The effect of delayed preconditioning on connexin 43 in ischemic myocardium.

The objective of this study is to investigate the effects of preconditioning on the restoration and distribution of connexin 43 (Cx43) in ischemic myocardium in dogs. In this study, 40 dogs were randomly divided into 5 groups of 8 as follows: control, 0hI-R (ischemia followed by 0 h reperfusion), 6hI-R (ischemia followed by 6 h reperfusion), 24hI-R (ischemia followed by 24 h reperfusion), and 48hI-R (ischemia followed by 48 h reperfusion). Four dogs in each group were preconditioned with brief episodes of ischemia prior to the respective treatments and were referred as the PC groups, while the other 4 were not preconditioned and were referred as the nonPC groups. The myocardial ischemia was induced by ligation of the left anterior descending coronary artery. The expression and distribution of Cx43 within the ischemic myocardium were measured by Western blot analysis and studied using laser confocal microscopy using a double-label immunohistochemistry technique. Compared with the control group, there was a significant reduction in Cx43 content within ischemic myocardium of all test groups both with and without PC (P < 0.01, P < 0.05). Within the 0hI-R, 6hI-R, and 24hI-R groups, an insignificant difference was found in the expression and distribution of Cx43 within the ischemic region between the PC and the nonPC groups. However, in the 48hI-R group, the area and intensity of Cx43 staining within the ischemic region of the PC dogs were significantly larger and more intense than those of the nonPC dogs (P < 0.01), and the ratio of Cx43 pixel density in intercalated disk areas to that in side-to-side junction areas in the PC dogs was significantly greater than that in nonPC dogs (P < 0.01). Our results suggest that preconditioning has a significant effect on the restoration and distribution of Cx43 in the ischemic myocardium in dogs after 48 h. Hence, preconditioning may be a plausible cause for the observed reductions in cardiac arrhythmias.     

Autophagy in ischemic preconditioning and hibernating myocardium

Autophagy is a major protective mechanism and has been identified in response to hypoxia and more recently, myocardial ischemia, but it is not known whether it is involved in mediating ischemic preconditioning, the most powerful intervention known to protect myocardium against lethal ischemic injury. We examined autophagy in several models of preconditioning induced by 6 repetitive episodes of ischemia every 12 hours versus classical first or second-window preconditioning in swine. The results indicated that autophagy is an important mechanism mediating cardioprotection following repetitive episodes of coronary stenosis or coronary occlusion, but less for traditional first or second window preconditioning.     

Activation of volume regulated chloride channels protects myocardium from ischemia/reperfusion damage in second-window ischemic preconditioning

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3(-/-)) mice and their age-matched heterozygous (Clcn3(+/-)) and wild-type (Clcn3(+/+)) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3(-/-) mice had significant slower heart rate (648±12 bpm) than Clcn3(+/+) littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3(-/-) mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3(+/+) (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3(+/-) mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3(-/-) littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3(-/-) myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.     

Noradrenaline dynamics in prolonged ischemia after ischemic preconditioning

AIM OF THE STUDY: To determine the effects of two-staged ischemic preconditioning on myocardial noradrenaline in prolonged ischemia and reperfusion. METHODS: Thirty-two male Wistar rats anesthetised with urethane randomly divided into 2 groups: group 1 (ischemic preconditioning group, n = 16), and group 2 (control, n = 16). Myocardial interstitial noradrenaline levels were measured using a microdialysis technique. Ischemic preconditioning was elicited by two episodes: 5 min of ischemia and 10 min of reperfusion. The intermittent occlusions were followed by prolonged occlusion (60 min) and reperfusion (60 min). RESULTS: An increase in interstitial noradrenaline was observed in 10 min of prolonged ischemia in group 2, and in 20 min in group 1. After 20 min of myocardial ischemia there was a significant difference between groups (p < 0.05) in interstitial noradrenaline levels. In control group, it was 60% higher. In reperfusion, noradrenaline levels decreased markedly in group 1. CONCLUSION: We suggest that ischemic preconditioning by two episodes: 5-min ischemia and 10-min reperfusion prevents excessive noradrenaline interstitial accumulation, perhaps, through protection of physiological uptake I carrier.     

