Chronic intermittent hypobaric hypoxia (CIHH) has been shown to attenuate intracellular Na+ accumulation and Ca2+ overload during ischemia and reperfusion (I/R), both of which are closely related to the outcome of myocardial damage. Na/K pump plays an essential role in maintaining the equilibrium of intracellular Na+ and Ca2+ during I/R. It has been shown that enhancement of Na/K pump activity by ischemic preconditioning may be involved in the cardiac protection. Therefore, we tested whether Na/K pump was involved in the cardioprotection by CIHH. We found that Na/K pump current in cardiac myocytes of guinea pigs exposed to CIHH increased 1.45-fold. The K1 and f1, which reflect the portion of α1-isoform of Na/K pump, dramatically decreased or increased, respectively, in CIHH myocytes. Western blot analysis revealed that CIHH increased the protein expression of the α1-isoform by 76%, whereas the protein expression of the α2-isoform was not changed significantly. Na/K pump current was significantly suppressed in simulated I/R, and CIHH preserved the Na/K pump current. CIHH significantly improved the recovery of cell length and contraction during reperfusion. Furthermore, inhibition of Na/K pump by ouabain attenuated the protective effect afforded by CIHH. Collectively, these data suggest that the increase of Na/K pump activity following CIHH is due to the upregulating α1-isoform of Na/K pump, which may be one of the mechanisms of CIHH against I/R-induced injury.
protection of the myocardium against ischemia and reperfusion (I/R) injury is the key to crucial interventions, such as coronary artery bypass graft surgery, angioplasty, and organ transplantation. Several different manipulations, including pharmacological intervention (27), ischemic preconditioning (IPC) (36), cardioplegia protection (19), or hypoxic adaptation (26), have been conducted to halt or retard irreversible injury. Investigations in our and other laboratories have shown that chronic intermittent hypobaric hypoxia (CIHH) adaptation can protect the myocardium I/R injury (10, 18). This cardiac protection lasts for a longer time than ischemic preconditioning (5, 47) and causes the less adverse effects, such as right ventricular hypertrophy, than those associated with chronic hypoxia (2, 31, 43). Therefore, the elucidation of the subcellular mechanisms through which CIHH exerts its beneficial effect may be of basic clinical importance. However, the precise mechanisms underlying the cardioprotective effects of CIHH are far from clear.
The Na/K pump (Na+,K+-ATPase) is ubiquitously expressed in mammalian tissues and is essential for cell survival. In cardiac myocytes, Na/K pump maintains the transmembrane [Na+] and [K+] gradients to protect myocytes excitability, therefore, myocytes contractility (35). It comprises a catalytic α-subunit and a glycosylated β-subunit (4). The α-subunit consists of three isoforms encoded by three distinct genes: α1, α2, and α3. The α-subunit is responsible for the catalytic activity of the enzyme for it contains the ATP and glycoside binding sites (6). Three isoforms vary in their affinity to cardiac glycosides; the α1-isoform exhibits a low affinity to ouabain, whereas α2- and α3-isoforms have a much greater affinity. The distribution of α-subunit is species specific. Only α1- and α2-isoforms of Na/K pump were found in guinea pig heart (14).
There is a substantial body of evidence that perturbation of [Na+]i and [Ca2+]i may play a crucial role in the pathophysiology of myocardial I/R injury (29). This rise in [Na+]i during ischemia has been attributed to a combination of an increased cellular influx of Na+ via the Na+ channel and the Na+/H+ exchanger, as well as a decreased Na+ extrusion by the Na/K pump (1, 21, 24). During reperfusion, Na+ entry is further more potential, and as Na/K pump activity remains depressed, this leads to elevated intracellular Ca2+ via the Na+/Ca2+ exchanger (Ca2+ overload). This failure of [Na+]i to recover completely during early reperfusion contributes to cardiac I/R injury (40) and may be attributed in part to be inadequate Na/K pump activity at this time (37). Stimulating the Na/K pump back to (or beyond) basal may be expected to limit injury resulting from high levels of [Na+]i. Some reports demonstrated that enhancement of Na/K pump activity by IPC may be involved in the cardiac protection (1, 38). Nawada et al. (28) proposed that IPC protects the rabbit heart against the I/R-induced reduction in Na/K pump activity, and inhibition of Na/K pump activity has been shown to attenuate the beneficial effect of IPC. CIHH has been shown to attenuate [Na+]i and Ca2+ overloaded during I/R (8, 48). Therefore, prevention of Ca2+ overload by CIHH may involve changes in Na/K pump activity. Whether CIHH exposure can affect cardiac Na/K pump has not been studied.
