2、Circulation 2019-m6A甲基化酶mettl3控制心脏稳态和肥大.pdf

Circulation.2019;139:533545.DOI:10.1161/CIRCULATIONAHA.118.036146 January 22,2019533Editorial,see p 546BACKGROUND:N6-Methyladenosine(m6A)methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA.The role of m6A mRNA methylation in the heart is not known.METHODS:To determine the role of m6A methylation in the heart,we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing.We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3(METTL3)both in culture and in vivo.We generated cardiac-restricted gain-and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function.RESULTS:We measured the level of m6A methylation on cardiomyocyte mRNA,and found a significant increase in response to hypertrophic stimulation,suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy.Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways.Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow,whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo.Finally,cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress,showing the necessity of RNA methylation for the maintenance of cardiac homeostasis.CONCLUSIONS:Our study identified METTL3-mediated methylation of mRNA on N6-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes.Enhanced m6A RNA methylation results in compensated cardiac hypertrophy,whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction,highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.2018 The Authors.Circulation is published on behalf of the American Heart Association,Inc.,by Wolters Kluwer Health,Inc.This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License,which permits use,distribution,and reproduction in any medium,provided that the original work is properly cited and is not used for commercial purposes.Lisa E.Dorn,BALior Lasman,BMSJing Chen,PhDXianyao Xu,MSThomas J.Hund,PhDMario Medvedovic,PhDJacob H.Hanna,MD,PhDJop H.van Berlo,MD,PhDFederica Accornero,PhDORIGINAL RESEARCH ARTICLEThe N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophyhttps:/www.ahajournals.org/journal/circCirculationKey Words:gene expression profiling hypertrophy mice,transgenic RNA processing,post-transcriptionalSources of Funding,see page 544Downloaded from http:/ahajournals.org by on January 22,2019Dorn et al METTL3 Controls Cardiac HomeostasisJanuary 22,2019 Circulation.2019;139:533545.DOI:10.1161/CIRCULATIONAHA.118.036146534ORIGINAL RESEARCH ARTICLEThe heart comprises long-lived cardiomyocytes that,in response to stress stimulation such as pressure overload or myocardial infarction,un-dergo hypertrophic growth.This hypertrophic re-sponse is initially an adaptive process to produce suf-ficient force to match an increase in wall tension or increased workload,but can ultimately lead to heart failure.1 Cardiac hypertrophy is mediated by increased gene expression and production of select proteins in cardiomyocytes.2 In the past,many studies have fo-cused on the signaling pathways leading to activation of prohypertrophic transcription factors that selective-ly augment gene expression in the heart.3 Although significant progress has been made in understanding the transcriptional control of gene expression during hypertrophy,it is now clear that posttranscriptional regulation of protein expression is a similarly critical mechanism for hypertrophic control.4 For example,RNA splicing factors and microRNA-mediated gene si-lencing are established mechanisms of posttranscrip-tional regulation of gene expression that directly alter protein levels in the heart.57 However,the extent to which modifications of the mRNA itself can regulate cardiac hypertrophy is not known.The most abundant internal mRNA posttranscrip-tional modification is methylation at the N6-Methy ladenosine(m6A),a modification catalyzed by the en-zyme methyltransferase-like 3(METTL3).810 m6A mRNA modification adds a new dimension to the developing landscape of posttranscriptional regulation of gene ex-pression.It is now clear that m6A methylation plays im-portant and diverse biological functions in plants,yeast,flies,and mammals.1116 For example,m6A manipula-tion via knockdown or deletion of METTL3 affects plant growth,yeast meiosis,body mass and metabolism,syn-aptic signaling,circadian clock regulation,and stem cell self-renewal and differentiation.1116 Despite the grow-ing appreciation of the biological significance of mRNA methylation,the mechanisms by which m6A might regulate gene expression are complex and appear to be context-and cell typedependent.Although m6A mRNA methylation has long been recognized as a posttranscriptional modification in mammalian cells,the roles of this posttranscriptional process and the functions of m6A mRNA methylation in cardiomyocytes