| Background:Cardiac fibrosis is characterized by the cardiac fibroblast activation,excessive proliferation and transition into myofibroblast,which lead to excessive deposition and abnormal distribution of extracellular matrix.Cardiac fibrosis usually happens post myocardial infarction and myocardial hypertrophy,causing chronic heart failure finally.Cardiac fibrosis is a common pathological process in the development of various cardiovascular diseases and a risk for sudden cardiac death.It is known that various cellular signaling pathways such as the renin-angiotensin system,inflammatory factors,and oxidative stress are involved in the process of cardiac fibrosis,whereas the underling mechanisms,especially myocardial fibroblast activation,is not fully understood.Therefore,further exploring its pathophysiologic mechanism may provide new insights and be helpful for clinical treatment.Disruption of endoplasmic reticulum(ER)homeostasis and accumulation of unfolded/misfolded proteins result in ER stress.It is mainly activated by three sensors which subsequently provoke downstream signaling pathways.These sensors contain protein kinase R-like ER kinase(PERK),inositol requiring enzyme 1α(IRE1α),and activated transcription factor 6(ATF6).Although previous studies have confirmed an involvement of ER stress in the pathogenesis of cardiac hypertrophy,how it regulating cardiac fibrosis remains unclear.Autophagy is critical for the development of cardiovascular diseases,such as cardiac hypertrophy and heart failure.Lysosome-mediated autophagy degrades and recycles cellular wastes,including proteins,lipids,and dysfunctional organelles.ATG-mediated autophagosomes/autophagolysosomes formation,and autophagosome contents degradation are key processes involved in autophagy.A variety of autophagy proteins are localized at ER,and autophagy originates from mitochondrial-associated endoplasmic reticulum membrane(MAM),the interface between ER and mitochondria.Sigma-1 receptor(Sig1R),a 223-amino acid ER chaperone lies at MAM is being investigated being related to autophagy and ER stress.Sig1R modulates ER stress,autophagy and apoptosis,and has been confirmed participating in neurodegenerative diseases and cardiac hypertrophy.Fluvoxamine,a selective serotonin reuptake inhibitor with high affinity for the Sig1R,ameliorates cardiac hypertrophy and dysfunction deriving from Sig1R activation.While this finding introduces a role of Sig1R in modulating cardiovascular disease,it raises many questions regarding the underline mechanisms,especially in cardiac fibrosis.In this study,we will explore how Sig1R regulates cardiac fibroblasts activation,as well as its roles in ER stress and autophagy.Objective1.To observe the expression of Sig1R in cardiac fibrosis and cardiac fibroblast activation in vitro.2.To explore how Sig1R modulates cardiac fibroblast activation.3.To investigate whether Sig1R regulates cardiac fibroblast activation via ER stress or autophagy.Methods1.The transverse aortic constriction(TAC)mice model was established to detect the expression of Sig1R and related pathway markers.Intraperitoneal injection of fluvoxamine was carried out for cardiac fibrosis treatment.(1)Mice were subjected to TAC operation to establish the pressureoverload induced cardiac fibrosis mice model.Cardiac functions of the mice were assessed by echocardiography at 4 weeks after surgery.Hematoxylineosin(HE),Sirius Red and Masson staining were performed to observe cardiac hypertrophy and cardiac fibrosis individually.RT-qPCR and western-blot were used to investigate the expression of fibrotic markers in the mice heart tissue.(2)In cardiac fibrosis mice model,the level of Sig1R in the heart tissue was detected by RT-qPCR and western-blot.(3)After TAC surgery,fluvoxamine(the known agonist of Sig1R)was intraperitoneal administrated(20mg/kg)everyday in the following 4 weeks.4 weeks later,the cardiac function of the mice was examined by echocardiography.HE,Sirius Red and Masson staining were carried out to test hypertrophy and fibrosis of the mice heart.RT-qPCR and western-blot were used to detect the levels of fibrotic markers in the mice heart tissue.Levels of ER stress and autophagic flux related proteins were evaluated via western blot.2.The expression of Sig1R was investigated in the primary cultured cardiac fibroblasts with transforming growth factor β1(TGF-β1)treatment.The role of Sig1R was determined both in vitro and in vivo.The potential underlying mechanism were explored in vitro.(1)Neonatal rat cardiac fibroblasts were isolated,cultured and then stimulated with TGF-β1(10ng/mL)for 24 hours.Levels of periostin(POSTN),connective tissue growth factor(CTGF)and TGF-β1,the activated cardiac fibroblast markers were detected by q-PCR and western-blot.We used confocal fluorescence microscope to investigate the expression of α-smooth muscle actin(α-SMA).The proliferation and migration rate of the cardiac fibroblasts were measured by EdU and wound healing assays respectively.mRNA and protein levels of Sig1R in cardiac fibroblasts were tested by RTqPCR,western-blot and confocal.