We hypothesized that increased Na/K pump activity contributes to the cardiac protection produced by CIHH treatment. The aim of our present study was to evaluate whether CIHH could change Na/K pump activity and whether Na/K pump was involved in the cardiac protection afforded by CIHH.
MATERIALS AND METHODS
Animals and CIHH treatment.
All experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996) and were reviewed and approved by the Ethics Committee for the Use of Experimental Animals at Hebei Medical University. Adult male guinea pigs (250 ± 20 g; provided by the Experimental Animal Center of Hebei Province, China) were divided into non-CIHH and CIHH groups. CIHH group guinea pigs were exposed to intermittent high-altitude hypoxia of 5,000 m (PB = 404 mmHg, PO2 = 84 mmHg) in a hypobaric chamber lasting 6 h/day for 28 days, and non-CHH group guinea pigs as the control were kept in the above environment except for the hypoxic exposure. All animals were used for further experiments on the next day following the pretreatment as described above. Both groups of guinea pigs were feeded at room temperature (20° to 24°C) with a natural light-dark cycle (12 h:12 h) and had free access to water and food throughout the pretreatment.
Preparation of adult guinea pig ventricular myocytes.
Myocytes were enzymatically isolated from non-CIHH or CIHH guinea pigs left ventricle by methods as previously described (19). Briefly, guinea pigs were anesthetized with the sodium pentobarbitone solution (60 mg/kg ip). The heart was quickly excised and perfused in a retrograde fashion with oxygenated Ca2+-free Tyrode's solution containing (in mM): 135.0 NaCl, 5.4 KCl, 1.0 MgCl2, 0.33 NaH2PO4, 5.0 HEPES, and 10.0 glucose, gassed with pure O2 (pH 7.4, 37°C) for 5 min, then the perfusion solution was switched to Ca2+-free Tyrode's solution containing 0.4 mg/ml collagenase II (Worthington Biochemical) for 15 min. The left ventricle was removed and agitated mechanically in high-K+ Kraft-Bruhe (KB) solution to obtain single ventricular myocytes which were used within 6 h after isolation. The composition of the high-K+ KB solution was (in mM): 80 KOH, 40 KCL, 3 MgSO4, 25 KH2PO4, 50 glutamic acid, 20 taurine, 10 HEPES, 1 EGTA, and 10 glucose at pH 7.2.
Measurement of Na/K pump current.
(46) The isolated ventricular myocytes were allowed to adhere to the bottom of a bath mounted on the stage of an inverted microscope (Nikon TE2000-S) and perfused with Ca2+-free tyrode solution containing 2 mM BaCl2 and 1 mM CdCl2 (at 1.5 ml/min). For whole cell current-clamp recordings, recording pipettes (1 to 3 M) were filled with intracellar solution that contained (in mM): 50 sodium aspartic acid, 20 potassium aspartic acid, 30 CsOH, 20 TEACl, 5 MgSO4, 5 HEPES, 11 EGTA, 10 glucose, 5 Na2-ATP, and 1 CaCl2 (pH to 7.2 with CsOH). The sampling rate was 200 μs/point, and the data were low pass filtered at 2 Hz. The free [Ca2+]i was 1.5 × 10−8 M in the intracellar solution containing 11 mM EGTA and 1 mM CaCl2. The ventricular myocytes were clamped at 0 mV, the saturating voltage for the Na/K pump, which could increase the signal-to-noise ratio and provide the better resolution.
To observe Na/K pump current (Ip; pA/Cm) density of cardiac myocyte, Strophanthidin (Str; 0.5 mM) was applied to generate an inward current (in pA). Signals were amplified using Axon 700B amplifier, and the data were acquired and analyzed using pCLAMP 9.0 software.
To examine the ouabain (Oua) dose-response curve, the bath solution was switched to the perfusing solution contained Oua (10−11-10−3 M), which resulted in an outward or inward shift of the membrane current at the whole cell mode (reference). START The Oua-sensitive currents indicated the change in Ip (▵Ip), and ▵Ip (stimulation or inhibition) were normalized to the maximal value of ▵Ip obtained in the same cell by total pump inhibition on application of 1 mM Oua. The basal ▵Ip before any application of Oua was defined as zero. When the Oua-induced current shifted above the basal level, due to the stimulation of Ip by low [Oua], we assigned ▵Ip a positive value. One the other hand, if the induced current shifted below the basal level, due to the inhibition of Ip by high [Oua], we assigned ▵Ip a negative value. Since 1 mM Oua is a saturating concentration that completely blocks Ip, we define the ▵Ip induced by 1 mM Oua as −1. A two-site binding model was developed to interpret our data. In guinea pig ventricular myocytes, IPT is the sum of the high Oua affinity Ip contributed by the α2-isoform (Ip2) and the low Oua affinity Ip contributed by the α1-isoform (Ip1). Since only the α2-isoform is involved in the stimulation of Ip by low concentrations of Oua, the parallel model was described by the following equation (16): ΔIpT = f2[kDK−2 − D(D + K+2)/(D + K−2)(D + K+2)] − f1[D/(D + K1)].
