The Roles Of Mitochondria Permeability Transition In Cardiomyocytes Against Anoxia-Reoxygenation Injury

Inhibition of Mitochondrial Permeability Transition Protects Cardiomyocytes Against Anoxia-Reoxygenation InjuryObjectiveMitochondria disfunction play a key role in determining cardiomyocytes fate during exposure to stress. The states of mitochondrial permeability transition pores (mPTP) is greatly associated with the mitochondria disfunction. Mitochondrial permeability transition (MPT) caused by the opening of mPTP is implicated as an important event in the control of cell death and survival and may be involved in both apoptosis and necrosis. The mPTP is considered to include the voltage-dependent anion channel (VDAC, located in mitochondrial outer-membrane), the adenine nucleotide translocator (ANT, across the mitochondrial outer and inner membrane at mitochondrial contact site), and the cydophilin D (CyP-D) in the matrix. Recent studies showed that peripheral benzodiazepine receptor, hexokinasc, and creatine kinase, may also be involved in the mPTP. MPT may act as a "central executioner" of cells subjected to a range of insults (such as oxidative stress, growth factor removal, or exposures to cytokines). MPT causes dissipation of the mitochondrial membrane potential, uncoupling of oxidative phosphorylation, ATP depletion, and equilibration of small solutes and ions between the cytosol and the mitochondrial matrix. Solute influx into the matrix can cause swelling followed by rupture of the outer membrane and consequent efflux of proteins from the intermembrane space, including Cyt c, procaspase 9, apoptosis-inducing factor, and endonuclease G. The regulation of the MPT plays a key role in maintaining the impermeability of the mitochondrial inner membrane to all but a few selected metabolites, thus helping to maintain the membrane potential, which drives ATP synthesis during oxidative phosphorylation.Mounting evidence indicates that MPT may play an important a role in cardiac reperfusion injury. As ischemic time increases, latent susceptibility of mitochondria to MPT increases (the MPT priming component), yet the conditions of reperfusion can modulate whether or not MPT is induced (the MPT trigger component). During the ischemic period, the elevating of Ca²⁺, decreasing of pH, accumulation of long-chain fatty acids and ROS cause MPT. The MPT trigger component, is influenced by the interplay between the MPT inducers/inhibitors present during reperfusion (particularly matrix-free Ca²⁺and ROS levels) and electron transport capacity for regenerating membrane potential. The latter is highly sensitive to the extent of Cyt c loss and IM leakiness occurring during the preceding ischemia. Because PTP open probability is voltage dependent, the rapidity with which electron transport regenerates membrane potentialwhen the PTP transiently closes will play a critical role in determining whether it remains closed or reopens. Intermembrane Cyt c content and IM leakiness are both major determinants of controlling the rate at which electron transport can regenerate and maintain membrane potential, so that Cyt c loss and IM leak, by depressing the membrane potential recovery rate, will increase the probability that a transiently closed mPTP will reopen. In addition, Cyt c depletion increases oxidant stress, promoting mPTP opening, Irreversible depolarization of mitochondria by MPT ultimately induces necrotic cell death by impairing energy production. For the heart to recover from ischemia/reperfusion, its mitochondria must return to full functionality. Our study is to investigate the effect of of inhibition of mitochondrial permeability transition on cardiomyocytes anoxia- reoxygenation injury.Materials and Methods1. Reagents and animal1-to 2-day-old Sprague-Dawley(SD) rats were supplied by the Animal Center of Xuzhou Medical College, China.Cyclosporin A was obtained from Fluka Biochemical company. Mouse monoclonal anti-Cyt c antibody was purchased from Santa Cruz Biotechnology. Anti-mouse IgG and Horseradish peroxidase(HRP)-linked antibody were purchased from Cell signaling Technology. Mitochondria Isolation Kit was purchased from Applygen company, mMHCα/βand TnI were purchased from Fuzhou steps newborn thing technical development company. FITC-Labeled Goat Anti-Mouse IgG and Rhodamine- Labeled Goat Anti-Mouse IgG were purchased from Santa Cruz Biotech. Annexin V-FITC Apoptosis Detection Kit were purchased from Nanjing KeyGen Biotech.2. Primary myocyte-rich cultures of the neonatal rat myocytesVentricles from hearts of 1- to 2-day-old rats were dissociated with trypsin and collagenase. The cells were resuspended