Maternal folic acid over-supplementation impairs cardiac function in mice offspring by inhibiting SOD1 expression.

BACKGROUND: Folic acid (FA) supplementation during pregnancy aims to protect foetal development. However, maternal over-supplementation of FA has been demonstrated to cause metabolic dysfunction and increase the risk of autism, retinoblastoma, and respiratory illness in the offspring. Moreover, FA supplementation reduces the risk of congenital heart disease. However, little is known about its possible adverse effects on cardiac health resulting from maternal over-supplementation. In this study, we assessed the detrimental effects of maternal FA over-supplementation on the cardiac health of the offspring. METHODS AND RESULTS: Eight-week-old C57BL/6J pregnant mice were randomly divided into control and over-supplemented groups. The offspring cardiac function was assessed using echocardiography. Cardiac fibrosis was assessed in the left ventricular myocardium by histological analysis. Proteomic, protein, RNA, and DNA methylation analyses were performed by liquid chromatography-tandem mass spectrometry, western blotting, real-time quantitative PCR, and bisulfite sequencing, respectively. We found that maternal periconceptional FA over-supplementation impaired cardiac function with the decreased left ventricular ejection fraction in the offspring. Biochemical indices and tissue staining further confirmed impaired cardiac function in offspring caused by maternal FA over-supplementation. The combined proteomic, RNA expression, and DNA methylation analyses suggested that key genes involved in cardiac function were inhibited at the transcriptional level possibly due to increased DNA methylation. Among these, superoxide dismutase 1 was downregulated, and reactive oxygen species (ROS) levels increased in the mouse heart. Inhibition of ROS generation using the antioxidant N-acetylcysteine rescued the impaired cardiac function resulting from maternal FA over-supplementation. CONCLUSIONS: Our study revealed that over-supplementation with FA during mouse pregnancy is detrimental to cardiac function with the decreased left ventricular ejection fraction in the offspring and provides insights into the mechanisms underlying the association between maternal FA status and health outcomes in the offspring.

High paternal homocysteine causes ventricular septal defects in mouse offspring.

Maternal hyperhomocysteinemia is widely considered as an independent risk of congenital heart disease (CHD). However, whether high paternal homocysteine causes CHD remains unknown. Here, we showed that increased homocysteine levels of male mice caused decreased sperm count, sperm motility defect and ventricular septal defect of the offspring. Moreover, high levels of paternal homocysteine decrease sperm DNMT3A/3B, accompanied with changes in DNA methylation levels in the promoter regions of CHD-related genes. Folic acid supplement could decrease the occurrence of VSD in high homocysteine male mice. This study reveals that increased paternal homocysteine level increases VSD risk in the offspring, indicating that decreasing paternal homocysteine may be an intervening target of CHD.

Nicotinamide Mononucleotide Alleviates Cardiomyopathy Phenotypes Caused by Short-Chain Enoyl-Coa Hydratase 1 Deficiency.

Short-chain enoyl-CoA hydratase 1 (ECHS1) deficiency plays a role in cardiomyopathy. Whether ECHS1 deficiency causes or is only associated with cardiomyopathy remains unclear. By using Echs1 heterogeneous knockout (Echs1 +/-) mice, we found that ECHS1 deficiency caused cardiac dysfunction, as evidenced by diffuse myocardial fibrosis and upregulated fibrosis-related genes. Mechanistically, ECHS1 interacts with the p300 nuclear localization sequence, preventing its nuclear translocation in fibroblasts. ECHS1 deficiency promotes p300 nuclear translocation, leading to increased H3K9 acetylation, a known risk factor for cardiomyopathy. Nicotinamide mononucleotide-mediated acetylation targeting suppressed ECHS1 deficiency-induced cardiomyopathy phenotypes in Echs1 +/- mice. Thus, enhancing p300-mediated H3K9ac is a potential interventional approach for preventing ECHS1 deficiency-induced cardiomyopathy.