(2)Cardiac fibroblasts were pre-treated with Sig1R-specific agonist fluvoxamine(5μM)and inhibitor NE-100(5μM)for 2 hrs,and their effects on cardiac fibroblasts activation was observed.Specific siRNA(100nM)of Sig1R was transfected into the cardiac fibroblasts,and the effect of Sig1R gene blocking on the activation of cardiac fibroblast was assessed.(3)Westernblot was performed to detect the ER stress signal pathway in cardiac fibroblasts activation.The role of Sig1R on ER stress was explored with Sig1R specific agonist and inhibitor treatments.To identify whether Sig1R regulates cardiac fibroblasts activation via ER stress,the classic ER stress inhibitor 4-phenyl butyric acid(4-PBA,500μM)was used to inhibit ER stress after NE-100(5μM)administration.Thapsigargin(100nM)was used to induce ER stress after fluvoxamine(5μM)administration.Whether Sig1R affects cardiac fibroblast activation by regulating the IRE 1α pathway was determined by use of the inhibitor of IRE1α(4μ8c,5 μM)pathway.(4)Western-blot and confocal were used to observe the autophagic flux in cardiac fibroblast activation.As well,autophagic flux were assessed after Sig1R agonist or inhibitor administration.The mRFP-GFP-LC3 adenovirus was transferred into the cardiac fibroblast to track the autophagic flux.Whether Sig1R regulates cardiac fibroblast activation by maintaining autophagic flux was also determined.Results1.Sig1R expression was reduced in fibrotic mice heart tissue and TGF-β1 treated cardiac fibroblasts.(1)Cardiac fibrosis mice model is well established.Compared with the Sham group,the mRNA and protein levels of Sig1R in the heart tissue were significantly reduced.(2)Compared with the control group,the mRNA and protein levels of Sig1R decreased markedly in activated cardiac fibroblasts.2.In vivo,fluvoxamine treatment alleviated cardiac fibrosis and cardiac dysfunction induced by TAC in mice.In vitro,specific activation of Sig1R activity alleviated the activation of cardiac fibroblasts,and specific inhibition of Sig1R activity or Sig1R mRNA silencing exacerbated the cardiac fibroblasts activation.(1)Specific activation of Sig1R activity with fluvoxamine alleviated the activation of cardiac fibroblasts induced by TGF-β1 and reduced the proliferation and migration capacity of activated cardiac fibroblasts.(2)Specific inhibition of Sig1R activity further promoted the activation of cardiac fibroblasts induced by TGF-β1 and increased the proliferation and migration capacity of activated cardiac fibroblasts.(3)Compared with the TAC group,the cardiac function of the mice treated with fluvoxamine(FLV)was significantly increased,the thickness of the interventricular septal and the left ventricular wall was markedly reduced,and the cardiac hypertrophy and fibrosis was significantly attenuated.3.Sig1R regulated cardiac fibroblast activation through inhibiting ER stress and maintaining autophagic flux.(1)Sig1R regulated cardiac fibroblast activation via blocking ER stress.a)The PERK/ATF4,IRE1α/XBP1 and ATF6 pathways were activated in the fibrotic mice cardiac tissue;The PERK/ATF4,IRE1α/XBP1 pathways were triggered in activated cardiac fibroblasts,but the ATF6 pathway was not affected.b)After activation of Sig1R activity,the PERK/ATF4,IRE1α/XBP1 and ATF6 pathways were inhibited in the fibrotic mice cardiac tissue;the IRE1α/XBP1 pathways were hindered in activated cardiac fibroblasts.Whereas the PERK/ATF4 and IRE1α/XBP1 were further activated after Sig1R depression.c)4-PBA administration after inhibition of Sig1R attenuated cardiac fibroblast activation.Sig1R agonist alleviated the cardiac fibroblast activation induced by thapsigargin.d)Sig1R inhibitor treatment after administration of IRE1α pathway inhibitor 4μ8c attenuated cardiac fibroblast activation.(2)Sig1R modulated cardiac fibroblast activation via maintaining autophagic flux.a)Inactivated cardiac fibroblast,ratio of LC3-Ⅱ/LC3-Ⅰ,the autophagosome marker was increased,and the degradation of p62 protein was decreased,indicating that autophagy flux is blocked.b)The activation of Sig1R activity alleviated the degree of obstruction of autophagic flux,whereas inhibition of Sig1R activity further hindered the autophagic flux.c)Transfection of mRFP-GFP-LC3 adenovirus further confirmed that the stimulation of Sig1R alleviated the obstruction of autophagic flux.Conclusions1.The level of Sig1R is lower in fibrotic mice heart tissue and activated cardiac fibroblasts.2.Sig1R agonist attenuates the activation of cardiac fibroblast,which indicates that Sig1R has a certain protective role in the process of cardiac fibroblast activation.3.Sig1 Ragonist blocks cardiac fibroblast activation through inhibiting ER stress and maintaining autophagic flux. |
PDF Full Text Request