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Following acute myocardial infarction, the recovery of blood flow leads to myocardial ischemia‑reperfusion (MI/R) injury, which is primarily characterized by the activation of inflammatory signals, microvascular obstruction, increased oxidative stress and excessive Ca 2+ overload. It has also been demonstrated that platelets can exacerbate MI/R injury by releasing reactive oxygen species, inflammatory factors and chemokines, while also obstructing microvessels through thrombus formation. As a bioactive molecule with proinflammatory and chemotactic properties, lipocalin 2 (LCN2) exhibits a positive correlation with obesity, hyperglycemia, hypertriglyceridemia and insulin resistance index, which are all significant risk factors for ischemic cardiomyopathy. Notably, the potential role of LCN2 in promoting atherosclerosis may be related to its influence on the function of macrophages, smooth muscle cells and endothelial cells, but its effect on platelet function has not yet been reported. In the present study, the effect of a high‑fat diet (HFD) on LCN2 expression was determined by detecting LCN2 expression levels in the liver and serum samples of mice through reverse transcription‑quantitative PCR and enzyme linked immunosorbent assay, respectively. The effect of LCN2 on platelet function was evaluated by examining whether LCN2 affected platelet activation, aggregation, adhesion, clot retraction and P‑selectin expression. To determine whether LCN2 aggravated MI/R injury in HFD‑fed mice by affecting platelet and inflammatory cell recruitment, wild‑type and LCN2 knockout mice fed a HFD were subjected to MI/R injury, then hearts were collected for hematoxylin and eosin staining and 2,3,5‑triphenyltetrazolium chloride staining, and immunohistochemistry was employed to detect the expression of CD42b, Ly6G, CD3 and B220. Based on observing the upregulation of LCN2 expression in mice fed a HFD, the present study further confirmed that LCN2 could accelerate platelet activation, aggregation and adhesion. Moreover, in vivo studies validated that knockout of LCN2 not only mitigated MI/R injury, but also inhibited the recruitment of platelets and inflammatory cells in myocardial tissue following ischemia‑reperfusion. In conclusion, the current findings suggested that the effect of HFD‑induced LCN2 on aggravating MI/R injury may totally or partially dependent on its promotion of platelet function.

Myocardial ischemia/reperfusion injury (MIRI) is a significant challenge in the management of myocardial ischemic disease. Extensive evidence suggests that the macrophage‑mediated inflammatory response may play a vital role in MIRI. Mesenchymal stem cells and, in particular, exosomes derived from these cells, may be key mediators of myocardial injury and repair. However, whether exosomes protect the heart by regulating the polarization of macrophages and the exact mechanisms involved are poorly understood. The present study aimed to determine whether exosomes secreted by bone marrow mesenchymal stem cells (BMSC‑Exo) harboring miR‑25‑3p can alter the phenotype of macrophages by affecting the JAK2/STAT3 signaling pathway, which reduces the inflammatory response and protects against MIRI. An in vivo MIRI model was established in rats by ligating the anterior descending region of the left coronary artery for 30 min followed by reperfusion for 120 min, and BMSC‑Exo carrying miR‑25‑3p (BMSC‑Exo‑25‑3p) were administered through tail vein injection. A hypoxia‑reoxygenation model of H9C2 cells was established, and the cells were cocultured with BMSC‑Exo‑25‑3p in vitro . The results of the present study demonstrated that BMSC‑Exo or BMSC‑Exo‑25‑3p could be taken up by cardiomyocytes in vivo and H9C2 cells in vitro . BMSC‑Exo‑25‑3p demonstrated powerful cardioprotective effects by decreasing the cardiac infarct size, reducing the incidence of malignant arrhythmias and attenuating myocardial enzyme activity, as indicated by lactate dehydrogenase and creatine kinase levels. It induced M1‑like macrophage polarization after myocardial ischemia/reperfusion (I/R), as evidenced by the increase in iNOS expression through immunofluorescence staining and upregulation of proinflammatory cytokines through RT‑qPCR, such as interleukin‑1β (IL‑1β) and interleukin‑6 (IL‑6). As hypothesized, BMSC‑Exo‑25‑3p inhibited M1‑like macrophage polarization and proinflammatory cytokine expression while promoting M2‑like macrophage polarization. Mechanistically, the JAK2/STAT3 signaling pathway was activated after I/R in vivo and in LPS‑stimulated macrophages in vitro , and BMSC‑Exo‑25‑3p pretreatment inhibited this activation. The results of the present study indicate that the attenuation of MIRI by BMSC‑Exo‑25‑3p may be related to JAK2/STAT3 signaling pathway inactivation and subsequent inhibition of M1‑like macrophage polarization.

Mitochondria‑mediated apoptosis is the primary cause of cardiomyocyte death. Therefore, mitochondria are a key target for treating myocardial injury. Mitochondrial calcium uniporter regulator 1 (MCUR1)‑mediated mitochondrial calcium homeostasis markedly promotes cell proliferation and resistance to apoptosis. However, whether MCUR1 is involved in regulation of cardiomyocyte apoptosis during myocardial ischaemia‑reperfusion remains unknown. microRNA‑124 (miR‑124) is upregulated in cardiovascular disease, suggesting a key role for miR‑124 in the cardiovascular system. Whether miR‑124 affects cardiomyocyte apoptosis and myocardial infarction is not well understood. Western blot showed that miR‑124 and MCUR1 were upregulated in cardiomyocyte apoptosis induced by hydrogen peroxide (H 2 O 2 ). Flow cytometry assay of cell apoptosis showed that miR‑124 inhibited cardiomyocyte apoptosis by activating MCUR1 following H2O2 treatment. The dual‑luciferase reporter assay confirmed binding of miR‑124 to MCUR1 3'‑UTR and subsequent activation of MCUR1. FISH assay revealed the entry of miR‑124 into the cell nucleus. Therefore, MCUR1 was identified as a novel target of miR‑124, and it was shown that the miR‑124‑MCUR1 axis modulated cardiomyocyte apoptosis induced by H 2 O 2 in vitro . The results indicated induced expression of miR‑124 during acute myocardial infarction and its transport to the nucleus. In the nucleus, miR‑124 transcriptionally activated MCUR1 by binding to its enhancers. These findings reveal a role of miR‑124 as a biomarker for myocardial injury and infarction.

