Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control
Charlotte Gineste,
Sonia Youhanna,
Sabine U. Vorrink,
Sara Henriksson,
Andrés Hernández,
Arthur J. Cheng,
Thomas Chaillou,
Andreas Buttgereit,
Dominik Schneidereit,
Oliver Friedrich,
Kjell Hultenby,
Joseph D. Bruton,
Niklas Ivarsson,
Linda Sandblad,
Volker M. Lauschke,
Håkan Westerblad
Affiliations
Charlotte Gineste
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Sonia Youhanna
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Sabine U. Vorrink
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Sara Henriksson
Umeå Core Facility for Electron Microscopy, Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
Andrés Hernández
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Arthur J. Cheng
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Thomas Chaillou
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Andreas Buttgereit
Institute of Medical Biotechnology, Department of Chemical and Biological Engineering, Friedrich-Alexander University of Erlangen-Nürnberg, 91052 Erlangen, Germany
Dominik Schneidereit
Institute of Medical Biotechnology, Department of Chemical and Biological Engineering, Friedrich-Alexander University of Erlangen-Nürnberg, 91052 Erlangen, Germany
Oliver Friedrich
Institute of Medical Biotechnology, Department of Chemical and Biological Engineering, Friedrich-Alexander University of Erlangen-Nürnberg, 91052 Erlangen, Germany
Kjell Hultenby
Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Huddinge, Sweden
Joseph D. Bruton
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Niklas Ivarsson
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Linda Sandblad
Umeå Core Facility for Electron Microscopy, Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
Volker M. Lauschke
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden; Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany; University of Tübingen, Tübingen, Germany; Corresponding author
Håkan Westerblad
Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden; Corresponding author
Summary: Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.
