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.     

Phosphorylation of nucleolin is indispensable to upregulate miR-21 and inhibit apoptosis in cardiomyocytes

Nucleolin is a multifunctional phosphoprotein and is involved in protecting from myocardial ischemia/reperfusion (I/R) injury. The function of nucleolin is regulated by posttranslational modifications, including phosphorylation and glycosylation. To study whether phosphorylation of nucleolin (P-nucleolin) was involved in the protection from myocardial I/R injury. We investigated the expression pattern of P-nucleolin (Thr-76 and 84) in hearts subjected to I/R injury, or rat cardiac myoblast cells (H9C2) subjected to hydrogen peroxide (H ₂O ₂). The results showed that the expression of P-nucleolin and the ratio of P-nucleolin/nucleolin were significantly increased both in vivo and in vitro. Mutant nucleolin was obtained by site directed mutagenesis in vitro: threonine at 76 and 84 was replaced by alanine, and we found that the protective effect of nucleolin on apoptosis induced by oxidative stress was dependent on its phosphorylation at 76 and 84 in H9C2 cells. Furthermore, the cardio-protective roles of P-nucleolin (Thr-76 and 84) in H9C2 cardiomyocytes, were attributable to the upregulation of microRNA (miR)-21. Further analysis found that P-nucleolin (Thr-76 and 84) could bind to miR-21, and P-nucleolin colocalized with argonaute 2 (Ago2) in cytoplasm and could interact with Ago2 in a RNA-independent manner under cell oxidative stress. The current study revealed that P-nucleolin (Thr-76 and 84) increased in I/R injury myocardium, P-nucleolin was indispensable to upregulate miR-21 and inhibited apoptosis induced by H ₂O ₂ in H9C2 cardiomyocytes. These findings provided new insight into the molecular mechanisms of nucleolin in myocardial I/R injury and oxidative stress cells.     

H2S protects from oxidative stress-driven ACE2 expression and cardiac aging

Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H₂S) plays an essential role in preserving cardiac functions. Angiotensin-converting enzyme 2 (ACE2) acts as the negative regulator of the renin-angiotensin system, exerting anti-oxidative stress and anti-inflammatory properties within the body. The interplays of CSE/H₂S signaling and ACE2 in cardiac aging are unclear. In this study, the regulatory roles of H₂S on ACE2 expression in mouse heart tissue and rat cardiomyocytes under different stress conditions were investigated. It was found that ACE2 protein level was lower in heart tissues from old mice (56-week-old) than young mice (8-week-old), and the knockout of CSE (CSE KO) induced moderate oxidative stress and further inhibited ACE2 protein level in mouse hearts at both young and old age. Incubation of rat cardiac cells (H9C2) with a low dose of H₂O₂ (50 µM) suppressed ACE2 protein level and induced cellular senescence, which was completely reversed by co-incubation with 30 µM NaHS (a H₂S donor). Prolonged nutrient excess is an increased risk of heart disorders by causing metabolic dysfunction and cardiac remodeling. We further found high-fat diet feeding stimulated ACE2 expression and induced severe oxidative stress in CSE KO heart in comparison with wild-type heart. Lipid overload in H9C2 cells to mimic a status of nutrient excess also enhanced the expression of ACE2 protein and induced severe oxidative stress and cell senescence, which were significantly attenuated by the supplementation of exogenous H₂S. Furthermore, the manipulation of ACE2 expression partially abolished the protective role of H₂S against cellular senescence. These results demonstrate the dynamic roles of H₂S in the maintenance of ACE2 levels under different levels of oxidative stress, pointing to the potential implications in targeting the CSE/H₂S system for the interruption of aging and diabetes-related heart disorders.