in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal calf serum. To selectively enrich the myocytes, dissociated cells were preplated to allow nonmyocytes to attach to the bottom of the culture dish. The resultant suspension of myocytes was transferred onto collagen-coated 90-mm culture dishes. Bromodeoxyuridine (100 mM) was added during the first 24～36 h to prevent proliferation of nonmyocytes3. Experimental protocolsMyocytes were divided into the following five groups: Control (con) group. The myocytes were incubated in Krebs-Ringer-HEPES (KRH) solution with glucose during the entire experimental period. Anoxia- reoxygenation(A/R)group, Myocytes subjected 3 hours anoxia and 2 hours reoxygenation. Cyclosporin A1 (CsA1) group: The myocytes were preincubated with cyclosporin A (1μmo1/1), an inhibitor of mitochondrial permeability transition, at 10 min before anoxia. Cyclosporin A2 (CsA2) group: The myocytes were incubated with cyclosporin A at 10 min before reoxygenation. Cyclosporin A3 (CsA3) group: The myocytes were incubated with cyclosporin A at 10 min after anoxia.4. Apoptosis was detected by flow cytometry Cyt c release from mitochondria into the cytosol was measured by Western blotting analysis. Calcein fluorescence(reflecting the opening of mPTP) was determined by using confocal microscopy.5. Statistical analysisStatistical analysis was performed by one-way analysis of variance, Least significant difference(LSD) was applied to test for the differences between individual groups. P＜0.05 was considered statistically significant.ResultsApoptosis index in CsA1,CsA2 group was lower than that in A/R group (P＜0.05). There were no significant diffierence between CsA3 and A/R group. The content of Cyt c in cytosol in CsA1 and CsA2 groupwas lower than that in A/R group (P＜0.05). The mitochondrial content of Cyt c in CsA1 and CsA2 group was higher than that in group A/R group (P＜0.05). There were no significant diffierence between CsA3 and A/R group. Green calcein fluorescence was maintained inside mitochondria in CsA1 and CsA2 group, while calcein release to the cytosol in A/R and CsA3 group.Conclusions1. Treatment of CsA before anoxia and reoxgenation can reduce cardiomyocytes apoptosis, maintained the content ofmitochondria Cyt c.2. Inhibition of MPT before anoxia and reoxgenation can mitigate cardiomyocytes anoxia-reoxygenation injury.3. Treatment of CsA after reoxygenation can not inhibit MPT, which is in irreversible state. The Involvement of Peripheral Benzodiazepine Ligallds in the Regulation of Rat Cardiac Mitochondria Permcability TransitionObjectiveThe peripheral benzodiazepine receptors (PBR) was discovered as benzodiazepine binding sites outside the CNS. Thereafter, PBR were found to be abundant in the cardiovascular system of rats. The structure and function of PBR is different from the central benzodiazepine receptor (CBR), which is coupled to GABA receptors and responsible to the classical sedative, anxiolytic and anticonvulsant effect. PBR is an evolutionarily conserved 18-kDa protein, which is highly hydrophobic, possesses five transmembrane domains a 169-amino acid protein associated with the the outer mitochondrial membrane within the cell. The mitochondria are well known as sites of electron transport and generators of cellular ATP. Because of their high level of expression in the mitochondria, initial studies of PBR function revolved around its effect on mitochondrial respiration. PBR ligands PK11195, Ro5-4864 and related compounds alter mitochondrial respiration with potencies correlated with their affinities for the PBR. Subsequent studies indicated that the PBR can regulate mitochondrial swelling, and are involved in succinate-Cyt c oxidoreductase (siteâ…¡) activity. PBR has been suggested to be involved in numerous physiological processes, such as steroid production, mitochondrial respiration, and ion channel activities,immunomodulation, porphyrin transport, heme biosynthesis, apoptosis and cell proliferation and in the regulation of responses to stress. Recently, PBR is reported to regulate the opening of the MPTP by directly modulating VDAC/ANT dynamics. Alternatively, PBR may modulate VDAC or ANT interaction with other regulators such as the Bcl-2 family proteins. Based on the entropy-driven and enthalpy-driven nature of ligand receptor interactions, Isoquinoline carboxamide PK11195 has been classified as an antagonist, the benzodiazepine Ro5-4864 (7-chloro-5- (4-chlorophenyl)-1, 3-dihydro-1-methyl-2H-1, 4-benzodiazepin-2-one) an agonist with specificity for the PBR. In the current study, Based on these considerations, we decided to investigate the role of PK11195 and Ro5-4864 in the regulation of cardiac MPT. Part OneThe Effect of Peripheral Benzodiazepine Receptor Antagonist PK11195 on the Regulation of Rat Cardiac Mitochondria Permeability TransitionMaterials and Methods1. Reagents and animal1- (2-Chlorophenyl-N-methyl-1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and Subtilisin Carlsberg were purchased from the Sigma company. Cyclosporin A was obtained from Fluka Biochemical company. Mouse monoclonal anti-Cyt c antibody was purchased from Santa Cruz Biotechnology. Anti-mouse IgG and Horseradish peroxidase (HRP) - linked antibody were purchased from Cell signaling Technology. 