Role of insulin in the anti-apoptotic effect of glucose-insulin-potassium in rabbits with acute myocardial ischemia and reperfusion

OBJECTIVE: To study the effects of glucose-insulin-potassium (GIK) cocktail on cardiac myocyte apoptosis and cardiac functional recovery following myocardial ischemia/reperfusion (MI/R), and to further determine the role of insulin in the GIK-induced cardioprotective effect in vivo . METHODS: Forty eight male rabbits were subjected to 40 min MI followed by R for 3 h and were randomly received one of the following treatments: saline, GIK (glucose: 150 g/L, insulin: 60 U/L and KCl: 80 mmol/L), or insulin (n = 16 in each group) at 1 ml x kg(-1) x h(-1), beginning 30 min before MI and continuing throughout the 3 h-reperfusion. Blood glucose, electrolytes, arterial blood pressure and left ventricular pressure (LVP) were monitored throughout the experiment. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activity were measured spectrophotometrically. Myocardial infarction and myocardial apoptosis (both DNA laddering and TUNEL analysis) were determined in a blinded manner. RESULTS: MI/R caused significant cardiac dysfunction and myocardial apoptosis (both strong DNA ladder formation and TUNEL-positive staining). Compared with vehicle, GIK-treated rabbits showed protection against MI/R as evidenced by reduced myocardial infarction (19.7% +/- 2.6% vs . 26.8% +/- 3.3% of vehicle, n = 10, P < 0.05), marked decrease in DNA fragmentation and apoptotic index (11.0% +/- 2.1% vs . 20.1% +/- 3.1% of vehicle, n = 6, P < 0.01), significant decrease of plasma CK and LDH and improved recovery of cardiac systolic/diastolic function at the end of R. Treatment with insulin alone decreased blood glucose significantly but still exerted cardioprotective effects comparable with that of GIK. CONCLUSIONS: GIK exerts cardioprotective effects against postischemic myocardial injury and improves cardiac functional recovery in vivo . Insulin, mainly through the anti-apoptotic effect, plays a key role in the GIK-elicited myocardial protection in MI/R.     

Cardioprotection by glucose-insulin-potassium: dependence on KATP channel opening and blood glucose concentration before ischemia

We tested the hypothesis that glucose-insulin-potassium (GIK)-induced protection against myocardial infarction depends on ATP-dependent K(+) (K(ATP)) channel activation and is abolished by hyperglycemia before the ischemia. Dogs were subjected to a 60-min coronary artery occlusion and 3-h reperfusion in the absence or presence of GIK (25% dextrose; 50 IU insulin/l; 80 mM/l KCl infused at 1.5 ml x kg(-1) x h(-1)) beginning 75 min before coronary artery occlusion or 5 min before reperfusion. The role of K(ATP) channels was evaluated by pretreatment with glyburide (0.1 mg/kg). The efficacy of GIK was investigated with increases in blood glucose (BG) concentrations to 300 or 600 mg/dl or experimental diabetes (alloxan/streptozotocin). Infarct size (IS) was 29 +/- 2% of the area at risk in control experiments. GIK decreased (P < 0.05) IS when administered beginning 5 min before reperfusion. This protective action was independent of BG (13 +/- 2 and 12 +/- 2% of area at risk; BG = 80 or 600 mg/dl, respectively) but was abolished in dogs receiving glyburide (30 +/- 4%), hyperglycemia before ischemia (27 +/- 4%), or diabetes (25 +/- 3%). IS was unchanged by GIK when administered before ischemia independent of BG (31 +/- 3, 27 +/- 2, and 35 +/- 3%; BG = 80, 300, and 600 mg/dl, respectively). The insulin component of GIK promotes cardioprotection by K(ATP) channel activation. However, glucose decreases K(ATP) channel activity, and this effect predominates when hyperglycemia is present before ischemia.     

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin‐interacting protein

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin‐interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG‐induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG‐induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG‐treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R.     

