Activation of the CaR-CSE/H2S pathway confers cardioprotection against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury is a multifactorial process triggered when an organ is subjected to transiently reduced blood supply. The result is a cascade of pathological complications and organ damage due to the production of reactive oxygen species following reperfusion. The present study aims to evaluate the role of activated calcium-sensing receptor (CaR)-cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway in I/R injury. Firstly, an I/R rat model with CSE knockout was constructed. Transthoracic echocardiography, TTC and HE staining were performed to determine the cardiac function of rats following I/R Injury, followed by TUNEL staining observation on apoptosis. Besides, with the attempt to better elucidate how CaR-CSE/H2S affects I/R, in-vitro culture of human coronary artery endothelial cells (HCAECs) was conducted with gadolinium chloride (GdCl3, a CaR agonist), H₂O₂, siRNA against CSE (siCSE), or W7 (a CaM inhibitor). The interaction between CSE and CaM was subsequently detected. Plasma oxidative stress indexes, H2S and CSE, and apoptosis-related proteins were all analyzed following cell apoptosis. We found that H2S elevation led to the improvement whereas CSE knockdown decreased cardiac function in rats with I/R injury. Moreover, oxidative stress injury in I/R rats with CSE knockout was aggravated, while the increased expression of H2S and CSE in the aortic tissues resulted in alleviated the oxidative stress injury. Moreover, increased H2S and CSE levels were found to inhibit cell apoptotic ability in the aortic tissues after I/R injury, thus attenuating oxidative stress injury, accompanied by inhibited expression of apoptosis-related proteins. In HCAECs following oxidative stress treatment, siCSE and CaM inhibitor were observed to reverse the protection of CaR agonist. Coimmunoprecipitation assay revealed the interaction between CSE and CaM. Taken together, all above-mentioned data provides evidence that activation of the CaR-CSE/H2S pathway may confer a potent protective effect in cardiac I/R injury.     

Inhibition of cathepsin S attenuates myocardial ischemia/reperfusion injury by suppressing inflammation and apoptosis

Myocardial ischemia/reperfusion (I/R) injury leads to high mortality and morbidity due to the incomplete understanding of the underlying mechanism and the consequent lack of effective therapy. The present study revealed and validated key candidate genes in relation to inflammation and apoptosis pathways underlying myocardial I/R injury. Cathepsin S was identified as the top hub protein based on the protein–protein interaction analysis, and, thus, its role during myocardial I/R injury was further investigated. Myocardial I/R in mice resulted in significantly increased levels of myocardial injury biomarkers (cardiac troponin I, lactic dehydrogenase, and creatinine kinase‐MB) and inflammatory cytokines (interleukin‐1β [IL‐1β], IL‐6, and tumor necrosis factor‐α), elevated apoptosis rate, and upregulated protein expression of cleaved caspase‐8, cleaved caspase‐3, and cleaved poly ADP‐ribose polymerase. These abovementioned changes were blocked by two different selective cathepsin S inhibitors, LY3000328 or MIV‐247. Moreover, Kaplan–Meier survival plot showed that cathepsin S inhibition improved 21‐day survival rate following myocardial I/R injury. This study demonstrated that the inhibition of cathepsin S alleviated myocardial I/R‐induced injury by suppressing inflammation and apoptosis, which may be used in clinical applications of cardioprotection.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Naoxintong attenuates Ischaemia/reperfusion Injury through inhibiting NLRP3 inflammasome activation

Naoxintong (NXT) is a Chinese Materia Medica standardized product extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinensis, Astragali Radix. Naoxintong is clinically effective in treating ischaemia heart disease. Nucleotide‐binding oligomerization domain‐Like Receptor with a Pyrin domain 3 (NLRP3) inflammasome has been critically involved in myocardial ischaemia/reperfusion (I/R) injury. Here, we have been suggested that NXT might attenuate myocardial I/R injury via suppression of NLRP3 inflammasome activation. Male C57BL6 mice were subjected to myocardial I/R injury via 45 min. coronary ligation and release for the indicated times. Naoxintong (0.7 g/kg/day) and PBS were orally administrated for 2 weeks before surgery. Cardiac function assessed by echocardiography was significantly improved in the NXT group compared to PBS group at day 2 after myocardial I/R. NLRP3 inflammasome activation is crucially involved in the initial inflammatory response after myocardial I/R injury, leading to cleaved caspase‐1, mature interleukin (IL)‐1β production, accompanying by macrophage and neutrophil infiltration. The cardioprotective effect of NXT was associated with a diminished NLRP3 inflammasome activation, decreased pro‐inflammatory macrophage (M1 macrophages) and neutrophil infiltration after myocardial I/R injury. In addition, serum levels of IL‐1β, indicators of NLRP3 inflammasome activation, were also significantly suppressed in the NXT treated group after I/R injury. Naoxintong exerts cardioprotive effects at least partly by suppression of NLRP3 inflammasome activation in this I/R injury model.     

