Cardiac Myoblast Stress Response Protocol

The NCMs were obtained from decapitated 0 to 3-day-old Sprague-Dawley rats by collagenase II (0.05%) (Gibco) and trypsin (0.05%) digestion according to the methodology of previous studies [17 (link)]. The culture medium consisted of DMEM (high glucose) (Gibco) and 10% (v/v) fetal bovine serum (FBS, Hyclone, USA). All cells were maintained in a humidified 5% CO₂/95% air incubator at 37°C. H9c2, rat embryonic cardiac myoblasts from American Type Culture Collection (ATCC), were grown in DMEM with 10% FBS at 37°C and 5% CO₂. When cells reached 70–80% confluence, they were incubated with 1% BSA-DMEM with or without PA (200 μM, Sigma) for 24 h. Thapsigargin (100 nM, Sigma) and pravastatin (10 μM, Squibb) were used as the agonist and antagonist for ER stress [18 (link)]. Nec-1 (10 nM, Selleck) was a known specific inhibitor for RIPK1, and rapamycin (1 μM, Sigma) was used to block the mTOR signaling activation. All these treatments were pretreated with the NCMs or H9c2 cells for 2 h before the PA stimulation.