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) was purchased from Molecular Probes. Male Sprague-Dawley(SD) rats weighing 200～250g were supplied by the Animal Center of Xuzhou Medical College.2. Preparation of rat heart mitochondriaRat's hearts were removed, homogenized in ice-cold buffer and incubated on ice for 5 min. mitochondria were separated and purified by differential centrifugation. Mitochondrial pellets were resuspended in buffer and kept in ice for the rest of experiement. All mitochondrial isolation procedures were carried out at 4℃. Protein concentration was determined by modified Bradford's method, using BSA as the standard.3. Experimental protocolsDifferent concentration of PK11195 (50, 100, 200μM) was added into incubation buffer, respectively. In additional experiments, 0.2μM cyclosporin A, an inhibitor of MPT was added 5 minutes before the addition of 100M PK11195. Calcium (150μM) induced MPT opening as positive control group.4. Determination of mitochondrial permeability transitionThe MPT was detected by the change of absorbance after chemical addition with spectrophotometer. MPT causes mitochondrial swelling, and a decrease in absorbance at 520 nm (Abs520 nm). Fresh heart mitochondria were added to a buffer containing 300 mM sucrose, 5 mM succinate and 10 mM MOPS, pH 7.4 with Tris, and the final volume is 1.0 ml and protein concentration is 1 mg/ml. The reference cuvette has the same buffer without mitochondria. The change of absorbance was measured for 10 min with spectrophotometer at 520 nm.5. Electron microscopy microscope analysisThe mitochondria were fixed with 2.5% glutaraldehyde and were postfixed in 1% cacodylate-buffered osmium tetroxide, and observed with H-600 transmission electron microscope at a magnification of x8 000.6. Mitochondrial Cyt c releaseThe mitochondrial pellet and supernatant was separated by centrifugation. Cyt c content in the pellet and supernatant was determined using Western Blotting. The bands were detected by NBT/BCIP, Laser scanning densitometry was used for the semi-quantitative determination of the proteins.7. Mitochondrial membrane potentialThe changes in mitochondrial membrane potential were monitored with the dye. The intensity of fluorescence was determined using a laser scanning confocal microscope. JC-1 monomer emits green fluorescence, JC-1 aggregate emits red fluorescence. The ratio of red to green fluorescence for each region was calculated. The decreased ratio was interpreted as a decrease in mitochondrial membrane potential.8. Statistical analysis Statistical analysis was performed by one-way analysis of variance, Newman-Keuls was applied to test for the differences between individual groups. P＜0.05 was considered statistically significant.Results1. Effects of PK11195 on mitochondrial permeability transitionTreatment mitochondria with 50, 100, or 200μM PK11195 led to a significant decrease in absorbance at 520 nm compared to non-chemical treated mitochondria (control group). Cyclosporin A (0.2μM) prevented MPT induced by PK11195 (100μM).2. Effects of PK11195 on ultrastructural observation of mitochondriaThe mitochondrial samples were observed with a transmission electron microscope. Control group showed well-preserved mitochondria with an electron-dense matrix and well-arranged cristae. In the specimens treated with Ca²⁺ or 100μM PK11195, most of mitochondria showed morphological change. In particular, they showed swelling, hypertrophy, cristolysis, and matrix dilution. In the presence of 100μM PK11195 plus cyclosporin A, the most of mitochondria displayed the characteristic ultrastructure of the intact organelle.3. Effects of PK11195 on Cyt c release from mitochondrialPK11195 resulted in the translocation of Cyt c from the mitochondria to the cytosol. Compared to untreated mitochondria, PK11195 (100μM) treatment markedly decreased Cyt c content in mitochondrial pellets (P＜0.01), whereas the content of Cyt c in cytosol was significantly increased (P＜0.01). Cyclosporin A treatment prevented