     

Beetroot juice reduces infarct size and improves cardiac function following ischemia–reperfusion injury: Possible involvement of endogenous H2S

Ingestion of high dietary nitrate in the form of beetroot juice (BRJ) has been shown to exert antihypertensive effects in humans through increasing cyclic guanosine monophosphate (cGMP) levels. Since enhanced cGMP protects against myocardial ischemia–reperfusion (I/R) injury through upregulation of hydrogen sulfide (H₂S), we tested the hypothesis that BRJ protects against I/R injury via H₂S. Adult male CD-1 mice received either regular drinking water or those dissolved with BRJ powder (10 g/L, containing ∼0.7 mM nitrate). Seven days later, the hearts were explanted for molecular analyses. Subsets of mice were subjected to I/R injury by occlusion of the left coronary artery for 30 min and reperfusion for 24 h. A specific inhibitor of H₂S producing enzyme – cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG, 50 mg/kg) was given i.p. 30 min before ischemia. Myocardial infarct size was significantly reduced in BRJ-fed mice (15.8 ± 3.2%) versus controls (46.5 ± 3.5%, mean ± standard error [SE], n = 6/group, P < .05). PAG completely blocked the infarct-limiting effect of BRJ. Moreover, BRJ significantly preserved ventricular function following I/R. Myocardial levels of H₂S and its putative protein target – vascular endothelial growth factor receptor 2 (VEGFR2) were significantly increased by BRJ intake, whereas CSE mRNA and protein content did not change. Interestingly, the BRJ-induced cardioprotection was not associated with elevated blood nitrate–nitrite levels following I/R nor induction of cardiac peroxiredoxin 5, a mitochondrial antioxidant enzyme previously linked to nitrate-induced cardioprotection. We conclude that BRJ ingestion protects against post-I/R myocardial infarction and ventricular dysfunction possibly through CSE-mediated endogenous H₂S generation. BRJ could be a promising natural and inexpensive nutraceutical supplement to reduce cardiac I/R injury in patients.     

Downregulation of p300/CBP-associated factor inhibits cardiomyocyte apoptosis via suppression of NF-κB pathway in ischaemia/reperfusion injury rats

Cardiomyocyte apoptosis is the main reason of cardiac injury after myocardial ischaemia-reperfusion (I/R) injury (MIRI), but the role of p300/CBP-associated factor (PCAF) on myocardial apoptosis in MIRI is unknown. The aim of this study was to investigate the main mechanism of PCAF modulating cardiomyocyte apoptosis in MIRI. The MIRI model was constructed by ligation of the rat left anterior descending coronary vessel for 30 min and reperfusion for 24 h in vivo. H9c2 cells were harvested after induced by hypoxia for 6 h and then reoxygenation for 24 h (H/R) in vitro. The RNA interference PCAF expression adenovirus was transfected into rat myocardium and H9c2 cells. The area of myocardial infarction, cardiac function, myocardial injury marker levels, apoptosis, inflammation and oxidative stress were detected respectively. Both I/R and H/R remarkably upregulated the expression of PCAF, and downregulation of PCAF significantly attenuated myocardial apoptosis, inflammation and oxidative stress caused by I/R and H/R. In addition, downregulation of PCAF inhibited the activation of NF-κB signalling pathway in cardiomyocytes undergoing H/R. Pretreatment of lipopolysaccharide, a NF-κB pathway activator, could blunt these protective effects of PCAF downregulation on myocardial apoptosis in MIRI. These results highlight that downregulation of PCAF could reduce cardiomyocyte apoptosis by inhibiting the NF-κB pathway, thereby providing protection for MIRI. Therefore, PCAF might be a promising target for protecting against cardiac dysfunction induced by MIRI.     

Dietary methionine restriction attenuates renal ischaemia/reperfusion‐induced myocardial injury by activating the CSE/H2S/ERS pathway in diabetic mice

Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)‐induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine‐restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R‐induced mediated myocardial injury and apoptosis were partially reversed by methionine‐restricted diet (MRD) via the down‐regulation of CSE expression and up‐regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose‐dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R‐induced myocardial injury.     

