Myocardial reperfusion injury exacerbation due to ALDH2 deficiency is mediated by neutrophil extracellular traps and prevented by leukotriene C4 inhibition.

BACKGROUND AND AIMS: The Glu504Lys polymorphism in the aldehyde dehydrogenase 2 (ALDH2) gene is closely associated with myocardial ischaemia/reperfusion injury (I/RI). The effects of ALDH2 on neutrophil extracellular trap (NET) formation (i.e. NETosis) during I/RI remain unknown. This study aimed to investigate the role of ALDH2 in NETosis in the pathogenesis of myocardial I/RI. METHODS: The mouse model of myocardial I/RI was constructed on wild-type, ALDH2 knockout, peptidylarginine deiminase 4 (Pad4) knockout, and ALDH2/PAD4 double knockout mice. Overall, 308 ST-elevation myocardial infarction patients after primary percutaneous coronary intervention were enrolled in the study. RESULTS: Enhanced NETosis was observed in human neutrophils carrying the ALDH2 genetic mutation and ischaemic myocardium of ALDH2 knockout mice compared with controls. PAD4 knockout or treatment with NETosis-targeting drugs (GSK484, DNase1) substantially attenuated the extent of myocardial damage, particularly in ALDH2 knockout. Mechanistically, ALDH2 deficiency increased damage-associated molecular pattern release and susceptibility to NET-induced damage during myocardial I/RI. ALDH2 deficiency induced NOX2-dependent NETosis via upregulating the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/leukotriene C4 (LTC4) pathway. The Food and Drug Administration-approved LTC4 receptor antagonist pranlukast ameliorated I/RI by inhibiting NETosis in both wild-type and ALDH2 knockout mice. Serum myeloperoxidase-DNA complex and LTC4 levels exhibited the predictive effect on adverse left ventricular remodelling at 6 months after primary percutaneous coronary intervention in ST-elevation myocardial infarction patients. CONCLUSIONS: ALDH2 deficiency exacerbates myocardial I/RI by promoting NETosis via the endoplasmic reticulum stress/microsomal glutathione S-transferase 2/LTC4/NOX2 pathway. This study hints at the role of NETosis in the pathogenesis of myocardial I/RI, and pranlukast might be a potential therapeutic option for attenuating I/RI, particularly in individuals with the ALDH2 mutation.

The role of deubiquitinases in cardiac disease.

Deubiquitinases are a group of proteins that identify and digest monoubiquitin chains or polyubiquitin chains attached to substrate proteins, preventing the substrate protein from being degraded by the ubiquitin-proteasome system. Deubiquitinases regulate cellular autophagy, metabolism and oxidative stress by acting on different substrate proteins. Recent studies have revealed that deubiquitinases act as a critical regulator in various cardiac diseases, and control the onset and progression of cardiac disease through a board range of mechanism. This review summarizes the function of different deubiquitinases in cardiac disease, including cardiac hypertrophy, myocardial infarction and diabetes mellitus-related cardiac disease. Besides, this review briefly recapitulates the role of deubiquitinases modulators in cardiac disease, providing the potential therapeutic targets in the future.

Mitochondrial transplantation combined with coenzyme Q10 induces cardioprotection and mitochondrial improvement in aged male rats with reperfusion injury.

Ischaemic heart diseases (IHD) are among the major causes of mortality in the elderly population. Although timely reperfusion is a common treatment for IHD, it causes additional damage to the ischaemic myocardium known as ischaemia-reperfusion (IR) injury. Considering the importance of preventing reperfusion injuries, we aimed to examine the combination effect of mitochondrial transplantation (MT) and coenzyme Q10 (CoQ10 ) in myocardial IR injury of aged male rats. Seventy-two aged male Wistar rats were randomly divided into six groups: Sham, IR, CoQ10 , MT, combination therapy (MT + CoQ10 ) and vehicle. Myocardial IR injury was established by occlusion of the left anterior descending coronary artery followed by reopening. Young male Wistar rats were used as mitochondria donors. Isolated mitochondria were injected intraventricularly (500 µL of a respiration buffer containing 6 × 106 ± 5 × 105  mitochondria/mL) in MT-receiving groups at the onset of reperfusion. CoQ10  (10 mg/kg/day) was injected intraperitoneally for 2 weeks before IR induction. Twenty-four hours after reperfusion, haemodynamic parameters, myocardial infarct size (IS), lactate dehydrogenase (LDH) release and cardiac mitochondrial function (mitochondrial reactive oxygen species (ROS) generation and membrane potential) were measured. The combination of MT and CoQ10  improved haemodynamic index changes and reduced IS and LDH release (P < 0.05). It also decreased mitochondrial ROS generation and increased membrane potential (P < 0.05). CoQ10 also showed a significant cardioprotective effect. Combination therapy displayed greater cardioprotective effects than single treatments. This study revealed that MT and CoQ10 combination treatment can be considered as a promising cardioprotective strategy to reduce myocardial IR injury in ageing, in part by restoring mitochondrial function.

