YTHDC1 regulates the postnatal development of heart

doi:10.21203/rs.3.rs-3301770/v1

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YTHDC1 regulates the postnatal development of heart

https://doi.org/10.21203/rs.3.rs-3301770/v1

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This study aimed to investigate the role of the N6-methyladenosine (m⁶A) reader protein YTHDC1 in heart development and its potential molecular mechanisms. Animal experiments were conducted using cardiac-specific Ythdc1 knockout (Ythdc1-CKO) mice, and human heart samples were collected from aborted fetuses. Echocardiography, immunoblotting, RNA-Seq, and ATAC-Seq were performed to assess cardiac function, gene expression, and chromatin accessibility. The results revealed that YTHDC1 expression was highest during embryonic and early postnatal stages and gradually decreased with age. Cardiac-specific deletion of Ythdc1 resulted in abnormal heart development, early dilated cardiomyopathy, and severe heart failure. RNA-Seq analysis revealed significant changes in gene expression profiles, particularly genes related to cardiac contraction and transmembrane transport. ATAC-Seq analysis demonstrated significant changes in chromatin accessibility, and the binding motifs of the transcription factors Mef2a, Mef2b, Mef2c, and Mef2d, which are essential for cardiac development, were switched off in Ythdc1-CKO mouse hearts. In conclusion, this study demonstrates that YTHDC1 plays a critical role in heart development and its deficiency leads to abnormal cardiac development and function. The findings provide insights into the molecular mechanisms underlying heart development and suggest potential therapeutic targets for heart diseases.

Biological sciences/Computational biology and bioinformatics

Biological sciences/Developmental biology

Biological sciences/Genetics

Health sciences/Cardiology

Health sciences/Diseases

YTHDC1

heart

development

dilated cardiomyopathy

The heart is the initial organ to form and acquire functionality during embryonic development. Abnormalities in heart development can lead to congenital heart diseases, which manifest as structural defects or functional abnormalities. Common diseases such as atrial septal defects, ventricular septal defects, tetralogy of Fallot, manifest as structural abnormalities, while dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and restrictive cardiomyopathy are examples of functional abnormalities.

Embryonic heart development is a complex process, precisely regulated by a variety of genes and signaling pathways in time and space. RNA-binding proteins (RBPs) play an important role in heart development. Mutations or deletions of certain RBPs, such as RBM24[1, 2] and RBM20[3–5], can lead to congenital cardiomyopathy. The N6-methyladenosine (m⁶A) modification is the most common mRNA modification in eukaryotes[6]. m⁶A modification related proteins play a significant role in several cardiac diseases[7–10]. Although previous studies have demonstrated that m⁶A modification related proteins play an essential role in regulating the development of brown adipose tissue[11, 12], liver[13], and nervous system[14], as well as the differentiation of embryonic stem cells[15], but their role in heart development is still largely unexplored.

YTHDC1 belongs to the m⁶A-modified direct reader family and is the only nuclear-located reader protein[16]. It is a member of the YTH (YT521-B homology) protein family, which contains 100–150 amino acids and is a highly conserved protein[17]. YTHDC1 is an important RBP that plays a critical regulatory role in many biological processes. Its primary function is to mediate mRNA splicing[18], epigenetic silencing mediated by the non-coding RNA XIST[19], and nuclear export of mRNA[20]. The deposition of m⁶A occurs co-transcriptionally[21], but recent studies have found that YTHDC1 directly affects transcription itself[22]. YTHDC1 forms YT-bodies with transcriptional active sites in a phase-separated form to regulate transcription and participate in transcriptional activation[16]. In addition, YTHDC1 plays a vital role in a variety of cardiac and cerebrovascular diseases, and YTHDC1 deficiency is associated with DCM[23]. It has been reported that YTHDC1 may also be involved in mitophagy and energy metabolism in HCM[24]. However, YTHDC1 overexpression can alleviate ischemic stroke[25]. Moreover, inhibition of YTHDC1 can reduce inflammation and improve cardiac function in sepsis-induced cardiomyopathy[26]. Therefore, it has potential therapeutic applications. YTHDC1 is also very important for growth and development. Previous studies have shown that YTHDC1 is required for male spermatogonial development and female oocyte growth and maturation. YTHDC1-deficient oocytes are blocked at the primary follicle stage, and its deletion can result in early embryonic lethality[27]. However, its regulatory role in heart development has yet to be investigated.

In this study, we found that the m⁶A reader protein YTHDC1 plays a vital role in heart development. Heart-specific deletion of YTHDC1 caused early DCM and severe heart failure in mice, all of which died early after birth. YTHDC1 may regulate cardiac development by affecting the expression of genes related to muscle contraction and transmembrane transport through alternative splicing. It may also regulate transcription factors and affect the expression of downstream target genes related to cardiac development. This study further reveals the critical role of YTHDC1 in regulating organ development and provides a basis for exploring disease treatment.

