Mitochondria-derived superoxide links to tourniquet-induced apoptosis in mouse skeletal muscle

Our previous study has reported that superoxide mediates ischemia-reperfusion (IR)-induced necrosis in mouse skeletal muscle. However, it remains poorly understood whether IR induces apoptosis and what factors are involved in IR-induced apoptosis in skeletal muscle. Using a murine model of tourniquet-induced hindlimb IR, we investigated the relationship between mitochondrial dysfunction and apoptosis in skeletal muscle. Hindlimbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Compared to sham treatment, tourniquet-induced IR significantly elevated mitochondria-derived superoxide production, activated opening of mitochondrial permeability transition pore (mPTP), and caused apoptosis in the gastrocnemius muscles. Pretreatment with a superoxide dismutase mimetic (tempol, 50 mg/kg) or a mitochondrial antioxidant (co-enzyme Q(10), 50 mg/kg) not only decreased mitochondria-derived superoxide production, but also inhibited mPTP opening and apoptosis in the IR gastrocnemius muscles. Additionally, an inhibitor of mPTP (cyclosporine A, 50 mg/kg) also inhibited both mPTP opening and apoptosis in the IR gastrocnemius muscles. These results suggest that mitochondria-derived superoxide overproduction triggers the mPTP opening and subsequently causes apoptosis in tourniquet-induced hindlimb IR.     

Lycopene Protects against Hypoxia/Reoxygenation-Induced Apoptosis by Preventing Mitochondrial Dysfunction in Primary Neonatal Mouse Cardiomyocytes

Background

Hypoxia/reoxygenation(H/R)-induced apoptosis of cardiomyocytes plays an important role in myocardial injury. Lycopene is a potent antioxidant carotenoid that has been shown to have protective properties on cardiovascular system. The aim of the present study is to investigate the potential for lycopene to protect the cardiomyocytes exposed to H/R. Moreover, the effect on mitochondrial function upon lycopene exposure was assessed.

Methods and Findings

Primary cardiomyocytes were isolated from neonatal mouse and established an in vitro model of H/R which resembles ischemia/reperfusion in vivo. The pretreatment of cardiomyocytes with 5 µM lycopene significantly reduced the extent of apoptosis detected by TUNEL assays. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and the process of mitochondria-mediated apoptosis were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of the mitochondrial permeability transition pore (mPTP) by reducing the intracellular reactive oxygen species (ROS) levels and inhibiting the increase of malondialdehyde (MDA) levels caused by H/R. Moreover, the loss of mitochondrial membrane potential, a decline in cellular ATP levels, a reduction in the amount of cytochrome c translocated to the cytoplasm and caspase-3 activation were observed in lycopene-treated cultures.

Conclusion

The present results suggested that lycopene possesses great pharmacological potential in protecting against H/R-induced apoptosis. Importantly, the protective effects of lycopene may be attributed to its roles in improving mitochondrial function in H/R-treated cardiomyocytes.     

5-Hydroxydecanoate Abolishes Cardioprotective Effects of a Structural Analogue of Apelin-12 in Ischemia/Reperfusion Injury

Chemically modified peptide apelin-12 (MA) with enhanced resistance to degradation by proteolytic enzymes is able to protect the heart against myocardial ischemia and reperfusion. This study was aimed to explore the role of mitochondrial ATP-sensitive K⁺-channels (mitoKATP) in effects of MA on myocardial energy state and membrane integrity in ischemia/reperfusion (I/R) injury. Isolated perfused working rat hearts were used to simulate global ischemia and reperfusion. Acute myocardial infarction was induced by coronary artery occlusion followed by restoration of coronary blood flow in anesthetized rats. Myocardial infarct size and cardiac dysfunction were used as indices of I/R injury at the end of reperfusion. Co-infusion of 5-hydroxydecanoate (5HD), the mitoKATP blocker, along with MA before ischemia significantly decreased functional recovery of isolated hearts as compared to administration of MA alone. These effects were accompanied by increased LDH release in the myocardial effluent, reduced restoration of myocardial ATP, AN, Cr, adenylate energy charge (AEC), and lactate accumulation. Coadministration of 5HD and MA at the onset of reperfusion substantially reduced infarct-limiting effect of the peptide in rats in vivo and increased the plasma LDH and CK-MB activity compared with MA treatment. Additionally, 5HD abolished MA influence on the metabolic state of the area at risk (AAR) at the end of reperfusion. In this case, the contents of metabolites and AEC in the AAR did not differ significantly from the values in control. Therefore, restoration of myocardial energy metabolism and sarcolemma integrity via activation of mitoKATP may be of critical importance for MA-induced protection against I/R injury.     