Myocardial ischemia‑reperfusion (I/R) serves a crucial role in myocardial infarction. C1q/TNF‑related protein 12 (CTRP12) is a secretory protein involved in metabolism. It has been reported that CTRP12 participates in the regulation of numerous cardiovascular diseases. However, its role in myocardial I/R injury remains unclear. In the present study, the left anterior descending coronary artery in mice was ligated to establish a mouse I/R model. A myocardial hypoxia‑reoxygenation (H/R) cell model was also established. Cardiomyocyte injury was evaluated using hematoxylin and eosin staining, Cell Counting Kit‑8 and a lactate dehydrogenase (LDH) kit. The expression levels of CTRP12 and Krueppel‑like factor 15 (KLF15) in murine myocardial tissues and H9c2 cells were determined using reverse transcription‑quantitative PCR and western blotting, as KLF15 was previously reported to protect against I/R‑induced cardiomyocyte damage. Furthermore, inflammatory factors TNF‑α, IL‑1β and IL‑6 were analyzed using ELISA while apoptosis was assessed using TUNEL assays and western blotting. Moreover, the activity of the CTRP12 promoter was determined using a dual‑luciferase reporter assay. The results demonstrated that I/R surgery markedly exacerbated myocardial tissue damage, whereas H/R treatment significantly reduced cell viability and significantly increased LDH activity as well as the release of inflammatory factors and apoptosis. I/R and H/R induction significantly reduced the expression levels of CTRP12 and KLF15. CTRP12 overexpression significantly alleviated H/R‑induced cell injury and significantly inhibited inflammation and apoptosis. Further analysis demonstrated that KLF15 could significantly promote the activity of the CTRP12 promoter. However, following CTRP12 knockdown, KLF15 overexpression exacerbated cell injury, inflammation and apoptosis. In conclusion, the present study demonstrated that CTRP12 may mitigate inflammation and apoptosis in H/R‑induced cardiomyocytes, possibly via the regulation of KLF15, which provided a theoretical basis for the potential treatment of I/R‑induced myocardial infarction.

The increasing rates of morbidity and mortality caused by ischemic heart disease pose a serious threat to human health. Long non‑coding (lnc)RNA small nucleolar RNA host gene 1 (SNHG1) has a protective effect on the myocardium. In the present study, the role of lncRNA SNHG1 in myocardial ischemia reperfusion injury (MIRI) and the underlying mechanisms were investigated. After hypoxia/reoxygenation (H/R) induction, the expression levels of lncRNA SNHG1 were detected using reverse transcription‑quantitative PCR. After lncRNA SNHG1 overexpression via cell transfection, cell viability was detected using an MTT assay, apoptotic rates were detected using TUNEL staining, apoptosis‑related protein expression levels were detected using western blotting and respective kits were used to measure the oxidative stress levels. The Encyclopedia of RNA Interactomes database predicted the presence of binding sites between lncRNA SNHG1 and microRNA (miR)‑450b‑5p, and between miR‑450b‑5p and insulin‑like growth factor 1 (IGF1). These interactions were then verified using luciferase reporter assays. Subsequently, the regulatory mechanism underlying the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis in MIRI was investigated by overexpressing miR‑450b‑5p and knocking down IGF1 expression in H/R‑induced cells. Finally, the expression of PI3K/Akt signaling pathway‑related proteins was detected using western blotting. lncRNA SNHG1 expression was significantly downregulated in H/R‑induced AC16 cells. lncRNA SNHG1 overexpression significantly inhibited apoptosis and decreased oxidative stress levels in H/R‑induced AC16 cells, which was mediated via regulation of the miR‑450b‑5p/IGF1 axis and activation of the PI3K/Akt signaling pathway. Therefore, the present study suggested that activation of the PI3K/Akt signaling pathway via the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis inhibited the apoptosis and oxidative stress levels of H/R‑induced AC16 cells.

Myocardial ischemia/reperfusion (MI/RI) syndrome is one of the leading causes of mortality and disability. Propofol postconditioning is known to improve myocardial ischemia/reperfusion injury (MI/RI). The present study aimed to explore the mechanism of propofol postconditioning in diabetic MI/RI. Diabetic MI/RI rat models were established and the rats were treated via propofol postconditioning. Staining with 2,3,5‑triphenyl‑2H‑tetrazolium chloride, H&E staining, TUNEL staining and ELISA were applied to detect infarct size, pathological changes, apoptosis and oxidative stress‑related factor and apoptotic factor levels, respectively. Subsequently, the effect of propofol on H9C2 cells was also assessed using the Cell Counting Kit‑8 assay. High‑glucose hypoxia/reperfusion (H/R) models of H9C2 cardiomyocytes were established. miR‑200c‑3p overexpression or AdipoR2 silencing combined with propofol postconditioning was performed in H/R‑induced H9C2 cells and STAT3 protein expression levels were determined. Propofol postconditioning significantly reduced myocardial infarct size, oxidative stress and apoptosis in diabetic MI/RI models. Furthermore, propofol postconditioning significantly reduced the oxidative stress and apoptosis of H9C2 cells in high‑glucose H/R models. Propofol postconditioning also significantly downregulated miR‑200c‑3p expression levels and promoted AdipoR2 expression levels. miR‑200c‑3p overexpression or AdipoR2 downregulation significantly reversed the effects of propofol postconditioning on its antioxidation and anti‑apoptotic effects in H9C2 cells and on decreasing STAT3 phosphorylation levels. Together, the results of the present study demonstrated that propofol postconditioning inhibited miR‑200c‑3p, upregulated AdipoR2 and activated the STAT3 signaling pathway, thus alleviating diabetic MI/RI and therefore highlighting its potential as a treatment of diabetic MI/RI.