and in animal models of cardiac func-tion are completely unknown.Here,we identified the m6A mRNA methylation sites in cultured cardiomyo-cytes,and show that inhibition of the methylase MET-TL3 blocks hypertrophy.We also found that reduction of m6A levels by deleting METTL3 in cardiomyocytes leads to long-term loss of normal cardiac structure and function in vivo.Conversely,enhancing m6A mRNA methylation in cardiomyocytes promotes spontaneous hypertrophic cardiomyocyte growth that results in com-pensated cardiac remodeling.METHODSAll data,material and methods are available on request.RNA sequencing data are accessible at the Gene Expression Omnibus database as also specified below.AnimalsThe generation of Mettl3 loxP-targeted(fl)mice(Mettl3 fl/fl)was previously described.12 Mettl3 fl/fl mice were crossed with mice expressing cre recombinase under the control of the cardiac-spe-cific Myh7 promoter(-myosin heavy chain-MHC)to obtain heart-restricted deletion of Mettl3(METTL3-cKO;cKO).Control mice for this group are Mettl3+/+-MHC cre.METTL3-cKO and control mice are on a C57BL6 background.A tetracycline/doxy-cycline-responsive binary-myosin heavy chain(-MHC)trans-gene system was used to express METTL3 in cardiomyocytes.This responder line was crossed with cardiac-restricted-MHC transgenic mice expressing the tetracycline transactivator pro-tein(all in the FVB/N background)to generate an overexpression Clinical PerspectiveWhat Is New?We discovered that methylation at the N6-Methy ladenosine(m6A),the most abundant mRNA modification,is increased under hypertrophic con-ditions in cardiomyocytes and enriched on genes involving protein kinases and intracellular signaling pathways,suggesting a regulatory role in the car-diac hypertrophic pathway.Increasing the expression of the m6A methyltrans-ferase-like 3(METTL3)in the heart drives sponta-neous,compensated hypertrophy but does not affect cardiac function,whereas METTL3 knock-down induces maladaptive eccentric remodeling and leads to morphological and functional signs of heart failure.METTL3,through m6A,helps modulate cardiac homeostasis and hypertrophic stress responses in mice.What Are the Clinical Implications?Our study demonstrates the importance of METTL3 and m6A modulation throughout the hearts lifes-pan,both in terms of cardiac homeostasis with aging and the hearts response to pressure-over-load stress.Our data determine that perturbing METTL3 and m6A levels induces spontaneous geometric changes in cardiomyocytes,thereby determining the adaptive or maladaptive nature of the resulting cardiac remodeling.Targeting m6A through its writer enzyme METTL3 may represent a novel therapeutic strategy for managing maladaptive cardiac hypertrophy and remodeling during the progression of heart failure.Downloaded from http:/ahajournals.org by on January 22,2019Dorn et al METTL3 Controls Cardiac HomeostasisCirculation.2019;139:533545.DOI:10.1161/CIRCULATIONAHA.118.036146 January 22,2019535ORIGINAL RESEARCH ARTICLEtransgenic system(METTL3-TG;M3-TG;TG).Controls for this group are tetracycline transactivator single transgenic mice.Male and female mice,10 to 32 weeks old,were used in this study.Echocardiographic measurements were taken using a Vevo2100 Visual Sonics(Visual Sonics)system and MS-400 transducer.The mice were lightly anesthetized(1.5%isoflurane)and the ejec-tion fraction,fractional shortening,and ventricular chamber dimensions were determined in the M-mode using the paraster-nal short-axis view at the level of the papillary muscles.Ejection fraction,fractional shortening,ventricular chamber dimensions,left ventricular mass,and heart rate were calculated automati-cally using the VevoLAB program.Relative wall thickness(RWT)was calculated using the formula RWTLVPWdLVIDd=2*,where LVPWd indicates left ventricular posterior wall dimension end diastole,and LVIDd indicates left ventricular internal diameter end diastole.All echocardiographic measurements are reported in Table I in the online-only Data Supplement.Cardiac injury was induced by transverse aortic constriction to produce pres-sure overload.In short,the transverse aortic arch was visualized through a median sternotomy,and a 7-0 silk ligature was tied around the aorta using a 27-gauge wire to obtain a defined degree of constriction between the right brachiocephalic and left common carotid arteries.Infusion of angiotensin II(432 g/kg of body weight/d)and phenylephrine(100 mgkg1d1)was performed with implantation of Alzet minipumps for 4 weeks(Durect Inc).All experiments involving animals were approved by the Institutional Animal Care and Use Committee at The Ohio State University.Cardiomyocyte Isolation and TreatmentsNeonatal rat ventricular cardiomyocytes were isolated