Vbit 4

Adult cardiomyocytes were isolated from Sprague Dawley rats (200–250 g, male, Shanghai Jiesijie) according to the standard enzymatic technique as described previously [24 ]. In brief, the heart was excised quickly from anesthetized rat, cannulated through the ascending aorta, mounted on a Langendorff equipment, and perfused with oxygenated Krebs-Henseleit Buffer (KHB) with collagenase II (Worthington, Cat No. LS004177) and hyaluronidase (Sigma, Cat No. H3506) at 37 °C. After 20 min, the perfused heart was cut into small pieces and gently swirl in 37 °C water bath. Rod shaped cardiomyocytes were collected and plated on the coverslip with pre-coated laminin (Thermo Fisher, Cat No. 23017-015). Cardiomyocytes cultured in M199 medium (Sigma, Cat. No. M2520) with 5.5 mmol/L glucose as fuel, and supplemented with 10 mmol/L glutathione, 26.2 mmol/L sodium bicarbonate, 0.02% bovine serum albumin and 50 U/ml penicillin–streptomycin for 72 h to allow adequate gene expression. Attached cardiomyocytes were infected with Ad-Drp1, Ad-Drp1 C607A, Ad-GFP-Drp1, Ad-GFP-Drp1 C607A, Ad-mt-SoNar and Ad-mt-cpYFP at a multiplicity of infection of 50–100. ISO (Sigma, Cat. No. I0599990, 10 μmol/L) incubated for 48 h. VBIT-4 (Selleck, Cat. No. S3544, 10 μmol/L) incubated for 48 h.