Dynamic Patterns of N6-Methyladenosine Profiles of Messenger RNA Correlated with the Cardiomyocyte Regenerability during the Early Heart Development in Mice

N6-Methyladenosine (m6A) plays important roles in regulating mRNA processing. Despite rapid progress in this field, little is known about the role and mechanism of m6A modification in myocardial development and cardiomyocyte regeneration. Existing studies have shown that the heart tissues of newborn mice have the capability of proliferation and regeneration, but its mechanism, particularly its relation to m6A methylation, remains unknown. Methods. To systematically profile the mRNA m6A modification pattern in the heart tissues of mice at different developmental stages, we jointly performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) of heart tissues of mice, respectively, aged 1 day old, 7 days old, and 28 days old. Results. We identified the linkages and association between differentially expressed mRNA transcripts and hyper or hypomethylated m6A peaks in C57BL/6J mice at different heart developmental stages. Results showed that the amount of m6A peaks and the level of m6A modification were the lowest in the heart of mice at 1 day old. By contrast, heart tissues from 7-day-old mice tended to possess the most m6A peaks and the highest global m6A level. However, the m6A characteristics of myocardial tissue changed little after 7 days old as compared to that of 1 day old. Specifically, we found 1269 downmethylated genes of 1434 methylated genes in 7-day-old mouse heart tissues as compared to those in 1-day-old mice. Hypermethylation of some specific genes may correlate with the heart’s strong proliferative and regenerative capability at the first day after birth. In terms of m6A density, the tendency shifted from coding sequences (CDS) to 3 ′ -untranslated regions (3 ′ UTR) and stop codon with the progression of heart development. In addition, some genes demonstrated remarkable changes both in methylation and expression, like kiss1, plekha6, and megf6, which may play important roles in proliferation. Furthermore, signaling pathways highly related to proliferation such as “Wnt signaling pathway,” “ECM-receptor interaction,” and “cardiac chamber formation” were significantly enriched in 1-day-old methylated genes. Conclusions. Our results reveal a pattern that different m6A modifications are distributed in C57BL/6J heart tissue at different developmental stages, which provides new insights into a novel function of m6A methylation of mRNA in myocardial development and regeneration.

Case Report: Novel Likely Pathogenic ACTN2 Variant Causing Heterogeneous Phenotype in a Korean Family With Left Ventricular Non-compaction

Left ventricular non-compaction (LVNC) is a very rare primary cardiomyopathy with a genetic etiology, resulting from the failure of myocardial development during embryogenesis, and it carries a high risk of left ventricular dysfunction, thromboembolic phenomenon, and malignant arrhythmias. Here, we report the first case of familial LVNC in Korea, caused by a novel ACTN2 missense variant. We performed duo exome sequencing (ES) to examine the genome of the proband and his father. A 15-year-old boy was admitted for the evaluation of exertional dyspnea for 2 weeks. He was diagnosed with LVNC with a dilated cardiomyopathy phenotype [left ventricular end-diastolic dimension 60 mm, interventricular septal dimension 8.2 mm by transthoracic echocardiography (TTE)]. For the screening of familial cardiomyopathy, TTE and cardiac magnetic resonance imaging (cMRI) were performed, which revealed hypertrophic and isolated LVNC in the proband's father and sister, respectively. In particular, the cMRI revealed dense hypertrabeculation with focal aneurysmal changes in the apical septal wall in the proband's father. ES of the father–son duo identified a novel heterozygous c.668T>C variant of the ACTN2 gene (NM_001103.3:c.668T>C, p.Leu223Pro; no rsID) as the candidate cause of autosomal dominant LVNC. Sanger sequencing confirmed this novel variant in the proband, his father, and sister, but not in the proband's grandmother. Even within families harboring the same variant, a variable risk of adverse outcomes is common. Therefore, familial screening for patients with LVNC associated with ACTN2 variant should be performed for early detection of the LVNC phenotype associated with poor outcomes, such as dilated LVNC.