as previ-ously published.17,18 In brief,hearts were incubated with trypsin at 4C overnight,followed by trypsin inhibitor and collagenase incubation at 37C for 1 hour(Worthington Biochemical).Hearts were mechanically dissociated and incubated for an additional 15 minutes,followed by resuspension in media sup-plemented with fetal bovine serum and preplating to remove noncardiomyocytes.Cardiomyocytes were plated on 0.1%gel-atincoated dishes in the presence of 10%serum.The follow-ing day,cells were washed twice and cultured in M199 media without serum(Corning).Hypertrophy was induced by cultur-ing cardiomyocytes for 48 hours in the presence of 2%serum.Knockdown of METTL3 was obtained by transfection of small interfering RNAtargeting METTL3(TriFECTa Mettl3 RNAi by Integrated DNA Technologies)or negative small interfering RNA control(TriFECTa negative control DS NC1 RNAi by Integrated DNA Technologies),and cells were analyzed 48 hours posttrans-fection.Overexpression of METTL3 or-galactosidase(control)was achieved by adenoviral infection,and cells were analyzed 48 hours postinfection.Adult mouse cardiomyocytes were iso-lated as previously described19 and imaged using bright field microscopy(magnification 20)on an EVOS FL Auto II micro-scope(Thermo Fisher).For analysis of cardiomyocyte number,freshly isolated cardiomyocytes from 3-month-old mice were counted(before the sedimentation steps to avoid cardiomyo-cyte loss)with a hemocytometer.For analysis of cardiomyo-cyte volume,isolated cardiomyocytes from 3-month-old mice were stained in suspension with fluorescein isothiocyanateconjugated wheat germ agglutinin(Sigma-Aldrich)and imaged with a Zeiss Confocal Microscope using a 40 objective.Z stack images were taken with a step size of 1 m,and cardiomyocyte cell volume was calculated using the length,width,and height of the cell as adapted from Mollova et al.20 Cardiomyocyte cell numbers in this case were determined using volume measure-ments in relation to heart weights of the animals from which individual cardiomyocytes were isolated;the total volume of the heart was determined from the heart weight divided by the spe-cific gravity of muscle(1.06 g/mL).m6A Quantification,Immunoprecipitation,RNA Sequencing,and BioinformaticsRNA was extracted from neonatal rat cardiomyocytes using Trizol(Life Technologies).Unstimulated or hypertrophic car-diomyocyte RNA samples were subjected to m6A quantifi-cation using the m6A RNA Methylation Quantification Kit(Colorimetric)(Abcam ab185912)in biological triplicate.Adult mouse cardiomyocytes from METTL3-cKO,METTL3-TG,and lit-termate controls were isolated,and RNA was extracted using Trizol.m6A quantification was performed using the Abcam m6A RNA Methylation Quantification Kit described above.For genome-wide m6A profiling,2.5 mg of total RNA was extracted from neonatal rat cardiomyocytes using Trizol and enriched for mRNAs using polyT columns.Unstimulated or hypertrophied cardiomyocyte RNA samples(in biological dupli-cates)were subjected to m6A immunoprecipitation as previ-ously described.9 In brief,mRNA was chemically fragmented and incubated with antibodies recognizing m6A-modified RNA(Synaptic Systems).Rabbit normal IgG was used as a negative control.Immunoprecipitated RNA was then submitted for RNA sequencing(University of Cincinnati sequencing and genome analysis core laboratory).Illumina reads were mapped to the rat reference genome(UCSC Rn4)using Tophat.The resulting bam files were individually analyzed for peak detection using MACS(version 1.4).21 The peaks from all repeats were merged to build the consensus peak regions,such that the peak is included if it appears in at least 1 repeat.The reads in each consensus peak region were counted in each samples bam file.The log2-transformed reads per kilobase millionnormalized read counts were used as the peak intensity for each consensus peak region in each sample.Negative control immunoprecipitation samples were used to control for unspecific binding:peaks that appeared in the negative control as well were excluded.Peaks whose intensities were significantly different between hypertrophy and normal samples were detected by Student t test comparing all normal and hypertrophy replicates with a false discovery rateadjusted P value 0.1.We used our own R scripts to create the analysis pipeline.This pipeline was based on the Nature Protocol article,“A computational pipeline for comparative ChIP-seq analyses,”which contained modi-fications to accommodate statistical analysis of replicates.22 Annotations of the consensus peak regions,including chromo-some number,start and end location on chromosomes,gene name and identification of the peak region,and the genomic feature of the peak(5-untranslate