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

Hypercholesterolemia Abrogates the Cardioprotection of Ischemic Postconditioning in Isolated Rat Heart: Roles of Glycogen Synthase Kinase-3β and the Mitochondrial Permeability Transition Pore

Ischemic postconditioning (IPO) reduces lethal reperfusion injury under normal conditions, but its effectiveness in hypercholesterolemia (HC) is disputed. We measured the cardioprotection of IPO in hypercholesterolemic rats and determined the roles of glycogen synthase kinase-3β (GSK-3β) and the mitochondrial permeability transition pore (mPTP). Isolated rat hearts underwent 30-min global ischemia and 120-min reperfusion. Postconditioning protocol induced six cycles of 10s ischemia and 10s reperfusion at the onset of the reperfusion. Myocardial infarct size was estimated by triphenyltetrazolium chloride staining and cardiomyocyte apoptosis was assessed by TUNEL staining. GSK-3β phosphorylation was measured by immunoblotting. The opening of mPTP was measured by NAD⁺ content in myocardium. In normocholesterolemia (NC) groups, infarct size and cardiomyocyte apoptosis were significantly reduced after IPO. These reductions were completely abolished by HC, as evidenced by a similar infarct size and cardiomyocyte apoptosis observed between the IPO-HC and IR (ischemia–reperfusion)-HC groups. GSK-3β phosphorylation was significantly higher in the IPO-NC than the IPO-HC group. In addition, NAD⁺ content in myocardium, a marker of mPTP opening, was higher in the IPO-NC group than the IPO-HC group. In conclusion, cardioprotection of IPO is blocked by hypercholesterolemia. This might be due to the impairment of phosphorylation of GSK-3β and attenuation of mPTP opening.     

Ischemia reperfusion injury, KATP channels, and exercise-induced cardioprotection against apoptosis

Exercise is a potent stimulus against cardiac ischemia reperfusion (IR) injury, although the protective mechanisms are not completely understood. The study purpose was to examine whether the mitochondrial or sarcolemmal ATP-sensitive potassium channel (mito K(ATP) or sarc K(ATP), respectively) mediates exercise-induced cardioprotection against post-IR cell death and apoptosis. Eighty-six, 4-mo-old male Sprague Dawley rats were randomly assigned to treadmill exercise (Ex; 30 m/min, 3 days, 60 min, ～70 maximal oxygen uptake) and sedentary (Sed) treatments. Rats were exposed to regional cardiac ischemia (50 min) and reperfusion (120 min) or Sham (170 min; no ligation) surgeries. Exercise subgroups received placebo (saline), 5-hydroxydecanoate (5HD; 10 mg/kg ip), or HMR1098 (10 mg/kg ip) to inhibit mito K(ATP) or sarc K(ATP) channel. Comprehensive outcome assessments included post-IR ECG arrhythmias, cardiac tissue necrosis, redox perturbations, and autophagy biomarkers. No arrhythmia differences existed between exercised and sedentary hearts following extended-duration IR (P < 0.05). The sarc K(ATP) channel was confirmed essential (P = 0.002) for prevention of antinecrotic tissue death with exercise (percent infarct, Sed = 42%; Ex = 20%; Ex5HD = 16%; ExHMR = 42%), although neither the mito K(ATP) (P = 0.177) nor sarc K(ATP) (P = 0.274) channel provided post-IR protection against apoptosis (terminal deoxynucleotidyl transferase deoxy UTP-mediated nick-end labeling-positive nuclei/mm(2), Sham = 1.8 ± 0.5; Sed = 19.4 ± 6.7; Ex = 7.5 ± 4.6; Ex5HD = 14.0 ± 3.9; ExHMR = 11.1 ± 1.8). Exercise preconditioning also appears to preserve basal autophagy levels, as assessed by Beclin 1 (P ≤ 0.001), microtubule-associated protein-1 light-chain 3B ratios (P = 0.020), and P62 (P ≤ 0.001), in the hours immediately following IR. Further research is needed to better understand these findings and corresponding redox changes in exercised hearts.     

