Yue-Min Wang

Physiologically tolerable insulin reduces myocardial injury and improves cardiac functional recovery in myocardial ischemic/reperfused dogs.

This study was designed to examine whether physiologically tolerable insulin, which maintains lower blood glucose, can protect the myocardium against ischemia/reperfusion (I/R) injury in a preclinical large animal model. Adult dogs were subjected to 50 minutes of myocardial ischemia (80% reduction in coronary blood flow) followed by 4 hours of reperfusion and treated with vehicle, glucose-insulin-potassium (GIK; glucose, 250 g/L; insulin, 60 U/L; potassium, 80 mmol/L), GK, or low-dose insulin (30 U/L) 10 minutes before reperfusion. Treatment with GIK exerted significant cardioprotective effects as evidenced by improved cardiac function, improved coronary blood flow, reduced infarct size, and myocardial apoptosis. In contrast, treatment with GK increased blood glucose level and aggravated myocardial I/R injury. It is interesting that treatment with insulin alone at the dose that reduced blood glucose to a clinically tolerable level exerted significant cardioprotective effects that were comparable to that seen in the GIK-treated group. This low-dose insulin had no effect on coronary blood flow after reperfusion but markedly reduced coronary reactive hyperemia and switched myocardial substrate uptake from fat to carbohydrate. Our results suggest that lower glucose supply to the ischemic myocardium at early reperfusion may create a “metabolic postconditioning” and thus reduce myocardial ischemia/reperfusion injury after prolonged reperfusion.

Resveratrol Attenuates Ischemia/Reperfusion Injury in Neonatal Cardiomyocytes and Its Underlying Mechanism

This study was designed to investigate whether Resveratrol (Res) could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions), the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion). To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na+-K+-ATPase and Ca2+-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na+-K+-ATPase and Ca2+-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor) or MB (a cGMP inhibitor), and partly by either DS (a PKC inhibitor) or Glybenclamide (a KATP inhibitor). Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and KATP may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.

Cardioprotective and antiarrhythmic effect of U50,488H in ischemia/reperfusion rat heart.

The objective of this study was to investigate the protective effect of U50,488H, a selective κ-opioid receptor agonist, in the ischemia/reperfusion (I/R) rat and to delineate the underlying mechanism. Rat heart I/R injury was induced by occluding the left anterior descending coronary artery for 45 min and restoring perfusion for 120 min. U50,488H or vehicle was intravenously injected before ischemia. Electrocardiogram, heart rate (HR), arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dp/dtmax), and diastolic function (−dp/dtmax) were monitored in the course of the experiment. Myocardial infarction size was evaluated. Plasma concentrations of cardiac troponin T (cTnT), creatine kinase (CK), and lactate dehydrogenase (LDH) were measured. Single rat ventricular myocyte was obtained by enzymatic dissociation method. The potassium currents (IK) of isolated ventricular myocytes were recorded with the whole-cell configuration of the patch-clamp technique. Compared with the sham control group, no significant change was found in HR, while ABP, LVP and ±dp/dtmax were significantly reduced in the I/R group. Administration of U50,488H significantly lowered HR in both control and I/R groups. Compared with the vehicle-treated I/R group, administration of U50,488H had no significant effect on I/R-induced reduction in ABP, LVP, and ±dp/dtmax. However, this treatment significantly reduced the myocardial infarction size, and markedly decreased the contents of plasma cTnT, CK and LDH. During ischemia and reperfusion, the incidence of ventricular arrhythmia in U50,488H-treated rats was significantly reduced. These effects were independent of the bradycardia induced by U50,488H, as the reducing infarct size and antiarrhythmic effect of U50,488H were still observed in animals in which heart rate was kept constant by electrical pacing. U50,488H and BRL-52537 still produced an antiarrhythmic effect when the rat heart was subjected to a shorter ischemic period of 10 min occlusion of coronary artery, which produced no infarction. IK of the myocytes were inhibited by U50,488H in a dose-dependent manner in normal and hypoxic rat ventricular myocytes. However, the effects of U50,488H on IK did not show any significant difference in normal and hypoxic myocytes. The above-described effects of U50,488H were totally blocked by nor-Binaltorphimine, a selective κ-opioid receptor antagonist. The results suggest that κ-opioid agonist U50,488H exerts its direct cardioprotective and antiarrhythmic effects against I/R via κ-opioid receptor, which participates in the regulation of potassium channels in normal and hypoxic ventricular myocytes.

