Title: Pharmacological inhibition of ryanodine receptors after eccentric contractions delays the regeneration process in rat skeletal muscle

Abstract: Muscle damage induced by eccentric contraction (ECC) is triggered by prolonged local accumulation of calcium ions (Ca 2+ ) in the cytoplasm. The lack of homeostasis in the regulation of intracellular Ca 2+ concentration ([Ca 2+ ]i) observed after ECC has been linked to a decrease in the function of the ryanodine receptor (RyR) which forms the Ca 2+ release channel in the sarcoplasmic reticulum. In contrast, transient and periodic Ca 2+ events mediated via RyR are essential for skeletal muscle differentiation and regeneration. However, the role of RyR in the dynamics of Ca 2+ events that signal the muscle regeneration process after ECC-induced muscle damage has not been elucidated. Purpose: This study tested the hypothesis that pharmacological inhibition of RyR during muscle regeneration suppresses [Ca 2+ ]i and delays myogenesis and growth of regenerating muscle cells. Methods: Tibialis anterior (TA) muscles of adult male Wistar rats either without ECC (CONT) or 5 (ECC5D) or 9 days (ECC9D) after ECC were imaged in vivo for [Ca 2+ ]i and subsequent in vitro histologic analysis. In addition an RyR inhibition group was treated with dantrolene (DAN) during the regeneration phase (intraperitoneal administration, 6-8 days) (ECC9D+DAN). TA muscles were loaded with the Ca 2+ indicator Fura2-AM and [Ca 2+ ]i distribution was evaluated at rest in each group by means of the 340/380 nm ratio. For muscle histology, satellite cell activation (Pax7, MyoD), inflammatory response (M2 macrophages), and muscle fiber cross-sectional area were evaluated by immunofluorescence and H&E staining. Results: Compared to control, [Ca 2+ ]i was significantly transiently decreased in ECC5D and recovered in ECC9D (Fura2 ratio values, CONT: 0.72 ± 0.05; ECC5D: 0.55 ± 0.07; ECC9D: 0.63 ± 0.06). The [Ca 2+ ]i of ECC9D+DAN (0.58 ± 0.08) was significantly lower than that of CONT. In the ECC9D+DAN group, TA muscle weight (right/left ratio, i.e., ECC leg/untreated leg) was lower than in the ECC9D group (ECC9D: 0.80 ± 0.06; ECC9D+DAN: 0.66 ± 0.06, p < 0.05). Furthermore, the proportion of muscle fibers with a cross-sectional area of less than 1000 μm 2 increased with dantrolene treatment (ECC9D: 48.0 ± 19.7, ECC9D+DAN: 65.8 ± 10.9%, p < 0.05). There were no significant differences between the two groups in the results of satellite cell activation (Pax7, MyoD) or inflammatory response (M2 macrophages). Conclusion: These findings support that decreased [Ca 2+ ]i consequent to RyR inhibition, 6 to 8 days post-ECC, delays the growth of regenerated skeletal muscle rather than preventing myogenesis per se. This study was supported in part by JSPS KAKENHI Grant (No. 20H04074, 23K24734). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.