Dysregulation of long non‑coding RNA (IncRNA) antisense non‑coding RNA in the INK4 locus (ANRIL) is associated with the risk of myocardial infarction (MI). Therefore, the present study aimed to determine the mechanisms underlying this association, which is currently poorly understood, to the best of our knowledge. The current study used an in vitro myocardial ischemia and reperfusion (MI/R) model, in which H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R), which demonstrated that ANRIL expression was downregulated and that ANRIL positively regulated sirtuin 1 (SIRT1) expression following H/R injury. Subsequently, it was demonstrated that ANRIL upregulated SIRT1 expression by sponging microRNA‑181a (miR‑181a). In addition, ANRIL overexpression reduced lactate dehydrogenase release and apoptosis of H9c2 cardiomyocytes exposed to H/R, indicating that ANRIL prevented H/R‑induced cardiomyocyte injury. Moreover, both miR‑181a overexpression and SIRT1 knockdown significantly decreased the protective effects of ANRIL on H/R‑induced cardiomyocyte injury, thus demonstrating that SIRT1 upregulation via sponging miR‑181a is a critical mechanism that mediates the function of ANRIL. These results provided a novel mechanistic insight into the role of ANRIL in H/R‑injured cardiomyocytes and suggested that the ANRIL/miR‑181a/SIRT1 axis may be a therapeutic target for reducing MI/R injury.

Myocardial ischemia‑reperfusion injury (MIRI) is a severe injury to the ischemic myocardium following the recovery of blood flow. Currently, there is no effective treatment for MIRI in clinical practice. Over the past two decades, biological studies of hypoxia and hypoxia‑inducible factor‑1α (HIF‑1α) have notably improved understanding of oxygen homeostasis. HIF‑1α is an oxygen‑sensitive transcription factor that mediates adaptive metabolic responses to hypoxia and serves a pivotal role in MIRI. In particular, previous studies have demonstrated that HIF‑1α improves mitochondrial function, decreases cellular oxidative stress, activates cardioprotective signaling pathways and downstream protective genes and interacts with non‑coding RNAs. The present review summarizes the roles and associated mechanisms of action of HIF‑1α in MIRI. In addition, HIF‑1α‑associated MIRI intervention, including natural compounds, exosomes, ischemic preconditioning and ischemic post‑processing are presented. The present review provides evidence for the roles of HIF‑1α activation in MIRI and supports its use as a therapeutic target.

The present study aimed to investigate the effect of the long non‑coding ribonucleic acid (lncRNA) HOX transcript antisense intergenic RNA (HOTAIR) on apoptosis induced by ischemia‑reperfusion injury. Differential lncRNAs in myocardial ischemia rats were screened by a lncRNA microarray and the expression levels of lncRNA HOTAIR and microRNA (miR)‑130a‑3p were analyzed using reverse transcription‑quantitative polymerase chain reaction in hypoxia‑induced cardiomyocytes. The mechanism of lncRNA HOTAIR in cardiotoxicity was investigated using cell transfection, lncRNA knockdown, Cell Counting Kit‑8, flow cytometry, western blotting, dual luciferase reporter assays and RNA immunoprecipitation. The expression level of lncRNA HOTAIR was significantly downregulated in the ischemic myocardium of rats. Overexpression of HOTAIR in H9c2 (rat cardiomyocyte line) cells could inhibit the apoptosis induced by H2O2. A direct interaction was found between HOTAIR and miR‑130a‑3p, and mouse double minute 4 (MDM4) was also found to be a potential target of miR‑130a‑3p. The overexpression of MDM4 in H9c2 cells transfected with miR‑130a‑3p mimics increased apoptosis, and miR‑130a‑3p targeted inhibition of MDM4 promoted H2O2‑induced apoptosis of H9c2 cells. Overall, HOTAIR was found to inhibit the apoptosis of H9c2 cells induced by H2O2 through the miR‑130a‑3p/MDM4 axis.

Long non‑coding RNA (lncRNA) ZNFX1 antisense RNA 1 (ZFAS1) is upregulated in acute myocardial infarction; however, the role of ZFAS1 in myocardial ischemia/reperfusion (I/R) injury remains unknown. The present study aimed to detect microRNA (miR)‑590‑3p expression levels in cardiomyocytes subjected to I/R, and to investigate the effects of ZFAS1 on myocardial I/R injury. An in vitro model of I/R injury was established using rat H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R). It was demonstrated that ZFAS1 was upregulated and miR‑590‑3p was downregulated in the in vitro model of cardiac I/R injury. Western blotting results indicated that the protein expression levels of p50, tumor necrosis factor‑α (TNF‑α), interleukin (IL)‑6, Bax and cleaved caspase‑3 were upregulated, and the expression levels of Bcl‑2 and pro‑caspase‑3 were downregulated. Flow cytometry results revealed that downregulation of ZFAS1 reduced H/R‑induced apoptosis in H9c2 cells. In addition, downregulation of ZFAS1 significantly increased the expression of miR‑590‑3p, and p50 was identified as a target gene of miR‑590‑3p. Furthermore, with 12 h of hypoxia followed by 2 h of reoxygenation in H9c2 cells, ZFAS1 knockdown increased the expression levels of miR‑590‑3p, Bax and cleaved‑caspase‑3, and decreased the expression levels of Bcl‑2 and pro‑caspase‑3. It was also found that the miR‑590‑3p‑mimic transfection increased the expression levels of Bax and cleaved‑caspase‑3, and decreased the protein expression levels of p50, TNF‑α, IL‑6, Bcl‑2 and pro‑caspase‑3. In addition, TNF‑α treatment induced apoptosis of H9c2 cells, and the changes in Bax, Bcl‑2, cleaved‑caspase‑3 and pro‑caspase‑3 expression levels in a dose‑dependent manner. Collectively, the present results suggested that ZFAS1 was upregulated in H9c2 cells subjected to I/R injury, and that ZFAS1 knockdown protected against I/R‑induced myocardial cell apoptosis by directly interacting with miR‑590‑3p, via the NF‑κB pathway.