In this equation, k is the increase in Ip2 when Oua is bound to the stimulatory site; K+2 and K−2 are the dissociation constants of the stimulatory and inhibitory Oua-binding sites on the α2-isoform, respectively; and K1 is the dissociation constant for the inhibitory Oua binding site on the α1-isoform. The symbols f2 and f1 represent the fractions of IpT due to Ip2 and Ip1, respectively.
To evaluate Ip by I/R, Str (0.5 mM) was bath applied and recorded the Na/K pump current. When the membrane current returned to the control level, simulated ischemia solution (SI) containing the following (in mM): 123 NaCl, 8 KCl, 6 NaHCO3, 0.9 NaH2PO4, 0.5 MgSO4, 20 Na-lactate, 1.8 CaCl2, 2 BaCl2, and 1 CdCl2, gassed with 95% N2-5% CO2 (pH 6.8), was applied for 5 min. When reperfusion happened, Str (0.5 mM) was given again.
Preparation of cell sarcolemmal membrane.
Harvested cells were homogenated in a lysis buffer containing (in mM) 5 Tris·HCl, 320 sucrose, 120 KCl, 1 EGTA, and 1 EDTA (4°C, pH 7.5) by the homogenizer (T 18 basic Ultra-Turrax; Mandel Scientific , Guelph, Canada). The homogenate was centrifuged for 5 min at 3,000 g to remove cellular debris. For cell membrane preparation, the supernatant was centrifuged at 100,000 g for 60 min and discarded. The pellet representing the sarcolemma-enriched fraction was suspended in the solution with (in mM) 1 EGTA, 1 EDTA, 20 HEPES, 10% glycerol, and 2% Triton X-100 (pH 7.4) for Western blot analysis. All solutions contained three protease inhibitors: soybean trypsin inhibitor (40 μg/ml), 0.1% PMSF, and leupeptin (0.5 μg/ml). Protein concentration was measured using the bicinchoninic acid method (Pierce, Rockford, IL) with bovine serum albumin as a standard.
Western blot analysis.
Cell sarcolemmal membrane samples were separated using SDS-PAGE on 10% acrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 5% nonfat dry milk in TBST [50 mM Tris·HCl, 150 mM NaCl, 0.1% Tween (pH 7.4)] for 1 h at room temperature. Membranes were then incubated with the polyclonal IgG for α1- or α2-isoform of Na/K pump (dilution 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, washed with TBST three times for 10 min each, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000) for 1 h at 37°C. After washing, blots were detected using an enhanced chemiluminescence plus system (ZhongShan Bioengineering Institute, Beijing, China). Quantification of Western blot signals was performed by densitometric measurements. The data were normalized to the ratios of β-actin (dilution 1:800; Santa Cruz Biotechnology, Santa Cruz, CA) detected on the same blot to control for possible variations in protein loading.
Measurement of cell length and contraction.
Myocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus IX-70) and perfused with the normal superfusate contained (in mM) 129 NaCl, 4 KCl, 20 NaHCO3, 0.9 NaH2PO4, 0.5 MgSO4, 10 glucose, and 1.8 CaCl2, gassed with 95%O2-5%CO2
All values in this text are means ± SE. Statistical significance was determined using Student's t-test or one-way ANOVA, as appropriate. Values of P < 0.05 were considered significant. Sigma Plot software was used to fit the curve.
Body weight and heart weight.
The body weight of guinea pigs in CIHH groups had no significant change compared with those in the non-CIHH groups. The ratios of the ventricle (including whole, left, and right) weight to body weight were not significantly different between non-CIHH and CIHH groups (Fig. 1), which indicated that the CIHH in this experimental condition did not result in heart hypertrophy.
The changes of Na/K pump current in CIHH cardiac myocytes.
We first determined the Na/K pump activity with voltage clamping. Figure 2A showed a representative current trace: 0.5 mM Str caused an inward shift of the holding current. After Str was washed out, the holding current returned to the initial level rapidly and completely, which is in accordance with the fact that Str dissociates from the Na/K pump α-subunit rapidly when Str is removed from the perfusate. The Ip in cardiac myocyte of 28-day CIHH exposure (1.09 ± 0.06 pA/pF) was much higher than that in the non-CIHH (0.75 ± 0.05 pA/pF; P < 0.01), which was increased by ∼45% (Fig. 2B).
