Muscle atrophy observed in several physio-pathological situations has harmful consequences for the patients and results from an imbalance between protein synthesis and proteolysis. Despite a thorough understanding of molecular events involved in muscle atrophy, primarily through using rodent and human models, no proven effective treatment exists to date (1). In our group, we use the brown bear model, which does not display muscle atrophy during hibernation, although prolonged fasting and physical inactivity. This muscle atrophy resistance is correlated with inhibition of the pro-atrophic TGF-β (Transforming Growth Factor) signalling and maintenance of the hypertrophic BMP (Bone Morphogenetic Protein) signalling (2). We also showed that winter-hibernating bear serum (WBS) induces hypertrophy in human myotubes (3).

The study aimed to further investigate WBS effects on human myotubes by analysing transcriptome changes (mRNA sequencing) after 48h cultivation with 5% SBS (summer-active bear serum) or WBS (n=3, DESEQ2 analysis). Subsequently, we selected some differentially expressed genes (DEGs) identified above within the BMP pathway and analysed their protein levels (n=8-9, ratio paired t-test). Finally, we investigated the impact of bear serum on human myotubes response (n=6) to increasing doses of BMP7 (0 to 2µg/ml) or TGF-β 3(0 to 100ng/ml) for 30 min. We thus analyzed SMAD3 or SMAD1/5 phosphorylation, indicative of TGF-β or BMP pathway activation, respectively (2-way ANOVA). Data are means +/-SEM and statistical significance threshold was set at 0.05.

We identified 352 DEGs in human myotubes cultivated with WBS versus SBS. Gene Ontology analysis revealed enrichment in pathways related to muscle function, extracellular matrix remodelling and regulation of BMP signalling. Specifically, mRNA levels for several BMP signalling inhibitors (SMAD6, CHRDL2, GREM1/2) and activators (ENG, SCUBE3) decreased by 20-58% in WBS compared to SBS conditions. In addition, several BMP-regulated genes were also downregulated by 40-70% in WBS (ID1, ID3, SMAD6, SAMD11). We further report here that ENG and GREM1 protein levels were also downregulated by 18-37% in WBS versus SBS conditions. We showed that BMP7 treatment induces dose-dependent SMAD1/5 phosphorylation in human myotubes, beginning at 0.1µg/ml in SBS conditions and 0.5µg/ml in WBS. The maximal induction was nevertheless identical in both conditions. Similarly, the response of human myotubes to TGF-β3 treatment was also lower in WBS conditions compared to SBS conditions. SMAD3 phosphorylation showed a dose-dependent increase, starting at 1ng/ml in SBS and 10 ng/ml in WBS. By contrast to the BMP challenge, the induction remained consistently lower across all concentrations, including the higher one (100ng/ml).

Overall, we show a transcriptomic reprogramming of human myotubes cultivated with WBS, including BMP signalling pathway regulation. The reduced responsiveness of both TGFb and BMP pathways to their ligands in the presence of WBS aligns with the hypometabolism previously reported in these myotubes (3) or hibernating brown bear muscle (4). Conversely, the lower induction of TGFβ signalling for all concentrations, contrasted with the consistent plateau of BMP induction at high doses, suggests that WBS may modulate the TGFβ/BMP balance towards the BMP pathway. These data are consistent with the specific TGF-β/BMP balance depicted in atrophy-resistant muscles from the hibernating brown bear (2).