Non‑coding RNAs, including long non‑coding RNAs (lncRNAs) and microRNAs (miRNAs/miRs), have significant regulatory effects on a number of biological processes in myocardial ischemia/reperfusion (I/R) injury, including cell differentiation, proliferation and apoptosis. In the present study, the expression levels of lncRNAs, miRNAs and mRNAs were evaluated in a mouse model of myocardial I/R injury. The potential functions of these differentially expressed genes were then analyzed via Gene Ontology and pathway analyses. Additionally, the interactions between lncRNA‑miRNA‑mRNA were predicted by constructing a competing endogenous RNA regulatory network. It was found that 14,366 lncRNAs, 151 miRNAs and 9,377 mRNAs were differentially expressed in mice hearts after I/R compared with the Sham group (fold change >2; P<0.05). The results indicated that these differentially expressed genes were involved in multiple molecular functions, including 'guanosine diphosphate binding', 'RNA polymerase II carboxy‑terminal domain kinase activity', 'TATA‑binding protein‑class protein binding', 'nicotinamide adenine dinucleotide binding' and 'protein phosphatase type 2A regulator activity'. The interactions between lncRNA‑miRNA‑mRNA, including five lncRNAs, 38 miRNAs and 196 mRNAs, were predicted, specifically Gm12040‑mmu‑miR‑125a‑5p‑decapping mRNA 1B, Rpl7l1‑ps1‑mmu‑miR‑124‑3p‑G protein‑coupled receptor 146, Gm11407‑mmu‑miR‑190a‑5p‑homeobox and leucine zipper encoding (HOMEZ), 1600029O15Rik‑mmu‑miR‑132‑3p‑HOMEZ and AK155692‑mmu‑miR‑1224‑3p‑activating transcription factor 6β. Collectively, these findings provided novel insights for future research on lncRNAs, miRNAs and mRNAs in myocardial I/R injury.

Morphine pre‑conditioning (MPC) can significantly reduce myocardial ischemic injury and inhibit cardiomyocyte apoptosis, but the underlying mechanism still remains unclear. The aim of the present study was to investigate the protective mechanism of MPC in myocardial hypoxia/reoxygenation (H/R) injury at the microRNA (miR) level. H9c2 cells were used as a model of H/R and subjected to morphine pre‑treatment. The protective effects of MPC on H/R injury in cardiomyocytes were evaluated using MTT and colorimetric assay, as well as flow cytometry. In addition, reverse transcription‑quantitative PCR, western blotting and dual‑luciferase reporter assay experiments were performed to determine the relationship between MPC, miR‑320‑3p and Akt3, and their effects on H/R injury. The present study demonstrated that MPC enhanced cell activity, decreased LDH content, and reduced apoptosis in rat cardiomyocytes, suggesting that MPC could protect these cells from H/R injury. Moreover, MPC partially reversed the increase in miR‑320‑3p expression and the decrease in Akt3 levels caused by H/R injury. Inhibition of miR‑320‑3p expression also attenuated the effects of H/R on cardiomyocyte activity, LDH content and apoptosis. Furthermore, Akt3 was predicted to be a target gene of miR‑320‑3p, and overexpression of miR‑320‑3p inhibited the expression of Akt3, blocking the protective effects of MPC on the cells. The current findings revealed that MPC could protect cardiomyocytes from H/R damage through targeting miR‑320‑3p to regulate the PI3K/Akt3 signaling pathway.

Toll-like receptor 2 (TLR2)-mediated myocardial inflammation serves an important role in promoting myocardial ischemic/reperfusion (I/R) injury. Previous studies have shown that miR‑499 is critical for cardioprotection after ischemic postconditioning (IPostC). Therefore, the present study evaluated the protective effect of IPostC on the myocardium by inhibiting TLR2, and also assessed the involvement of microRNA (miR)‑499. Rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The IPostC was 3 cycles of 30 sec of reperfusion and 30 sec of re‑occlusion prior to reperfusion. In total, 90 rats were randomly divided into six groups (n=15 per group): Sham; I/R; IPostC; miR‑499 negative control adeno‑associated virus (AAV) vectors + IPostC; miR‑499 inhibitor AAV vectors + IPostC; and miR‑499 mimic AAV vectors + IPostC. It was identified that IPostC significantly decreased the I/R‑induced cardiomyocyte apoptotic index (29.4±2.03% in IPostC vs. 42.64±2.27% in I/R; P<0.05) and myocardial infarct size (48.53±2.49% in IPostC vs. 66.52±3.1% in I/R; P<0.05). Moreover, these beneficial effects were accompanied by increased miR‑499 expression levels (as demonstrated by reverse transcription‑quantitative PCR) in the myocardial tissue and decreased TLR2, protein kinase C (PKC), interleukin (IL)‑1β and IL‑6 expression levels (as demonstrated by western blotting and ELISA) in the myocardium and serum. The results indicated that IPostC + miR‑499 mimics significantly inhibited inflammation and the PKC signaling pathway and enhanced the anti‑inflammatory and anti‑apoptotic effects of IPostC. However, IPostC + miR‑499 inhibitors had the opposite effect. Therefore, it was speculated that IPostC may have a miR‑499‑dependent cardioprotective effect. The present results suggested that miR‑499 may be involved in IPostC‑mediated ischemic cardioprotection, which may occur via local and systemic TLR2 inhibition, subsequent inhibition of the PKC signaling pathway and a decrease in inflammatory cytokine release, including IL‑1β and IL‑6. Moreover, these effects will ultimately lead to a decrease in the myocardial apoptotic index and myocardial infarct size via the induction of the anti‑apoptotic protein Bcl‑2, and inhibition of the pro‑apoptotic protein Bax in myocardium.

Previous studies have established that proteinase‑​activated receptor 2 (PAR2) activation protects against myocardial ischemia/reperfusion injury (MI/RI). However, the role of PAR2 deficiency in MI/RI remains unclear. The aim of the present study was to examine the effect of PAR2 deficiency on cardiomyocyte apoptosis and to clarify the potential molecular mechanisms for its protective effect against MI/RI. Using a mouse model of MI/RI, cardiac function was evaluated by echocardiography, infarct size was assessed by triphenyltetrazolium chloride staining, and myocardial cell apoptosis was measured by terminal deoxynucleotide transferase‑mediated dUTP nick end‑labeling staining. Annexin V/propidium iodide staining, and expression of Bcl‑2 and cleaved PARP were determined to assess apoptosis in myocardial H9c2 cells exposed to hypoxia/reoxygenation (H/R) injury‑simulating MI/RI. Phosphorylated ERK1/2, JNK, and p38 MAPK protein expression levels were analyzed by western blotting. The findings indicated that PAR2 deficiency markedly reduced cardiomyocyte apoptosis in the MI/RI mouse model, as well as in myocardial H9c2 cells exposed to H/R. Furthermore, PAR2 knockdown clearly prevented phosphorylation of ERK1/2 and JNK in myocardial H9c2 cells. The results revealed that PAR2 deficiency alleviated MI/RI‑associated apoptosis by inhibiting phosphorylation of ERK1/2 and JNK. Therefore, targeted PAR2 silencing may be a potential therapeutic approach for alleviation of MI/RI.

