Fig 2: Proposed pathway of TMZ effects on HFD-induced muscle dysfunction. Mitochondrial quality control (MQC) including mitochondrial biogenesis, dynamics, and autophagy cooperate to ensure mitochondrial homeostasis. Mitochondrial dysfunction arising from impaired mitochondrial quality control is critically involved in the pathogenesis of muscle atrophy. During high-fat diet (HFD)-induced obesity, adversely alteration in atrophic factors (i.e. Atrogin-1/MAFbx and MuRF1) and MQC were observed. However, our data shows exercise training or TMZ treatment enhances MQC and suppresses atrophic factors to alleviate HFD-induced muscle dysfunction. Thus, we conclude that TMZ might be a potential therapy in the treatment of muscle atrophy in addition to exercise against HFD-induced muscle dysfunction.

Fig 3: Blocking STAT3 activation with Stattic, a STAT3 inhibitor, prevents GDF11 mediated atrophy in vitro. A Bright-field images of myotubes treated with rGDF11 (50ng/ml), with or without STAT3 inhibitor Stattic for 48 h. Scale bars = 50 µm. The fiber widths were measured and calculated (right panel). B Myotubes treated with rGDF11 then with Stattic for 48h were used for Western blot analysis with antibodies against iNOS, pY-STAT3, total STAT3, socs3, and GAPDH. C Total protein content of rGDF11-treated myotubes, with or without STAT3 inhibitor Stattic for 48 h. D NO levels were measured in supernatant from the myotubes described in the panel. E Representative western blotting images of ubiquitin from myotubes. F Protein expression of trim63, fbx32, and foxo1 in myotubes treated with rGDF11 then with Stattic for 48h. G Representative Western blots of phosphorylation and total STAT3 from myotubes treated with rGDF11, siALK5, or AcvRIIb. Data presented as mean ± SEM. Versus vehicle control, *P < 0.05, **P < 0.01, ***P < 0.001; versus rGDF11 control, #P < 0.05, ##P < 0.01, ###P < 0.001; n=3

Fig 4: Assessment of ubiquitin proteasome levels in a mouse model of PVLD. A: Levels of Fbxo32 and Trim63 mRNA from RNAseq analysis at 49 d after SeV infection or SeV-UV control (n = 5 mice per condition in each group). B: Levels of Fbxo32 and Trim63 proteins at 8, 12, 21, and 49 d after SeV infection or SeV-UV control (n = 3–4 mice per condition per group). ns=not significant (P>0.05) by ANOVA.

Fig 5: The P4HB inhibitor inhibits apoptosis in vitro and in vivo. (a and b) For apoptosis analysis, C2C12 myoblasts were treated with 0.1% DMSO (vehicle control), or 0 µM, 0.5 µM, 1 µM, 3 µM, and 5 µM CCF642 in combination with 35 µM cisplatin for 24 h. The cells were collected and stained with Annexin V and PI. (c) Western blot analysis of apoptotic markers in C2C12 myoblasts treated with indicated concentrations of CCF642 in combination with 35 µM cisplatin for 24 h. (d) Body weight of YES2-bearing mice after intraperitoneal injections of albumin vehicle (10 mg/kg, n = 9) or CCF642 (10 mg/kg, n = 9) accompanied with cisplatin (5 mg/kg) three times a week. (e) Tumour volume of YES2-bearing mice after intraperitoneal injections of albumin vehicle (10 mg/kg, n = 9) or CCF642 (10 mg/kg, n = 9) accompanied with cisplatin (5 mg/kg) three times a week. (f) Representative micrographs of H&E histology of GA muscle in YES2-bearing mice injected with cisplatin and CCF642 (n = 9), relative to YES2-bearing mice injected with cisplatin and albumin (n = 9). Scale bars, 100 µm. (g) Quantification of the average myofiber cross-sectional areas of GA muscle after injections of cisplatin with CCF642 or cisplatin with albumin into YES2-bearing mice. (h) Western blotting in vivo of MHC, MURF1 and LC3 protein in GA muscle of YES2-bearing mice injected with cisplatin and CCF642 (n = 4), compared to YES2-bearing mice injected with cisplatin and albumin (n = 4)