YTHDC1 expression is highest in embryonic and early postnatal stages and gradually decreases with age

We assayed the temporal and spatial expression profiles of each transcriptome during mouse heart development from a published Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo) with accession number GSE51483. Within this gene series, it was observed that the expression of Ythdc1 was highest during the embryonic stage. However, as age progresses from birth to adulthood, the expression gradually declined (Figure 1A/B), with a similar expression profile in both the left and right ventricles. To verify this expression pattern, mouse heart tissues were collected at various developmental stages, including the embryonic period. After Western-blot verification, the expression level of YTHDC1 was highest during embryonic development and the early postnatal phase, while its expression was significantly decreased in adulthood (Figure 1C/D), consistent with the data in the database. This expression profile suggests that YTHDC1 potentially plays a critical role in mouse cardiac development.

Cardiac-specific deletion of Ythdc1 causes abnormal heart development

To clarify the role YTHDC1 plays in heart development, we gathered human fetal hearts from the hospital aborted because of congenital heart disease. We also collected hearts without heart disease from aborted fetuses with similar gestational age as a control group for comparison. We observed a decrease in YTHDC1 expression in the heart tissue of a fetus with ventricular septal defect, aortic stenosis, and aortic arch dysplasia (Figure 2A). Additionally, we created heart specific Ythdc1 knockout mice (Ythdc1-CKO) by inserting loxp sites at both ends of Ythdc1 exons 5-7 and breeding them with Myh6-Cre^+/-mice (Figure 2B). The results of the Western blot analysis have confirmed that Ythdc1 exhibited a specific knockdown of approximately 50% in the heart (Figure 2C). We collected liver, muscle, kidney, brain and lung tissues from Ythdc1-CKO and Ythdc1^flox/floxmice, Western-blot analysis demonstrated that the YTHDC1 ablation had no significant impact on the expression level in these tissues except the heart (Figure 2D). We observed that the Ythdc1-CKO mice were born without any problems and followed the expected Mendelian inheritance patterns. However, 90% of Ythdc1-CKO mice died within 20 days after born (Figure 2E). To determine the cause of death, we dissected the mice and discovered that the hearts of the Ythdc1-CKO mice were enlarged (Figure 2F). We killed mice 15 days after birth, collected ventricular muscle tissue, and measured the body weight, heart weight, and heart weight/body weight ratio. Statistical analyses revealed a considerable decrease in body weight and a substantial increase in heart weight of the Ythdc1-CKO mice. Moreover, there was a significant increase in the ratio of heart weight to body weight (Figure 2G). The tissues were then fixed and examined using H&E staining and electron microscopy. H&E staining showed enlargement of the entire heart with thinning of the ventricular wall, similar to the characteristics of DCM (Figure 2H). Transmission electron microscopy reveals partial disorganization of muscle fibers, with certain Z-lines (Z) fractured and a minor absence of H-band (H). Additionally, certain mitochondria exhibit vacuolar degeneration (Figure 2I). To further assess cardiac function quantitatively, we performed echocardiography on days 1 and 15 after birth using a small animal ultrasound device (Vevo3100) to measure left ventricular diameter, thickness and other indicators. No significant differences between Ythdc1-CKO and Ythdc1^flox/floxmice were observed in any echocardiographic or physiological parameters at 1 day of age. However, on day 15 after birth, a significant decrease in ejection fraction (EF) and fractional shortening (FS) was observed, along with a significant decrease in the end-diastolic diameter of the interventricular septum (IVS, d), while the end-diastolic diameter of the left ventricle (LVID, d) significantly increased (Figure 2J/K/L). In summary, our findings indicate that Ythdc1-CKO mice are born with normal hearts, then gradually develop enlarged hearts with severe heart failure similar to DCM, which directly results in the premature death of KO mice. These results demonstrate that YTHDC1 is of great importance in heart development, and specific deletion of YTHDC1 in the heart causes structural and functional changes in both human and mouse hearts.

Cardiac-specific deletion of Ythdc1 largely changes gene expression profile in the heart

