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The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy

The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy September122018 Circulation ORIGINAL RESEARCH ARTICLE The N -Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy Editorial, see p 546 Lisa E. Dorn, BA Lior Lasman, BMS Jing Chen, PhD BACKGROUND: N -Methyladenosine (m6A) methylation is the most Xianyao Xu, MS prevalent internal posttranscriptional modification on mammalian mRNA. Thomas J. Hund, PhD The role of m6A mRNA methylation in the heart is not known. Mario Medvedovic, PhD Jacob H. Hanna, MD, PhD METHODS: To determine the role of m6A methylation in the Jop H. van Berlo, MD, PhD heart, we isolated primary cardiomyocytes and performed m6A Federica Accornero, PhD 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 Key Words: gene expression profiling ◼ hypertrophy ◼ mice, transgenic stress, showing the necessity of RNA methylation for the maintenance of ◼ RNA processing, post-transcriptional cardiac homeostasis. Sources of Funding, see page 544 CONCLUSIONS: Our study identified METTL3-mediated methylation © 2018 The Authors. Circulation is of mRNA on N -adenosines as a dynamic modification that is published on behalf of the American Heart Association, Inc., by Wolters enhanced in response to hypertrophic stimuli and is necessary for a Kluwer Health, Inc. This is an open normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA access article under the terms of the Creative Commons Attribution Non- methylation results in compensated cardiac hypertrophy, whereas Commercial License, which permits diminished m6A drives eccentric cardiomyocyte remodeling and use, distribution, and reproduction in any medium, provided that the original dysfunction, highlighting the critical importance of this novel stress- work is properly cited and is not used response mechanism in the heart for maintaining normal cardiac function. for commercial purposes. https://www.ahajournals.org/journal/circ Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 533 Dorn et al METTL3 Controls Cardiac Homeostasis lencing are established mechanisms of posttranscrip- tional regulation of gene expression that directly alter Clinical Perspective 5–7 protein levels in the heart. However, the extent to What Is New? which modifications of the mRNA itself can regulate cardiac hypertrophy is not known. • We discovered that methylation at the N -Methy The most abundant internal mRNA posttranscrip- ladenosine (m6A), the most abundant mRNA tional modification is methylation at the N -Methy modification, is increased under hypertrophic con- ladenosine (m6A), a modification catalyzed by the en- ditions in cardiomyocytes and enriched on genes 8–10 zyme methyltransferase-like 3 (METTL3). m6A mRNA involving protein kinases and intracellular signaling pathways, suggesting a regulatory role in the car- modification adds a new dimension to the developing diac hypertrophic pathway. landscape of posttranscriptional regulation of gene ex- • Increasing the expression of the m6A methyltrans- pression. It is now clear that m6A methylation plays im- ferase-like 3 (METTL3) in the heart drives sponta- portant and diverse biological functions in plants, yeast, neous, compensated hypertrophy but does not 11–16 flies, and mammals. For example, m6A manipula- affect cardiac function, whereas METTL3 knock- tion via knockdown or deletion of METTL3 affects plant down induces maladaptive eccentric remodeling growth, yeast meiosis, body mass and metabolism, syn- and leads to morphological and functional signs of aptic signaling, circadian clock regulation, and stem cell heart failure. 11–16 self-renewal and differentiation. Despite the grow- • METTL3, through m6A, helps modulate cardiac ing appreciation of the biological significance of mRNA homeostasis and hypertrophic stress responses methylation, the mechanisms by which m6A might in mice. regulate gene expression are complex and appear to be What Are the Clinical Implications? context- and cell type–dependent. Although m6A mRNA methylation has long been • Our study demonstrates the importance of METTL3 recognized as a posttranscriptional modification in and m6A modulation throughout the heart’s lifes- mammalian cells, the roles of this posttranscriptional pan, both in terms of cardiac homeostasis with process and the functions of m6A mRNA methylation in aging and the heart’s response to pressure-over- cardiomyocytes and in animal models of cardiac func- load stress. • Our data determine that perturbing METTL3 tion are completely unknown. Here, we identified the and m6A levels induces spontaneous geometric m6A mRNA methylation sites in cultured cardiomyo- changes in cardiomyocytes, thereby determining cytes, and show that inhibition of the methylase MET- the adaptive or maladaptive nature of the resulting TL3 blocks hypertrophy. We also found that reduction cardiac remodeling. of m6A levels by deleting METTL3 in cardiomyocytes • Targeting m6A through its writer enzyme METTL3 leads to long-term loss of normal cardiac structure and may represent a novel therapeutic strategy for function in vivo. Conversely, enhancing m6A mRNA managing maladaptive cardiac hypertrophy and methylation in cardiomyocytes promotes spontaneous remodeling during the progression of heart failure. hypertrophic cardiomyocyte growth that results in com- pensated cardiac remodeling. he heart comprises long-lived cardiomyocytes that, in response to stress stimulation such as METHODS Tpressure overload or myocardial infarction, un- All data, material and methods are available on request. dergo hypertrophic growth. This hypertrophic re- RNA sequencing data are accessible at the Gene Expression sponse is initially an adaptive process to produce suf- Omnibus database as also specified below. ficient force to match an increase in wall tension or increased workload, but can ultimately lead to heart Animals failure. Cardiac hypertrophy is mediated by increased The generation of Mettl3 loxP-targeted (fl) mice (Mettl3 fl/fl) was gene expression and production of select proteins in previously described. Mettl3 fl/fl mice were crossed with mice cardiomyocytes. In the past, many studies have fo- expressing cre recombinase under the control of the cardiac-spe- cused on the signaling pathways leading to activation cific Myh7 promoter (β -myosin heavy chain [β-MHC]) to obtain of prohypertrophic transcription factors that selective- heart-restricted deletion of Mettl3 (METTL3-cKO; cKO). Control ly augment gene expression in the heart. Although +/+ mice for this group are Mettl3 β-MHC cre. METTL3-cKO and significant progress has been made in understanding control mice are on a C57BL6 background. A tetracycline/doxy- the transcriptional control of gene expression during cycline-responsive binary α-myosin heavy chain (α-MHC) trans- hypertrophy, it is now clear that posttranscriptional gene system was used to express METTL3 in cardiomyocytes. regulation of protein expression is a similarly critical This responder line was crossed with cardiac-restricted α-MHC mechanism for hypertrophic control. For example, transgenic mice expressing the tetracycline transactivator pro- RNA splicing factors and microRNA-mediated gene si- tein (all in the FVB/N background) to generate an overexpression January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis transgenic system (METTL3-TG; M3-TG; TG). Controls for this with a Zeiss Confocal Microscope using a 40× objective. Z stack group are tetracycline transactivator single transgenic mice. Male images were taken with a step size of 1 µm, and cardiomyocyte and female mice, 10 to 32 weeks old, were used in this study. cell volume was calculated using the length, width, and height Echocardiographic measurements were taken using a Vevo2100 of the cell as adapted from Mollova et al. Cardiomyocyte cell numbers in this case were determined using volume measure- Visual Sonics (Visual Sonics) system and MS-400 transducer. The ments in relation to heart weights of the animals from which mice were lightly anesthetized (1.5% isoflurane) and the ejec- individual cardiomyocytes were isolated; the total volume of the tion fraction, fractional shortening, and ventricular chamber heart was determined from the heart weight divided by the spe- dimensions were determined in the M-mode using the paraster- cific gravity of muscle (1.06 g/mL). 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- m6A Quantification, cally using the VevoLAB program. Relative wall thickness (RWT) Immunoprecipitation, RNA Sequencing, 2* LVPWd was calculated using the formula RWT = , where and Bioinformatics LVIDd LVPWd indicates left ventricular posterior wall dimension end RNA was extracted from neonatal rat cardiomyocytes using diastole, and LVIDd indicates left ventricular internal diameter Trizol (Life Technologies). Unstimulated or hypertrophic car- end diastole. All echocardiographic measurements are reported diomyocyte RNA samples were subjected to m6A quantifi- in Table I in the online-only Data Supplement. Cardiac injury cation using the m6A RNA Methylation Quantification Kit was induced by transverse aortic constriction to produce pres- (Colorimetric) (Abcam ab185912) in biological triplicate. Adult sure overload. In short, the transverse aortic arch was visualized mouse cardiomyocytes from METTL3-cKO, METTL3-TG, and lit- through a median sternotomy, and a 7-0 silk ligature was tied termate controls were isolated, and RNA was extracted using around the aorta using a 27-gauge wire to obtain a defined Trizol. m6A quantification was performed using the Abcam degree of constriction between the right brachiocephalic and m6A RNA Methylation Quantification Kit described above. left common carotid arteries. Infusion of angiotensin II (432 μg/ For genome-wide m6A profiling, 2.5 mg of total RNA was –1 –1 kg of body weight/d) and phenylephrine (100 mg·kg ·d ) was extracted from neonatal rat cardiomyocytes using Trizol and performed with implantation of Alzet minipumps for 4 weeks enriched for mRNAs using polyT columns. Unstimulated or (Durect Inc). All experiments involving animals were approved by hypertrophied cardiomyocyte RNA samples (in biological dupli- the Institutional Animal Care and Use Committee at The Ohio cates) were subjected to m6A immunoprecipitation as previ- State University. ously described. In brief, mRNA was chemically fragmented and incubated with antibodies recognizing m6A-modified RNA (Synaptic Systems). Rabbit normal IgG was used as a negative Cardiomyocyte Isolation and Treatments control. Immunoprecipitated RNA was then submitted for RNA Neonatal rat ventricular cardiomyocytes were isolated as previ- sequencing (University of Cincinnati sequencing and genome 17,18 ously published. In brief, hearts were incubated with trypsin analysis core laboratory). Illumina reads were mapped to the rat at 4°C overnight, followed by trypsin inhibitor and collagenase reference genome (UCSC Rn4) using Tophat. The resulting bam incubation at 37°C for 1 hour (Worthington Biochemical). files were individually analyzed for peak detection using MACS Hearts were mechanically dissociated and incubated for an (version 1.4). The peaks from all repeats were merged to build additional 15 minutes, followed by resuspension in media sup- the consensus peak regions, such that the peak is included if plemented with fetal bovine serum and preplating to remove it appears in at least 1 repeat. The reads in each consensus noncardiomyocytes. Cardiomyocytes were plated on 0.1% gel- peak region were counted in each sample’s bam file. The log2- atin–coated dishes in the presence of 10% serum. The follow- transformed reads per kilobase million–normalized read counts ing day, cells were washed twice and cultured in M199 media were used as the peak intensity for each consensus peak