Endoplasmic reticulum (ER) stress and autophagy are involved in myocardial ischemia‑reperfusion (I/R) injury; however, their roles in this type of injury remain unclear. The present study investigated the roles of ER stress and autophagy, and their underlying mechanisms, in H9c2 cells during hypoxia/reoxygenation (H/R) injury. Cell viability was detected by CCK‑8 assay. The autophagy flux was monitored with mCherry‑GFP‑LC3‑adenovirus transfection. The expression levels of autophagy‑related proteins and ER stress‑related proteins were measured by western blotting. Apoptosis was detected by flow cytometry and western blotting. The results indicated that autophagy was induced, ER stress was activated and apoptosis was promoted in H9c2 cells during H/R injury. The inhibition of ER stress by 4‑phenylbutyrate or C/EBP homologous protein (CHOP)‑targeting small interfering RNA (siRNA) decreased autophagy and ameliorated cell apoptosis during H/R injury. Activation of autophagy by rapamycin attenuated ER stress and ameliorated cell apoptosis. Inhibition of autophagy by 3‑methyladenine or Beclin1‑targeting siRNA aggravated ER stress and exacerbated cell apoptosis, and activation of ER stress by thapsigargin decreased autophagy and induced cell apoptosis. Collectively, the findings of the present study demonstrated that H/R induced apoptosis and autophagy via ER stress in H9c2 cells, and that CHOP may serve an important role in ER stress‑induced autophagy and apoptosis. Autophagy, as an adaptive response, was activated by ER stress and alleviated ER stress‑induced cell apoptosis during H/R injury.

Acute myocardial infarction induces ventricular remodeling, which is implicated in dilated heart and heart failure. The pathogenical mechanism of myocardium remodeling remains to be elucidated. The aim of the present study was to identify key genes and networks for myocardium remodeling following ischemia‑reperfusion (IR). First, the mRNA expression data from the National Center for Biotechnology Information database were downloaded to identify differences in mRNA expression of the IR heart at days 2 and 7. Then, weighted gene co‑expression network analysis, hierarchical clustering, protein‑protein interaction (PPI) network, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were used to identify key genes and networks for the heart remodeling process following IR. A total of 3,321 differentially expressed genes were identified during the heart remodeling process. A total of 6 modules were identified through gene co‑expression network analysis. GO and KEGG analysis results suggested that each module represented a different biological function and was associated with different pathways. Finally, hub genes of each module were identified by PPI network construction. The present study revealed that heart remodeling following IR is a complicated process, involving extracellular matrix organization, neural development, apoptosis and energy metabolism. The dysregulated genes, including SRC proto‑oncogene, non‑receptor tyrosine kinase, discs large MAGUK scaffold protein 1, ATP citrate lyase, RAN, member RAS oncogene family, tumor protein p53, and polo like kinase 2, may be essential for heart remodeling following IR and may be used as potential targets for the inhibition of heart remodeling following acute myocardial infarction.

In a previous study using a microRNA (miRNA/miR) microarray assay, we demonstrated that miR-133b-5p was upregulated in response to hypoxic preconditioning (HPC). The present study was designed to investigate the role of the miR‑133b‑5p in HPC‑induced cardioprotection and the underlying mechanisms involving caspase‑8 and caspase‑3 apoptotic signaling. Adult rats were subjected to myocardial ischemia/reperfusion (I/R) injury with or without ischemic preconditioning (IPC), and the level of miR‑133b‑5p in myocardium was measured. Neonatal rat cardiomyocytes were isolated and subjected to hypoxia/reoxygenation (H/R) injury, with or without HPC. miR‑133b‑5p antagomir was transfected into the cardiomyocytes to observe whether it could block HPC‑induced cardioprotection. Cellular injury was evaluated by detecting cell viability, lactate dehydrogenase (LDH) activity and apoptotic rate. Reverse transcription‑quantitative polymerase chain reaction was used to measure the level of miR‑133b‑5p. The activation of caspase‑8 and caspase‑3 were measured by western blot analysis to detect the cleaved fragments as well as a colorimetric assay. Following myocardial I/R injury, the expression of miR‑133b‑5p was decreased in myocardium, while this decrease was restored by IPC. HPC protected neonatal rat cardiomyocytes against H/R injury by increasing cell viability, while reducing LDH release and cell apoptosis. These protective effects were coupled with the upregulation of miR‑133b‑5p. However, the knockdown of miR‑133b‑5p in the cardiomyocytes blocked HPC‑mediated cardioprotection as reflected by the aggravation of cell injury and apoptosis. HPC upregulated miR‑133b‑5p level was markedly suppressed by the antagomir. In addition, the cleavage and activities of caspase‑8 and caspase‑3 were inhibited by HPC while reversed by knockdown of miR‑133b‑5p. Upregulation of miR‑133b‑5p contributes to HPC‑mediated cardioprotection in cardiomyocytes, and the mechanism may be associated with inhibition of caspase‑8 and caspase‑3 apoptotic signaling.

The gravitational field is an important determinant of cardiovascular function. Exposure to microgravity during spaceflight may lead to a series of maladaptive alterations in the cardiovascular system. The authors have previously demonstrated that microgravity can increase the susceptibility to myocardial ischemia‑reperfusion (IR) injury under simulated microgravity. Although Notch1 signaling protects against myocardial IR injury, whether Notch1 protects against myocardial IR injury under simulated weightlessness remains unknown. The present study is designed to investigate the role of the Notch1 receptor in myocardial IR injury under simulated weightlessness. The differences in Notch signaling expression and myocardial infarct size following myocardial IR were compared between normal rats and tail‑suspended rats that were kept in 30˚ head‑down tilt and hindlimb unloading position. The data revealed low expression levels of Notch1 receptor and its endogenous ligand Jagged1 in normal adult rat hearts. However, significantly higher expression of Notch1 was observed in the border zone compared with the infarcted area and the remote zone following myocardial IR. Notch1 expression was notably reduced in the infarcted hearts of tail‑suspended rats compared with the control group. Conversely, the myocardial infarct size was significantly increased in tail‑suspended rats compared with the control rats. In conclusion, these data suggested that the proper function of Notch signaling may be hampered under simulated microgravity.

