A Novel MyD88 Inhibitor Significantly Reverses Ischemia And Reperfusion Injury Following Acute Myocardial Infarction

Part I Inhibition of dimerization of MyD88 by MyD88 inhibitor TJ-M2010-5 and TJ-M2010-5 reduced myocardial ischemia and reperfusion injury in mice [Objective] To explore the effect of MyD88 inhibitor TJ-M2010-5 on dimer of MyD88,and its effect and mechanism on myocardial ischemia and reperfusion injury.[Methods] The full sequence length-expressing plasmids pcDNA3.1-(Flag-MyD88)for FLAG-tagged MyD88,pcDNA3.1-(HA-MyD88)for HA-tagged MyD88,pcDNA3.1-(Flag-TIRAP)for FLAG-tagged TIRAP,pcDNA 3.1-(Flag-con)for FLAG-tagged control,and pcDNA 3.1-(HA-con)for HA-tagged control were transfected to H9C2 cells.Then H9C2 cells were treated with different doses of TJ-M2010-5(0,15,or 30 ?mol/L).After 48 h,cells were harvested for co-immunoprecipitation assays to determine whether various concentrations of TJ-M2010-5 could inhibit MyD88 dimerization.By establishing a mouse model of myocardial ischemia and reperfusion injury,the left anterior descending coronary arteries were ligated by tying slipknots with a 7-0 silk suture around PE-10 tubing.After 30 min,reperfusion was permitted for various time periods by untying the slipknots.Sham-operated animals were subjected to the aforementioned surgical procedures except that the sutures were passed under the left anterior descending arteries without being tied.Mice were divided into four groups:(1)Sham group,whereby mice were subjected to intraperitoneal injection(i.p.)with dd H2O(50 mg/kg);(2)dd H₂O group,whereby mice were injected with dd H2O(50 mg/kg,i.p.)as a positive control plus an equal volume of TJ-M-2010-5;(3)TJ-M2010-5 group,whereby mice were injected with TJ-M2010-5(50 mg/kg,i.p.);and(4)MyD88-/-group,whereby mice were injected with dd H2O(50 mg/kg,i.p.).All groups were treated once daily for 2 d before surgery.The mRNA and protein expression levels of MyD88 in heart tissues were detected.The expression of MyD88 in-actinin+ cells,f4/80+ cells and vimentin+ cells in the myocardium was observed by immunofluorescence.After 1,3,7,and 14 d of reperfusion,mice in each group were examined by cardiac ultrasound for left ventricle(LV)dimensions at end-diastole and at end-systole,and ejection fraction and fractional shortening were calculated to evaluate cardiac function.Serum LDH,CK-MB and c TNI in blood samples were detected respectively.Evans blue-TTC double staining was used to calculate the ischemic area(AAR),infarcted area(AI)and AI/AAR.HE staining was used to observe the pathological changes of the heart.The expression of MPO and F4/80 in heart was observed by immunohistochemistry.TUNEL fluorescence was used to observe the level of the apoptosis rate of ?-Actinin+ cardiomyocytes.We isolated single mouse heart cells 24 h after MIRI and evaluated neutrophil and macrophage activation in each group by FCM.The levels of IL-1?,IL-6,and TNF-? were detected by ELISA and q RT-PCR.TLR4,TLR2,p-P38,p-JNK,p-ERK,NF-?B nuclear translocation,AP-1,COX2,Cleaved caspase-3,Bax,and Bcl-2 protein levels were analyzed by western blotting in heart tissues after MIRI.We examined Masson's trichrome,collagen I,collagen III,fibronectin,and ?-SMA staining via IHC 28 d post-MIRI.We further studied the effect of TJ-M2010-5 on collagens I and III,fibronectin,and ?-SMA expression at the protein and mRNA level.[Results] MyD88 expression highly at various reperfusion time points after MIRI.Doubleand triple-immunofluorescence analyses revealed that nearly all MyD88-expressing cardiac cells were ?