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Remote ischaemic preconditioning (RIPC) is well known to protect the myocardium against ischaemia/reperfusion injury (IRI). Exosomes are small extracellular vesicles that have become the key mediators of intercellular communication. Various studies have confirmed that circulating exosomes mediate RIPC. However, the underlying mechanisms for RIPC-induced exosome-mediated cardioprotection remain elusive. In our study, we found that the expression level of miR-24 was higher in exosomes derived from the plasma of rats subjected to RIPC than in exosomes derived from the plasma of control rats in vivo. The rat plasma exosomes could be taken up by H9c2 cells. In addition, miR-24 was present in RIPC-induced exosomes and played a role in reducing oxidative stress-mediated injury and decreasing apoptosis by downregulating Bim expression in H2O2-treated H9c2 cells in vitro. In vivo, miR-24 in RIPC-induced exosomes reduced cardiomyocyte apoptosis, attenuated the infarct size and improved heart function. Furthermore, the apoptosis-reducing effect of miR-24 was counteracted by miR-24 antagomirs or inhibitors both in vitro and in vivo. Therefore, we provided evidence that RIPC-induced exosomes could reduce apoptosis by transferring miR-24 in a paracrine manner and that miR-24 in the exosomes plays a central role in mediating the protective effects of RIPC.

Myocardial cell apoptosis is the main pathophysiological process underlying ischemia-reperfusion (I/R) injury. FoxO4, which was initially identified as a tumor suppressor that limits cell proliferation and induces apoptosis, plays diverse roles in cardiovascular diseases. However, its contribution to myocardial I/R injury remains unclear. The present study was undertaken to explore the role of FoxO4 in apoptosis during myocardial I/R injury and its underlying mechanisms in vivo. Rats were subjected to ligation/restoration of the left anterior descending branch of the coronary artery and 30 min of ischemia, followed by 4 h of reperfusion. Then, triphenyltetrazolium chloride (TTC) staining was performed to evaluate the infarct size. Transthoracic echocardiography was performed to evaluate cardiac function. Terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) staining was performed to assess cell death in the myocardium. Real-time PCR was performed to measure FoxO4 mRNA expression. Western blots were performed to assess expression levels of the FoxO4 and cleaved caspase 3 proteins. Immunofluorescence staining was performed to measure cleaved caspase 3 expression levels. The hydroxylamine and TBA methods were performed to evaluate malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity, respectively. Dihydroethidium (DHE) staining was performed to measure reactive oxygen species (ROS) generation. We successfully established a rat model of myocardial I/R injury and observed an increase in FoxO4 expression in the myocardium. FoxO4 knockdown significantly protected rats from myocardial I/R injury, as indicated by a marked decrease in infarct sizes and improvements in cardiac function. Mechanistically, I/R induced excessive oxidative stress in rat hearts, most likely as a result of increased FoxO4 levels, and these effects contributed to inducing apoptosis. In conclusion, the FoxO4/ROS pathway represents a potentially novel mechanism underlying apoptosis during myocardial I/R injury. Therapeutic strategies targeting FoxO4 might represent new treatments for myocardial I/R injury.

Objectives To determine the effects of co-exposure to ischemia and cold stress on cardiac injury and whether Bim is involved in this situation. Methods Myocardial ischemia model was established by permanent ligation of left coronary artery. Sprague–Dawley rats were randomly divided to four groups (n=12): sham+normal temperature (S group); sham+cold stress (SC group); myocardial ischemia+normal temperature (I group); myocardial ischemia+cold stress (IC group). At the condition of 26°C, SC and IC groups were kept in a 4°C artificial chamber for 8 h (8:00–16:00) for 4 consecutive days. Cardiac function was assessed by echocardiography; pathological changes were analysed by HE staining; myocardial infarct size was determined by TTC staining; myocardial apoptosis was detected by TUNEL assay; Bim, caspase-3 expression in myocardium were determined by western blot. Results It was demonstrated that, compared with single myocardial ischemia, co-exposure of mycardial ischemia and cold stress could create significant abnormalities in both appearance and pathology of cardiac muscle; compared to group I, apoptosis of myocardium and infarct size were increased markedly in IC group ((27.47±0.47)% vs (19.02±0.30)%, (47.33±2.73) % vs (35.69±1.50), p Conclusion Co-exposure to myocardial ischemia and cold stress aggravates the cardiac injury and increases the expression of pro-apoptosis protein Bim which may play a detrimental role in this situation.

