YTHDC1 regulates the postnatal development of heart

YTHDC1 regulates the postnatal development of heart | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article YTHDC1 regulates the postnatal development of heart Lei Shi, Quanwei Wang, Meiwei Zhang, Cong Qin, Dongpu Shao, Mengling Peng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3301770/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to investigate the role of the N6-methyladenosine (m 6 A) 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction 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 (m 6 A) modification is the most common mRNA modification in eukaryotes[ 6 ]. m 6 A modification related proteins play a significant role in several cardiac diseases[ 7 – 10 ]. Although previous studies have demonstrated that m 6 A 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 m 6 A-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 m 6 A 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 m 6 A 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. 2. Results 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/flox mice, 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/flox mice 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). 3. Discussion 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 m 6 A-modified writer protein are completely different in different organs during development. The temporal and spatial expression profile of the m 6 A 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. 2 A). 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 Ythdc1 flox/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. 4. Conclusions 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. 5. Materials and Methods 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/flox and 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. Declarations 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. References 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3301770","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":230067628,"identity":"be90cc4d-09c6-4be0-8fad-de3fd4708e0b","order_by":0,"name":"Lei Shi","email":"","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Shi","suffix":""},{"id":230067629,"identity":"cb4d2b59-781f-47e3-ae0e-1320c58af041","order_by":1,"name":"Quanwei Wang","email":"","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Quanwei","middleName":"","lastName":"Wang","suffix":""},{"id":230067630,"identity":"aee1630f-cad8-46fe-a568-c14e8af52fb1","order_by":2,"name":"Meiwei Zhang","email":"","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Meiwei","middleName":"","lastName":"Zhang","suffix":""},{"id":230067631,"identity":"04ca7d33-8031-4f50-bf56-3f0a84d3fac1","order_by":3,"name":"Cong Qin","email":"","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Qin","suffix":""},{"id":230067632,"identity":"1978e02b-27c0-489d-9dfc-f174f94e4a65","order_by":4,"name":"Dongpu Shao","email":"","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongpu","middleName":"","lastName":"Shao","suffix":""},{"id":230067633,"identity":"9474b9f2-0097-4c37-aedf-c5a7fe4d9c6f","order_by":5,"name":"Mengling Peng","email":"","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengling","middleName":"","lastName":"Peng","suffix":""},{"id":230067634,"identity":"b2f3c301-8538-485d-bfb3-a6f34c08728f","order_by":6,"name":"Ying Yang","email":"","orcid":"","institution":"Harbin Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Yang","suffix":""},{"id":230067635,"identity":"386f6418-a479-41a6-9f11-e4d1cc239c75","order_by":7,"name":"Wei Shi","email":"","orcid":"","institution":"Sunshine Union Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Shi","suffix":""},{"id":230067636,"identity":"e329ac7f-c577-423e-8af4-140ef6d90187","order_by":8,"name":"Zhiguo Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie3NsYrCQBCA4Q2B2MwDjETZV1gJJI3oc9htELSxFsuAhd3VJ8lDpLIeWdDm3uAsYpPKwuI4LATd9QE2ue7A/YthFuZjGXO5/mP4mgSsk6WkNy9rT4Dk34heJGtHeL6uzz/3U49PqkoBG/ZL8uvKRrzikEQ9qGHwLaUms6ikIBE24qOMQ0QFg/xFVFoSBGgjAc5/QxSabMmQRzMBXMTdqz7myAyhZoK4WIaMFAiQcl+IabRVQWwl/HO+697uasw3X2l1WY36H8d1bSUmH/QQBFJP82y613k3813WoRa3LpfL9Y49AUgMS4N+DHITAAAAAElFTkSuQmCC","orcid":"","institution":"the first hospital of Jilin University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-08-28 04:14:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3301770/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3301770/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42677677,"identity":"fe69bcf7-f936-47d3-a1d7-5d3905879858","added_by":"auto","created_at":"2023-09-06 01:11:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1108421,"visible":true,"origin":"","legend":"\u003cp\u003eYTHDC1 expression is highest in embryonic and early postnatal stages and gradually decreases with age. \u003cstrong\u003e(A-B)\u003c/strong\u003e: Temporal expression profiles of \u003cem\u003eYthdc1\u003c/em\u003e in left ventricle and right ventricle myocardial tissues by RNA-seq analysis of public database data. (GSE51483, n=3). \u003cstrong\u003e(C)\u003c/strong\u003e. Western blot was used to detect the expression of YTHDC1 protein in the heart of mice at different developmental stages. \u003cstrong\u003e(D)\u003c/strong\u003e. Bar graph for statistical analysis of YTHDC1 protein expression levels in mouse hearts at different developmental times detected by western assay. The expression level of YTHDC1 at prenatal 14 days was set as the baseline (normalized to 1), while the expression levels at other ages were compared to it. (n=3). *, p\u0026lt; 0.05. **, p\u0026lt; 0.01. ns, no significance. Data represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3301770/v1/d44482d0d54db45a49b6eab9.jpg"},{"id":42677678,"identity":"584ae762-0782-41f1-bf8c-785dc1496a90","added_by":"auto","created_at":"2023-09-06 01:11:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2359627,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac-specific deletion of \u003cem\u003eYthdc1\u003c/em\u003e causes abnormal heart development.\u003cstrong\u003e (A)\u003c/strong\u003e. YTHDC1 expression is decreased in the heart tissue of a miscarried fetus with ventricular septal defect, aortic stenosis, and aortic arch hypoplasia (CHD), in comparison to the heart tissue of a miscarried fetus of similar age with normal heart development (CON). \u003cstrong\u003e(B)\u003c/strong\u003e. Schematic of cardiomyocyte-specific \u003cem\u003eYthdc1\u003c/em\u003e knockout mouse model. \u003cstrong\u003e(C)\u003c/strong\u003e. Typical Western blot analysis of YTHDC1 expression in \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox \u003c/sup\u003eand \u003cem\u003eYthdc1-\u003c/em\u003eCKO mice. (n=4). \u003cstrong\u003e(D)\u003c/strong\u003e. YTHDC1 and Tubulin protein levels were measured using Western blot analysis in the heart, skeletal muscle, liver, kidney, brain, and lung of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox \u003c/sup\u003eand \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 15 days of age. \u003cstrong\u003e(E)\u003c/strong\u003e. Survival curve of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice during postnatal development. \u003cstrong\u003e(F)\u003c/strong\u003e. Representative pictures of male \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox \u003c/sup\u003eand \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 15 days old.\u003cstrong\u003e (G)\u003c/strong\u003e. Body weight, heart weight, and the ratio of heart weight to body weight for \u003cem\u003eYthdc1\u003c/em\u003e-CKO (n=10) and \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e (n=10) mice at 15 days old.\u003cstrong\u003e (H)\u003c/strong\u003e. Representative H\u0026amp;E staining of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eYthdc1\u003c/em\u003e-CKO heart at 15 days old (scale bar: 1mm).\u003cstrong\u003e (I)\u003c/strong\u003e. Representative transmission electron microscopy (TEM) of sarcomere structure from \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 15 days old (scale bar: 1μm). TEM reveals partial disorganization of muscle fibers, with certain Z-lines fractured and a minor absence of H-band. Additionally, certain mitochondria exhibit vacuolar degeneration (▲).\u003cstrong\u003e (J).\u003c/strong\u003e Representative echocardiographic images of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e and \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 1 day old.\u003cstrong\u003e (K).\u003c/strong\u003e Representative echocardiographic images of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003eand \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 15 days old.\u003cstrong\u003e (L).\u003c/strong\u003e Echocardiographic quantification of left ventricular end-diastolic diameter (LVID, d), interventricular septum end-diastolic diameter (IVS, d), ejection fraction (EF) and fractional shortening (FS) from \u003cem\u003eYthdc1\u003c/em\u003e-CKO and \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003emice at 1 day and 15 days old (n=5). *, p\u0026lt; 0.05. **, p\u0026lt; 0.01. Data represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3301770/v1/f3dfc1aa94ce6115f4377feb.jpg"},{"id":42677675,"identity":"b2a86701-7b0d-498b-89ab-3fac505ad5cb","added_by":"auto","created_at":"2023-09-06 01:11:57","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2290972,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac-specific deletion of \u003cem\u003eYthdc1\u003c/em\u003e largely changes gene expression profile in the heart. \u003cstrong\u003e(A) \u003c/strong\u003eRNA-seq analysis was performed in hearts from \u003cem\u003eYthdc1-\u003c/em\u003eCKO and \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice at 15 days of age (n=3 independent mice). The differentially expressed genes (DEGs) (CKO VS flox/flox) including 437 downregulated genes and 599 upregulated genes were illustrated in a volcanoplot (|log2 FoldChange|\u0026gt;1, Padj\u0026lt; 0.05).