Effect of Serial Cell Passaging in the Retention of Fiber Type and Mitochondrial Content in Primary Human Myotubes Article

International Collaboration

cited authors

  • Covington, Jeffrey D., Myland, Cassandra K., Rustan, Arild C., Ravussin, Eric, Smith, Steven R., Bajpeyi, Sudip

funding text

  • This work was supported by an unrestricted research grant from Novartis, Novartis Clinical Innovation Fund (to S.R.S.); an unrestricted research grant from Takeda Pharmaceuticals North America; and NIH 1R01AG030226-01A2 (to S.R.S.) and was partially supported by the NORC Center grant # 2P20DK072476-06 sponsored by NIDDK and COBRE (NIH 2P20-RR021945). This work used the facilities of the Cell Biology and Bioimaging Core facilities that are supported in part by COBRE (NIH P20-RR021945) and NORC (NIH 1P30-DK072476) center grants from the NIH.

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

Publication Date

  • December 1, 2015

webpage

published in

category

start page

  • 2414

end page

  • 2420

volume

  • 23

issue

  • 12

WoS Citations

  • 1

WoS References

  • 37