Characteristics of ▵Ip-[oua] relation curve in CIHH cardiac myocytes.
To determine which isoform of Na/K pump was changed by CIHH, the Oua dose-response curve was constructed. In the ▵Ip-[Oua] relation curve of the non-CIHH group (Fig. 3A), the ▵Ip values produced by each concentration of Oua from 10−10 to 10−3 mol/l were 0.088 ± 0.030, 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 × 10−11 M, 5.2 × 10−8 M, and 1.1 × 10−5 M, respectively (f2 = 0.31, f1 = 0.68), whereas in the CIHH group (Fig. 3B), the ▵Ip values 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 × 10−10 M, 4.2 × 10−8 M, 2.8 × 10−6 M, f2 = 0.20, f1 = 0.80. When compared with the non-CIHH group, the K1 and f1 were much more decreased or increased, respectively, in the CIHH group, indicating that CIHH may affect the α1-isoform of Na/K pump.
Expression of Na/K pump isoforms protein in CIHH and non-CIHH cardiac myocytes.
To gain further insight into the molecular basis of CIHH on Na/K pump, we examined the content of the α1- and α2-isoform of Na/K pump by using Western blot analysis. We found that the expression of α1-isoform protein was increased significantly in CIHH cardiac myocytes. The expression of α1-isoform protein in CIHH cardiac myocytes was 1.85 ± 0.21, which was higher than 1.05 ± 0.21 (n = 5; P < 0.01) in non-CIHH cardiac myocytes (Fig. 4). However, the protein expression of the α2-isoform in CIHH cardiac myocytes did not change significantly. This suggests that CIHH increases the activities of Na/K pump by upregulating the protein expression of the α1-isoform.
Effects of SI and reperfusion on Na/K pump current.
The Ip in cardiac myocytes after 28-day CIHH exposure was much higher than that in the corresponding non-CIHH (Fig. 5; P < 0.01). The Ip were decreased during SI and reperfusion in non-CIHH and CIHH cardiac myocytes (P < 0.01); however, the Ip in CIHH myocytes were still higher than those in non-CIHH myocytes (P < 0.01). The result indicates that CIHH can preserve the Na/K pump current during I/R.
Effects of CIHH on cell length during reperfusion.
After 20 min ischemia followed by 30 min reperfusion, diastolic cell length shortened in all groups (Fig. 6). However, CIHH significantly improved the recovery of cell length comparing with that of non-CIHH myocytes (96.3 ± 0.9% vs. 86.8 ± 2.9%; P < 0.01). Oua administered at the preischemia 5 min to the end of reperfusion completely abolished this beneficial effect in CIHH myocytes.
Effects of CIHH on myocytes contraction during reperfusion.
I/R injury resulted in a remarkable decrease in the amplitude of contraction in all groups (Fig. 7A). However, CIHH adaptation improved the recovery of contraction amplitude (P < 0.01). I/R injury produced a considerable decrease in the maximal velocity of shortening (+dL/dt) and maximal velocity of relengthening (−dL/dt) in non-CIHH and CIHH groups. In contrast, CIHH significantly increased it compared with non-CIHH (Fig. 7, B and C). When CIHH myocytes were treated with Oua from the preischemia 5 min to the end of reperfusion, all the beneficial effects except −dL/dt were completely eliminated. These results indicated that Na/K pump might be involved in cardiac protection of CIHH.
This is the first study to find that the activity of Na/K pump was increased in CIHH-treated guinea pigs. The data suggest that the increase of Na/K pump activity followed by CIHH is due to the upregulating of α1-isoform of Na/K pump, which may be one of the mechanisms of CIHH against I/R-induced injury. Our findings provide novel insights into the cellular and molecular mechanisms underlying the cardioprotection by CIHH against I/R-induced injury, which may help in the development of a preventive therapeutic regimen against ischemic injury.
There are many researches about the mechanisms of CIHH-induced cardioprotection from I/R, such as upregulation of antioxidative enzymes (18), activation of ATP-sensitive K+ channels (48), inhibition of mitochondrial permeability transition pores (49), changes in cardiac adrenergic receptors (17, 41), alterations in sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) and the Na/Ca exchanger, ryanodine receptors, calmodulin kinase II, and activation of protein kinase C (PKC) (8, 10, 20). Certainly, like IPC, CIHH does not only involve one or two mechanisms in cardioprotective effects. We think the mechanisms may have an internal relationship. For example, PKC exerts its protective effect by activating mitoKATP channels (39). On the other hand, PKC is downstream of the mitoKATP channels activation and reactive oxygen species (ROS) production (7). PKC exerts a key role in the modulation of Na/K pump activity (13, 15). Na/K pump can affect Na/Ca exchanger activity (12). Adrenergic receptors can regulate Na/K pump activity and expression (3, 15). We will further study and demonstrate which is the primary mechanism.