\u003cstrong\u003e (B)\u003c/strong\u003e. Top GO biological process terms enriched in downregulated genes.\u003cstrong\u003e (C)\u003c/strong\u003e. Top GO biological process terms enriched in upregulated genes. \u003cstrong\u003e(D)\u003c/strong\u003e. KEGG analysis of the downregulated gene.\u003cstrong\u003e (E)\u003c/strong\u003e. KEGG analysis of the upregulated gene.\u003cstrong\u003e (F)\u003c/strong\u003e. Pie chart showing the distribution of different types of AS events.\u003cstrong\u003e (G)\u003c/strong\u003e. GO analysis of genes that were alternatively spliced in response to\u003cem\u003e Ythdc1\u003c/em\u003e-CKO.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3301770/v1/f7f9f99815998be96e3ffe8a.jpg"},{"id":42677676,"identity":"40687628-93c4-41e0-81ea-9186342949a8","added_by":"auto","created_at":"2023-09-06 01:11:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1585552,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac-specific deletion of \u003cem\u003eYthdc1\u003c/em\u003e significantly alters chromatin accessibility in the heart. \u003cstrong\u003e(A)\u003c/strong\u003e. Reads heatmap around TSS. \u003cstrong\u003e(B).\u003c/strong\u003e Reads profile around TSS. \u003cstrong\u003e(C)\u003c/strong\u003e. ATAC-seq analysis was performed in the hearts of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 15 days old. The differentially peak-related genes (CKO VS flox/flox) including 5566 downregulated peak-related genes and 2326 upregulated peak-related genes were illustrated in a heatmap. \u003cstrong\u003e(D)\u003c/strong\u003e. Significantly differential transcription factor (TF) binding motifs were identified by analyzing ATAC-seq data collected from hearts of \u003cem\u003eYthdc1\u003c/em\u003e-CKO and \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e mice at 15 days old. \u003cstrong\u003e(E).\u003c/strong\u003e ATAC-seq and RNA-seq were performed in the hearts of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003eand \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice at 15 days old. Combined analysis of the downregulated genes in both ATAC-seq and RNA-seq identified 141 downregulated genes in \u003cem\u003eYthdc1\u003c/em\u003e-CKO hearts, which were illustrated in a Venn diagram plot. \u003cstrong\u003e(F)\u003c/strong\u003e. KEGG analysis of the 141 downregulated genes.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3301770/v1/56a7121fcdc28b59158eaf63.jpg"},{"id":43921393,"identity":"f82d252d-ba2d-47c0-8ebf-84e9cc5a9e47","added_by":"auto","created_at":"2023-09-30 06:52:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1095411,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3301770/v1/a7a90bb0-c72e-4477-85db-53a4932bce8c.pdf"},{"id":42677679,"identity":"d80a5c0d-4356-4776-9910-4c2ab628ab49","added_by":"auto","created_at":"2023-09-06 01:11:57","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":173101,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-3301770/v1/a61fafe5dbf7ec11bcec23e5.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"YTHDC1 regulates the postnatal development of heart","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eEmbryonic 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[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and RBM20[\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], can lead to congenital cardiomyopathy. The N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) modification is the most common mRNA modification in eukaryotes[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. m\u003csup\u003e6\u003c/sup\u003eA modification related proteins play a significant role in several cardiac diseases[\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although previous studies have demonstrated that m\u003csup\u003e6\u003c/sup\u003eA modification related proteins play an essential role in regulating the development of brown adipose tissue[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], liver[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and nervous system[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], as well as the differentiation of embryonic stem cells[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], but their role in heart development is still largely unexplored.