MCC-134, a blocker of mitochondrial and opener of sarcolemmal ATP-sensitive K+ channels, abrogates cardioprotective effects of chronic hypoxia

We examined the effect of MCC-134, a novel inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels and activator of sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels, on cardioprotection conferred by adaptation to chronic hypoxia. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 5-6 weeks) and susceptibility of their hearts to ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion on the day after the last hypoxic exposure. MCC-134 was administered intravenously 10 min before ischemia and 5 min before reperfusion in a total dose of 0.3 mg/kg or 3 mg/kg divided into two equal boluses. The infarct size (tetrazolium staining) was reduced from 59.2+/-4.4 % of the area at risk in normoxic controls to 43.2+/-3.3 % in the chronically hypoxic group. Chronic hypoxia decreased the reperfusion arrhythmia score from 2.4+/-0.5 in normoxic animals to 0.7+/-0.5. Both doses of MCC-134 completely abolished the antiarrhythmic protection (score 2.4+/-0.7 and 2.5+/-0.5, respectively) but only the high dose blocked the infarct size-limiting effect of chronic hypoxia (54.2+/-3.7 %). MCC-134 had no effect in the normoxic group. These results support the view that the opening of mitoKATP channels but not sarcKATP channels plays a crucial role in the mechanism by which chronic hypoxia improves cardiac tolerance to ischemia/reperfusion injury.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.     

Effect of prenatal hypoxia on heat stress-mediated cardioprotection in adult rat heart

Fetal programming has profound effects on cardiovascular function in later adult life. We tested the hypothesis that chronic hypoxic exposure during fetal development downregulates endogenous cardioprotective mechanisms in adult rats. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O2 from days 15 to 21 of gestation) groups. The male progeny were studied at 2 mo of age. Rats were subjected to heat stress (42 degrees C for 15 min). After 24 h, hearts were excised and subjected to 30 min of global ischemia and 1 h of reperfusion. Prenatal hypoxia did not change adult rat body weight and heart weight, but significantly increased the cross-sectional area of a left ventricular (LV) myocyte. Heat stress significantly improved postischemic recovery of LV function in normoxic control rats, but not in prenatally hypoxic rats. The infarct size in the LV resulting from ischemia-reperfusion was reduced by the heat stress pretreatment in control rats, but not in prenatally hypoxic rats. In accordance, heat stress significantly increased LV myocardial content of heat shock protein 70 only in normoxic control rats. In addition, there was a significant decrease in the LV myocardial content of the PKC-epsilon isoform in prenatally hypoxic rats compared with control rats. We conclude that prenatal hypoxia causes in utero programming of hsp70 gene in the LV, leading to an inhibition of its response to heat stress and a loss of cardioprotection in later adult life.     

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia

Exposure to chronic hypoxia results in hypoxic pulmonary hypertension (HPH). In rats HPH develops during the first two weeks of exposure to hypoxia, then it stabilizes and does not increase in severity. We hypothesize that free radical injury to pulmonary vascular wall is an important mechanism in the early days of the hypoxic exposure. Thus antioxidant treatment just before and at the beginning of hypoxia should be more effective in reducing HPH than antioxidant therapy of developed pulmonary hypertension. We studied adult male rats exposed for 4 weeks to isobaric hypoxia (F(iO2) = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given "early" (7 days before and the first 7 days of hypoxia) or "late" (last two weeks of hypoxic exposure). These experimental groups were compared with normoxic controls and untreated hypoxic rats (3-4 weeks hypoxia). All animals kept in hypoxia had significantly higher mean pulmonary arterial blood pressure (PAP) than normoxic animals. PAP was significantly lower in hypoxic animals with early (27.1 +/- 0.9 mmHg) than late NAC treatment (30.5 +/- 1.0 mmHg, P < 0.05; hypoxic without NAC 32.6 +/- 1.2 mmHg, normoxic controls 14.9 +/- 0.7 mmHg). Early but not late NAC treatment inhibited hypoxia-induced increase in right ventricle weight and muscularization of distal pulmonary arteries assessed by quantitative histology. We conclude that release of free oxygen radicals in early phases of exposure to hypoxia induces injury to pulmonary vessels that contributes to their structural remodeling and development of HPH.     

