Elliott, B.

Getting, S.J.

MacKenzie, R.

Renshaw, D.

Watt, P.W.

WestminsterResearch

In vitro – Does hypoxia directly increase myostatin & induce atrophy? C2C12 myoblasts were either scratched and exposed to hypoxia or differentiated into myotubes and exposed to hypoxia.                          Despite alterations in total protein content and cell size, no change in cellular myostatin expression was measured (data not shown). In vivo – Does hypoxia increase myostatin? Participants (N = 8, ♂) were     exposed to hypoxic (12 % O2) or control (21 % O2) conditions in a counter-balanced design.  606570758085900 2 4 6 8 10SpO2 at 120 minutes (%) Plasma myostatin at 120 minutes (ng.mL-1) p = 0.044 050100150200250-30 120 240 320% change from baseline Time (min) Introduction Skeletal muscle is the largest tissue in humans and is key in thermoregulation, locomotion, glycolysis and homeostasis. Muscle atrophy increases mortality in a variety of disorders. Myostatin is  a powerful regulator of muscle mass1 and acts via multiple pathways to induce atrophy2.           Hypoxia is a poorly understood inducer of muscle atrophy. Atrophy is seen in mountaineering humans3 (figure 1). Rats exposed to hypoxia for 5 weeks show increased myostatin expression4, as do COPD patients who are chronically (at least 6 months) hypoxemic. Both COPD patients and mountaineering individuals are difficult models to study, as they present with several confounding factors.   We therefore aimed to investigate the effect of hypoxia upon myostatin expression in vitro and in vivo in healthy humans, hypothesizing that hypoxia alone would be sufficient to induce increased myostatin expression and therefore be causative of muscle atrophy.  6065707580859095100-30 0 30 60 90 120SaO2 (%) Time (min) HypoxiaControl* * * * * Acute Hypoxia Rapidly Alters Myotube Size in vitro & Myostatin Signaling in vivo Bradley Elliott1, Derek Renshaw1, Stephen Getting1, Peter Watt2 & Richard Mackenzie1 1. Faculty of Science & Technology, University of Westminster & 2. Chelsea School, University of Brighton Discussion Hypoxia rapidly induces atrophy of myotubes in vitro, with decreased protein content and decreased size, in a time-dependent manner. Hypoxia decreased muscle myostatin content in vivo relative to resting control, suggesting myostatin was either degraded or left the muscle tissue to act in its endocrine role.   These results suggest hypoxia alone is sufficient to induce atrophy. This may in part explain hypoxic conditions or disorders where muscle atrophy is seen. Current work is examining the dose- and time-dependent effect of hypoxia in vivo (figures 7 & 8), before attempts to inhibit the effect of hypoxia upon muscle mass in vivo can be attempted in a similar manner to our in vitro work.      Financial support was provided by the Society for Endocrinology (UK), The Physiological Society & the University of Westminster. Figure 1: Healthy individuals at altitude lose muscle mass. Figure 6: A) SpO2 is reduced during 12 % O2 exposure. B) Muscle myostatin is reduced 320 minutes after hypoxic exposure relative to control time point. Representative Western blot shown. C) Plasma myostatin is not altered by 2 hours hypoxia. D) Drop in SpO2 correlates with increase in plasma myostatin immediately following hypoxic exposure.  1.  McPherron, A. C., Lawler, A. M. & Lee, S. J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387, 83-90 (1997). 2. Elliott, B., Renshaw, D., Getting, S. & Mackenzie, R. The central role of myostatin in skeletal muscle and whole body homeostasis. Acta Physiol (Oxf) 205, 324-340, (2012).  3. Hoppeler, H. et al. Morphological adaptations of human skeletal muscle to chronic hypoxia. Int J Sports Med 11 Suppl 1, S3-9 (1990). 4. Hayot, M. et al. Myostatin up-regulation is associated with the skeletal muscle response to hypoxic stimuli. Molecular and cellular endocrinology (2010). Figure 2:. C2C12 myoblasts were plated in growth media and grown to confluence. Cells were then  either scratched for chemotaxis (top) or differentiated into myotubes (bottom) before being exposed to 1 % or 21 % O2 for 2, 24 or 48 hours. Western blot Hypoxia (O2 12 %) Time (24 hour clock, not to scale) 08:00          10:00                                                  18:00 Hypoxia (O2 ~ 12 % / 10 %) Time (hours) 0                          2                                              4 Plasma 1 % / 21 % O2  2, 24 or 48 hours Figure 5:. Healthy males were biopsied pre (-30 min), immediately post (120 min), 300 min and 320 minutes post 2 hours of 12 % or 21 % O2. N = 8. MB = MB, BS = blood sample. Figure 3: 1% O2 hypoxic inhibition reduces chemotaxis in a NF-κB dependent manner. Representative images shown. Figure 7: Healthy males (N = 8) biopsied pre- and post-exposure to 10 hours of 12 % O2. Plasma collected pre- and post-exposure. Figure 8: Healthy males (N = 8) will have plasma collected pre-, post- and 2 hours post-exposure to 2 hours of 12 % or 10 % O2 in a counter-balanced design, with a 7 day washout between exposures. 020406080100120Ctrl HypoxiaScratch Diameter (% control) Condition pre post post+PS1145A A B B D C Figure 4: 1 % O2 reduces myotube A) protein content & B) diameter in a time-dependent manner which is offset by PS1145.  0204060801001202 24 48Myotube diameter (% control) Time (hours) A A B B C C C C A A A) B) D) C) A) B) Plasma / muscle * 320 -30 120 300 320 -30 120 300 Control Hypoxic 050100150200250300350400450500-30 120 300 320∆ myostatin (% baseline) Time (min) CONTROLHYPOXICPre Post Post + PS1145 012345672 24 48total protein concentration (ug/mL) Time (hours) Control Hypoxia Hypoxia +PS1145A    A    A A    B     A  A C    C 

Acute hypoxia alters myotube size in vitro and myostatin signalling in vivo

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Acute hypoxia alters myotube size in vitro and myostatin signalling in vivo

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