\u003c/p\u003e \u003cp\u003eYTHDC1 belongs to the m\u003csup\u003e6\u003c/sup\u003eA-modified direct reader family and is the only nuclear-located reader protein[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It is a member of the YTH (YT521-B homology) protein family, which contains 100\u0026ndash;150 amino acids and is a highly conserved protein[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. YTHDC1 is an important RBP that plays a critical regulatory role in many biological processes. Its primary function is to mediate mRNA splicing[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], epigenetic silencing mediated by the non-coding RNA XIST[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and nuclear export of mRNA[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The deposition of m\u003csup\u003e6\u003c/sup\u003eA occurs co-transcriptionally[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], but recent studies have found that YTHDC1 directly affects transcription itself[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. YTHDC1 forms YT-bodies with transcriptional active sites in a phase-separated form to regulate transcription and participate in transcriptional activation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, YTHDC1 plays a vital role in a variety of cardiac and cerebrovascular diseases, and YTHDC1 deficiency is associated with DCM[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been reported that YTHDC1 may also be involved in mitophagy and energy metabolism in HCM[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, YTHDC1 overexpression can alleviate ischemic stroke[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, inhibition of YTHDC1 can reduce inflammation and improve cardiac function in sepsis-induced cardiomyopathy[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 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[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, its regulatory role in heart development has yet to be investigated.\u003c/p\u003e \u003cp\u003eIn this study, we found that the m\u003csup\u003e6\u003c/sup\u003eA 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003eYTHDC1 expression is highest in embryonic and early postnatal stages and gradually decreases with age\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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 \u003cem\u003eYthdc1\u003c/em\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCardiac-specific deletion of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eYthdc1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;causes abnormal heart development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 \u003cem\u003eYthdc1\u003c/em\u003e knockout mice (\u003cem\u003eYthdc1\u003c/em\u003e-CKO) by inserting \u003cem\u003eloxp\u0026nbsp;\u003c/em\u003esites at both ends of \u003cem\u003eYthdc1\u003c/em\u003e exons 5-7 and breeding them with \u003cem\u003eMyh6-Cre\u003c/em\u003e\u003csup\u003e+/-\u0026nbsp;\u003c/sup\u003emice (Figure 2B). The results of the Western blot analysis have confirmed that \u003cem\u003eYthdc1\u003c/em\u003e exhibited a specific knockdown of approximately 50% in the heart (Figure 2C). We collected liver, muscle, kidney, brain and lung tissues from \u003cem\u003eYthdc1\u003c/em\u003e-CKO and \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003emice, 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 \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice were born without any problems and followed the expected Mendelian inheritance patterns. However, 90% of \u003cem\u003eYthdc1\u003c/em\u003e-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\u003cem\u003e\u0026nbsp;Ythdc1-\u003c/em\u003eCKO 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 \u003cem\u003eYthdc1\u003c/em\u003e-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\u0026amp;E staining and electron microscopy. H\u0026amp;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 \u003cem\u003eYthdc1-\u003c/em\u003eCKO and \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003emice 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 \u003cem\u003eYthdc1\u003c/em\u003e-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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCardiac-specific deletion of Ythdc1 largely changes gene expression profile in the heart\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 \u003cem\u003eYthdc1\u003c/em\u003e-CKO and\u003cem\u003e\u0026nbsp;Ythdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e 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 \u003cem\u003eYthdc1\u003c/em\u003e-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-\u0026beta; signaling pathway, HCM, and p53 signaling (Figure 3E). Previous studies have shown that \u003cem\u003eYthdc1\u003c/em\u003e 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\u0026apos; splice site (A3SS) accounted for 8% and alternative 5\u0026apos; 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCardiac-specific deletion of Ythdc1 significantly alters chromatin accessibility in the heart\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious 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 \u003cem\u003eYthdc1\u003c/em\u003e-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 \u003cem\u003eYthdc1\u003c/em\u003e-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).