Responses of chronically hypoxic rat hearts to ischemia: KATP channel blockade does not abolish increased RV tolerance to ischemia

Chronic hypoxia may precondition the myocardium and protect from ischemia-reperfusion damage. We therefore examined the recovery of left and right ventricular function after ischemia and reperfusion (15 min each) in isolated blood-perfused working hearts from normoxic (Norm) and hypoxic (Hypo; 14 days, 10.5% O(2)) adult rats. In addition, the mRNA expression of hypoxia-inducible factor (HIF)-1alpha and the protein expression of endothelial nitric oxide synthase (eNOS) were measured. Postischemic left ventricular function recovered to 66 +/- 6% and 67 +/- 5% of baseline in Norm and Hypo, respectively. In contrast, postischemic right ventricular function was 93 +/- 2% of baseline in Hypo vs. 67 +/- 3% in Norm (P < 0.05). Improved postischemic right ventricular function in Hypo (93 +/- 2% and 96 +/- 2% of baseline) was observed with 95% O(2) or 21% O(2) in the perfusate, and it was not attenuated by glibenclamide (5 and 10 micromol/l) (86 +/- 4% and 106 +/- 6% recovery). HIF-1alpha mRNA and eNOS protein expression were increased in both left and right hypoxic ventricles. In conclusion, postischemic right, but not left, ventricular function was improved by preceding chronic hypoxia. ATP-sensitive K(+) channels are not responsible for the increased right ventricular tolerance to ischemia after chronic hypoxia in adult rat hearts.     

Effect of neonatal hypoxia on the development of hepatic lipase in the rat

Increases in plasma lipids occur during hypoxia in suckling but not in weaned rats and may result from altered hepatic enzyme activity. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Hypoxia led to an increase in hepatic lipid content in the suckling rat only. Hepatic lipase was decreased to approximately 45% of control in 7-day-old rats exposed to hypoxia but not in hypoxic 35-day-old rats. Hypoxic suckling rats also had a 50% reduction in lactate dehydrogenase activity, whereas transaminase activity and CYP1A and CYP3A protein content were not different between hypoxic and normoxic groups. Additional rats were studied 7 and 14 days after recovery from hypoxic exposure from birth to 7 days of age; hepatic lipase activity had recovered to 85% by 7 days and to 100% by 14 days in the rats previously exposed to hypoxia. Administration of dexamethasone to neonatal rats to simulate the hyperglucocorticoid state found in hypoxic 7-day-old rats led to a moderate decrease ( approximately 75% of control) in hepatic lipases. Developmentally, in the normoxic state, hepatic lipases increased rapidly after birth and reached levels more than twofold that of the newborn by 7 days of age. Hypoxia delays the maturation of hepatic lipases. We suggest that the decrease in hepatic lipase activity contributes to hyperlipemia in the hypoxic newborn rats.     

Intermittent hypoxic training protects canine myocardium from infarction

This investigation examined cardiac protective effects of normobaric intermittent hypoxia training. Six dogs underwent intermittent hypoxic training for 20 consecutive days in a normobaric chamber ventilated intermittently with N2 to reduce fraction of inspired oxygen (FiO2) to 9.5%-10%. Hypoxic periods, initially 5 mins and increasing to 10 mins, were followed by 4-min normoxic periods. This hypoxia-normoxia protocol was repeated, initially 5 times and increasing to 8 times. The dogs showed no discomfort during intermittent hypoxic training. After 20 days of hypoxic training, the resistance of ventricular myocardium to infarction was assessed in an acute experiment. The left anterior descending (LAD) coronary artery was occluded for 60 mins and then reperfused for 5 hrs. At 30 mins of LAD occlusion, radioactive microspheres were injected through a left atrial catheter to assess coronary collateral blood flow into the ischemic region. After 5 hrs reperfusion, the heart was dyed to delineate the area at risk (AAR) of infarction and stained with triphenyl tetrazolium chloride to identify infarcted myocardium. During LAD occlusion and reperfusion, systemic hemodynamics and global left ventricular function were stable. Infarction was not detected in 4 hearts and was 1.6% of AAR in the other 2 hearts. In contrast, 6 dogs sham-trained in a chamber ventilated with compressed air and 5 untrained dogs subjected to the same LAD occlusion/reperfusion protocol had infarcts of 36.8% +/- 5.8% and 35.2% +/- 9.5% of the AAR, respectively. The reduction in infarct size of four of the six hypoxia-trained dogs could not be explained by enhanced collateral blood flow to the AAR. Hypoxia-trained dogs had no ventricular tachycardia or ventricular fibrillation. Three sham-trained dogs had ventricular tachycardia and two had ventricular fibrillation. Three untrained dogs had ventricular fibrillation. In conclusion, intermittent hypoxic training protects canine myocardium from infarction and life-threatening arrhythmias during coronary artery occlusion and reperfusion. The mechanism responsible for this potent cardioprotection merits further study.     