Gestational Leucylation Suppresses Embryonic T-Box Transcription Factor 5 Signal and Causes Congenital Heart Disease.

Dysregulated maternal nutrition, such as vitamin deficiencies and excessive levels of glucose and fatty acids, increases the risk for congenital heart disease (CHD) in the offspring. However, the association between maternal amino-acid levels and CHD is unclear. Here, it is shown that increased leucine levels in maternal plasma during the first trimester are associated with elevated CHD risk in the offspring. High levels of maternal leucine increase embryonic lysine-leucylation (K-Leu), which is catalyzed by leucyl-tRNA synthetase (LARS). LARS preferentially binds to and catalyzes K-Leu modification of lysine 339 within T-box transcription factor TBX5, whereas SIRT3 removes K-Leu from TBX5. Reversible leucylation retains TBX5 in the cytoplasm and inhibits its transcriptional activity. Increasing embryonic K-Leu levels in high-leucine-diet fed or Sirt3 knockout mice causes CHD in the offspring. Targeting K-Leu using the leucine analogue leucinol can inhibit LARS activity, reverse TBX5 K-Leu modification, and decrease the occurrence of CHD in high-leucine-diet fed mice. This study reveals that increased maternal leucine levels increases CHD risk in the offspring through inhibition of embryonic TBX5 signaling, indicating that leucylation exerts teratogenic effects during heart development and may be an intervening target of CHD.

High maternal blood lipid levels during early pregnancy are associated with increased risk of congenital heart disease in offspring.

INTRODUCTION: This study aimed to investigate whether maternal blood lipid levels during early pregnancy are associated with the occurrence of congenital heart disease (CHD) in their offspring. MATERIAL AND METHODS: In this single-center case-control study, mothers of offspring with CHD (n = 230) and without CHD (n = 381) were included. Maternal lipid levels were determined on fasting blood samples taken in the first trimester. Relevant demographic and clinical data were extracted from the medical records. Maternal lipid profile was compared between the two groups, and regression analysis was performed to evaluate the association between lipid profile and CHD risk in offspring. RESULTS: Compared with the control group, levels of triglyceride, apolipoprotein-A1, and apolipoprotein-B in early pregnancy were significantly higher in the CHD group. Multivariate analyses showed that triglyceride (odds ratio [OR] 2.46, 95% CI 1.62-3.73, p < 0.01), total/high-density lipoprotein cholesterol (OR 2.10, 95% CI 1.07-4.13, p = 0.03), and apolipoprotein-A1 (OR 2.73, 95% CI 1.16-6.40, p = 0.02) were positively associated with CHD risk in offspring. CONCLUSIONS: Elevated maternal lipid profile was associated with increased risk of CHD in offspring.

[Effect of dhfr gene overexpression on ethanol-induced abnormal cardiovascular development in zebrafish embryos].

OBJECTIVE: To study the effect of dhfr gene overexpression on ethanol-induced abnormal cardiac and vascular development in zebrafish embryos and underlying mechanisms. METHODS: dhfr mRNA was transcribed in vitro and microinjected into zebrafish fertilized eggs to induce the overexpression of dhfr gene, and the efficiency of overexpression was verified. Wild-type zebrafish were divided into a control group, an ethanol group, and an ethanol+dhfr overexpression group (microinjection of 6 nL dhfr mRNA). The embryonic development was observed for each group. The transgenic zebrafish Tg (cmlc2:mcherry) with heart-specific red fluorescence was used to observe atrial and ventricular development. Fluorescence microscopy was performed to observe the development of cardiac outflow tract and blood vessels. Heart rate and ventricular shortening fraction were used to assess cardiac function. Gene probes were constructed, and embryo in situ hybridization and real-time PCR were used to measure the expression of nkx2.5, tbx1, and flk-1 in the embryo. RESULTS: Compared with the ethanol group, the ethanol+dhfr overexpression group had a significant reduction in the percentage of abnormal embryonic development and a significant increase in the percentage of embryonic survival (P<0.05), with significant improvements in the abnormalities of the atrium, ventricle, outflow tract, and blood vessels and cardiac function. Compared with the control group, the ethanol group had significant reductions in the expression of nkx2.5, tbx1, and flk-1 (P<0.05), and compared with the ethanol group, the ethanol+dhfr overexpression group had significant increases in the expression of nkx2.5, tbx1, and flk-1 (P<0.05), which were still lower than their expression in the control group. CONCLUSIONS: The overexpression of the dhfr gene can partially improve the abnormal development of embryonic heart and blood vessels induced by ethanol, possibly by upregulating the decreased expression of nkx2.5, tbx1, and flk-1 caused by ethanol.