During ischemia, loss of Na+, K+, and Ca2+ gradients leads to membrane depolarization, cell swelling, glutamate release, and cell death. The Na/K pump is critical for restoring these gradients. Some reports have demonstrated the importance of Na/K pump in I/R (1, 38). Tian et al. found that Na/K pump activity significantly increased 24 h after the preconditioning treatment in hippocampal slice, and elevated Na/K pump was accompanied by increased surface expression of the α1- and α2-isoforms of the Na/K pump (37a). Our result also demonstrated that CIHH adaptation can increase Ip significantly compared with corresponding non-CIHH.
CIHH adaptation can increase the composing proportion of α1-isoform, which is relatively resistant to oxidative stress (44, 45), suggesting CIHH adaptation can increase the resistance of Na/K pump to oxidative stress. It is clear that myocardial I/R induces an injurious cascade of ROS (22). Many reports have demonstrated that ROS can inhibit Na/K pump (23, 30). Thus it is generally considered that manipulations that attenuate the level of oxidative stress of heart tissue may be useful in preserving ischemia and I/R-induced inhibition of Na/K pump. In our previous study, we have demonstrated that CIHH could protect the heart against I/R injury through upregulation of antioxidant enzymes. The antioxidative cardiac protection mechanism of CIHH may be partly mediated by Na/K pump.
Recently, several groups have proposed that Na/K pump has dual functions. In addition to pumping Na+ and K+ across cell membranes, it also relays an extracellular ouabain signal to intracellular compartments via activation of different protein kinases (45). Pierre and coauthors (33) have provided evidence supporting the possibility that ouabain preconditioning may be cardioprotective in the therapeutic dose range. Ouabain preconditioning at concentrations without inotropic response before ischemia initiated a cardioprotective signaling pathway that induced ROS production and required mitoKATP channel opening (32). On the other hand, digoxin also has been shown to trigger preconditioning in ischemia brain (34). However, long term pretreated with digoxin had evidence of enhanced I/R injury in dog (25). Generally, Na/K pump inhibitor preconditioning can transiently activate signal transduction, which may cause induction of cardiac tolerance to ischemic stress. However, Na/K pump inhibitor exists with the certain dose during I/R, which can enhance I/R injury. Endogenous digitalis-like compound (EDLC) is an endogenous ligand of the digitalis receptor and can remarkably inhibit Na/K pump activity. Antidigoxin antiserum (ADA) can prevent EDLC-mediated reperfusion injury (42). Whether digoxin/Ouabain is potentially harmful in I/R states is not in conflict with our results. CIHH can upregulate Na/K pump activity and expression and attenuate I/R injury.
The single cardiomyocyte does not contain neutrophils, endothelial cells, and so on. Molecular and cellular approaches can exclude some interference factor. They are conducive to mechanistic studies, although they fail to reveal the complexity of isolated heart and in vivo responses. It is difficult to accurately simulate clinical myocardial I/R in a cellular model. We applied the model that was previously used to describe alterations in ventricular myocytes in the course of I/R (9), and many researchers used it (24, 48).
I/R injury is a complication that depresses the cardiac function and expands myocardial infarction. Although some research demonstrated adverse effects or controversial results of intermittent hypoxia (3), the present findings imply that CIHH in this experimental condition may be potentially clinically useful for preventing I/R injury in ischemic diseases and can induce beneficial consequences. As a therapeutic strategy it might be difficult to implement as most episodes of I/R are unscheduled. However, cardiac surgery is perhaps an exception.
In summary, the present study has provided evidence for the first time that CIHH increase Na/K pump activity by upregulating α1-isoform of Na/K pump. These alterations of Na/K pump may contribute to cardiac protection in CIHH guinea pigs.
This study was supported by the Natural Science Foundation of Hebei Province of China (No. 301360), the Foundation for the Youth in High Educational Institute of Hebei (No. 2010148), and the Research Foundation of Hebei Medical University (No. M2009011).
No conflicts of interest, financial or otherwise, are declared by the author(s).
We thank Dr. Yi Zhang (Department of Physiology, Hebei Medical University) for providing hypobaric chamber.
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