ATP-dependent potassium channels involved in the cardiac protection induced by intermittent hypoxia against ischemia/reperfusion injury

The aim of this study was to investigate the protection afforded by intermittent hypoxia (IH) against ischemia/reperfusion injury and its effects on calcium homeostasis during ischemia/reperfusion. The roles of KATP channels in these two actions were to be explored. Isolated hearts from IH and normoxic rats were subjected to 30 min global ischemia followed by 30 min reperfusion. Cardiac function was less deteriorated during ischemia and reperfusion in the IH rat hearts compared to normoxia rat hearts. Amplitude of the maximal contracture during ischemia was lower, while time to maximal contracture was extended in IH hearts. Post-ischemic recovery of left ventricular developed pressure and +/-dP/dtmax were higher in IH hearts than in normoxic hearts. KATP antagonist glibenclamide (10 microM) completely abolished these protective effects of IH, but had no appreciable influence on normoxic hearts. In cardiomyocytes isolated from normoxic hearts, [Ca2+]i, measured as arbitrary units of fluorescence ratio (340 nm/380 nm) of fura-2, gradually increased during 20 min simulated ischemia and kept at high level during 30 min reperfusion (1.081 +/- 0.004 and 1.088 +/- 0.006 respectively, p<0.01 vs pre-ischemia perfusion). However, in cardiomyocytes isolated from IH hearts, [Ca2+]i kept at normal level during ischemia and reperfusion (1.012 +/- 0.006 and 1.021 +/- 0.002 respectively, P>0.05 vs pre-ischemia perfusion). 10 microM glibenclamide and 100 microM 5-hydroxydecanoate (a selective mitochondria KATP antagonist) respectively abolished this effect of IH; calcium overloading reappeared during ischemia (1.133 +/- 0.007 and 1.118 +/- 0.007 respectively, P<0.01) and reperfusion (1.091 +/- 0.004 and 1.095 +/- 0.012 respectivly, P<0.01). However they had no effects on simulated ischemia and reperfusion-induced calcium overloading in normoxic myocytes. 50 microM pinacidil, a KATP opener, attenuated calcium overloading during ischemia and reperfusion in normoxic myocytes, but had no effect on [Ca2+]i change in IH myocytes. These results suggested that KATP channels contributed to the cardiac protection induced by IH against ischemia/reperfusion injury; the elimination of calcium overloading during ischemia/reperfusion by IH might underlie the mechanism of protection.     

Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice

Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h (protocol 1) or 30 days (protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca(2+) retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca(2+) retention capacity (132 +/- 13 and 153 +/- 31 vs. 53 +/- 16 nmol Ca(2+)/mg protein in control) and reduced infarct size to 35 +/- 4 and 32 +/- 7% of area at risk vs. 61 +/- 6% in control (P < 0.05). At 30 days, ejection fraction averaged 74 +/- 6 and 77 +/- 6% in postconditioned and Debio-025 groups, respectively, vs. 62 +/- 12% in the control group (P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.     