胰岛素保护缺血/再灌注心脏:PI3-K/Akt和JNKs信号通路间的交互作用

我们前期研究表明胰岛素可激活细胞内信号转导机制如磷脂酰肌醇3．激酶．蛋白激酶B．内皮型一氧化氮合酶．一氧化氮（P13-K-Akt-eNOS-NO）信号通路,减轻心肌缺血／再灌注（ischemia／reperfusion,I／R）损伤,改善缺血后心肌功能恢复。然而c-Jun氨基末端激酶（c-JunNH2-terminal kinase,JNK）信号通路在胰岛素保护I／R心肌中的作用尚不清楚,本研究旨在探讨JNK信号通路在胰岛素保护I／R心肌中的作用及其与P13．K／Akt信号通路间的相互关系。离体Sprague-Dawley大鼠心脏缺血30min后施行2h或4h的再灌注,缺血前用LY294002（15mmol／L）和SP600125（10mmol／L）灌注15min,分别阻断P13．K／Akt和磷酸化JNK（phosphorylated．JNK,p-JNK）活化,观测心脏功能、心肌梗死、细胞凋亡和蛋白磷酸化水平。与对照组相比,胰岛素再灌注2h后,心率、左心室发展压和左心室收缩／舒张最大速率均明显增加,梗死面积减少约16．1％[（28．9±2．0）％vs（45．0±4．0）％,n=6,P〈O．01],细胞凋亡指数从（27．6±113）％减少到（16．0±0．7）％（n=6,P〈O．01）,Akt的活性增加1．7倍（n=6,P〈0．05）,同时JNK活性增加1．5倍铆=6,P〈O．05）。用LY294002处理后,胰岛素对I／R心肌的保护作用消失;而用SP600125处理可增强胰岛素的保护作用,且可部分逆转LY294002的抑制作用。进一步观察发现SP600125减弱了Akt的磷酸化m=6,P〈0．05）。上述结果表明,在I／R心肌中,胰岛素可同时激活P13．K／Akt及JNK信号通路,且通过后者进一步增加Akt活化,从而减轻I／R损伤,改善心肌功能。这种P13．K／Akt与JNK信号通路交互机制对胰岛素保护I／R心肌有重要意义。     

Cardioprotective effects of nitric oxide-aspirin in myocardial ischemia-reperfused rats

In this study, the cardioprotective effects of nitric oxide (NO)-aspirin, the nitroderivative of aspirin, were compared with those of aspirin in an anesthetized rat model of myocardial ischemia-reperfusion. Rats were given aspirin or NO-aspirin orally for 7 consecutive days preceding 25 min of myocardial ischemia followed by 48 h of reperfusion (MI/R). Treatment groups included vehicle (Tween 80), aspirin (30 mg.kg(-1).day(-1)), and NO-aspirin (56 mg.kg(-1).day(-1)). NO-aspirin, compared with aspirin, displayed remarkable cardioprotection in rats subjected to MI/R as determined by the mortality rate and infarct size. Mortality rates for vehicle (n = 23), aspirin (n = 22), and NO-aspirin groups (n = 22) were 34.8, 27.3, and 18.2%, respectively. Infarct size of the vehicle group was 44.5 +/- 2.7% of the left ventricle (LV). In contrast, infarct size of the LV decreased in the aspirin- and NO-aspirin-pretreated groups, 36.7 +/- 1.8 and 22.9 +/- 4.3%, respectively (both P < 0.05 compared with vehicle group; P < 0.05, NO-aspirin vs. aspirin ). Moreover, NO-aspirin also improved ischemia-reperfusion-induced myocardial contractile dysfunction on postischemic LV developed pressure. In addition, NO-aspirin downregulated inducible NO synthase (iNOS; 0.37-fold, P < 0.01) and cyclooxygenase-2 (COX-2; 0.61-fold, P < 0.05) gene expression compared with the vehicle group after 48 h of reperfusion. Treatment with N(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg), a nonselective NOS inhibitor, aggravated myocardial damage in terms of mortality and infarct size but attenuated effects when coadministered with NO-aspirin. L-NAME administration did not alter the increase in iNOS and COX-2 expression but did reverse the NO-aspirin-induced inhibition of expression of the two genes. The beneficial effects of NO-aspirin appeared to be derived largely from the NO moiety, which attenuated myocardial injury to limit infarct size and better recovery of LV function following ischemia and reperfusion.     