Susceptibility to congenital heart defects associated with a polymorphism in TBX2 3' untranslated region in the Han Chinese population.

BACKGROUND: Tbx2 plays a critical role in determining fates of cardiomyocytes. Little is known about the contribution of TBX2 3' untranslated region (UTR) variants to the risk of congenital heart defect (CHD). Thus, we aimed to determine the association of single-nucleotide polymorphisms (SNPs) in TBX2 3' UTR with CHD susceptibility. METHODS: We recruited 1285 controls and 1241 CHD children from China. SNPs identification and genotyping were detected using Sanger Sequencing and SNaPshot. Stratified analysis was conducted to explore the association between rs59382073 polymorphism and CHD subtypes. Functional analyses were performed by luciferase assays in HEK-293T and H9c2 cells. RESULTS: Among five TBX2 3'UTR variants identified, rs59382073 minor allele T carriers had a 1.89-fold increased CHD risk compared to GG genotype (95% CI = 1.48-2.46, P = 4.48 × 10-7). The most probable subtypes were right ventricular outflow tract obstruction, conotruncal, and septal defect. G to T variation decreased luciferase activity in cells. This discrepancy was exaggerated by miR-3940 and miR-708, while their corresponding inhibitors eliminated it. CONCLUSION: T allele of rs59382073 in TBX2 3'UTR contributed to greater CHD risk in the Han Chinese population. G to T variation created binding sites for miR-3940 and miR-708 to inhibit gene expression.

A regulatory variant in TBX2 promoter is related to the decreased susceptibility of congenital heart disease in the Han Chinese population.

BACKGROUND: Tbx2 plays a vital role in the cardiac cushion development. In this study, we aimed to determine the relationship between common genetic variants in the promoter region of TBX2 gene and the risk of congenital heart disease (CHD). METHODS: Blood samples of 516 CHD patients and 587 control subjects were enrolled. Sanger sequencing and SNaPshot analysis were performed for genotyping in our case-control cohort. Luciferase and electrophoretic mobility shift assay (EMSA) were conducted to uncover the potential modulatory mechanism of the related variants. RESULTS: Variant rs4455026(c.-1028G>C) in TBX2 promoter region was found to be associated with significantly lower CHD susceptibility. The risk of CHD in C allele carriers (GC and CC genotypes) decreased by 30% compared to the wild-type GG genotype subjects (OR = 0.70, 95% CI = 0.55-0.89, p = 0.0038). It was revealed that G to C variation resulted in a decrease in the transcriptional activity of luciferase gene, and a potential change in binding affinity with certain nucleoproteins in EMSA data. CONCLUSION: The minor C allele of rs4455026 in TBX2 promoter region was related with lower CHD susceptibility in the Han Chinese population via repressing its transcriptional activity.

[Research advances in the pathogenesis of familial Kawasaki disease].

Kawasaki disease has become the leading cause of acquired heart disease in children in North America and Japan. The incidence rate of Kawasaki disease varies significantly across regions and races. The first-degree relatives of patients with Kawasaki disease have a significantly higher risk of this disease than the general population. This article reviews the onset of familial Kawasaki disease and possible pathogenesis.

