INTRODUCTION

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SYSTEMIC HYPOXIA (SH) represents a common pathophysiological condition that may be caused by environmental factors (such as high altitude) or by a number of cardiopulmonary and hematological diseases (such as heart failure, apnea, and anemia). These factors may interfere or disrupt any stage(s) of the oxygen supply, transport, and utilization process, resulting in tissue oxygen deficiency and impairment of the normal cellular function fueled mainly by ATP generated through oxidative phosphorylation. In contrast, SH may also elicit a complex physiological response that leads to adaptation at system, organ, and tissue levels to compensate the lower availability of oxygen. Hurtado (15) reported that people living in high altitude areas had extremely low incidence of myocardial infarction and hypertension. The high altitude population from various ethnic groups also possessed greater vascularity and vasodilatation in all major organs. These unique observations were confirmed in laboratory animal models where a paradoxical improvement in myocardial ischemic tolerance was demonstrated in immature or adult mammals after exposure to continuous or intermittent chronic SH (21, 23, 27, 28, 33). Thus the discovery of SH-induced myocardial protection well preceded the description of "ischemic preconditioning" (24), an endogenous cardioprotective phenomenon first described in 1986.

Despite the convincing clinical and experimental evidence for SH-induced cardioprotection, it remains unclear whether acute exposure of SH would also result in cardioprotection, where the adverse effects of chronic SH, such as pulmonary hypertension and right ventricular hypertrophy (27), can be effectively avoided. Furthermore, the mechanism of SH-induced cardioprotection is not fully understood. Several studies have attempted to establish a temporal profile of the myocardial enzymes that regulate antioxidant defense (25) and energy metabolism (26) in adult rats exposed to chronic hypobaric hypoxia. There is mounting evidence suggesting the role of sarcolemmal and/or mitochondrial ATP-sensitive potassium (K_ATP) channels in SH-induced cardioprotection in immature or adult mammals (1, 2, 8). An increase in nitric oxide (NO) production after exposure to chronic SH was also reported (3, 20, 30). In the immature rabbit model of chronic SH, Baker and colleagues (3, 30) reported upregulation of endothelial NO synthase (NOS3) rather than inducible NO synthase (NOS2) in hypoxic hearts. In contrast, several studies in adult rodents demonstrated that acute SH enhances NOS2 expression in various organs and tissues (11, 17), and chronic SH may actually downregulate NOS3 expression in vivo (36). We and others (4, 14, 16, 34, 39, 41, 45, 46, 47) have demonstrated that NOS2 is an essential mediator for delayed pharmacological or ischemic preconditioning. Similar to the heart, an NO-dependent mechanism has been proposed in acute SH-induced neuronal protection against ischemic injury in the brain (12). Recently, it has been suggested that cyclooxygenase 2 (COX-2) also plays a critical role in the late phase of ischemic preconditioning in rabbits (31) and mice (13). However, no data are available to show the effect of SH on myocardial COX-2 expression, although this protein is induced by hypoxia in the lungs (5). Therefore, the present study was focused on the following three specific goals: 1) to determine whether moderate acute SH induces delayed cardioprotection in the mouse; 2) to elucidate the role of NOS2 as a potential trigger or mediator of the SH-induced cardioprotection; and 3) to determine the possible involvement of COX-2 in delayed protection induced by acute SH. The preliminary results of this work were presented at "Experimental Biology 2001" (Orlando, FL) and published in abstract form (42).

	METHODS

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Animals. Adult male outbred ICR mice were supplied by Harlan Sprague Dawley (Indianapolis, IN). Adult male homozygous (/) NOS2 gene knockout mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by the National Institutes of Health (No. 85-23, Revised 1996).

Chemicals and drugs. Chemicals for preparing the heart perfusion buffer as well as the selective inhibitors of NOS2 (S-methylisothiourea sulfate, SMT) or COX-2 (NS-398) were purchased from Sigma-Aldrich (St. Louis, MO).

