Ca²⁺sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers

Abstract

Ca(+) sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca(2+) sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca(2+) sparks were rare in these fibers. At 5-7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca(2+) sparks. The average rate of occurrence of Ca(2+) sparks is >10-fold higher after 5-7 days in culture than in days 1-3. With the use of fibers cultured for 7 days, application of the Ca(2+) channel blockers Co(2+) or nifedipine almost completely suppressed the occurrence of Ca(2+) sparks, as previously shown in embryonic fibers, suggesting that Ca(2+) sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca(2+) sparks, suggesting that Ca(2+) sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling.

Dysferlin stabilizes stress-induced Ca 2+ signaling in the transverse tubule membrane

“…Single myofibers were enzymatically isolated in DMEM with 2% (wt/vol) BSA, 1 μL/mL gentamicin, and 2 mg/mL type II collagenase (Gibco, Life Technologies) for 2 h at 37°C, as adapted from ref. 45. Myofibers were incubated at 37°C for 12-24 h and plated on glass coverslips coated with Matrigel (BD Biosciences) or Geltrex (Invitrogen) before experimentation.…”

Enhanced Excitation-Coupled Calcium Entry in Myotubes Expressing Malignant Hyperthermia Mutation R163C Is Attenuated by Dantrolene

“…FDB muscles were dissected from adult mice (C57/B6), and single intact FDB myofibers were enzymatically isolated as described previously (Brown et al, 2007). After isolation, FDBs were plated on ECM (Sigma, St. Louis, MO)-coated glass-bottomed imaging dishes (Matek Corporation Ashland, MD) and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 0.2% bovine serum albumin and 100 mM gentamicin (Sigma).…”

The Na+/Ca2+Exchange Inhibitor 2-(2-(4-(4-Nitrobenzyloxy)phenyl)ethyl)isothiourea Methanesulfonate (KB-R7943) Also Blocks Ryanodine Receptors Type 1 (RyR1) and Type 2 (RyR2) Channels

“…Preparation of Isolated Fiber Cultures from Adult Mouse Skeletal Muscle. Flexor digitorum brevis (FDB) were dissected from adult mice (C57BL/6), and single intact FDB myofibers were enzymatically isolated as described previously (Brown et al, 2007). After isolation, the fibers were plated on Matrigel-coated plates (BD Biosciences, San Jose, CA) and maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% bovine serum albumin and 0.1 mg/ml penicillin/streptomycin (Sigma).…”