Mechanism of decreased adenosine protection in reperfusion injury of aging rats

The purpose of this study was to determine whether the protective effects of adenosine on myocardial ischemia-reperfusion injury are altered with age, and if so, to clarify the mechanisms that underlie this change related to nitric oxide (NO) derived from the vascular endothelium. Isolated perfused rat hearts were exposed to 30 min of ischemia and 60 min of reperfusion. In the adult hearts, administration of adenosine (5 micromol/l) stimulated NO release (1. 06 +/- 0.19 nmol. min(-1). g(-1), P < 0.01 vs. vehicle), increased coronary flow, improved cardiac functional recovery (left ventricular developed pressure 79 +/- 3.8 vs. 57 +/- 3.1 mmHg in vehicle, P < 0.001; maximal rate of left ventricular pressure development 2,385 +/- 103 vs. 1,780 +/- 96 in vehicle, P < 0.001), and reduced myocardial creatine kinase loss (95 +/- 3.9 vs. 159 +/- 4.6 U/100 mg protein, P < 0.01). In aged hearts, adenosine-stimulated NO release was markedly reduced (+0.42 +/- 0.12 nmol. min(-1). g(-1) vs. vehicle), and the cardioprotective effects of adenosine were also attenuated. Inhibition of NO production in the adult hearts significantly decreased the cardioprotective effects of adenosine, whereas supplementation of NO in the aged hearts significantly enhanced the cardioprotective effects of adenosine. The results show that the protective effects of adenosine on myocardial ischemia-reperfusion injury are markedly diminished in aged animals, and that the loss in NO release in response to adenosine may be at least partially responsible for this age-related alteration.     

Hydrogen sulfide contributes to cardioprotection during ischemia-reperfusion injury by opening K ATP channels

The present study was undertaken to investigate the protective effect of H2S against myocardial ischemia-reperfusion (I/R) injury and its possible mechanism by using isolated heart perfusion and patch clamp recordings. Rat isolated hearts were Langendorff-perfused and subjected to a 30-minute ischemia insult followed by a 30-minute reperfusion. The heart function was assessed by measuring the LVDP, +/-dP/dt max, and the arrhythmia score. The results showed that the treatment of hearts with a H2S donor (40 micromol/L NaHS) during reperfusion resulted in significant improvement in heart function compared with the I/R group (LVDP recovered to 85.0% +/- 6.4% vs. 35.0% +/- 6.1%, +dP/dt max recovered to 80.9% +/- 4.2% vs. 43.0% +/- 6.4%, and -dP/dt max recovered to 87.4% +/- 7.3% vs. 53.8% +/- 4.9%; p < 0.01). The arrhythmia scores also improved in the NaHS group compared with the I/R group (1.5 +/- 0.2 vs. 4.0 +/- 0.4, respectively; p < 0.001). The cardioprotective effect of NaHS during reperfusion could be blocked by an ATP-sensitive potassium channel (K ATP) blocker (10 micromol/L glibenclamide). In single cardiac myocytes, NaHS increased the open probability of K ATP channels from 0.07 +/- 0.03 to 0.15 +/- 0.08 after application of 40 mumol/L NaHS and from 0.07 +/- 0.03 to 0.36 +/- 0.15 after application of 100 mumol/L NaHS. These findings provide the first evidence that H2S increases the open probability of K ATP in cardiac myocytes, which may be responsible for cardioprotection against I/R injury during reperfusion.     

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.     

Distinct effects of acute pretreatment with lipophilic and hydrophilic statins on myocardial stunning, arrhythmias and lethal injury in the rat heart subjected to ischemia/reperfusion