without serum (Corning). Hypertrophy was induced by cultur- region in each sample. Negative control immunoprecipitation ing cardiomyocytes for 48 hours in the presence of 2% serum. samples were used to control for unspecific binding: peaks Knockdown of METTL3 was obtained by transfection of small that appeared in the negative control as well were excluded. interfering RNA–targeting METTL3 (TriFECTa Mettl3 RNAi by Peaks whose intensities were significantly different between Integrated DNA Technologies) or negative small interfering RNA hypertrophy and normal samples were detected by Student t control (TriFECTa negative control DS NC1 RNAi by Integrated test comparing all normal and hypertrophy replicates with a DNA Technologies), and cells were analyzed 48 hours posttrans- false discovery rate–adjusted P value <0.1. We used our own R fection. Overexpression of METTL3 or β-galactosidase (control) scripts to create the analysis pipeline. This pipeline was based was achieved by adenoviral infection, and cells were analyzed on the Nature Protocol article, “A computational pipeline 48 hours postinfection. Adult mouse cardiomyocytes were iso- for comparative ChIP-seq analyses,” which contained modi- 19 22 lated as previously described and imaged using bright field fications to accommodate statistical analysis of replicates. microscopy (magnification 20×) on an EVOS FL Auto II micro- Annotations of the consensus peak regions, including chromo- scope (Thermo Fisher). For analysis of cardiomyocyte number, some number, start and end location on chromosomes, gene freshly isolated cardiomyocytes from 3-month-old mice were name and identification of the peak region, and the genomic counted (before the sedimentation steps to avoid cardiomyo- feature of the peak (5ʹ-untranslated region, intron, exon, and cyte loss) with a hemocytometer. For analysis of cardiomyo- 3ʹ-untranslated region) were added according to UCSC Rn4 cyte volume, isolated cardiomyocytes from 3-month-old mice gene definitions. RNA sequencing data sets were submitted to were stained in suspension with fluorescein isothiocyanate– the Gene Expression Omnibus database (series GSE119170). conjugated wheat germ agglutinin (Sigma-Aldrich) and imaged Peaks were visualized using the IGV browser. Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 535 Dorn et al METTL3 Controls Cardiac Homeostasis that the percentage of m6A RNA modification increas- Western Blotting, Histology, es substantially in response to a hypertrophic stimulus Immunostaining, and mRNA Expression (Figure 1B). To define the specific mRNA sequences that Analysis were dynamically affected by m6A modification in re- Western blotting was performed from neonatal rat cardio- sponse to a hypertrophic stimulus, we performed m6A myocytes using standard procedures. Antibodies used were sequencing of m6A-modified mRNA from hypertrophic METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), cardiomyocytes in comparison with unstimulated condi- MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling tions. Our analysis revealed differential m6A methyla- Technology), and GAPDH (Fitzgerald Industries). Masson tri- tion during hypertrophy on 713 cardiomyocyte mRNAs chrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining (Table III in the online-only Data Supplement). It is inter- was performed using α-actinin antibodies (Sigma-Aldrich), esting to note that we found that, in hypertrophic car- and cell area was quantified using CellProfiler following pub- diomyocytes, m6A changes are not randomly distributed lished methods. Detection of the cell membrane was per- throughout the genome, but are specifically increased in formed using fluorescein isothiocyanate–conjugated wheat select classes of mRNAs (Figure 1C). Within these spe- germ agglutinin (Sigma-Aldrich). The cross-sectional area was cific functional categories, m6A peaks in mRNAs encod- measured using ImageJ. RNA was extracted from neonatal ing for protein kinases and modifiers were the most rat cardiomyocytes or mouse hearts using Trizol, and reverse significantly enriched during hypertrophy (Figure  1C). transcription was performed using the High Capacity cDNA Representative m6A peaks in the ryanodine receptor Reverse Transcription kit (Applied Biosystems). Selected genes (Ryr2) and mitogen-activated protein kinase kinase ki- and m6A peaks were analyzed by real-time polymerase chain nase 6 (Map3k6) 3ʹ-untranslated regions are shown as reaction using SYBR green (Applied Biosystems). Quantified examples of m6A-methylated mRNA that showed either mRNA expression was normalized to Rpl7 (ribosomal protein L7) and expressed relative to controls. no change with hypertrophy (Ryr2) or increased levels of m6A with hypertrophy (Map3k6) (Figure 1D and 1E). To validate the results of m6A sequencing and quantify Statistics the extent of enhanced methylation in response to a All results are presented as mean±SEM. Statistical analysis hypertrophic stimulus, we performed m6A immunopre- was performed with an unpaired 2-tailed t test (for 2 groups) cipitation followed by real-time polymerase chain reac- and 1-way ANOVA with Bonferroni correction (for groups of tion using gene-specific primers flanking the identified ≥3). P values <0.05 were considered significant. m6A peaks from RNA sequencing (Table 1). We assessed increased m6A levels following hypertrophic stimulus in RESULTS 16 genes from the functional category of protein-mod- ifying enzymes that showed increased m6A levels fol- m6A Is a Dynamic Modification in lowing hypertrophic stimulus in our genome-wide anal- Cardiomyocytes ysis, and identified 15 to be significantly enriched for To determine the mRNAs that are modified by m6A m6A. These results clearly show that m6A modifications on mRNA are enhanced on specific classes of proteins in methylation in cardiomyocytes, we performed m6A response to a hypertrophic stimulus. sequencing on cultured rat neonatal cardiomyocytes. Purified mRNAs were fragmented to allow for analy- sis of the specific location of m6A peaks, pulled down METTL3 Drives Cardiomyocyte using a specific antibody recognizing m6A-modified Hypertrophy In Vitro and In Vivo RNA, and processed for sequencing (m6A sequencing, meRIP-seq). Using this strategy, we identified 3922 m6A To determine whether enhancing m6A modification peaks that showed a general distribution throughout on mRNA was sufficient to cause cardiomyocyte hy- the coding region of genes (Figure  1A and Table II in pertrophy, we tested how overexpression of the m6A- the online-only Data Supplement). We also detected catalyzing enzyme METTL3 affects cardiomyocytes. relative enrichment on the untranslated regions, consis- Specifically, we generated an adenoviral vector to in- tent with previous reports (Figure 1A and Table II in the crease METTL3 expression in cardiomyocytes. We in- online-only Data Supplement). To assess a possible role fected cardiomyocytes under unstimulated conditions, for m6A modifications on mRNA in the development of and used adenovirus encoding for β-galactosidase as cardiomyocyte hypertrophy, we performed an indepen- a negative control. We first verified that METTL3 was dent m6A quantification experiment where we assessed indeed expressed at higher levels (Figure  2A). We also if the overall percentage of m6A in RNA changes during tested if METTL3 overexpression affected protein lev- cardiomyocyte hypertrophy. We used serum as a prohy- els of m6A-methylated mitogen-activated protein ki- pertrophic stimulus and isolated RNA from neonatal rat nases and found that METTL3 enhanced the levels of cardiomyocytes. Using an antibody-mediated capture of MAP3K6, MAP4K5, and MAPK14 in cardiomyocytes m6A followed by colorimetric analysis, we determined (Figure 2A and 2B). It is remarkable that upregulation January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Figure 1. m6A is a dynamic modification in cardiomyocytes. A, Distribution of m6A peaks throughout mRNAs. B, Percentage of m6A-methylated RNA in relation to unmodified adenosine as quantified using an antibody-medi- ated m6A capture assay in unstimulated (Normal) and hypertrophic (Hyper.) neonatal rat cardiomyocytes (n=3 each). C, PANTHER analysis of enriched functional gene categories showing differential m6A peaks during hypertrophy. D and E, Visualization of representative m6A peaks under unstimulated or hypertrophic conditions from the indicated mRNAs using Integrative Genomics Viewer. *P≤0.05 versus normal. Intracell. indicates intracellular; IP, immunoprecipitation; Map3k6, mitogen- activated protein kinase kinase kinase 6; m6A, N -Methyladenosine; PANTHER, Protein Analysis through Evolutionary Relationships; phosphor., phosphorylation; Ryr2, ryanodine receptor; and UTR, untranslated region. of METTL3 caused a significant increase in cardiomyo- age (Figure 2G and 2H). The lowest expressing line was cyte size, indicating that METTL3 expression is sufficient then further characterized to follow the progression of to cause cardiomyocyte hypertrophy (Figure  2C and cardiac remodeling over time. We found that this line 2D). We also validated that the m6A mRNA targets we Table 1. m6A Modification on Specific mRNAs in Response to identified using isolated neonatal rat cardiomyocytes Hypertrophy were also relevant in the adult heart. To this end, we Gene Normal Hypertrophy P Value performed m6A immunoprecipitations from neonatal Dapk1 1.04±0.11 9.87±0.77 <0.001 and adult mouse heart RNA followed by real-time poly- Dclk2 1.01±0.12 36.59±11.76 0.039 merase chain reaction. With the exception of STE20- Grk4 1.82±1.06 25.49±5.98 0.018 like protein kinase 4 (Mst4), we found that all m6A Ikbkap 1.01±0.13 5.23±0.82 0.007 peaks identified in the isolated neonatal cardiomyocyte screen were also present in the adult heart (Table  2). Ikbkb 1.08±0.27 9.14±0.85 0.001 This experiment also revealed that m6A methylation of Krs1 1.17±0.47 9.60±2.73 0.038 the tested mRNAs was, in many cases, higher in adult Map3k14 1.03±0.21 16.30±3.59 0.013 hearts than in the neonatal state, in agreement with Map3k6 1.03±0.19 15.85±1.89 0.001 overall higher m6A levels in adult hearts (Table  2 and Map4k5 0.97±0.27 5.77±2.81 0.164 Figure IA in the online-only Data Supplement). These Mapk14 1.13±0.37 8.19±1.52 0.011 findings suggest a role for m6A mRNA methylation in Mst4 1.01±0.13 18.75±2.98 0.004 adult cardiac homeostasis. Nuak2 1.00±0.07 8.16±2.16 0.030 To further verify a role for m6A in regulating car- Rps6ka2 1.04±0.20 8.32±1.79 0.015 diomyocyte homeostasis and hypertrophy in the adult Rps6ka4 1.04±0.20 8.32±1.79 0.015 heart, we generated a METTL3-overexpressing (MET- TL3-TG or M3-TG) mouse line by cross-breeding a trans- Rps6ka5 1.03±0.19 16.12±1.92 0.001 genic αMHC promoter–driven tetO responder with the Sgk1 1.07±0.26 13.05±2.56 0.010 αMHC–tetracycline transactivator driver to impart gene m6A immunoprecipitation was performed followed by qPCR using primers expression specifically in cardiomyocytes (Figure 2E and designed around preidentified m6A peaks. The qPCR data were corrected to overall gene expression level under the same conditions (normal versus 2F). METTL3-overexpressing mice showed a significant hypertrophy). Data represented are average±SEM from 3 biological replicates increase in m6A levels in cardiomyocyte RNA and dose- -Methyladenosine; and qPCR, quantitative polymerase each. m6A indicates N dependent cardiac hypertrophic growth at 3 months of chain reaction. Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 537 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 2. Generation and characterization of METTL3 overexpression models. A, Western blot from cardiomyocytes overexpressing β-galactosidase (Ad-Control) or METTL3 (Ad-Mettl3) using antibodies against the indicated proteins and GAPDH loading control. B, Densitometry quantification of the expression for the indicated proteins relative to GAPDH control. C, Quantification of cardiomyocyte cell area based on pixel size from cardiomyocytes overexpressing β-galactosidase control or METTL3 (n=3 each). D, Representative images of cardiomyocytes from the indicated treatments stained for α-actinin (green). Scale bar=20 µm. E, Schematic of cardiomyocyte-specific METTL3 gain-of-function mouse model. F, Western blot from cardiac extracts from control (Ctrl) or METTL3 transgenic (TG) line 1 and 2. GAPDH was used as loading control. G, percentage of m6A-methylated RNA relative to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in isolated cardiomyocytes from control mice and METTL3 TG line 1 mice (n=3 each). H, Gravimetric analysis of heart weight normalized to body weight (HW/BW) in the indicated genotypes at 3 months of age (n≥6 each). *P<0.05 versus control. A.U. indicates arbitrary units; m6A, N -Methyladenosine; and METTL3, methyltransferase-like 3. expresses, on average, 19-fold higher levels of METTL3 3C). Analysis of isolated adult cardiomyocytes revealed in the heart than control mice (Figure IB and IC in the physiological growth of cardiomyocytes in both width online-only Data Supplement). and length, resulting in a preserved length-to-width ra- By 8 months of age, METTL3-overexpressing mice tio (Figure 3D and 3E). However, despite the structural exhibit cardiac hypertrophy, as demonstrated by in- changes METTL3-TG hearts undergo, no histopatholog- creased heart weight to body weight ratios (Figure 3A). ic changes were observed in these mice (Figure 3F). It is Cardiac hypertrophic growth was accompanied by a important to note that cardiac function, as measured larger cardiomyocyte cross-sectional area (Figure 3B and by echocardiographic analysis of the percentage frac- January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Table 2. m6A Modification on Specific mRNAs in the Neonatal and changes in cardiomyocyte size. However, on stimulation Adult Heart of cardiomyocyte hypertrophy through the addition of Gene Neonatal Adult P Value serum, we noticed a normal hypertrophic response in control small interfering RNA–treated cardiomyocytes, Dapk1 1.01±0.08 2.00±0.40 0.053 but a complete block of hypertrophy when METTL3 Dclk2 1.83±0.78 1.07±0.55 0.459 was knocked down (Figure 4B and 4C). Grk4 1.09±0.25 0.78±0.13 0.273 Ikbkap 1.10±0.30 0.90±0.27 0.642 Ikbkb 1.16±0.34 1.25±0.38 0.863 Cardiomyocyte-Specific METTL3 Krs1 1.03±0.16 1.67±0.37 0.162 Knockout Induces Cardiac Structural Map3k14 1.06±0.24 3.11±0.82 0.075 and Functional Changes With Aging and Map3k6 1.11±0.21 3.73±0.48 0.0005 Stress Map4k5 1.18±0.34 0.95±0.26 0.602 To determine the necessity of METTL3 and m6A in the Mapk14 1.10±0.26 1.89±0.17 0.043 heart, we generated a cardiomyocyte-specific MET - Mst4 N/A N/A N/A TL3 knockout (METTL3-cKO) mouse line using a typi- Nuak2 1.00±0.01 3.92±0.72 0.018 cal (β-MHC promoter driven) cardiomyocyte-specific cre-loxP system (Figure  5A and 5B). Consistent with Rps6ka2 1.12±0.29 2.17±0.40 0.075 their METTL3 knockout status, METTL3-cKO mice Rps6ka4 1.09±0.32 4.69±0.40 0.002 have significantly decreased m6A levels in compari- Rps6ka5 1.03±0.14 3.75±0.76 0.012 son with their littermate controls (Figure 5C). Three- Sgk1 1.03±0.13 2.02±0.46 0.086 month-old METTL3-cKO animals do not show cardiac m6A immunoprecipitation was performed followed by qPCR using primers morphological or functional changes, indicating that designed around preidentified m6A peaks. The qPCR data were corrected METTL3 knockout does not impair cardiac develop- to overall gene expression level under the same conditions (neonatal versus ment (Figure  5D through 5H). Specifically, no histo- adult). Data represented are average±SEM from 4 biological replicates each. m6A indicates N -Methyladenosine; N/A, not available; and qPCR, quantitative pathologic and hypertrophic changes were observed polymerase chain reaction. (Figure 5D and 5E). Also, cardiac function was unaf- fected as measured by echocardiographic analysis of tional shortening, was preserved (Figure 3G). To further the percentage fractional shortening (Figure  5F). To confirm the adaptive nature of the cardiac remodeling confirm the absence of remodeling at the cellular lev- observed in METTL3-TG mice and ensure that it does el, we then measured cardiomyocyte cross-sectional not accelerate dysfunction during stress, we performed area and observed no changes in METTL3-cKO mice pressure overload stimulation on 3-month-old METTL3- (Figure 5G and 5H). To further exclude developmental TG and control mice through transverse aortic constric- defects in METTL3-cKO mice, we assessed postnatal tion. We found that increased expression of METTL3 is proliferative and hypertrophic growth (Figure II in the not detrimental post stress (Figure 3H). Also, the hyper- online-only Data Supplement). Expression analysis trophic response to transverse aortic constriction was of proliferation markers revealed no abnormalities not exacerbated by METTL3 upregulation at the organ in METTL3-cKO hearts (Figure IIA through IIC in the level or in terms of cardiomyocyte cross-sectional area online-only Data Supplement). Similarly, markers of (Figure 3I through 3K). Therefore, enhancing m6A mod- hypertrophy and the postnatal switch between β- and ification of mRNA through cardiomyocyte-specific MET - α-MHCs (Myh7 and Myh6, respectively) were also TL3 overexpression induces compensated hypertrophic unaffected (Figure IID through IIF in the online-only remodeling of the heart without inducing cardiac func- Data Supplement). Considering that, within the first tional deficits either at baseline or under cardiac stress. week of life, murine cardiomyocytes lose their ability to grow by hyperplasia and instead activate a post- natal hypertrophic program, we tested cardiomyocyte METTL3 Inhibition Prevents the cross-sectional area in day 7 hearts from METTL3-cKO Development of Cardiomyocyte and control mice. This analysis revealed that postnatal Hypertrophy in Vitro growth is unaffected in the absence of METTL3 (Fig- To determine if m6A methylation plays a primary role in ure IIG and IIH in the online-only Data Supplement). the development of cardiac hypertrophy, we inhibited Consequently, cardiomyocyte numbers and cell vol- expression of the m6A-catalyzing enzyme METTL3 in umes in 3-month-old METTL3-cKO and control mice isolated neonatal rat cardiomyocytes. We observed a were also unchanged (Figure II I through IIK in the significant knockdown of METTL3 by delivery of MET - online-only Data Supplement). Overall, these results TL3-targeting small interfering RNA (Figure 4A). In the suggest that METTL3 is dispensable for postnatal absence of hypertrophic stimuli, we did not observe any heart development. Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 539 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 3. METTL3 drives compensated hypertrophy in vivo. A, Heart weight to body weight ratios (HW/BW) in 8-month-old METTL3-TG animals or littermate controls (n≥7 each group). B and C, Wheat germ agglutinin (green)–stained cardiac cross-sections of 8-month-old METTL3-TG or littermate control mice with quantification of cardiomyocyte cross-sectional area using ImageJ software. Scale bar=50 µm (n≥100 cells/animal, n=6 mice each). D, Representative images of isolated cardiomyocytes from 8-month-old hearts from the indicated genotypes. Scale bar=50 µm. E, Length/width ratios of isolated cardiomyocytes from 8-month-old control and TG mice (n≥100 cells/animal; n=3 mice each). F, Representative images of Masson trichrome staining of cardiac cross-sections of 8-month-old METTL3-TG or littermate control mice. Scale bar=1 mm. G, Echocardiographic quantification of percentage fractional shortening (FS) for 8-month-old METTL3-TG animals or littermate controls (n=12 each). H, Echocardiographic quantification of percentage fractional shortening for Sham or TAC-operated METTL3-TG animals or littermate controls (n≥ 5 each). I, HW/BW in TAC-operated METTL3-TG animals or littermate controls (n≥5 each group). J and K, Wheat germ agglutinin (green)–stained cardiac cross-sections of TAC-operated METTL3-TG or littermate control mice with quantification of cardiomyocyte cross-sectional area using ImageJ software. Scale bar=100 µ m (n≥100 cells/animal, n=5 mice each). *P≤0.05 versus Ctrl. Ctrl indicates control; METTL3, methyltransferase-like 3; TAC, transverse aortic constriction; and TG, transgenic. However, when METTL3-cKO animals are aged were not accompanied by overall heart weight altera- to 8 months, they begin to show cardiac abnormali- tions (Figure  6C). To further understand these results, ties consistent with a progression toward heart failure we isolated adult cardiomyocytes and found eccentric (Figure 6). Cardiomyocyte cross-sectional area was sig- cardiomyocyte remodeling in the absence of METTL3 nificantly reduced in 8-month-old METTL3-cKO mice (Figure  6D through 6F). Indeed, cardiomyocytes from (Figure 6A and 6B). It is interesting that these changes METTL3-cKO hearts were elongated and showed an January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Figure 4. METTL3 inhibition prevents the development of cardiomyocyte hypertrophy. A, qPCR analysis for METTL3 expression in response to Ctrl or METTL3 siRNA-mediated knockdown (n=3 each). B, Representative images of cardiomyocytes treated with control siRNA (si-Ctrl) or siRNA targeting METTL3 (si-M3) stained for α-actinin (green). Scale bar=20 µm. C, Quantification of cardiomyocyte cell area (n≥50 cells/well, n=3 independent experiments/treatment). *P<0.05 versus baseline; #P<0.05 versus si-Ctrl hypertrophy. Ctrl indicates control; METTL3, methyl- transferase-like 3; siRNA, small interfering RNA; and qPCR, quantitative polymerase chain reaction. overall increase in their length-to-width ratio (Fig- METTL3 in controlling cardiomyocyte geometry and ure  6F). Consistent with maladaptive eccentric car- adaptation to stress. diomyocyte remodeling, cardiac knockout of METTL3 Altogether, our data demonstrate a critical role for caused a significant increase in left ventricular chamber METTL3 and m6A modifications of mRNA for the main- tenance of cardiac homeostasis, function, and stress dimensions and ventricular dilation (Figure 6G through responses. 