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 O₂ 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 Ca²⁺, mitochondrial Ca²⁺, 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 O₂^−· and protected cardiomyocytes against H₂O₂- and I/R-induced cell death. Moreover, H₂O₂-induced Ca²⁺ imbalance and mitochondrial membrane depolarization were attenuated by IH, which also reduced the I/R-induced Ca²⁺ 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 Ca²⁺ homeostasis and antioxidant defense capacity. Taken together, our findings suggest that IH protected the cardiomyocytes against H₂O₂- and I/R-induced oxidative stress and cell death through maintaining Ca²⁺ homeostasis as well as the mitochondrial membrane potential, and upregulation of antioxidant enzymes.

Ferroptosis is an iron‐dependent form of cell death caused by the inactivation of glutathione peroxidase 4 (GPX4) and accumulation of lipid peroxides. Ferroptosis has been found to participate in the ischemia‐reperfusion (I/R) injury, leading to heart dysfunction and myocardial cell death. Xanthohumol (XN), a prenylated flavonoid isolated from Humulus lupulus, has multiple pharmacological activities, such as anti‐inflammatory and antioxidant. This study is aimed at investigating whether XN could attenuate the I/R‐induced ferroptosis in cardiomyocytes and the underlying mechanisms. Cardiomyocytes were treated with Fe‐SP and RSL3, and the rat hearts were treated with I/R. The results from the present study show that XN was able to protect cardiomyocytes against Fe‐SP‐ and RSL3‐induced ferroptotic cell death by decreasing the production of lipid peroxidation and ROS, chelating iron, reducing the NRF2 protein level, and modulating the protein levels of GPX4. Moreover, XN significantly decreased the mRNA levels of ferroptosis markers, Ptgs2 and Acsl4, and the protein levels of ACSL4 and NRF2 and modulated the protein levels of GPX4 in I/R‐treated hearts. The findings from the present study suggest that XN might have the therapeutic potential for the I/R‐induced ferroptosis injury.

Myocardial ischemia/reperfusion (I/R) injury has been associated with ferroptosis, which is characterized by an iron-dependent accumulation of lipid peroxide to lethal levels. Gossypol acetic acid (GAA), a natural product taken from the seeds of cotton plants, prevents oxidative stress. However, the effects of GAA on myocardial I/R-induced ferroptosis remain unclear. This study investigated the ability of GAA to attenuate I/R-induced ferroptosis in cardiomyocytes along with the underlying mechanisms in a well-established rat model of myocardial I/R and isolated neonatal rat cardiomyocytes. H9c2 cells and cardiomyocytes were treated with the ferroptosis inducers erastin, RSL3, and Fe-SP. GAA could protect H9c2 cells against ferroptotic cell death caused by these ferroptosis inducers by decreasing the production of malondialdehyde and reactive oxygen species, chelating iron content, and downregulating mRNA levels of Ptgs2. GAA could prevent oxygen-glucose deprivation/reperfusion-induced cell death and lipid peroxidation in the cardiomyocytes. Moreover, GAA significantly attenuated myocardial infarct size, reduced lipid peroxidation, decreased the mRNA levels of the ferroptosis markers Ptgs2 and Acsl4, decreased the protein levels of ACSL4 and NRF2, and increased the protein levels of GPX4 in I/R-induced ex vivo rat hearts. Thus, GAA may play a cytoprotectant role in ferroptosis-induced cardiomyocyte death and myocardial I/R-induced ferroptotic cell death.

Intermittent hypoxia (IH), characterized as cyclic episodes of short-period hypoxia followed by normoxia, occurs in many physiological and pathophysiological conditions such as pregnancy, athlete, obstructive sleep apnea, and asthma. Hypoxia can induce autophagy, which is activated in response to protein aggregates, in the proteotoxic forms of cardiac diseases. Previous studies suggested that autophagy can protect cells by avoiding accumulation of misfolded proteins, which can be generated in response to ischemia/reperfusion (I/R) injury. The objective of the present study was to determine whether IH-induced autophagy can attenuate endoplasmic reticulum (ER) stress and cell death. In this study, H9c2 cell line, rat primary cultured cardiomyocytes, and C57BL/6 male mice underwent IH with an oscillating O₂ concentration between 4 and 20% every 30 min for 1–4 days in an incubator. The levels of LC3, an autophagy indicator protein and CHOP and GRP78 (ER stress-related proteins) were measured by Western blotting analyses. Our data demonstrated that the autophagy-related proteins were upregulated in days 1–3, while the ER stress-related proteins were downregulated on the second day after IH. Treatment with H₂O₂ (100 μM) for 24 h caused ER stress and increased the level of ER stress-related proteins, and these effects were abolished by pre-treatment with IH condition. In response to the autophagy inhibitor, the level of ER stress-related proteins was upregulated again. Taken together, our data suggested that IH could increase myocardial autophagy as an adaptive response to prevent the ER stress and apoptosis.

Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH). Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death.