Cyt c loss from mitochondria induced by PK11195 (P＜0.05).4. Effects of PK11195 on mitochondrial membrane potentialThe exposure of mitochondria to PK11195 caused dissipation of mitoc ondrial membrane potential significantly. Mitochondrial membrane potential in 100μM PK11195 treated mitochondria is 0.34±0.09, much lower than that in untreated group (0.94±0.23) (P＜0.01). The mitochondrial membrane potential was maintained in PK 11195 with cyclosporin A (0.62±0.13) and higher than that in 100μM group.Conclusions1. PBR antagonist PK11195 induced MPT without any calcium addition.2. PBR antagonist PK11195 induce mitochondrial permeability transition in a concentration- dependent manner.3. PBR antagonist PK11195caused mitochondria ultrastructural abnormalities and Cyt c release via induction of MPT.4. Modulation of peripheral benzodiazepine receptor may represent an novel way to regulate MPT during pathophysiological condition. Part TwoThe Effect of Peripheral Benzodiazepine Receptor Agonist Ro5-4864 on the Regulation of Rat Cardiac Mitochondria Permeability TransitionMaterials and Methods1. Reagents and animal7-chloro-5-(4-chlorophenyl)-1, 3-dihydro-1-methy1-2H-1,4-benzodiazepin-2-one (Ro5-4864) and Atractyloside(ATR) were obtained from the Sigma company. Animal and other reagents are the same as that in part one.2. Preparation of rat heart mitochondriaThe same as that in part one.3. Experimental protocolsMitochondria were incubated with 50, 100, 200μmol/L Ro5-4864 (50, 100, 200μM group). In additional experiments, ATR(20μmol/L), an opener of MPT was added 5 minutes before the addition of 100μmol/L Ro5-4864 (ATR group). Swelling was initiated by addition of 150μmol/L Ca²⁺ as positive control group (Ca²⁺ group), negative control group (Con group) was given none treatment.4. Determination of mitochondrial permeability transitionThe same as that in part one.5. Electron microscopy microscope analysisThe same as that in part one.6. Mitochondrial Cyt c releaseThe same as that in part one. 7. Mitochondrial Membrane PotentialThe same as that in part one.8. Statistical AnalysisThe same as that in part one.Results1. Cardiac mitochondrial swellingThe exposure of mitochondria to 50, 100, or 200μmol/L Ro5-4864 significantly inhibited the decrease of absorbance at 520 nm compared to that in Ca²⁺ group (P＜0.01, P＜0.05). Compared with 50μM group, The ratio of decreasing of absorbance at 520 nm in 100μM and 200μM group were higher(P＜0.05), but there were no significantly difference between 100μM and 200μM group(P＞0.05). ATR, an opener of mPTP, abolished the effect of 100μmol/L Ro5-4864 against mitochondria swelling, there was significantly difference between 100μM group and ATR+Ro group(P＜0.01).2. Ultrastructural observation of PBR antagonists Ro5-4864 exposed mitochondriaControl specimens showed well-preserved mitochondria with an electron-dense matrix and well-arranged cristae. In the specimens treated with Ca²⁺ the majority of mitochondria were grossly damaged. In particular, they showed swelling, hypertrophy, cristolysis, and matrix dilution. In the presence of 100μM Ro5-4864, the most of mitochondria displayed the characteristic ultrastructure of the intact organelle. The mitochondrial ultrastructure abnormality in ATR+Ro group is similar to Ca²⁺ group.3. Mitochondrial Cyt c releaseCa²⁺ resulted in the translocation of Cyt c from the mitochondrial pellet to the cytosol. The release of mitochondrial Cyt c was significantly inhibited by Ro5-4864, while the effect of Ro5-4864 was abolished by ATR, an opener of mPTP. Compared with Ca²⁺ group, the Cyt c in cytosol in 100μM group was lower(P＜0.05), while the Cyt c in mitochondria was higher(P＜0.05). In ATR+Ro group, the Cyt c in cytosol was higher(P＜0.05), the Cyt c in mitochondria was lower(P＜0.05), compared with 100μM group.4. Effects of PBR agonists Ro5-4864 on mitochondrial membrane potentialThe exposure of mitochondria to Ca²⁺ caused dissipation of mitochondrial membrane potential, while this effect was prevented by Ro5-4864. ATR(an opener of mPTP) abolished the effect of Ro5-4864 against dissipation of mitochondrial membrane potential. Mitochondrial membrane potential in 100μM Ro5-4864 group (0.62±0.13) is higher than that in Ca²⁺ group(0.25±0.07) and ATR +Ro group (0.31±0.09) (P＜0.05).Conclusions1. PBR agonists Ro5-4864 inhibit MPT.2. Ro5-4864 resultes in a significant protection against mitochondria injury induced by Ca²⁺.3. Ro5-4864 prevent the release of mitochondria Cyt c and dissipation of miotochondria potential in part via induction of MPT.4. PBR involves in the regulation of MPT, represent a novel therapeutic target against cardiac mitochondrial damage.