6I). The structural changes we observed in METTL3-cKO cardiomyocytes were also associated with a decrease in overall cardiac function as measured by echocardiogra- DISCUSSION phy (Figure 6J). To test if METTL3 is necessary for adaptation to The control of protein synthesis is achieved by complex stress, we then subjected 3-month-old METTL3-cKO and still poorly defined mechanisms that regulate the and control mice to pressure overload stimulation (or posttranscriptional processing of mRNAs. Understand- transverse aortic constriction). We found that the ab- ing how specific classes of functionally related mRNAs sence of METTL3 accelerates heart failure progression are coregulated in the heart is crucial to elucidate the and leads to reduced cardiomyocyte cross-sectional molecular mechanisms controlling the characteristic area (Figure 7A through 7C). These data suggest that increase in cardiac mass that defines cardiac hypertro- METTL3 is critical for adaptive cardiac remodeling af- phy within myocytes. Here, we discovered METTL3 as ter injury. To further test if maladaptive cardiomyo- a critical RNA-modifying protein that drives cardiomyo- cyte remodeling occurs before dysfunction and could cyte hypertrophy by catalyzing methylation of m6A on indeed be a pathological driver of cardiomyopathy, specific subsets of mRNAs. Our genome-wide m6A-se- we applied a milder stress through the infusion of quencing analysis in cardiomyocytes represents the first angiotensin and phenylephrine for 4 weeks. In this report demonstrating the occurrence of m6A in cardiac condition in which cardiac function was still intact, cells, the dynamic changes occurring in hypertrophic we observed reduction in the cross-sectional area of conditions, and the specific mRNA targets of this modi- stressed cardiomyocytes lacking METTL3 (Figure  7D fication. An interesting result from our analysis was the discovery of protein kinases as the most affected cat- through 7F). This result indicates a direct role for Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 541 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 5. Generation and characterization of METTL3 cardiac-restricted knockout mice. A, Schematic of METTL3 loss-of-function mouse model. B, Western blot for METTL3 from cardiac extracts from control (Ctrl) or cardiomyocyte-specific METTL3 knockout mice (cKO). GAPDH and Ponceau staining were used as loading control. C, percentage of m6A-methylated RNA relative to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in isolated cardiomyocytes from control and METTL3-cKO mice (n=3 each). D, Representative Masson trichrome–stained cardiac cross-sections from 3-month-old METTL3-cKO mice and controls. Scale bar=1 mm. E, Quantification of heart weight to body weight ratio (HW/BW) for 3-month-old METTL3-cKO or control mice (n=5 each). F, Echocardiographic quantification of percentage fractional shortening (FS) for 3-month-old METTL3-cKO or control mice (n=12 each). G and H, Wheat germ agglutinin (green)–stained cardiac cross-sections of 3-month-old METTL3-cKO or control mice with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µ m (n≥100 cells/animal, n≥4 mice each). *P≤0.05 versus Ctrl. m6A indicates N -Methyladenosine; and METTL3, methyltransferase-like 3. egory of genes dynamically controlled by the METTL3- of METTL3 as a master controller of large subsets of m6A pathway during hypertrophy. Indeed, many stud- signaling molecules through posttranscriptional regu- ies have recognized the importance of kinase-regulated lation of mRNA processing might further explain how signaling pathways for cardiomyocyte hypertrophic stress responses are coordinated at a posttranscription- growth, and mitogen-activated protein kinases specifi- al level in cardiomyocytes. Our findings open intriguing cally have well-established roles in hypertrophy. In ad- new possibilities for controlling cardiac remodeling and dition, we also found hypertrophy-dependent methyla- pathological stress responses in the heart. tion on mRNAs encoding for kinases converging on the It is striking to observe how m6A levels increase nuclear factor kB, which has been shown to cooperate in specific functional clusters of mRNAs in response not only with mitogen-activated protein kinases, but to hypertrophic growth signals in cardiomyocytes. also with nuclear factor of activated T cells in regulating Although the origin of this specificity is currently un- cardiac hypertrophy. Last, our m6A profile revealed known, the fact that increased expression of METTL3 dynamic methylation in mRNAs encoding for members is sufficient to cause hypertrophy might indicate that of the ribosomal S6 family of kinases. Ribosomal S6 the activity of this enzyme could be a limiting factor kinases are downstream effectors of the extracellular under homeostatic conditions. In addition, it is im- regulated kinase branch of mitogen-activated protein portant to consider that specific demethylases might kinases and have established roles in adaptive cardiac act with different specificity, and their activity could growth through the regulation of mammalian target of also be controlled by hypertrophic signals in cardio- 27,28 rapamycin–dependent protein synthesis. Consider- myocytes. Indeed, the m6A epigenetic mark has been ing the complexity and intersection of multiple signaling shown to be reversible. The protein that removes the mechanisms driving cardiac hypertrophy, the discovery methyl group m6A was identified as fat mass and obe- January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Figure 6. METTL3 loss-of-function causes maladaptive cardiomyocyte remodeling and cardiac dysfunction with aging. A and B, Wheat germ agglutinin–stained cardiac cross-sections of 8-month-old METTL3-cKO or control mice with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥5 mice each). C, Quantification of heart weight to body weight ratio (HW/BW) for 8-month-old METTL3-cKO or control mice (n≥13 each). D and F, Representative bright field image of isolated cardiomyocytes from the indicated genotypes at 8 months of age (D) and mea- surements of length (E) and length/width ratios (F) (n≥100 cells/animal; n=3 animals each). Scale bar=50 µm. G, Representative Masson trichrome–stained cardiac cross-sections from 8-month-old METTL3-cKO mice and control mice. Scale bar=1 mm. H through J, Representative images of short-axis (M-mode) and long-axis (B-mode) echocardiographic analysis (H), and echocardiographic quantification of left ventricular chamber end-diastolic dimensions (LVEDd) (I) and percentage fractional shortening (FS) (J) in 8-month-old METTL3-cKO and control mice (n=18 each). *P≤0.05 versus Ctrl. cKO indicates cardiomyocyte-specific METTL3 knock- out mice; Ctrl, control; and METTL3, methyltransferase-like 3. sity associated protein (FTO), a member of the alkyla- beginning to be explored in the heart. Similar to the tion repair homologs (ALKBH) family. Furthermore, deposition of epigenetic marks on DNA, methylation METTL3 acts in a complex composed of methyltrans- of mRNAs can impact gene expressivity and define the ferase-like 14 (METTL14) and additional regulatory fate of subgroups of mRNAs. This is especially impor- subunits, Wilms tumor 1–associating protein (WTAP) tant for stress-response pathways that need to rapidly 30,31 and KIAA1429. It is becoming clear that, although react to environmental challenges. Indeed, transcrip- the METTL3-containing protein complex is ubiqui- tion is time-consuming, and cells have devised meth- ods to preserve pools of mRNAs that can be readily tously responsible for the deposition of the m6A epi- genetic mRNA mark, different ALKBH proteins have and dynamically available for translation. Our study acquired some level of tissue specificity, and the rela- shows the importance of m6A mRNA modification as tive contribution of each specific member in demeth- a dynamic epigenetic mark that cardiomyocytes use to ylating m6A from RNA is still unclear. In addition to respond to hypertrophic stimuli. It is also interesting to m6A writers and erasers, m6A recognition factors (or note that enhancing the METTL3-m6A pathway was readers) have also been discovered and are members sufficient to induce cardiomyocyte hypertrophy in the of the YT521-B homology domain family. Readers, absence of additional stimuli, and that inhibition of together with writers and erasers, contribute to the METTL3 was sufficient to block hypertrophy in vitro. overall regulation of m6A-mRNA biology. It is intriguing that our data on newly generated gain- The m6A modification is part of the larger field of and loss-of-function mouse models revealed that RNA epigenetics, which is a growing field that is only modulation of METTL3 is sufficient to govern cardio- Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 543 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 7. METTL3 loss-of-function causes maladaptive cardiomyocyte remodeling and cardiac dysfunction poststress. A, Echocardiographic quantification of percentage fractional shortening (FS) in 3-month-old METTL3-cKO and control mice subjected to sham or TAC surgery for the indicated times (n=8 each). B and C, Wheat germ agglutinin–stained cardiac cross-sections of METTL3-cKO or control mice subjected to 6 weeks of TAC with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n=7 mice each). D, Echocardiographic quantification of percentage FS in 3-month-old METTL3-cKO and control mice subjected to vehicle (Veh) or angiotensin/phenylephrine infusion (Ang/PE) for 4 weeks (n=5 each). E and F, Wheat germ agglutinin–stained cardiac cross-sections of METTL3-cKO or control mice subjected to 4 weeks of vehicle orAng/PE with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥3 mice each). *P≤0.05 versus Ctrl. cKO indicates cardiomyocyte-specific METTL3 knockout mice; Ctrl, control; METTL3, methyltransferase-like 3; and TAC, transverse aortic constriction. myocyte geometry and function with aging and dur- for METTL3 in the heart and a potential therapeutic ing 2 forms of cardiac stress, pointing to the METTL3- benefit of targeting this pathway for pathological car - m6A pathway as a novel critical mediator of cardiac diac remodeling. homeostasis. Preservation of physiological cardiomyocyte geom- ARTICLE INFORMATION etry is critical for cardiac function. Cardiomyocytes Received May 25, 2018; accepted October 11, 2018. initially grow in a concentric manner following he- The online-only Data Supplement is available with this article at https:// modynamic stress, but elongate eccentrically during www.ahajournals.org/doi/suppl/10.1161/circulationaha.118.036146. 1,36 cardiomyopathic dilation and heart failure. Growth of cardiomyocytes is also present during physiological Correspondence stimuli of the heart, such as exercise, helping maintain Federica Accornero, PhD, Davis Heart and Lung Research Institute, Department 1,36 cardiac output in the face of increased afterload. of Physiology and Cell Biology, The Ohio State University, 473 W 12th Ave, Columbus, OH 43210. Email [email protected] We found that perturbing the level of METTL3 and m6A is sufficient to induce spontaneous cardiomyo- Affiliations cyte remodeling, because increased m6A led to adap- Department of Physiology and Cell Biology (L.E.D., F.A.), Department of Bio- tive growth, whereas decreased m6A induced eccen- medical Engineering (X.X., T.J.H.), Dorothy M. Davis Heart and Lung Research tric and detrimental cardiomyocyte geometry. Before Institute, The Ohio State University, Columbus. Department of Molecular Ge- our study, members of the Mapk family of kinases netics, Weizmann Institute of Science, Rehovot, Israel (L.L., J.H.H.). Division of Biostatistics and Bioinformatics, Department of Environmental Health, were one of the few examples of such a spontaneous University of Cincinnati, OH (J.C., M.M.). Cardiovascular Division, Lillehei regulation of cardiomyocyte shape. Our findings are Heart Institute and Stem Cell Institute, University of Minnesota, Minneapolis in line with these previous results, because we found (J.H.v.B.). that m6A modification is specifically occurring on pro- Sources of Funding tein kinase mRNAs, including several members of the Mapk signaling cascade. This indicates that METTL3 is This work was supported by grants from the National Institutes of Health (HL112852 and HL130072 to Dr van Berlo, HL121284 and HL136951 to Dr able to control a gene expression program in cardio- Accornero, and HL134616 to L.E. Dorn); the American Heart Association (AHA myocytes that is responsible for the regulation of car- 17IRG33460198 to Dr Accornero); The Israel Science Foundation (355/17 and diac remodeling and function, suggesting a key role 107/14), the Flight Attendant Medical Research Council, the New York Stem January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Cell Foundation, and the European Research Council Consolidator Grant Cell- 16. Lin S, Choe J, Du P, Triboulet R, Gregory RI. The m(6)A methyltrans- Naivety (to Dr Hanna); and The United States-Israel Binational Science Founda- ferase METTL3 promotes translation in human cancer cells. Mol Cell. tion (2017094 to Drs Accornero and Hanna). 2016;62:335–345. doi: 10.1016/j.molcel.2016.03.021 17. Oka T, Dai YS, Molkentin JD. 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The N6-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy

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September122018 Circulation ORIGINAL RESEARCH ARTICLE The N -Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy Editorial, see p 546 Lisa E. Dorn, BA Lior Lasman, BMS Jing Chen, PhD BACKGROUND: N -Methyladenosine (m6A) methylation is the most Xianyao Xu, MS prevalent internal posttranscriptional modification on mammalian mRNA. Thomas J. Hund, PhD The role of m6A mRNA methylation in the heart is not known. Mario Medvedovic, PhD Jacob H. Hanna, MD, PhD METHODS: To determine the role of m6A methylation in the Jop H. van Berlo, MD, PhD heart, we isolated primary cardiomyocytes and performed m6A Federica Accornero, PhD 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 Key Words: gene expression profiling ◼ hypertrophy ◼ mice, transgenic stress, showing the necessity of RNA methylation for the maintenance of ◼ RNA processing, post-transcriptional cardiac homeostasis. Sources of Funding, see page 544 CONCLUSIONS: Our study identified METTL3-mediated methylation © 2018 The Authors. Circulation is of mRNA on N -adenosines as a dynamic modification that is published on behalf of the American Heart Association, Inc., by Wolters enhanced in response to hypertrophic stimuli and is necessary for a Kluwer Health, Inc. This is an open normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA access article under the terms of the Creative Commons Attribution Non- methylation results in compensated cardiac hypertrophy, whereas Commercial License, which permits diminished m6A drives eccentric cardiomyocyte remodeling and use, distribution, and reproduction in any medium, provided that the original dysfunction, highlighting the critical importance of this novel stress- work is properly cited and is not used response mechanism in the heart for maintaining normal cardiac function. for commercial purposes. https://www.ahajournals.org/journal/circ Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 533 Dorn et al METTL3 Controls Cardiac Homeostasis lencing are established mechanisms of posttranscrip- tional regulation of gene expression that directly alter Clinical Perspective 5–7 protein levels in the heart. However, the extent to What Is New? which modifications of the mRNA itself can regulate cardiac hypertrophy is not known. • We discovered that methylation at the N -Methy The most abundant internal mRNA posttranscrip- ladenosine (m6A), the most abundant mRNA tional modification is methylation at the N -Methy modification, is increased under hypertrophic con- ladenosine (m6A), a modification catalyzed by the en- ditions in cardiomyocytes and enriched on genes 8–10 zyme methyltransferase-like 3 (METTL3). m6A mRNA involving protein kinases and intracellular signaling pathways, suggesting a regulatory role in the car- modification adds a new dimension to the developing diac hypertrophic pathway. landscape of posttranscriptional regulation of gene ex- • Increasing the expression of the m6A methyltrans- pression. It is now clear that m6A methylation plays im- ferase-like 3 (METTL3) in the heart drives sponta- portant and diverse biological functions in plants, yeast, neous, compensated hypertrophy but does not 11–16 flies, and mammals. For example, m6A manipula- affect cardiac function, whereas METTL3 knock- tion via knockdown or deletion of METTL3 affects plant down induces maladaptive eccentric remodeling growth, yeast meiosis, body mass and metabolism, syn- and leads to morphological and functional signs of aptic signaling, circadian clock regulation, and stem cell heart failure. 11–16 self-renewal and differentiation. Despite the grow- • METTL3, through m6A, helps modulate cardiac ing appreciation of the biological significance of mRNA homeostasis and hypertrophic stress responses methylation, the mechanisms by which m6A might in mice. regulate gene expression are complex and appear to be What Are the Clinical Implications? context- and cell type–dependent. Although m6A mRNA methylation has long been • Our study demonstrates the importance of METTL3 recognized as a posttranscriptional modification in and m6A modulation throughout the heart’s lifes- mammalian cells, the roles of this posttranscriptional pan, both in terms of cardiac homeostasis with process and the functions of m6A mRNA methylation in aging and the heart’s response to pressure-over- cardiomyocytes and in animal models of cardiac func- load stress. • Our data determine that perturbing METTL3 tion are completely unknown. Here, we identified the and m6A levels induces spontaneous geometric m6A mRNA methylation sites in cultured cardiomyo- changes in cardiomyocytes, thereby determining cytes, and show that inhibition of the methylase MET- the adaptive or maladaptive nature of the resulting TL3 blocks hypertrophy. We also found that reduction cardiac remodeling. of m6A levels by deleting METTL3 in cardiomyocytes • Targeting m6A through its writer enzyme METTL3 leads to long-term loss of normal cardiac structure and may represent a novel therapeutic strategy for function in vivo. Conversely, enhancing m6A mRNA managing maladaptive cardiac hypertrophy and methylation in cardiomyocytes promotes spontaneous remodeling during the progression of heart failure. hypertrophic cardiomyocyte growth that results in com- pensated cardiac remodeling. he heart comprises long-lived cardiomyocytes that, in response to stress stimulation such as METHODS Tpressure overload or myocardial infarction, un- All data, material and methods are available on request. dergo hypertrophic growth. This hypertrophic re- RNA sequencing data are accessible at the Gene Expression sponse is initially an adaptive process to produce suf- Omnibus database as also specified below. ficient force to match an increase in wall tension or increased workload, but can ultimately lead to heart Animals failure. Cardiac hypertrophy is mediated by increased The generation of Mettl3 loxP-targeted (fl) mice (Mettl3 fl/fl) was gene expression and production of select proteins in previously described. Mettl3 fl/fl mice were crossed with mice cardiomyocytes. In the past, many studies have fo- expressing cre recombinase under the control of the cardiac-spe- cused on the signaling pathways leading to activation cific Myh7 promoter (β -myosin heavy chain [β-MHC]) to obtain of prohypertrophic transcription factors that selective- heart-restricted deletion of Mettl3 (METTL3-cKO; cKO). Control ly augment gene expression in the heart. Although +/+ mice for this group are Mettl3 β-MHC cre. METTL3-cKO and significant progress has been made in understanding control mice are on a C57BL6 background. A tetracycline/doxy- the transcriptional control of gene expression during cycline-responsive binary α-myosin heavy chain (α-MHC) trans- hypertrophy, it is now clear that posttranscriptional gene system was used to express METTL3 in cardiomyocytes. regulation of protein expression is a similarly critical This responder line was crossed with cardiac-restricted α-MHC mechanism for hypertrophic control. For example, transgenic mice expressing the tetracycline transactivator pro- RNA splicing factors and microRNA-mediated gene si- tein (all in the FVB/N background) to generate an overexpression January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis transgenic system (METTL3-TG; M3-TG; TG). Controls for this with a Zeiss Confocal Microscope using a 40× objective. Z stack group are tetracycline transactivator single transgenic mice. Male images were taken with a step size of 1 µm, and cardiomyocyte and female mice, 10 to 32 weeks old, were used in this study. cell volume was calculated using the length, width, and height Echocardiographic measurements were taken using a Vevo2100 of the cell as adapted from Mollova et al. Cardiomyocyte cell numbers in this case were determined using volume measure- Visual Sonics (Visual Sonics) system and MS-400 transducer. The ments in relation to heart weights of the animals from which mice were lightly anesthetized (1.5% isoflurane) and the ejec- individual cardiomyocytes were isolated; the total volume of the tion fraction, fractional shortening, and ventricular chamber heart was determined from the heart weight divided by the spe- dimensions were determined in the M-mode using the paraster- cific gravity of muscle (1.06 g/mL). 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- m6A Quantification, cally using the VevoLAB program. Relative wall thickness (RWT) Immunoprecipitation, RNA Sequencing, 2* LVPWd was calculated using the formula RWT = , where and Bioinformatics LVIDd LVPWd indicates left ventricular posterior wall dimension end RNA was extracted from neonatal rat cardiomyocytes using diastole, and LVIDd indicates left ventricular internal diameter Trizol (Life Technologies). Unstimulated or hypertrophic car- end diastole. All echocardiographic measurements are reported diomyocyte RNA samples were subjected to m6A quantifi- in Table I in the online-only Data Supplement. Cardiac injury cation using the m6A RNA Methylation Quantification Kit was induced by transverse aortic constriction to produce pres- (Colorimetric) (Abcam ab185912) in biological triplicate. Adult sure overload. In short, the transverse aortic arch was visualized mouse cardiomyocytes from METTL3-cKO, METTL3-TG, and lit- through a median sternotomy, and a 7-0 silk ligature was tied termate controls were isolated, and RNA was extracted using around the aorta using a 27-gauge wire to obtain a defined Trizol. m6A quantification was performed using the Abcam degree of constriction between the right brachiocephalic and m6A RNA Methylation Quantification Kit described above. left common carotid arteries. Infusion of angiotensin II (432 μg/ For genome-wide m6A profiling, 2.5 mg of total RNA was –1 –1 kg of body weight/d) and phenylephrine (100 mg·kg ·d ) was extracted from neonatal rat cardiomyocytes using Trizol and performed with implantation of Alzet minipumps for 4 weeks enriched for mRNAs using polyT columns. Unstimulated or (Durect Inc). All experiments involving animals were approved by hypertrophied cardiomyocyte RNA samples (in biological dupli- the Institutional Animal Care and Use Committee at The Ohio cates) were subjected to m6A immunoprecipitation as previ- State University. ously described. In brief, mRNA was chemically fragmented and incubated with antibodies recognizing m6A-modified RNA (Synaptic Systems). Rabbit normal IgG was used as a negative Cardiomyocyte Isolation and Treatments control. Immunoprecipitated RNA was then submitted for RNA Neonatal rat ventricular cardiomyocytes were isolated as previ- sequencing (University of Cincinnati sequencing and genome 17,18 ously published. In brief, hearts were incubated with trypsin analysis core laboratory). Illumina reads were mapped to the rat at 4°C overnight, followed by trypsin inhibitor and collagenase reference genome (UCSC Rn4) using Tophat. The resulting bam incubation at 37°C for 1 hour (Worthington Biochemical). files were individually analyzed for peak detection using MACS Hearts were mechanically dissociated and incubated for an (version 1.4). The peaks from all repeats were merged to build additional 15 minutes, followed by resuspension in media sup- the consensus peak regions, such that the peak is included if plemented with fetal bovine serum and preplating to remove it appears in at least 1 repeat. The reads in each consensus noncardiomyocytes. Cardiomyocytes were plated on 0.1% gel- peak region were counted in each sample’s bam file. The log2- atin–coated dishes in the presence of 10% serum. The follow- transformed reads per kilobase million–normalized read counts ing day, cells were washed twice and cultured in M199 media were used as the peak intensity for each consensus peak without serum (Corning). Hypertrophy was induced by cultur- region in each sample. Negative control immunoprecipitation ing cardiomyocytes for 48 hours in the presence of 2% serum. samples were