LncRNA TUG1 attenuates ischaemia-reperfusion-induced apoptosis of renal tubular epithelial cells by sponging miR-144-3p via targeting Nrf2

Renal ischaemia/reperfusion (I/R) injury may induce kidney damage and dysfunction, in which oxidative stress and apoptosis play important roles. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are reported to be closely related to renal I/R, but the specific molecular mechanism is still unclear. The purpose of this research was to explore the regulatory effect of lncRNA TUG1 on oxidative stress and apoptosis in renal I/R injury. This research revealed that in renal I/R injury and hypoxia/reperfusion (H/R) injury in vitro, the expression level of lncRNA TUG1 was upregulated, and oxidative stress levels and apoptosis levels were negatively correlated with the expression level of lncRNA TUG1. Using bioinformatics databases such as TargetScan and microRNA.org, microRNA-144-3p (miR-144-3p) was predicted to be involved in the association between lncRNA TUG1 and Nrf2. This study confirmed that the level of miR-144-3p was significantly reduced following renal I/R injury and H/R injury in vitro, and miR-144-3p was determined to target Nrf2 and inhibit its expression. In addition, lncRNA TUG1 can reduce the inhibitory effect of miR-144-3p on Nrf2 by sponging miR-144-3p. In summary, our research shows that lncRNA TUG1 regulates oxidative stress and apoptosis during renal I/R injury through the miR-144-3p/Nrf2 axis, which may be a new treatment target for renal I/R injury.     

Cardioprotective effect of isorhamnetin against myocardial ischemia reperfusion (I/R) injury in isolated rat heart through attenuation of apoptosis

In this study, we investigated the effects of isorhamnetin on myocardial ischaemia reperfusion (I/R) injury in Langendorff-perfused rat hearts. Isorhamnetin treatment (5, 10 and 20 μg/mL) significantly alleviated cardiac morphological injury, reduced myocardial infarct size, decreased the levels of marker enzymes (LDH and CK) and improved the haemodynamic parameters, reflected by the elevated levels of the left ventricular developed pressure (LVDP), coronary flow (CF) and the maximum up/down velocity of left ventricular pressure (+dp/dt_max). Moreover, isorhamnetin reperfusion inhibited apoptosis of cardiomyocytes in the rats subjected to cardiac I/R in a dose-dependent manner concomitant with decreased protein expression of Bax and cleaved-caspase-3, as well as increased protein expression of Bcl-2. In addition, I/R-induced oxidative stress was manifestly mitigated by isorhamnetin treatment, as showed by the decreased malondialdehyde (MDA) level and increased antioxidant enzymes activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). These results indicated that isorhamnetin exerts a protective effect against I/R-induced myocardial injury through the attenuation of apoptosis and oxidative stress.     

Downregulation of p300/CBP‐associated factor inhibits cardiomyocyte apoptosis via suppression of NF‐κB pathway in ischaemia/reperfusion injury rats

Cardiomyocyte apoptosis is the main reason of cardiac injury after myocardial ischaemia‐reperfusion (I/R) injury (MIRI), but the role of p300/CBP‐associated factor (PCAF) on myocardial apoptosis in MIRI is unknown. The aim of this study was to investigate the main mechanism of PCAF modulating cardiomyocyte apoptosis in MIRI. The MIRI model was constructed by ligation of the rat left anterior descending coronary vessel for 30 min and reperfusion for 24 h in vivo. H9c2 cells were harvested after induced by hypoxia for 6 h and then reoxygenation for 24 h (H/R) in vitro. The RNA interference PCAF expression adenovirus was transfected into rat myocardium and H9c2 cells. The area of myocardial infarction, cardiac function, myocardial injury marker levels, apoptosis, inflammation and oxidative stress were detected respectively. Both I/R and H/R remarkably upregulated the expression of PCAF, and downregulation of PCAF significantly attenuated myocardial apoptosis, inflammation and oxidative stress caused by I/R and H/R. In addition, downregulation of PCAF inhibited the activation of NF‐κB signalling pathway in cardiomyocytes undergoing H/R. Pretreatment of lipopolysaccharide, a NF‐κB pathway activator, could blunt these protective effects of PCAF downregulation on myocardial apoptosis in MIRI. These results highlight that downregulation of PCAF could reduce cardiomyocyte apoptosis by inhibiting the NF‐κB pathway, thereby providing protection for MIRI. Therefore, PCAF might be a promising target for protecting against cardiac dysfunction induced by MIRI.     