Systemic hypoxia pretreatment. A cage containing three to four conscious, unrestrained mice was placed in a normobaric Plexiglas chamber (100 × 60 × 50 cm) with 10% inspiratory oxygen fraction (i.e., 0.10 FIO₂) by introducing pure N₂ into the chamber. The oxygen concentration was continuously monitored with a Beckman oxygen analyzer and precisely maintained at 10% by adjusting influx flow of N₂. Normoxia control experiments were conducted in the identical way except that compressed air, instead of N_2, was introduced into the chamber for maintaining a condition of 21% inspiratory oxygen fraction. A thermohygrometer (Fisher Scientific) was placed inside the chamber for continuously monitoring the temperature and humidity.

Mouse model of myocardial ischemia-reperfusion injury. The methodology of the Langendorff isolated buffer-perfused mouse heart preparation was previously described in detail (38-41). Ventricular contractile function was measured periodically during the 20 min of no-flow global ischemia and 30 min of reperfusion (I/R) with a force-displacement transducer (model FT03, Grass) attached to the apex through a no. 5 surgical thread and a metal hook. The resting tension was initially adjusted to ~0.30 g and kept at this level without readjustment thereafter. Ventricular developed force was recorded with a Beckman R-511A polygraph connected to the force transducer. Infarct size was determined at the end of I/R as describe previously (38-41). The coronary flow rate (expressed as ml · min¹ · g wet wt¹ of the heart) was calculated by timed collection of the efflux perfusate.

Experimental groups. Mice were randomized into the following 12 experimental groups (n = 6-8 mice each). Normoxia: ICR mice were kept in the chamber under normoxic condition for 4 h followed by 24-h intervals before I/R; H30min & H4h: ICR mice were pretreated with hypoxia for 30 min or 4 h followed by 24 h reoxygenation before I/R; H2hx2 & H4hx2: ICR mice were pretreated with two cycles of either 2 or 4 h of hypoxia followed by 24 h of reoxygenation before I/R; SMT+H4hx2 & H4hx2+SMT: SMT was given (3 mg/kg ip) either 30 min before two cycles of 4 h of hypoxia followed by 24 h of reoxygenation or 30 min before I/R following SH pretreatment in ICR mice; NOS2-KO+H4hx2: NOS2 knockout mice were pretreated with the repetitive SH before I/R; NS398+H4hx2 & H4hx2+NS398: NS398 was given (10 mg/kg ip) either 30 min before the repetitive SH or 30 min before I/R following SH pretreatment in ICR mice; Normoxia+NS398 & Normoxia+SMT: ICR mice were pretreated under normoxia and received NS398 or SMT 30 min before I/R. In addition, we used our previously reported data (40) for serving as the NOS2-KO normoxic control group, which was collected under the similar experimental conditions and protocol.

Western blot analysis. In parallel series of experiments, ventricular tissue samples (n = 3-8 samples/group) were collected following the various pretreatments (see Experimental groups). Tissue samples were ground under liquid nitrogen and homogenized with the Polytron at 4°C in 1 ml of phosphate-buffered saline (pH 7.4). The homogenate was then centrifuged at 12,000 g for 10 min, and the supernatant was recovered. For measurement of COX-2 expression in subcellular compartments, cytosolic and membranous fractions were prepared by the method of Shinmura et al. (31). In brief, the heart tissue was homogenized in ice-cold lysis buffer using a glass pestle and single burst of 5 s with a Polytron homogenizer. Under 4°C, the homogenate was centrifuged at 45,000 g for 1 h, and the supernatant was recovered as the cytosolic samples. The pellets were then incubated in lysis buffer + 1% Triton X-100 for 1 h, rehomogenized, and centrifuged at 45,000 g for 1 h, as described above. The supernatant representing the membranous samples was finally recovered. Either 40 µg (for COX-2) or 60 µg (for NOS2 and NOS3) of protein from each sample were separated by SDS-PAGE on 10% acrylamide gels. Proteins were then transferred to a nitrocellulose membrane and blocked with 5% nonfat dry milk in Tris buffer saline. The membrane was subsequently incubated with a rabbit polyclonal antibody reacting specifically to NOS2 or NOS3 (dilution 1:500 for NOS2 and NOS3; Santa Cruz) or with a goat polyclonal antibody reacting specifically to COX-2 (1:100 dilution; Santa Cruz). The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:1,000 or 2,000 dilution; Amersham). The membranes were developed using enhanced chemiluminescence and exposed to X-ray film. Optical density for each Western blot band was determined with a scanner/densitometer system (Molecular Dynamics 4.0) and normalized against background density for each gel.