used to control for unspecific binding: peaks Knockdown of METTL3 was obtained by transfection of small that appeared in the negative control as well were excluded. interfering RNA–targeting METTL3 (TriFECTa Mettl3 RNAi by Peaks whose intensities were significantly different between Integrated DNA Technologies) or negative small interfering RNA hypertrophy and normal samples were detected by Student t control (TriFECTa negative control DS NC1 RNAi by Integrated test comparing all normal and hypertrophy replicates with a DNA Technologies), and cells were analyzed 48 hours posttrans- false discovery rate–adjusted P value <0.1. We used our own R fection. Overexpression of METTL3 or β-galactosidase (control) scripts to create the analysis pipeline. This pipeline was based was achieved by adenoviral infection, and cells were analyzed on the Nature Protocol article, “A computational pipeline 48 hours postinfection. Adult mouse cardiomyocytes were iso- for comparative ChIP-seq analyses,” which contained modi- 19 22 lated as previously described and imaged using bright field fications to accommodate statistical analysis of replicates. microscopy (magnification 20×) on an EVOS FL Auto II micro- Annotations of the consensus peak regions, including chromo- scope (Thermo Fisher). For analysis of cardiomyocyte number, some number, start and end location on chromosomes, gene freshly isolated cardiomyocytes from 3-month-old mice were name and identification of the peak region, and the genomic counted (before the sedimentation steps to avoid cardiomyo- feature of the peak (5ʹ-untranslated region, intron, exon, and cyte loss) with a hemocytometer. For analysis of cardiomyo- 3ʹ-untranslated region) were added according to UCSC Rn4 cyte volume, isolated cardiomyocytes from 3-month-old mice gene definitions. RNA sequencing data sets were submitted to were stained in suspension with fluorescein isothiocyanate– the Gene Expression Omnibus database (series GSE119170). conjugated wheat germ agglutinin (Sigma-Aldrich) and imaged Peaks were visualized using the IGV browser. Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 535 Dorn et al METTL3 Controls Cardiac Homeostasis that the percentage of m6A RNA modification increas- Western Blotting, Histology, es substantially in response to a hypertrophic stimulus Immunostaining, and mRNA Expression (Figure 1B). To define the specific mRNA sequences that Analysis were dynamically affected by m6A modification in re- Western blotting was performed from neonatal rat cardio- sponse to a hypertrophic stimulus, we performed m6A myocytes using standard procedures. Antibodies used were sequencing of m6A-modified mRNA from hypertrophic METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), cardiomyocytes in comparison with unstimulated condi- MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling tions. Our analysis revealed differential m6A methyla- Technology), and GAPDH (Fitzgerald Industries). Masson tri- tion during hypertrophy on 713 cardiomyocyte mRNAs chrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining (Table III in the online-only Data Supplement). It is inter- was performed using α-actinin antibodies (Sigma-Aldrich), esting to note that we found that, in hypertrophic car- and cell area was quantified using CellProfiler following pub- diomyocytes, m6A changes are not randomly distributed lished methods. Detection of the cell membrane was per- throughout the genome, but are specifically increased in formed using fluorescein isothiocyanate–conjugated wheat select classes of mRNAs (Figure 1C). Within these spe- germ agglutinin (Sigma-Aldrich). The cross-sectional area was cific functional categories, m6A peaks in mRNAs encod- measured using ImageJ. RNA was extracted from neonatal ing for protein kinases and modifiers were the most rat cardiomyocytes or mouse hearts using Trizol, and reverse significantly enriched during hypertrophy (Figure  1C). transcription was performed using the High Capacity cDNA Representative m6A peaks in the ryanodine receptor Reverse Transcription kit (Applied Biosystems). Selected genes (Ryr2) and mitogen-activated protein kinase kinase ki- and m6A peaks were analyzed by real-time polymerase chain nase 6 (Map3k6) 3ʹ-untranslated regions are shown as reaction using SYBR green (Applied Biosystems). Quantified examples of m6A-methylated mRNA that showed either mRNA expression was normalized to Rpl7 (ribosomal protein L7) and expressed relative to controls. no change with hypertrophy (Ryr2) or increased levels of m6A with hypertrophy (Map3k6) (Figure 1D and 1E). To validate the results of m6A sequencing and quantify Statistics the extent of enhanced methylation in response to a All results are presented as mean±SEM. Statistical analysis hypertrophic stimulus, we performed m6A immunopre- was performed with an unpaired 2-tailed t test (for 2 groups) cipitation followed by real-time polymerase chain reac- and 1-way ANOVA with Bonferroni correction (for groups of tion using gene-specific primers flanking the identified ≥3). P values <0.05 were considered significant. m6A peaks from RNA sequencing (Table 1). We assessed increased m6A levels following hypertrophic stimulus in RESULTS 16 genes from the functional category of protein-mod- ifying enzymes that showed increased m6A levels fol- m6A Is a Dynamic Modification in lowing hypertrophic stimulus in our genome-wide anal- Cardiomyocytes ysis, and identified 15 to be significantly enriched for To determine the mRNAs that are modified by m6A m6A. These results clearly show that m6A modifications on mRNA are enhanced on specific classes of proteins in methylation in cardiomyocytes, we performed m6A response to a hypertrophic stimulus. sequencing on cultured rat neonatal cardiomyocytes. Purified mRNAs were fragmented to allow for analy- sis of the specific location of m6A peaks, pulled down METTL3 Drives Cardiomyocyte using a specific antibody recognizing m6A-modified Hypertrophy In Vitro and In Vivo RNA, and processed for sequencing (m6A sequencing, meRIP-seq). Using this strategy, we identified 3922 m6A To determine whether enhancing m6A modification peaks that showed a general distribution throughout on mRNA was sufficient to cause cardiomyocyte hy- the coding region of genes (Figure  1A and Table II in pertrophy, we tested how overexpression of the m6A- the online-only Data Supplement). We also detected catalyzing enzyme METTL3 affects cardiomyocytes. relative enrichment on the untranslated regions, consis- Specifically, we generated an adenoviral vector to in- tent with previous reports (Figure 1A and Table II in the crease METTL3 expression in cardiomyocytes. We in- online-only Data Supplement). To assess a possible role fected cardiomyocytes under unstimulated conditions, for m6A modifications on mRNA in the development of and used adenovirus encoding for β-galactosidase as cardiomyocyte hypertrophy, we performed an indepen- a negative control. We first verified that METTL3 was dent m6A quantification experiment where we assessed indeed expressed at higher levels (Figure  2A). We also if the overall percentage of m6A in RNA changes during tested if METTL3 overexpression affected protein lev- cardiomyocyte hypertrophy. We used serum as a prohy- els of m6A-methylated mitogen-activated protein ki- pertrophic stimulus and isolated RNA from neonatal rat nases and found that METTL3 enhanced the levels of cardiomyocytes. Using an antibody-mediated capture of MAP3K6, MAP4K5, and MAPK14 in cardiomyocytes m6A followed by colorimetric analysis, we determined (Figure 2A and 2B). It is remarkable that upregulation January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Figure 1. m6A is a dynamic modification in cardiomyocytes. A, Distribution of m6A peaks throughout mRNAs. B, Percentage of m6A-methylated RNA in relation to unmodified adenosine as quantified using an antibody-medi- ated m6A capture assay in unstimulated (Normal) and hypertrophic (Hyper.) neonatal rat cardiomyocytes (n=3 each). C, PANTHER analysis of enriched functional gene categories showing differential m6A peaks during hypertrophy. D and E, Visualization of representative m6A peaks under unstimulated or hypertrophic conditions from the indicated mRNAs using Integrative Genomics Viewer. *P≤0.05 versus normal. Intracell. indicates intracellular; IP, immunoprecipitation; Map3k6, mitogen- activated protein kinase kinase kinase 6; m6A, N -Methyladenosine; PANTHER, Protein Analysis through Evolutionary Relationships; phosphor., phosphorylation; Ryr2, ryanodine receptor; and UTR, untranslated region. of METTL3 caused a significant increase in cardiomyo- age (Figure 2G and 2H). The lowest expressing line was cyte size, indicating that METTL3 expression is sufficient then further characterized to follow the progression of to cause cardiomyocyte hypertrophy (Figure  2C and cardiac remodeling over time. We found that this line 2D). We also validated that the m6A mRNA targets we Table 1. m6A Modification on Specific mRNAs in Response to identified using isolated neonatal rat cardiomyocytes Hypertrophy were also relevant in the adult heart. To this end, we Gene Normal Hypertrophy P Value performed m6A immunoprecipitations from neonatal Dapk1 1.04±0.11 9.87±0.77 <0.001 and adult mouse heart RNA followed by real-time poly- Dclk2 1.01±0.12 36.59±11.76 0.039 merase chain reaction. With the exception of STE20- Grk4 1.82±1.06 25.49±5.98 0.018 like protein kinase 4 (Mst4), we found that all m6A Ikbkap 1.01±0.13 5.23±0.82 0.007 peaks identified in the isolated neonatal cardiomyocyte screen were also present in the adult heart (Table  2). Ikbkb 1.08±0.27 9.14±0.85 0.001 This experiment also revealed that m6A methylation of Krs1 1.17±0.47 9.60±2.73 0.038 the tested mRNAs was, in many cases, higher in adult Map3k14 1.03±0.21 16.30±3.59 0.013 hearts than in the neonatal state, in agreement with Map3k6 1.03±0.19 15.85±1.89 0.001 overall higher m6A levels in adult hearts (Table  2 and Map4k5 0.97±0.27 5.77±2.81 0.164 Figure IA in the online-only Data Supplement). These Mapk14 1.13±0.37 8.19±1.52 0.011 findings suggest a role for m6A mRNA methylation in Mst4 1.01±0.13 18.75±2.98 0.004 adult cardiac homeostasis. Nuak2 1.00±0.07 8.16±2.16 0.030 To further verify a role for m6A in regulating car- Rps6ka2 1.04±0.20 8.32±1.79 0.015 diomyocyte homeostasis and hypertrophy in the adult Rps6ka4 1.04±0.20 8.32±1.79 0.015 heart, we generated a METTL3-overexpressing (MET- TL3-TG or M3-TG) mouse line by cross-breeding a trans- Rps6ka5 1.03±0.19 16.12±1.92 0.001 genic αMHC promoter–driven tetO responder with the Sgk1 1.07±0.26 13.05±2.56 0.010 αMHC–tetracycline transactivator driver to impart gene m6A immunoprecipitation was performed followed by qPCR using primers expression specifically in cardiomyocytes (Figure 2E and designed around preidentified m6A peaks. The qPCR data were corrected to overall gene expression level under the same conditions (normal versus 2F). METTL3-overexpressing mice showed a significant hypertrophy). Data represented are average±SEM from 3 biological replicates increase in m6A levels in cardiomyocyte RNA and dose- -Methyladenosine; and qPCR, quantitative polymerase each. m6A indicates N dependent cardiac hypertrophic growth at 3 months of chain reaction. Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 537 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 2. Generation and characterization of METTL3 overexpression models. A, Western blot from cardiomyocytes overexpressing β-galactosidase (Ad-Control) or METTL3 (Ad-Mettl3) using antibodies against the indicated proteins and GAPDH loading control. B, Densitometry quantification of the expression for the indicated proteins relative to GAPDH control. C, Quantification of cardiomyocyte cell area based on pixel size from cardiomyocytes overexpressing β-galactosidase control or METTL3 (n=3 each). D, Representative images of cardiomyocytes from the indicated treatments stained for α-actinin (green). Scale bar=20 µm. E, Schematic of cardiomyocyte-specific METTL3 gain-of-function mouse model. F, Western blot from cardiac extracts from control (Ctrl) or METTL3 transgenic (TG) line 1 and 2. GAPDH was used as loading control. G, percentage of m6A-methylated RNA relative to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in isolated cardiomyocytes from control mice and METTL3 TG line 1 mice (n=3 each). H, Gravimetric analysis of heart weight normalized to body weight (HW/BW) in the indicated genotypes at 3 months of age (n≥6 each). *P<0.05 versus control. A.U. indicates arbitrary units; m6A, N -Methyladenosine; and METTL3, methyltransferase-like 3. expresses, on average, 19-fold higher levels of METTL3 3C). Analysis of isolated adult cardiomyocytes revealed in the heart than control mice (Figure IB and IC in the physiological growth of cardiomyocytes in both width online-only Data Supplement). and length, resulting in a preserved length-to-width ra- By 8 months of age, METTL3-overexpressing mice tio (Figure 3D and 3E). However, despite the structural exhibit cardiac hypertrophy, as demonstrated by in- changes METTL3-TG hearts undergo, no histopatholog- creased heart weight to body weight ratios (Figure 3A). ic changes were observed in these mice (Figure 3F). It is Cardiac hypertrophic growth was accompanied by a important to note that cardiac function, as measured larger cardiomyocyte cross-sectional area (Figure 3B and by echocardiographic analysis of the percentage frac- January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Table 2. m6A Modification on Specific mRNAs in the Neonatal and changes in cardiomyocyte size. However, on stimulation Adult Heart of cardiomyocyte hypertrophy through the addition of Gene Neonatal Adult P Value serum, we noticed a normal hypertrophic response in control small interfering RNA–treated cardiomyocytes, Dapk1 1.01±0.08 2.00±0.40 0.053 but a complete block of hypertrophy when METTL3 Dclk2 1.83±0.78 1.07±0.55 0.459 was knocked down (Figure 4B and 4C). Grk4 1.09±0.25 0.78±0.13 0.273 Ikbkap 1.10±0.30 0.90±0.27 0.642 Ikbkb 1.16±0.34 1.25±0.38 0.863 Cardiomyocyte-Specific METTL3 Krs1 1.03±0.16 1.67±0.37 0.162 Knockout Induces Cardiac Structural Map3k14 1.06±0.24 3.11±0.82 0.075 and Functional Changes With Aging and Map3k6 1.11±0.21 3.73±0.48 0.0005 Stress Map4k5 1.18±0.34 0.95±0.26 0.602 To determine the necessity of METTL3 and m6A in the Mapk14 1.10±0.26 1.89±0.17 0.043 heart, we generated a cardiomyocyte-specific MET - Mst4 N/A N/A N/A TL3 knockout (METTL3-cKO) mouse line using a typi- Nuak2 1.00±0.01 3.92±0.72 0.018 cal (β-MHC promoter driven) cardiomyocyte-specific cre-loxP system (Figure  5A and 5B). Consistent with Rps6ka2 1.12±0.29 2.17±0.40 0.075 their METTL3 knockout status, METTL3-cKO mice Rps6ka4 1.09±0.32 4.69±0.40 0.002 have significantly decreased m6A levels in compari- Rps6ka5 1.03±0.14 3.75±0.76 0.012 son with their littermate controls (Figure 5C). Three- Sgk1 1.03±0.13 2.02±0.46 0.086 month-old METTL3-cKO animals do not show cardiac m6A immunoprecipitation was performed followed by qPCR using primers morphological or functional changes, indicating that designed around preidentified m6A peaks. The qPCR data were corrected METTL3 knockout does not impair cardiac develop- to overall gene expression level under the same conditions (neonatal versus ment (Figure  5D through 5H). Specifically, no histo- adult). Data represented are average±SEM from 4 biological replicates each. m6A indicates N -Methyladenosine; N/A, not available; and qPCR, quantitative pathologic and hypertrophic changes were observed polymerase chain reaction. (Figure 5D and 5E). Also, cardiac function was unaf- fected as measured by echocardiographic analysis of tional shortening, was preserved (Figure 3G). To further the percentage fractional shortening (Figure  5F). To confirm the adaptive nature of the cardiac remodeling confirm the absence of remodeling at the cellular lev- observed in METTL3-TG mice and ensure that it does el, we then measured cardiomyocyte cross-sectional not accelerate dysfunction during stress, we performed area and observed no changes in METTL3-cKO mice pressure overload stimulation on 3-month-old METTL3- (Figure 5G and 5H). To further exclude developmental TG and control mice through transverse aortic constric- defects in METTL3-cKO mice, we assessed postnatal tion. We found that increased expression of METTL3 is proliferative and hypertrophic growth (Figure II in the not detrimental post stress (Figure 3H). Also, the hyper- online-only Data Supplement). Expression analysis trophic response to transverse aortic constriction was of proliferation markers revealed no abnormalities not exacerbated by METTL3 upregulation at the organ in METTL3-cKO hearts (Figure IIA through IIC in the level or in terms of cardiomyocyte cross-sectional area online-only Data Supplement). Similarly, markers of (Figure 3I through 3K). Therefore, enhancing m6A mod- hypertrophy and the postnatal switch between β- and ification of mRNA through cardiomyocyte-specific MET - α-MHCs (Myh7 and Myh6, respectively) were also TL3 overexpression induces compensated hypertrophic unaffected (Figure IID through IIF in the online-only remodeling of the heart without inducing cardiac func- Data Supplement). Considering that, within the first tional deficits either at baseline or under cardiac stress. week of life, murine cardiomyocytes lose their ability to grow by hyperplasia and instead activate a post- natal hypertrophic program, we tested cardiomyocyte METTL3 Inhibition Prevents the cross-sectional area in day 7 hearts from METTL3-cKO Development of Cardiomyocyte and control mice. This analysis revealed that postnatal Hypertrophy in Vitro growth is unaffected in the absence of METTL3 (Fig- To determine if m6A methylation plays a primary role in ure IIG and IIH in the online-only Data Supplement). the development of cardiac hypertrophy, we inhibited Consequently, cardiomyocyte numbers and cell vol- expression of the m6A-catalyzing enzyme METTL3 in umes in 3-month-old METTL3-cKO and control mice isolated neonatal rat cardiomyocytes. We observed a were also unchanged (Figure II I through IIK in the significant knockdown of METTL3 by delivery of MET - online-only Data Supplement). Overall, these results TL3-targeting small interfering RNA (Figure 4A). In the suggest that METTL3 is dispensable for postnatal absence of hypertrophic stimuli, we did not observe any heart development. Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 539 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 3. METTL3 drives compensated hypertrophy in vivo. A, Heart weight to body weight ratios (HW/BW) in 8-month-old METTL3-TG animals or littermate controls (n≥7 each group). B and C, Wheat germ agglutinin (green)–stained cardiac cross-sections of 8-month-old METTL3-TG or littermate control mice with quantification of cardiomyocyte cross-sectional area using ImageJ software. Scale bar=50 µm (n≥100 cells/animal, n=6 mice each). D, Representative images of isolated cardiomyocytes from 8-month-old hearts from the indicated genotypes. Scale bar=50 µm. E, Length/width ratios of isolated cardiomyocytes from 8-month-old control and TG mice (n≥100 cells/animal; n=3 mice each). F, Representative images of Masson trichrome staining of cardiac cross-sections of 8-month-old METTL3-TG or littermate control mice. Scale bar=1 mm. G, Echocardiographic quantification of percentage fractional shortening (FS) for 8-month-old METTL3-TG animals or littermate controls (n=12 each). H, Echocardiographic quantification of percentage fractional shortening for Sham or TAC-operated METTL3-TG animals or littermate controls (n≥ 5 each). I, HW/BW in TAC-operated METTL3-TG animals or littermate controls (n≥5 each group). J and K, Wheat germ agglutinin (green)–stained cardiac cross-sections of TAC-operated METTL3-TG or littermate control mice with quantification of cardiomyocyte cross-sectional area using ImageJ software. Scale bar=100 µ m (n≥100 cells/animal, n=5 mice each). *P≤0.05 versus Ctrl. Ctrl indicates control; METTL3, methyltransferase-like 3; TAC, transverse aortic constriction; and TG, transgenic. However, when METTL3-cKO animals are aged were not accompanied by overall heart weight altera- to 8 months, they begin to show cardiac abnormali- tions (Figure  6C). To further understand these results, ties consistent with a progression toward heart failure we isolated adult cardiomyocytes and found eccentric (Figure 6). Cardiomyocyte cross-sectional area was sig- cardiomyocyte remodeling in the absence of METTL3 nificantly reduced in 8-month-old METTL3-cKO mice (Figure  6D through 6F). Indeed, cardiomyocytes from (Figure 6A and 6B). It is interesting that these changes METTL3-cKO hearts were elongated and showed an January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Figure 4. METTL3 inhibition prevents the development of cardiomyocyte hypertrophy. A, qPCR analysis for METTL3 expression in response to Ctrl or METTL3 siRNA-mediated knockdown (n=3 each). B, Representative images of cardiomyocytes treated with control siRNA (si-Ctrl) or siRNA targeting METTL3 (si-M3) stained for α-actinin (green). Scale bar=20 µm. C, Quantification of cardiomyocyte cell area (n≥50 cells/well, n=3 independent experiments/treatment). *P<0.05 versus baseline; #P<0.05 versus si-Ctrl hypertrophy. Ctrl indicates control; METTL3, methyl- transferase-like 3; siRNA, small interfering RNA; and qPCR, quantitative polymerase chain reaction. overall increase in their length-to-width ratio (Fig- METTL3 in controlling cardiomyocyte geometry and ure  6F). Consistent with maladaptive eccentric car- adaptation to stress. diomyocyte remodeling, cardiac knockout of METTL3 Altogether, our data demonstrate a critical role for caused a significant increase in left ventricular chamber METTL3 and m6A modifications of mRNA for the main- tenance of cardiac homeostasis, function, and stress dimensions and ventricular dilation (Figure 6G through responses. 