Peli1 Regulates Pressure Overload-induced Cardiac Fibrosis Via Exosomal MiRNA Derived From Cardiomyocytes

BackgroundHeart failure（HF）is the leading cause of death in patients with cardiovascular diseases.Clinically,increased pressure overload induced-cardiac fibrosis caused by hypertension and valvular disease is the main risk factor that promotes the development of patients’cardiac function from decompensated to decompensated,which leads to HF.However,the mechanism has not been elucidated.Pressure overload can significantly increase the mechanical stretch（MS）tension of cardiomyocytes（CMs）and cardiac fibroblasts（CFs）in myocardium,and then induce CFs activation directly or through the paracrine mechanism of signal transmission between cells in myocardium,leading to cardiac fibrosis and dysfunction.In particular,the paracrine effect of CMs is the critical factor,which affects the development of cardiac fibrosis,but the molecular mechanism of paracrine of cardiomyocytes and the CM-CF communication is unclear.Exosomes（Exos）can be used as a biologically active intercellular carrier,which can mediate the cell-cell communication through their shuttled DNA,m RNA,micro RNA（miRNA）,long noncoding RNA（Lnc RNA）and proteins.It has been reported that CM derived miR-378 and exosomal miR-208a play an important role in regulating the activation of CFs,suggesting that CMs can regulate CFs activation through the miRNAs containing exosomes.However,how does CMs-exosomal miRNA change when the CMs subjected to MS?Can CMs regulate CFs activation via the changes in exosomal miRNAs?What is the regulated signal mechanism of changed exosomal miRNAs derived from MS induced-CMs?These scientific problems have not yet been clarified.Reports by us and other scholars have shown that Toll-like receptors/Interleukin-1 receptors（TLRs/IL-1R）mediated intracellular signal transduction played an importantly regulatory role in pressure overload induced-cardiac hypertrophy and fibrosis.Pellino1（Peli1）is a vital protein in the TLRs/IL-1R signaling pathway.Our previous research found that CM conditional knockout of Peli1 could improve myocardial fibrosis and collagen deposition after myocardial infarction,suggesting that Peli1 was involved the development process of post-myocardial infarction-induced cardiac fibrosis.Based on the above researches,it is speculated that CM Peli1 may regulate the CFs activation through paracrine,which in turn affects the development of myocardial fibrosis.This study intends to use a method of organically combining two aspects of in vivo animals and in vitro cells to determine whether Peli1 can mediate CFs activation and pressure overload-induced myocardial fibrosis via cardiomyocyte derived exosomes.To clarify the regulatory effect of Peli1 on the changes of CM-derived exosomal miRNA content,and reveal what kind of miRNAs carried by the MS-CMs-derived exosomes mediating the CM-to-CF paracrine pathway.To further elucidate the signal mechanism in which the changes of the key miRNA regulating CFs activation.ObjectiveTo determine whether Peli1 can regulate the development process of CFs activation and pressure overload induced-cardiac fibrosis through the paracrine mechanism of CMs derived exosomes;To expound whether the regulated mechanism of cardiac fibrosis is related to miR-494-3p mediated the activation of CFs;To provide the new clues for prevention and treatment of myocardial fibrosis and heart failure.Methods and observation targets1.In vitro experimentsWild type（WT）and Peli1 systemic knockout(Peli1^-/-)mouse CMs and WT CFs were cultured,and the CMs were stimulated with MS at a peak elongation of 15%for24 hours to replicate the cardiomyocyte hypertrophy model.First,to confirm that the Peli1 regulated the paracrine function of CF activation through CM-exosomes.CM conditioned medium was used to stimulate myocardial fibroblasts.q RT-PCR was used to detect the level of collagenⅠandⅢm RNA in CFs.Identification of CM derived exosomes by transmission electron microscopy,NTA,Western blot,immunofluorescence;PKH67 labeling the CM-exosomes,and observing the uptake of CFs to CM-exosomes with confocal microscopy.Ed U staining reflecting the proliferation of CFs