Icariin (ICA) has been implicated in certain biological and pathological processes, including myocardial ischemia/reperfusion (I/R) injury. The aim of the present study was to investigate the role of ICA in I/R‑induced cardiomyocyte injury and the potential underlying mechanism. Cell proliferation and apoptosis of H9C2 cells was determined by cell counting kit‑8 and flow cytometry assays. In addition, reactive oxygen species (ROS) production in H9C2 cells was measured by flow cytometry. Reverse transcription‑quantitative polymerase chain reaction and western blot assay were performed to examine the expression levels of proteins, including HSP20, B‑cell lymphoma 2 (Bcl‑2), cytochrome complex (Cyt‑c), apoptotic protease activating factor 1 (APAF1), caspase‑9 andcaspase‑3, and the phosphorylation of Akt (p‑Akt) in H9C2 cells. The present results demonstrated that, compared with the control group, the I/R group demonstrated significantly reduced levels of HSP20 expression and cell proliferation, and increased apoptosis and ROS production in H9C2 cells. In parallel, the expression levels of Cyt‑c, APAF1, caspase‑9 and caspase‑3 were significantly increased in the I/R group, although Bcl‑2 and p‑Akt/Akt expression levels were decreased. Furthermore, compared with the I/R group, ICA treatment and/or HSP20 overexpression significantly improved cardiac function, as evidenced by promoted cell proliferation and inhibited apoptosis of H9C2 cells. The current study indicates that ICA exerts a cardioprotective effect against I/R injury, which is associated with the upregulation of HSP20.

Ischemic reperfusion injury (IRI) contributes to morbidity and mortality worldwide and results in a poor outcome for patients suffering from myocardial infarction. Ischemic post‑conditioning (IPostC), consisting of one or several brief periods of ischemia and reperfusion, generates powerful protection against IRI. The mechanism of IPostC initiation and development has previously been investigated, however still remains to be fully elucidated. Notably, long non‑coding (lnc) RNAs have previously been demonstrated to be important in cardiovascular diseases. However, there is little information about the systematic analysis of IRI‑associated lncRNA expression signature. The present study used microarrays to analyze the lncRNA expression characters of ischemic IPostc (corresponding to IRI), and demonstrated that 2,292 lncRNAs were observed to be upregulated and 1,848 lncRNAs downregulated. Gene ontology (GO) and Pathway analysis subsequently demonstrated that dysregulated lncRNAs participated in various biological processes, which are upregulated or downregulated in IPostC tissues. Finally, the present study verified that AK144818, ENS​MUS​T00000156637, ENS​MUS​T00000118342, ENS​MUS​T00000118149, uc008ane.1, ENS​MUS​T00000164933, ENS​MUS​T00000162347, ENS​MUS​T00000135945, and ENS​MUS​T00000176338, ENS​MUST00000120587, END​MUST00000155271, ENS​MUS​T00000125121 and Uc008thl.1 were associated with the initiation and development of IPostC. The present study may aid in the understanding of the initiation and development mechanisms of IPostC and provide novel and potential biomarkers that may be used in the diagnosis or as therapeutic targets in the treatment of IRI.

DJ‑1 protein, as a multifunctional intracellular protein, has been demonstrated to serve a critical role in regulating cell survival and oxidative stress. To provide in vivo evidence that DJ‑1 is involved in the delayed cardioprotection induced by ischemic preconditioning (IPC) against oxidative stress caused by ischemia/reperfusion (I/R), the present study subjected male Sprague‑Dawley rats to IPC (3 cycles of 5‑min coronary occlusion/5‑min reperfusion) 24 h prior to I/R (30‑min coronary occlusion/120‑min reperfusion). A lentiviral vector containing short hairpin RNA was injected into the left ventricle three weeks prior to IPC, to knockdown DJ‑1 in situ. Lactate dehydrogenase (LDH) and creatine kinase‑MB (CK‑MB) release, infarct size, cardiac function, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, malondialdehyde (MDA), intracellular reactive oxygen species (ROS), and DJ‑1 protein expression levels were assessed. IPC caused a significant increase in the expression levels of DJ‑1 protein. In addition, IPC reduced LDH and CK‑MB release, attenuated myocardial infarct size, improved cardiac function following I/R, and inhibited the elevation of ROS and MDA and the decrease in activities of the antioxidant enzymes SOD, CAT and GPx. However, in situ knockdown of DJ‑1 attenuated the IPC‑induced delayed cardioprotection, and reversed the inhibitory effect of IPC on I/R‑induced oxidative stress. The present study therefore provided novel evidence that DJ‑1 is involved in the delayed cardioprotection of IPC against I/R injury in vivo. Notably, DJ‑1 is required for IPC to inhibit I/R‑induced oxidative stress.

Trimetazidine is a piperazine-derived metabolic agent, which exerts cell protective effects and has been reported to be efficient in the treatment of chronic stable angina pectoris. In addition, it has been shown to exert protection against acute myocardial infarction. The present study aimed to investigate whether trimetazidine protects against cardiac ischemia/reperfusion (I/R) injury, and to determine whether its curative effects are associated with microRNA (miRNA)‑21 expression, Akt, and the B‑cell lymphoma 2 (Bcl‑2)/Bcl‑2‑associated X protein (Bax) pathway. Cardiac I/R injury was induced by ligating the left anterior descending coronary artery in adult rats. Subsequently, cardiac function was evaluated, and the expression levels of miRNA‑21, Bcl‑2, Bax and phosphorylated‑Akt were detected using quantitative polymerase chain reaction and western blotting. The results indicated that trimetazidine was able to significantly protect cardiac function and reduce infarct size in rats following cardiac I/R injury. Furthermore, trimetazidine significantly promoted miRNA‑21 expression and phosphorylated‑Akt protein expression, and reduced the Bcl‑2/Bax ratio in rats following cardiac I/R injury. Knockdown of miRNA‑21 using anti‑miR‑21 plasmids was able to reverse the protective effects of trimetazidine against cardiac I/R injury. These results indicated that miRNA‑21 serves a protective role in cardiac I/R injury via Akt and the Bcl‑2/Bax pathway. In addition, trimetazidine exerts protective effects against cardiac I/R injury through cardiac miRNA‑21 expression, Akt, and the Bcl‑2/Bax pathway. Therefore, the present study provided evidence regarding the protective effects of miRNA‑21 on cardiac I/R injury following treatment with trimetazidine in vivo.