6I). The structural changes we observed in METTL3-cKO cardiomyocytes were also associated with a decrease in overall cardiac function as measured by echocardiogra- DISCUSSION phy (Figure 6J). To test if METTL3 is necessary for adaptation to The control of protein synthesis is achieved by complex stress, we then subjected 3-month-old METTL3-cKO and still poorly defined mechanisms that regulate the and control mice to pressure overload stimulation (or posttranscriptional processing of mRNAs. Understand- transverse aortic constriction). We found that the ab- ing how specific classes of functionally related mRNAs sence of METTL3 accelerates heart failure progression are coregulated in the heart is crucial to elucidate the and leads to reduced cardiomyocyte cross-sectional molecular mechanisms controlling the characteristic area (Figure 7A through 7C). These data suggest that increase in cardiac mass that defines cardiac hypertro- METTL3 is critical for adaptive cardiac remodeling af- phy within myocytes. Here, we discovered METTL3 as ter injury. To further test if maladaptive cardiomyo- a critical RNA-modifying protein that drives cardiomyo- cyte remodeling occurs before dysfunction and could cyte hypertrophy by catalyzing methylation of m6A on indeed be a pathological driver of cardiomyopathy, specific subsets of mRNAs. Our genome-wide m6A-se- we applied a milder stress through the infusion of quencing analysis in cardiomyocytes represents the first angiotensin and phenylephrine for 4 weeks. In this report demonstrating the occurrence of m6A in cardiac condition in which cardiac function was still intact, cells, the dynamic changes occurring in hypertrophic we observed reduction in the cross-sectional area of conditions, and the specific mRNA targets of this modi- stressed cardiomyocytes lacking METTL3 (Figure  7D fication. An interesting result from our analysis was the discovery of protein kinases as the most affected cat- through 7F). This result indicates a direct role for Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 541 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 5. Generation and characterization of METTL3 cardiac-restricted knockout mice. A, Schematic of METTL3 loss-of-function mouse model. B, Western blot for METTL3 from cardiac extracts from control (Ctrl) or cardiomyocyte-specific METTL3 knockout mice (cKO). GAPDH and Ponceau staining were used as loading control. C, percentage of m6A-methylated RNA relative to unmodified adenosine as quantified using an antibody-mediated m6A capture assay in isolated cardiomyocytes from control and METTL3-cKO mice (n=3 each). D, Representative Masson trichrome–stained cardiac cross-sections from 3-month-old METTL3-cKO mice and controls. Scale bar=1 mm. E, Quantification of heart weight to body weight ratio (HW/BW) for 3-month-old METTL3-cKO or control mice (n=5 each). F, Echocardiographic quantification of percentage fractional shortening (FS) for 3-month-old METTL3-cKO or control mice (n=12 each). G and H, Wheat germ agglutinin (green)–stained cardiac cross-sections of 3-month-old METTL3-cKO or control mice with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µ m (n≥100 cells/animal, n≥4 mice each). *P≤0.05 versus Ctrl. m6A indicates N -Methyladenosine; and METTL3, methyltransferase-like 3. egory of genes dynamically controlled by the METTL3- of METTL3 as a master controller of large subsets of m6A pathway during hypertrophy. Indeed, many stud- signaling molecules through posttranscriptional regu- ies have recognized the importance of kinase-regulated lation of mRNA processing might further explain how signaling pathways for cardiomyocyte hypertrophic stress responses are coordinated at a posttranscription- growth, and mitogen-activated protein kinases specifi- al level in cardiomyocytes. Our findings open intriguing cally have well-established roles in hypertrophy. In ad- new possibilities for controlling cardiac remodeling and dition, we also found hypertrophy-dependent methyla- pathological stress responses in the heart. tion on mRNAs encoding for kinases converging on the It is striking to observe how m6A levels increase nuclear factor kB, which has been shown to cooperate in specific functional clusters of mRNAs in response not only with mitogen-activated protein kinases, but to hypertrophic growth signals in cardiomyocytes. also with nuclear factor of activated T cells in regulating Although the origin of this specificity is currently un- cardiac hypertrophy. Last, our m6A profile revealed known, the fact that increased expression of METTL3 dynamic methylation in mRNAs encoding for members is sufficient to cause hypertrophy might indicate that of the ribosomal S6 family of kinases. Ribosomal S6 the activity of this enzyme could be a limiting factor kinases are downstream effectors of the extracellular under homeostatic conditions. In addition, it is im- regulated kinase branch of mitogen-activated protein portant to consider that specific demethylases might kinases and have established roles in adaptive cardiac act with different specificity, and their activity could growth through the regulation of mammalian target of also be controlled by hypertrophic signals in cardio- 27,28 rapamycin–dependent protein synthesis. Consider- myocytes. Indeed, the m6A epigenetic mark has been ing the complexity and intersection of multiple signaling shown to be reversible. The protein that removes the mechanisms driving cardiac hypertrophy, the discovery methyl group m6A was identified as fat mass and obe- January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Figure 6. METTL3 loss-of-function causes maladaptive cardiomyocyte remodeling and cardiac dysfunction with aging. A and B, Wheat germ agglutinin–stained cardiac cross-sections of 8-month-old METTL3-cKO or control mice with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥5 mice each). C, Quantification of heart weight to body weight ratio (HW/BW) for 8-month-old METTL3-cKO or control mice (n≥13 each). D and F, Representative bright field image of isolated cardiomyocytes from the indicated genotypes at 8 months of age (D) and mea- surements of length (E) and length/width ratios (F) (n≥100 cells/animal; n=3 animals each). Scale bar=50 µm. G, Representative Masson trichrome–stained cardiac cross-sections from 8-month-old METTL3-cKO mice and control mice. Scale bar=1 mm. H through J, Representative images of short-axis (M-mode) and long-axis (B-mode) echocardiographic analysis (H), and echocardiographic quantification of left ventricular chamber end-diastolic dimensions (LVEDd) (I) and percentage fractional shortening (FS) (J) in 8-month-old METTL3-cKO and control mice (n=18 each). *P≤0.05 versus Ctrl. cKO indicates cardiomyocyte-specific METTL3 knock- out mice; Ctrl, control; and METTL3, methyltransferase-like 3. sity associated protein (FTO), a member of the alkyla- beginning to be explored in the heart. Similar to the tion repair homologs (ALKBH) family. Furthermore, deposition of epigenetic marks on DNA, methylation METTL3 acts in a complex composed of methyltrans- of mRNAs can impact gene expressivity and define the ferase-like 14 (METTL14) and additional regulatory fate of subgroups of mRNAs. This is especially impor- subunits, Wilms tumor 1–associating protein (WTAP) tant for stress-response pathways that need to rapidly 30,31 and KIAA1429. It is becoming clear that, although react to environmental challenges. Indeed, transcrip- the METTL3-containing protein complex is ubiqui- tion is time-consuming, and cells have devised meth- ods to preserve pools of mRNAs that can be readily tously responsible for the deposition of the m6A epi- genetic mRNA mark, different ALKBH proteins have and dynamically available for translation. Our study acquired some level of tissue specificity, and the rela- shows the importance of m6A mRNA modification as tive contribution of each specific member in demeth- a dynamic epigenetic mark that cardiomyocytes use to ylating m6A from RNA is still unclear. In addition to respond to hypertrophic stimuli. It is also interesting to m6A writers and erasers, m6A recognition factors (or note that enhancing the METTL3-m6A pathway was readers) have also been discovered and are members sufficient to induce cardiomyocyte hypertrophy in the of the YT521-B homology domain family. Readers, absence of additional stimuli, and that inhibition of together with writers and erasers, contribute to the METTL3 was sufficient to block hypertrophy in vitro. overall regulation of m6A-mRNA biology. It is intriguing that our data on newly generated gain- The m6A modification is part of the larger field of and loss-of-function mouse models revealed that RNA epigenetics, which is a growing field that is only modulation of METTL3 is sufficient to govern cardio- Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 January 22, 2019 543 Dorn et al METTL3 Controls Cardiac Homeostasis Figure 7. METTL3 loss-of-function causes maladaptive cardiomyocyte remodeling and cardiac dysfunction poststress. A, Echocardiographic quantification of percentage fractional shortening (FS) in 3-month-old METTL3-cKO and control mice subjected to sham or TAC surgery for the indicated times (n=8 each). B and C, Wheat germ agglutinin–stained cardiac cross-sections of METTL3-cKO or control mice subjected to 6 weeks of TAC with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n=7 mice each). D, Echocardiographic quantification of percentage FS in 3-month-old METTL3-cKO and control mice subjected to vehicle (Veh) or angiotensin/phenylephrine infusion (Ang/PE) for 4 weeks (n=5 each). E and F, Wheat germ agglutinin–stained cardiac cross-sections of METTL3-cKO or control mice subjected to 4 weeks of vehicle orAng/PE with quantification of cardiomyocyte cross-sectional area. Scale bar=50 µm (n≥100 cells/animal, n≥3 mice each). *P≤0.05 versus Ctrl. cKO indicates cardiomyocyte-specific METTL3 knockout mice; Ctrl, control; METTL3, methyltransferase-like 3; and TAC, transverse aortic constriction. myocyte geometry and function with aging and dur- for METTL3 in the heart and a potential therapeutic ing 2 forms of cardiac stress, pointing to the METTL3- benefit of targeting this pathway for pathological car - m6A pathway as a novel critical mediator of cardiac diac remodeling. homeostasis. Preservation of physiological cardiomyocyte geom- ARTICLE INFORMATION etry is critical for cardiac function. Cardiomyocytes Received May 25, 2018; accepted October 11, 2018. initially grow in a concentric manner following he- The online-only Data Supplement is available with this article at https:// modynamic stress, but elongate eccentrically during www.ahajournals.org/doi/suppl/10.1161/circulationaha.118.036146. 1,36 cardiomyopathic dilation and heart failure. Growth of cardiomyocytes is also present during physiological Correspondence stimuli of the heart, such as exercise, helping maintain Federica Accornero, PhD, Davis Heart and Lung Research Institute, Department 1,36 cardiac output in the face of increased afterload. of Physiology and Cell Biology, The Ohio State University, 473 W 12th Ave, Columbus, OH 43210. Email [email protected] We found that perturbing the level of METTL3 and m6A is sufficient to induce spontaneous cardiomyo- Affiliations cyte remodeling, because increased m6A led to adap- Department of Physiology and Cell Biology (L.E.D., F.A.), Department of Bio- tive growth, whereas decreased m6A induced eccen- medical Engineering (X.X., T.J.H.), Dorothy M. Davis Heart and Lung Research tric and detrimental cardiomyocyte geometry. Before Institute, The Ohio State University, Columbus. Department of Molecular Ge- our study, members of the Mapk family of kinases netics, Weizmann Institute of Science, Rehovot, Israel (L.L., J.H.H.). Division of Biostatistics and Bioinformatics, Department of Environmental Health, were one of the few examples of such a spontaneous University of Cincinnati, OH (J.C., M.M.). Cardiovascular Division, Lillehei regulation of cardiomyocyte shape. Our findings are Heart Institute and Stem Cell Institute, University of Minnesota, Minneapolis in line with these previous results, because we found (J.H.v.B.). that m6A modification is specifically occurring on pro- Sources of Funding tein kinase mRNAs, including several members of the Mapk signaling cascade. This indicates that METTL3 is This work was supported by grants from the National Institutes of Health (HL112852 and HL130072 to Dr van Berlo, HL121284 and HL136951 to Dr able to control a gene expression program in cardio- Accornero, and HL134616 to L.E. Dorn); the American Heart Association (AHA myocytes that is responsible for the regulation of car- 17IRG33460198 to Dr Accornero); The Israel Science Foundation (355/17 and diac remodeling and function, suggesting a key role 107/14), the Flight Attendant Medical Research Council, the New York Stem January 22, 2019 Circulation. 2019;139:533–545. DOI: 10.1161/CIRCULATIONAHA.118.036146 ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Dorn et al METTL3 Controls Cardiac Homeostasis Cell Foundation, and the European Research Council Consolidator Grant Cell- 16. Lin S, Choe J, Du P, Triboulet R, Gregory RI. The m(6)A methyltrans- Naivety (to Dr Hanna); and The United States-Israel Binational Science Founda- ferase METTL3 promotes translation in human cancer cells. Mol Cell. tion (2017094 to Drs Accornero and Hanna). 2016;62:335–345. doi: 10.1016/j.molcel.2016.03.021 17. Oka T, Dai YS, Molkentin JD. 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Journal

Circulation – Pubmed Central

Published: Nov 28, 2018

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