Intermittent Hypoxia Prevents Myocardial Mitochondrial Ca(2+) Overload and Cell Death during Ischemia/Reperfusion: The Role of Reactive Oxygen Species

It has been documented that reactive oxygen species (ROS) contribute to oxidative stress, leading to diseases such as ischemic heart disease. Recently, increasing evidence has indicated that short-term intermittent hypoxia (IH), similar to ischemia preconditioning, could yield cardioprotection. However, the underlying mechanism for the IH-induced cardioprotective effect remains unclear. The aim of this study was to determine whether IH exposure can enhance antioxidant capacity, which contributes to cardioprotection against oxidative stress and ischemia/reperfusion (I/R) injury in cardiomyocytes. Primary rat neonatal cardiomyocytes were cultured in IH condition with an oscillating O(2) concentration between 20% and 5% every 30 min. An MTT assay was conducted to examine the cell viability. Annexin V-FITC and SYTOX green fluorescent intensity and caspase 3 activity were detected to analyze the cell death. Fluorescent images for DCFDA, Fura-2, Rhod-2, and TMRM were acquired to analyze the ROS, cytosol Ca(2+), mitochondrial Ca(2+), and mitochondrial membrane potential, respectively. RT-PCR, immunocytofluorescence staining, and antioxidant activity assay were conducted to detect the expression of antioxidant enzymes. Our results show that IH induced slight increases of O(2)(−)(·) and protected cardiomyocytes against H(2)O(2)- and I/R-induced cell death. Moreover, H(2)O(2)-induced Ca(2+) imbalance and mitochondrial membrane depolarization were attenuated by IH, which also reduced the I/R-induced Ca(2+) overload. Furthermore, treatment with IH increased the expression of Cu/Zn SOD and Mn SOD, the total antioxidant capacity, and the activity of catalase. Blockade of the IH-increased ROS production abolished the protective effects of IH on the Ca(2+) homeostasis and antioxidant defense capacity. Taken together, our findings suggest that IH protected the cardiomyocytes against H(2)O(2)- and I/R-induced oxidative stress and cell death through maintaining Ca(2+) homeostasis as well as the mitochondrial membrane potential, and upregulation of antioxidant enzymes.