Measurement of caspase-3 activity

Myocardial caspase-3 activity was determined using a caspase-3 activity assay kit according to the manufacturer’s instructions (c1116; Beyotime Biotech, Shanghai, China). Frozen myocardial tissues were crushed in liquid nitrogen and homogenized in an ice-cold lysis buffer. The homogenates were then centrifuged for 15 min at 4°C in a microcentrifuge (16,000×g). After collecting the supernatant, the protein level in each sample was assayed. Subsequently, 40 µL of reaction buffer with 10 µL of Ac-DEVD-pNA was added to 50 µL of protein samples, followed by incubation at 37°C for 6 h. Finally, the release of p-nitroaniline was measured at 405 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Caspase-3 activity was expressed as unit/h/mg protein.