The Effect of Mechanical Stretch on Myotube Growth Suppression by Tumor‐derived Factors

Abstract

The mechanisms of cancer-induced muscle wasting are widely investigated with in vivo preclinical models and in vitro experiments examining effects of tumor-derived factors. Myogenic differentiation is associated with rapid increases in myotube size and contractile properties, which tumor-derived factors can suppress. Specifically, colon-26 (C26) conditioned media induces catabolic signaling in cultured myotubes. While mechanical stimuli can induce anabolic signaling and myotube growth, the effect of mechanical stretch on myotubes treated with C26 conditioned media (CM) is not known. Therefore, we examined if uniaxial mechanical stretch could reverse myotube growth suppression caused by C26 conditioned media. Cachectic C26 or non-cachectic EL4 lymphoma CM was collected after 48h (~90% confluence). C2C12 myoblasts were seeded on type I collagen-coated polystyrene or Flexcell uniaxial stretch plates, proliferated, then placed in differentiation media for 5-7 days. On day 5 of differentiation, myotubes were treated with 50% control, C26, or EL4 CM for 48h. An additional experiment stretched myotubes 5% for the last 24h of the C26 incubation, and myotubes were collected immediately post stretch. Myotube diameter quantification used digital images. For all comparisons, at least three replicates from 2 independent experiments were used. Myotubes increased diameter (28%, p<0.0001) and myosin heavy chain-fast (MHC-F) protein expression (IOD/ug protein; 127%, p<0.0001) between days 5 and 7 of differentiation. Myotube growth was associated with increased AktT308 phosphorylation and suppressed E3 ligase expression. C26, but not EL4 suppressed myotube diameter (C26: -18%, p<0.0001; EL4: 1%, p=0.708) and MHC-F protein expression (C26: -62%, p<0.0001; EL4: 0%, p=0.999). C26 suppressed AktS473, ERK1/2, and 4E-BP1phosphorylation and increased MuRF1 and Atrogin1 expression. The C26 decrease in myotube diameter was not rescued by 24h of stretch (C26 control: -18%, p<0.0001; C26 stretch: -12%, p=0.0005). Stretch prevented C26 suppression of MHC-F protein expression (C26 control: -18%, p=0.001; C26 stretch: 11%, p=0.389). In summary, MHC-F protein concentration was rescued by mechanical stretch in C26 treated myotubes independent of myotube size. Additional research is warranted to determine how mechanical stretch prevents myosin's catabolic regulation in myotubes by tumor-derived factors.