Inhibiting Mitochondrial Permeability Transition Pore Opening Protects Rat Heart From Ischaemia

IntroductionThe heart must be undergoing a period of ischemia arrest during open heart surgery. Reperfusion is a prerequisite for salvaging viable myocardium from an episode of ischemia. Paradoxically, the act of reperfusion itself can induce further myocyte damage—a phenomenon termed ischemia reperfusion injury. Despite recent developments in myocardial protection techniques, reperfusion damage still occurs, and significant morbidity remains a problem and further improvement in myocardial protection is needed. Clinically, how to protect heart from ischaemia reperfusion injury is an important task to be resolved.Cardioplegic arrest during cardiac surgery has been traditionally accomplished through administration of a hyperkalemic cardioplegic solution. However, heart dysfunction has been shown to occur after hyperkalemic cardioplegic arrest which may involve alterations in ionic homeostasis and intracellular pH changes. The Langendorff perfusion model and isolated myocyte model of simulated cardioplegic arrest may be useful to determine mechanisms responsible for the heart dysfunctionwith reperfusion as well as potential strategies to prevent these consequences.In recent years the central role of mitochondria in apoptotic and necrotic cell death has become apparent, and a major player in this arena is the mitochondrial permeability transition pore (MPTP). A critical determinant of cell in the setting of ischemia reperfusion injury is the opening of the MPTP. MPTP is a Voltage dependent, high conductance channel located in the inner mitochondrial membrane. In the fully open state, the apparent pore diameter is 3 nm, allowing passive diffusion of solutes up to 1.5 kDa in size. This results in swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane, leading to the release of cytochrome C and other pro-apoptotic molecules into the cytosol. These then activate the caspase cascade that sets apoptosis in motion. Furthermore, the opening of the MPTP uncouples mitochondrial oxidative phosphorylation, resulting in the collapse of the mitochondrial membrane potential (Δ ψ m), which leads to cellular death by necrosis if the MPTP opening is prolonged and ATP levels are depleted.The balance between the number of closed and open MPTP within any cell will be critical in determining whether a cell lives or dies. Inhibited the MPTP opening might be a target for protecting hearts from reperfusion injury. Heart protection may be mediated either through direct inhibition of the pore with agents such as cyclosporin A and SFA, or through the indirect effect associated with a decreased in the factors responsible for MPTP opening such as mitochondrial calcium overload and oxidative stress. Mitochondrial K_ATP channel opener, diazoxide hasbeen reported to inhibit the phase precede MPTP opening. The mechanism for the action of diazoxide on MPTP is poor understood.The aim of this study was to investigate the effect of hyperkalemic cardioplegic solution enriched with mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on rat heart protection. Furthermore in order to demonstrate the mechanism by which cyclosporin A and diazoxide may induce protection, a myocyte model was used to measure intracellular Ca²⁺, Δ ψ m and isolated ventricular myocyte viability, another model was used for detecting MPTP opening in isolated adult rat cardiac mitochondria subjected ischemia.Part 1The effect of mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on rat heart protectionObjective To observe the cardioprotective effect of hyperkalemic cardioplegic solution enriched with mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on rat heart subjected ischemia.Methods Forty-five adult male Sprague-Dawley rat hearts were randomly divided into 5 groups (9 rats in each group): St.Thomas group;30 μ mol/L diazoxide+St.Thomas group; 0. 2 μ mol/L cyclosporin A + St.Thomas group; 0. 2 μ mol/L cyclosporin A + 20 μ mol/L atractyloside + St.Thomas group and 30 μ mol/L diazoxide+100 μ mol/L 5-HD + St.Thomas group. After control perfusion in modified Langendorff mode with Krebs-Henseleit bicarbonate buffer for 20 minutes, the isolated rat hearts were infused with different cardioplegia 20 ml. During global ischemia, cardioplegia was reinfused every 30 minutes and hearts were preserved in 4℃. After two hours' global ischemia, the hearts were reperfused with Krebs-Henseleit bicarbonate buffer for 60 minutes. The hemodynamic parameters, coronary flow and the level of lactate dehydrogenase (LDH), creatine kinase (CK), aspartate aminotransferase (AST) in coronary sinus venous effluent were measured before ischemia and during reperfusion. Myocardial ultrastructures were observed under electronmicroscope.Results Before myocardial ischemia, there were no significant differences among all above mentioned parameters in all groups. After ischemia, hyperkalemic cardioplegic solution enriched with mitochondrial permeability transition pore inhibitor cyclosporin A or ATP-sensitive K⁺ channel opener diazoxide improved significantly recovery of cardiac function, increased coronary flow and decreased the level of LDH, CK and AST in coronary sinus venous effluent (P<0.05), and