C 0780

For isolation of arCMs, hearts were digested using a Langendorff system in perfusion buffer (130 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 0.5 mM NaH₂PO₄, 10 mM HEPES, 10 mM glucose, 10 mM 2,3-butanedione monoxime, 10 mM taurine, at pH 7.5) with 1 mg/ml collagenase II (LS004177, Worthington). The left ventricle became soft and flaccidness after an ~20-min digestion. Cardiomyocytes from the left ventricle were then isolated by gravity three times and then plated onto laminin-coated culture dishes at a density of 1×10⁴ cells/cm². Adult cardiomyocytes were cultured in M199 (M7528, Sigma) supplemented with 5 mM creatine (C-0780, Sigma), 5 mM taurine (T-7146, Sigma), 2 mM L-carnitine (C-0283, Sigma) and 1% penicillin–streptomycin.
For isolation of neonatal rat cardiomyocytes (nrCMs), ventricles of the hearts were removed from 1-day-old Sprague Dawley rats, and minced, followed by complete digestion with 1 mg/ml collagenase II (LS004177, Worthington) and 0.125% trypsin. nrCMs were separated from fibroblasts by differential plating and then cultured in gelatin-coated tissue culture plates at a density of 2 × 10⁴ cells/cm² in medium containing DMEM/F12, 10% FBS, 1% penicillin–streptomycin, and 1% L-glutamine.

C2C12 cells (Korean Type Culture Collection, Seoul, Korea) were cultured in DMEM medium containing 10% fetal bovine serum, 1% antibiotic–antimycotic solution, and 3.7 g/L NaHCO₃ at 37^oC in a 5% CO₂/95% air humidified incubator [16 (link)]. Confluent myoblasts (80%) were differentiated in DMEM medium containing 2% horse serum for 4 days. Probiotics (1 × 10⁴ cells/mL) or creatine (Cr, 5 mM, C0780, Sigma) were treated 10 h after treatment with dexamethasone (10 µM, D4902, Sigma) or lipopolysaccharide (LPS, 100 ng/mL) in differentiated C2C12 cells (1 × 10⁵ cells/mL).