To further explore the effect of heart-specific YTHDC1 deletion on the molecular mechanisms associated with cardiac development abnormalities, we conducted RNA-Seq analysis on heart tissue samples obtained from Ythdc1-CKO and Ythdc1^flox/flox mice, aiming to examine the transcriptional profile comprehensively. Figure 3A illustrates that 437 genes were down-regulated and 599 genes were up-regulated in the Ythdc1-CKO heart (Figure 3A). GO analysis revealed that: The down-regulated genes were mainly associated with heart contraction, heart processes, regulation of ion transmembrane transport and regulation of ion transmembrane transport, regulation of blood circulation, calcium ion transport, cardiac muscle contraction, regulation of heart contraction, divalent metal ion transport, and divalent inorganic cation transport (Figure 3B), while the upregulated gene was associated with extracellular structure organization, extracellular matrix organization, positive regulation of the cell migration, positive regulation of cell motility, cartilage development, positive regulation of response to external stimulus, regulation of ossification, skeletal system development, and biomineral tissue development (Figure 3C). KEGG pathway enrichment analysis showed that pathways such as adrenergic signaling in cardiomyocytes, cardiac muscle contraction, neuroactive ligand-receptor interaction, arrhythmogenic right ventricular cardiomyopathy (ARVC), calcium signaling pathway, HCM, vascular smooth muscle contraction, the MAPK signaling pathway, DCM, and the serotonergic synapse were downregulated. (Figure 3D). The up-regulated genes were mainly related to the biosynthesis of amino acids, HIF-1 signaling pathway, ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, ErbB signaling pathway, TGF-β signaling pathway, HCM, and p53 signaling (Figure 3E). Previous studies have shown that Ythdc1 can regulate alternative splicing[18], so we used rMATS software to analyze alternative splicing events in RNA-seq data, and there were 250 significantly different alternative splicing events, among which: skipped exon (SE) accounted for 65.2%, retained intron (RI) accounted for 11.6%, mutually exclusive exon (MXE) accounted for 9.2%. alternative 3' splice site (A3SS) accounted for 8% and alternative 5' splice site (A5SS) for 6% (Figure 3F). GO analysis was performed on 250 differential alternative splicing events. The differentially spliced genes were mainly related to muscle contraction, muscle filament sliding, regulation of phagocytosis, sarcomere organization, chromatin organization, cardiac muscle contraction, ventricular cardiac muscle tissue morphogenesis, endocytosis, and actin cytoskeleton organization (Figure 3G). These data suggest that heart-specific YTHDC1 deletion dramatically alters gene expression profiles in the heart.

Cardiac-specific deletion of Ythdc1 significantly alters chromatin accessibility in the heart

Previous reports have indicated that several RBPs can participate in transcriptional regulation and influence chromatin through direct or indirect mechanisms[16, 28-30]. To further investigate whether YTHDC1 regulates chromatin accessibility and gene transcription in the heart, the transposase accessible chromatin sequencing (ATAC-seq) technique was used to map the open chromatin in the heart of Ythdc1-CKO mice. As expected, significant enrichments of open chromatin were observed near gene promoters and transcription start sites (TSS) within ATAC-seq peaks (Figure 4A/B). Analysis of the ATAC-seq data revealed that 2326 peak-related genes were up-regulated and 5566 peak-related genes were down-regulated (Figure 4C, Supplementary material online, Table S1). We identified several transcription factor (TF) binding motifs using the motif analysis software HOMER. The binding motifs of five transcription factors (NF1, Mef2a, Mef2b, Mef2c, and Mef2d) were switched off in Ythdc1-CKO mouse hearts (Figure 4D). These data suggest that YTHDC1 regulates chromatin accessibility in the heart. The following analysis of the ATAC-seq and RNA-seq data showed that 141 genes were down-regulated in both datasets (Figure 4E and Supplementary material online, Table S2). The KEGG analysis revealed the down-regulation of genes associated with adrenergic signaling in cardiomyocytes, MAPK signaling pathway, cardiac muscle contraction, calcium signaling pathway, and glutamatergic synapse (Figure 4F).

Accurate regulation of the heart development is extremely significant. Abnormal heart development leads to several congenital heart diseases. Thus, elucidating the potential molecular mechanisms of heart development constitute urgent priorities. Previous studies have shown that proteins associated with m6A RNA modification play a key role in embryonic development regulation[11, 31, 32]. However, whether they regulate cardiac development remains unclear. In this study, we demonstrated for the first time that YTHDC1 is an essential element for cardiac development, and heart-specific deletion of YTHDC1 results in early postnatal mortality accompanied by the phenotype resembling DCM. The expression profile of WTAP and METTL3 in the mouse cerebellum decreases gradually with age[33]. It has been reported that the spatio-temporal expression profile of WTAP in brown adipose tissue is exactly the opposite, and the expression level gradually increases after birth[12]. However, the spatial and temporal expression profiles of the same m⁶A-modified writer protein are completely different in different organs during development. The temporal and spatial expression profile of the m⁶A reader protein YTHDC1 in the heart has not been studied. Our research has revealed that YTHDC1 is highly expressed during the embryonic period and two weeks after birth and then decreases with age during cardiac development. This expression profile indicates its crucial role in heart development. Therefore, we collected fetal hearts with heart disease from aborted fetuses and observed a significant decrease in the expression level of YTHDC1 in the heart tissue of a fetus with ventricular septal defect, aortic stenosis, and hypoplastic aortic arch (Fig. 2A). We used the Loxp-Cre knockout system to knockout YTHDC1 in the heart, and 90% of Ythdc1-CKO mice died within 20 days after birth due to severe heart failure. Thus, we have found for the first time that YTHDC1 is indeed critical to heart development.