induced by mechanical stretched CM-exosomes（MS-Exos）;the expression ofα-SMA,which evaluated the differentiation of MS-Exos treated CFs detected by Western blot;q RT-PCR was used to detect the effect of MS-Exos on the changes of CFsα-SMA,collagenⅠ/Ⅲand connective tissue growth factor（CTGF）m RNA levels.Secondly,to screen and clarify the key CM-exosomal miRNA-miR-494-3p on the regulation of CFs activation.Screened and identified the key miRNAs in changed CM-Exos under the stimulation of MS by miRNA microarray and q RT-PCR;after screening,it was preliminarily determined that CM-exosomal miR-494-3p may be the important factor that regulated the activation of CFs;miR-494-3p mimics were further constructed,and the effect of overexpression of miR-494-3p on the activation of myocardial fibroblasts was detected by Ed U staining,Western blot and q RT-PCR.Third,to explore the specific molecular mechanism of CM-exosomal miR-494-3p on CFs activation.Through the reporter assay,the direct target of miR-494-3p on PTEN was proved.Constructed miR-494-3p mimics and inhibitors,using MS-Exos as the inducing factor,and detected the expression of PTEN and AKT（T308/S473）,Smad2（S465/467）,Smad3（S423/425）and ERK（T202/Y204）phosphorylation levels of the above sites.2.In vivo experimentsConstruction of cardiomyocytes-conditional knockout of Peli1 mice(Peli1^cko)and control littermate mice(Peli1^Flox/^Flox,Peli1^F/^F);miR-494-3p was specifically inhibited in myocardium when C57BL/6 mice were injected with r AAV9-anti-miR-494-3p in the tail vein（r AAV9-NC:control group）;To replicate the pressure overload induced myocardial fibrosis model through transverse aortic constriction.First,to prove the effect of Peli1 on the development of myocardial fibrosis caused by pressure overload,the Langendorff perfusion system was used to extract the left ventricular CMs of each group of mice,and pressure overload induced Peli1 was detected by Western blot;morphological observation of heart size,calculation of mouse heart weight/body weight,heart weight/tibia length,H&E staining to detect the severity of cardiac hypertrophy caused by TAC in mice;Masson staining detection of interstitial and perivascular collagen deposition in myocardial tissue,q RT-PCR detection of m RNA expression levels of typeⅠandⅢcollagen and connective tissue growth factor in myocardial tissue,detection of hydroxyproline to calculate total collagen content in myocardial tissue,and assessment of TAC induced cardiac fibrosis.Second,to clarify the effect of miR-494-3p in CM-Exos on cardiac fibrosis and dysfunction caused by pressure overload and explore its specific molecular mechanism.r AAV9-anti-miR-494-3p was used to specifically inhibit the expression of miR-494-3p in myocardial tissue for in vivo experiments in mice.Observed the green fluorescence intensity of GFP through stereo fluorescence microscope and the method of q RT-PCR to detect the level of miR-494-3p m RNA in myocardium to identify the efficiency of r AAV9-anti-miR-494-3p or r AAV9-NC transduction into myocardium;morphological observation of heart size,calculation of mouse heart weight/body weight,heart weight/tibia length,H&E staining to detect the severity of hypertrophy;Masson staining,detection of interstitial and perivascular collagen deposition in myocardial tissue;q RT-PCR detection of myocardial tissueⅠandⅢcollagen and connective tissue growth factor m RNA expression levels;Hydroxyproline was used to detect the total collagen content of myocardial tissue to assess the severity of myocardial fibrosis caused by TAC;echocardiography was used to detect changes in cardiac function;Western blot was used to detect the expression of PTEN and AKT（T308/S473）,Smad2（S465/467）,Smad3（S423/425）and ERK（T202/Y204）phosphorylation levels of the above sites.Results1.Peli1 promoted pressure overload-induced cardiac fibrosis through CM-mediated paracrine mechanism.1.1 TAC could obviously induce the expression of Peli1 in CMs of Peli1^F/F mice.CM conditional knockout of Peli1(Peli1^cko mice)could knock out Peli1 expression in CMs;At the same time,TAC could increase the size of heart in Peli1^F/F mice,increase HW/BW,HW/TL and LW/BW,increase the cross-sectional area