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.

[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.

[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.

[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.

[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.

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.

The impacts of maternal gestational diabetes mellitus (GDM) on fetal hearts.

OBJECTIVE: To evaluate the fetal cardiac function in gestational diabetes mellitus (GDM) pregnancies under different maternal glycemic controls. METHODS: Forty four GDM mothers received 78 fetal echocardiographic evaluations at three gestational periods (<28, 28-34 and >34 weeks) and were divided into poorly-(DM1) and well-(DM2) controlled groups according to their glycemic control at examination. Seventy uncomplicated mothers were selected as controls. Parameters of fetal cardiac anatomy and function were measured and analyzed. RESULTS: GDM fetuses' cardiac ventricular walls were thicker than controls', and the differences between DM1 and DM2 were not significant except for end-diastolic left ventricular walls. In both GDM groups, the aortic flow velocities increased earlier than pulmonary artery and DM1 fetuses changed earlier than DM2 ones. GDM fetuses' left atrial shortening fraction was smaller than the controls' in the period of ⩾34 weeks and negatively correlated with thicknesses of left ventricular walls and interventricular septum in DM1 fetuses (r=-0.438 and -0.506). The right ventricular diastolic function in DM1 and DM2 fetuses decreased after the period of 28-34 weeks and in the period of >34 weeks respectively. Tei index of both left and right ventricles increased in DM1 group after the period of <28 weeks and in DM2 group only in the period of ⩾34 weeks, with no significant differences between DM1 and DM2 groups in this period. CONCLUSION: Fetuses of GDM mothers showed cardiac function impairments. Good maternal glycemic control may delay the impairments, but cannot reduce the degree. Some cardiac changes in GDM fetuses were similar to those in pregestational diabetic pregnancies except for several parameters and their changing time.

[Post-transcriptional protein modification of Gata4].

Gata4 is an important transcription factor in heart development. Gata4 post-transcriptional protein modification regulates transcriptional activity and DNA binding, which in turn affects expression of downstream genes and transcription factors, differentiation of embryonic stem cells and cardiogenesis. This article summarizes the effect of post-transcriptional protein modification on transcriptional activity of Gata4 and the relationship between this effect and congenital heart disease. It was shown that acetylation, phosphorylation and SUMOylation upregulate transcriptional activity, DNA binding, downstream gene expression and embryonic stem cell differentiation. On the other hand, methylation and deacetylation downregulate Gata4 transcriptional activity. Post-transcriptional protein modification of Gata4 is very important in clinical research on congenital and other heart diseases.

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.

[Lipoprotein lipase gene mutations and the risk of cardiovascular diseases in children with obesity].

OBJECTIVE: To inquire into the relationship between lipoprotein lipase (LPL) gene D9N, N291S and S447X polymorphisms and the development of cardiovascular diseases in children with obesity. METHODS: The polymerase chain reaction (PCR) and restriction fragment length polymorphism (RLFP) techniques were used to detect three common mutations of LPL gene exon D9N, N291S and S447X in 157 obese children and 175 normal controls. Plasma lipid and lipoprotein levels between children with different genotypes were compared. RESULTS: The D9N and N291S gene mutations were not detected in either the obese or the control groups. There were no significant differences in the frequency of S447X gene mutation between the two groups. There were no significant differences in the levels of plasma lipid and lipoprotein between children with S447 and X447 genotypes. CONCLUSIONS: D9N and N291S gene mutations may not be risk factors associated with cardiovascular diseases in children with obesity. S447X gene mutation might not play an important role in the development of cardiovascular diseases in childhood.

[Evaluation of cardiac function in fetuses from pregnant women with abnormal blood glucose levels by brain natriuretic peptide in umbilical cord blood].