Next, we sought to find the underlying molecular mechanism of YTHDC1 in the regulation of heart development. RNA-seq analysis revealed that deficiency of cardiac YTHDC1 significantly altered gene expression profiles. The differentially expressed genes were mainly related to biological processes such as myocardial contraction and transmembrane transport. Previous studies have indicated that YTHDC1 regulates alternative splicing[18, 27]. The alternative splicing events in RNA-seq data were analyzed using rMATS software. And 250 significantly different alternative splicing genes were found after Ythdc1 deletion. According to GO analysis, the 250 genes were mainly related to biological processes such as muscle contraction and filament sliding. It's worth noting that Mef2d is one of the mutually exclusive events of differential alternative splicing exons, while ATAC-Seq motif analysis indicated that the binding motif of Mef2d was turned off in Ythdc1-cKO mice. The combined analysis revealed that YTHDC1 may regulate the alternative splicing of transcription factor Mef2d and further influence the expression of the downstream target genes. Previous studies have demonstrated that the transcription factor Mef2d is an essential factor in cell signal transduction that regulates the differentiation of various cell types, and it plays a vital role in the differentiation of myocardium and skeletal muscle[34–36]. Alternative splicing of Mef2d may lead to abnormal myocardial differentiation result in cardiac insufficiency during development. Nevertheless, the deletion of Mef2d in mice does not cause early postnatal heart failure and death[37]. Therefore, there may be other possible mechanisms leading to abnormal heart development in Ythdc1-CKO mice.

Studies have shown that a variety of RBPs can participate in transcriptional regulation and modify chromatin either directly or indirectly[16, 22, 28]. Oliver Nayler et al. found that YTHDC1 forms YT-bodies with transcription active sites in the form of phase separation and participates in transcriptional activation[16]. Liu J et al. revealed that YTHDC1 plays a crucial role in chromatin modification[22]. Consistently, our ATAC-seq motif enrichment analysis performed on Ythdc1flox/flox and Ythdc1-CKO mice heart tissue showed that the downstream target genes of transcription factors Mef2a, Mef2b, Mef2c, and Mef2d were significantly down-regulated in the Ythdc1-CKO group. Previous studies have shown that MEF2 plays a crucial role in the regulation of cardiac development[38, 39]. It has been reported that the Mef2a-null mice experienced right ventricular dilatation as well as sudden death during postnatal days 3–8[40]. Although the absence of Mef2b does not cause obvious defects in mice, MEF2B binds to the same DNA sequence as other members of the MEF2 family and acts as an effective trans-activator through this sequence[41]. Mef2c controls cardiac morphogenesis and myogenesis in mice, and its deletion results in embryonic lethality[42]. As previously described, Mef2d plays an important role in muscle differentiation[34–36]. Even though motif analysis demonstrated that the binding motifs of Mef2a, Mef2b, Mef2c, and Mef2d were significantly switched off after Ythdc1-CKO, RNA-seq revealed that the transcription level of Mef2a, Mef2b, Mef2c, and Mef2d did not change, except that Mef2d existed alternative splicing. It is possible that Ythdc1 has other mechanisms to regulate Mef2a, Mef2b, and Mef2c to influence heart development. Through the joint analysis of RNA-seq and ATAC-seq, 141 genes in both datasets were down-regulated, and KEGG enrichment analysis showed that down-regulated genes are mainly involved in the calcium signaling pathway, cardiac muscle contraction, and adrenergic signaling in cardiomyocytes. Disruption of these signaling pathways can seriously affect the function of the heart and may lead to early death after birth. In other words, YTHDC1 may regulate the transcription of genes related to the above-mentioned pathway, and affect cardiac function during heart development.

We discovered the temporal expression profile of YTHDC1 in the heart and identified its crucial function in heart development for the first time. YTHDC1 may regulate the alternative splicing of genes related to cardiac contraction and transmembrane transport. Alternatively, YTHDC1 may regulate transcription factors related to cardiac development and change the chromatin accessibility of its downstream target genes, resulting in abnormal cardiac development.

Animal experiments：

Mice were housed at the Center for Life Sciences, Harbin Institute of Technology. The use of animals was assessed and approved by the Animal Experimental Ethics Committee of the Harbin Institute of Technology (IACUC-2022066). All animal experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by the Institutional Animal Care and Use Committee. The authors complied with the ARRIVE guidelines.The Ythdc1 gene is flanked by two Loxp sites in exons 5-7. Ythdc1^flox/flox mice and Myh6-Cre^+/- mice were mated to produce cardiomyocyte specific Ythdc1 knockout (Ythdc1-CKO) mice. Mice were housed under stable conditions (temperature 22–25̊ C, relative humidity 50–70%, 12-h light/dark cycle) with unrestricted access to water and diet.

Human heart samples：

Fetal heart tissues were collected from aborted fetuses terminated pregnancies carried out at the First Hospital of Jilin University. After matching for gestational age, the tissues were divided into the congenital heart disease and normal heart development groups. This study was approved by the Research Ethics Committee of the First Hospital of Jilin University and complied with the detailed regulations on the management of Human Genetic Resources issued by the Ministry of Science and Technology of China. Individual consent was obtained following standard informed consent procedures. Studies conformed to the principles outlined in the Declaration of Helsinki regarding the use of human tissues.

