Bcaa mixture

Neonatal rat cardiomyocytes were isolated from new-born SD rats as previously reported [11 (link)]. Briefly, the left ventricles were harvested, minced and digested with collagenase Ⅱ (Biofrox, Hessen, Germany, #9001-12-1). Cells were cultured in Dulbecco’s Modified Eagle Medium-F12 with 10% fetal bovine serum (BI Co., Kibbutz Beit Haemek, Israel, #04-001-1ACS) and 1% penicillin-streptomycin (HyClone Co., Logan, UT, USA, #SV30010) at 37 °C in 5% CO₂. Hypoxia (12 h) was induced by culturing the cells in serum- and glucose-free medium in an air-tight Plexiglas chamber as previously described [26 (link)]. Normoxic cells were cultured without intervention. Cells were treated with BCAA mixture (weight ratio, leucine:valine:isoleucine = 2:1:1; Sigma-Aldrich, St. Louis, MO, USA) with a concentration of 3 mM at 10 min before hypoxia, and BCAA mixture were maintained in the culture medium during hypoxia. Recombinant adenovirus-Ppm1k (Ad-Ppm1k) and the negative control adenovirus (Ad-NC) were constructed and packaged (Han Bio Co., Shanghai, China), and the titer of adenovirus is 1 × 10⁹. Once the cells reached 70% confluence, they were infected with Ad-Ppm1k or Ad-NC (2 μL/mL). Cells were exposed to normoxia or hypoxia at 60 h post infection.

All animal studies were carried out in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Animal Care Committee of Air Force Medical University. PP2Cm global knockout (KO) mice are widely used as animal models with BCAA catabolic defects 10 (link). KO mice were obtained and maintained as we previously described 13 (link). Both KO and their wild-type (WT) littermates (aged from 10-12 weeks) were housed in a constant-temperature vivarium at 22°C with a 12-h light/dark cycle. Food and water were available ad libitum. BCAA mixture (weight ratio, leucine: valine: isoleucine=2:1:1; Sigma-Aldrich, St. Louis, MO, USA) were given into mice by oral gavage (1.5 mg/g/day) for 7 d before these mice received sham or I/R operation, as described by Li et al 8 (link). Vehicle or Etomoxir (Eto) (5 mg/kg body weight, Sigma-Aldrich) was intraperitoneally injected at 15 min before I/R procedure 16 (link). BCKA, including α-ketoisovaleric acid (αKIV), α-ketoisocaproate (αKIC) and α-keto-β-methylvalerate (αKMV), were commercially obtained from Sigma-Aldrich. A customized BCAA-free DMEM was used to exclude the impact of culture medium-contained BCAA. The detailed composition of BCAA-free DMEM is showed in Table S4.