Differential role of PI3K/Akt pathway in the infarct size limitation and antiarrhythmic protection in the rat heart

Endogenous cardiac protection against prolonged ischemic insult can be achieved by repeated brief episodes of ischemia (hypoxia) or by cardiac adaptation to various stresses such as chronic hypoxia. Activation of phosphatidylinositol 3-kinase (PI3K)/Akt is involved in antiapoptotic effects, however, it is not clear whether it is required for overall heart salvage including protection against myocardial infarction and arrhythmias. We focussed on the potential common role of PI3K/Akt in anti-infarct protection, in the experimental settings of long-term adaptation to chronic intermittent hypobaric hypoxia (IHH; 8 h/day, 25–30 exposures, in vivo rats) and acute ischemic preconditioning (IP; Langendorff-perfused hearts). In addition, we explored the role of PI3K/Akt in susceptibility to ischemic ventricular arrhythmias. In normoxic open-chest rats, PI3K/Akt inhibitor LY294002 (LY; 0.3 mg/kg) given 5 min before test occlusion/reperfusion (I/R) did not affect infarct size (IS) normalized to the size of area at risk (AR). In hypoxic rats, LY partially attenuated IS-limiting effect of IHH (IS/AR 59.7 ± 4.1% vs. 51.8 ± 4.4% in the non-treated rats; p > 0.05) and increased IS/AR to its value in normoxic rats (64.9 ± 5.1%). In the isolated hearts, LY (5 μM) applied 15 min prior to I/R completely abolished anti-infarct protection by IP (IS/AR 55.0 ± 4.9% vs. 15.2 ± 1.2% in the non-treated hearts and 42.0 ± 5.5% in the non-preconditioned controls; p < 0.05). In the non-preconditioned hearts, PI3K/Akt inhibition did not modify IS/AR, on the other hand, it markedly suppressed arrhythmias. In the LY-treated isolated hearts, the total number of ventricular premature beats and the incidence of ventricular tachycardia (VT) was reduced from 518 ± 71 and 100% in the controls to 155 ± 15 and 12.5%, respectively (p < 0.05). Moreover, bracketing of IP with LY did not reverse antiarrhythmic effect of IP. These results suggest that activation of PI3K/Akt cascade plays a role in the IS-limiting mechanism in the rat heart, however, it is not involved in the mechanisms of antiarrhythmic protection.     

Increased expression and altered subcellular distribution of PKC-delta in chronically hypoxic rat myocardium: involvement in cardioprotection

We examined the role of protein kinase C (PKC) in the cardioprotective mechanism induced by long-term adaptation to chronic intermittent hypoxia. Adult male Wistar rats were exposed to hypobaric hypoxia of 7,000 m for 8 h/day, 5 days/wk; the total number of exposures was 24-32. A control group was kept under normoxic conditions. Western blot analysis of PKC isoforms-delta and -epsilon was performed in the cytosol and three particulate fractions of left ventricular myocardium. Infarct size was determined in open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion. The PKC inhibitors chelerythrine (1 or 5 mg/kg) or rottlerin (selective for PKC-delta isoform; 0.3 mg/kg) were administered intravenously as a single bolus 15 min before ischemia. Chronic hypoxia had no effect on the expression and distribution of PKC-epsilon. The relative amount of PKC-delta increased in the cytosol and nuclear-cytoskeletal, mitochondrial, and microsomal fractions of chronically hypoxic myocardium by 100%, 212%, 237%, and 146%, respectively, compared with corresponding normoxic values. Chronic hypoxia decreased the size of myocardial infarction (normalized to the area at risk) by about one-third on the average (P < 0.05). Both doses of chelerythrine tended to reduce infarction in controls, and only the high dose completely abolished the improvement of ischemic tolerance in hypoxic hearts (P < 0.05). Rottlerin attenuated the infarct size-limiting effect of chronic hypoxia (P < 0.05), and it had no effect in controls. These results suggest that chronic intermittent hypoxia-induced cardioprotection in rats is partially mediated by PKC-delta; the contribution of other isoforms remains to be determined.     

Cardioprotective effect of chronic hypoxia is blunted by concomitant hypercapnia

The effect of chronic hypercapnia on cardioprotection induced by chronic hypoxia was investigated in adult male Wistar rats exposed to isobaric hypoxia (10 % O(2)) for three weeks. In the first experimental group, CO(2) in the chamber was fully absorbed; in the second group, its level was increased to 4.1 %. Normoxic controls were kept in atmospheric air. Anesthetized open-chest animals were subjected to 20-min LAD coronary artery occlusion and 3-h reperfusion for infarct size determination (TTC staining). Chronic hypoxia alone reduced body weight and increased hematocrit; these effects were significantly attenuated by hypercapnia. The infarct size was reduced from 61.9+/-2.2 % of the area at risk in the normoxic controls to 44.5+/-3.3 % in the hypoxic group (P<0.05). Hypercapnia blunted the infarct size-limiting effect of hypoxia (54.8+/-2.4 %; P<0.05). It is concluded that increased CO(2) levels in the inspired air suppress the development of the chronic hypoxia-induced cardioprotective mechanism, possibly by interacting with ROS signalling pathways.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Role of oxidative stress in PKC-delta upregulation and cardioprotection induced by chronic intermittent hypoxia