\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAccurate 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[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. 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[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the spatial and temporal expression profiles of the same m\u003csup\u003e6\u003c/sup\u003eA-modified writer protein are completely different in different organs during development. The temporal and spatial expression profile of the m\u003csup\u003e6\u003c/sup\u003eA 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We used the Loxp-Cre knockout system to knockout YTHDC1 in the heart, and 90% of \u003cem\u003eYthdc1\u003c/em\u003e-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.\u003c/p\u003e \u003cp\u003eNext, 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[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The alternative splicing events in RNA-seq data were analyzed using rMATS software. And 250 significantly different alternative splicing genes were found after \u003cem\u003eYthdc1\u003c/em\u003e 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 \u003cem\u003eMef2d\u003c/em\u003e 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 \u003cem\u003eYthdc1-\u003c/em\u003ecKO 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[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Alternative splicing of \u003cem\u003eMef2d\u003c/em\u003e may lead to abnormal myocardial differentiation result in cardiac insufficiency during development. Nevertheless, the deletion of \u003cem\u003eMef2d\u003c/em\u003e in mice does not cause early postnatal heart failure and death[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, there may be other possible mechanisms leading to abnormal heart development in \u003cem\u003eYthdc1-\u003c/em\u003eCKO mice.\u003c/p\u003e \u003cp\u003eStudies have shown that a variety of RBPs can participate in transcriptional regulation and modify chromatin either directly or indirectly[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Liu J et al. revealed that YTHDC1 plays a crucial role in chromatin modification[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consistently, our ATAC-seq motif enrichment analysis performed on \u003cem\u003eYthdc1\u003c/em\u003eflox/flox and \u003cem\u003eYthdc1\u003c/em\u003e-CKO mice heart tissue showed that the downstream target genes of transcription factors Mef2a, Mef2b, Mef2c, and Mef2d were significantly down-regulated in the \u003cem\u003eYthdc1\u003c/em\u003e-CKO group. Previous studies have shown that MEF2 plays a crucial role in the regulation of cardiac development[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It has been reported that the \u003cem\u003eMef2a\u003c/em\u003e-null mice experienced right ventricular dilatation as well as sudden death during postnatal days 3\u0026ndash;8[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Although the absence of \u003cem\u003eMef2b\u003c/em\u003e 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[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eMef2c\u003c/em\u003e controls cardiac morphogenesis and myogenesis in mice, and its deletion results in embryonic lethality[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As previously described, \u003cem\u003eMef2d\u003c/em\u003e plays an important role in muscle differentiation[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Even though motif analysis demonstrated that the binding motifs of Mef2a, Mef2b, Mef2c, and Mef2d were significantly switched off after \u003cem\u003eYthdc1\u003c/em\u003e-CKO, RNA-seq revealed that the transcription level of \u003cem\u003eMef2a, Mef2b, Mef2c\u003c/em\u003e, and \u003cem\u003eMef2d\u003c/em\u003e did not change, except that \u003cem\u003eMef2d\u003c/em\u003e existed alternative splicing. It is possible that \u003cem\u003eYthdc1\u003c/em\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe 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.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice 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\u0026nbsp;Ythdc1\u0026nbsp;gene is flanked by two Loxp sites in exons 5-7.\u0026nbsp;Ythdc1\u003csup\u003eflox/flox\u003c/sup\u003e mice and\u0026nbsp;Myh6-Cre\u003csup\u003e+/-\u003c/sup\u003e mice were mated to produce cardiomyocyte specific\u0026nbsp;Ythdc1\u0026nbsp;knockout (Ythdc1-CKO) mice. Mice were housed under stable conditions (temperature 22\u0026ndash;25̊\u0026nbsp;C, relative humidity 50\u0026ndash;70%, 12-h light/dark cycle) with unrestricted access to water and diet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHuman heart samples\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFetal 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEchocardiography:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExaminations were performed with the Vevo 3100 High Resolution Imaging System (VisualSonics, Toronto, Canada) to assess cardiac function on postnatal days 1 and 15,\u0026nbsp;as described previously[43, 44].