The importance of hypothermia in the preconditioning increase of ischemic tolerance to global cerebral ischemia

Various types of preconditioning including the main hypoxia (hypoxic, circulatory, hematic/hypemic and tissue/histotoxic), agonists of adenosine A-receptors and openers of K(ATP)-channels induce a hypothermia. A-agonists act through A1-receptors, CoCl2 and NiCl2--via endogenous adenosine and activation by it A1-receptors. The developing hypothermia correlates with neuroprotective effect and is important, but not the only mechanism of tolerance increase to global ischemia. At the similar hypothermia the preconditioning effect excels more frequently an influence of external cooling.     

Redistribution of connexin43 in regional acute ischemic myocardium: influence of ischemic preconditioning

Connexins are known to play an essential role in the ischemic preconditioning (IP) of the heart; their functional role in this process, however, has not been clearly defined. For this reason, anesthetized rats were subjected to regional myocardial ischemia, with or without IP or reperfusion. In frozen sections of hearts, fluorescence immunohistochemical staining for connexin43 (Cx43) was performed. In contrast to undisturbed zones, tissue that had been subjected to ischemia revealed Cx43 immunostaining not only in the gap junctions but also in a conspicuous pattern in the free cellular membranes of the myocytes. In myocardium that was exposed to IP only, the ratio of immunofluorescence intensity in the free cellular membrane to that in the interior of the cell was 1.22 +/- 0.04 (ratio in non-ischemia-exposed area = 1.04 +/- 0.01). When 15 or 45 min of permanent ischemia followed IP, the effect became more evident (ratio = 1.31 +/- 0.03 and 1.46 +/- 0.03, respectively) and proved to be significantly greater than in the corresponding non-IP groups (ratio = 1.16 +/- 0.03 and 1.30 +/- 0.03, respectively, P < 0.01). Reperfusion led to an overall weakening of fluorescence intensities and a disappearance of the observed IP-specific differences. We conclude that IP initiates a redistribution of Cx43 from its natural position in the gap junctions toward the free plasma membrane, thereby improving the cell's chance of survival during the subsequent phase of prolonged ischemia by an unknown, supposedly gap junction-independent, mechanism.     

Role of cellular energetics in ischemia-reperfusion and ischemic preconditioning of myocardium

A short period of ischemia followed by reperfusion produces a state of affairs in which the cells' potential for surviving longer ischemia is enhanced. This is called ischemic preconditioning. The effects of preconditioning are also related to the reperfusion damage which ensues upon tissue oxygenation. The role of the cellular energy state in reperfusion damage remains an enigma, although ischemic preconditioning is known to trigger mechanisms which contribute to the prevention of unnecessary ATP waste. In some species up to 80% of ATP hydrolysis in ischemia can be attributed to mitochondrial F₁-F₀-ATPase (ATP synthase), and a role for its inhibitor protein (IF₁) in ATP preservation has been proposed. Although originally regarded as limited to large animals with a slow heart beat, inhibition by IF₁ is probably a universal phenomenon. Coincidentally with ATPase inhibition, the decline in cellular ATP slows down, but even so the difference in ATP concentration between preconditioned and non-conditioned hearts is still small at the final stages of a long ischemia, when the beneficial effect of preconditioning is observable, although the energy state during reperfusion remains low in hearts which do not recover.     

Cardio-protective determinants are conserved in aged human myocardium after ischemic preconditioning

Ischemic preconditioning (IPrec) improves post-ischemic dysfunctions of the myocardium along with activation of protein kinase C isozymes including PKCdelta. Moreover, expression of cardio-protective determinants can reduce ischemic damages. Because IPrec is limited in aged hearts, we assessed in an experimental model the impact of aging on PKCdelta and selected protective proteins in the preconditioned myocardium from adult (< or =55) and older (> or =70 years) humans. Adult myocardium showed PKCdelta up-regulation after IPrec along with improved post-ischemic contractility. Although there was no functional benefit, PKCdelta increased in older myocardium as well. Subsequent mRNA analyses demonstrated that IPrec stabilizes the mRNA expression of protective proteins (Hsp70, Bcl-2/-xL, IAPs) in both aging groups. Moreover, older hearts revealed increase in post-ischemic Hsp90beta. Our study indicates, that IPrec conserves the expression of cardio-protective determinants in aged hearts despite limited functional recovery.     