myocardial ultrastructural changes were better than those of the hyperkalemic cardioplegic solution. However, atractyloside and 5-HD attenuated their effects (P<0.05).Conclusion As an adjunct in hyperkalemic cardioplegic solution, mitochondrial permeability transition pore inhibitor cyclosporin A andATP-sensitive K⁺ channel opener diazoxide can significantly improve the myocardial protection on adult rat hearts.Part 2Section AThe cardioprotection of mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on rat ventricular myocytesObjective To study the cardioprotective mechanism of hyperkalemic cardioplegic solution enriched with mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on rat ventricular myocytes.Methods Isolated cardiac myocytes were prepared by enzymatic dissociation from adult male Sprague-Dawley rat hearts during retrograde perfusion using a modified Langendorff technique. Isolated cardiac myocytes were randomly divided into 6 groups: K-H group; St.Thomas group; 30 μ mol/L diazoxide + St.Thomas group; 0. 2 μ mol/L cyclosporin A + St.Thomas group; 0. 2 μ mol/L cyclosporin A + 20 μ mol/L atractyloside+St.Thomas group and 30 μ mol/L diazoxide+100 μ mol/L 5-HD+St.Thomas group. Cardiac myocytes were preserved indifferent treated cardioplegia for 2 hours at 4℃. Cardioplegia was replaced every 30 minutes. Intracellular Ca²⁺ was measured by the fluorescent dye Fluo-3, and mitochondrial membrane potential (Δ ψ m) was measured by the fluorescent dye tetramethyl-rhodamine ethyl ester (TMRE) and the trypan blue exclusion was used as an index of the isolated ventricular myocyte viability.Results The Fluo-3 fluorescence intensity presented intracellular Ca²⁺ concentration was observed has no significant differences among all groups (P>0.05). It is well known that the MPTP opens will cause collapse of the Δ ψ m. The fluorescent intensity in TMRE-loaded cardiomyocytes was measured and a significantly increased of the fluorescent intensity in K-H group and St.Thomas group were observed, which indicated that the Δ ψ m was depolarized. While hyperkalemic cardioplegic solution enriched with cyclosporin A or diazoxide group prevented the depolarization of Δ ψ m (P<0.05), and co-treated with atractyloside or 5-HD attenuated the effect of cyclosporin A or diazoxide (P<0.05). The protective effects of hyperkalemic cardioplegic solution enriched with cyclosporin A or diazoxide were confirmed by the isolated ventricular myocyte viability. The rate of cell death was 61.7 ± 2.1% in K-H group and 62.0 ± 3.1% in St.Thomas group. Treated with cyclosporin A or diazoxide was significantly decreased the cell death rate to 53.8 ± 3.1% or 54.5±3.2% (P<0.05), respectively, and co-treated with atractyloside or 5-HD attenuated the effect of cyclosporin A or diazoxide, the cell death rate increased to 59.2 ± 3.0% or 59.9±2.5% (P<0.05), respectively.Conclusion Hyperkalemic cardioplegic solution enriched withmitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide could inhibite the MPTP opening and prevent the depolarization of Δ ψ m, significantly improved the myocardial protection of hyperkalemic cardioplegic solution on adult rat hearts, but there was no effect on intracellular Ca²⁺.Section BThe effect of mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on mitochondrial permeability transition of rat heartObjective To study the cardioprotective mechanism of hyperkalemic cardioplegic solution enriched with mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide on rat ventricular myocytes in mitochondrial level.Methods Eighteen adult male Sprague-Dawley rat hearts were randomly divided into 6 groups (3 rats in each group): K-H group; St.Thomas group; 30 μ mol/L diazoxide+St.Thomas group; 0.2 μ mol/L cyclosporin A + St.Thomas group; 0.2 μ mol/L cyclosporin A + 20 μ mol/L atractyloside+St.Thomas group and 30 μ mol/L diazoxide + 100μ mol/L 5-HD+St.Thomas group. After control perfusion in modified Langendorff mode with Krebs-Henseleit bicarbonate buffer for 20 minutes, the isolated rat hearts were infused with different cardioplegia 20ml. During global ischemia, cardioplegia was reinfused every 30 minutes and hearts were preserved in 4℃. After two hours' global ischemia, mitochondria were isolated from rat hearts by differential centrifugation. 200 μ mol/L CaCl₂ was added to induced MPTP opening. The absorbance at 520nm (A520) was measured spectro-photometrically 15 minutes, a decreased at A520 was used to indicate mitochondria swelling as a result of MPTP opening.Results In mitochondria isolated from St.Thomas + 30 μ mol/L diazoxide group and St.Thomas + 0.2 μ mol/L cyclosporin A group, a large decrease at A520 was observed, there was a significant differences compared with those of mitochondria from K-H group and St.Thomas group (P<0.001). Co-treated with 20 μ mol/L atractyloside or 100 μ mol/L 5-HD attenuated the effect of cyclosporin or diazoxide on mitochondria (P<0.001).Conclusion Hyperkalemic cardioplegic solution enriched with mitochondrial permeability transition pore inhibitor cyclosporin A and mitochondrial ATP-sensitive K⁺ channel opener diazoxide could inhibite the MPTP opening, and significantly improve the myocardial protection of hyperkalemic cardioplegic solution on adult rat mitochondrial.