Although both lipophilic and more hydrophilic statins share the same pathway of the inhibition of HMG-CoA reductase, their pleiotropic cardioprotective effects associated with the ability to cross cellular membranes, including membranes of heart cells, may differ. To test this hypothesis, isolated rat hearts were Langendorff-perfused either with simvastatin (S, 10 micromol/l) or pravastatin (P, 30 micromol/l), 15 min prior to ischemia. Control untreated hearts (C) were perfused with perfusion medium only. Postischemic contractile dysfunction, reperfusion-induced ventricular arrhythmias and infarct size were investigated after exposure of the hearts to 30-min global ischemia and 2-h reperfusion. Both lipophilic S and hydrophilic P reduced the severity of ventricular arrhythmias (arrhythmia score) from 4.3 +/- 0.2 in C to 3.0 +/- 0 and 2.7 +/- 0.2 in S and P, respectively, (both P < 0.05), decreased the duration of ventricular tachycardia and suppressed ventricular fibrillation. Likewise, the extent of lethal injury (infarct size) determined by tetrazolium staining and expressed in percentage of risk area, was significantly lower in both treated groups, moreover, the effect of P was more pronounced (27 +/- 2 % and 10 +/- 2 % in S and P groups, respectively, vs. 42 +/- 1 % in C; P < 0.05). In contrast, only S, but not P, was able to improve postischemic recovery of left ventricular developed pressure (LVDP; 48 +/- 12 % of preischemic values vs. 25 +/- 4 % in C and 21 +/ -7 % in P groups; P < 0.05). Our results suggest that differences in water solubility of statins indicating a different ability to cross cardiac membranes may underlie their distinct cardioprotective effects on myocardial stunning and lethal injury induced by ischemia/reperfusion.     

Glucose-insulin-potassium preserves systolic and diastolic function in ischemia and reperfusion in pigs

Clinical and experimental studies have suggested benefit of treatment with intravenous glucose-insulin-potassium (GIK) in acute myocardial infarction. However, patients hospitalized with acute coronary syndromes often experience recurrent myocardial ischemia without infarction that may cause progressive left ventricular (LV) dysfunction. This study tested the hypothesis that anticipatory treatment with GIK attenuates both systolic and diastolic LV dysfunction resulting from ischemia and reperfusion without infarction in vivo. Open-chest, anesthetized pigs underwent 90 min of moderate regional ischemia (mean subendocardial blood flow 0.3 ml x g(-1) x min(-1)) and 90 min reperfusion. Eight pigs were treated with GIK (300 g/l glucose, 50 U/l insulin, and 80 meq/l KCl; infused at 2 ml x kg(-1) x h(-1)) beginning 30 min before ischemia and continuing through reperfusion. Eight untreated pigs comprised the control group. Regional LV wall area was measured with orthogonal pairs of sonomicrometry crystals. GIK significantly increased myocardial glucose uptake and lactate release during ischemia. After reperfusion, indexes of regional systolic function (external work and fractional systolic wall area reduction), regional diastolic function (maximum rate of diastolic wall area expansion), and global LV function (LV positive and negative maximum rate of change in pressure with respect to time) recovered to a significantly greater extent in GIK-treated pigs than in control pigs (all P < 0.05). The findings suggest that the clinical utility of GIK may extend beyond treatment of acute myocardial infarction to anticipatory metabolic protection of myocardium in patients at risk for recurrent episodes of ischemia.     

Attenuation of irreversible rat heart injury by reperfusion with metabolic protectors

The effects of intravenous infusion of potassium-magnesium aspartate (K-Mg-Asp), a glucoseinsulin-potassium cocktail (GIK), a combination of glucose, insulin and potassium aspartate (GIKAsp), and insulin (I) alone on metabolism of the risk area (AR) and cardiomyocyte membrane damage have been investigated in rats during reperfusion after myocardial regional ischemia. Acute myocardial infarction (MI) was induced by a 40-min occlusion of the anterior descending coronary artery followed by a 60-min reperfusion. During reperfusion, K-Mg-Asp, GIK, GIKAsp, I or the physiological solution (control) was infused into the jugular vein at a rate of 1 ml/kg/h. After reperfusion, the MI sizes were significantly lower than in control and reduced in the following order: K-Mg-Asp > GIKAsp > I > GIK. By the end of reperfusion with metabolic protectors, ATP and phosphocreatine levels in the AR were 2–2.5 times higher that in the control (56.3 ± 3.4 and 81.8 ± 7.9% of the initial values, respectively). The losses of aspartate and glutamate pool and lactate and glucose accumulation in AR were significantly lower in the experimental groups than in control. At the end of the reperfusion, the total creatine content in the AR decreased to 32.3 ± 2.3% of the initial value in control, but restored after perfusion with GIK, I and K-Mg-Asp to 78.0 ± 5.7, 76.7 ± 5.5, and 62.4 ± 5.6% (of the initial value), respectively. The recovery of most parameters of aerobic metabolism and cell membrane integrity was maximal in the GIK and I groups and insignificantly lower after reperfusion with K-Mg-Asp.The metabolic efficacy of these protectors corresponded to MI size limitation induced by their infusion. The results suggest that myocardial reperfusion with GIK, I and K-Mg-Asp is a promising adjunctive therapy in patients with acute MI.     