Myocardial TGFβ2 Is Required for Atrioventricular Cushion Remodeling and Myocardial Development

Among the three transforming growth factor beta (TGFβ) ligands, TGFβ2 is essential for heart development and is produced by multiple cell types, including myocardium. Heterozygous mutations in TGFB2 in patients of connective tissue disorders result in congenital heart defects and adult valve malformations, including mitral valve prolapse (MVP) with or without regurgitation. Tgfb2 germline knockout fetuses exhibit multiple cardiac defects but the role of myocardial-TGFβ2 in heart development is yet to be elucidated. Here, myocardial Tgfb2 conditional knockout (CKO) embryos were generated by crossing Tgfb2flox mice with Tgfb2+/−; cTntCre mice. Tgfb2flox/− embryos were normal, viable. Cell fate mapping was done using dual-fluorescent mT/mG+/− mice. Cre-mediated Tgfb2 deletion was assessed by genomic PCR. RNAscope in situ hybridization was used to detect the loss of myocardial Tgfb2 expression. Histological, morphometric, immunohistochemical, and in situ hybridization analyses of CKOs and littermate controls at different stages of heart development (E12.5–E18.5) were used to determine the role of myocardium-derived TGFβ2 in atrioventricular (AV) cushion remodeling and myocardial development. CKOs exhibit a thin ventricular myocardium, AV cushion remodeling defects and developed incomplete AV septation defects. The loss of myocardial Tgfb2 resulted in impaired cushion maturation and dysregulated cell death. Phosphorylated SMAD2, a surrogate for TGFβ signaling, was “paradoxically” increased in both AV cushion mesenchyme and ventricular myocardium in the CKOs. Our results indicate that TGFβ2 produced by cardiomyocytes acting as cells autonomously on myocardium and via paracrine signaling on AV cushions are required for heart development.

Transcriptional regulation of structural and functional adaptations in a developing adulthood myocardium

The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adult hood stages. Herein, we used C57/Bl6/J mice at 4 (28-days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis and metabolic pathways was noticed in both PN and AH hearts. Echocardiography reveals significant structural and functional (i.e. systolic/diastolic) changes during the transition of PN to adult stage. Particularly, a progressive decline in ejection fraction (EF) and cardiac output was observed in AH hearts. These structural adaptations are in line with critical signaling pathways that drive the maturation of heart during AH. Overall, we have presented a comprehensive transcriptomic analysis along with structural-functional relationship during the myocardial development in adult mice.

IGF1 Knockdown Hinders Myocardial Development through Energy Metabolism Dysfunction Caused by ROS-Dependent FOXO Activation in the Chicken Heart

Insulin-like growth factor 1 (IGF1) is a multifunctional cellular regulatory factor that can regulate cell growth and development by mediating growth hormone stimulation. However, the mechanism of IGF1 dysfunction in cardiomyocyte development is seldom reported. To study this, we employed the models of IGF1 knockdown in chicken embryo in vivo and in cardiomyocytes in vitro. We detected the antioxidant capacity, PI3K/Akt pathway, energy metabolism-related genes, and myocardial development-related genes. Our results revealed that the low expression of IGF1 can significantly suppress the antioxidant capacity and increase the ROS (P<0.05) levels, activating the AMPK and PI3K pathway by inhibiting the expression of IRS1. We also found that myocardial energy metabolism is blocked through IGF1, GLUT, and IGFBP inhibition, further inducing myocardial developmental disorder by inhibiting Mesp1, GATA, Nkx2.5, and MyoD expression. Altogether, we conclude that low IGF1 expression can hinder myocardial development through the dysfunction of energy metabolism caused by ROS-dependent FOXO activation.

Selenium deficiency inhibits myocardial development and differentiation by targeting the mir-215-5p/CTCF axis in chicken

Selenium (Se) is imperative for normal myocardial differentiation and development, and these basic cellular functions can be regulated by miRNA during cardiogenesis.

Myocardial Notch-Rbpj deletion does not affect heart development or function

During vertebrate cardiac development NOTCH signaling activity in the endocardium is essential for the crosstalk between endocardium and myocardium that initiates ventricular trabeculation and valve primordium formation. This crosstalk leads later to the maturation and compaction of the ventricular chambers and the morphogenesis of the cardiac valves, and its alteration may lead to disease. Although endocardial NOTCH signaling has been shown to be crucial for heart development, its physiological role in the myocardium has not been clearly established. Here we have used a genetic strategy to evaluate the role of NOTCH in myocardial development. We have inactivated the unique and ubiquitous NOTCH effector RBPJ in the early cardiomyocytes progenitors, and examined its consequences in cardiac development and function. Our results demonstrate that mice with cTnT-Cre-mediated myocardial-specific deletion of Rbpj develop to term, with homozygous mutant animals showing normal expression of cardiac development markers, and normal adult heart function. Similar observations have been obtained after Notch1 deletion with cTnT-Cre. We have also deleted Rbpj in both myocardial and endocardial progenitor cells, using the Nkx2.5-Cre driver, resulting in ventricular septal defect (VSD), double outlet right ventricle (DORV), and bicuspid aortic valve (BAV), due to NOTCH signaling abrogation in the endocardium of cardiac valves territories. Our data demonstrate that NOTCH-RBPJ inactivation in the myocardium does not affect heart development or adult cardiac function.