Low temperature affects the morphology and impairs glutamine-induced hypertrophic response in human primary myotubes

• Cold water immersion and other strategies are extensively used by athletes to reduce muscle temperature (TEMP) and are believed to limit muscle soreness and improve recovery after strenuous exercise. However, it remains unclear whether low TEMP affects skeletal muscle hypertrophy and protein synthesis in response to anabolic stimuli. In this study, we used an in vitro model to investigate whether a reduced TEMP (32°C, TEMP in skeletal muscle during cold water immersion vs. 37°C, core TEMP) affects the growth and impairs glutamine (GLUT)-induced hypertrophic response in human myotubes. Myoblasts from human muscle biopsies (n=8) were first differentiated into myotubes during 48h at 37°C. Then, myotubes were treated with GLUT to induce hypertrophy and were incubated at either 37 or 32°C during 1h (T1), 8h (T8) or 48h (T48). Myotube area (a marker of myotube size) was assessed at T48 by using immunocytology. Protein synthesis (puromycin incorporation) and phosphorylation levels of two components of the mechanistic target of rapamycin (mTOR) pathway [P70 S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E (4E-BP1] were assessed at T1 and T8 from western blot analysis. Ribosome biogenesis was evaluated at T8 by determining total RNA concentration, and by assessing the levels of 18S ribosomal RNA (18S rRNA), precursor 45S rRNA (pre-45S rRNA) and MYC mRNA from quantitative polymerase chain reaction. Data were analysed by a two-way ANOVA for repeated measures. A first major observation was that low TEMP induced morphological alterations (short myotube segments and larger myotube area). Importantly, GLUT-induced myotube hypertrophy was only observed at 37°C (+40%, P<0,01 at 37°C; +4%, NS at 32°C). Similarly, GLUT stimulated protein synthesis at T1 (main effect, P<0,01), with a 53% increase at 37°C (P<0,05) and a 23% increase at 32°C (NS). Protein synthesis was reduced at 32°C at T8 (main effect, P<0,01), while GLUT increased protein synthesis at 37°C (+17%, P<0,01) but not at 32°C (-7%, NS). GLUT-induced 4E-BP1 phosphorylation at T1 was found at 37°C (+16%, P<0,01) but not at 32°C (-1%, NS). Moreover, GLUT increased the phosphorylation of P70S6K at T8 by 50% at 37°C (P<0,01) and by 26% at 32°C (P<0,05). Overall, total RNA concentration, levels of MYC mRNA, 18S rRNA and pre-45S rRNA were reduced at 32°C. In conclusion, this study indicates that low TEMP affects myotube morphology. In addition, we demonstrate that low TEMP impairs GLUT-induced myotube hypertrophy and protein synthesis, a result accompanied by a minored activation of the mTOR pathway. The impaired ribosome biogenesis observed at 32°C at T8 may also contribute to the reduced protein synthesis at this time point. These findings suggest that reducing muscle TEMP after resistance exercise may be detrimental for muscle hypertrophy and training adaptations. 

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MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Fysiologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Physiology (hsv//eng)

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