The role of endogenous nitric oxide in inhibition of ischemia/reperfusion-induced cardiomyocyte apoptosis

The effect of nitric oxide (NO) synthase inhibition on apoptosis of cardiomyocytes during ischemia/reperfusion was investigated. Isolated perfused guinea-pig hearts were subjected to 35 min ischemia (I) followed by 30 min reperfusion (IR) in the presence or absence of NO synthase inhibitors, L-NAME or L-NMMA or a superoxide scavenger, SOD. Apoptosis was assessed by immunohistochemistry (TUNEL assay, Bax protein staining), by spectrophotometric measurement of cytochrome oxidase activity (COX), and by ultrastructural analysis. Inhibition of NOS significantly increased apoptosis with activation of Bax protein and decrease of COX. SOD infusion had a protective effect on these apoptotic markers. The results suggest that endogenous NO synthesis during I/R protects the heart against apoptotic cell death.     

乙酰胆碱对离体大鼠缺血/复灌心脏的保护作用及其机制

目的:探讨乙酰胆碱(ACh)预处理抗心肌缺血复灌(I/R)损伤作用及其与线粒体渗透性转换孔和/或线粒体ATP敏感性钾通道的关系。方法:采用离体大鼠心脏Langendorff灌流方法进行全心停灌30min,复灌120min复制I/R模型。测定心室力学指标和复灌各时间点冠脉流出液中乳酸脱氢酶(LDH)含量。实验结束测定心肌组织formazan含量的变化。结果:与单纯I/R组相比,ACh(0.1μmol/L,5min)预处理明显提高心肌细胞的formazan含量,降低复灌期间冠脉流出液中LDH含量,明显改善I/R所致的左室发展压、左心室内压最大上升和下降速率、心率与发展压乘积和左室舒张末压力的下降,缓解冠脉流量的减少。线粒体渗透性转换孔开放剂苍术苷(20μmol/L,复灌前给药20min)和线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,缺血前给药20min)能明显减弱ACh的保护作用。结论:在大鼠离体心脏灌流模型上,ACh预处理具有抗心脏缺血/复灌损伤的作用,这种保护作用可能与其抑制线粒体渗透性转换孔的开放和促进线粒体ATP敏感性钾通道的开放有关。     

Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O₂ during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X_L in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X_L, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Catecholamine-induced cardiac mitochondrial dysfunction and mPTP opening: protective effect of curcumin

The present study was designed to characterize the mitochondrial dysfunction induced by catecholamines and to investigate whether curcumin, a natural antioxidant, induces cardioprotective effects against catecholamine-induced cardiotoxicity by preserving mitochondrial function. Because mitochondria play a central role in ischemia and oxidative stress, we hypothesized that mitochondrial dysfunction is involved in catecholamine toxicity and in the potential protective effects of curcumin. Male Wistar rats received subcutaneous injection of 150 mg·kg(-1)·day(-1) isoprenaline (ISO) for two consecutive days with or without pretreatment with 60 mg·kg(-1)·day(-1) curcumin. Twenty four hours after, cardiac tissues were examined for apoptosis and oxidative stress. Expression of proteins involved in mitochondrial biogenesis and function were measured by real-time RT-PCR. Isolated mitochondria and permeabilized cardiac fibers were used for swelling and mitochondrial function experiments, respectively. Mitochondrial morphology and permeability transition pore (mPTP) opening were assessed by fluorescence in isolated cardiomyocytes. ISO treatment induced cell damage, oxidative stress, and apoptosis that were prevented by curcumin. Moreover, mitochondria seem to play an important role in these effects as respiration and mitochondrial swelling were increased following ISO treatment, these effects being again prevented by curcumin. Importantly, curcumin completely prevented the ISO-induced increase in mPTP calcium susceptibility in isolated cardiomyocytes without affecting mitochondrial biogenesis and mitochondrial network dynamic. The results unravel the importance of mitochondrial dysfunction in isoprenaline-induced cardiotoxicity as well as a new cardioprotective effect of curcumin through prevention of mitochondrial damage and mPTP opening.     