Data analysis and statistics. The data are presented as the group means ± SE. The difference among experimental groups was compared by unpaired t-test or one-way ANOVA followed by Student-Newman-Keuls post hoc test. Linear regression analysis and the Spearman rank-order correlation test were used to evaluate the correlation coefficients between NOS2 protein expression and infarct size. P < 0.05 was considered as statistically significant.

	RESULTS

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Exclusions. A total number of 74 hearts were subjected to the I/R protocol for assessment of ventricular function and infarct size. Five hearts (i.e., 6.2% of the 81 perfused hearts) were excluded due to either time delay (>3 min) or aortic damage during aortic cannulation.

Experimental conditions. Morphometric characteristics of the animals and conditions for the SH pretreatment and I/R experiment are summarized in Table 1. There were no significant differences in body and heart weights of the animals in various treatment groups except for the smaller body weights in NOS2 knockout mice (P < 0.05). Similarly, the changes of temperature in the hypoxia chamber during treatment sessions were nominal (<1.0°C) with limited variations in different groups. SH treatment resulted in a gradual increase in humidity compared with the normoxia group (Table 1), which may be due to hyperventilation in the mice in response to hypoxic stimulus. Inhibition of NOS2 (SMT+H4hx2 and NOS2-KO+H4hx2) augmented such a hypoxic response, whereas inhibition of COX-2 (NS398+H4hx2) suppressed the response. Also, there were no differences in pH, PCO₂, and PO₂ of the perfusion buffer for all the experiment groups, indicating that the isolated perfused heart experiments were conducted under tightly controlled conditions.

Cardiac contractile function. There was no significant difference in preischemic cardiac function among the groups except that the baseline heart rate was lower in the NOS2-KO+H4hx2 (Table 2) groups. After 20 min of global ischemia, ventricular developed force and rate-force product during the reperfusion period were depressed in all the groups, whereas heart rate remained reasonably constant and was comparable to the preischemic values. The only exception was the H4hx2+SMT group, which possessed both positive inotropic and chronotropic effects especially during the early stage of reperfusion. Furthermore, the resting tension was not significantly different among all the experimental groups (data not shown).

Infarct size. Pretreatment with 4 h of SH resulted in a significant reduction in infarct size 24 h after SH (i.e., H4h group, 21.4 ± 2.2%) compared with normoxia (29.7 ± 2.6%; P < 0.05; Fig. 1). Two cycles of the SH further reduced infarct size to 16.0 ± 3.1% (H4hx2; P < 0.05). Shorter durations of SH (30 min or 2 h) were insufficient to reduce infarct size (H30min: 27.5 ± 6.0%; H2hx2: 29.0 ± 4.2%; P > 0.05 vs. normoxia). The infarct-limiting effect of SH in H4hx2 group was abolished by SMT, the NOS2 inhibitor, given either before or after SH (SMT+H4hx2: 24.8 ± 3.3%; H4hx2+SMT: 31.8 ± 5.9%; P > 0.05 vs. normoxia). SMT had no significant effect on infarct size in the normoxic hearts (Normoxia+SMT: 26.8 ± 4.2%). Furthermore, H4hx2 failed to reduce the infarct size in NOS2 gene knockout mice (28.5 ± 3.8%) compared with the nonpretreated NOS2-KO mice (23.5 ± 3.8%; n = 10; P > 0.05) as reported previously (40). In contrast, NS398, an inhibitor of COX-2, given either before or after SH, did not block the SH-induced protection (NS398+H4hx2: 20.8 ± 4.0%; H4hx2+NS398: 18.4 ± 3.4%; P < 0.05 vs. normoxia). NS398 had no effect on infarct size in normoxic controls (Normoxia+NS398: 29.4 ± 4.7%).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Effect of acute systemic hypoxia on myocardial infarct size (A) and coronary flow (B). Values are means ± SE (see METHODS for abbreviations of experiment groups).

