Experimental protocols

The cells were incubated at 37°C in a humidified 5% CO₂ atmosphere, the experiments were divided into 5 groups as follows: i) Normal control group, where H9c2 cardiac cells were cultured in DMEM supplemented with 10% fetal bovine serum; ii) a hypertonic control group (HPG), where H9c2 cardiac cells were subjected to 35 mM mannitol as an osmotic control for 24 h; iii) a high glucose group (HG), where H9c2 cardiac cells were subjected to 35 mM glucose stress for 24 h to induce cell injury; iv) an HG+Alda-1 group where H9c2 cardiac cells, which were subjected to HG stress were treated with 20 µM Alda-1 (a specific activator of ALDH2) (9) for 24 h and v) an HG+Nec-1 group, where H9c2 cardiac cells that were subjected to HG stress were treated with 100 µM Nec-1 for 24 h.

The hypertonic control group was used to exclude the role of the hypertonic effect. The aim of the HG+Alda-1 group was to investigate whether the activation of ALDH2 can attenuate HG induced-cardiac cell injury. The purpose for the HG+Nec-1 group was to observe whether inhibiting necroptosis can attenuate HG-induced cardiac cell injury.