Hypercholesterolemia blunts NO donor-induced late preconditioning against myocardial infarction in conscious rabbits

Experimental preparation

The experimental preparation is similar to that described in detail previously [7, 9, 35, 44] except that rabbits were rendered hypercholesterolemic. Briefly, New Zealand White male rabbits (weight, 2.0~2.5 kg; age, 3–4 months) were instrumented under sterile conditions with a balloon occluder around a major branch of the left coronary artery, a 10-MHz pulsed Doppler ultrasonic crystal in the center of the region to be rendered ischemic, and bipolar ECG leads on the chest wall. The rabbits were fed a standard rabbit chow until the cholesterol-enriched diet began. Beginning on day 3 after surgery, the animals were assigned to two groups: an age-matched normocholesterolemic group (NC), which was continuously fed a normal diet, and a hypercholesterolemic group (HC), which was fed a diet enriched with 1% cholesterol (Purina Test Diets, Richmond, IN) for 4 weeks. All rabbits were restricted to 100 g of chow per day and had free access to drinking water at all times. Two blood samples (2 ml each) for measurement of the plasma lipid profile were collected from each rabbit, one during open chest surgery and the other during sacrifice. The response of arterial pressure to endothelium-dependent (bradykinin) and endothelium-independent (nitroglycerin) vasodilators was tested in some cholesterol-fed rabbits at the beginning of the cholesterol diet and at the completion of the experiment (just prior to sacrifice). Arterial pressure was measured by cannulating the ear dorsal artery with a 22-gauge angiocatheter as previously described [7, 9, 35, 44]. After sacrifice of the HC rabbits, a segment of descending thoracic aorta was collected and stained with oil red O stain [13] to verify the absence of atherosclerotic lesions.

Experimental protocol

At the end of the 4-week diet feeding, each diet group of rabbits was divided into two sub-groups, generating a total of four groups, i.e., NC-control (group I), NC-DETA/NO (group II), HC-control (group III), and HC-DETA/NO (group IV). All experiments were performed while the rabbits were conscious. Groups I and III received vehicle (PBS), whereas groups II and IV received 4 consecutive intravenous boluses of DETA/NO (0.1 mg/kg each) every 25 minutes (total dose, 0.4 mg/kg). This dose was selected because in our previous studies [34, 44, 45, 51] it induced a robust late PC protection and triggered the signaling cascade of late PC [34, 51]. DETA/NO (Alexis Corp) was dissolved in PBS (total volume infused, 4 ml). To remove oxygen from the solution, the PBS was bubbled with nitrogen for at least 30 minutes before dissolving DETA/NO. Twenty-four hours later, rabbits were subjected to a 30-min coronary artery occlusion followed by 3 days of reperfusion. Diazepam was administered 20 min before the onset of ischemia (4 mg/kg i.p.) to relieve the stress caused by the coronary occlusion. Successful coronary occlusion was verified by observing the development of ST-segment elevation and the changes in the QRS complex on the ECG and the appearance of paradoxical systolic wall thinning on the ultrasonic crystal recordings.

Measurement of region at risk and infarct size

At the conclusion of the study, the rabbits were given heparin (1,000 U i.v.), after which they were anesthetized with sodium pentobarbital (50 mg/kg i.v.) and euthanized with KCl. The heart was excised and perfused with Krebs-Henseleit solution through an aortic cannula using a Langendorff apparatus. To delineate infarcted from viable myocardium, the heart was then perfused with a 1% solution of 2,3,5-triphenyltetrazolium chloride in phosphate buffer (pH 7.4, 37 °C) at a pressure of 60 mmHg (~30 ml over 5 min). To delineate the occluded-reperfused coronary vascular bed, the coronary artery was then tied at the site of the previous occlusion and the aortic root was perfused with a 5% solution of phthalo blue dye (Heucotech, Fairless Hill, PA) in normal saline (~10 ml over 3 min). As a result of this procedure, the portion of the left ventricle (LV) supplied by the previously occluded coronary artery (region at risk) was identified by the absence of blue dye, whereas the rest of the LV was stained dark blue. The heart was then cut into 6-7 transverse slices, and all atrial and right ventricular tissues were excised. The slices were weighed, fixed in a 10% neutral buffered formaldehyde solution, and photographed (Nikon D100 Digital Camera with a Nikkor AF28-105 mm lens plus Promaster Spectrum 7 close-up lenses). Color pictures of heart slices were projected onto a paper screen at a ten-fold magnification and the borders of the infarcted, ischemic-reperfused, and non-ischemic regions were traced. The tracings were then scanned and the corresponding areas measured by computerized planimetry (Adobe Photoshop, version 6.0). From these measurements infarct size was calculated as a percentage of the region at risk using methods analogous to those employed in previous studies [35, 40, 44].