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

Mitochondria are targets for geranylgeranylacetone-induced cardioprotection against ischemia-reperfusion in the rat heart

It has been shown that orally administered geranylgeranylacetone (GGA), an anti-ulcer drug, induces expression of heat shock protein 72 (HSP72) and provides protection against ischemia-reperfusion in rat hearts. The underlying protective mechanisms, however, remain unknown. Mitochondria have been shown to be a selective target for heat stress-induced cardioprotection. Therefore, we hypothesized that preservation of mitochondrial function, owing to an opening of a putative channel in the inner mitochondrial membrane, the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, could be involved in GGA- or heat stress-induced cardioprotection against ischemia-reperfusion. Rats were treated with oral GGA or vehicle. Twenty-four hours later, each heart was isolated and perfused with a Langendorff apparatus. GGA-treated hearts showed better functional recovery, and less creatine kinase was released during a 30-min reperfusion period, after 20 min of no-flow ischemia. Concomitant perfusion with 5-hydroxydecanoate (5-HD, 100 microM) or glibenclamide (10 microM) abolished the GGA-induced cardioprotective effect. GGA also showed preserved mitochondrial respiratory function, isolated at the end of the reperfusion period, which was abolished with 5-HD treatment. GGA prevented destruction of the mitochondrial structure by ischemia-reperfusion, as shown by electron microscopy. In cultured cardiomyocytes, GGA induced HSP72 expression and resulted in less damage to cells, including less apoptosis in response to hypoxia-reoxygenation. Treatment with 5-HD abolished the GGA-induced cardioprotective effects but did not affect HSP72 expression. Our results indicate that preserved mitochondrial respiratory function, owing to GGA-induced HSP72 expression, may, at least in part, have a role in cardioprotection against ischemia-reperfusion. These processes may involve opening of the mitoK(ATP) channel.     

Reversible Cyclosporin A-sensitive Mitochondrial Depolarization Occurs within Minutes of Stroke Onset in Mouse Somatosensory Cortex in Vivo: A TWO-PHOTON IMAGING STUDY*

Neuronal structure and function are rapidly damaged during global ischemia but can in part recover during reperfusion. Despite apparent recovery in the hours/days following an ischemic episode, delayed cell death can be initiated, making it important to understand how initial ischemic events affect potential mediators of apoptosis. Mitochondrial dysfunction and the opening of the mitochondrial permeability transition pore (mPTP) are proposed to link ischemic ionic imbalance to mitochondrially mediated cell death pathways. Using two-photon microscopy, we monitored mitochondrial transmembrane potential (Δψ_m) in vivo within the somatosensory cortex during ischemia and reperfusion in a mouse global ischemia model. Our results indicated a synchronous loss of Δψ_m within 1–3 min of ischemic onset that was linked to within seconds of plasma membrane potential (Δψ_p) depolarization. Δψ_m recovered rapidly upon reperfusion, and no delayed depolarization was observed over 2 h. Cyclosporin A treatment largely blocked Δψ_m collapse during ischemia, suggesting a role for the mPTP. Blocking Δψ_m depolarization did not affect structural damage to dendrites, indicating that the opening of the mPTP and damage to dendrites are separable pathways that are activated during Δψ_p depolarization. Our findings using in vivo imaging suggest that mitochondrial dysfunction and specifically the activation of the mPTP are early reversible events during brain ischemia that could trigger delayed cell death.     

Diazoxide-induced cardioprotection via DeltaPsim loss depending on timing of application

Although the role of mitochondrial ATP-sensitive potassium (mitoKATP) channels in cardioprotection is widely accepted, it remains unclear when their opening is critical for protection. We tested the hypothesis that the mitoKATP channel acts as a trigger or mediator of protection against apoptosis through loss of mitochondrial inner membrane potential (DeltaPsim). Exposure of neonatal rat cardiomyocytes to H2O2 (0.5 mmol/L) resulted in apoptosis associated with severe DeltaPsim loss. Pretreatment with diazoxide (20 to 100 micromol/L) prevented H2O2-induced apoptosis and DeltaPsim loss at 2 but not 18 h after exposure, while the latter was prevented by cotreatment with diazoxide. Lack of protection by pretreatment with diazoxide was observed in cardiomyocytes cultured in a medium containing H2O2 for 2 h and then not containing for 16 h. The slopes of the regression lines of the relationship between the proportion of apoptotic cells and DeltaPsim loss (y = -0.89 vs. -0.42) and the proportion of cells with high side scatter signal differed between cardiomyocytes exposed H2O2 for 2 and 18 h. Diazoxide per se caused a transient DeltaPsim loss (within 30 min) with a recovery followed by persistent DeltaPsim loss (after 6 h). Inhibition of the former by 5-hydroxydecanoate (5-HD, 0.5 mmol/L) abolished protection of pretreatment with diazoxide (trigger phase), while that of the latter prevented the protection of cotreatment with diazoxide (mediator phase). Our results suggest that mitoKATP channels act as a trigger and mediator of cardioprotection through a transient or persistent DeltaPsim loss depending on phenotypic consequence in response to oxidants.     