Myocardial Apoptosis and Infarction after Ischemia/Reperfusion Are Attenuated by κ-Opioid Receptor Agonist

BACKGROUND AND AIMS It remains unclear whether U50488H (a selective kappa-opioid receptor agonist) produces anti-apoptotic effect during ischemia and reperfusion (I/R). Therefore, the effect of U50488H on myocardial apoptosis was investigated in the present study. METHODS Rats were subjected to 45min coronary artery occlusion and 180min of reperfusion. U50488H (1.5mg/kg IV) was given prior to occlusion. Nor-Binaltorphimine (nor-BNI) (2mg/kg IV), a selective kappa-opioid receptor antagonist, was given 10min prior to U50488H. Cardiac apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay and in situ identification of nuclear DNA fragmentation. RESULTS The ultrastructure injury of myocardium, myocardial infarct size, and plasma CK and LDH were reduced significantly with administration of U50488H before I/R, whereas the effects of U50488H were abolished by nor-BNI. DNA fragments were visualized by agarose electrophoresis, and clear DNA ladder formation was observed in myocardial tissue from hearts subjected to I/R. Administration of U50488H before ischemia exerted a significant anti-apoptotic effect as evidenced by markedly weaker DNA ladder formation. TUNEL staining showed U50488H treatment before I/R significantly reduced the percentage of apoptotic cells, which was blocked by 5-HD, a mitochondrial k(ATP) channel blocker. In accordance, U50488H treatment significantly inhibited I/R-induced elevated activities of caspase-3 and caspase-9. U50488H also produced an increase in Bcl-2 and a decrease in Bax protein expression in the I/R heart, and the anti-apoptotic effects of U50488H were all blocked by nor-BNI. CONCLUSIONS U50488H reduces myocardial necrosis and apoptosis after I/R and activation of kappa-opioid receptor may mediate a role in U50488H-induced myocardial protection.

Effects of U50,488H on hypoxia pulmonary hypertension and its underlying mechanism

The aim of the present study was to determine whether U50,488H, a selective kappa-opioid receptor agonist, inhibits the remodeling of the pulmonary artery (PA). In addition, changes in the concentrations of nitric oxide (NO), endothelin (ET) and angiotensin II (AngII) in hypoxic pulmonary hypertensive (HPH) rats were investigated to explore the mechanisms underlying the effects of U50, 488H on HPH. We found that intraperitoneal administration of U50,488H (every other day) during hypoxia depressed mean pulmonary arterial pressure (mPAP) and attenuated right ventricular pressure (RVP) and right ventricular hypertrophy, at the same time it inhibited remodeling of the PA compared with hypoxia for 2 wk. Moreover, U50,488H also inhibited proliferation of the pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia for 48 h in a dose-dependent manner. Compared with the 2 wk hypoxia group, U50,488H increased the concentration of NO and decreased the production of ET and AngII (P<0.01). In addition, acute intravenous administration of U50,488H after hypoxia for 4 wk decreased mPAP. Our results suggest that effects of anti-remodeling of the PA and anti-proliferation of the PASMC, and regulation of the vasomotor factors in both blood and pulmonary tissues of HPH rats may be critical mechanisms underlying the preventive and therapeutic effects of U50,488H in HPH rats.

U50,488H depresses pulmonary pressure in rats subjected to chronic hypoxia.

