title: Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.
abstract: A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation.
authors.affiliation: Department of Physiology, Vegetative Physiology 4220, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Kubis
authors.affiliation: Department of Biochemistry, Institute of Physiological Chemistry, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Renate J
authors.initials: RJ
authors.lastname: Scheibe
authors.affiliation: Department of Anatomy, Institute of Cell Biology, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Decker
authors.affiliation: Department of Physiology, Vegetative Physiology 4220, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Hufendiek
authors.affiliation: Department of Physiology, Vegetative Physiology 4220, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Nina
authors.initials: N
authors.lastname: Hanke
authors.affiliation: Department of Physiology, Vegetative Physiology 4220, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Gerolf
authors.initials: G
authors.lastname: Gros
authors.affiliation: Department of Physiology, Vegetative Physiology 4220, Hannover Medical School, 30625, Hannover, Germany.
authors.forename: Joachim D
authors.initials: JD
authors.lastname: Meissner
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry; D003402:Creatine Kinase / Q000378:metabolism; D004338:Drug Combinations; D005780:Gelatin / Q000737:chemistry*; D007797:Laminin / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011509:Proteoglycans / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004338:Drug Combinations; D007797:Laminin; D020033:Protein Isoforms; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D005780:Gelatin; D003094:Collagen; D003402:Creatine Kinase; D018995:Myosin Heavy Chains
keywords: T-tubules; creatine kinase; myofibrils; myosin heavy chain