title: Effect of serial cell passaging in the retention of fiber type and mitochondrial content in primary human myotubes.
abstract: OBJECTIVE
  The purpose of the study was to determine the effects of passaging on retention of donor phenotypic characteristics in primary human myotubes.
  
  
  METHODS
  Primary muscle cultures and serial passaged myotubes from physically active, sedentary lean, and individuals with type 2 diabetes were established. Maximal ATP synthesis capacity (ATPmax) and resting ATP flux (ATPase) in vivo were measured by (31) P magnetic resonance spectroscopy, type-I fibers and intramyocelluar lipid (IMCL) in vastus lateralis tissue were determined using immunohistochemistry techniques, and oxidative phosphorylation complexes (OXPHOS) were measured by Western immunoblotting. Similar in vitro measures for lipid and type-I fibers were made in myotubes, along with mitochondrial content measured by MitoTracker.
  
  
  RESULTS
  Passage 4 and 5 measures for myotubes correlated positively with in vivo measurements for percent type-I fibers (P4: R(2)  = 0.39, p = 0.02; P5: R(2)  = 0.48, p = 0.01), ATPmax (P4: R(2)  = 0.30, p = 0.03; P5: R(2)  = 0.22, p = 0.05), and OXPHOS (P4: R(2)  = 0.44, p = 0.04; P5: R(2)  = 0.59, p = 0.006). No correlations were observed for IMCL. However, passage 4 measures for myotubes correlated with passage 5 measures for percent type-I fibers (R(2)  = 0.49, p = 0.01), IMCL (R(2)  = 0.80, p < 0.001), and mitochondrial content (R(2)  = 0.26, p = 0.03).
  
  
  CONCLUSIONS
  Myotubes through the first two passages following immunopurification (referred to as passage 4 and 5) reflect the mitochondrial and type-I fiber content in vivo phenotype of the donor.
authors.affiliation: Laboratory of Skeletal Muscle Physiology, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA.|School of Medicine, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Covington
authors.affiliation: Laboratory of Skeletal Muscle Physiology, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA.
authors.forename: Cassandra K
authors.initials: CK
authors.lastname: Myland
authors.affiliation: Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway.
authors.forename: Arild C
authors.initials: AC
authors.lastname: Rustan
authors.affiliation: Laboratory of Skeletal Muscle Physiology, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Ravussin
authors.affiliation: Translational Research Institute for Metabolism and Diabetes, Florida Hospital, Sanford-Burnham Medical Research Institute, Winter Park, Florida, USA.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Smith
authors.affiliation: Laboratory of Skeletal Muscle Physiology, Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA.|Department of Kinesiology, University of Texas at El Paso, El Paso, Texas, USA.
authors.forename: Sudip
authors.initials: S
authors.lastname: Bajpeyi
mesh_terms: D000251:Adenosine Triphosphatases / Q000096:biosynthesis; D000328:Adult; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D005260:Female; D006801:Humans; D050356:Lipid Metabolism; D008297:Male; D008875:Middle Aged; D008931:Mitochondria, Muscle / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism; D010085:Oxidative Phosphorylation; D010641:Phenotype; D052097:Quadriceps Muscle / Q000378:metabolism
chemical_list: D000251:Adenosine Triphosphatases