Mutation In NPPA Causes Atrial Fibrillation By Activating Inflammation And Cardiac Fibrosis In A Knockin Rat Model

Atrial fibrillation(AF)affects >30 million individuals worldwide.Despite advances in the management of patients with AF,this arrhythmia remains one of the major causes of stroke,and increases risk of heart failure,sudden death,and cardiovascular morbidity in the world.AF is rare in the young,but its prevalence rate increases with age.Both genetic and environmental factors as well as their interactions contribute to the pathogenesis of AF.Genetic mutations in many ion channels such as KCNQ1,KCNH2,SCN5 A,SCN3B and GJA5 cause AF.Interestingly,mutations in non-ion channels such as NPPA,NUP155,CAV1 and TNNI3 also cause or increase susceptibility to AF.Structural remodeling results in electrical dissociation between muscle bundles and local conduction heterogeneities,which favors re-entry and perpetuation of the arrhythmia.One hallmark of AF is its progressive nature due to the electrical and structural remodeling of the cardiac tissue at the cellular and subcellular levels.NPPA encodes the atrial natriuretic peptide(ANP),which is a circulating hormone that belongs to the natriuretic peptide family(NPS).ANP has potent natriuretic,diuretic,vasodilator,sympatholytic and renin-and aldosterone-suppressing activities that are also involved in the regulation of volume and electrolyte balance and blood pressure.The main receptor for ANP is the natriuretic peptide receptor A(NPRA),which is a guanylate cyclase that catalyzes the production of the important second massager cyclic guanosine 3'5' monophosphate(cGMP)from GTP.The second messenger cGMP and its downstream effector protein kinase G(PKG)mediate a cascade of intracellular signaling processes involved in stress and physiological responses.Our lab previously reported a mutational analysis of the NPPA gene in a panel of 384 lone AF patients.Screening of all exons and exon–intron boundaries of NPPA identified a novel variant of unknown significance,p.Ile138 Thr.ANP has 28 amino acids,which are connected by a disulfide bond between two cysteine residues at positions 7 and 23.The p.I138 residue lies in the cyclic loop of ANP,which is responsible for receptor selectivity and is highly conserved during evolution.It is the first single-amino-acid change in the mature ANP and located in a highly conserved domain.However,more evidence is needed to establish the pathogenicity of the variant.Moreover,the molecular mechanism by which the p.Ile138 Thr variant cuases AF is unknown.Therefore,in this study,we developed a knockin(KI)line of rats with this mutation.The physiological characterization of the KI rats in vivo and molecular and cellular studies in vitro provided unequivocal evidence that p.Ile138 Thr is a mutation that causes AF,and uncovered a novel,fibroblast-based molecular mechanism by which ANP mutations cause AF.The details of our data are describe below.Firstly,we used multiple computational programs to predict the pathogenicity of the p.Ile138 Thr variant.Multiple computational programs predict that p.Ile138 Thr is damaging.Based on 2015 ACMG(American College of Medical Genetics and Genomics)standards and guidelines,p.Ile138 Thr may be classified as a pathogenic mutation.Secondly,to identify the ability of the mANP to activate the receptor,we characterized the interaction between ANP and its receptor NPRA using coimmunoprecipitation(Co-IP)analysis and detected the level of cGMP stiumluated by wANP and mANP.Co-IP analysis revealed that the p.Ile138 Thr mutation reduces the interaction between ANP and its receptor NPRA,which decreases levels of second messenger cGMP.Thirdly,the p.Ile137 Thr mutation of NPPA was introduced into rat genomic DNA by the TALEN technique.The atria of male and female rats aged between 200 g and 250 g at 10 weeks were rapidly stimulated through the esophagus and the surface electrocardiogram was recorded.The cardiac function and diastolic function of rats were measured by ultrasound.Immunohistochemistry was used to detect the expression of related proteins and markers in heart sections.KI rats are more susceptible to the development of AF compared with WT rats.Compared to wild type(WT)rats,KI rats showed a longer duration and higher susceptibility to the development of AF.Forthly,to determine whether there is atrial structural remodeling in KI rats,we carried out Masson's trichrome staining for different parts of the heart.Significantly more fibrosis was identified in atria in KI rats than in WT rats,however,no difference was found in ventricular fibrosis compared with WT rats.The ratio of heart to body weight was significantly increased in KI rats compared with WT rats.Echocardiography showed that KI rats have hyperdynamic LV function(i.e.increased EF% and FS%)as compared with WT rats.Moreover,immunostaining analysis of the cardiac sections further showed that the expression level of IL-1?,Col1,MMP9 and CD45 was significantly increased in KI rats compared with WT rats.The myocardium of KI rats presented with more severe inflammation and cardiac remodeling than WT rats.The expression of phosphorylatedp65 and p65 in cardiac fibroblasts isolated from KI rats was higher than that in WT rats.Fifthly,to detect the effect of mANP on target cells in the heart,we pretreated rat neonatal fibroblasts with angiotensin II combined with wANP or mANP.Moreover,highthroughput RNAseq and pathway analyses were performed.We performed RNAseq analysis with neonatal rat cardiac fibroblasts treated with either mANP with the p.Ile138 Thr mutation or control wANP in combination with angiotenson II(AngII,1 mg/mL).Interestingly,the TNF-? pathway and NF-?B pathways were enriched in the mutant ANP group.Follow-up real-time RT-PCR analysis confirmed that the expression level of Tnfa mRNA was significantly up-regulated by 4-fold by mANP compared with wANP.?-SMA expression in KI rat cardiac fibroblasts is higher than the WT in rats.We analyzed the effects of mANP vs.wANP on cardiac fibroblasts of neonatal rats together with or without AngII(1 mg/mL).Compared with wANP,the mANP significantly increased proliferation of cardiac fibroblasts in a concentration–dependent manner.These data suggest that the p.Ile138 Thr is a functional mutation that promotes proliferation of cardiac fibroblasts.Immunofluorescence experiments with staining with fibroblast marker vimentin and proliferation marker Ki67 showed that p.Ile138 Thr mutation could promote the proliferation of cardial fibroblasts.Our study establishes the first KI rat model for an ANP mutation and demonstrates that the p.Ile138 Thr mutation of NPPA causes AF in vivo and further mechanistic studies identify a cardiac fibroblast-based molecular mechanism for the pathogenesis of AF.The p.Ile138 Thr mutation may cause AF by inducing inflammation,aggravating cardiac remodeling,and increasing the proliferation of cardiac fiborblasts by inhibiting the interaction between ANP and NPRA,which reduces intracellular cGMP levels and activates TNF-? and NF-?B pathways.Finally,the occurrence or susceptibility of KI animal atrial fibrillation was caused.Together,these studies indeitify a new molecular mechainism for the pathogenesis of AF.