| Summary: | 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<sub>2</sub> 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<sup>2+</sup>, mitochondrial Ca<sup>2+</sup>, 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<sub>2</sub><sup>−</sup><sup>·</sup> and protected cardiomyocytes against H<sub>2</sub>O<sub>2</sub>- and I/R-induced cell death. Moreover, H<sub>2</sub>O<sub>2</sub>-induced Ca<sup>2+</sup> imbalance and mitochondrial membrane depolarization were attenuated by IH, which also reduced the I/R-induced Ca<sup>2+</sup> 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<sup>2+</sup> homeostasis and antioxidant defense capacity. Taken together, our findings suggest that IH protected the cardiomyocytes against H<sub>2</sub>O<sub>2</sub>- and I/R-induced oxidative stress and cell death through maintaining Ca<sup>2+</sup> homeostasis as well as the mitochondrial membrane potential, and upregulation of antioxidant enzymes.
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