Synbiotic supplementation ameliorates anxiety and myocardial ischaemia-reperfusion injury in hyperglycaemic rats by modulating gut microbiota.

Hyperglycaemia, hyperlipidaemia, hypertension and obesity are the main risk factors affecting the development and prognosis of ischaemic heart disease, which is still an important cause of death today. In our study, male Sprague-Dawley rats were fed either a standard diet (SD) or a high fat and high carbohydrate diet (HF-HCD) for 8 weeks and streptozotocin (STZ) was injected at the seventh week of the feeding period. In one set of rats, a mixture of a prebiotic and probiotics (synbiotic, SYN) was administered by gavage starting from the beginning of the feeding period. Experimental myocardial ischaemia-reperfusion (30 min/60 min) was induced at the end of 8 weeks. Hyperglycaemia, hypertension and increased serum low-density lipoprotein levels occurred in SD- and HF-HCD-fed and STZ-treated rats followed for 8 weeks. Increased density of the Proteobacteria phylum was observed in rats with increased blood glucose levels, indicating intestinal dysbiosis. The severity of cardiac damage was highest in the dysbiotic HF-HCD-fed hyperglycaemic rats, which was evident with increased serum creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), tumour necrosis factor-α, and interleukin-6 levels, along with a decrease in ST-segment resolution index. SYN supplementation to either a normal or a high-fat high-carbohydrate diet improved gut dysbiosis, reduced anxiety, decreased CK-MB and cTnI levels, and alleviated myocardial ischaemia-reperfusion injury in hyperglycaemic rats.

Inhibition of inflammation and apoptosis through the cyclic GMP-AMP synthase-stimulator of interferon genes pathway by stress granules after ALKBH5 demethylase activation during diabetic myocardial ischaemia-reperfusion injury.

AIM: Post-transcriptional modifications and their specific mechanisms are the focus of research on the regulation of myocardial damage. Stress granules (SGs) can inhibit the inflammatory response by inhibiting the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. This study investigated whether alkylation repair homologue protein 5 (ALKBH5) could affect myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion injury (IRI) through the cGAS-STING pathway via SGs. METHODS: A diabetes ischaemia-reperfusion rat model and a high glucose hypoxia/reoxygenation cell model were established. Adeno-associated virus (AAV) and lentivirus (LV) were used to overexpress ALKBH5, while the SG agonist arsenite (Ars) and the SG inhibitor anisomycin were used as interventions. Then, the levels of apoptosis and related indicators in the cell and rat models were measured. RESULTS: In the in vivo experiment, compared with the normal sham group, the degree of myocardial tissue damage, creatine kinase-MB and cardiac troponin I in serum, and myocardial apoptosis, the infarcted area of myocardium, and the level of B-cell lymphoma 2 associated X protein, cGAS-STING pathway and inflammatory factors in the diabetes ischaemia-reperfusion group were significantly increased. However, the expression of SGs and the levels of ALKBH5, rat sarcoma-GTPase-activating protein-binding protein 1, T-cell intracellular antigen-1 and Bcl2 were significantly decreased. After AAV-ALKBH5 intervention, the degree of myocardial tissue damage, degree of myocardial apoptosis, and extent of myocardial infarction in myocardial tissue were significantly decreased. In the in vitro experiment, compared with those in the normal control group, the levels of lactate dehydrogenase, inflammation and apoptosis were significantly greater, and cell viability and the levels of ALKBH5 and SGs were decreased in the high glucose and hypoxia/reoxygenation groups. In the high glucose hypoxia/reoxygenation cell model, the degree of cell damage, inflammation, and apoptosis was greater than those in the high glucose and hypoxia/reoxygenation models, and the levels of ALKBH5 and SGs were further decreased. LV-ALKBH5 and Ars alleviated the degree of cell damage and inhibited inflammation and cell apoptosis. The inhibition of SGs could partly reverse the protective effect of LV-ALKBH5. The cGAS agonist G140 antagonized the inhibitory effects of the SG agonist Ars on cardiomyocyte apoptosis, inflammation and the cGAS-STING pathway. CONCLUSION: Both ALKBH5 and SGs inhibited myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion. Mechanistically, ALKBH5 might inhibit the apoptosis of cardiomyocytes by promoting the expression of SGs through the cGAS-STING pathway.