-actinin+ and F4/80+ on day 1 after MIRI and vimentin+ on day 28 after ischemia reperfusion.MyD88 mRNA levels significantly increased as soon as 2 h after reperfusion and continued to rise until 28 d after reperfusion.Moreover,western blotting using heart tissues of mupregulated in the treatdimerization in a dose-TJ-M2010-5 or knock-Echocardiography of Tfraction compared wipretreatment attenuated and serum concentratioTJ-M2010-5 treatment gshowed that the dd H₂Ocellular degeneration aHowever,these patholpretreatment.IHC indiF4/80+ macrophages ingroup 24 h after MIRI using heart tissues of mice with induced MIRI confirmed that MyD88 was substantially upregulated in the treatment groups relative to the control.TJ-M2010-5 inhibited MyD88 dimerization in a dose-dependent manner.Compared to untreated mice,pretreatment with TJ-M2010-5 or knock-out of MyD88 reversed nearly two-thirds of the infarct area.Echocardiography of TJ-M2010-5-treated mice revealed significant increases in ejection fraction compared with wild-type mice after ischemia reperfusion.TJ-M2010-5 pretreatment attenuated the LDH,CK-MB,and c Tn I levels at 24 h.The mRNA expression and serum concentrations of IL-1?,IL-6,and TNF-? were substantially lower in the TJ-M2010-5 treatment group than in the dd H₂O group.Histopathological heart tissue slices showed that the dd H₂O group presented with a wide range of focal myocardial lesions,cellular degeneration and necrosis,and inflammatory cell infiltration 24 h after MIRI.However,these pathological alterations were significantly ameliorated by TJ-M2010-5 pretreatment.IHC indicated that there were considerably fewer MPO+ neutrophils and F4/80+ macrophages in heart tissues of TJ-M2010-5-pretreated mice than in the dd H₂O group 24 h after MIRI.TJ-M2010-5 pretreatment significantly reduced the number of apoptotic cardiomyocytes 24 h after MIRI.FCM demonstrated that levels of CD45+CD11b+Ly-6G+ neutrophils and CD45+CD11b+F4/80+ macrophages were substantially lower in TJ-M2010-5-pretreated mice than in the dd H₂O group.TJ-M2010-5 pretreatment markedly downregulated TLR4,TLR2,MyD88,p-P38,p-JNK,p-ERK,NF-?B nuclear translocation,AP-1,COX-2,cleaved caspase-3 and Bax in heart tissues.Collagens I and III,fibronectin,and ?-SMA mRNA and protein levels were significantly downregulated by TJ-M2010-5 treatment.[Conclusion] MyD88 is significantly upregulated in mouse heart tissues subjected to induced MIRI.TJ-M2010-5 inhibits MyD88-MyD88 homodimerization and TIRAP-MyD88 heterodimerization.TJ-M2010-5 can reduce the inflammation and remodeling of myocardial ischemia reperfusion injury.This protective effect is mainly due to the inhibition of NF-?B and MAPK signaling pathway.Part II The effect and mechanism of MyD88 inhibitor TJ-M2010-5 on primary cardiomyocytes,BMDMs and fibroblasts [Objective] To investigate the effect and mechanism on primary myocardiocytes after A/R,the activation and migration of BMDMs and fibroblasts.[Methods] Primary cardiomyocytes and fibroblasts from 1-day-old C57BL/6 mice,and BMDMs from 6–8-week-old male C57BL/6 mice were prepared.CCK-8 assay was used to detect the cytotoxic effects of MyD88 inhibitor TJ-M2010-5 at different concentrations on primary cardiomyocytes,fibroblasts and BMDMs,respectively.The protein levels of MyD88 in cardiomyocytes after A/R,BMDMs and primary fibroblasts stimulated by supernatant culture of cardiomyocytes after A/R were detected.The primary cardiomyocytes underwent A/R,and different concentrations of TJ-M2010-5 