The optimization conditions of establishing an H9c2 cardiomyocyte hypoxia/reoxygenation injury model based on an AnaeroPack System

Abstract Ischemia–reperfusion (I/R) injury is a major cause of cardiomyocyte apoptosis after vascular recanalization, which was mimicked by a hypoxia/reoxygenation (H/R) injury model of cardiomyocytes in vitro. In this study, we explored an optimal H/R duration procedure using the AnaeroPack System. To study the H/R procedure, cardiomyocytes were exposed to the AnaeroPack System with sugar and serum‐free medium, followed by reoxygenation under normal conditions. Cell injury was detected through lactate dehydrogenase (LDH) and cardiac troponin (c‐Tn) release, morphological changes, cell apoptosis, and expression of apoptosis‐related proteins. The results showed that the damage to H9c2 cells increased with prolonged hypoxia time, as demonstrated by increased apoptosis rate, LDH and c‐Tn release, HIF‐1α expression, as well as decreased expression of Bcl‐2. Furthermore, hypoxia for 10 h and reoxygenation for 6 h exhibited the highest apoptosis rate and damage and cytokine release; in addition, cells were deformed, small, and visibly round. After 12 h of hypoxia, the majority of the cells were dead. Taken together, this study showed that subjecting H9c2 cells to the AnaeroPack System for 10 h and reoxygenation for 6 h can achieve a practicable and repeatable H/R injury model.