Objective The purpose of this study was to investigate the potential cardioprotection roles of autophagy induced by ischemia/reperfusion (I/R) against apopotosis in cardiac myocytes, and tries to clarify the effects of PI3K in this process. Methods We employed simulated I/R of neonatal rat ventricular myocytes as an in vitro model of I/R injury to the heart. Cardiac myocytes were exposed to 3h hypoxia followed by 14h of reoxygenation, and during the reoxygenation cardiac myocytes were treated with 10mM 3-Methyladenine (3-MA) to inhibit autophagy or 50uM rapamycin to enhance autophagy. Then, we used real-time quantitative PCR (qPCR) to investigate the expression levels of Beclin 1, Bim, and caspase-3. Western blot analysis was used to examine variation in the expression of LC3-II/I (a marker of autophagy), Bim, caspase-3 and PI3K. Results Our results demonstrated that the mRNA expression of Beclin 1 and the ratio of LC3II/I were significantly increased when myocytes followed sI/R (simulated I/R). Moreover, autophagy enhanced by rapamycin during sI/R, significantly reduced the mRNA expression of Bim and caspase-3 , and downregulation of the protein expression level of Bim, caspase-3 and PI3K. In contrast, the mRNA expression of Bim and caspase-3 was increased significantly, and the expression of Bim, caspase-3 and PI3K elevated when 3-MA inhibited the activation of autophagy. Conclusions Our results demonstrate that autophagy induced by sI/R constitutes a powerful and previously uncharacterized protective mechanism against apopotosis in cardiac myocytes via downregulating the pro-apopototic protein Bim and caspase-3, which is downregulated by PI3K. Rapamycin, the autophagic induction, can be used to reduced damage of fatal I/R injury via protecting against apoptosis, which may provide novel strategies for clinical treatment of I/R injury.

Objective This study aims to investigate the potential roles of autophagy induced by ischemia and tries to clarify the effects of HIF-1 in this process. Methods We employed simulated hypoxia of neonatal rat ventricular myocytes as an in vitro model of ischemia injury to the heart. Cardiac myocytes were exposed to 2, 4, 8, 14, 24 h ischemia in hypoxic pouches. Cardiac myocytes were pretreated with 10mM 3-Methyladenine (3-MA) to inhibits autophagy. Western blot analysis was used to examine variation in the expression of LC3-II/I (a marker of autophagy), Bim, caspase-3 and HIF-1. Results Our results demonstrated that the ratio of LC3-II/I was significantly increased when cardiac myocytes were exposed to hypoxia for 8h. Moreover, our results also demonstrated that significant a autophagy was reduced by pretreating with 3-Methyladenine (3-MA) in hypoxia, and the expression of Bim and caspase-3 was significantly increased. Western blot analysis showed that the expression of HIF-1 was increased significantly in hypoxia group and reduced in hypoxia+3-MA group. Conclusions These results demonstrate that inhibition of autophagy induced by hypoxia constitutes a powerful and previously uncharacterized apopotosis in cardiac myocytes via upregulating the pro-apopototic protein Bim and caspase-3, which is regulated by HIF-1. Thus, manipulation of autophagy may represent a potential future therapeutic target to treat or prevent development of ischemic heart disease.

Objective To investigate the role of atorvastatin in myocardium of rats following co-stress of myocardial ischemia and cold stress, and the relative mechanism. Methods Seventy male Sprague–Dawley rats were randomly divided to five groups: sham+normal temperature (S group); sham+cold stress (SC group); myocardial ischemia+normal temperature (I group); myocardial ischemia+cold stress (IC group); atorvastatin treatment group (A group). Co-stress model was induced by ligation of left coronary artery combined with cold stress of 4°C,8 h/day for four consecutive days. Rats in A group were gavaged with atorvastatin 20 mg/kg/d for 3 days before co-stress was induced and thereafter for four days. Excluding the dead animals of anaesthetic accident, arrhythmia, acute heart failure after surgery, 12 rats were in each group eventually. Cardiac function was assessed by echocardiography; myocardial infarct size was determined by TTC staining; p- PI3K, p-GSK3β, Bim and caspase-3 expression in myocardium was determined by western blotting. Results It was demonstrated that co-exposure to myocardial ischemia and cold stress could significantly deteriorate the cardiac function and increase the infarct size (p Conclusion It is possible that atorvastatin attenuates cardiac injury induced by co-stress of myocardial ischemia and cold stress through activating PI3K/Akt/GSK3β pathway and decreasing expression of Bim.