Salvia miltiorrhiza and ligustrazine are traditional Chinese medicines that have been used in combination for treatment of cardiovascular disease, including coronary heart disease, cardiac angina and atherosclerosis in Asia, in particular, China. The present study aimed to determine the effect of S. miltiorrhiza and ligustrazine injection (SLI) on myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R) injuries via the Akt serine/threonine kinase (Akt)‑endothelial nitric oxide synthase (eNOS) signaling pathway. Male Sprague‑Dawley rats were randomly assigned into six groups: i) Sham group; ii) I/R group; iii) Low‑SLI group (6.8 mg/kg/day, i.p.); iv) Medium‑SLI group (20.4 mg/kg/day, i.p.); v) High‑SLI group (61.2 mg/kg/day, i.p.); vi) verapamil group (6 mg/kg/day, i.p.). Prior to surgery, the aforementioned groups were pretreated with a homologous drug once per day for 3 days. The effect of SLI following 35 min coronary artery occlusion and 2 h reperfusion was evaluated by determining infarct size, hemodynamics, biochemical values and histological observations. Additionally, cell viability, caspase‑3 expression, B cell leukemia/lymphoma‑2 (Bcl‑2)/Bcl‑2‑associated X protein (Bax) ratio, phosphorylated (p‑)Akt and p‑eNOS were also investigated following 2 h simulated ischemia and 2 h simulated reperfusion in H9C2 cardiomyocyte cells. Pretreatment with SLI significantly improved cardiac function in a dose‑dependent manner and reduced myocardial infarct size, creatine kinase, lactate dehydrogenase and malondialdehyde levels in blood serum. Additionally, myocardial histopathology changes in the rat model were also alleviated in SLI treatment groups. The present in vitro study revealed that treatment with SLI reduced the apoptotic rate of H9C2 cells by inhibiting the activation of caspase‑3 and increasing the Bcl‑2/Bax ratio. The effect of SLI was associated with increased phosphorylation of the survival kinase Akt at Ser473 and its downstream target eNOS following H/R. The present study determined that SLI may alleviate I/R injury in cardiomyocytes and inhibit apoptosis in rats by the activation of the Akt‑eNOS signaling pathway, and downregulation of the expression levels of proapoptotic factors, including caspase-3.

The aim of the present study was to investigate the effects of microRNA (miR)-128 inhibition on the targeted activation of peroxisome proliferator-activated receptor gamma (PPARG) and on cardiomyocyte apoptosis induced by myocardial ischemia/reperfusion (I/R) injury. In vitro, the expression of PPARG was detected by reverse transcription-quantitative polymerase chain reaction and western blotting in neonatal rat ventricular myocytes (NRVMs) and HEK293 cells transfected with the mimics or inhibitors of miR‑128 or control RNA. Luciferase reporter assays were used to identify whether PPARG is a direct target of miR‑128. In vivo, miR‑128 was knocked down via ear vein injection of antagomir‑128 in a rabbit myocardial I/R injury model. Western blotting investigated the activation of Akt [phosphorylated (p)‑Akt] and the expression of total‑Akt, PPARG and myeloid leukemia cell differentiation protein‑1 (Mcl‑1) in the myocardium. Cardiomyocyte apoptosis was examined with transmission electron microscropy and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. PPARG mRNA and protein were downregulated in NRVMs transfected with miR‑128 mimics, but upregulated by antagomir‑128 compared with control. This indicates that PPARG is a direct miR‑128 target. Activation of Akt (p‑Akt), Mcl‑1 and PPARG expression in the myocardium were increased by miR‑128 inhibition. Furthermore, miR‑128 antagomirs significantly reduced apoptosis in hearts subjected to I/R injury, which was blocked by the PPARG inhibitor GW9662. In conclusion, miR‑128 inhibition attenuated I/R injury‑induced cardiomyocyte apoptosis by the targeted activation of PPARG signaling.

Amniotic fluid-derived mesenchymal stem cells (AFMSCs) are an attractive cell source for applications in regenerative medicine, due to characteristics such as proliferative capacity and multipotency. In addition, Akt, a serine‑threonine kinase, maintains stem cells by promoting viability and proliferation. Whether the transplantation of Akt-overexpressing AFMSCs protects the heart against ischemia‑reperfusion (I/R) injury has yet to be elucidated. Accordingly, the Akt gene was overexpressed in AFMSCs using lentiviral transduction, and Akt‑AFMSCs were transplanted into the ischemic myocardium of rabbits prior to reperfusion. Any protective effects resulting from this procedure were subsequently sought after three weeks later. A histological examination revealed that there was a decrease in intramyocardial inflammation and ultrastructural damage, and an increase in capillary density and in the levels of GATA binding protein 4, connexin 43 and cardiac troponin T in the Akt‑AFMSC group compared with the control group. A significant decrease in cardiomyocyte apoptosis, accompanying an increase in phosphorylated Akt and B‑cell lymphoma 2 (Bcl-2) and a decrease in caspase‑3, was also observed. Furthermore, the left ventricular function was markedly augmented in the Akt‑AFMSC group compared with the control group. These observations suggested that the protective effect of AFMSCs may be due to the delivery of secreted cytokines, promotion of neoangiogenesis, prevention of cardiomyocyte apoptosis, transdifferentiation into cardiomyocytes and promotion of the viability of AFMSCs, which are assisted by Akt gene modification. Taken together, the results of the present study have indicated that transplantation of Akt-AFMSCs is able to alleviate myocardial I/R injury and improve cardiac function.

