Molecular Mechanisms And Intervention Study Of TGF-β1Regulating CTGF In Atrial Structural Remodeling

Background and Objective:Atrial fibrillation (AF) is a common arrhythmia with high-rate asynchronous atrialelectrical activation. It is also a strongly age-related disease with high morbidity andmortality. With the aging trend of population accelerated, AF has become an importantmedical issue affecting public health. Its etiology and pathogenesis remains unclear, leadingto the lack of safe and effective clinical treatment. Recent studies have shown that atrialstructural remodeling is responsible for the development, maintenance, and recurrence ofAF. Atrial fibrosis is the main manifestation of atrial structural remodeling.Transforming growth factor-β1(TGF-β1), an important pro-fibrotic factors in atrialfibrillation, may regulate its downstream gene connective tissue growth factor (CTGF) viathe TGF-β1/Smad pathway and subsequently induce fibrosis and structural remodeling.Angiotensin II (Ang II) is considered to be a fibrosis promoter. It can regulate theexpression of TGF-β1pathway through angiotensin type I receptor (AT1receptor), playsan important role in promoting fibrosis. Ang II also can induce the expression of CTGF,leading to ventricular fibrosis and ventricular remodeling. However, whether Ang II causesatrial fibrosis and atrial structural remodeling by upregulating CTGF in atrial fibrillation,and whether this regulation is via TGF-β1/Smad pathway, still need further investigation.The aim of this study is to observe the expressions of TGF-β1/Smad/CTGF pathway inchronic atrial fibrillation patients, Ang II-treated mice and Ang II-treated primary atrialfibroblasts, study the effect of pathway’s intervention and discuss their roles in atrialfibrosis and atrial structural remodeling.Methods:Part1: Observe the fibrosis of right atrial tissues from chronic atrial fibrillation (cAF)group and Sinus rhythm (SR) group. Histological features were studied by Masson’strichrome staining and sirius red staining. The mRNA expression of TGF-β1, Smad3and CTGF was measured by real time quantitative PCR (RT-PCR). Proteins of TGF-β1, Smad3and CTGF was measured by Western blotting and immunofluorescence.Part2: Heart rhythm changes of Ang II-treated mice were observed and recorded.Histological features of left atrial tissues from Ang II-treated mice were studied byMasson’s trichrome staining and sirius red staining. The mRNA expression of TGF-β1,CTGF and collagen I from left atrial tissues of Ang II-treated mice was measured byRT-PCR. Then primary atrial fibroblasts were isolated and cultured from left atrial tissuesof Ang II-treated mice, and the mRNA expression of TGF-β1, Smad3and CTGF of thefibroblasts was measured by RT-PCR.Part3: Primary atrial fibroblasts were isolated and cultured from left atrial tissues ofneonatal rats. Ang II, AT1receptor blockers, small interfering RNA of TGF-β1and CTGFwere used to interfere the signaling pathway. The impact of the intervention factors on cellmorphology was observed. The mRNA and protein expression of TGF-β1, Smad3, CTGF,collagen I and fibronectin of the fibroblasts was measured by RT-PCR and Western blotting.Results:1. Compare to SR group, cAF group had higher content of collagen and higher CVF inright atrial tissue (P<0.05).2. The mRNA and protein levels of TGF-β1and Smad3were significantly higher incAF group than SR group (P<0.05).3. The mRNA and protein levels of CTGF were significantly higher in cAF group thanSR group (P<0.05). The protein expression of CTGF strongly correlated with the TGF-β1level. The correlation between Smad3and TGF-β1was also significant (CTGF protein: r=0.528, p <0.05; Smad3protein: r=0.757, p <0.05).4. The protein levels of TGF-β1and CTGF in the cAF group correlated positively withCVF (TGF-β1protein: r=0.580, p <0.01; CTGF protein: r=0.626, p <0.05).5. The ECG of Ang II-treated mice showed persistent atrial fibrillation or paroxysmalatrial fibrillation3weeks after treatment.6. Compare to control group, pathological staining results showed higher content ofcollagen in left atrial tissues of Ang II-treated group.7. Compare to control group, The mRNA levels of TGF-β1, CTGF and collagen I weresignificantly higher in left atrial tissues of Ang II-treated group.(all P<0.01). 8. Compare to control group, The mRNA levels of TGF-β1, Smad3and CTGF weresignificantly higher in the primary atrial fibroblasts which were isolated from mice’s leftatrial tissues(all P<0.01).9. Ang II induced proliferation of atrial fibroblasts. Pretreatment of selective AT1receptor antagonist, TGF-β1siRNA or CTGF siRNA interference inhibited the Ang II-induced proliferation.10. Ang II induced upregulation of TGF-β1, p-Smad3, CTGF, fibronectin and collagenI in atrial fibroblasts. TGF-β1siRNA can reduced this upregulation. SiRNA interferenceagainst CTGF gene can down-regulate the expression of fibronectin and collagen I.11. Pretreatment with losartan reduced the upregulation of TGF-β1, p-Smad3, CTGF,fibronectin and collagen I which were induced by Ang II.Conclusions:1. Atrial fibrosis and structural remodeling occurred in patients with atrial fibrillation.The mRNA and protein levels of TGF-β1, Smad3and CTGF is significantly elevatedin the atrial tissues of patients with cAF, indicate that the upregulation of CTGF maycontribute to atrial structural remodeling, play an important role in AF recurrence andmaintainance.2. By using Ang II-treated mice model, we preliminarily confirmed that Ang II mayinduce upregulation of CTGF and TGF-β1/Smad signaling pathway in atrial fibroblasts, andplays an important role in atrial fibrosis and atrial fibrillation.3. Based on the research of Ang II induced and intervention effect of atrial fibroblasts,we further confirmed that Ang II/AT1-R/TGF-β1/Smad/CTGF pathway may be animportant signaling pathway in the process of atrial fibrosis. CTGF, as a downstreampro-fibrotic factor of TGF-β1, may involve in the AF process of fibrosis and atrial structuralremodeling.