Primary cardiomyocytes were subjected to hypoxia for 1 h followed by 1 h reperfusion with or without ginsenoside Rb1 pretreatment. The time point was selected with reference to previous reports 10, 30. After H/R treatment, the conditioned medium was collected for a lactate assay (Jiancheng Bioengineering Institute, Nanjing, China) and the cells were harvested for ATP contents detection by ATP Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. For the mitochondrial membrane potential (Δψm) assay, treated cardiomyocytes were loaded with a fluorescent indicator tetramethylrhodamine ethyl ester (TMRE) for 30 min at 37 °C. TMRE staining was examined by confocal microscopy (Zeiss LSM 700). MPTP opening was assayed with Mitochondrial Transition Pore Assay Kit (Life Technology, USA) according to the manufacturer's protocol. Cell viability was measured using Cell Counting Kit-8 (CCK-8) (Yeasen).

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