Cell culture and study design

C2C12 myoblasts were purchased from Cobioer Biosciences Co., Ltd. (Nanjing, China) and maintained in growth medium [Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin–streptomycin) in 5% CO₂ at 37 °C for proliferation. For myogenesis, C2C12 myoblasts cultured in 6‐well plates were transferred into differentiation medium (DM; containing DMEM and 2% horse serum, 1% penicillin–streptomycin) for 4 days after reaching 80–90% confluence. The C2C12 myotubes were then treated with different drug interventions, respectively, for 24 h in DM for related analysis.

NNC‐55 (Sigma‐Aldrich Co, St. Louis, MO, USA), ERS inhibitors including sodium 4‐phenylbutyrate (4PBA, No.1716‐12‐7; MedChemExpress, Monmouth Junction, NJ, USA) and tauroursodeoxycholic acid (TUDAC, CAS No.35807‐85‐3, MedChemExpress), and intracellular Ca²⁺ chelator of BAPTA‐AM (CAS No.126150‐97‐8; MedChemExpress) were all dissolved in DMSO (Sigma‐Aldrich) and stored at −20 C or −80 °C.

For transfection, C2C12 myoblasts were transfected with negative‐control siRNA (NC) and three small interfering RNAs against TH including siRNA‐1, 2 and 3 (si‐1/si‐m‐Cacna1h‐001 5′‐GGGTAAACATCATGTACGA‐3′, si‐2/si‐m‐Cacna1h‐002 5′‐GGAATGTGGTTCTTTACAA‐3′ and si‐3/si‐m‐Cacna1h‐003 5′‐GTCGCATTGTAGACAGCA‐3′, Ribobio, Guangzhou, China) using riboFECT™CP Transfection Kit at a final concentration of 50 nm according to the manufacturer's protocol. After 48‐h transduction, the efficiency of TH knockdown was verified by quantitative real‐time PCR (qRT‐PCR), and the cells were cultured in DM for another 4 days to fuse into myotubes for subsequent analysis.