The aim was to determine whether increased oxidative stress during the adaptation to chronic intermittent hypoxia (CIH) plays a role in the induction of improved cardiac ischemic tolerance. Adult male Wistar rats were exposed to CIH in a hypobaric chamber (7,000 m, 8 h/day, 5 days/wk, 24-30 exposures). Half of the animals received antioxidant N-acetylcysteine (NAC; 100 mg/kg) daily before the exposure; the remaining rats received saline. Control rats were kept under normoxia and treated in a corresponding manner. One day after the last exposure (and/or NAC injection), anesthetized animals were subject to 20 min of coronary artery occlusion and 3 h of reperfusion for determination of infarct size. In parallel subgroups, biochemical analyses of the left ventricular myocardium were performed. Adaptation to CIH reduced infarct size from 56.7 +/- 4.5% of the area at risk in the normoxic controls to 27.7 +/- 4.9%. NAC treatment decreased the infarct size in the controls to 42.0 +/- 3.4%, but it abolished the protection provided by CIH (to 41.1 +/- 4.9%). CIH decreased the reduced-to-oxidized glutathione ratio and increased the relative amount of PKC isoform-delta in the particulate fraction; NAC prevented these effects. The expression of PKC-epsilon was decreased by CIH and not affected by NAC. Activities of superoxide dismutase, catalase, and glutathione peroxidase were affected by neither CIH nor NAC treatment. It is concluded that oxidative stress associated with CIH plays a role in the development of increased cardiac ischemic tolerance. The infarct size-limiting mechanism of CIH seems to involve the PKC-delta-dependent pathway but apparently not the increased capacity of major antioxidant enzymes.     

热休克蛋白90在缺氧心肌细胞中表达的初步研究

观察热休克蛋白90(HSP90)在缺氧心肌细胞中的表达变化规律,并初步阐明HSP90在早期心肌缺氧损害中的作用。方法:原代培养SD大鼠乳鼠心肌细胞,随机分为正常对照组、缺氧组和加HSP90阻断剂格尔霉素(geldanamycin,GA)预处理后再缺氧组(GA 缺氧组),蛋白免疫印迹法(western blot)及间接免疫荧光法检测缺氧1、3、6、12、24h后心肌细胞中HSP90蛋白分布表达情况,并检测细胞上清中肌酸激酶同工酶(CK-MB)及乳酸脱氢酶(LDH)的含量变化。结果:心肌细胞缺氧3h后HSP90蛋白在胞浆内表达量升高,持续到12h达到峰值。与单纯缺氧组相比,GA 缺氧组中LDH,CK-MB含量在不同时相点均有显著升高。结论:缺氧早期即可引起心肌细胞胞浆中HSP90蛋白表达增强,HSP90可能在早期心肌缺氧损害中发挥其内源性抗损伤机制。     

Myocardial ischemia-reperfusion injury is enhanced in a model of systemic allergy and asthma

Despite epidemiological evidence of cardiovascular complications in asthmatics, the direct contribution of asthmatic pathophysiology to cardiovascular effects is unknown. Considering parallels in underlying pathophysiology, we tested the hypothesis that presence of systemic allergy and asthma worsens the outcome of myocardial ischemia-reperfusion injury. Systemic allergy and asthma were created in rabbits by repeated intraperitoneal injections of allergen with adjuvant, followed by an airway challenge in two groups. Nonsensitized animals served as controls. In situ myocardial ischemia-reperfusion was induced in anesthetized animals by a 30-min ligation of a coronary artery, followed by 3 h of reperfusion. Ischemia-reperfusion was done at 24 h after intraperitoneal boost (1 DB) and 7 days (7 DB) after the last intraperitoneal injection and at 24 h (1DAWCH) and 7 days (7DAWCH) after airway challenge. The infarct size (determined by 2,3,5-triphenyltetrazolium chloride staining, normalized to area at risk) was significantly higher in all sensitized groups compared with control (1DB, 31 +/- 4; 7DB, 28.9 +/- 2.6; 1DAWCH, 66.1 +/- 4.1; 7DAWCH, 28.9 +/- 9.2; control, 16.7 +/- 3.2; means +/- SE; P < 0.01 by ANOVA; n = 6). The 1DAWCH group showed significantly greater infarct than all other groups (P < 0.05). Myocardial neutrophil infiltration was significantly higher in the sensitized groups compared with control (P < 0.01). Tissue neutrophil counts showed a strong positive correlation to infarct sizes (r2 = 0.9). These observations indicate that the presence of systemic allergy and asthma is associated with increased myocardial neutrophil infiltration during acute ischemia-reperfusion and increased size of the resulting infarct.     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Hyperbaric oxygen preconditioning alleviates myocardial ischemic injury in rats