OBJECTIVE: To study the feasibility of umbilical cord brain natriuretic peptide (BNP) level measurement for the evaluation of perinatal cardiac function in fetuses from pregnant women with abnormal blood glucose levels and the influence of abnormal blood glucose on fetal cardiac function. METHODS: Twenty-four mothers with gestational diabetes mellitus (n=18) or gestational impaired glucose tolerance (n=6) (diabetic group) were classified into two subgroups according to blood glucose level before delivery: good (n=17) and poor (n=7) glucose control. They underwent fetal echocardiography in their late pregnant periods and fetal cardiac sizes and function were measured. Twenty-five normal pregnant mothers served as the control group. Umbilical cord blood BNP concentrations were measured at delivery. RESULTS: The umbilical cord blood BNP concentrations in the diabetic group were significantly higher than in the control group(114.0+/-39.0 pg/mL vs 80.6+/-13.7 pg/mL; p<0.01). The poor glucose control subgroup demonstrated higher umbilical cord blood BNP concentrations than the good glucose control subgroup (142.1+/-44.1 pg/mL vs 102.4+/-31.2 pg/mL; p<0.01). No difference was found between the gestational diabetes mellitus and the impaired glucose tolerance groups. The BNP concentration was positively correlated to the thicknesses of fetal left ventricular walls and the peak velocities of mitral A wave (r=0.715, 0.491 respectively, p<0.05), and negatively correlated to the mitral E/A ratio (r=-0.507, p<0.05). CONCLUSIONS: The fetuses of pregnant women with abnormal blood glucose levels have an increased BNP level in umbilical cord blood. Umbilical cord BNP level is related to maternal blood glucose control and the changes in fetal cardiac function. It may reflex the latent impairments of fetal cardiac function. A good glucose control may decrease the impact of abnormal maternal blood glucose on fetal hearts.

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.

[Evaluation of the prenatal screening mode for fetal congenital heart diseases by ultrasound].

OBJECTIVE: To assess prenatal heart disease screening program by ultrasound. METHODS: A total of 11,544 second-trimester screening scans were performed before 24 weeks' gestation on 11,410 women between February 2004 and May 2007 in Obstetrics and Gynecology Hospital of Fudan University. Fetal heart screening was based on four-chamber and outflow tract views (left ventricular outflow + three vessel view). The sensitivity and specificity of different views were evaluated. Follow-up data of newborns was obtained. RESULTS: (1) Among 11,544 cases,48 cases of congenital heart disease (CHD) were diagnosed in utero. Six cases were false negative, and 2 cases were false positive. The incidence of CHD was 0.47% (54/11 544). (2) Thirty-three CHDs were detected based on the four-chamber view, including 18 ventricular septal defect (9 with conotruncal anomalies), 6 anomalous atrioventricle valve, 9 disproportion of left/right ventricle. The sensitivity of the four-chamber view alone was 61.11% (33/54), and the specificity was 99.98% (11 488/11 490). Fifteen CHDs were detected based on the left ventricular outflow and three vessel views, including 1 pulmonary atresia, 3 pulmonary valve stenosis, 2 transposition of the great arteries (TGA), 1 pulmonary stenosis with TGA, 6 tetralogy of Fallot, and 2 pulmonary stenosis. The sensitivity of the combination of the four-chamber and outflow tract views was 88.89% (48/54), and the specificity was 99.98% (11 488/11 490). (3) Of 48 CHDs, 11 cases were accompanied by other malformations. Eleven cases were performed amniocentesis, among whom 5 cases were trisomy 21. CONCLUSION: The screening program based on four-chamber and outflow tract views shows good sensitivity and excellent specificity. Our prenatal heart screening program is clinically feasible.

[Association of nitric oxide and eNOS with the pathogenesis of vasovagal syncope].

OBJECTIVE: To investigate the roles of nitric oxide (NO) and eNOS in the pathogenesis of vasovagal syncope (VVS). METHODS: Fourteen children with VVS (group A), 10 children with syncope other than vasovagal (group B) and 20 healthy volunteers (group C) were enrolled. Plasma NO levels in groups A and B were determined before and at the termination of the head-up tilt table test (HUT). The G894T polymorphism within the eNOS gene was determined in the three groups. RESULTS: Plasma NO levels in group A increased significantly when syncope attacked from 76.7+/-9.6 micromol/L (before HUT) to 90.0+/-11.4 micromol/L (P<0.05). After the syncope attack was improved, plasma NO level in group A was significantly reduced. There were no statistical differences in plasma NO levels before and after the HUT in group B. Determining the G894T polymorphism within the eNOS gene showed that group A was associated with a higher incidence of the GT gene type as compared to groups B and C (42.9% vs 10%; P<0.05). CONCLUSIONS: Plasma NO may be involved in the pathogenesis of VVS. The increased plasma NO level may be associated with the G894T polymorphism of the eNOS gene.