of CMs（H&E staining）,myocardial interstitial and perivascular collagen deposition,increaseⅠandⅢcollagen and CTGF m RNA expression,increase hydroxyproline content in myocardium;while Peli1^cko could significantly improve the cardiac hypertrophy and fibrosis caused by TAC.It showed that the expression of Peli1 in myocardial tissue is closely related to pressure overload induced cardiac hypertrophy and fibrosis.CM conditional knockout of Peli1 could effectively improve cardiac hypertrophy and fibrosis caused by pressure overload.1.2 Conditioned medium obtained from MS stimulated CM（MS-C）could significantly increase the expression ofⅠandⅢcollagen m RNA in CFs,while Peli1knock out inhibited above effect.It is suggested that MS can affect the activation of CFs by Peli1 regulating paracrine.2.Peli1 mediated its paracrine regulation of CFs activation through CM-exosomes2.1 Transmission electron microscopy showed that MS-Exos were cup-shaped;NTA showed MS-Exos particle size between 30-150nm;The biomarkers（CD9,CD63,TSG101）of MS-Exos and Con-Exos were expressed,but calnexin,a negative control,was not expressed in CM-Exos determined by Western blot;GW4869 as exosome specificity Inhibitors could significantly aggregate CD63（immunofluorescence）in CMs,suggesting that GW4869 could make MS-Exos secretion blocked.The above results suggested that MS could induce the expression of Peli1 and promote the secretion of CM-Exos.2.2 Compared with the negative control group,more PKH67-labeled MS-Exos（immunofluorescence）were be seen in CFs with positive marker vimentin,confirming that CFs could effectively take up CM-Exos;MS-Exos significantly led to increased Ed U incorporation（Ed U staining）in CFs,increased expression ofα-SMA protein in CFs,increased expression of collagen m RNA ofⅠandⅢcollagens in CFs,and knockout of CM Peli1 Obviously inhibited the proliferation,differentiation and collagen synthesis of CFs caused by MS-Exos.The above results indicated that MS could regulated CFs activation via Peli1 mediated paracrine of CM-Exos.3.Peli1 mediated its paracrine regulation of CFs activation through CM-exosomal miR-494-3p3.1 Using miRNA microarray and a fold change>2 and p<0.05 as the threshold cutoff,we identified 17 miRNAs that were differentially expressed between WT Con-Exos and WT MS-Exos group,and 37 miRNAs between WT MS-Exos and Peli1-/-MS-Exos,8 eligible miRNAs were picked out.Combining the fibrotic signal pathway,myocardial remodeling related molecules and miRNA target gene prediction database,it was speculated that miR-494-3p may target PTEN to mediate its regulatory effect on CFs activation;MS significantly increased miR-494-3p m RNA expression in CMs,MS-Exos and MS-Exos-treated CFs while CM Peli1 knockout could inhibit the above changes.The results suggested that Peli1 had a regulatory effect on the up-regulation of miR-494-3p in MS-Exos,and in turn up-regulated the expression level of miR-494-3p m RNA in CFs.3.2 Compared with NC control group,CFs transfected with miR-494-3p mimics could increase Ed U incorporation,the expression ofα-SMA m RNA and protein,ⅠandⅢcollagen and CTGF m RNA levels.The above findings indicated that overexpression of miR-494-3p in cardiac fibroblasts could promote their activation.3.3 Compared with the Sham group,TAC significantly increased the expression of miR-494-3p m RNA in myocardium of Peli1^F/F mice.CM Conditional knockout of Peli1 could inhibit the high-expressed miR-494-3p m RNA caused by TAC.It was suggested that the CM Peli1 may regulate the upregulation of miR-494-3p m RNA level in pressure overload induced myocardium.3.4 Observation of myocardial GFP green fluorescence after two weeks in vivo transduction of r AAV9-anti-miR-494-3p or r AAV9-NC,which confirmed that r AAV9reached the transduction efficiency for experimental requirement;TAC could induce a significant increase in miR-494-3p m RNA levels in r AAV9-NC mice,while the r AAV9-anti-miR-494-3p myocardial tissue-specific transduced mice showed almost no expression of miR-494-3p m RNA after sham or TAC surgery.The results showed that r AAV9-anti-miR-494-3p myocardial specific transduction could inhibit the high expression of miR-494-3p in pressure overload-induced