Improved cardioprotective effect of 3-nitro-N-methyl salicylamide solution after a prolonged preservation time of rat heart.

Ischemic reperfusion injury, caused by oxidative stress during reperfusion, is an inevitable outcome of organ transplantation, especially when the organ preservation time is prolonged. Prolonged ischaemic preservation is a valuable technique for improving the success of organ transplantation, but numerous challenges remain. 3-nitro-N-methyl salicylamide (3-NNMS), an inhibitor of mitochondrial electron transport chain complex III, can be used to reduce reactive oxygen species production during blood reperfusion by slowing the electron flow rate of the respiratory chain. Based on this property, a novel preservation solution was developed for the preservation of isolated rat heart and its cardioprotective effect was investigated during an 8-h cold ischaemia preservation time for the first time. For comparison, 3-NNMS was also included in the histidine-tryptophan-ketoglutarate (HTK) solution. Compared to HTK, HTK supplemented with 3-NNMS significantly improved the heart rate of isolated rat hearts after 8 h of cold storage. Both 3-NNMS solution and HTK supplemented with 3-NNMS solution decreased cardiac troponin T and lactate dehydrogenase levels in perfusion fluid and reduced reactive oxygen species and malondialdehyde levels in the myocardium. The 3-NNMS also maintained the membrane potential of myocardial mitochondria and significantly increased superoxide dismutase levels. These results showed that the new 3-NNMS solution can protect mitochondrial and cardiomyocyte function by increasing antioxidant capacity and reducing oxidative stress in cryopreserved rat hearts during a prolonged preservation time, resulting in less myocardial injury and better heart rate.

Decreased MFN2 activates the cGAS-STING pathway in diabetic myocardial ischaemia-reperfusion by triggering the release of mitochondrial DNA.

BACKGROUND: The cause of aggravation of diabetic myocardial damage is yet to be elucidated; damage to mitochondrial function has been a longstanding focus of research. During diabetic myocardial ischaemia-reperfusion (MI/R), it remains unclear whether reduced mitochondrial fusion exacerbates myocardial injury by generating free damaged mitochondrial DNA (mitoDNA) and activating the cGAS-STING pathway. METHODS: In this study, a mouse model of diabetes was established (by feeding mice a high-fat diet (HFD) plus a low dose of streptozotocin (STZ)), a MI/R model was established by cardiac ischaemia for 2 h and reperfusion for 30 min, and a cellular model of glycolipid toxicity induced by high glucose (HG) and palmitic acid (PA) was established in H9C2 cells. RESULTS: We observed that altered mitochondrial dynamics during diabetic MI/R led to increased mitoDNA in the cytosol, activation of the cGAS-STING pathway, and phosphorylation of the downstream targets TBK1 and IRF3. In the cellular model we found that cytosolic mitoDNA was the result of reduced mitochondrial fusion induced by HG and PA, which also resulted in cGAS-STING signalling and activation of downstream targets. Moreover, inhibition of STING by H-151 significantly ameliorated myocardial injury induced by MFN2 knockdown in both the cell and mouse models. The use of a fat-soluble antioxidant CoQ10 improved cardiac function in the mouse models. CONCLUSIONS: Our study elucidated the critical role of cGAS-STING activation, triggered by increased cytosolic mitoDNA due to decreased mitochondrial fusion, in the pathogenesis of diabetic MI/R injury. This provides preclinical insights for the treatment of diabetic MI/R injury. Video Abstract.