[Research advances in the mechanism of congenital heart disease induced by pregestational diabetes mellitus].

Congenital heart disease (CHD) is the most common birth defect at present and has a complex etiology which involves the combined effect of genetic and environmental factors. Pregestational diabetes mellitus is significantly associated with the development of CHD, but the detailed mechanism remains unknown. This article reviews the research advances in the molecular mechanism of CHD caused by pregestational diabetes mellitus.

A TBX5 3'UTR variant increases the risk of congenital heart disease in the Han Chinese population.

TBX5 is a vital transcription factor involved in cardiac development in a dosage-dependent manner. But little is known about the potential association of TBX5 3' untranslated region (UTR) variations with congenital cardiac malformations. This study aimed to investigate the relationship between TBX5 3'UTR variants and risk for congenital heart disease (CHD) susceptibility in two Han Chinese populations, and to reveal its molecular mechanism. The relationship between TBX5 3'UTR variants and CHD susceptibility was examined in 1 177 CHD patients and 990 healthy controls in two independent case-control studies. Variant rs6489956 C>T was found to be associated with increased CHD susceptibility in both cohorts. The combined CHD risk for the CT and TT genotype carriers was 1.83 times higher than that of CC genotype, while the risk for CT or TT genotype was 1.94 times and 2.31 times higher than that of CC carriers, respectively. Quantitative real-time PCR and western blot analysis showed that T allele carriers exhibited reduced TBX5 mRNA and protein levels in CHDs tissues. Compared with C allele, T allele showed increased binding affinity to miR-9 and miR-30a in both luciferase assays and surface plasmon resonance analysis. Functional analysis confirmed that miR-9 and miR-30a downregulated TBX5 expression at the transcriptional and translational levels, respectively. The assays in zebrafish model were in support of the interaction of miR-9/30a and TBX5 3'UTR (C and T allele). We concluded that TBX5 3'UTR variant rs6489956 increased susceptibility of CHD in the Han Chinese population because it changes the binding affinity of two target miRNAs that specifically mediate TBX5 expression.

[Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis].

Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber, and participates in heart excitation-contraction coupling process. Here, we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique, and performed phenotypic analysis of the identified tnnt2a mutants. We observed that there was no significant difference between heterozygous mutant and wild type zebrafish, and the homozygous mutants displayed significant malformations in heart, including cardiac arrest, atrium and ventricle enlargement, pericardium effusion, and the individuals usually died before 7 day post fertilization (dpf). We further analyzed the expression alternations of heart sarcomere genes (tnnt2a, actc1a, tpm4a, myl7, vmhc) at transcriptional level in tnnt2a-/-(Δ2) zebrafish by performing real time RT-PCR, and found that the RNA expression level of tnnt2a in tnnt2a-/- zebrafish decreased constantly at each time point of developmental stages, and actc1a, tpm4a, myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages, in comparison with those of wild type zebrafish. Lastly, electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a-/-(Δ2) zebrafish, which was further confirmed by F-actin and Tpm4 immunofluorescence staining. The tnnt2a-/- zebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy, and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.

Mild decrease in TBX20 promoter activity is a potentially protective factor against congenital heart defects in the Han Chinese population.

Congenital heart defects (CHDs) are one of the most common human birth defects worldwide. TBX20 is a crucial transcription factor for the development of embryonic cardiovascular system. Previous studies have demonstrated that mutations in the TBX20 coding region contribute to familial and sporadic CHD occurrence. However, it remains largely unknown whether variants in the TBX20 regulatory region are also related to CHDs. In this study, we sequenced the 2 kb region upstream of the TBX20 transcription start site in 228 CHD patients and 292 controls in a Han Chinese population. Among the 8 single nucleotide polymorphisms (SNPs) identified, six SNPs are in strong linkage disequilibrium and the minor alleles are associated with lower CHD risk (for rs10235849 chosen as tag SNP, p = 0.0069, OR (95% CI) = 0.68 (0.51-0.90)). Functional analysis showed that the minor alleles have lower transcriptional activity than major alleles in both human heart tissues and three cell lines. The electrophoretic mobility shift assay suggested that TBX20 minor alleles may exhibit higher binding affinity with certain transcription repressors. Our results indicate that a moderately lower TBX20 activity potentially reduces CHD risk in the Han Chinese population, providing new insight in the study of CHD etiology.