The optimization conditions of establishing an H9c2 cardiomyocyte hypoxia/reoxygenation injury model based on an AnaeroPack System

Ischemia–reperfusion (I/R) injury is a major cause of cardiomyocyte apoptosis after vascular recanalization, which was mimicked by a hypoxia/reoxygenation (H/R) injury model of cardiomyocytes in vitro. In this study, we explored an optimal H/R duration procedure using the AnaeroPack System. To study the H/R procedure, cardiomyocytes were exposed to the AnaeroPack System with sugar and serum-free medium, followed by reoxygenation under normal conditions. Cell injury was detected through lactate dehydrogenase (LDH) and cardiac troponin (c-Tn) release, morphological changes, cell apoptosis, and expression of apoptosis-related proteins. The results showed that the damage to H9c2 cells increased with prolonged hypoxia time, as demonstrated by increased apoptosis rate, LDH and c-Tn release, HIF-1α expression, as well as decreased expression of Bcl-2. Furthermore, hypoxia for 10 h and reoxygenation for 6 h exhibited the highest apoptosis rate and damage and cytokine release; in addition, cells were deformed, small, and visibly round. After 12 h of hypoxia, the majority of the cells were dead. Taken together, this study showed that subjecting H9c2 cells to the AnaeroPack System for 10 h and reoxygenation for 6 h can achieve a practicable and repeatable H/R injury model.     

Cardioprotective effect of polydatin against ischemia/reperfusion injury: Roles of protein kinase C and mito KATP activation

Polydatin preconditioning (PPC) has been reported to be protective against brain and intestine ischemia/reperfusion injury (I/R injury), but whether polydatin exerts cardioprotective effect against myocardial ischemia/reperfusion and the underlying mechanisms remain unclear. Previous studies have demonstrated that oxidative stress plays an important role in the process of I/R. Elevation of oxidative agents and decline in anti-oxidant substance would promote I/R. Meanwhile, the activation of PKC signaling seems to mediate the cardioprotective effects of many drugs by alleviating Ca²⁺ influx. In the present study, we reported for the first time that intravenous administration of polydatin before I/R significantly limited the infarct size, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) leakage from the damaged myocardium after I/R. The activity of SOD and the content of MDA remarkably changed in the presence of polydatin as well. However, the cardiac function-preserving and myocardial enzymes leakage-limiting effects of polydatin vanished in the presence of PKC inhibitors and mito K_ATP channel blockers. But there was not a significant change in the activity of SOD and MDA content. We therefore conclude that PPC exerts cardioprotective effect by the activation of PKC-K_ATP-dependent signaling and the direct anti-oxidative stress mechanisms.     

Protective effects of Araloside C against myocardial ischaemia/reperfusion injury: potential involvement of heat shock protein 90