It has been shown that after ischemia-reperfusion, application of hyperbaric oxygen (HBO) reduces cardiac injury. In this study we tested the hypothesis that HBO preconditioning reduces injury to the ischemic myocardium. One hundred and eight adult male Sprague-Dawley rats (250-280 g) were randomly divided into four groups: normoxia + sham surgery (CS), normoxia + permanent occlusion of the left anterior descending (LAD) coronary artery (CMI), HBO preconditioning + sham surgery (HS), and HBO preconditioning + permanent LAD occlusion (HMI). Rats receiving HBO preconditioning were intermittently exposed to 100% O(2) at 2.5 atmosphere absolute (ATA) for 60 min, twice daily for 2 days followed by 12 hrs of recovery in room air prior to the myocardial ischemic insult induced by LAD ligation. Rats in the normoxia group were time-matched with the HBO group and maintained under normoxic conditions prior to LAD occlusion. At 3 and 7 days after LAD occlusion, heart function parameters were measured by inserting a catheter into the left ventricle, infarct size was calculated using the method of TTC staining, myocardial capillary density was determined by immunohistochemical staining with a monoclonal anti-CD(31)/PECAM-1 antibody, and VEGF protein level was determined by Western blot analysis. At 3 days after LAD ligation, the infarct size of the HMI group was significantly smaller than that of the CMI group (26 +/- 2.5% vs. 38 +/- 3%, P < 0.05). The heart function parameters including left ventricular systolic pressure (LVSP), +dP/dt(max) and -dP/dt(max) were significantly improved in the HMI group compared to the CMI group at 3 and 7 days after LAD occlusion. Capillary density and VEGF protein levels were significantly increased in the ischemic myocardium pre-exposed to HBO. We conclude that HBO preconditioning alleviates myocardial ischemia in rat model.     

Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 μM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by N(G)-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 μM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 μM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area (P < 0.001) and limited the postischemic myocardial contracture (P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.     

Suppression of ischemic and reperfusion ventricular arrhythmias by inhalational anesthetic-induced preconditioning in the rat heart

Inhalational anesthetic-induced preconditioning (APC) has been shown to reduce infarct size and attenuate contractile dysfunction caused by myocardial ischemia. Only a few studies have reported the effects of APC on arrhythmias during myocardial ischemia-reperfusion injury, focusing exclusively on reperfusion. Accordingly, the aim of the present study was to examine the influence of APC on ventricular arrhythmias evoked by regional no-flow ischemia. APC was induced in adult male Wistar rats by 12-min exposures to two different concentrations (0.5 and 1.0 MAC) of isoflurane followed by 30-min wash-out periods. Ventricular arrhythmias were assessed in the isolated perfused hearts during a 45-min regional ischemia and a subsequent 15-min reperfusion. Myocardial infarct size was determined after an additional 45 min of reperfusion. The incidence, severity and duration of ventricular arrhythmias during ischemia were markedly reduced by APC. The higher concentration of isoflurane had a larger effect on the incidence of ventricular fibrillation than the lower concentration. The incidence of ventricular tachycardia and reversible ventricular fibrillation during reperfusion was also significantly reduced by APC; the same was true for myocardial infarct size. In conclusion, we have shown that preconditioning with isoflurane confers profound protection against myocardial ischemia- and reperfusion-induced arrhythmias and lethal myocardial injury.