The mechanical stimulation of myotubes counteracts the effects of tumor-derived factors through IL-4 secretion and the modulation of the activin/follistatin ratio

Exercise counteracts cachexia, but it is unclear to which extent the exercise-dependent mechanical stimulation of muscle per se plays a role in exercise beneficial effects. To study the mechanisms underlying mechanical stimulation, we cultured C2C12 myotubes in the absence or in the presence of a cyclic mechanical stretching stimulus (MS) and in the absence or presence of C26 tumour-derived factors (C26-CM), so as to mimic the mechanical stimulation of exercise and cancer cachexia, respectively. We found that C26-CM contains activin and induces activin release by myotubes, further exacerbating its negative effects, consisting in myotube atrophy and in hampering myoblast recruitment and fusion into myotubes. A high level of circulating activin is an adverse prognostic factor in cancer patients, and our in vitro results demonstrate that activin may be a direct player and not just a marker of cachexia. We also found that MS is sufficient to counteract the adverse tumour-mediated effects on muscle cells, in association with an increased follistatin/activin ratio in the cell culture medium, indicating that myotubes actively release follistatin upon stretching. In addition, MS induces IL- 4 secretion by muscle cells. Recombinant follistatin counteracts C26 tumour effects on myotubes exclusively by rescuing fusion index, while recombinant IL-4 ameliorates fusion index, as well as the myotube size, both in terms of myotube diameter and number of nuclei per myotube. Our results indicate that tumour cells negatively affect muscle cells by releasing soluble factors and that MS is sufficient to counteract these effects, by affecting the muscle secretome with autocrine/paracrine pathways. Activin and Act-R ligands are becoming increasingly important as triggers of muscle wasting and as pharmacological targets to treat cachexia; however, since follistatin alone is incapable to entirely block the C26-CM effects, the development of novel activintargeted approaches should consider the existence of further significant tumour-secreted factors mediating cachexia.