Molecular Mechanisms And Intervention Study Of AngⅡ And TGF-β1 Inducing Atrial Fibrosis

Atrial fibrillation(AF)is the most common arrhythmia in the clinical setting,and traditional pharmacological approaches have proved to have important weaknesses.Structural remodeling has been observed in both clinical and experimental AF paradigms,and is an important feature of the AF substrate,producing fibrosis that alters atrial tissue composition and function.The precise mechanisms underlying atrial fibrosis are not fully elucidated.A variety of signaling systems seem to be centrally involved in the promotion of fibrosis.Firstly,angiotensin Ⅱ(Angll)-induced atrial fibrosis has long been thought to be a potential etiology of AF.And the agonist of nuclear receptor peroxisome proliferator-activated receptor-γ(PPAR-γ),Pioglitazone(Pio),is considered to improve atrial structural remodeling.However,the mechanisms responsible for protective effects of Pio on Angll-induced atrial fibrosis are not completely understood.This paper reports our findings on the proliferation of atrial fibrobalsts induced by Angll via NF-κB/TGF-β1/TRIF/TRAF6 signaling pathway.And our research also provides the new evidence that Pio can inhibit above signaling pathway,which might be at least in part associated with its protective effect on Angll-induced atrial fibrosis.Secondly,studies have established a strong association between inflammation and atrial fibrosis in AF.And Angll is considered to be with cytokine-like,leukocyte-activating properties.However,the mechanisms of interaction between atrial fibroblasts and macrophages are not completely understood.This manuscript reports the findings that patients with AF have higher level of Angll,more serious atrial fibrosis,higher TRIF expression and more macrophage migration in atria compared with patients with sinus rhythm.And our research also provides the new evidence that Angll-treated atrial fibroblasts induced chemotaxis of macrophages in a TRIF-dependent manner by enhancing the expressions of some chemokines.Meanwhile,we also found that macrophages increased the proliferation of atrial fibroblasts.As a result,the interaction between atrial fibroblasts and macrophages created a long-term positive feedback loop,leading to atrial fibrosis.Thirdly,both of AF and atrial fibrosis are independent of ventricular changes.However,there are still no effective medicines to treat atrial fibrosis and very few studies have investigated atrial fibrosis in animal models with selective atrial fibrosis.This manuscript reported that atrial fibrosis was inhibited by antagonist of nuclear receptor mineralocorticoid receptors(MR),eplerenone(EPL),in mutant TGF-β1 transgenic mice,which showed selective atrial fibrosis.Our research also provided new evidence that TGF-β1-treated fibroblasts showed high expression of fibrosis-related molecules that was attenuated by EPL.We also found that TGF-β1-treated fibroblasts induced macrophages migration likely via IL-6 and EPL suppressed these effects of TGF-β1.Thus,EPL may inhibit the TGF-β1/IL-6 axis,and then suppresses the interaction between fibroblasts and macrophages,and thus reduces atrial fibrosis.Taken together,our research highlights emerging therapeutic approaches,antagonist or agonist of nuclear receptor,aimed at attenuating structural remodeling to prevent AFSection one[Objective]The exact mechanisms underlying inhibitory effects of pioglitazone(Pio),PPAR-γ(nuclear recepter)agonist,on Angiotensin Ⅱ(Angll)-induced atrial fibrosis are complex and remain largely unknown.[Methods]In the present study,we examined the effect of Pio on Angll-induced mice atrial fibrosis in vivo and atrial fibroblasts proliferation in vitro.[Results]Our in vivo study showed that Angll infusion induced atrial fibrosis and increased expressions of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β(TRIF)and tumor necrosis factor receptor associated factor 6(TRAF6)in mice models.However,those effects could be attenuated by Pio(P<0.01).As for in vitro experiment,Pio suppressed Angll-induced atrial fibroblasts proliferation via nuclear factor-κB/transforming growth factor-β1/TRIF/TRAF6 signaling pathway in primary cultured mice atrial fibroblasts(P<0.01).[Conclusions]In conclusion,suppression of Pio on Angll-induced atrial fibrosis might be related with its inhibitory effects on above signaling pathway.Section two[Objective]Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation(AF).However,which specific mediator may regulate the interaction between them remains unclear.[Methods]We tested human samples and cell experiments providing the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β(TRIF),an important inflammation-related molecule,in the pathophysiology of AF.[Results]Patients with AF showed higher levels of AngⅡ and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm(SR).In the cell study,AngⅡ induced chemokines expressions in mouse atrial fibroblasts and AngⅡ-stimulated atrial fibroblasts induced the chemotaxis of macrophages,which were reduced by losartan and TRIF siRNA.Meanwhile,AngⅡ-stimulated atrial fibroblasts proliferation was enhanced by macrophages.[Conclusions]Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages,leading to atrial fibrosisSection three[Objective]To study the impacts of eplerenone(EPL),an antagonist of mineralocorticoid receptors(MR)(nuclear recepter),on atrial fibrosis in a mouse model with selective fibrosis in the atrium,and to explore the possible underlying molecular mechanisms[Methods]In order to test the effect of EPL on atrial fibrosis and atria macrophage accumulation,we used mutant TGF-β1 transgenic(Tx)mice with immunohistochemistry and western blotting.Also,we tested the expression of fibrosis-related moleculars in fibroblasts stimulated by TGF-β1.Further,co-culture system of macrophages and fibroblasts was used to detect the inhibitory effect of EPL on the interaction between macrophages and fibroblasts[Results]Using mutant TGF-β1 transgenic(Tx)mice,we first demonstrated that EPL inhibited atrial fibrosis specifically and decreased macrophage accumulation in the atria of these mice.Results from immunohistochemistry and western blotting showed that EPL attenuated protein expression of fibrosis-related molecules such as CTGF and fibronectin in the atria of Tx mice.In culture,EPL inhibited gene expression of fibrosis-related molecules such as TGF-β1,α-SMA,and CTGF in TGF-β1-stimulated fibroblasts.Finally,using a co-culture system,we showed that TGF-β1-stimulated fibroblasts induced migration of macrophages and this was blocked by EPL.EPL also blocked TGF-β1-induced gene expression of IL-6 in fibroblasts[Conclusions]Our data demonstrated that EPL attenuated atrial fibrosis and macrophage infiltration specifically.TGF-β1 and IL-6 were involved in the impacts of EPL on activation of fibroblasts and interactions between fibroblasts and macrophages.