Long non-coding RNA KCNQ1OT1/microRNA-204-5p/LGALS3 axis regulates myocardial ischemia/reperfusion injury in mice

Myocardial ischemia/reperfusion (IR) injury is one of the most prevalent cardiovascular diseases, known for its high mortality and morbidity worldwide. Based on pre-existing evidence, LGALS3 has been found to be closely associated with cardiac diseases. Hence, the objective of our study is to explore the potential function of KCNQ1OT1/microRNA-204-5p (miR-204-5p)/ LGALS3 axis on myocardial IR injury and the underlying mechanism. A myocardial IR injury mouse model was established in vivo and an in vitro cardiomyocyte model was induced by hypoxia/Reoxygenation exposure. Next, gain- and loss-of-function experiments were employed in order to measure the viability and apoptosis of cardiomyocytes and the area of ischemic infarct by CCK-8, TUNEL staining and Evans blue/TTC double staining. LGALS3 was found to be highly expressed in myocardial IR injury. The downregulation of LGALS3 resulted in the alleviation of myocardial IR injury in mouse models. In addition, KCNQ1OT1 could promote the LGALS3 expression by binding to miR-204-5p, which led to aggravated myocardial IR injury. In conclusion, KCNQ1OT1 binds to miR-204-5p to exacerbate myocardial IR injury in mice through the up-regulation of LGALS3, providing a novel insight for myocardial IR injury treatment.     

Cardioprotection of the aged rat heart by GSK-3beta inhibitor is attenuated: age-related changes in mitochondrial permeability transition pore modulation

It is well established that inhibition of glycogen synthase kinase (GSK)-3β in the young adult myocardium protects against ischemia-reperfusion (I/R) injury through inhibition of mitochondrial permeability transition pore (mPTP) opening. Here, we investigated age-associated differences in the ability of GSK-3β inhibitor [SB-216763 (SB)] to protect the heart and to modulate mPTP opening during I/R injury. Fischer 344 male rats were assigned from their respective young or old age groups. Animals were subjected to 30 min ischemia following 120 min reperfusion to determine myocardial infarction (MI) size in vivo. Ischemic tissues were collected 10 min after reperfusion for nicotinamide adenine dinucleotide (NAD(+)) measurements and immunoblotting. In parallel experiments, ventricular myocytes isolated from young or old rats were exposed to oxidative stress through generation of reactive oxygen species (ROS), and mPTP opening times were measured by using confocal microscopy. Our results showed that SB decreased MI in young SB-treated rats compared with young untreated I/R animals, whereas SB failed to significantly affect MI in the old animals. SB also significantly increased GSK-3β phosphorylation in young rats, but phosphorylation levels were already highly elevated in old control groups. There were no significant differences observed between SB-treated and untreated old animals. NAD(+) levels were better maintained in young SB-treated animals compared with the young untreated group during I/R, but this relative improvement was not observed in old animals. SB also significantly prolonged the time to mPTP opening induced by ROS in young cardiomyocytes, but not in aged cardiomyocytes. These results demonstrate that this GSK-3β inhibitor fails to protect the aged myocardium in response to I/R injury or prevent mPTP opening following a rise in ROS and suggest that healthy aging alters mPTP regulation by GSK-3β.     