Echocardiography:

Examinations were performed with the Vevo 3100 High Resolution Imaging System (VisualSonics, Toronto, Canada) to assess cardiac function on postnatal days 1 and 15, as described previously[43, 44]. Briefly, mice were placed in the supine position on a heating pad. Two-dimensional and M-mode echocardiography was used to assess wall motion, chamber dimensions, and cardiac function. Data were analyzed using Vevo LAB software (version 3.2.0).

Immunoblotting：

Heart tissue was homogenized in RIPA lysis buffer, and cells were harvested in RIPA lysis buffer. Tissue or cell extracts were immunoblotted with the indicated antibodies and visualized using ECL. Immunoblotting antibodies were as follows: YTHDC1 (77422S, CST, 1:2000 dilution); β-actin (60008-1-lg, Proteintech, 1:500 dilution) and α-tubulin (sc-5286, Santa Cruz, 1:500).

ATAC-Sequence：

Each heart sample was obtained from five Ythdc1-CKO and five Ythdc1^flox/flox mice at 15 days old. Nuclei were extracted from heart samples，and the nuclear pellet was resuspended in the Tn5 transposase reaction mixture. The transposition reaction was incubated at 37 ° C for 30 min, then, equimolar amounts of Adapter1 and Adapter2 were added. The libraries were amplified by PCR and purified with AMPure beads. The quality of library was assessed by Qubit. Index-coded samples were clustered on the cBot cluster generation system using TruSeq PE Clustering Toolkit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, library preparation was sequenced on an Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads. ATAC-seq analysis was performed using standard protocols[45, 46].

RNA-Sequence：

Total RNA was extracted from the heart of Ythdc1^flox/floxand Ythdc1-CKO mice at 15 days old using Tripure Isolation Reagent (94015120, Roche). RNA-sequence was performed by using Illumina NovaSeq 6000 platform. Paired-end clean reads were aligned to the mouse reference genome (Ensemble_GRCm38.90) with TopHat (version 2.0.12), and the aligned reads were used to quantify mRNA expression by using HTSeq-count (version 0.6.1). Alternative splicing was analyzed by rMATS software (version 3.2.5). False Discovery Rate (FDR)<0.05 was considered statistically significant.

Transmission Electron Microscope (TEM)：

Careful selection of fresh tissue is performed to minimize mechanical damage, ensuring that the tissue size does not exceed 1mm × 1mm × 1mm. The tissue is promptly fixed in an electron microscope fixative at a temperature of 4 ℃ for 2-4 hours. Subsequently, the tissue is rinsed three times with 0.1M phosphate buffer PB (PH7.4), with each rinse lasting 15 minutes. Next, the tissue undergoes post-fixation by immersing it in a solution of 1% osmic acid and 0.1M phosphate buffer PB (PH7.4) for 2 hours at room temperature (20°C). It is then rinsed three times with 0.1M phosphate buffer PB (PH7.4) for 15 minutes each. The tissue is then dehydrated through a sequential process using alcohol and acetone solutions of increasing concentration. Each step lasts for 15 minutes, starting from 50% alcohol and progressing to 70%, 80%, 90%, 95%, and finally 100% alcohol. This is followed by 100% acetone and another round of 100% acetone. After dehydration, the tissue is infiltrated by incubating it in a mixture of acetone and 812 embedding agent in a ratio of 1:1 for 2-4 hours. It is then transferred to a mixture of acetone and 812 embedding agent in a ratio of 1:2 and left overnight. Finally, the tissue is incubated in pure 812 embedding agent for 5-8 hours. The sample is placed in an embedding plate containing pure 812 embedding agent and polymerized overnight in a 37 ℃ oven. Once the tissue block is polymerized, it is subjected to embedding by placing it in a 60 ℃ oven for 48 hours. Ultra-thin sections with a thickness ranging from 60-80nm are obtained by using an ultra-thin sectioning machine. The sections are then stained using uranium-lead double staining. Each staining agent, 2% uranyl acetate saturated alcoholic solution and lead citrate, is applied for 15 minutes. The stained sections are left to dry at room temperature overnight. Finally, the prepared sections are ready for observation and analysis using a Transmission Electron Microscope (TEM).

Statistical analysis:

Data were presented as mean ± SEM. Differences between groups were analyzed using two-tailed Student's t-tests. All statistical analyses were conducted using GraphPad Prism version 8.0.1 (GraphPad Software Inc., San Diego, CA, USA). P values＜0.05 were considered statistically significant.

Data availability: The data that support the findings of this study are available within the article and its supplementary materials files. The RNA-seq and ATAC-seq data of this study have been deposited in the Gene Expression Omnibus database with accession number GSE241272 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241272, The security token used for censorship purposes is srshsagezbqbjkn).

Author Contributions: Conceptualization, Zhiguo Zhang; methodology, Zhiguo zhang, Lei Shi; software, Lei Shi, Quanwei Wang; validation, Wei Shi, Cong Qin; formal analysis, Meiwei Zhang, Ying Yang; investigation, Dongpu Shao; writing, Lei Shi, Mengling Peng; All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: All animal studies carried out at the at the Center for Life Sciences, Harbin Institute of Technology. The use of animals was assessed and approved by the Animal Experimental Ethics Committee of the Harbin Institute of Technology (IACUC-2022066).

Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Acknowledgments: We acknowledge Novogene for assistance in ATAC-seq, RNA-seq experiments.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Zhang, M.; Zhang, Y.; Xu, E.; Mohibi, S.; de Anda, D.M.; Jiang, Y.; Zhang, J.; Chen, X. Rbm24, a target of p53, is necessary for proper expression of p53 and heart development. Cell Death Differ 2018, 25, 1118-1130, doi:10.1038/s41418-017-0029-8.
Poon, K.L.; Tan, K.T.; Wei, Y.Y.; Ng, C.P.; Colman, A.; Korzh, V.; Xu, X.Q. RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility. Cardiovasc Res 2012, 94, 418-427, doi:10.1093/cvr/cvs095.
Guo, W.; Schafer, S.; Greaser, M.L.; Radke, M.H.; Liss, M.; Govindarajan, T.; Maatz, H.; Schulz, H.; Li, S.; Parrish, A.M.; et al. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med 2012, 18, 766-773, doi:10.1038/nm.2693.
Brauch, K.M.; Karst, M.L.; Herron, K.J.; de Andrade, M.; Pellikka, P.A.; Rodeheffer, R.J.; Michels, V.V.; Olson, T.M. Mutations in ribonucleic acid binding protein gene cause familial dilated cardiomyopathy. J Am Coll Cardiol 2009, 54, 930-941, doi:10.1016/j.jacc.2009.05.038.
Li, D.; Morales, A.; Gonzalez-Quintana, J.; Norton, N.; Siegfried, J.D.; Hofmeyer, M.; Hershberger, R.E. Identification of novel mutations in RBM20 in patients with dilated cardiomyopathy. Clin Transl Sci 2010, 3, 90-97, doi:10.1111/j.1752-8062.2010.00198.x.
Cantara, W.A.; Crain, P.F.; Rozenski, J.; McCloskey, J.A.; Harris, K.A.; Zhang, X.; Vendeix, F.A.; Fabris, D.; Agris, P.F. The RNA Modification Database, RNAMDB: 2011 update. Nucleic Acids Res 2011, 39, D195-201, doi:10.1093/nar/gkq1028.
Mathiyalagan, P.; Adamiak, M.; Mayourian, J.; Sassi, Y.; Liang, Y.; Agarwal, N.; Jha, D.; Zhang, S.; Kohlbrenner, E.; Chepurko, E.; et al. FTO-Dependent N(6)-Methyladenosine Regulates Cardiac Function During Remodeling and Repair. Circulation 2019, 139, 518-532, doi:10.1161/CIRCULATIONAHA.118.033794.
Dorn, L.E.; Lasman, L.; Chen, J.; Xu, X.; Hund, T.J.; Medvedovic, M.; Hanna, J.H.; van Berlo, J.H.; Accornero, F. The N(6)-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy. Circulation 2019, 139, 533-545, doi:10.1161/CIRCULATIONAHA.118.036146.
Wang, L.; Wang, J.; Yu, P.; Feng, J.; Xu, G.E.; Zhao, X.; Wang, T.; Lehmann, H.I.; Li, G.; Sluijter, J.; et al. METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury. Nat Commun 2022, 13, 6762, doi:10.1038/s41467-022-34434-y.
Chien, C.S.; Li, J.Y.; Chien, Y.; Wang, M.L.; Yarmishyn, A.A.; Tsai, P.H.; Juan, C.C.; Nguyen, P.; Cheng, H.M.; Huo, T.I.; et al. METTL3-dependent N(6)-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium. Proc Natl Acad Sci U S A 2021, 118, e2025070118, doi:10.1073/pnas.2025070118.
Wang, Y.; Gao, M.; Zhu, F.; Li, X.; Yang, Y.; Yan, Q.; Jia, L.; Xie, L.; Chen, Z. METTL3 is essential for postnatal development of brown adipose tissue and energy expenditure in mice. Nat Commun 2020, 11, 1648, doi:10.1038/s41467-020-15488-2.
Wang Y, L.X.; Liu C, Z.L.; Shi L, Z.Z.; Chen L, G.M.; Gao L, X.Y.; Huang H, L.J.; Chen, Z. WTAP Regulates
Postnatal Development of Brown Adipose Tissue by stabilizing METTL3 in mice. Life Metabolism 2022. ,
Wang, S.; Chen, S.; Sun, J.; Han, P.; Xu, B.; Li, X.; Zhong, Y.; Xu, Z.; Zhang, P.; Mi, P.; et al. m(6)A modification-tuned sphingolipid metabolism regulates postnatal liver development in male mice. Nat Metab 2023, 5, 842-860, doi:10.1038/s42255-023-00808-9.
Wang, C.X.; Cui, G.S.; Liu, X.; Xu, K.; Wang, M.; Zhang, X.X.; Jiang, L.Y.; Li, A.; Yang, Y.; Lai, W.Y.; et al. METTL3-mediated m6A modification is required for cerebellar development. PLoS Biol 2018, 16, e2004880, doi:10.1371/journal.pbio.2004880.