Regular exercise is associated with a protective metabolic phenotype in the rat heart

Adaptation of myocardial energy substrate utilization may contribute to the cardioprotective effects of regular exercise, a possibility supported by evidence showing that pharmacological metabolic modulation is beneficial to ischemic hearts during reperfusion. Thus we tested the hypothesis that the beneficial effect of regular physical exercise on recovery from ischemia-reperfusion is associated with a protective metabolic phenotype. Function, glycolysis, and oxidation of glucose, lactate, and palmitate were measured in isolated working hearts from sedentary control (C) and treadmill-trained (T: 10 wk, 4 days/wk) female Sprague-Dawley rats submitted to 20 min ischemia and 40 min reperfusion. Training resulted in myocardial hypertrophy (1.65 +/- 0.05 vs. 1.30 +/- 0.03 g heart wet wt, P < 0.001) and improved recovery of function after ischemia by nearly 50% (P < 0.05). Glycolysis was 25-30% lower in T hearts before and after ischemia (P < 0.05), whereas rates of glucose oxidation were 45% higher before ischemia (P < 0.01). As a result, the fraction of glucose oxidized before and after ischemia was, respectively, twofold and 25% greater in T hearts (P < 0.05). Palmitate oxidation was 50-65% greater in T than in C before and after ischemia (P < 0.05), whereas lactate oxidation did not differ between groups. Alteration in content of selected enzymes and proteins, as assessed by immunoblot analysis, could not account for the reduction in glycolysis or increase in glucose and palmitate oxidation observed. Combined with the studies on the beneficial effect of pharmacological modulation of energy metabolism, the present results provide support for a role of metabolic adaptations in protecting the trained heart against ischemia-reperfusion injury.     

Early anti-apoptosis treatment reduces myocardial infarct size after a prolonged reperfusion

OBJECTIVE : Significant myocardial apoptosis occurs in ischemia/reperfused hearts. However, the contribution of apoptosis to the development of myocardial injury remains controversial. The present study attempted to obtain evidence that inhibition of apoptosis at early reperfusion can reduce myocardial infarction after prolonged reperfusion. METHODS : Adult male rats were subjected to 30 min ischemia and 4 (apoptosis assay) or 24 h (myocardial infarction determination) of reperfusion and treated with vehicle, SB 239063, insulin or insulin plus wortmannin. RESULTS : Treatment with SB 239063 or insulin markedly decreased myocardial apoptosis (10.6 +/- 1.5% and 7.9 +/- 0.9% respectively, P < 0.01 vs. vehicle) and significantly reduced infarct size (43 +/- 3.6% and 35 +/- 2.9%, respectively, P < 0.01 vs. vehicle). Most interestingly, inhibition of insulin signaling with wortmannin to block insulin signaling not only blocked insulin's anti-apoptotic effect, but also abolished its infarct reduction property. CONCLUSION : These data indicate that apoptosis contributes to the development of myocardial infarction, and inhibition of apoptosis at early reperfusion reduces the myocardial infarction.     

Cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2 is mediated by the MAPK cascade

Our laboratory showed previously that cardiac-specific overexpression of FGF-2 [FGF-2 transgenic (Tg)] results in increased recovery of contractile function and decreased infarct size after ischemia-reperfusion injury. MAPK signaling is downstream of FGF-2 and has been implicated in other models of cardioprotection. Treatment of FGF-2 Tg and wild-type hearts with U-0126, a MEK-ERK pathway inhibitor, significantly reduced recovery of contractile function after global low-flow ischemia-reperfusion injury in FGF-2 Tg (86 +/- 2% vehicle vs. 66 +/- 4% U-0126; P < 0.05) but not wild-type (61 +/- 7% vehicle vs. 67 +/- 7% U-0126) hearts. Similarly, MEK-ERK inhibition significantly increased myocardial infarct size in FGF-2 Tg (12 +/- 3% vehicle vs. 31 +/- 2% U-0126; P < 0.05) but not wild-type (30 +/- 4% vehicle vs. 36 +/- 7% U-0126) hearts. In contrast, treatment of FGF-2 Tg and wild-type hearts with SB-203580, a p38 inhibitor, did not abrogate FGF-2-induced cardioprotection from postischemic contractile dysfunction. Instead, inhibition of p38 resulted in decreased infarct size in wild-type hearts (30 +/- 4% vehicle vs. 11 +/- 2% SB-203580; P < 0.05) but did not alter infarct size in FGF-2 Tg hearts (12 +/- 3% vehicle vs. 14 +/- 1% SB-203580). Western blot analysis of ERK and p38 activation revealed signaling alterations in FGF-2 Tg and wild-type hearts during early ischemia or reperfusion injury. In addition, MEK-independent ERK inhibition by p38 was observed during early ischemic injury. Together these data suggest that activation of ERK and inhibition of p38 by FGF-2 is cardioprotective during ischemia-reperfusion injury.     