\u0026nbsp;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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeart 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); \u0026beta;-actin (60008-1-lg, Proteintech, 1:500 dilution) and \u0026alpha;-tubulin (sc-5286, Santa Cruz, 1:500).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATAC-Sequence\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach heart sample was obtained from five \u003cem\u003eYthdc1\u003c/em\u003e-CKO and five\u003cem\u003e\u0026nbsp;Ythdc1\u003c/em\u003e\u003csup\u003eflox/flox\u003c/sup\u003e 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 \u0026deg; 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\u0026apos;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].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-Sequence\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the heart of \u003cem\u003eYthdc1\u003c/em\u003e\u003csup\u003eflox/flox\u0026nbsp;\u003c/sup\u003eand \u003cem\u003eYthdc1-\u003c/em\u003eCKO 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)\u0026lt;0.05 was considered statistically significant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransmission Electron Microscope (TEM)\u003c/em\u003e\u003cem\u003e：\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCareful selection of fresh tissue is performed to minimize mechanical damage, ensuring that the tissue size does not exceed 1mm \u0026times; 1mm \u0026times; 1mm. The tissue is promptly fixed in an electron microscope fixative at a temperature of 4\u0026nbsp;℃\u0026nbsp;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\u0026deg;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\u0026nbsp;℃\u0026nbsp;oven. Once the tissue block is polymerized, it is subjected to embedding by placing it in a 60\u0026nbsp;℃\u0026nbsp;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).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were presented as mean \u0026plusmn; SEM. Differences between groups were analyzed using two-tailed Student\u0026apos;s t-tests. All statistical analyses were conducted using GraphPad Prism version 8.0.1 (GraphPad Software Inc., San Diego, CA, USA). \u003cem\u003eP\u003c/em\u003e values＜0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe 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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eAll 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). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We acknowledge Novogene for assistance in ATAC-seq, RNA-seq experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang, M.; Zhang, Y.; Xu, E.; Mohibi, S.; de Anda, D.M.; Jiang, Y.; Zhang, J.; Chen, X. 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An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 959-962, doi:10.1038/nmeth.4396.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"YTHDC1, heart, development, dilated cardiomyopathy","lastPublishedDoi":"10.21203/rs.3.rs-3301770/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3301770/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to investigate the role of the N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) reader protein YTHDC1 in heart development and its potential molecular mechanisms. Animal experiments were conducted using cardiac-specific \u003cem\u003eYthdc1\u003c/em\u003e knockout (\u003cem\u003eYthdc1-\u003c/em\u003eCKO) 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 \u003cem\u003eYthdc1\u003c/em\u003e 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 \u003cem\u003eYthdc1-\u003c/em\u003eCKO 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.\u003c/p\u003e","manuscriptTitle":"YTHDC1 regulates the postnatal development of heart","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-06 01:11:52","doi":"10.21203/rs.3.rs-3301770/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc6999f5-bfcb-4565-a218-933542fb86d3","owner":[],"postedDate":"September 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":24366571,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":24366572,"name":"Biological sciences/Developmental biology"},{"id":24366573,"name":"Biological sciences/Genetics"},{"id":24366574,"name":"Health sciences/Cardiology"},{"id":24366575,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2023-09-30T06:44:24+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-06 01:11:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3301770","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3301770","identity":"rs-3301770","version":["v1"]},"buildId":"oE6Zbj460LM0Up2FdVbMZ","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}