STAT-3 activation is necessary for ischemic preconditioning in hypertrophied myocardium

The JAK-STAT pathway is activated in the early and late phases of ischemic preconditioning (IPC) in normal myocardium. The role of this pathway and the efficacy of IPC in hypertrophied hearts remain largely unknown. We hypothesized that phosphorylated STAT-3 (pSTAT-3) is necessary for effective IPC in pressure-overload hypertrophy. Male Sprague-Dawley rats 8 wk after thoracic aortic constriction (TAC) or sham operation underwent echocardiography and Langendorff perfusion. Randomized hearts were subjected to 30 min of global ischemia and 120 min of reperfusion with or without IPC in the presence or absence of the JAK-2 inhibitor AG-490 (AG). Functional recovery and STAT activation were assessed. TAC rats had a 31% increase in left ventricular mass (1,347 +/- 58 vs. 1,028 +/- 43 mg, TAC vs. sham, P < 0.001), increased anterior and posterior wall thickness but no difference in ejection fraction compared with sham-operated rats. In TAC, IPC improved end-reperfusion maximum first derivative of developed pressure (+dP/dt(max); 4,648 +/- 309 vs. 2,737 +/- 343 mmHg/s, IPC vs. non-IPC, P < 0.05) and minimum -dP/dt (-dP/dt(min); -2,239 +/- 205 vs. -1,215 +/- 149 mmHg/s, IPC vs. non-IPC, P < 0.05). IPC increased nuclear pSTAT-1 and pSTAT-3 in sham-operated rats but only pSTAT-3 in TAC. AG in TAC significantly attenuated +dP/dt(max) (4,648 +/- 309 vs. 3,241 +/- 420 mmHg/s, IPC vs. IPC + AG, P < 0.05) and -dP/dt(min) (-2,239 +/- 205 vs. -1,323 +/- 85 mmHg/s, IPC vs. IPC + AG, P < 0.05) and decreased only nuclear pSTAT-3. In myocardial hypertrophy, JAK-STAT signaling is important in IPC and exhibits a pattern of STAT activation distinct from nonhypertrophied myocardium. Limiting STAT-3 activation attenuates the efficacy of IPC in hypertrophy.     

Resistance of the myocardium to ischemia and efficacy of myocardial preconditioning in experimental diabetes mellitus

Data on myocardial tolerance of ischemia in the animals with experimental diabetes are controversial. In our study, myocardial sensitivity to ischemia and infarction-limiting effect of ischemic preconditioning have been investigated in the in vivo rat model of myocardial infarction in alloxan-induced insulin-dependent diabetes mellitus. It has been shown that in 6 weeks after alloxan injection in the diabetic rats infarction size as determined by TTC staining was significantly smaller than in healthy controls (39.8 +/- 8.8 and 62.3 +/- 6.6%, respectively, p < 0.01). Also, occurrence of ischemic tachyarrhythmias was more rare in diabetic rats than in controls. A single episode of ischemic preconditioning in diabetic rats showed a much lesser protection against infarction than in controls. Therefore, the data obtained support the existence of endogenous protective myocardial phenotype in diabetes, although the effectiveness of ischemic preconditioning in diabetes is reduced.     

预热处理对缺血心肌的保护作用及蛋白激酶C信号转导通路机制

缺血心肌的保护一直是心血管研究领域的热点问题。目前,除了继续从外源性药物途径进行研究外,有一个崭新的领域越来越受到人们的重视,即通过心肌细胞本身的内源性抗损伤能力,产生自身保护作用来减轻缺血的损害。研究发现,适当的预处理可以有效调动该保护机制。本文就近年来发展较快的预热处理(HP)对缺血心肌的保护作用及其蛋白激酶C(PKC)信号转导通路机制作一简要概述。     

Effect of ischemic preconditioning on free radical centers of the isolated rat heart during ischemia and early reperfusion

