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Ex Vivo Measurements of Ca2+ Transients in Intracellular Compartments of Skeletal Muscle Fibers by Means of Genetically Encoded Probes

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Calcium Signalling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1925))

Abstract

We report a method for ex vivo measurements of Ca2+ transients in skeletal muscle fibers, both in the sarcoplasma and into the mitochondria. These measurements are based on the use of genetically encoded probes. Addition of targeting DNA sequences, in frame with the probe encoding sequence, ensures protein expression in specific compartments. The use of probes with different excitation spectra allows the simultaneous determination of cytosolic and mitochondrial Ca2+ transients in the same fiber. Probe encoding plasmids are expressed in flexor digitorum brevis (FDB) muscles by means of the in vivo electroporation technique. Measurements are then performed ex vivo in isolated single myofibers.

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Acknowledgments

Research is supported by fundings from the Italian Ministry of Education, University, and Research (PRIN 2015W2N883_003) and the French Muscular Dystrophy Association AFM-Téléthon (18857).

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Authors and Affiliations

  1. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Gaia Gherardi & Cristina Mammucari

Authors
  1. Gaia Gherardi
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  2. Cristina Mammucari
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Correspondence to Cristina Mammucari .

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Editors and Affiliations

  1. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Anna Raffaello

  2. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Denis Vecellio Reane

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Gherardi, G., Mammucari, C. (2019). Ex Vivo Measurements of Ca2+ Transients in Intracellular Compartments of Skeletal Muscle Fibers by Means of Genetically Encoded Probes. In: Raffaello, A., Vecellio Reane, D. (eds) Calcium Signalling. Methods in Molecular Biology, vol 1925. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9018-4_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9018-4_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9017-7

  • Online ISBN: 978-1-4939-9018-4

  • eBook Packages: Springer Protocols

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