Adiponectin protects skeletal muscle from ischaemia-reperfusion injury in mice through miR-21/PI3K/Akt signalling pathway.

Previous studies have confirmed that adiponectin (APN) plays a protective role in myocardial ischaemia-reperfusion (IR) injury, and the aim of this study was to investigate its effect on skeletal muscle. ELISA was used to detect the levels of Creatinine Kinase (CK), LDH, SOD and MDA in the plasma of the lower limbs of mice, and the levels of IL-6, IL-1ß and TNF-α in the gastrocnemius. Quantitative PCR was used to detect the expression level of miR-21. TUNEL staining was used to detect the apoptosis of the gastrocnemius. The expression levels of apoptosis proteins, autophagy marker proteins and downstream target genes of miR-21 in gastrocnemius were detected by Western Blot. The results of this study revealed that APN levels were significantly reduced in gastrocnemius of IR mice. The oxidative stress, inflammatory response, apoptosis and autophagy induced by IR were significantly ameliorated by APN injection. The above effects of APN may be achieved through miR-21/PI3K signalling pathway, as found by interfering gene expression levels with miRNA antagomir and lentiviral injection. Taken together, our study revealed that APN protects skeletal muscle from IR injury through miR-21 /PI3K/Akt signalling pathway through inhibiting inflammatory response, apoptosis and autophagy.

Therapeutic hypothermia alleviates myocardial ischaemia-reperfusion injury by inhibiting inflammation and fibrosis via the mediation of the SIRT3/NLRP3 signalling pathway.

Therapeutic hypothermia (TH) may attenuate myocardial ischaemia-reperfusion injury, thereby improving outcomes in acute myocardial infarction. However, the specific mechanism by which TH alleviates MIRI has not been elucidated so far. In this study, 120 healthy male Sprague-Dawley rats were randomly divided into five groups. Haemodynamic parameters, myocardial infarction area, histological changes and the levels of cardiac enzymes, caspase-1 and inflammatory cytokines were determined. In addition, the extent of myocardial fibrosis, the degree of cardiomyocyte apoptosis and the expression levels of SIRT3, GSDMD-N, fibrosis-related proteins and inflammation-related proteins were estimated.TH reduced myocardial infarct area and cardiac enzyme levels, improved cardiomyopathic damage and haemodynamic indexes, and attenuated myocardial fibrosis, the protein expression levels of collagen I and III, myocardial apoptosis, the levels of inflammatory cytokines and inflammation-related proteins. Notably, the immunofluorescence and protein expression levels of SIRT3 were upregulated in the 34H+DMSO group compared to the I/R group, but this protective effect was abolished by the SIRT3 inhibitor 3-TYP. After administration of Mcc950, the reversal effects of 3-TYP were significantly abolished, and TH could protect against MIRI in a rat isolated heart model by inhibiting inflammation and fibrosis. The SIRT3/NLRP3 signalling pathway is one of the most important signalling pathways in this regard.

PGC-1α protects from myocardial ischaemia-reperfusion injury by regulating mitonuclear communication.

The recovery of blood supply after a period of myocardial ischaemia does not restore the heart function and instead results in a serious dysfunction called myocardial ischaemia-reperfusion injury (IRI), which involves several complex pathophysiological processes. Mitochondria have a wide range of functions in maintaining the cellular energy supply, cell signalling and programmed cell death. When mitochondrial function is insufficient or disordered, it may have adverse effects on myocardial ischaemia-reperfusion and therefore mitochondrial dysfunction caused by oxidative stress a core molecular mechanism of IRI. Peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) is an important antioxidant molecule found in mitochondria. However, its role in IRI has not yet been systematically summarized. In this review, we speculate the role of PGC-1α as a key regulator of mitonuclear communication, which may interacts with nuclear factor, erythroid 2 like -1 and -2 (NRF-1/2) to inhibit mitochondrial oxidative stress, promote the clearance of damaged mitochondria, enhance mitochondrial biogenesis, and reduce the burden of IRI.