Coronary flow. The preischemic coronary flow was not different among the groups (Fig. 1; P > 0.05). However, postischemic coronary flow was generally higher in all SH-treated groups compared with normoxia. Administration of SMT before SH blocked the flow improvement in H4hx2, whereas SMT given after SH did not have effect on the increased coronary flow (P < 0.05 vs. normoxia). The improvement in coronary flow was partially blocked in the NS398-treated groups.

NOS2 expression. SH exposure resulted in consistent upregulation in NOS2 protein expression in all the hypoxic groups compared with normoxia (n = 4-8/group; Fig. 2). The densitometric analysis indicated a significant increase in NOS2 in the H4h, H2hx2, and H4hx2 groups(P < 0.05 vs. Normoxia), but not in the H30min group. However, no significant linear correlation was observed between NOS2 protein content and myocardial infarct size (r = 0.44, P > 0.05), despite the general trend of NOS2 upregulation in all SH-treated hearts. Administration of SMT before SH completely abolished the induction of NOS2 by SH (see SMT+H4hx2).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Inducible nitric oxide synthase (NOS2) expression in the mouse heart. A: two representative Western blot pictures demonstrating NOS2 protein expression in mouse heart following various pretreatments. B: densitometric quantification of NOS2 expression.

NOS3 expression. NOS3 was constitutively expressed in both normoxic and hypoxic mouse hearts (Fig. 3). Acute SH of various durations caused a slight decrease in NOS3 protein expression in the heart (P > 0.05 vs. normoxia). SMT treatment had no effect on the expression of NOS3.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Murine cardiac expression of endothelial NOS (NOS3). A: representative Western blot picture showing NOS3 expression following various pretreatments. B: densitometric quantification of NOS3 expression.

COX-2 expression. COX-2 was primarily detected in the membranous fraction in both normoxic and hypoxic hearts (Fig. 4). The densitometric results indicated a rather slight nonsignificant downregulation of COX-2 expression in the membranous fraction of H4hx2 hearts.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Cyclooxygenase-2 (COX-2) expression in mouse heart. A: Western blots of COX-2 protein expression in cytosolic and membranous fractions of heart tissue following normoxic or hypoxic (H4hx2) pretreatment. B: densitometric quantification of COX-2 expression.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study was aimed at determining the role of acute SH in adaptation of heart to ischemia. A second goal was to demonstrate the intrinsic molecular mechanism of this adaptation against I/R in the heart. Our results show that 1) acute SH induced significant cardioprotection against myocardial infarction in mice in a duration-sensitive manner; 2) the infarct-limiting effect of acute SH was associated with significant improvement in postischemic coronary flow without any effect on ventricular contractile function; 3) the cardioprotection was abolished by pharmacological inhibition of NOS2 (either before SH or ischemia) and was absent in mice with targeted ablation of NOS2, and COX-2 inhibitor NS398 failed to block the cardioprotective effect of acute SH; and 4) SH pretreatment resulted in a significant increase in expression of NOS2 but failed to increase NOS3 and COX-2. Taken together, for the first time, these results demonstrate that NOS2 plays a trigger and mediator role in SH-induced delayed cardioprotection in adult mice.