myocardium.3.5 Compared with the corresponding Sham group,TAC increased the heart size of r AAV9-NC transduced mice,increased HW/BW and HW/TL,increased the cross-sectional area of CMs,increased myocardial interstitial and perivascular collagen deposition,increasedⅠandⅢcollagen and CTGF m RNA levels in myocadium,increased hydroxyproline content.IVS,LVPW,LVID,LW/BW increased,while EF and FS decreased by 33%（42.571±5.584 VS 63.540±3.021,P<0.001,n=6）and38.9%（20.774±3.036 VS 34.002±2.140,P<0.001,n=6）.r AAV9-anti-miR-494-3p significantly improved myocardial hypertrophy,myocardial fibrosis and cardiac dysfunction caused by TAC.The above findings suggested that specific inhibition of myocardial miR-494-3p could effectively improve pressure overload induced cardiac hypertrophy and fibrosis cardiac dysfunction.4.CM derived exosomal miR-494-3p regulated CF activation via inhibiting CF PTEN expression and activating AKT,Smad2/3 and ERK signaling pathway4.1 Luciferase reporter assay showed that miR-494-3p mimics could reduce the luciferase activity of the wild-type PTEN 3’UTR reporter plasmid（WT-PTEN）,however,after mutating the WT-PTEN-3’UTR region,the downregulated PTEN luciferase activity recovered to basal level,which confirmed that miR-494-3p could directly target PTEN.4.2 Overexpression of miR-494-3p mimics or MS-Exos could significantly inhibit the expression of CFs PTEN,and up-regulate the phosphorylation levels of AKT at threonine 308（T308）and AKT at 473 serine sites（T308/S473）,Smad2 at 465 and 467serine sites（S465/467）,Smad3 at 423 and 425 serine sites（S423/425）,and ERK at202 threonine and 204 tyrosine sites（T202/Y204）,while pretreatment of CFs with miR-494-3p inhibitors effectively reversed the above effect.It was suggested that MS-Exos could down-regulate PTEN and activate AKT,Smad2/3 and ERK signaling pathways through miR-494-3p.4.3 Compared with the Sham group,TAC significantly blunted the expression of CFs PTEN,and upregulated the phosphorylation levels of myocardial AKT（T308/S473）,Smad2（S465/467）,Smad3（S423/425）and ERK（T202/Y204）;Specific inhibition of myocardial miR-494-3p could reverse the downregulation of PTEN and the high level phosphorylation of AKT（T308/S473）,Smad2（S465/467）,Smad3（S423/425）and ERK（T202/Y204）.The above results showed that myocardial miR-494-3p played an important role in the pressure overload evoked the downregulation of PTEN expression,and the activation of AKT,Smad2/3 and ERK signal pathway.Conclusion1.CM conditional knock out of Peli1 improves pressure overload-induced cardiac fibrosis;MS can affect CF activation via Peli1 regulated CM paracrine.It is suggested that Peli1 can promote pressure overload-induced cardiac fibrosis through the CM-paracrine mechanism.2.Knockout of CM Peli1 can effectively inhibit the CFs activation caused by MS-Exos,suggesting that Peli1 promotes CF activation by exosome-mediated paracrine.3.CM Peli1 knockout can significantly inhibit the increase of miR-494-3p expression MS-Exos and MS-Exos treated CFs,while overexpression of miR-494-3p in CFs induce their activation;CM conditional knockout of Peli1 or r AAV9-anti-miR-494-3p myocardium-specific transduction can significantly suppress the pressure overload induced high expressed miR-494-3p in myocardium;r AAV9-anti-miR-494-3p can also alleviate pressure overload-induced cardiac fibrosis and improve cardiac dysfunction.The above results suggest that Peli1 can promote the activation of CFs through the paracrine pathway mediated by CM-exosomal miR-494-3p,and thus play a critical role in the development of myocardial fibrosis induced by pressure overload.4.MS-Exos can reduce CFs PTEN expression and activate AKT,Smad2/3 and ERK signaling pathways through the miR-494-3p;Specific inhibition of myocardial miR-494-3p significantly reverses the pressure overload-induced down-regulated PTEN expression in myocardium and activated AKT,Smad2/3 and ERK signaling axis.These findings reveal that CM derived miR-494-3p-containing exosomes regulates CF activation via targeting CF PTEN expression and activating AKT,Smad2/3 and ERK signaling pathway,which is closely related to the occurrence and development of pressure overload induced cardiac fibrosis.