PAR-2 activation at the time of reperfusion salvages myocardium via an ERK1/2 pathway in in vivo rat hearts

Protease-activated receptor-2 (PAR-2) may have proinflammatory effects in some tissues and protective effects in other tissues. The role of PAR-2 in in vivo myocardial ischemia-reperfusion has not yet been determined. This study tested the hypothesis that PAR-2 activation with the PAR-2 agonist peptide SLIGRL (PAR-2 AP) reduces myocardial infarct size when given at reperfusion in vivo, and this cardioprotection involves the ERK1/2 pathway. Anesthetized rats were randomly assigned to the following groups with 30 min of regional ischemia and 3 h reperfusion: 1) control with saline; 2) vehicle (DMSO); 3) PAR-2 AP, 1 mg/kg given intravenously 5 min before reperfusion; 4) scrambled peptide (SP), 1 mg/kg; 5) the ERK1/2 inhibitor PD-98059 (PD), 0.3 mg/kg given 10 min before reperfusion; 6) the phosphatidylinositol 3-kinase inhibitor LY-294002 (LY), 0.3 mg/kg given 10 min before reperfusion; 7) PD + PAR-2 AP, 0.3 mg/kg PD given 5 min before PAR-2 AP; 8) LY + PAR-2 AP, 0.3 mg/kg LY given 5 min before PAR-2 AP; 9) chelerythrine (Chel) alone, 5 mg/kg given 10 min before reperfusion; and 10) Chel + PAR-2 AP, Chel was given 5 min before PAR-2 AP (10 min before reperfusion). Activation of ERK1/2, ERK5, Akt, and the downstream targets of ERK1/2 [P90 RSK and bcl-xl/bcl-2-associated death promoter (BAD)] was determined by Western blot analysis in separate experiments. PAR-2 AP significantly reduced infarct size compared with control (36 +/- 2% vs. 53 +/- 1%, P < 0.05), and SP had no effect on infarct size (53 +/- 3%). PAR-2 AP significantly increased phosphorylation of ERK1/2, p90RSK, and BAD but not Akt or ERK5. Accordingly, the infarct-size sparing effect of PAR-2 AP was abolished by PD (PAR-2 AP, 36 +/- 2% vs. PD + PAR-2 AP, 50 +/- 1%; P < 0.05) and by Chel (Chel + PAR-2 AP, 58 +/- 2%) but not by LY (PAR-2 AP, 36 +/- 2% vs. LY + PAR-2 AP, 38 +/- 3%; P > 0.05). Therefore, PAR-2 activation is cardioprotective in the in vivo rat heart ischemia-reperfusion model, and this protection involves the ERK1/2 pathway and PKC.     

κ-Opioid receptor stimulation modulates TLR4/NF-κB signaling in the rat heart subjected to ischemia–reperfusion

