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.

Oxidative stress is one of the major causes of cell death. Using time-lapse confocal recording of live cardiomyocytes, we showed that H2O2 (OH*) caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na]cyt, [Ca]cyt) and mitochondria ([Na]m, [Ca]m). The H2O2-induced intracellular Na+ ([Na]i) overload contributed to the H2O2-induced [Ca]cyt/[Ca]m overload via activation of the reverse mode of the Na-Ca exchanger. When myocytes were treated for 40 min with 100 microM H2O2 in normal medium, then returned to H2O2-free medium, the percentage of apoptotic cells increased from 4% at 0 h to 55 and 85% at 4.5 and 16 h, respectively. H2O2-induced apoptosis was completely prevented by using Na-free, but not Ca-free, medium. When a Na+ ionophore cocktail in Ca-free medium was used instead of H2O2 to increase the [Na]i by more than 30 mM without any change in the [Ca]i, cytochrome c release and caspase 3-dependent apoptosis occurred, showing that [Na]i overload per se induced apoptosis. We also showed that the increase in the mitochondrial, but not the cytosolic, Na+ levels resulted in the opening of the permeation transition pore, followed by cytochrome c release. Our findings therefore suggest that H2O2-induced [Na]m overload is an important upstream signal for the apoptotic machinery, and the prevention of [Na]m overload thus represents a particularly attractive target for strategies aimed at preventing oxidative stress-induced cell death.

Arachidonic acid (AA) exerts multiple physiological and pathophysiological effects in the brain. By continuously measuring the intracellular pH (pHi) and Ca2+ levels ([Ca2+]i) in primary cultured rat cerebellar granule cells, we have found, for the first time, that 20 min treatment with 10 μm AA resulted in marked increases in Ca2+ and H+ levels in both the cytosol and nucleus. A much higher concentration (40 mm) of another weak acid, propionic acid, was needed to induce a similar change in pHi. The [Ca2+]i increase was probably caused by AA‐induced activation of Ni2+‐sensitive cationic channels, but did not involve NMDA channels or the Na+‐Ca2+ exchanger. AA‐induced acidosis occurs by a different mechanism involving predominantly the passive diffusion of the un‐ionized form of AA, rather than a protein carrier, as proposed by Kamp & Hamilton for fatty acids (FAs) in artificial phospholipid bilayers (the ‘flip‐flop’ model). The following results, which are similar to those observed in lipid bilayers, support this conclusion: (1) FAs containing a ‐COOH group (AA, linoleic acid, α‐linolenic acid, and docosahexaenoic acid) induced intracellular acidosis, whereas a FA with a ‐COOCH3 group (AA methyl ester) had little effect on pHi, (2) a FA amine, tetradecylamine, induced intracellular alkalosis, and (3) the AA‐/FA‐induced pHi changes were reversed by bovine serum albumin. Further evidence in support of a passive diffusion model, rather than a membrane protein carrier, is that: (1) there was a linear relationship between the initial rate of acid flux and the concentration of AA (2‐100 μm), (2) acidosis was not inhibited by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid, a potent inhibitor of the plasma membrane FA carrier protein, and (3) the involvement of most known H+‐related membrane carriers and H+ conductance has been ruled out. Since AA can be released under both physiological and pathophysiological conditions, the possible significance of the AA‐evoked increases in H+ and Ca2+ in both the cytoplasm and nucleoplasm is discussed.

Background/Aims: Intermittent hypoxia (IH) may exert pre-conditioning-like cardioprotective effects and alter Ca²⁺ regulation; however, the exact mechanism of these effects remains unclear. Thus, we examined Ca²⁺-handling mechanisms induced by IH in rat neonatal cardiomyocytes. Methods: Cardiomyocytes were exposed to repetitive hypoxia-re-oxygenation cycles for 1-4 days. Mitochondrial reactive oxygen species (ROS) generation was determined by flow cytometry, and intracellular Ca²⁺ concentrations were measured using a live-cell fluorescence imaging system. Protein kinase C (PKC) isoforms and Ca²⁺-handling proteins were analysed using immunofluorescence and western blotting. Results: After IH exposure for 4 days, the rate of Ca²⁺ extrusion from the cytosol to the extracellular milieu during 40-mM KCl-induced Ca²⁺ mobilization increased significantly, whereas ROS levels increased mildly. IH activated PKC isoforms, which translocated to the membrane from the cytosol, and Na⁺/Ca²⁺ exchanger-1, leading to enhanced Ca²⁺ efflux capacity. Simultaneously, IH increased sarcoplasmic reticulum (SR) Ca²⁺-ATPase and ryanodine receptor 2 (RyR-2) activities and RyR-2 expression, resulting in improved Ca²⁺ uptake and release capacity of SR in cardiomyocytes. Conclusions: IH-induced mild elevations in ROS generation can enhance Ca²⁺ efflux from the cytosol to the extracellular milieu and Ca²⁺-mediated SR regulation in cardiomyocytes, resulting in enhanced Ca²⁺-handling ability.