Background: Toll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury. Methods: Male C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1. h prior to myocardial ischemia (60. min) followed by reperfusion. Untreated mice served as I/R control (n. =10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14. days. Results: CpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14. days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection. Conclusion: CpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.

Background: Toll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury. Methods: Male C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1. h prior to myocardial ischemia (60. min) followed by reperfusion. Untreated mice served as I/R control (n. =10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14. days. Results: CpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14. days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection. Conclusion: CpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.

AimsThe present study examined the role of microRNA-125b (miR-125b) in myocardial ischaemia/reperfusion (I/R) injury. We constructed lentivirus-expressing miR-125b (LmiR-125b) and developed transgenic mice with overexpression of miR-125b.Methods and resultsLmiR-125b was transfected into mouse hearts through the right common carotid artery. Lentivirus vector (LmiR-Con) served as vector control. Untreated mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (45 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by 2,3,5-triphenyltetrazolium chloride staining. In separate experiments, hearts were subjected to ischaemia (45 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography before, as well as 3 and 7 days after myocardial I/R. Increased expression of miR-125b significantly decreased I/R-induced myocardial infarct size by 60 and prevented I/R-induced decreases in ejection fraction (EF) and fractional shortening (FS). Transgenic mice with overexpression of miR-125b also showed the protection against myocardial I/R injury. Increased expression of miR-125b attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. Western blot showed that increased expression of miR-125b suppresses p53 and Bak1 expression in the myocardium. In addition, transfection of LmiR-125b decreased the levels of TNF receptor-associated factor 6 (TRAF6) and prevented I/R-induced NF-κB activation.ConclusionmiR-125 protects the myocardium from I/R injury by preventing p53-mediated apoptotic signalling and suppressing TRAF6-mediated NF-κB activation.

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3-/-) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3-/- and WT mice were subjected to myocardial ischemia (45min) followed by reperfusion for up to 3days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3-/- mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3-/- mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.

AimsWe have reported that either toll-like receptor 4 deficiency (TLR4 -/-) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.Methods and resultsWe constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.ConclusionsMicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A-/- and WT mice were subjected to ischemia (45min) followed by reperfusion for up to 7days. SR-A-/- mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A-/- heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A-/-) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A-/- macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A-/- macrophages. The levels of miR-125b in SR-A-/- macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A-/- macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.

AimsWe have reported that either toll-like receptor 4 deficiency (TLR4 -/-) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.Methods and resultsWe constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.ConclusionsMicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A-/- and WT mice were subjected to ischemia (45min) followed by reperfusion for up to 7days. SR-A-/- mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A-/- heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A-/-) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A-/- macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A-/- macrophages. The levels of miR-125b in SR-A-/- macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A-/- macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.