| Ischemic preconditioning is a phenonmenon in which brief exposures of the myocardium to ischemia protect potently the heart against subsequent severe ischemia and many researchers try to study its mechanism. A number of mediators such as adenosine, acetylcholine, catecholamines, and opioid peptides, and their receptors have been implicated to be involved in the cardioprotective mechanism of ischemic preconditioning. But its mechanism remains unclear up-to-date. Recent reports indicated that gap junction is intimately related to the cardioprotection of ischemic preconditioning, and ischemic preconditioning delayed the onset of electrical uncoupling during subsequent prolonged ischemia. It is important for the ischemic myocardium because electrical uncoupling indicates an onset of irreversible injury. But it is not clear whether similar changes exist in cardioprotection induced by other agonists or methods. Therefore, the aims of the present study are to investigate the effect of gap junction uncoupler heptanol, a non selective opioid receptor agonist morphine, and a selective K-opioid receptor agonist U50,488H on myocardium during ischemia/reperfusion in isolated perfused rat heart or in the intact rat, and on electrical coupling during myocardial ischemia in isolated perfused ratheart, and to assess whether changes in electrical coupling correlate with these cardiac protections, and to explore the possible mechanism.OBJECTIVES1. To investigate the effect of heptanol on myocardium during ischemia/reperfusion in isolated perfused rat heart and in the intact rat, on electrical coupling during myocardial ischemia in isolated perfused rat heart, and on action potential parameters of right papillary muscle in isolated rat heart, and to determine the correlation and mechanism of heptanol-induced cardioprotection with changes in electrical coupling during ischemia in heptanol-treated rat heart.2. To evaluate the effect of morphine on myocardium during ischemia/reperfusion in isolated perfused rat heart, and on electrical coupling during myocardial ischemia in isolated perfused rat heart, and to explore the correlation of morphine-induced cardioprotection with changes in electrical coupling during ischemia in morphine-treated rat heart.3. To observe the effect of U50,488H on myocardium during ischemia/reperfusion in isolated perfused rat heart, on electrical coupling during myocardial ischemia in isolated perfused rat heart, and on changes in distribution of connexin-43 (Cx43) in cardiomyocyte membrane of U50,488H-treated rat heart during 30 min ischemia, and to determine the correlation and mechanism of U50,488H-induced cardioprotection with changes in electrical coupling during ischemia in heptanol-treated rat heart.METHODS1. Ischemia/reperfusion model in the intact rat heart: measurements of arrhythmia scores, infarct size, heart rate, and mean arterial blood pressure.2. Isolated perfuesd rat heart: measurements of hemodynamic parameters,arrhythmia scores, infarct size, formazan content, heart rate, coronary flow, lactate dehydrogenase (LDH), eletrical coupling parameters (including onset of uncoupling, plateau time, slope, and fold increase in rt), and immunohistochemistry.3. Isolated rat right papillary muscle: observation of resting potential (RP), action potential amplitude (APA), maximal upstroke of depolarization (Vmax), action potential duration at 90% repolarization (APD90), effective refractory period (ERP), and action potential conduction velocity.RESULTSPart 1: Effect and mechanism of heptanol on myocardium during ischemia/reperfusion and electrical coupling during myocardial ischemia1. Ischemic preconditioning markedly decreased arrhythmia scores and infarct size relative to controls during the subsequent prolonged ischemia and reperfusion in isolated rat heart. Heptanol at concentrations of 1.0, 0.5 or 0.1 mmol/L markedly decreased arrhythmia scores during the subsequent ischemia and reperfusion in isolated rat heart. Heptanol also markedly reduced infarct size and decreased heart rate during perfusion with the drug in isolated rat heart.2. Heptanol at concentrations of 0.06 and 0.3 mg/kg markedly decreased arrhythmia scores during the subsequent ischemia and reperfusion in the intact rat, but heptanol of 0.03-0.6 mg/kg could produce occasional premature ventricular contraction. Although heptanol of 0.6 mg/kg markedly decreased arrhythmia scores during the subsequent reperfusion in the intact rat, it markedly increased arrhythmia scores during ischemia. Heptanol of 0.06-0.6 mg/kg markedly reduced infarct size in the intact rat.3. Heptanol treatment decreased the maximal rate of uncoupling during ischemia by 27% or more, as determined by measuring the slopes of the rt curves during the rapid uncoupling phase. Heptanol also significantly increased the timerequired for rt to reach a plateau. The onset of uncoupling occurred later in heptanol-treated hearts. While heptanol-treated hearts eventually reached a maximum level