It is well documented that the Toll-like receptor 4 (TLR4)/NF-κB signaling mediates early inflammation during myocardial ischemia and reperfusion. Our previous study has demonstrated that κ-opioid receptor stimulation with U50,488H produces cardioprotective and anti-inflammatory effects. The aim of the present study was to investigate whether κ-opioid receptor stimulation could modulate the TLR4/NF-κB signaling and reduce neutrophil accumulation and TNF-α induction in an ischemia–reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia and reperfusion (MI/R), and MI/R + U50,488H in the absence or presence of Nor-BNI, a selective κ-opioid receptor antagonist. The results demonstrated that after MI/R, the expressions of myocardial TLR4 and NF-κB increased significantly both in ischemia area and risking area. Compared with MI/R, κ-opioid receptor stimulation with U50,488H significantly attenuated the expressions of TLR4 and NF-κB. At the mean time, it also reduced myeloperoxidase (MPO) levels, both serum and myocardial TNF-α production, myocardial infarct sizes (INF/AAR%) and myocardial apoptosis induced by MI/R, all the effects of U50,488H were abolished by Nor-BNI. These data provide evidence for the first time that κ-opioid receptor stimulation inhibits TLR4/NF-κB signaling in the rat heart subjected to MI/R.     

Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been demonstrated to possess cardioprotective properties during ischemia-reperfusion. However, this notion remains controversial as recent evidence has suggested an increased risk in cardiac events associated with long-term use of RGZ in patients with type 2 diabetes. In this study, we tested the hypothesis that acute RGZ treatment is beneficial during I/R by modulating cardioprotective signaling pathways in a nondiabetic mouse model. RGZ (1 μg/g) was injected intravenously via the tail vein 5 min before reperfusion. Myocardial infarction was significantly reduced in mice treated with RGZ compared with vehicle controls (8.7% ± 1.1% vs. 20.2% ± 2.5%, P < 0.05). Moreover, isolated hearts were subjected to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system. Postischemic recovery was significantly improved with RGZ treatment administered at the onset of reperfusion compared with vehicle (P < 0.001). Immunoblot analysis data revealed that the levels of both phospho-AMP-activated protein kinase (Thr(172)) and phospho-Akt (Ser(473)) were significantly upregulated when RGZ was administered 5 min before reperfusion compared with vehicle. On the other hand, inflammatory signaling [phospho-JNK (Thr(183)/Tyr(185))] was significantly downregulated as a result of RGZ treatment compared with vehicle (P < 0.05). Intriguingly, pretreatment with the selective PPAR-γ inhibitor GW-9662 (1 μg/g iv) 10 min before reperfusion significantly attenuated these beneficial effects of RGZ on the ischemic heart. Taken together, acute treatment with RGZ can reduce ischemic injury in a nondiabetic mouse heart via modulation of AMP-activated protein kinase, Akt, and JNK signaling pathways, which is dependent on PPAR-γ activation.     

Hydrogen sulfide and its possible roles in myocardial ischemia in experimental rats.

The role of hydrogen sulfide (H(2)S) in myocardial infarction (MI) has not been previously studied. We therefore investigated the effect of H(2)S in a rat model of MI in vivo. Animals were randomly divided into three groups (n = 80) and received either vehicle, 14 micromol/kg of sodium hydrosulfide (NaHS), or 50 mg/kg propargylglycine (PAG) everyday for 1 wk before surgery, and the treatment was continued for a further 2 days after MI when the animals were killed. The mortality was 35% in vehicle-treated, 40% in PAG-treated, and 27.5% in NaHS-treated (P < 0.05 vs. vehicle) groups. Infarct size was 52.9 +/- 3.5% in vehicle-treated, 62.9 +/- 7.6% in PAG-treated, and 43.4 +/- 2.8% in NaHS-treated (P < 0.05 vs. vehicle) groups. Plasma H(2)S concentration was significantly increased after MI (59.2 +/- 7.16 microM) compared with the baseline concentration (i.e., 38.2 +/- 2.07 microM before MI; P < 0.05). Elevated plasma H(2)S after MI was abolished by treatment of animals with PAG (39.2 +/- 5.02 microM). We further showed for the first time cystathionine-gamma-lyase protein localization in the myocardium of the infarct area by using immunohistochemical staining. In the hypoxic vascular smooth muscle cells, we found that cell death was increased under the stimuli of hypoxia but that the increased cell death was attenuated by the pretreatment of NaHS (71 +/- 1.2% cell viability in hypoxic vehicle vs. 95 +/- 2.3% in nonhypoxic control; P < 0.05). In conclusion, endogenous H(2)S was cardioprotective in the rat model of MI. PAG reduced endogenous H(2)S production after MI by inhibiting cystathionine-gamma-lyase. The results suggest that H(2)S might provide a novel approach to the treatment of MI.