In this study, we determined the effect of U50,488H (a selective κ-opioid receptor agonist) on pulmonary artery in rats and investigated its prevention and treatment effects on hypoxic pulmonary hypertension (HPH). Isolated pulmonary arterial rings were superfused and the tension of the vessel was measured. The model of HPH was developed and indexes for hemodynamics and right ventricular hypertrophy were measured. We found that U50,488H relaxed the pulmonary artery rings in a dose-dependent manner and the effect was abolished by nor-binaltorphimine, a selective κ-opioid receptor antagonist. Intravenous administration of U50,488H significantly lowered mean pulmonary artery pressure (mPAP) in normal rats and this effect was also abolished by nor-binaltorphimine. Hypoxia induced severe HPH in rats and intraperitoneal administration of U50,488H (every other day) during chronic hypoxia reduced mPAP and attenuated right ventricular hypertrophy compared with the control group. Moreover, acute intravenous administration of U50,488H after the rats subjected to chronic hypoxia for 4 weeks significantly lowered mPAP. Thus, U50,488H has significant vasorelaxant effect in rat pulmonary artery and has certain preventive and therapeutic application in HPH.

Antiarrhythmic effect mediated by κ-opioid receptor is associated with Cx43 stabilization.

Objective:Acute myocardial ischemia induces electrical and chemical uncoupling of gap junctions, which contributes to conduction abnormalities and re-entrant arrhythmias. We tested the hypothesis that structure and function of Connexin43 may vibrate during acute myocardial ischemia and reperfusion and &kgr;-opioid receptor stimulation may stabilize the alteration of Connexin43. Design:An animal intervention study was conducted with comparison to a control group. Setting:University preclinical research laboratory. Subjects:Age-, weight-, and sex-matched Sprague-Dawley rats. Interventions:Adult rat hearts were subjected to ischemia or ischemia/reperfusion, which was induced by temporary occlusion of the left main coronary artery. U50488H was given 10 mins before tissue specimens were taken or before ischemia (1.5 mg/kg, intravenous) and nor-BNI was given 15 mins before tissue specimens were taken or before ischemia (2 mg/kg, intravenous). Tissue samples came from left ventricular myocardium of the rat hearts. Measurements and Main Results:Electrocardiogram, immunohistochemistry, immunoblotting, and reverse transcription–polymerase chain reaction were used to measure changes of arrhythmias, protein, and gene expression of Connexin43, respectively. &kgr;-opioid receptor activation with U50 decreased arrhythmia in a model of myocardial ischemia and reperfusion. In normal hearts, immunohistochemical data showed reduced amount and lateralization of Connexin43 induced by &kgr;-opioid receptor activation, whereas immunoblotting data demonstrated no significant changes between control and U50 group. During ischemia, however, Connexin43 protein underwent dephosphorylation and degradation, and Connexin43 mRNA was upregulated. These alterations were significantly attenuated on &kgr;-opioid receptor stimulation. During ischemia and reperfusion, Connexin43 protein underwent dephosphorylation and degradation and recovered slowly during reperfusion. Activation of &kgr;-opioid receptor accelerated recovery of phosphorylated and total Connexin43. Conclusions:In normal rat hearts, Connexin43 translocates from intercellular junctions to intracellular locations on &kgr;-opioid receptor activation. In rat hearts experiencing acute myocardial ischemia and reperfusion, protein and gene expression of Connexin43 undergo vibration. This phenomenon is stabilized when &kgr;-opioid receptor is activated and by the fact that &kgr;-opioid receptor produces antiarrhythmic effects.