Hydrogen sulfide protects rat lung from ischemia-reperfusion injury

Recent studies have indicated that hydrogen sulfide (H(2)S) is capable of modulating many physiological processes, which prompted us to investigate the potential of H(2)S as a lung protective agent. To explore changes in the generation of endogenous H(2)S and the role of H(2)S in the pathogenesis of pulmonary ischemia-reperfusion (I/R) injury in rats, we built an isolated rat lung I/R model. Lungs were subjected to 45 min ischemia followed by reperfusion (45 min) and were pretreated with H(2)S (50 micromol/l or 100 micromol/l) or an irreversible inhibitor of cystathionine-gamma-lyase (CSE), propargylglycine (PPG; 2 mmol/l). We examined indices of lung injury: lung histological change, perfusion flow rate, ratio of lung wet weight to dry weight (w/d), and lung compliance. H(2)S content and CSE protein expression in lung tissues were measured. Malondialdehyde (MDA) content, activities of superoxide dismutase (SOD) and catalase (CAT), and restraint of superoxide anion (O(2)(-)) production in lung tissues were measured to reflect oxidative stress. In the current study, we demonstrated that H(2)S content and CSE activity in lungs after I/R were significantly higher than those in the control group. Preperfusion with H(2)S attenuated the lung I/R injury while preperfusion with PPG aggravated the lung I/R injury. H(2)S preperfusion reduced I/R-induced MDA production and potentiated SOD and CAT activities and the restraint of O(2)(-) production in the lungs under I/R, which attenuated lung oxidative injury. These findings suggest that endogenous CSE/H(2)S pathway might be involved in the pathogenesis of lung I/R injury and that administration of H(2)S might be of clinical benefit in lung I/R injury.     

Cinaciguat, a novel activator of soluble guanylate cyclase, protects against ischemia/reperfusion injury: role of hydrogen sulfide

Cinaciguat (BAY 58-2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H(2)S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H(2)S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H(2)S generation and a powerful protection against I/R injury in heart.     

Poly (I:C) therapy decreases cerebral ischaemia/reperfusion injury via TLR3‐mediated prevention of Fas/FADD interaction

Toll‐like receptor (TLR)‐mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre‐treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)‐induced protection was lost in TLR3 knockout mice. In poly (I:C)‐treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF‐κB activation in the brain. Poly (I:C) also decreased I/R‐induced apoptosis by attenuation of Fas/FasL‐mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase‐3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)‐induced interaction between Fas and FADD as well as caspase‐3 and ‐8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co‐association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase‐3 and ‐8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.     

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.     

MCU Up‐regulation contributes to myocardial ischemia‐reperfusion Injury through calpain/OPA‐1–mediated mitochondrial fusion/mitophagy Inhibition

Mitochondrial dynamic disorder is involved in myocardial ischemia/reperfusion (I/R) injury. To explore the effect of mitochondrial calcium uniporter (MCU) on mitochondrial dynamic imbalance under I/R and its related signal pathways, a mouse myocardial I/R model and hypoxia/reoxygenation model of mouse cardiomyocytes were established. The expression of MCU during I/R increased and related to myocardial injury, enhancement of mitochondrial fission, inhibition of mitochondrial fusion and mitophagy. Suppressing MCU functions by Ru360 during I/R could reduce myocardial infarction area and cardiomyocyte apoptosis, alleviate mitochondrial fission and restore mitochondrial fusion and mitophagy. However, spermine administration, which could enhance MCU function, deteriorated the above‐mentioned myocardial cell injury and mitochondrial dynamic imbalanced. In addition, up‐regulation of MCU promoted the expression and activation of calpain‐1/2 and down‐regulated the expression of Optic atrophy type 1 (OPA1). Meantime, in transgenic mice (overexpression calpastatin, the endogenous inhibitor of calpain) I/R model and OPA1 knock‐down cultured cell. In I/R models of transgenic mice over‐expressing calpastatin, which is the endogenous inhibitor of calpain, and in H/R models with siOPA1 transfection, inhibition of calpains could enhance mitochondrial fusion and mitophagy, and inhibit excessive mitochondrion fission and apoptosis through OPA1. Therefore, we conclude that during I/R, MCU up‐regulation induces calpain activation, which down‐regulates OPA1, consequently leading to mitochondrial dynamic imbalance.     