Despite the large body of evidence on chronic SH-induced cardioprotection in several mammalian species including humans (15, 21, 23, 27, 28, 33), it remains unknown if acute or subacute SH could also produce similar protection. Our results show that acute SH induced delayed protection in a duration-sensitive manner (Fig. 1). A minimum of 4 h of SH exposure was necessary to achieve significant infarct size reduction (H4h, H4hx2) and improvement in postischemic coronary flow, whereas the shorter SH durations were ineffective (H30min, H2hx2). Only mild improvement in ventricular contractile function during reperfusion was observed following SH (Table 2; P > 0.05 vs. normoxia). Such a disassociation between functional improvement and infarct reduction has been reported previously by us (38) and others (6) following classic ischemic preconditioning or delayed pharmacological preconditioning (34, 39, 41). On the other hand, a good association between infarct reduction and functional improvement was observed with other preconditioning modalities, including whole body hyperthermia in mice (35).

Previous studies have suggested that NOS2 is an essential mediator of the late phase of myocardial preconditioning induced by brief episodes of I/R (13, 16) or by a number of pharmacological agents (34, 39, 41, 45, 46, 47). A unique observation in the present study is that NOS2 served as the trigger and mediator of SH-induced late cardioprotection. The role of NOS2 in the protective process was supported by: 1) blockade of SH-induced cardioprotection with SMT (NOS2 inhibitor), given either before or after SH as shown in Fig. 1; 2) a significant upregulation of NOS2 protein content in myocardium by acute SH (Fig. 2); and 3) absence of SH-induced delayed cardioprotection in NOS2 gene knockout mice; i.e., no difference in infarct size was found between NOS2-KO+H4hx2 group (Fig. 1) and the control NOS2-KO group (40). Furthermore, inhibition of NOS2 with SMT did not reverse the improvement of postischemic coronary flow in H4hx2 (Fig. 1). These results indicate that SH could improve vascular endothelial function independent of NOS2. It is also conceptually consistent with the previous findings of ours (39) and others (19) on monophosphoryl lipid A-induced cardioprotection. Inhibition of NOS2 may block the signaling pathway via NOS2-generated superoxide and/or peroxynitrite (43), which are well-known triggers of preconditioning. Previous studies have argued against the triggering role of NOS2 in delayed ischemic preconditioning in rabbits (44). Because NOS2 was constitutively expressed in normal mouse hearts (Fig. 2 and Ref. 45), such a role of this enzyme may be species specific. Another surprising observation in the present study was the lack of linear correlation between reduction in infarct size and the level of NOS2 expression following the shorter duration of SH, i.e., H2hx2 (Figs. 1 and 2). These results suggest that longer duration of SH (i.e., 4 h) may activate certain unknown cofactor(s) that may potentially control the posttranslational modifications of NOS2, which in turn regulate its activity.

It is suggested that NOS3 plays an important role in the chronic SH-induced cardioprotection in immature rabbits (3, 28). In the present study, NOS3 expression was not increased following SH pretreatment (Fig. 3), suggesting that this protein is unlikely to be involved in acute SH-induced delayed cardioprotection. These differences may be attributed to the species (rabbit vs. mouse), age (immature vs. adult), and duration of hypoxia (chronic vs. acute). However, the triggering role of NOS3 in the SH-induced protection cannot be ruled out completely in the present study. This is because the blockade of SH-induced infarct size reduction was relatively partial when SMT was given before SH (SMT+H4hx2: 24.8 ± 3.3% vs. normoxia: 29.7 ± 2.6%; Fig. 1) compared with the complete blockade when SMT was given before I/R (H4hx2+SMT: 31.8 ± 5.9%). In addition, like most pharmacological inhibitors, the selectivity of SMT in inhibiting NOS2 is far from perfect (10, 32). SMT is a non-amino acid analog of L-arginine and is reported to be ~600-fold more potent than aminoguanidine for inhibiting mouse NOS2 in vitro (10). The in vivo drug dose of 3 mg/kg used in the present study has been reported previously in the rat (32), rabbit (37), and mouse (39, 41, 45). Wildhirt et al. (37) showed that this dose of SMT caused no significant inhibition of NOS3 activity in the rabbit ventricular myocardium. Furthermore, there is no evidence to show that SMT inhibits NOS3 activity in the mouse tissue at this dose level. Nevertheless, the questions concerning the role of NOS3 in acute SH-induced late cardioprotection need to be addressed in the future investigations that would include the use of NOS3-KO mice and other more selective NOS2 inhibitors, including those structurally distinct from SMT.

