| Intermittent hypoxia, or periodic exposure to hypoxia interrupted by return to normoxia or less hypoxic conditions, is encountered more frequently in life than sustained hypoxia. Many studies showed that chronic intermittent hypobaric hypoxia (CIHH) adaptation had the cardioprotective effects similar to those observed in ischemic preconditioning (IPC). However, the cardioprotective effect of CIHH lasts longer than that of IPC and it is easy to manipulate the CIHH model, thus it has significance in the study of CIHH. A number of studies have attempted to define the mechanisms of this phenomenon and several potential factors have been proposed to be involved in the protective mechanism afforded by CIHH, however, the precise mechanisms underlying the cardioprotective effects of CIHH are far from clear. It is well known that oxidative stress and oxygen-derived free radicals (mainly ROS) contribute to I/R injury. There are different reports on the role of antioxidation in cardioprotection of CIHH. Whether antioxidation contributed to the cardioprotection of CIHH and the detail mechanism of antioxidation in CIHH need further study.Na+,K+-ATPase (sodium pump) is a heterodimer protein composed ofα- andβ-subunits that plays a key role in regulating membrane potential and cation transport in the myocardium. When myocardial ischemia, Ca2+ and Na+ influx into myocardium, and Ca2+ overload is regarded as a crucial factor in the development of ischemic myocardial damage. Na+,K+-ATPase facilitates transportation of Na+ from the intracellular space. The decrease in the intracellular Na+ concentration enhances removal of Ca+ from the intracellular space by facilitating Na+/Ca2+ exchange mechanism. Some researches demonstrated that ischemia and I/R injury can depress the activity of the cardiac Na+,K+-ATPase. Therefore, prevention of Ca2+ overload by CIHH may involve changes in Na+,K+-ATPase activity. Whether CIHH treatment can affect cardiac Na+,K+-ATPase have not been studied. Some reports demonstrated that ROS and Na+,K+-ATPase have a crosstalk relationship. ROS can inhibit Na+,K+-ATPase activity. As oxidative stress has been shown to occur during development of I/R injury, it is likely that the depression of Na+,K+-ATPase activity in I/R hearts may be due to oxidative stress. The aim of the study is to investigate whether CIHH has a protective effect on guinea pig heart, and to explore the underlying mechanism. Our study consists of three parts: (1) Protective effect of CIHH against ischemia/reperfusion injury inguinea pig heart. (2) The role of antioxidant enzymes in the cardioprotection of CIHH. (3) The role of the sodium pump in the cardioprotection of CIHH.Part 1 Protective effect of CIHH against ischemia/reperfusion injury in guinea pig heartObjective: The aim of this study was to investigate the effect of CIHH on myocardial ischemia/reperfusion injury in guinea pigMethods: Adult male guinea pigs (n=132) were divided randomly into six groups: non-CIHH 14 days group (non-CIHH14), non-CIHH 28 days group (non-CIHH28), non-CIHH 42 days group (non-CIHH42), CIHH 14 days group (CIHH14), CIHH 28 days group (CIHH28), and CIHH 42 days group (CIHH42). In CIHH groups, guinea pigs were exposed to CIHH mimicking 5000m high altitude (PB=404 mmHg, PO2=84 mmHg) in a hypobaric chamber lasting 6 hrs/day for 14 days, 28 days and 42 days respectively. The animals in non-CIHH groups were kept in the same environment as the CIHH guinea pigs except for hypoxic exposure. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia, and reperfusion period. The parameters of cardiac function including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of LVDP (±LVdp/dtmax) and coronary flow (CF) were measured. Myocardium was stained by TTC to measure infarct size. Results: 1 The body weight of guinea pigs in CIHH group had no significant change compared with non-CIHH animals. No differences in the ratio of heart weight to body weight, ratio of right ventricular weight to left ventricular plus inter-ventricular septum weight, ratio of right ventricular weight to body weight were observed between CIHH and non-CIHH groups.2 The basic CF in CIHH28 and CIHH42 guinea pigs was significant higher than that in corresponding non-CIHH guinea pigs, while other parameters of cardiac function didn't change. During the whole period of reperfusion, the recoveries of LVDP, LVEDP,±dp/dtmax, and CF in guinea pigs after 28 and 42 d of CIHH exposure were much better than those in the corresponding non-CIHH guinea pigs.3 The myocardial infarct size induced by ischemia and reperfusion was markedly reduced in CIHH28 and CIHH42 groups compared with those in corresponding non-CIHH guinea pigsConclusion: The results suggest that CIHH has a protective effect against ischemia/reperfusion injury on guinea pig heart.Part 2 The role of antioxidant enzymes in the cardioprotection of CIHHObjective: The aim of the present study was to evaluate: whether antioxidation was involved in the cardiacprotection afforded by CIHH.Methods: Adult male guinea pigs were exposed to CIHH mimicking 5000m high altitude (PB=404 mmHg, PO2=84 mmHg) in a hypobaric chamber lasting 6 hrs/day for 28 days. Langendorff-perfused isolated guinea pig hearts were used to measure variables of left ventricular function during baseline perfusion, ischemia, and reperfusion period. The activities and protein expressions of antioxidant enzymes in left ventricle were evaluated using biochemical methods and Western blotting, respectively. Intracellular reactive oxygen species (ROS) were assessed using