca2+). This study shows mitochondrial Ca2+ dynamics closely follow cytoplasmic changes during zebrafish muscle activity.
Area of Science:
- Cellular Biology
- Mitochondrial Physiology
- Skeletal Muscle Physiology
Background:
- Mitochondria are implicated in regulating intracellular calcium (Ca2+) transients during skeletal muscle contraction.
- Uncertainty exists regarding whether mitochondrial matrix Ca2+ dynamics mirror cytoplasmic changes in vivo.
Purpose of the Study:
- To characterize cytosolic and mitochondrial Ca2+ signals during spontaneous skeletal muscle contractions in zebrafish embryos.
- To investigate the relationship between mitochondrial and cytoplasmic Ca2+ dynamics in vivo.
Main Methods:
- Utilized bioluminescent genetically encoded calcium indicators: GFP-aequorin (cytosolic) and mitoGFP-aequorin (mitochondrial matrix).
- Measured Ca2+ transients alongside transmitted light imaging of muscle contractions.
- Employed mitochondrial uncoupler FCCP and mitochondrial calcium uniporter (MCU) inhibitor DS16570511 to validate tool specificity.
- Compared bioluminescent data with fluorescent Ca2+ indicator Twitch-4 for spatio-temporal resolution.
Main Results:
- Mitochondrial Ca2+ transients closely followed cytoplasmic Ca2+ changes during muscle contractions, with a slower decay.
- FCCP and DS16570511 confirmed mitoGFP-aequorin localization and demonstrated their impact on Ca2+ signaling and contraction frequency.
- GFP-aequorin enabled continuous measurements, resolving kinetic parameters comparable to higher-resolution fluorescent indicators.
Conclusions:
- Mitochondrial Ca2+ dynamics are tightly coupled to cytoplasmic Ca2+ transients during skeletal muscle activity in vivo.
- Bioluminescent indicators like GFP-aequorin are valuable tools for long-term in vivo studies of cellular Ca2+ signaling.
- This study provides in vivo evidence for the role of mitochondria in modulating skeletal muscle Ca2+ handling.