[Role of the canonical Wnt signaling pathway in heart valve development].

Formation of the heart valves is one of critical steps in vertebrate cardiac development. Valvular heart anomaly can induce severe cardiac impairment, which is one of most common symptoms for congenital heart defects (CHD). The canonical Wnt/ß-catenin signaling pathway, which is essential for numerous developmental processes, has also been suggested to be involved in the regulation of proliferation, differentiation, and migration of myocardium, endocardium and valve primordia at different stages. The canonical Wnt signaling also regulates the endocardial-mesenchymal transformation (EMT) process during the endocardial cushion formation. This paper reviews current knowledge about the canonical Wnt signaling pathway in heart valve development, including the functional diversities of Wnt activity in heart valve development at different stages and its interaction with other valve-relevant signaling pathways and the potential role of canonical Wnt activity in heart valve mesenchymal stem cells at the late developmental stage.

miR-10a and miR-10b target the 3'-untranslated region of TBX5 to repress its expression.

As a well-known transcription factor, TBX5 is involved in embryonic cardiac development. Although TBX5 functions in a dose-dependent manner, the posttranscriptional regulation of human TBX5 is poorly understood. Thus, this study aimed to identify microRNAs that modulate TBX5 expression. Luciferase assays were used to screen miRNAs predicted to modulate TBX5 expression. Using quantitative reverse transcriptase-polymerase chain reaction and Western blot analysis, the authors found that miR-10a and miR-10b significantly repressed TBX5 expression and decreased TBX5 protein levels by targeting the TBX5 3'-untranslated region. In addition, miR-10a and miR-10b expression levels were respectively 2.77 and 3.51 times higher in the heart tissues of congenital heart disease patients than in healthy control subjects, suggesting that they are potential diagnostic biomarkers. In conclusion, the study results indicate that miR-10a and miR-10b inhibit TBX5 expression at the level of translation. Higher levels of miR-10a and miR-10b expression are associated with a higher risk of congenital heart defects.

A genetic variant in vitamin B12 metabolic genes that reduces the risk of congenital heart disease in Han Chinese populations.

BACKGROUND: Genome-wide association studies on components of the one-carbon metabolic pathway revealed that human vitamin B12 levels could be significantly influenced by variations in the fucosyltransferase 2 (FUT2), cubilin (CUBN), and transcobalamin-I (TCN1) genes. An altered vitamin B12 level is an important factor that disturbs the homeostasis of the folate metabolism pathway, which in turn can potentially lead to the development of congenital heart disease (CHD). Therefore, we investigated the association between the variants of vitamin B12-related genes and CHD in Han Chinese populations. METHODS AND RESULTS: Six variants of the vitamin B12-related genes were selected for analysis in two independent case-control studies, with a total of 868 CHD patients and 931 controls. The variant rs11254363 of the CUBN gene was associated with a decreased risk of developing CHD in both the separate and combined case-control studies. Combined samples from the two cohorts had a significant decrease in CHD risk for the G allele (OR = 0.48, P = 1.7×10⁻5) and AG+GG genotypes (OR = 0.49, P = 4×10⁻5), compared with the wild-type A allele and AA genotype, respectively. CONCLUSIONS: Considering the G allele of variant rs11254363 of the CUBN gene was associated with an increased level of circulating vitamin B12. This result suggested that the carriers of the G allele would benefit from the protection offered by the high vitamin B12 concentration during critical heart development stages. This finding shed light on the unexpected role of CUBN in CHD development and highlighted the interplay of diet, genetics, and human birth defects.