## 4 Discussion
However, it remains unknown whether TOP2B is involved in DOX-induced myotoxicity in skeletal muscle, particularly in oxidative SOL muscle. In this regard, our results for the first time showed that DOX-induced TOP2B upregulation was associated with myotoxicity. Based on this observation, we postulated if EXE-mediated TOP2B hindrance might be associated with myocyte protection against DOX. However, our postulation was rejected since EXE treatment failed to prevent DOX-induced TOP2B upregulation. Thus, it seemsreasonable for us to exclude the TOP2B scenario from possible protective mechanisms of EXE against DOX in SOL muscle at least. Recent studies have demonstrated that DOX-induced mitochondrial damage provokes cellular dysfunction [17]. Given the notion that EXE enhances mitochondrial function and promotes mitochondrial biogenesis [37; 38], we postulated that EXE-mediated mitochondrial enrichment might rescue myocytes from DOX toxicity. To confirm the alterations of mitochondrial volume, we measured the CS levels since they have been used as a prime index of mitochondrial volume estimation, especially in oxidative muscle [39; 40]. We found that SED-DOX treatment did not affect the mitochondrial volume; more surprisingly, EXE-SAL (8 weeks of treadmill running for 60 min/day at 70-80% VO2 max) failed to increase the CS levels (mitochondrial volume), which contradicts the canonical concept of EXE as a potent inducer of mitochondrial biogenesis. Nevertheless, EXE-DOX significantly increased the CS levels, suggesting that EXE may promote mitochondrial biogenesis upon DOX toxicity to accelerate mitochondrial turnover (more biosynthesis of mitochondria in parallel with selective removal of injured mitochondria) for myocyte protection. Indeed, this presumption corresponds to our histological data shown in Figure 2A,B, showing EXE-induced myocyte protection. Besides mitochondria, MRFs have been reported to be involved in DOX-induced myotoxicity. A previous in vitro study has shown that downregulation of MRFs in C2 myoblasts in response to DOX administration impairs myogenic differentiation [41], suggesting that restoration of DOX-induced MRFs loss would be a potential protective mechanism. In support of this concept, a recent study using rats has implicated that EXE-induced upregulation of MRFs may protect skeletal muscle against DOX treatment [14]. However, our study does not support the above study's finding because EXE did not alter MRFs, such as PAX7 (activation of satellite cells), MYOD1 (commitment to differentiation), and MYOG (fusion into myotube). This conflicting result may be due to different study designs in which we used a chronic EXE protocol (eight weeks of treadmill exercise whereas the other study used an acute EXE protocol (two weeks of treadmill exercise). Moreover, the mode of DOX treatment could have affected the results: a chronic model of DOX treatment (e.g., five injections on a bi-weekly basis during which EXE was administered) vs. an acute model of DOX (e.g., one injection immediately after two weeks of EXE). Although these seemingly different study designs might cause the discrepancy, further studies are warranted to define whether EXE-induced MRFs modulation is necessary for myocyte protection against DOX-mediated myotoxicity. protein synthesis, leading to more muscle loss. This proposition was not the case because neither SED-DOX nor EXE-DOX altered a p-mTORSer2448 state. We next were prompted to seek to explain why EXE-DOX promoted more muscle loss but maintained healthy muscle morphology from autophagy because autophagy is another catabolic process comparable to the UPS. While the UPS degrades short-lived and soluble proteins, autophagy catholizes long-lived and insoluble proteins via lysosome-dependent pathways to maintain skeletal muscle homeostasis [44; 45; 46; 47]. Recent studies have shown that dysregulated autophagy is associated with skeletal muscle atrophy and myopathy [48; 49; 50].