Statistical analysis

Data are reported as means ± SEM. For intragroup comparisons, arterial pressure response was analyzed by a two-way repeated-measures ANOVA (time and dose) followed by Student’s t-tests for paired data with the Bonferroni correction. For intergroup comparisons, data were analyzed by a one-way ANOVA followed by unpaired Student’s t-tests with the Bonferroni correction. The relationship between infarct size and risk region size was compared among groups with an ANCOVA using the size of the risk region as the covariate [35, 40, 44]. The correlation between infarct size and risk region size was assessed by linear regression analysis using the least-squares method. ANOVA was performed using the SigmaStat for Windows version 2.0 and ANCOVA using SPSS for Windows 10.

Exclusions and heart rate

A total of 28 rabbits were instrumented for this study (14 for each of the two diets). Seven rabbits were assigned to each of the four groups. Five out of the 28 rabbits were excluded from the study: 4 died of ventricular fibrillation during coronary occlusion (2 in group I, 1 in group III, and 1 in group IV), and 1 (in group III) was excluded because of malfunction of the balloon occluder. Therefore, a total of 5 rabbits completed the experimental protocol in groups I and III, and 6 in group IV. All 7 rabbits in group II were included into the analysis.

As seen in Table 1, there were no appreciable differences in heart rate among groups, either during administration of DETA/NO (groups II and IV) on day 1 or during coronary occlusion and reperfusion on day 2.

Plasma cholesterol levels and atherosclerotic lesions

As seen in Table 2, the plasma lipid profile was similar at baseline between groups and did not change in rabbits fed a normal diet for four weeks. In contrast, a marked increase in plasma levels of total cholesterol, % LDL-cholesterol and the LDL/HDL ratio and a decrease in % HDL-cholesterol were noted in rabbits fed a 1% cholesterol-enriched diet for four weeks, although triglycerides were not significantly altered. Despite the marked changes in lipid profile, postmortem oil red O staining did not reveal any macroscopic atherosclerotic lesion in the descending thoracic aorta of any HC rabbit (data not shown).

Vasodilator response

Studies were conducted in six cholesterol diet-fed rabbits to confirm that HC impairs NO activity. The response of arterial blood pressure to vasodilators was tested at baseline (before starting the cholesterol diet) and after 4 weeks on the cholesterol diet (just prior to sacrifice of the animal). As shown in Fig. 1, endothelium-dependent (bradykinin) and -independent (nitroglycerin) vasodilators decreased mean arterial pressure dose-dependently at baseline. The cholesterol diet reduced the response of arterial pressure to bradykinin (Fig. 1A) but not to nitro-glycerin (Fig. 1B), suggesting that NO bioactivity is impaired in HC rabbits.

Region at risk and infarct size

There were no significant differences among the 4 groups with respect to the weight of the region at risk (0.90 ± 0.18 g [18.2 ± 3.2% of LV weight], 0.87 ± 0.07 g [22.3 ± 2.5% of LV weight], 1.09 ±0.15 g [23.1 ± 3.2% of LV weight], and 1.10 ± 0.16 g [21.9 ± 3.3% of LV weight], respectively, in groups I, II, III, and IV). The average infarct size was 52% smaller in group II (NC-DETA/NO group) compared with group I (NC-control group) (29.7 ± 3.4% vs. 62.4 ± 4.0% of the region at risk, respectively; P < 0.05 [Fig. 2]), indicating that DETA/NO induced delayed protection against myocardial infarction in healthy rabbits, which confirms our previous observations [34, 44]. Infarct size in group III (HC-control group) was comparable to that in group I (NC-control group) (68.1 ± 4.5% vs. 62.4 ± 4.0% of the region at risk, respectively, P = NS [Fig. 2]). However, although group IV (HC-DETA/NO group) received the same dose of DETA/NO, infarct size was not smaller (61.0 ± 5.7% of the region at risk) than in group III (Fig. 2), suggesting that DETA/NO failed to induce a delayed protective effect against myocardial infarction in HC rabbits.

In all groups, the size of the infarction was positively and linearly related to the size of the region at risk (r = 0.95 in group I [NC-control group], 0.80 in group II [NC-DETA/NO group], 0.93 in group III [HC-control group], and 0.90 in group IV [HC-DETA/NO group]) (Fig. 3). As expected [44], the regression line was shifted to the right in the NC-DETA/NO group as compared with the NC-control group (P < 0.05 by ANCOVA) (Fig. 3), indicating that for any given size of the region at risk, the resulting infarct size was reduced by pretreatment with DETA/NO in NC rabbits. In contrast, in the HC-DETA/NO group (group IV), the regression line did not differ significantly from that observed in the HC-control group (group III) (Fig. 3), demonstrating that, in HC rabbits, DETA/NO fails to induce the infarct size-limiting effect seen in healthy rabbits.

