Introduction: Damped amplitude of circadian rhythm in locomotor activity and clock gene expression is strongly suggested to initiate or exacerbate skeletal muscle catabolism in, e.g., aging, obesity and diabetes [1]. However, functional drugs or nutrients to counteract/reverse circadian rhythm disruption have not been identified yet. A bio-inspired approach could help fill this gap, by taking advantage of the peculiar ability of hibernating brown bears (Ursus arctos) not to develop muscle atrophy and metabolic diseases although they stay torpid (inactive and fasting with a reduced metabolic rate) for 5-7 months. We have shown previously that the hibernating bear serum contains components able to inhibit proteolysis in human muscle cells in vitro [2]. We therefore assessed the potential of bear serum to modulate muscular circadian rhythms and metabolic efficiency.

Method: To monitor the molecular clock oscillation, soleus and extensor digitorum longus (EDL) muscles, and the hypothalamic suprachiasmatic nucleus (SCN) from Period2-luciferase knockin mice [3] were dissected and cultured with 5% bear serum (BS, pool from 15 females and 4 males, see [4] for details about bear immobilization) collected either during summer (SBS, n=10) or winter (WBS, n=11). 5% fetal bovine serum (FBS, n=15) was used as a control. In parallel, mouse skeletal muscle C2C12 cells were cultured with SBS (n=6), WBS (n=6), or horse serum (HS, n=6) to evaluate mitochondrial oxygen consumption rate. All data was analyzed using a one-way ANOVA followed by Tukey-Kramer multiple comparison tests.

Results: We observed that both SBS and WBS increased the amplitude of Per2 independently of muscle type (202% increase p=0.043 in SBS-soleus, 231% increase p=0.003 in WBS-soleus, 680% increase p<0.001 in SBS-EDL, and 690% increase p<0.001 in WBS-EDL). The circadian period was shortened in soleus cultured with SBS (p=0.031), but not in EDL. The robustness of the rhythm was higher in soleus cultured with WBS (p=0.019) and in EDL cultured with SBS (p<0.001) or WBS (p=0.011) than in FBS-treated muscles. Neither SBS nor WBS affected any of the circadian parameters tested in the central clock, i.e. SCN.  On the other hand, mitochondrial oxygen consumption in mouse skeletal muscle cells was not affected by either SBS or WBS (p=0.688 and p=0.664, respectively).

Conclusions: Previous reports showed that the amplitude of clock gene oscillation was higher in skeletal muscle of healthy people than in diabetic patients, suggesting that an ample oscillation is linked to the maintenance of proper metabolic function [5]. In our study, both SBS and WBS increased the amplitude of Per2 oscillation in skeletal muscles, but not in the SCN. However, bear serum had no effect on energy metabolism in myocytes. The effect of bear serum on skeletal muscle clock therefore appears to be independent of energy metabolism, at least in in vitro conditions. Further studies are needed to determine (i) whether bear serum coordinates the circadian clock and energy metabolism under specific culture conditions, such as low temperature, and (ii) to what extent bear serum modifies the circadian transcriptome/proteome of exposed cells.