Ca ²⁺ , ROS, IL-6, and p38 MAPK signaling loops underlying alterations in myotube formation induced by a severe MH/CCD mutation in RyR1

Mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) can result in muscle diseases, termed RyR1-related myopathies (RyR1-RM). Examples include malignant hyperthermia (MH), central core disease (CCD), and centronuclear myopathy (CNM). The muscles involved often have more (and mispositioned) nuclei than normal. A subset of the corresponding mutant proteins shows an overactive or leaky sarcoplasmic reticulum (SR) channel behavior that depletes the SR Ca 2+ content and increases the level of cytosolic Ca 2+ . In addition, two remarkable effects of these RyR1 variants have been reported in cultured myogenic cells: enhanced expression of interleukin-6 (IL-6) and stimulation of myoblast fusion (myonuclei accretion). Here, we have investigated whether the latter effect is due to a possible IL-6-dependent autocrine loop. Toward this goal, we analyzed the impact of the overactive Y523S mutant compared to the wild-type RyR1 after expression in C2C12 cells. The results show that this mutation indeed drastically promotes myoblast fusion up to ~300%. Moreover, this action depends on the sequential activation of SR Ca 2+ release, store-operated Ca 2+ channels, reactive oxygen species (ROS, cytosolic and mitochondrial), calpain, and calcineurin. Additionally, a neutralizing antibody directed against IL-6 and a p38 inhibitor completely suppressed the stimulation of myoblast fusion. Furthermore, in RyR1-expressing cells, myotube formation was promoted by either exogenous IL-6 or conditioned medium obtained from the Y523S-expressing cells. These findings suggest an autocrine mechanism involving the interplay between Ca 2+ , ROS, IL-6, and p38 signaling pathways in controlling myonuclei density, which could be essential to explain the pathogenesis of RyR1-RM.