were administered at the same time.The apoptosis level of cardiomyocytes was detected by flow cytometry,and the transcription level of inflammatory cytokines IL-1? and IL-6 the secretion level of cell culture supernatant were detected.Phosphorylation levels of P38,ERK,JNK,apoptotic protein levels of Bcl-2 and cleaved Caspase3,NF-?B nuclear translocation were detected.Macrophages were incubated with TJ-M2010-5 for 12 h before adding the supernatants of cardiomyocytes subjected to A/R and incubating the cells for another 24 h.The activation of macrophages was detected by FCM.The expression of TNF-?,IL-1? and IL-6 in cell culture supernatant were detected.Phosphorylation levels of P38,ERK,JNK and NF-?B nuclear translocation were detected.Furthermore,we incubated fibroblasts with the supernatant of cardiomyocytes subjected to A/R and then analyzed the effect of TJ-M2010-5 on ?-SMA expression.Macrophage and fibroblast migrations were monitored using a Transwell migration assay.[Results] MyD88 was substantially upregulated in A/R-treated cardiomyocytes,BMDMs and fibroblasts stimulated by the supernatants of A/R-treated cardiomyocytes.TJ-M2010-5 had no cytotoxic effect on mouse primary cardiomyocytes,fibroblasts and BMDMs at a concentration of 30?mol/L.FCM showed that TJ-M2010-5 pretreatment reduced neonatal rat cardiomyocyte apoptosis in a dose-dependent manner.After A/R,levels of cleaved caspase-3 decreased whereas Bcl-2 levels increased in cardiomyocytes pretreated with TJ-M2010-5 compared with the dd H₂O group.TJ-M2010-5 dramatically downregulated phosphorylation of P38,JNK,and ERK,as well as NF-?B nuclear translocation in cardiomyocytes.Moreover,IL-1? and IL-6 mRNA expression and concentration in the supernatants were markedly lower in the TJ-M2010-5 pretreatment group than in the A/R group.TJ-M2010-5 downregulated CD80,CD86,and MHCII in BMDMs in a dose-dependent manner.TJ-M2010-5 at 30 ?mol/L significantly inhibited BMDM activation.TJ-M2010-5 also downregulated p-P38,p-JNK,and p-ERK,as well as NF-?B nuclear translocation in macrophagocytes.We further employed ELISA to measure IL-1?,TNF-?,and IL-6 levels 24 h after stimulation with the supernatants of A/R-exposed cardiomyocytes with or without TJ-M2010-5 pretreatment.We found that these proinflammatory factors were downregulated by TJ-M2010-5 in a dose-dependent manner.We incubated fibroblasts with the supernatant of cardiomyocytes subjected to hypoxia and reoxygenation and then analyzed the effect of TJ-M2010-5 on ?-SMA expression.Both ?-SMA protein and mRNA levels increased in the A/R group and decreased in response to TJ-M2010-5 pretreatment.The effect of TJ-M2010-5 on macrophagocyte and fibroblasts migration was observed by Transwell migration assays,which indicated that TJ-M2010-5 significantly suppressed macrophages and fibroblasts migration in a dose-dependent manner,especially at 30 ?mol/L.[Conclusion] MyD88 was substantially upregulated in A/R-treated cardiomyocytes,macrophagocytes and fibroblasts stimulated by the supernatants of A/R-treated cardiomyocytes.TJ-M2010-5 pretreatment reduced neonatal rat cardiomyocyte apoptosis in a dose-dependent manner.After A/R,levels of cleaved caspase-3 decreased whereas Bcl-2 levels increased in cardiomyocytes pretreated with TJ-M2010-5 compared with the dd H₂O group.TJ-M2010-5 dramatically downregulated phosphorylation of P38,JNK,and ERK,as well as NF-?B nuclear translocation in cardiomyocytes and macrophages.TJ-M2010-5 attenuates macrophages and fibroblast activation and migration.