Effect of HC on myocardial infarction in the nonpreconditioned state

The studies reported herein were performed in conscious animals in an effort to test the potential cardioprotective action of NO donors under physiological conditions. Since HC is one of the most prevalent risk factors for coronary artery disease, examining the PC phenomenon in the presence of HC is clinically relevant. Given that the focus of the present study was to examine the role of NO in triggering late PC, the use of a chronically instrumented conscious rabbit model was particularly important because the trauma and inflammatory reaction associated with a thoracotomy may elicit release of cytokines which, in turn, could modulate the activity of NOS and confound NO generation. Accordingly, we felt that studying late PC in a conscious hypercholesterolemic rabbit model would be both physiologically and clinically relevant. The duration of the cholesterol diet (4 weeks) was selected to avoid the development of atherosclerotic lesions while achieving high and steady plasma cholesterol levels so as to enable us to test the effect of HC in the absence of coexisting atherosclerosis. Indeed, this protocol induced high plasma cholesterol levels without histological evidence of atherosclerosis as demonstrated by postmortem oil red O staining of the aortas. As shown in Fig. 1, this hypercholesterolemic rabbit model exhibited impaired NO bioactivity, whereby the hypotensive response to the endothelium-dependent vasodilator bradykinin was blunted.

Despite the high cholesterol levels and the blunted pressure response to bradykinin, however, infarct size was not significantly different from that in normocholesterolemic rabbits (Fig. 2), suggesting that HC per se did not affect the susceptibility to ischemia/reperfusion injury. This finding is apparently in contrast with studies by Jung et al. [24] and Wang et al. [52] in anesthetized open-chest rabbit preparations, which concluded that hypercholesterolemic myocardium is more susceptible to ischemia/reperfusion injury. In the study by Jung et al. [24], rabbits were fed an atherogenic diet (0.25% cholesterol and 3% coconut oil) for 4 weeks, after which myocardial infarction was induced by a 30-min coronary occlusion followed by 2 h of reperfusion. Infarct size in their model was increased by 52% compared with normal diet-fed rabbits. Similar findings have been reported by Wang et al. [52] in rabbits fed 0.5% cholesterol and 10% coconut oil for 8 weeks followed by a 30-min coronary occlusion/4-h reperfusion sequence. The reason for the apparent discrepancy between these findings and our present results is unknown. The divergent results may be secondary to differences in experimental preparations (open-chest vs. conscious animals), diet components (cholesterol plus coconut oil vs. cholesterol only), or experimental protocols (2–4 h vs. 24 h reperfusion).

Effect of HC on PC

A number of reports [15, 17, 24–27, 37, 39, 48, 50] have examined the PC response in hypercholesterolemic animals. These few studies have dealt mainly with the early phase of PC [15, 17, 24–27, 39, 50] and have yielded conflicting results. Szilvassy et al. [39] first reported that HC abrogated the early protective effects induced by rapid cardiac pacing in a conscious rabbit model. Interestingly, normalization of serum cholesterol by re-feeding normal chow over 6 additional weeks reinstated the PC phenomenon even though intimal lesions were still present [39]. This finding was subsequently confirmed in hearts isolated from hypercholesterolemic rats, in which the early PC protection induced by rapid ventricular pacing was lost [17]. These studies [17, 39] indicate that HC per se, without atherosclerosis, inhibits the early PC response. Komamura et al. [26] and Ueda et al. [50] reported that the infarct-limiting effect of early PC was blunted by HC in rabbits fed a cholesterol diet. Kocic et al. [25] found that the protective effect of early PC on contractility and responsiveness to phenylephrine was prevented by experimental hyperlipidemia in isolated stunned rat papillary muscle. A recent study [29] in patients found that hyperlipidemia prevented the expected decrease in ST-segment elevation after repeated balloon inflations during coronary angioplasty, suggesting that HC blocks early PC in humans as well. In contrast, Kremastinos et al. [27] and Jung et al. [24] reported that the infarct size-limiting effect of early ischemic PC was not altered in hypercholesterolemic rabbits. Thus, data on the effect of HC on early PC are conflicting.

