Pellinol Promotes Myocardial Ischemia/Reperfusion Injury In The Early Phase By Regulating TRAF3 Degradation

BackgroundWith modern percutaneous coronary intervention,the overall mortality rate still reaches 25%3 years following ST-segment elevation myocardial infarction.Myocardial ischemia/reperfusion(I/R)injury seems to be the majory limitation for the therapeutic efficacy of MI.During the reperfusion course inflammation is one of the majory causes of myocardial I/R injury.Activation of the innate immunity is an essential component of the acute inflammatory response in the setting of I/R.So far,we learned that TLR2/TLR4 expressed in cardiomyocyte mediated signaling pathway plays an important role in the innate immunity.Recent studys show that Pellinol plays a role in regulating TLR2/TLR4 mediated signaling pathway.With the previous found of our group that silencing of Pellinol significantly attenuated MI-induced cardiac dysfunction and remodeling through down-regulation of TLR-/IL-1R-mediated NF-kB activation signaling,we want to study whether Pellinol promotes myocardial I/R injury in the early phase,and if it does,we also want to know the molecular mechanism.ObjectiveTo clarify whether Pellinol promotes myocardial I/R injury in the early phase and regulates H/R(hypoxia/reoxygenation)induced cardiomyocytes injury,and to illuminate the molecular mechanism.Methods1.Animal models in vivo We establish myocardial ischemia/reperfusion injury model by ligation left anterior descending coronary artery(LAD)for 45min and followed by reperfusion.To study the relevance between Pellinol and myocardial I/R injury in the early phase,7-8 weeks C57/BL6 male mice were randomly divided into two groups,including sham and I/R.To clarify whether Pellinol promotes myocardial I/R injury in the early phase through cardiomyocytes,we establish Pellinol cardiomyocytes conditional knockout mice.The mice were randomly divided into four groups,including Peli1Flox/Flox sham,Peli1Flox/Flox I/R,Peli1cKO sham,Peli1cKO I/R.2.Cell models in vitroNeonatal rats ventricular myocytes were isolated from neonatal Sprague-Dawley rats.When in good condition,the incubated cells were subjected to H/R.To clarify whether Pellinol regulates H/R induced cardiomyocytes injury,we transfected cells with adv-shPellinol or adv-shScr.The cells were divided into four groups,including Con,H/R,H/R advshScr,H/R advshPelil.Results1.Pellinol promotes myocardial I/R injury in the early phase through cardiomyocytes.The expression levels of Pellino1 protein and mRNA were increased in myocardium after myocardial ischemia 45min followed by reperfusion 3d,indicating that there has a tight connection between Pellino1 and myocardial I/R injury in the early phase.The myocardial infarction area was very obvious and cardiac function was decreased after myocardial I/R.When compared with PelilFlox/Flox I/R,Pellinol cardiomyocyte conditional knockout decreased the myocardial infarction area obviously and ameliorated cardiac diastolic dysfunction,indicating that Pellinol cardiomyocyte conditional knockout protects heart from myocardial I/R injury in the early phase.MPO activity in myocardium was increased notably after myocardial ischemia 45min followed by reperfusion 24h,suggesting that there were increased neutrophils infiltration in myocardium after myocardial I/R 24h.When compared with Peli 1Flox/Flox I/R,Pellino 1 cardiomyocyte conditional knockout decreased MPO activity in myocardium significantly,indicating that Pellino 1 cardiomyocyte conditional knockout decreased neutrophils infiltration in myocardium after myocardial I/R 24h.Macrophage infiltration in myocardium was increased notably after myocardial ischemia 45min followed by reperfusion 3d.When compared with Peli 1Flox/Flox I/R,Pellino 1 cardiomyocyte conditional knockout reduced macrophage infiltration in myocardium significantly.There was notably cell apoptosis augmentation in myocardium after myocardial I/R.When compared with Peli 1Flox/Flox I/R,Pellino 1 cardiomyocyte conditional knockout reduced cell apoptosis noticeably,indicating that Pellino 1 cardiomyocyte conditional knockout protects cells from myocardial I/R injury in the early phase.2.Pellinol promotes H/R induced cardiomyocytes injury through regulating cIAP2/TRAF3/MAPK signaling pathway.Pellinol protein expression level was increased significantly after H/R.The mRNA levels of inflammatory factors IL-1?IL-6?TNF-a in cardiomyocytes and cardiomyocytes apoptosis were also augmented after H/R.When compared with H/R,Pellinol knockout decreased the mRNA levels of inflammatory factors IL-1?IL-6?TNF-a in cardiomyocytes and reduced cardiomyocytes apoptosis notably,indicating that Pellino1 knockout protects cardiomyocytes from H/R.H/R induced the ubiquitination of cIAP2 and Lys48 ubiquitination of TRAF3,however Pellinol knockout decreased the ubiquitination of cIAP2 and Lys48 ubiquitination of TRAF3.TRAF3 protein level was decreased after H/R,and Pellino1 knockout increased the protein level of TRAF3,indicating that Pellinol knockout inhibited the degradation of TRAF3.H/R promoted the phosphorylation of JNK,p-38,ERK,the MAPKs in cardiomyocytes,however Pellinol knockout decreased the phosphorylation of p-38 and JNK,indicating that Pellinol knockout inhibited the activation of MAPK signaling.ConclusionWith the founds from in vivo and in vitro experiments,we suggests that Pellinol can promote myocardial I/R injury in the early phase and regulates H/R induced cardiomyocytes injury via its regulation of cIAP2/TRAF3/MAPK signaling pathway.