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 We established primary muscle cultures and serial passaged myotubes from physically active, sedentary lean, and individuals with type 2 diabetes (T2D). We measured maximal ATP synthesis capacity (ATPmax) and resting ATP flux (ATPase) in vivo by 31P magnetic resonance spectroscopy, type-I fibers and intramyocelluar lipid (IMCL) in vastus lateralis tissue using immunohistochemistry techniques, and oxidative phosphorylation complexes (OXPHOS) 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=0.62, p=0.02; P5: r=0.69, p=0.01), ATPmax (P4: r=0.54, p=0.03; P5: r=0.47, p=0.05), and OXPHOS (P4: r=0.66, p=0.04; P5: r=0.77, 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=0.70, p=0.01), IMCL (r=0.89, p<0.001), and mitochondrial content (r=0.51, p=0.03). Conclusions Myotubes through the first two passages following immunopurification (referred to as passage 4 and 5) reflect mitochondrial and type-I fiber content in vivo phenotype of the donor.

Greater Oxidative Capacity in Primary Myotubes from Endurance-trained Women

“…However, we show that primary muscle cells isolated from exercise-trained skeletal muscle and grown in vitro maintain greater oxidative capacity compared to untrained donors cells. Our findings are in line with previous observations demonstrating that some exercise adaptations persist in primary myotubes grown in vitro (6,7). Together, these data suggest that primary myotubes isolated from exercise-trained individuals maintain the “exercise metabolic program” for enhanced oxidative metabolism ex vivo .…”

“…It is worth noting that all of our experiments were carried out at passage four. Prior work has shown that the metabolic characteristics of myotubes gradually fade after each passage (7,19). Thus, it would be of use to identify how many passages the “exercise metabolic program” can persist in these myotubes.…”

“…In one study, exercise-induced alterations in skeletal muscle mitochondrial content persisted in myotubes, but they did not perform functional assays to assess oxidative capacity (7). In another study, exercise training enhanced capacity for myotubes to oxidize some but not all substrates, but their exercise training protocol did not promote a robust increase in mitochondrial biogenesis in muscle biopsy samples (6).…”

Altered dynamics of lipid metabolism in muscle cells from patients with idiopathic inflammatory myopathy is ameliorated by 6 months of training

“…Importantly, imprinting of muscle satellite cells appears to be relatively stable, and it can persist in vitro across many passages of cells (Covington et al . 2015; Gaster, 2019). Seaborne et al .…”

“…So far, only a very limited number of studies have explored the impact of relatively short term in vivo interventions on the in vitro phenotypes of cultured muscle cells (Sparks et al 2013;Hinkley et al 2017;Lund et al 2017). Importantly, imprinting of muscle satellite cells appears to be relatively stable, and it can persist in vitro across many passages of cells (Covington et al 2015;Gaster, 2019). Seaborne et al (2018a,b) showed that even acute exercise-induced changes in methylation of specific genes related to exercise adaptive response can persist in skeletal muscle for as long as 22 weeks.…”