ROS- sensitive fluorescence.Results:1 There was no significant difference in MDA contents between non-CIHH and CIHH group before ischemia/reperfusion. After reperfusion, MDA content increased significantly in both non-CIHH and CIHH hearts (p < 0.01), whereas MDA content in CIHH hearts was still lower than that in non-CIHH hearts (p < 0.01).2 The baseline activities of total SOD, SOD-2, and CAT in CIHH hearts were higher than those in non-CIHH hearts (p < 0.01), but SOD-1 and GPX activity did not significantly change. After reperfusion, SOD-2 and CAT activities decreased in both non-CIHH and CIHH hearts (p < 0.01), whereas the activities of SOD-2 and CAT in CIHH hearts were still higher than those in non-CIHH hearts (p < 0.01).3 Western blot analysis demonstrated that the baseline expressions of SOD-2 and CAT protein in CIHH hearts were higher than those in non-CIHH hearts; however, the expression of SOD-1 protein did not change. The protein expressions of SOD-2 and CAT were not significantly changed after reperfusion in both non-CIHH and CIHH hearts, whereas the expressions of SOD-2 and CAT in CIHH hearts were still higher than those in non-CIHH hearts (p < 0.01).4 Treatment with CAT inhibitor ATZ (1.0 g/kg) completely eliminated the protective effect of CIHH on cardiac function (p < 0.01), whereas it had no effect on non-CIHH hearts during reperfusion. A similar improvement in these cardiac function parameters was also observed in hearts treated with the antioxidant mixture containing SOD + CAT.5 Cardiac contractile dysfunction and oxidative stress induced by exogenous hydrogen peroxide (H2O2) were attenuated by CIHH and CAT. Conclusion: These data suggest that CIHH can protect heart against I/R injury through upregulation of antioxidant enzymes in guinea pig.Part 3 The role of sodium pump in the cardioprotection of CIHHObjective: whether Na+,K+-ATPase was involved the cardiacprotection afforded by CIHH.Methods: Adult male guinea pigs were exposed to CIHH mimicking 5000m high altitude (PB=404 mmHg, PO2=84 mmHg) in a hypobaric chamber lasting 6 hrs/day for 14, 21, 28, and 42days. The left ventricular myocytes were enzymatically isolated. The sodium pump current was recorded using whole cell patch clamp technique. The cell length and contraction were assessed by a video-based, motion-edge detection system.Results:1 After 20 min of ischemia followed by 30 min of reperfusion, cell length shortened in each group. CIHH significantly improved the recovery of cell length compared to that of non-CIHH myocytes (96.3±0.9% vs. non-CIHH 86.8±2.9%, P < 0.01). While Oua administered at 5 min before the ischemia completely abolished this beneficial effect in CIHH myocytes.2 Ischemia–reperfusion injury resulted in a marked decrease in the amplitude of contraction in each group. CIHH adaptation improved the recovery of contraction amplitude. When CIHH myocytes were treated with Oua at 5 min before the ischemia, all the beneficial effects were completely eliminated.3 The sodium pump currents in CIHH21, CIHH28, and CIHH42 guinea pigs were significant higher than those in corresponding non-CIHH guinea pigs.4 In the△Ip-[Oua] relation curve of non-CIHH, the△Ip values produced by each concentration of Oua from 10-10 to 10-3 mol/L were 0.088±0.03, 0.150±0.03, 0.060±0.01, -0.145±0.02, -0.391±0.06, -0.670±0.02, -0.98±0.01, and -1.000±0.00, respectively. K+2, K-2 and K1 were 8.5 x 10-11M, 5.2 x 10-8M, and 1.1 x 10-5M, repectively. f2 = 0.31, f1 = 0.68. In the△Ip-[Oua] relation curve of CIHH (Fig.5F), the△Ip values produced by each concentration of Oua from 10-10 to 10-3 mol/L were 0.069±0.02, -0.036±0.03, -0.181±0.02, -0.202±0.05, -0.459±0.03, -0.770±0.02, -0.978±0.01, and -1.000±0.00, respectively. K+2 , K-2 and K1 were 2.4 x 10-10M, 4.2 x 10-8M, 2.8 x 10-6M, f2 = 0.20, f1 = 0.80.5 Ip in cardiac myocytes after 28 CIHH was much higher than that in the corresponding non-CIHH (p < 0.01). After H2O2 (1mM) perfusion for 5 min, Ip decreased significantly in both non-CIHH and CIHH myocytes (p < 0.01 or p < 0.05), whereas the Ip in CIHH myocytes were still higher than those in non-CIHH myocytes (p < 0.01). 6 0.1mM H2O2 can inhibit Ip significantly in non-CIHH myocytes, but have no significantly effect on Ip in CIHH and CAT myocytes. 1mM H2O2 can inhibit Ip significantly in all myocytes, whereas the Ip in CIHH or CAT myocytes were still higher than those in non-CIHH myocytes. 10mM H2O2 can maximally inhibit Ip in all myocytes, whereas the Ip in CIHH myocytes was still higher than those in non-CIHH. CAT make the curve of concentration-dependence of H2O2 -induced inhibition of Ip move to the right parallelly. CIHH make the curve of concentration-dependence of H2O2 -induced inhibition of Ip move to the right, whereas maximal inhibition induced by H2O2 was smaller than those in non-CIHH or CAT myocytes.Conclusion: Sodium pump may play an important role in the cardioprotection of CIHH against I/R injury in guinea pig.SUMMARY1 CIHH has a protective effect against ischemia/reperfusion injury on guinea pig heart.2 CIHH upregulates the activity and protein expressions of antioxidant enzymes leading to an increase in antioxidant capacity, which may play an important role in the cardiac protection of CIHH against I/R injury in guinea pig.3 CIHH increase the sodium pump current and resistance to oxidative stress in cardiac myocytes. Sodium pump was involved in the cardiacprotection afforded by CIHH and may be an intermedium in the anti-oxidative cardiac protection mechanism of CIHH.
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