High fat diet reduces the expression of miRNA-29b in heart and increases susceptibility of myocardium to ischemia/reperfusion injury

Several studies have shown the role of microRNAs (miRNAs) in myocardial dysfunction in response to ischemia/reperfusion (I/R). In this study, we investigated the impact of high fat (HF) diet in the myocardial susceptibility to I/R injury, as well as in the expression of miRNA-29b. Isolated heart experiments using the ex vivo Langendorff perfusion model were used to induce cardiac I/R injury. HF diet-induced cardiac hypertrophy and impaired cardiac functional recovery after I/R. miRNA-29b, which targets Col1, was reduced in the heart of HF diet-fed mice, whereas the cardiac expression of Col1 was increased. In addition, hypoxia–reoxygenation (H/R) reduced the expression of miRNA-29b in cardiomyoblasts cultures. However, the overexpression of miRNA-29b in cardiomyoblasts reduced p53 mRNA levels and H/R injury, suggesting that downregulation of miRNA-29b may be involved in I/R injury. Together, our findings suggest that the reduced expression of miRNA-29b may be involved in the deteriorated cardiac functional recovery following I/R in obese mice.     

Ozone alleviates ischemia/reperfusion injury by inhibiting mitochondrion‐mediated apoptosis pathway in SH‐SY5Y cells

Cerebral ischemia/reperfusion (I/R) injuries are common and often cause severe complications. Ozone has been applied for protecting I/R injury in animal models of several organs including cerebra, but the detailed mechanism remains unclear. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase measurement were used to determine the influence of ozone on cell activity and damage of SH‐SY5Y cells. Some redox items such as catalase (CAT), malondialdehyde (MDA), glutathione peroxidase (GSH‐Px), and superoxide dismutase (SOD) were measured by enzyme‐linked immunosorbent assay. The mitochondrial membrane potential (ΔΨ_m) was determined by JC‐1 assay. Cytochrome‐c (cyt‐c) level in the cytoplasm and mitochondrion was measured by western blotting. Apoptosis was determined by flow cytometry, and some apoptosis‐related molecules were detected by quantitative real‐time polymerase chain reaction and western blotting. Ozone alleviated oxidative damage by increasing GSH‐Px, SOD, CAT, and decreasing MDA. Ozone decreased mitochondrial damage caused by I/R injury and inhibited the release of cyt‐c from mitochondrion to cytoplasm in SH‐SY5Y cells. The cell apoptosis caused by I/R was inhibited by ozone, and ozone could decrease apoptosis by increasing the ratio of Bcl‐2/Bax and inhibiting caspase signaling pathway in SH‐SY5Y cells. Ozone has the ability of maintaining redox homeostasis, decreasing mitochondrion damage, and inhibiting neurocytes apoptosis induced by I/R. Therefore, ozone may be a promising protective strategy against cerebral I/R injury.     

Sulodexide attenuates endoplasmic reticulum stress induced by myocardial ischaemia/reperfusion by activating the PI3K/Akt pathway

Acute myocardial ischaemia/reperfusion (MI/R) injury causes severe arrhythmias with a high rate of lethality. Extensive research focus on endoplasmic reticulum (ER) stress and its dysfunction which leads to cardiac injury in MI/R Our study evaluated the effects of sulodexide (SDX) on MI/R by establishing MI/R mice models and in vitro oxidative stress models in H9C2 cells. We found that SDX decreases cardiac injury during ischaemia reperfusion and decreased myocardial apoptosis and infarct area, which was paralleled by increased superoxide dismutase and reduced malondialdehyde in mice plasm, increased Bcl‐2 expression, decreased BAX expression in a mouse model of MI/R. In vitro, SDX exerted a protective effect by the suppression of the ER stress which induced by tert‐butyl hydroperoxide (TBHP) treatment. Both of the in vivo and in vitro effects were involved in the phosphatidylinositol 3‐kinase (PI3K)/Akt signalling pathway. Inhibition of PI3K/Akt pathway by specific inhibitor, LY294002, partially reduced the protective effect of SDX. In short, our results suggested that the cardioprotective role of SDX was related to the suppression of ER stress in mice MI/R models and TBHP‐induced H9C2 cell injury which was through the PI3K/Akt signalling pathway.     

Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis,mitochondrial biogenesis and insulin sensitivity

Oncostatin M (OSM) exhibits many unique biological activities by activating Oβ receptor. However, its role in myocardial I/R injury in diabetic mice remains unknown. The involvement of OSM was assessed in diabetic mice which underwent myocardial I/R injury by OSM treatment or genetic deficiency of OSM receptor Oβ. Its mechanism on cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity were further studied. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through inhibition of inositol pyrophosphate 7 (IP7) production, thus activating PI3K/Akt/BAD pathway, decreasing Bax expression while up‐regulating Bcl‐2 expression and decreasing the ratio of Bax to Bcl‐2 in db/db mice. OSM enhanced mitochondrial biogenesis and mitochondrial function in db/db mice subjected to cardiac I/R injury. On the contrary, OSM receptor Oβ knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in cardiac I/R injured diabetic mice. Inhibition of IP7 production by TNP (IP6K inhibitor) exerted similar effects of OSM. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and adenine mononucleotide protein kinase/PGC‐1α pathway. OSM protects against cardiac I/R Injury by regulating apoptosis, insulin sensitivity and mitochondrial biogenesis in diabetic mice through inhibition of IP7 production.     

Hydrogen sulfide protects against acetaminophen-induced acute liver injury by inhibiting apoptosis via the JNK/MAPK signaling pathway

Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic. It can cause hepatotoxicity. Recent studies demonstrated that hydrogen sulfide (H₂S) exhibits cell protection in several cell types. This study was designed to investigate whether H ₂S ameliorated APAP-induced acute liver injury and to elucidate its mechanisms. In this study, we analyzed the detailed biological and molecular processes of APAP-induced hepatotoxicity using a bioinformatics analysis, which showed that apoptosis and the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase pathway were confirmed to play critical roles in these processes. We further investigated the protective effects of H ₂S on APAP-induced hepatotoxicity. In vivo, we observed that the exogenous supplement of H ₂S ameliorated APAP-induced liver injury. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) systems were the endogenous pathway of H ₂S. The expression of CBS/CSE was decreased in APAP-treated mice, while H ₂S could significantly restore it. In addition, APAP-induced JNK activation was inhibited by H ₂S in vivo. In vitro, H ₂S abolished the active effects of APAP on caspase3, Bax, and Bcl-2 expressions as well as JNK phosphorylation in hepatocytes. It was found through flow cytometry that the amount of APAP-induced apoptotic hepatocytes was decreased in the presence of H ₂S. In conclusion, our results suggested that H ₂S attenuated APAP-induced apoptosis in hepatocytes through JNK/MAPK siganaling pathway.     

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.     

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia–reperfusion injury in hypertrophic heart

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91^phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.     

内质网过度应激在肺缺血/再灌注小鼠心肌损伤中的作用

目的：探讨内质网过度应激与肺缺血/再灌注小鼠心肌损伤的关系。方法：雄性健康SPF级C57BL/6J小鼠40只,随机将其分为4组（n=10）：假手术组（Sham组）、肺缺血/再灌注组（I/R组）、ERS通路激动剂衣霉素（TM）组、ERS通路抑制剂4-苯基丁酸（4-PBA）组。采用夹闭左侧肺门30 min再灌注180 min的方法制备肺缺血/再灌注损伤模型。Sham组仅行开胸处理,不夹闭肺门,机械通气210 min;TM组、4-PBA组分别于造模前30 min腹腔注射衣霉素1 mg/kg和4-苯基丁酸400 mg/kg。于再灌注180 min时眼眶取血行心肌酶检测,处死后取心肌组织,行光镜、TUNEL Caspase 3酶活性、RT-PCR和Western blot检测。结果：与Sham组比较,光镜下I/R组、TM组和4-PBA组心肌细胞均有损伤性变化,肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）活性及心肌细胞凋亡指数、天冬氨酸特异性半胱氨酸蛋白酶3（Caspase 3）酶活性升高,p-Jun氨基末端激酶（p-JNK）、Caspase 12、CCAAT增强子结合蛋白同源蛋白（CHOP）、葡萄糖调节蛋白78（GRP78）及其mRNA表达上调（P<0.01）;与I/R组比较,TM组光镜下心肌细胞损伤加重,血清CK-MB、LDH活性及心肌细胞凋亡指数、Caspase 3酶活性升高,p-JNK、Caspase 12、CHOP和GRP78蛋白及mRNA表达增加（P<0.01）,4-PBA组以上指标均下降,光镜下心肌细胞损伤减轻（P<0.01）;与TM组比较,4-PBA组光镜下心肌细胞损伤减轻,血清CK-MB、LDH活性及心肌细胞凋亡指数、Caspase 3酶活性降低,p-JNK、Caspase 12、CHOP和GRP78蛋白及mRNA表达下降（P<0.01）。结论：内质网过度应激参与肺I/R诱发的心肌损伤,抑制内质网过度应激能减轻心脏损伤。