| Obstructive sleep apnea(OSA) patients and animals exposed to chronic intermittent hypoxia(CIH) had been shown to result in cardiac dysfunction and vascular endothelium injury. OSA demonstrates recurrent episodes of pharyngeal collapse occurring during sleep, which directly causes chronic intermittent hypoxia(CIH), become one of the most important risks of cardiovascular diseases in OSA patients. Evidences indicate that CIH can induce left ventricular dysfunction of OSA patients. Moreover,the incidence of coronary artery disease in OSA patients was increased. As we all know, impairment of coronary arteries directly induce cardiac dysfunction in many cardiovascular disease. But in OSA patients, whether the decreased cardiac function is associated with coronary dysfunction, and the mechanisms of coronary arteries dysfunction induced by CIH remain to be determined.Endothelin-1(ET-1), one of the biologically active substances produced by endothelium, plays an important role in vascular injury induced by CIH. As a potent endogenous vasoconstrictor, ET-1 acts via two receptors, ETA and ETB. In rats exposed to CIH, circulating ET-1 levels and the sensitivity of the vasoconstrictor response to ET-1 were increased. Moreover, previous studies have shown that ET-1and ET-1 receptors play a key role in CIH-induced endothelial dysfunction and augmented vasoconstriction of aortas and pulmonary arteries in rats. Nitric oxide(NO), another important biologically active substances produced by endothelium, is a primary endothelium-derived relaxing factor regulating vascular resistance. Activation of endothelial ETB receptors results mostly in increased endothelial nitric oxide synthase(e NOS) activity and release of NO. We suspect that CIH induced endothelial dysfunction in coronary arteries may mediate by ET-1 and ET receptors. Furthermore, the ET-1 mediated endothelial injury will be deteriorated by the change of NO release.In the present study, we evaluated the effects of CIH on coronary arteries and cardiac function, investigating whether the decreased cardiac function was associated with coronary arteries injury, especially endothelia injury. In addition, we analyzed the mechanisms of ET-1 and ET receptors mediated coronary injury in CIH rats.Objective: This study was aimed at evaluating the effects of CIH on coronary arteries and cardiac function in isolated hearts from normoxia and CIH rats, and then analyzing endothelial mechanisms of coronary arteries and cardiac dysfunction by different methods.Methods:Male Sprague-Dawley(SD) rats, weight 250-300 g, were purchased from the Laboratory Animal Center(Hebei Medical University, China). The rats were separated randomly into two groups,including CIH group and normoxia group. During hypoxic exposure, animals were placed daily in commercial hypoxic chambers that were flushed with 100% N2 to inspired O2 fraction(FIO2) nadir of 9% for 1.5 min, the FIO2 gradually returned to 21% over the remainder of each cycle. The exposure cycle was repeated every 3 min for 8 h/day, 7 days/wk for 3 wk during the animal’s sleeping hours. After the exposure cycle was completed, animals were randomly assigned to different part of the experiment.Rats were anesthetized and hearts were rapidly removed into ice-cold perfusion buffer, cannulated via the aorta, and then perfused in the Langendorff mode with Krebs–Henseleit(KH) solution at constant perfusion pressure of 80 mm Hg. After equilibration period, drugs were injected into coronary arteries from aortas of the isolated hearts. Then the index of cardiac function was recorded and coronary resistance(CR) was calculated. In another part of the experiment, hearts were perfused at constant flow conditions, for which the flow rate was adjusted in order to obtain the same coronary flow as in the preparation at constant pressure. The index of cardiac function was recorded as already described. In addition, hematoxylin and eosin(H&E) staining was used to assessment the extent of coronary arteries injury after CIH, the expression of ET-1, ETA and ETB receptors after CIH was observed by immunohistochemistry and western blot. Moreover, the changes of NO production and e NOS expression induced by CIH were also been examined.Results:1 Effects of CIH on cardiac functionThe left ventricular developed pressure( LVDP), +d P/dtmax,-d P/dtmax and Coronary flow(CF) were decreased in the CIH group(7.5%,17.8%,31.5% and 26.2%, compared to the normoxia group respectively, P < 0.05). Conversely, the coronary resistance was significantly increased in CIH rats(26.5% compared to the normoxia group, P < 0.05).2 Effects of CIH on coronary resistanceCoronary resistance(CR) was significantly higher in animals exposed to CIH than that in normoxia rats. After stimulus by endothelium-dependent vasodilator acetylcholine(Ach), the percent decrease of CR was significantly lower in CIH rats compared with normoxia group(CIH 9.75+1.03%; Normoxia 24.38+1.82%; P < 0.01). Conversely, the endothelium-independent vasodilator sodium nitroprusside(SNP) was effective in both groups of animals, and the decrease percent in CR was not affected by CIH exposure(CIH 34.25+3.57%; Normoxia 32.75+4.41%; P >0.05).3 Effects of CIH on ET-1-mediated CR and LVDP changesET-1 caused a significant decrease of LVDP and an obvious increase of CR in both groups, and the change percentage of LVDP(CIH 38.51+2.82%; Normoxia 14.87+1.17%; P < 0.01) and CR(CIH 170.01+15.32%; Normoxia 44.86+6.63%; P < 0.01) was markedly higher in CIH rats than in normoxia rats.4 Effects of ETA and ETB receptor antagonists on ET-1-induced CR and LVDP changesThe LVDP and CR changes after ET-1 administration were mostly inhibited by the ETA receptor antagonist BQ123 in both groups(P < 0.05), with no significant difference in the percentage inhibition between the two expressionThere was a significant decrease in the e NOS expression levels in CIH group compared with normoxia group(P < 0.01). Meanwhile, the NO levels of coronary arteries were reduced in CIH group(P < 0.01).Conclusions: These results demonstrated CIH induced cardiac dysfunction was associated with coronary injury. The increased expression of ETA receptors, which mediate a potent vasoconstrictor response, plays an important role in coronary pathogenesis of CIH. Moreover, the damage of coronary artery endothelial cell, the decreased endothelial ETB receptor expression and the reduction in NO release play an important role in the enhanced coronary vasoconstriction after CIH exposure. groups(CIH 90.46+3.99%; Normoxia 91.32+3.06%; P >0.05). However, the ETB receptor antagonist BQ788 markedly augmented ET-1-mediated LVDP decrease and CR increase in normoxia rats(P < 0.05). The ET-1-induced decrease in LVDP and increase in CR was not affected by BQ788 in CIH group.5 Effects of L-NAME on ET-1-induced CR and LVDP changesThe NO synthase(NOS) inhibitor L–NAME significantly enhanced ET-1 induced LVDP decrease and CR increase in normoxia group(P < 0.05). However, in CIH group, there were no significantly differences of LVDP and CR changes after injected L-NAME compared with only injected ET-1.6 Effects of ET-1 on LVDP at constant coronary flowThere was no significant difference in LVDP level between the two groups in baseline at constant coronary flow. And this experiment observed that 20 pmol ET-1 can induce a little increase of LVDP in normoxia rats, but have no significant difference compared with baseline(P > 0.05). In CIH group, 20 pmol ET-1 markedly increased the LVDP from isolated hearts(P < 0.05).7 Effects of CIH on histological changes in coronary arteries of ratsCompared with normoxia group, coronary arteries in CIH rats exhibited obvious injury in endothelial layer and smooth muscle cells.8 Effects of CIH on expression of ET-1, ETA and ETB in coronary arteriesCoronary arteries in CIH rats showed a strong positive staining pattern of ET-1 on the endothelial layer and smooth muscle cell layer compared with than normoxia rats(P < 0.05). Positive ETA receptors staining was predominantly distribute in the smooth muscle cells of coronary arties from normoxia rats, which increased after CIH exposure(P < 0.05). Coronary vessels from normoxia and CIH rats showed a similar positive staining pattern of ETB on the smooth muscle cell layer. However, CIH treated vessels showed very light staining for ETB located on endothelium compared with normoxia group(P < 0.05).9 Effect of CIH on Rat coronary arteries NO production and e NOS... |
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