The effect of ischemic preconditioning on the free-radical state of isolated rat myocardium fixed by rapid freezing at the 25th min of normothermic total ischemia and the 3rd min of reperfusion was studied by the EPR method. It was shown that EPR spectra registered at -40 degrees C consist of two free-radical signals: of the semireduced forms of ubiquinone and flavine coynzymes. It was found that during ischemia and at the beginning of reperfusion, the preconditioning results in a narrowing of the spectra (as compared with control) due to an increase in the narrow ubisemiquinone EPR signal portion, and a decrease in the total concentration of free-radical centers: by 16% in the case of ischemia, and 23% in the case of reperfusion. It was concluded that in both cases the changes were due to a decrease in the concentration of myocardial flavosemiquinones as a result of ischemic preconditioning. We registered the microvawe power saturation curves for these two stages, which corresponded to control and ischemic preconditioning. In the case of ischemia these dependences had similar shapes; however, in the case of reperfusion they differ from each other due to changes in the relative intensities of the EPR signals from ubisemiquinone and flavosemiquinones in the integral myocardial free-radical spectra.     

肢体缺血预处理减轻大鼠海马缺血/再灌注损伤

目的:探讨肢体缺血预处理(LIP)对大鼠全脑缺血/再灌注损伤的影响.方法: 36只大鼠椎动脉凝闭后随机分为假手术(Control)组、脑缺血组、肢体缺血组、LIP 0 d组(LIP后即刻行脑缺血)、LIP 1 d组(LIP后1 d行脑缺血)和LIP 2 d组(LIP后2 d行脑缺血).重复夹闭大鼠双侧股动脉3次(每次10 min,间隔10 min)作为LIP,夹闭颈总动脉进行全脑缺血8 min后再灌注.硫堇染色观察海马CA1区组织学分级及锥体神经元密度以判断海马损伤程度.结果:脑缺血组海马CA1区锥体神经元损伤严重,与Control组比较,组织学分级明显升高,神经元密度明显降低(P<0.01).LIP 0 d组海马CA1区神经元损伤较脑缺血组明显减轻,组织学分级明显降低,神经元密度明显升高(P<0.01).而LIP 1 d组和LIP 2 d组大鼠海马CA1区锥体细胞缺失较多,仍有明显的组织损伤.结论:LIP可减轻随后立即发生的脑缺血/再灌注损伤,但对间隔1 d后的脑缺血/再灌注损伤无显著对抗作用.     

Cardiac interstitial bradykinin release during ischemia is enhanced by ischemic preconditioning

Ischemic preconditioning is known to protect the myocardium from ischemia-reperfusion injury. We examined the transmural release of bradykinin during myocardial ischemia and the influence of ischemic preconditioning on bradykinin release during subsequent myocardial ischemia. Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery in anesthetized cats. Cardiac microdialysis was performed by implantation and perfusion of dialysis probes in the epicardium and endocardium. In eight animals, bradykinin release was greater in the endocardium than in the epicardium (14.4 +/- 2.8 vs. 7.3 +/- 1.7 ng/ml, P < 0.05) during 30 min of ischemia. In seven animals subjected to preconditioning, myocardial bradykinin release was potentiated significantly from 2.4 +/- 0.6 ng/ml during the control period to 23.1 +/- 2.5 ng/ml during 30 min of myocardial ischemia compared with the non-preconditioning group (from 2.7 +/- 0.6 to 13.4 +/- 1.9 ng/ml, P < 0.05, n = 6). Thus this study provides further evidence that transmural gradients of bradykinin are produced during ischemia. The results also suggest that ischemic preconditioning enhances bradykinin release in the myocardial interstitial fluid during subsequent ischemia, which is likely one of the mechanisms of cardioprotection of ischemic preconditioning.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

缺血预处理对缺血再灌注后兔脊髓磷酸腺苷代谢的影响

目的：研究缺血参处理对缺血再灌注后兔脊髓磷酸腺苷代谢的影响。方法：往置入腹主动脉的Swan-Ganz导管气囊内注气造成兔腰髓缺血模型。将实验兔分为假手术组、缺血组和预处理组。应用反相高效液相色谱方法（reverse phase HPLC),对缺血再灌注后不同时间点腰髓组织中磷酸腺苷（ATP、ADP、AMP）的含量进行检测。结果：和假手术组相比,缺血组兔再灌后各时间点腰髓组织ATP含量有明显下降（P＜0．01）。与缺血组相应时间点相比,预处理组兔再灌注后腰髓组织ATP含量明显提高（P＜0．01）。结论：缺血预处理显著提高缺血再灌注后兔脊髓组织ATP含量,这可能是缺血预处理对脊髓缺血再灌注损伤产生保护作用的机制之一。