Batista, P.J.; Molinie, B.; Wang, J.; Qu, K.; Zhang, J.; Li, L.; Bouley, D.M.; Lujan, E.; Haddad, B.; Daneshvar, K.; et al. m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 2014, 15, 707-719, doi:10.1016/j.stem.2014.09.019.
Nayler, O.; Hartmann, A.M.; Stamm, S. The ER repeat protein YT521-B localizes to a novel subnuclear compartment. J Cell Biol 2000, 150, 949-962, doi:10.1083/jcb.150.5.949.
Zhang, Z.; Theler, D.; Kaminska, K.H.; Hiller, M.; de la Grange, P.; Pudimat, R.; Rafalska, I.; Heinrich, B.; Bujnicki, J.M.; Allain, F.H.; et al. The YTH domain is a novel RNA binding domain. J Biol Chem 2010, 285, 14701-14710, doi:10.1074/jbc.M110.104711.
Xiao, W.; Adhikari, S.; Dahal, U.; Chen, Y.S.; Hao, Y.J.; Sun, B.F.; Sun, H.Y.; Li, A.; Ping, X.L.; Lai, W.Y.; et al. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell 2016, 61, 507-519, doi:10.1016/j.molcel.2016.01.012.
Patil, D.P.; Chen, C.K.; Pickering, B.F.; Chow, A.; Jackson, C.; Guttman, M.; Jaffrey, S.R. m(6)A RNA methylation promotes XIST-mediated transcriptional repression. Nature 2016, 537, 369-373, doi:10.1038/nature19342.
Roundtree, I.A.; Luo, G.Z.; Zhang, Z.; Wang, X.; Zhou, T.; Cui, Y.; Sha, J.; Huang, X.; Guerrero, L.; Xie, P.; et al. YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife 2017, 6, e31311, doi:10.7554/eLife.31311.
Huang, H.; Weng, H.; Chen, J. The Biogenesis and Precise Control of RNA m(6)A Methylation. Trends Genet 2020, 36, 44-52, doi:10.1016/j.tig.2019.10.011.
Liu, J.; Gao, M.; He, J.; Wu, K.; Lin, S.; Jin, L.; Chen, Y.; Liu, H.; Shi, J.; Wang, X.; et al. The RNA m(6)A reader YTHDC1 silences retrotransposons and guards ES cell identity. Nature 2021, 591, 322-326, doi:10.1038/s41586-021-03313-9.
Gao, S.; Sun, H.; Chen, K.; Gu, X.; Chen, H.; Jiang, L.; Chen, L.; Zhang, S.; Liu, Y.; Shi, D.; et al. Depletion of m(6) A reader protein YTHDC1 induces dilated cardiomyopathy by abnormal splicing of Titin. J Cell Mol Med 2021, 25, 10879-10891, doi:10.1111/jcmm.16955.
Li, Y.; Zhang, W.; Dai, Y.; Chen, K. Identification and verification of IGFBP3 and YTHDC1 as biomarkers associated with immune infiltration and mitophagy in hypertrophic cardiomyopathy. Front Genet 2022, 13, 986995, doi:10.3389/fgene.2022.986995.
Zhang, Z.; Wang, Q.; Zhao, X.; Shao, L.; Liu, G.; Zheng, X.; Xie, L.; Zhang, Y.; Sun, C.; Xu, R. YTHDC1 mitigates ischemic stroke by promoting Akt phosphorylation through destabilizing PTEN mRNA. Cell Death Dis 2020, 11, 977, doi:10.1038/s41419-020-03186-2.
Xie, W.; Zhang, A.; Huang, X.; Zhou, H.; Ying, H.; Ye, C.; Ren, M.; Qian, M.; Liu, X.; Mo, Y. SILENCING M 6 A READER YTHDC1 REDUCES INFLAMMATORY RESPONSE IN SEPSIS-INDUCED CARDIOMYOPATHY BY INHIBITING SERPINA3N EXPRESSION. Shock 2023, 59, 791-802, doi:10.1097/SHK.0000000000002106.
Kasowitz, S.D.; Ma, J.; Anderson, S.J.; Leu, N.A.; Xu, Y.; Gregory, B.D.; Schultz, R.M.; Wang, P.J. Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development. PLoS Genet 2018, 14, e1007412, doi:10.1371/journal.pgen.1007412.
Li, X.; Ding, K.; Li, X.; Yuan, B.; Wang, Y.; Yao, Z.; Wang, S.; Huang, H.; Xu, B.; Xie, L.; et al. Deficiency of WTAP in hepatocytes induces lipoatrophy and non-alcoholic steatohepatitis (NASH). Nat Commun 2022, 13, 4549, doi:10.1038/s41467-022-32163-w.
Van Nostrand, E.L.; Freese, P.; Pratt, G.A.; Wang, X.; Wei, X.; Xiao, R.; Blue, S.M.; Chen, J.Y.; Cody, N.; Dominguez, D.; et al. A large-scale binding and functional map of human RNA-binding proteins. Nature 2020, 583, 711-719, doi:10.1038/s41586-020-2077-3.
Xiao, R.; Chen, J.Y.; Liang, Z.; Luo, D.; Chen, G.; Lu, Z.J.; Chen, Y.; Zhou, B.; Li, H.; Du, X.; et al. Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription. Cell 2019, 178, 107-121.e18, doi:10.1016/j.cell.2019.06.001.