With regard to the effect of HC on late PC, only two reports [37, 38] are available in addition to our recent study [48]. In the first report, Szekeres et al. [37] found that the protection induced by rapid ventricular pacing against the hemodynamic and electrophysiological changes caused by subsequent rapid overpacing was still present in cholesterol-fed rabbits but a greater PC stimulus was required. These results suggest that HC elevates the threshold for the PC response. In another study, Szilvassy et al. [38] reported that the LPS-induced late PC effect on the hemodynamic and electrophysiological changes caused by ventricular overpacing was not abrogated in cholesterol-fed conscious rabbits. However, in both studies [37, 38], the experiments were performed in HC rabbits with atherosclerotic lesions, which may have confounded the effect of HC per se; furthermore, the endpoints were ST-segment elevation, left ventricular end-diastolic pressure, and shortening of the ventricular effective refractory period, not infarct size. Therefore, the findings are difficult to evaluate because the animal model was not one of pure hypercholesterolemia and because the significance of the end-points selected is unclear. Using a more straightforward experimental protocol and a HC rabbit model without significant atherosclerotic alterations, we have found that a sequence of six 4-min coronary occlusion/4-min reperfusion cycles, which induces delayed protection 24 h later against stunning [7, 9, 11, 12, 34, 41, 47, 53] and infarction [34, 35, 42] in healthy rabbits, failed to induce delayed protection in rabbits fed a 1% cholesterol-enriched diet for 6 weeks [48], indicating that HC abrogates the cardioprotective effect of the late phase of ischemic PC. The present study is the first to deal with the effect of HC on NO donor-induced late PC. Because the NO donor DETA/NO releases NO spontaneously and triggers the development of late PC in healthy rabbits [34, 44, 45], we expected that exogenous NO would reinstate the late PC effect in HC rabbits. Surprisingly, our present data show that administration of the NO donor DETA/NO that induces the late PC effect in NC rabbits failed to do so in HC rabbits, suggesting that HC blunts NO donor-induced late PC as well. However, because our present study did not examine another NO donor or a higher dose of DETA/NO, it is unknown whether other NO donors or higher doses of DETA/NO would reinstate the late PC effect in HC rabbits.

Possible mechanism whereby HC blunts NO donor-induced late preconditioning

Since it has been well documented that HC impairs NO bioactivity [5, 10, 21, 36] and that NO acts as a trigger of late PC [8], it is plausible that HC could impair the triggering role of NO in PC. If loss of late PC protection in hypercholesterolemic animals results from an impairment of the NO triggering mechanism, one would expect that NO donors would overcome this block and elicit the cardioprotection of late PC; that is, the loss of late PC should be rescued by exogenous NO. Our present results, however, show that administration of an NO donor fails to induce late PC, suggesting that the loss of PC protection in HC is not due, or at least is not due solely, to loss of the trigger. Since iNOS-derived NO plays an essential role in mediating the cardioprotection of late PC [8, 9, 20], one possibility is that HC may interfere with the role of NO as a mediator in lieu of or in addition to the role of NO as a trigger. That is, even though exogenous NO (via administration of an NO donor) initiates the late PC response, cardioprotection would not be manifest because HC also impairs the formation of the NO that mediates late PC. In this regard, it is well known that tetrahydrobiopterin (BH₄) is an essential cofactor for iNOS to generate NO [18]. Since HC is known to be associated with increased production of superoxide anion [33], which may inhibit the biosynthesis or prevent recycling of BH₄ [32], we speculate that HC may undermine the bioavailability of BH₄, leading to impairment of the iNOS-derived NO formation that is required to mediate NO donor-induced late PC protection. In addition, we have previously found that administration of N-acetyl-serotonin, a BH₄ synthesis inhibitor, to ischemically pre-conditioned rabbits abrogates late PC against both myocardial stunning [43] and infarction [48] and that ischemic PC elevates myocardial levels of BH₄ in NC rabbits, but not in HC rabbits [48], indicating that BH₄ plays an essential role in mediating ischemia-induced late PC and that HC prevents ischemic PC-induced upregulation of myocardial BH₄. However, it remains to be determined whether and how, by undermining the bioavailability of BH₄, HC affects the formation of NO that mediates NO donor-induced late PC.

Conclusions

In conclusion, the present study demonstrates that administration of the NO donor DETA/NO, which induces late PC protection against myocardial ischemia/reperfusion injury in healthy animals, fails to elicit cardioprotection in hypercholesterolemic rabbits. This finding suggests that the inhibitory effect of HC on the late PC response elicited by either ischemia or NO donors are due, at least in part, to a disruption of the signaling cascades and/or mediator proteins downstream of the NO that triggers these two adaptive responses.