Nutrient restriction preserves calcium cycling and mitochondrial function in cardiac myocytes during ischemia and reperfusion

Nutrient restriction (NR) prolongs longevity via enhanced mitochondrial function. We tested the hypothesis that NR enhances resistance to ischemia/reperfusion (IR) arrhythmias via preserved calcium (Ca) cycling and mitochondrial function. We examined the protective effects of NR on regional IR in cultured neonatal rat ventricular myocyte monolayers. Optical mapping of intracellular Ca and mitochondrial membrane potential Δψ(m) was performed using Rhod 2-AM and TMRE, respectively. Regional ischemia was mimicked by covering a portion of monolayer with a glass coverslip until loss of Ca propagation, and reperfusion was mimicked by removing the coverslip. NR was mimicked by culture in serum- and glucose-free medium for 24 h. Relative to controls, NR monolayers: (1) sustained Ca oscillations during longer periods of ischemia (19.2 ± 1.8 min vs 10.4 ± 1.4 min, p<0.001); (2) had attenuated increases in Ca transient duration (CaD) and time decay constant (Tau) during ischemia; (3) had preserved conduction velocity (CV) during early reperfusion, leading to protection against reperfusion arrhythmias; (4) had minimal "rebound" decreased CaD and Tau during reperfusion; and (5) had no depolarization of Δψ(m) during IR. NR attenuates IR arrhythmias via (1) stable calcium cycling and (2) prevention of Δψ(m) depolarization during IR. Enhanced mitochondrial resistance to IR arrhythmias may play a role in NR-induced longevity prolongation.     

Role of mitochondrial permeability transition pore and mitochondrial ATP-sensitive potassium channels in the protective effects of ischemic preconditioning in isolated hearts from fed and fasted rats

The aim of the present study was to assess whether the protective effects of ischemic preconditioning (PC) are associated with activation of the mitochondrial ATP-sensitive potassium channels (mitoKATP) and if there is any relationship between the activity of these channels and the mitochondrial permeability transition pore (MPTP) opening in ischemic-reperfused rat hearts under different nutritional conditions. Langendorff-perfused hearts of fed and 24-h fasted rats were exposed to 25 min of no-flow global ischemia plus 30 min of reperfusion. Fasting accelerated functional recovery and attenuated MPTP opening. The mitoKATP blocker, 5-hydroxydecanoic (HD), did not influence functional recovery and MPTP opening induced by ischemia–reperfusion in the fed hearts but partially reversed the beneficial effects of fasting. PC and the mitoKATP opener, diazoxide (DZ), improved functional recovery, preserved cell viability, and inhibited MPTP opening in both fed and fasted hearts. The protection elicited by PC and DZ on contractile recovery and MPTP opening was reversed by HD, which did not affect cell viability. Altogether, these results argue for a role of mitoKATP and its impact on preservation mitochondrial inner membrane permeability as a relevant factor in the improvement of contractile function in the ischemic-reperfused rat heart. They also suggest that the functional protection elicited by PC may be related to this mechanism.     

Chloride channel inhibition prevents ROS-dependent apoptosis induced by ischemia-reperfusion in mouse cardiomyocytes.

Apoptosis of cardiomyocytes following ischemia and Apoptosis of cardiomyocytes following ischemia and known about the mechanism by which it is induced. Recently, essential roles of a Cl- channel whose activity triggers the apoptotic volume decrease and of reactive oxygen species (ROS) in activation of this channel have been identified in mitochondrion-mediated apoptosis. Therefore, in this study, involvement of Cl- channels and ROS in apoptosis was studied in primary mouse cardiomyocyte cultures subjected to ischemia-reperfusion. Apoptotic cell death as measured by caspase-3 activation, chromatin condensation, DNA laddering, and cell viability reduction was observed tens of hours after reperfusion but never immediately after ischemia. A non-selective Cl-channel blocker (DIDS or NPPB) rescued cells from apoptotic death when applied during the reperfusion, but not ischemia, period. Another blocker relatively specific to the volume-sensitive outwardly rectifying (VSOR) Cl-channel (phloretin) was also effective in protecting ischemic cardiomyocytes from apoptosis induced by reperfusion. A profound increase in intracellular ROS was detected in cardiomyocytes during the reperfusion, but not ischemia, period. Scavengers for ROS, H2O2 and superoxide all inhibited apoptosis induced by ischemia-reperfusion. Thus, it is concluded that the mechanism by which cardiomyocyte apoptosis is induced by ischemia-reperfusion involves VSOR Cl- channel activity and intracellular ROS production.     

Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro

Activation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoKATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoKATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoKATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoKATP channels was usually below 80 pS ( approximately 70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoKATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 microM) increased the cumulative open probability of the mitoKATP channel previously inhibited by ATP (P < 0.05), but its inactive analog 4alpha-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K+ channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-delta in the IMM. These data provide the first characterization of the human cardiac mitoKATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning.