Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

Preconditioning in the absence or presence of sustained ischemia modulates myocardial Cx43 protein levels and gap junction distribution

In the heart, brief repeated episodes of ischemia prior to a sustained occlusion (ischemic preconditioning; PC) significantly delay the onset of necrosis and arrhythmogenesis. Ischemia has been reported to influence gap junction organization and connexin43 (Cx43) content, but whether PC affects these structures is not known. We investigated the effect of PC (2 cycles of 5-min ischemia plus 10-min reperfusion) followed by prolonged reperfusion without concomitant regional coronary occlusion on the myocardial Cx43 content and its spatial distribution in rabbit hearts. We also compared the effect of sustained ischemia with or without PC on Cx43 spatial distribution. In experiments with PC only, there was an initial decrease in Cx43 levels within the ischemic zone followed by a progressive increase after 48 h reperfusion. End-to-end immunolabeling of Cx43 was augmented in the ischemic region between 24 and 48 h reperfusion; labeling was not uniquely confined to myocyte abutments, but was also dispersed along the sarcolemma. Cx43 immunolabelling was more intense and diffuse in hearts subjected to PC before sustained coronary occlusion (compared to non-PC). These data indicate that gap junctions are significantly altered during brief episodes of ischemia. Reorganization of the gap junction complex could contribute to PC-mediated reductions in cardiac arrhythmias.     

No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning

Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1alpha, PP2Aalpha, and PP2Balpha and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of G?ttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 +/- 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 +/- 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2Aalpha and PP1alpha, but not PP2Balpha, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2Aalpha but not with PP1alpha. Although the total PP2Aalpha immunoreactivity (confocal laser scan) was increased to 154 +/- 24% and 194 +/- 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2Aalpha coimmunoprecipitated with Cx43 remained unchanged. Only PP2Aalpha coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2Aalpha is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.     

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.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.     

Selenium status as determinant of connexin-43 dephosphorylation in ex vivo ischemic/reperfused rat myocardium.

Recent studies have demonstrated that electrical uncoupling at gap junctions during ischemia is associated with cardiac Connexin-43 (Cx43) dephosphorylation. Whether oxidative stress is involved in this phenomenon still remains unclear. In the present study, we examined the influence of selenium intake on reperfusion-induced Cx43 dephosphorylation. Male Wistar rats were fed a diet containing either 0.05 mg/kg (Low-Se, n = 13) or 1.5 mg/kg (High-Se, n = 11) selenium for 8 weeks. At the end of this diet, hearts were isolated and subjected to 10 min regional ischemia followed by 10 min reperfusion. The level of dephosphorylated Cx43 was determined in tissue samples from ischemic/reperfused and non-ischemic regions of the hearts. At the end of the experiemental diet, the activity of the antioxidant enzyme glutathione peroxidase (GSH-Px) was increased in high-Se hearts compared with low-Se hearts (+ 13%; p < 0.05). After ischemia/reperfusion, in low-Se hearts, Cx43 dephosphorylation appeared significantly increased in the left ventricle compared to the non-ischemic right ventricle (+ 149%; p < 0.05). The high-Se diet significantly reduced Cx43 dephosphorylation in the left ventricle (p < 0.05 vs. low-Se diet). In conclusion, our results suggest that oxidative stress may be involved in Cx43 dephosphorylation during myocardial ischemia/reperfusion, thereby contributing to arrhythmogenesis.     

Cholesterol diet leads to attenuation of ischemic preconditioning-induced cardiac protection: the role of connexin 43

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.     

Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43

Connexin 43 (Cx43) is localized at left ventricular (LV) gap junctions and in cardiomyocyte mitochondria. A genetically induced reduction of Cx43 as well as blockade of mitochondrial Cx43 import abolishes the infarct size (IS) reduction by ischemic preconditioning (IP). With progressing age, Cx43 content in ventricular and atrial tissue homogenates is reduced. We now investigated whether or not 1) the mitochondrial Cx43 content is reduced in aged mice hearts and 2) IS reduction by IP is lost in aged mice hearts in vivo. Confirming previous results, sarcolemmal Cx43 content was reduced in aged (>13 mo) compared with young (<3 mo) C57Bl/6 mice hearts, whereas the expression levels of protein kinase C epsilon and endothelial nitric oxide synthase remained unchanged. Also in mitochondria isolated from aged mice LV myocardium, Western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. In young mice hearts, IP by one cycle of 10 min ischemia and 10 min reperfusion reduced IS (% of area at risk) following 30 min regional ischemia and 120 min reperfusion from 67.7 +/- 3.3 (n = 17) to 34.2 +/- 6.6 (n = 5, P < 0.05). In contrast, IP's cardioprotection was lost in aged mice hearts, since IS in nonpreconditioned (57.5 +/- 4.0, n = 10) and preconditioned hearts (65.4 +/- 6.3, n = 8, P = not significant) was not different. In conclusion, mitochondrial Cx43 content is decreased in aged mouse hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection by IP.     

Protection afforded by ischemic preconditioning is not mediated by effects on cell-to-cell electrical coupling during myocardial ischemia-reperfusion

The end-effectors of ischemic preconditioning (IPC) are not well known. It has been recently shown that transgenic mice underexpressing the gap junction protein connexin43 (Cx43) cannot be preconditioned. Because gap junctions allow spreading of cell death during ischemia-reperfusion in different tissues, including myocardium, we hypothesized that the protection afforded by IPC is mediated by effects on gap junction-mediated intercellular communication. To test this hypothesis, we analyzed the effect of IPC (5 min ischemia-5 min reperfusion x 2) on the changes in electrical impedance (four electrode probe) and impulse propagation velocity (transmembrane action potential) induced by ischemia (60 min) and reperfusion (60 min) in isolated rat hearts. IPC (n = 8) reduced reperfusion-induced lactate dehydrogenase release by 65.8% with respect to control hearts (n = 9) (P = 0.04) but had no effect on the time of onset of rigor contracture (increase in diastolic tension), electrical uncoupling (sharp changes in tissue resistivity and phase angle in impedance recordings), or block of impulse propagation during ischemia. Normalization of electrical impedance during reperfusion was also unaffected by IPC. The lack of effect of IPC on ischemic rigor contracture and on changes in tissue impedance during ischemia-reperfusion were validated under in vivo conditions in pigs submitted to 48 min of coronary occlusion and 120 min of reperfusion. IPC (n = 12) reduced infarct size (triphenyltetrazolium) by 64.9% (P = 0.01) with respect to controls (n = 17). We conclude that the protection afforded by IPC is not mediated by effects on electrical coupling. This result is consistent with recent findings suggesting that Cx43 could have effects on cell survival independent on changes in cell-to-cell communication.     

Hypercholesterolemia Abrogates the Cardioprotection of Ischemic Postconditioning in Isolated Rat Heart: Roles of Glycogen Synthase Kinase-3β and the Mitochondrial Permeability Transition Pore

Ischemic postconditioning (IPO) reduces lethal reperfusion injury under normal conditions, but its effectiveness in hypercholesterolemia (HC) is disputed. We measured the cardioprotection of IPO in hypercholesterolemic rats and determined the roles of glycogen synthase kinase-3β (GSK-3β) and the mitochondrial permeability transition pore (mPTP). Isolated rat hearts underwent 30-min global ischemia and 120-min reperfusion. Postconditioning protocol induced six cycles of 10s ischemia and 10s reperfusion at the onset of the reperfusion. Myocardial infarct size was estimated by triphenyltetrazolium chloride staining and cardiomyocyte apoptosis was assessed by TUNEL staining. GSK-3β phosphorylation was measured by immunoblotting. The opening of mPTP was measured by NAD⁺ content in myocardium. In normocholesterolemia (NC) groups, infarct size and cardiomyocyte apoptosis were significantly reduced after IPO. These reductions were completely abolished by HC, as evidenced by a similar infarct size and cardiomyocyte apoptosis observed between the IPO-HC and IR (ischemia–reperfusion)-HC groups. GSK-3β phosphorylation was significantly higher in the IPO-NC than the IPO-HC group. In addition, NAD⁺ content in myocardium, a marker of mPTP opening, was higher in the IPO-NC group than the IPO-HC group. In conclusion, cardioprotection of IPO is blocked by hypercholesterolemia. This might be due to the impairment of phosphorylation of GSK-3β and attenuation of mPTP opening.     

Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart

Brief, nonlethal episodes of ischemia in the mammalian heart provide cardioprotection against the detrimental effects of a longer duration ischemia. The manifestation of this preconditioning (PC) phenomenon is initiated by the enhanced phosphorylation state of signal transduction proteins. We reported previously that PC is decreased in the aged rat myocardium. Although the mechanism responsible for this loss is not understood, a reduction in the phosphorylation of critical proteins associated with PC may be postulated. Experiments were conducted to investigate whether PC in the aged heart can be restored with the inhibition of endogenous protein phosphatases thereby enhancing phosphorylation of signaling proteins. Levels of phosphatase activities were also assessed with adult heart aging. Hearts from young adult (3-4 mo.) and aged (21-22 mo.) Fischer-344 rats were perfused in the presence or absence of okadaic acid (OKA; 0.1 microM). Aged adult hearts were either not preconditioned or were preconditioned with two PC cycles (5 min ischemia/5 min reperfusion). Myocardial cellular death that developed with a subsequent ischemia was determined with triphenyltetrazolium. With PC, 55% of the aged heart after ischemia was no longer viable. OKA administered before or after ischemia reduced this ischemia-induced cellular death by 29%. Without PC, OKA reduced viability 18% only when present before and after the ischemic episode. OKA in the ischemic young heart during reperfusion reduced the loss of viability 31%. The Protein Phosphatase 2A (PP2A) activity was found to be up to 82% greater in ventricular myocardium of aged rats. In conclusion, aging-induced changes in protein dephosphorylation may be one mechanism reducing the manifestation of preconditioning in the aged heart.     

Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3 beta

The aim of this study was to determine whether erythropoietin (EPO) affords additional cardioprotection to the preconditioned myocardium by enhanced phosphorylation of Akt, STAT3, or glycogen synthase kinase-3beta (GSK-3 beta). Preconditioning (PC) with 5-min ischemia/5-min reperfusion and EPO (5,000 U/kg iv) reduced infarct size (as % of area at risk, %IS/AR) after 20-min ischemia in rat hearts in situ from 56.5 +/- 1.8% to 25.2 +/- 2.1% and to 36.2 +/- 2.8%, respectively. PC-induced protection was significantly inhibited by a protein kinase C inhibitor, chelerythrine (5 mg/kg), and slightly blunted by a phosphatidylinositol-3-kinase inhibitor, wortmannin (15 microg/kg). The opposite pattern of inhibition was observed for EPO-induced protection. The combination of PC and EPO further reduced %IS/AR to 8.9 +/- 1.9%, and this protection was inhibited by chelerythrine and wortmannin. The additive effects of PC and EPO on infarct size were mirrored by their effects on the level of phosphorylated GSK-3 beta at 5 min after reperfusion but not their effects on the level of phospho-Akt or phospho-STAT3. To mimic phosphorylation-induced inhibition of GSK-3 beta activity, SB-216763 (SB), a GSK-3 beta inhibitor, was administered before ischemia or 5 min before reperfusion. Infarct size was significantly reduced by preischemic injection (%IS/AR = 40.4 +/- 2.2% by 0.6 mg/kg SB and 34.0 +/- 1.8% by 1.2 mg/kg SB) and also by prereperfusion injection (%IS/AR = 32.0 +/- 2.0% by 1.2 mg/kg SB). These results suggest that EPO and PC afford additive infarct size-limiting effects by additive phosphorylation of GSK-3beta at the time of reperfusion by Akt-dependent and -independent mechanisms.     

Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits

Nicorandil has been shown to induce an infarct-limiting effect similar to that induced by the early phase of ischemic preconditioning (PC). The goals of this study were to determine whether nicorandil induces a delayed cardioprotection that is analogous to the late phase of ischemic PC and, if so, whether nicorandil-induced late PC is associated with upregulation of cardioprotective proteins. Chronically instrumented, conscious rabbits received vehicle (intravenous normal saline; control group, n = 10), nicorandil (100 microg/kg bolus + 30 microg x kg(-1) x min(-1) i.v. for 60 min; nicorandil group, n = 10), or ischemic PC (6 cycles of 4-min coronary occlusion/4-min reperfusion; PC group, n = 8). Twenty-four hours later, rabbits underwent a 30-min coronary occlusion, followed by 3 days of reperfusion. Myocardial infarct size was significantly reduced in rabbits pretreated with nicorandil (27.5 +/- 5.3% of the risk region) or with ischemia (30.3 +/- 4.2%) versus controls (59.1 +/- 4.7%, P < 0.05 vs. both). Furthermore, the expression of cyclooxygenase-2 (COX-2) and Bcl-2 was significantly elevated (+38% and +126%, respectively; P < 0.05) in myocardium of rabbits given nicorandil 24 h earlier versus controls. We conclude that nicorandil induces delayed cardioprotection against myocardial infarction similar to that afforded by the late phase of ischemic PC, possibly by upregulating COX-2 and Bcl-2.     

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.     

beta1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.     

Cardiac preconditioning with 4-h, 17 degrees C ischemia reduces [Ca(2+)](i) load and damage in part via K(ATP) channel opening

Brief ischemia before normothermic ischemia protects hearts against reperfusion injury (ischemic preconditioning, IPC), but it is unclear whether it protects against long-term moderate hypothermic ischemia. We explored in isolated guinea pig hearts 1) the influence of two 2-min periods of normothermic ischemia before 4 h, 17 degrees C hypothermic ischemia on cardiac cytosolic [Ca(2+)], mechanical and metabolic function, and infarct size, and 2) the potential role of K(ATP) channels in eliciting cardioprotection. We found that IPC before 4 h moderate hypothermia improved myocardial perfusion, contractility, and relaxation during normothermic reperfusion. Protection was associated with markedly reduced diastolic [Ca(2+)] loading throughout both hypothermic storage and reperfusion. Global infarct size was markedly reduced from 36 +/- 2 (SE)% to 15 +/- 1% with IPC. Bracketing ischemic pulses with 200 microM 5-hydroxydecanoic acid or 10 microM glibenclamide increased infarct size to 28 +/- 3% and 26 +/- 4%, respectively. These results suggest that brief ischemia before long-term hypothermic storage adds to the cardioprotective effects of hypothermia and that this is associated with decreased cytosolic [Ca(2+)] loading and enhanced ATP-sensitive K channel opening.     

First molecular evidence that inositol trisphosphate signaling contributes to infarct size reduction with preconditioning

Considerable attention has focused on the role of protein kinase C (PKC) in triggering the profound infarct-sparing effect of ischemic preconditioning (PC). In contrast, the involvement of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], the second messenger generated in parallel with the diacylglycerol-PKC pathway, remains poorly understood. We hypothesized that, if Ins(1,4,5)P(3) signaling [i.e., release of Ins(1,4,5)P(3) and subsequent binding to Ins(1,4,5)P(3) receptors] contributes to PC-induced cardioprotection, then the reduction of infarct size achieved with PC would be attenuated in mice that are deficient in Ins(1,4,5)P(3) receptor protein. To test this concept, hearts were harvested from 1) B6C3Fe-a/a-Itpr-1(opt+/-)/J mutants displaying reduced expression of Ins(1,4,5)P(3) receptor-1 protein, 2) Itpr-1(opt+/+) wild types from the colony, and 3) C57BL/6J mice. All hearts were buffer-perfused and randomized to receive two 5-min episodes of PC ischemia, pretreatment with d-myo-Ins(1,4,5)P(3) [sodium salt of native Ins(1,4,5)P(3)], the mitochondrial ATP-sensitive K(+) channel opener diazoxide, or no intervention (controls). After the treatment phase, all hearts underwent 30-min global ischemia followed by 2 h of reperfusion, and infarct size was delineated by tetrazolium staining. In both wild-type and C57BL/6J cohorts, area of necrosis in hearts that received PC, d-myo-Ins(1,4,5)P(3), and diazoxide averaged 28-35% of the total left ventricle (LV), significantly smaller than the values of 52-53% seen in controls (P < 0.05). In contrast, in Itpr-1(opt+/-) mutants, protection was only seen with diazoxide: neither PC nor d-myo-Ins(1,4,5)P(3) limited infarct size (52-58% vs. 56% of the LV in mutant controls). These data provide novel evidence that Ins(1,4,5)P(3) signaling contributes to infarct size reduction with PC.     

PI3K/Akt信号通路在白藜芦醇抗大鼠缺血/再灌注性心律失常中的作用及机制

目的：探讨磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt）信号通路在白藜芦醇抗缺血/再灌注性心律失常中的作用及机制。方法：48只健康雄性SD大鼠,取心电图正常者随机分为4组（n=10）：假手术（SC组）组、缺血/再灌注（I/R组）组、白藜芦醇处理（Res处理组）组、PI3K抑制剂LY294002（LY294002组）组。建立大鼠在体心肌缺血/再灌注模型,观察各组心律失常的发生情况及左室血流动力学变化,Western blot法测定心肌组织中蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）、缝隙连接蛋白43（Cx43）蛋白表达水平,以RT-PCR法从转录水平检测Cx43的表达水平。结果：与I/R组相比,Res处理组心律失常的发生率（心律失常评分）明显降低、左室舒缩功能明显升高,同时心肌Akt、Cx43蛋白表达及Cx43mRNA水平也明显升高;使用PI3K抑制剂LY294002后,心肌Akt、Cx43蛋白表达及Cx43mRNA水平下降的同时心律失常的发生率明显升高、左室舒缩功能明显降低。结论：白藜芦醇的抗再灌注性心律失常作用可能是通过激活PI3K/Akt信号通路,改变Cx43活性及分布实现的。     

Late preconditioning enhances recovery of myocardial function after infarction in conscious rabbits

It is unknown whether late preconditioning (PC) enhances the recovery of left ventricular (LV) function after a myocardial infarction. Thus 25 conscious rabbits were subjected to a 30-min coronary occlusion followed by 28 days of reperfusion after PC 24 h earlier with either ischemia or nitric oxide donor administration [S-nitroso-N-acetylpenicillamine (SNAP)]. The recovery of wall thickening (WTh) after reperfusion was significantly improved in the ischemic PC and SNAP PC groups compared with controls, both at rest and during dobutamine stress. Interestingly, neither ischemia- nor SNAP-induced late PC attenuated myocardial stunning from day 1 through day 14. Infarct size was smaller in the ischemic PC and SNAP PC groups compared with controls. In all groups, WTh at 28 days was positively and linearly related to the percentage of viable tissue in the region underlying the ultrasonic crystal (r = 0.90), indicating that the improvement in LV function after both ischemia-induced and NO donor-induced late PC can be fully explained by the reduction in infarct size; a separate effect of late PC on LV remodeling or LV contractility need not be invoked. In conclusion, in conscious rabbits late PC, induced either by ischemia or pharmacologically, not only limits infarct size but also enhances the recovery of LV function after myocardial infarction. This finding has important clinical implications and provides triphenyltetrazolium chloride-independent evidence that late PC limits myocellular death after sustained ischemia.     

Fluctuations of Contraction Rhythm During Simulated Ischemia/Reperfusion in Cultured Cardiac Myocytes from Neonatal Rats

Cardiac ischemia results in a rapid decrease of intracellular pH and in the rise of intracellular Ca 2+ , changes that have been shown to reduce intercellular communication via gap junctions (GJ) between cardiac myocytes. Ischemia also results in electrical instability probably caused by the reduced GJ permeability contributing to an increased vulnerability to arrhythmias. This study aims at elucidating whether the fluctuations of contraction rhythm of spontaneously beating cardiac myocytes in culture changes during simulated ischemia/reperfusion. The coefficient of variation (CV) of contraction intervals, reflecting the fluctuation of contraction rhythm, increased significantly during simulated ischemia/reperfusion. However, the contraction rhythm of the cardiac myocytes in an aggregate remained synchronized during simulated ischemia/reperfusion. In contrast, pharmacological blockade of GJ with 12-doxyl stearic acid, a blocker of GJ permeability, resulted in the de-synchronization of contraction rhythm and in an increase in the CV of contraction intervals in normoxic conditions. The present findings lead to the suggestion that GJ remained open during simulated ischemia/reperfusion, and that a mechanism other than electrical uncoupling between myocytes contributed to the observed increase in the fluctuation of beating rhythm during ischemia.