of uncoupling equal to that of non-preconditioned hearts, the procedures significantly slowed the rate of uncoupling and lengthened the time required for complete uncoupling. There were no significant differences in fold increase in rt.4. Heptanol significantly reduced the conduction velocity of the action potential in a dose-dependent manner, but had no significant effect on the other parameters of transmembrane potential.Part 2: Effect of morphine on myocardium during ischemia/reperfusion and electrical coupling during myocardial ischemia1. Pretreatment with morphine at concentration of 3xlO"7 increased formazan content. Similarly, morphine also reduced LDH release at 10 min of reperfusion. And the effect of the agonist was abolished by a nonselective opioid receptor antagonist naloxone at 10"6 mol/L, which alone had no effects on formazan content and LDH release. Morphine also significantly increased LVDP, LVDPxHR and +dP/dtmax during late reperfusion. Naloxone itself also significantly increased LVDP, LVDPxHR during late reperfusion.2. Morphine of 3x10"7 mol/L decreased the maximal rate of uncoupling during ischemia by 55%. Morphine significantly increased the plateau time. Compared to control group, the onset of uncoupling was delayed in the isolated perfused hearts pretreated with morphine. The effect of morphine on the plateau time and maximal rate of uncoupling was abolished by naloxone of 10"6 mol/L. The delaying effect of morphine on the onset of uncoupling was not only abolished, but also advanced by naloxone. The naloxone alone also advanced the onset of uncoupling compared with that of control group. There were no significant differences in fold increase among these groups.Part 3: Effect and mechanism of U50,488H on myocardium duringischemia/reperfusion and electrical coupling during myocardial ischemia1. U50,488H concentration dependently increased formazan content. Similarly, pretreatment with U50,488H also reduced LDH release at 1 and 5 min of reperfusion. And the effect of the agonist at concentration of 10'5 mol/L was abolished by a selective K-opioid receptor antagonist nor-BNI at 5x1 (T6 mol/L, which alone had no effects on formazan content and LDH release.The effects of U50,488H on formazan content and LDH release were also abolished on blockade of mitochondrial KAtp channel with its inhibitor, 5-HD, which itself had no effect at all. U50,488H of 10"5 mol/L significantly increased LVDP, LVDPxHR and +dP/dtmax during late reperfusion.2. U50,488H decreased the maximal rate of uncoupling during ischemia by 34% or more (determined by measuring the slopes of the rt curves during the rapid uncoupling phase). High concentrations of U50,488H also significantly increased the time required to reach a plateau in rt. Compared to control group, the onset of uncoupling was delayed in the isolated perfused hearts by high concentrations of U50,488H. The effect of 10"5 mol/L U50,488H on the plateau time and maximal rate of uncoupling was abolished by a selective K-opioid receptor antagonist nor-BNI of 5xlO~6 mol/L or by a selective mitochondrial KAtp channel blocker 5-HD of 10"4 mol/L. The delaying effect of U50,488H on the onset of uncoupling was not only abolished, but also advanced by nor-BNI or 5-HD. The nor-BNT alone also advanced the onset of uncoupling, but 5-HD alone did not have such effect. There were no significant differences in fold increase among these groups.3. A linear positive relation between formazan content and onset of the uncoupling within the concentrations of 10~7-10"5mol/L of U50,488H was observed. The correlation was significant (PO.0001), with R2=0.6409. A similar linear negative relations between the LDH release at 1 or 5 min of reperfusion and the time of onset of uncoupling during prolonged ischemia in the isolated rat heart pretreatedwith U50,488H at different concentrations were also observed. The correlations were significant (PO.0001), with R2=0.6235, or R2=0.7405, respectively.4. The amount of Cx43 immunoreactive signal in gap junctions was greatly reduced after 30 min of ischemia in the hearts that did not undergo U50,488H preconditioning. In contrast, U50,488H preconditioning resulted in a marked preservation of Cx43 signal in gap junctions. These effects were abolished by nor-BNI or 5-HD.CONCLUSIONS1. Heptanol could induce cardioprotection in isolated perfused rat heart or in the intact rat. The protective effect was related to delayed electrical uncoupling during myocardial ischemia, but having nothing to do with the active electrical properties of cardiomyocyte membrane.2. Morphine could induce cardioprotection in isolated perfused rat heart. The protective effect was related to delayed electrical uncoupling during myocardial ischemia.3. U50,488H-induced cardioprotection was intimately related to delayed electrical uncoupling during myocardial ischemia. These effects probably were correlated with preservation of Cx43 by U50,488H.
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