Fukusumi, Y.; Naruse, C.; Asano, M. Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo. Dev Dyn 2008, 237, 618-629, doi:10.1002/dvdy.21444.
Wang, Y.; Li, X.; Liu, C.; Zhou, L.; Shi, L.; Zhang, Z.; Chen, L.; Gao, M.; Gao, L.; Xu, Y.; et al. WTAP regulates postnatal development of brown adipose tissue by stabilizing METTL3 in mice. Life Metabolism 2022, 1, 270-284, doi:10.1093/lifemeta/loac028.
Ma, C.; Chang, M.; Lv, H.; Zhang, Z.W.; Zhang, W.; He, X.; Wu, G.; Zhao, S.; Zhang, Y.; Wang, D.; et al. RNA m(6)A methylation participates in regulation of postnatal development of the mouse cerebellum. Genome Biol 2018, 19, 68, doi:10.1186/s13059-018-1435-z.
Sebastian, S.; Faralli, H.; Yao, Z.; Rakopoulos, P.; Palii, C.; Cao, Y.; Singh, K.; Liu, Q.C.; Chu, A.; Aziz, A.; et al. Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation. Genes Dev 2013, 27, 1247-1259, doi:10.1101/gad.215400.113.
Breitbart, R.E.; Liang, C.S.; Smoot, L.B.; Laheru, D.A.; Mahdavi, V.; Nadal-Ginard, B. A fourth human MEF2 transcription factor, hMEF2D, is an early marker of the myogenic lineage. Development 1993, 118, 1095-1106, doi:10.1242/dev.118.4.1095.
Lilly, B.; Zhao, B.; Ranganayakulu, G.; Paterson, B.M.; Schulz, R.A.; Olson, E.N. Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila. Science 1995, 267, 688-693, doi:10.1126/science.7839146.
Kim, Y.; Phan, D.; van Rooij, E.; Wang, D.; McAnally, J.; Qi, X.; Richardson, J.A.; Hill, J.A.; Bassel-Duby, R.; Olson, E.N. The MEF2D transcription factor mediates stress-dependent cardiac remodeling in mice. JOURNAL OF CLINICAL INVESTIGATION 2008, 118, 124-132, doi:10.1172/JCI33255.
Black BL, Cripps RM. Chapter 9.5 - Myocyte Enhancer Factor 2 Transcription Factors in Heart Development and Disease. 2010 : 673-699. 2010 ,
The Function of the MEF2 Family of Transcription Factors in Cardiac Development, C.; Reprogramming, a.D. Desjardins CA, Naya FJ. Journal of Cardiovascular Development and Disease 2016 ,
Naya, F.J.; Black, B.L.; Wu, H.; Bassel-Duby, R.; Richardson, J.A.; Hill, J.A.; Olson, E.N. Mitochondrial deficiency and cardiac sudden death in mice lacking the MEF2A transcription factor. Nat Med 2002, 8, 1303-1309, doi:10.1038/nm789.
Molkentin, J.D.; Firulli, A.B.; Black, B.L.; Martin, J.F.; Hustad, C.M.; Copeland, N.; Jenkins, N.; Lyons, G.; Olson, E.N. MEF2B is a potent transactivator expressed in early myogenic lineages. Mol Cell Biol 1996, 16, 3814-3824, doi:10.1128/MCB.16.7.3814.
Lin, Q.; Schwarz, J.; Bucana, C.; Olson, E.N. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science 1997, 276, 1404-1407, doi:10.1126/science.276.5317.1404.
Liu, X.; Kim, A.J.; Reynolds, W.; Wu, Y.; Lo, C.W. Phenotyping cardiac and structural birth defects in fetal and newborn mice. Birth Defects Res 2017, 109, 778-790, doi:10.1002/bdr2.1048.
Le, V.P.; Wagenseil, J.E. Echocardiographic Characterization of Postnatal Development in Mice with Reduced Arterial Elasticity. Cardiovasc Eng Technol 2012, 3, 424-438, doi:10.1007/s13239-012-0108-4.
Buenrostro, J.D.; Giresi, P.G.; Zaba, L.C.; Chang, H.Y.; Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 2013, 10, 1213-1218, doi:10.1038/nmeth.2688.
Corces, M.R.; Trevino, A.E.; Hamilton, E.G.; Greenside, P.G.; Sinnott-Armstrong, N.A.; Vesuna, S.; Satpathy, A.T.; Rubin, A.J.; Montine, K.S.; Wu, B.; et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 2017, 14, 959-962, doi:10.1038/nmeth.4396.

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YTHDC1 regulates the postnatal development of heart

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