κ-Opioid Receptor Stimulation Improves Endothelial Function in Hypoxic Pulmonary Hypertension

The present study was designed to investigate the effect of κ-opioid receptor stimulation with U50,488H on endothelial function and underlying mechanism in rats with hypoxic pulmonary hypertension (HPH). Chronic hypoxia-induced HPH was simulated by exposing the rats to 10% oxygen for 2 wk. After hypoxia, mean pulmonary arterial pressure (mPAP), right ventricular pressure (RVP) and right ventricular hypertrophy index (RVHI) were measured. Relaxation of pulmonary artery in response to acetylcholine (ACh) was determined. Expression and activity of endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) with NO production, total antioxidant capacity (T-AOC), gp91phox expression and nitrotyrosine content were measured. The effect of U50,488H administration during chronic hypoxia was investigated. Administration of U50,488H significantly decreased mPAP and right ventricular hypertrophy as evidenced by reduction in RVP and RVHI. These effects were mediated by κ-opioid receptor. In the meantime, treatment with U50,488H significantly improved endothelial function as evidenced by enhanced relaxation in response to ACh. Moreover, U50,488H resulted in a significant increase in eNOS phosphorylation, NO content in serum, and T-AOC in pulmonary artery of HPH rats. In addition, the activity of eNOS was enhanced, but the activity of iNOS was attenuated in the pulmonary artery of chronic hypoxic rats treated with U50,488H. On the other hand, U50,488H markedly blunted HPH-induced elevation of gp91phox expression and nitrotyrosine content in pulmonary artery, and these effects were blocked by nor-BNI, a selective κ-opioid receptor antagonist. These data suggest that κ-opioid receptor stimulation with U50,488H improves endothelial function in rats with HPH. The mechanism of action might be attributed to the preservation of eNOS activity, enhancement of eNOS phosphorylation, downregulation of iNOS activity and its antioxidative/nitrative effect.

Distribution of κ-Opioid Receptor in the Pulmonary Artery and its Changes During Hypoxia

The present study evaluated the distribution of κ‐opioid receptors (κ‐ORs) in pulmonary arteries (PAs) in rats and investigated whether κ‐ORs are altered in PAs during hypoxia. An animal model of hypobaric/hypoxic pulmonary hypertension and a pulmonary artery smooth muscle cell (PASMC) model of hypoxia were utilized. Distribution of κ‐ORs was determined by fluorescence immunohistochemistry and changes in κ‐ORs expression in PAs and PASMCs were determined by fluorescence immunohistochemistry or Western blot techniques. The κ‐ORs were primarily distributed in the smooth muscle layer of the PAs and in the nucleus of PASMCs. The expression of the κ‐ORs were increased in PAs of rats subjected to hypoxia for 1–4 week (P < 0.01). Accordingly, the expression of κ‐ORs in PASMCs were also increased when subjected to hypoxia for 12–36 hr (P < 0.05). The present study has provided evidence for the first time of the precise location of κ‐ORs in PAs and PASMCs of rats and that hypoxia upregulates expression of κ‐ORs. Anat Rec, 2009.

U50,488H inhibits neutrophil accumulation and TNF-α induction induced by ischemia–reperfusion in rat heart

The role of the κ-opioid receptor in inflammation is not well understood. The aim of this study was to investigate whether the κ-opioid receptor agonist U50,488H modulates neutrophil accumulation and TNF-α induction in an ischemia-reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia-reperfusion (MI/R) alone, MI/R+U50,488H, MI/R+U50,488H+Wortmannin, and MI/R+U50,488H+L-NAME. The results demonstrated that compared to MI/R, U50,488H reduced myocardial infarction area, myocardial myeloperoxidase (MPO) levels, serum creatinine kinase (CK) levels, and both serum and myocardial TNF-α production. Increases were seen in NOx levels in the myocardium subjected to MI/R injury. All demonstrated effects of U50,488H were abolished by Nor-BNI, a selective κ-opioid receptor antagonist; Wortmannin, a specific PI3K inhibitor; or L-NAME, a nitric oxide synthase (NOS) inhibitor. In summary, κ-opioid receptor stimulation with U50,488H produces both cardioprotective and anti-inflammatory effects. These effects may be associated with an increase in NO production and the inhibition of neutrophil accumulation and TNF-α induction via a PI3K sensitive pathway in myocardium subjected to MI/R.