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In Vivo Monitoring of Ca2+ Uptake into Subcellular Compartments of Mouse Skeletal Muscle

  • Rüdiger RudolfEmail author
  • Sofie Trajanovska
  • David Grant Allen
  • Tullio Pozzan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1925)

Abstract

Ca2+ regulates many functions of skeletal muscle, including excitation-contraction coupling, energy homeostasis, and fiber-type-specific gene expression. However, microscopic observation of Ca2+ signalling in live skeletal muscle tissue has been hampered, in particular, by the combination of the high speed of Ca2+ transients and the contractile properties that are inherent to muscle. The present chapter describes methods to visualize Ca2+ signals during relaxation-contraction cycles in different subcellular compartments at high spatiotemporal resolution or at the global muscle level in combination with simultaneous measurements of muscle force. These protocols employ transfection of genetically encoded ratiometric Ca2+ sensors and two-photon microscopy as well as force transducers and associated hardware for data acquisition. Information on how to determine subcellular localization of the genetically encoded Ca2+ sensors and on how to calibrate the ratiometric data in a semiquantitative manner is given in the final paragraphs.

Key words

Cameleon Force transducer FRET Mitochondria Myoplasm Ratiometric measurement Sarcoplasmic reticulum Two-photon microscopy 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Rüdiger Rudolf
    • 1
    • 2
    • 3
    Email author
  • Sofie Trajanovska
    • 4
  • David Grant Allen
    • 4
  • Tullio Pozzan
    • 5
    • 6
  1. 1.Institute of Molecular and Cell Biology, Mannheim University of Applied SciencesMannheimGermany
  2. 2.Institute of Toxicology and Genetics, Karlsruhe Institute of TechnologyEggenstein-LeopoldshafenGermany
  3. 3.Interdisciplinary Center for Neurosciences, Heidelberg UniversityHeidelbergGermany
  4. 4.School of Medical Sciences and Bosch Institute, School of Molecular Bioscience, University of SydneySydneyAustralia
  5. 5.Neuroscience InstituteItalian National Research Council (CNR)PaduaItaly
  6. 6.Department of Biomedical SciencesUniversity of PaduaPaduaItaly

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