The present study was designed to investigate whether Araloside C, one of the major triterpenoid compounds isolated from Aralia elata known to be cardioprotective, can improve heart function following ischaemia/reperfusion (I/R) injury and elucidate its underlying mechanisms. We observed that Araloside C concentration‐dependently improved cardiac function and depressed oxidative stress induced by I/R. Similar protection was confirmed in isolated cardiomyocytes characterized by maintaining Ca²⁺ transients and cell shortening against I/R. Moreover, the potential targets of Araloside C were predicted using the DDI‐CPI server and Discovery Studio software. Molecular docking analysis revealed that Araloside C could be stably docked into the ATP/ADP‐binding domain of the heat shock protein 90 (Hsp90) protein via the formation of hydrogen bonds. The binding affinity of Hsp90 to Araloside C was detected using nanopore optical interferometry and yielded KD values of 29 μM. Araloside C also up‐regulated the expression levels of Hsp90 and improved cell viability in hypoxia/reoxygenation‐treated H9c2 cardiomyocytes, whereas the addition of 17‐AAG, a pharmacologic inhibitor of Hsp90, attenuated Araloside C‐induced cardioprotective effect. These findings reveal that Araloside C can efficiently attenuate myocardial I/R injury by reducing I/R‐induced oxidative stress and [Ca²⁺]_i overload, which was possibly related to its binding to the Hsp90 protein.     

microRNA-340-5p inhibits hypoxia/reoxygenation-induced apoptosis and oxidative stress in cardiomyocytes by regulating the Act1/NF-κB pathway

MicroRNAs (miRNAs) have been reported to play critical roles in the occurrence, progression, and treatment of many cardiovascular diseases. However, the molecular mechanism by which miRNA regulates target gene expression in ischemia-reperfusion (I/R) injury in acute myocardial infarction (AMI) is not entirely clear. MiR-340-5p was reported to be downregulated in acute ischemic stroke. However, it still remains unknown whether miR-340-5p is mediated in the pathogenesis process of I/R injury after AMI. In the present study, male C57BL/6 J mice and H9C2 cardiomyocytes were used as experimental models. Real-time polymerase chain reaction analysis, Western blot analysis, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling immunofluorescence staining assay were conducted to examine related indicators in the study. We confirmed that the expression of miR-340-5p is downregulated after I/R in AMI mice and hypoxia/reperfusion (H/R)-induced cardiomyocytes. miR-340-5p could inhibit apoptosis and oxidative stress in H/R-induced H9C2 cells via downregulating activator 1 (Act1). The inhibiting action of miR-340-5p on H/R-induced apoptosis and oxidative stress in cardiomyocytes was partially reversed after Act1 overexpression. Moreover, the results showed that the NF-κB pathway may be mediated in the role of miR-340-5p on H/R-induced cardiomyocyte apoptosis and oxidative stress. We demonstrated that upregulation of miR-340-5p suppresses apoptosis and oxidative stress induced by H/R in H9C2 cells by inhibiting Act1. Therapeutic strategies that target miR-340-5p, Act1, and the NF-κB pathway could be beneficial for the treatment of I/R injury after AMI.     

Donepezil provides neuroprotective effects against brain injury and Alzheimer's pathology under conditions of cardiac ischemia/reperfusion injury

Cardiac ischemia/reperfusion (I/R) injury induces brain pathology. Donepezil, a well-known acetylcholine esterase (AChE) inhibitor, has been proven to exert neuroprotective effects against several neurodegenerative diseases. However, the comprehensive mechanism regarding the therapeutic potential of donepezil on the brain under cardiac I/R injury remains obscure. Here, we hypothesized that treatment with donepezil ameliorates brain pathology following cardiac I/R injury by decreasing blood brain barrier (BBB) breakdown, oxidative stress, neuroinflammation, mitochondrial dysfunction, mitochondrial dynamics imbalance, microglial activation, amyloid-beta (Aβ) accumulation, neuronal apoptosis, and dendritic spine loss. Forty-eight adult male Wistar rats were subjected to surgery for cardiac I/R injury. Then, rats were randomly divided into four groups to receive either (1) saline (vehicle group), donepezil 3 mg/kg via intravenously administered (2) before ischemia (pretreatment group), (3) during ischemia (ischemia group), or (4) at the onset of reperfusion (reperfusion group). At the end of cardiac I/R paradigm, the brains were evaluated for BBB breakdown, brain inflammation, oxidative stress, mitochondrial function, mitochondrial dynamics, microglial morphology, Aβ production, neuronal apoptosis, and dendritic spine density. Administration of donepezil at all time points equally showed an attenuation of brain damage in response to cardiac I/R injury, as indicated by increased expression of BBB junction protein, reduced brain inflammation and oxidative stress, improved mitochondrial function and mitochondrial dynamics, and alleviated Aβ accumulation and microglial activation, resulting in protection of neuronal apoptosis and preservation of dendritic spine number. These findings suggest that donepezil potentially protects brain pathology caused by cardiac I/R injury regardless the timing of treatment.     