The growth of the heart during mammalian embryonic development is primarily dependent on an increase in the number of cardiomyocytes (CM). However, shortly following birth, CMs cease proliferating and further growth of the myocardium is achieved via hypertrophic expansion of the existing CM population. The cyclin-dependent kinase inhibitor 2A (Cdkn2a) locus encodes overlapping genes for two tumor suppressor proteins, p16INK4a and p19 alternative reading frame (ARF). To determine whether decreased Cdkn2a gene expression results in improved cardiac regeneration in vitro and in vivo following cardiac injury, the proliferation of CMs isolated from Cdkn2a knockout (KO) and wild‑type (WT) mice in vitro and in vivo were evaluated following generation of ischemia reperfusion (IR) injury. The KO mice demonstrated enhanced CM proliferation not only in vitro, but also in vivo. Furthermore, heart function was improved and scar size was decreased in the KO mice compared with that of the WT mice. The results also indicated that microRNA (miR)‑1 and miR‑195 expression levels associated with cell proliferation were reduced following IR injury in KO mice compared with those of WT mice. These results suggested that the inactivation of INK4a and ARF stimulated CM proliferation and promoted cardiac repair.

Several studies have recently demonstrated that G protein-coupled estrogen receptor (GPER) 30 directly binds to estrogen and mediates its action. The aim of the present study was to investigate the effects of GPER on cardiocyte apoptosis following ischemia/reperfusion injury (MIRI) in H9C2 myocardial cells. H9C2 cells were treated with a specific GPER agonist (G1), 17β-estradiol (E2) or the vehicle. The cells were subjected to 20 min of myocardial ischemia followed by 120 min of reperfusion. They were then randomly assigned to three experimental groups: Control, G1, E2. B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) levels were measured, Hoechst 33258 staining was performed to assess apoptosis, and superoxide dismutase (SOD), tumor necrosis factor (TNF)-α and adenosine triphosphatase (ATPase) levels were determined. To test the specificity of G1, GPER-knockout cells were treated with G1 and analyzed as stated above. Compared with the vehicle-treated groups, G1 and E2-treated groups exhibited elevated Bcl-2 levels, decreased Bax levels and cell apoptosis, significantly increased SOD and ATP levels and decreased TNF-α levels following ischemia-reperfusion. However, G1 had no evident effects on the GPER-knockout cells. In conclusion, the present study suggested that GPER activation provided a cardioprotective effect following ischemia-reperfusion by inhibiting cardiocyte apoptosis.

Ischemic postconditioning (IPostC) is a promising protective mechanism for combating reperfusion injury. However, the role of autophagy in the protective effects of IPostC and the associated signaling pathways have remained to be elucidated. Male Sprague Dawley rats were subjected to 30 min ischemia and 1, 2, 3, 6, 12 and 24 h of reperfusion, with or without IPostC treatment. Autophagic flux was evaluated by detecting mRNA and protein expression levels of microtubule associated protein 1 light chain 3 and p62. Phosphorylated (p)-P70S6 kinase (P70S6K), p-adenosine monophosphate-activated kinase (AMPK) and Beclin 1 protein levels were measured by western blot analysis. Myocardial infarct size was measured using staining with Evans blue dye and myocardial apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Autophagic activity was observed to be inhibited within the first hour of reperfusion, increased during 2-6 h and reduced from 12-24 h following IPostC compared with reperfusion without IPostC. Inhibition of autophagy by chloroquine significantly reversed the effects of IPostC on myocardial infarct size and the levels of apoptosis. IPostC significantly increased Beclin 1 levels, inhibited AMPK activation and increased P70S6K activation within the first hour compared with reperfusion without IPostC. IPostC attenuated the upregulation of Beclin 1 levels, increased AMPK activation and reduced P70S6K activation between 2 and 12 h, and subsequently exerted the opposite effects on these molecules between 12 and 24 h. IPostC was demonstrated to regulate autophagic activity in a time‑dependent manner. The Beclin 1 and AMPK-mammalian target of rapamycin signaling pathways are suggested to be involved in the regulation of IPostC in autophagy.

Myocardial tissue injury caused by ischemia and hypoxia is a major cause of fatal diseases, including coronary atherosclerosis resulting from myocardial infarction and stroke. A number of microRNAs have been demonstrated to function as protectors against ischemia-reperfusion (I/R) and/or hypoxia-reperfusion (H/R)-induced myocardial injury, including microRNA-21 (miR-21). However, the protective mechanism of miR-21 has not been fully elucidated. The present study demonstrated that miR-21 had an anti-apoptotic role in I/R-induced myocardial damage in vivo and in H/R-induced H9C2 cell death in vitro. Of note, the present study indicates that a common molecular mechanism is likely to exist in I/R- and H/R-induced cardiocyte apoptosis. During I/R and H/R, forced expression of miR-21 upregulated the Akt signaling activity via suppressing the expression of phosphatase and tensin homolog (PTEN) and the increased activity of Akt signaling further inhibited apoptosis partially by increasing the ratio of B-cell lymphoma 2(Bcl-2)/Bcl-2-associated X protein, which further suppressed the expression of caspase-3. In conclusion, to the best of our knowledge, it was shown for the first time that miR-21 had a protective role in I/R- and H/R-induced cardiocyte apoptosis via the PTEN/Akt-dependent mechanism. The present study indicates that miR-21 may be a promising agent for the treatment of I/R and H/R-induced myocardial injury.

The aim of this study was to investigate the effects of edaravone on lung injury caused by myocardial ischemia reperfusion (I/R) in rats. Wistar rats (n=24) were randomly divided into 4 groups: the sham operation (S group) and myocardial I/R groups (C group) and two edaravone‑treated groups (E1 and E2 groups). Rats in the E1 and E2 groups were injected with 3 or 10 mg/kg edaravone, respectively, 1 min before reperfusion. The rats were sacrificed and the lung tissue, bronchoalveolar lavage (BAL) fluid and serum were obtained. The concentration of serum creatine kinase isoenzyme (CK-MB) was determined, the lung permeability index (PPI) was calculated and β-defensin-2 (BD-2) mRNA expression in the lung tissue and BD-2 and TNF-α protein content levels were determined. Serum CK-MB activity and the PPI were increased, while BD-2 mRNA and BD‑2 and TNF-α protein levels in the lung tissue were upregulated in the C, E1 and E2 groups compared with the S group. The above‑mentioned indicators were decreased in the E1 and E2 groups compared with the IR group. The level of the decrease for indicators in the E2 group was significantly different compared with that in the E1 group. In conclusion, edaravone reduced the lung injury caused by myocardial I/R in rats. Its mechanism of action was not only oxygen free radical scavenging, but was also associated with a suppression of the inflammatory response of the lung tissue.