Recent studies proposed that COX-2 could serve as a critical mediator of delayed ischemic preconditioning in rabbits (31) and mice (13). However, our results show a lack of the role for this enzyme because NS398 did not block SH-induced cardioprotection, regardless of the timings of its administration, i.e., before or after hypoxia pretreatment (Fig. 1). COX-2 was preferentially expressed in the membranous fraction (Fig. 4), similar to the previously described rabbit study (31). However, no increase in the membranous COX-2 protein expression was found in the preconditioned hearts (H4hx2) compared with normoxia. These results suggest that COX-2 may not have any role in SH-induced cardioprotection. The possible explanation for the discrepancy between this study and the rabbit study could be that the moderate SH stimulus (0.10 FIO₂) may not be powerful enough to activate the COX-2 dependent proinflammatory pathways triggered with multiple cycles of coronary occlusion-reperfusion (13, 31). Thus, unlike the essential role of NOS2 in delayed cardioprotection with pharmacological (34, 39, 41, 45, 46, 47), ischemic (14, 16), or hypoxic preconditioning in the present study (Figs. 1 and 2), the involvement of COX-2 may be dependent on the type of preconditioning stimulus. In support of this hypothesis, a recent study has also demonstrated that COX-2 did not mediate the delayed pharmacological preconditioning induced by adenosine A₁ or A₃ receptor agonists in the rabbit heart (18).

SH is a clinically relevant condition with potential importance in cardiovascular pathophysiology. For example, the antiischemic and antihypertensive beneficial effects have been observed in the human subjects adapted to SH under the simulated high altitude environment (22). Furthermore, recent studies demonstrated that the gene expression and enzyme activity of NOS2 was upregulated, whereas NOS3 activity and gene expression were downregulated in the right atrial tissues in children with cyanotic congenital heart defects (9). The NOS2 upregulation may represent an adaptive response to the condition of SH persisting in the sick children. These clinical observations further support the importance of a NOS2-dependent adaptive mechanism in SH. The exact upstream regulation and downstream targets of NOS2 induced by SH need to be further elucidated. However, hypoxia inducible factor 1 appears to be a critical transcription factor controlling the upregulation of NOS2 gene expression in response to SH (17). As proposed previously (39, 45), upregulation of NOS2 may increase the myocardial NO level that may open mitochondrial K_ATP channels (29, 46), which have been considered as the potential end effector for SH-induced cardioprotection (1, 2, 8). In addition, pretreatment of neonatal rats with acute SH rendered protection against cerebral ischemic injury (12). Therefore, one could expect that an adequate degree and duration of SH may induce protective effect in multiple organs, which may be more advantageous than ischemic preconditioning where the remote protection has not been consistently demonstrated. For example, a brief ischemia in kidneys or skeletal muscle could reduce myocardial infarct size caused by a subsequent coronary occlusion, a transient cerebral ischemia failed to produce the similar cardioprotection (7).

In summary, we have demonstrated that pretreatment with moderate acute SH can induce delayed protection against myocardial infarction in mice. The length of SH exposure influenced the degree of cardioprotection, with repetition of the exposure conferring more significant protection. Using pharmacological inhibition and gene knockout mice, we have demonstrated that the infarct-limiting effect of acute SH is triggered and mediated by NOS2. In contrast, NOS3 and COX-2 may only have insignificant role in the cardioprotection in this model. Further studies are needed to identify the upstream and downstream signaling cascade that leads to the activation of NOS2, which in turn plays a major role in acute SH-induced delayed cardioprotection.