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.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Diabetes aggravates myocardial ischaemia reperfusion injury via activating Nox2‐related programmed cell death in an AMPK‐dependent manner

Cardiovascular diseases such as myocardial ischaemia have a high fatality rate in patients with diabetes. This study was designed to expose the crosstalk between oxidative stress and AMPK, a vital molecule that controls biological energy metabolism, in myocardial ischaemia reperfusion injury (I/RI) in diabetic rats. Diabetes was stimulated in rats using streptozotocin injection. Rats were separated on random into control, control + I/R, Diabetes, Diabetes + I/R, Diabetes + I/R + N‐acetylcysteine and Diabetes + I/R + Vas2870 groups. Myocardial infarct size was determined, and the predominant Nox family isoforms were analysed. In vitro, the H9C2 cells were administered excess glucose and exposed to hypoxia/reoxygenation to mimic diabetes and I/R. The AMPK siRNA or AICAR was used to inhibit or activate AMPK expression in H9C2 cells, respectively. Then, myocardial oxidative stress and programmed cell death were measured. Diabetes or high glucose levels were found to aggravate myocardial I/RI or hypoxia/reoxygenation in H9C2 cells, as demonstrated by an increase in myocardial infarct size or lactate dehydrogenase levels, oxidative stress generation and induction of programmed cell death. In diabetic rat hearts, cardiac Nox1, Nox2 and Nox4 were all heightened. The suppression of Nox2 expression using Vas2870 or Nox2‐siRNA treatment in vivo or in vitro, respectively, protected diabetic rats from myocardial I/RI. AMPK gene knockout increased Nox2 protein expression while AMPK agonist decreased Nox2 expression. Therefore, diabetes aggravates myocardial I/RI by generating of Nox2‐associated oxidative stress in an AMPK‐dependent manner, which led to the induction of programmed cell death such as apoptosis, pyroptosis and ferroptosis.     

Stomatin-like protein-2 relieve myocardial ischemia/reperfusion injury by adenosine 5′-monophosphate-activated protein kinase signal pathway

Previous studies have shown that stomatin-like protein-2 (SLP-2) could regulate mitochondrial biogenesis and function. The study was designed to explore the contribution of SLP-2 to the myocardial ischemia and reperfusion (I/R) injury. Anesthetized rats were treated with SLP-2 and subjected to ischemia for 30 minutes before 3 hours of reperfusion. An oxygen-glucose deprivation/reoxygenation model of I/R was established in H9C2 cells. In vivo, SLP-2 significantly improved cardiac function recovery of myocardial I/R injury rats by increasing fractional shortening and ejection fraction. SLP-2 pretreatment alleviated infarct area and myocardial apoptosis, which was paralleled by decreasing the level of cleaved caspase-3 and the ratio of Bax/Bcl-2, increasing the content of superoxide dismutase and reducing oxidative stress damage in serum. In addition, SLP-2 increased the level of ATP and stabilized mitochondrial potential (Ψm). The present in vitro study revealed that overexpression with SLP-2 reduced H9C2 cells apoptosis, accompanied by an increased level of ATP, the ratio of mitochondrial DNA/nuclear DNA, activities of complex II and V, and decreased the production of mitochondrial reactive oxygen species. Simultaneously, SLP-2 activated the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway in myocardial I/R injury rats and H9C2 cells. This study revealed that SLP-2 mediates the cardioprotective effect against I/R injury by regulating AMPK signaling pathway.