Ventricular Septal Defects: Molecular Pathways and Animal Models.

Ventricular septation is a complex process which involves the major genes of cardiac development, acting on myocardial cells from first and second heart fields, and on mesenchymal cells from endocardial cushions. These genes, coding for transcription factors, interact with each other, and their differential expression conditions the severity of the phenotype. In this chapter, we will describe the formation of the ventricular septum in the normal heart, as well as the molecular mechanisms leading to the four main anatomic types of ventricular septal defects: outlet, inlet, muscular, and central perimembranous, resulting from failure of development of the different parts of the ventricular septum. Experiments on animal models, particularly transgenic mouse lines, have helped us to decipher the molecular determinants of ventricular septation. However, a precise description of the anatomic phenotypes found in these models is mandatory to achieve a better comprehension of the complex mechanisms responsible for the various types of VSDs.

Human Genetics of Ventricular Septal Defect.

Ventricular septal defects (VSDs) are recognized as one of the commonest congenital heart diseases (CHD), accounting for up to 40% of all cardiac malformations, and occur as isolated CHDs as well as together with other cardiac and extracardiac congenital malformations in individual patients and families. The genetic etiology of VSD is complex and extraordinarily heterogeneous. Chromosomal abnormalities such as aneuploidy and structural variations as well as rare point mutations in various genes have been reported to be associated with this cardiac defect. This includes both well-defined syndromes with known genetic cause (e.g., DiGeorge syndrome and Holt-Oram syndrome) and so far undefined syndromic forms characterized by unspecific symptoms. Mutations in genes encoding cardiac transcription factors (e.g., NKX2-5 and GATA4) and signaling molecules (e.g., CFC1) have been most frequently found in VSD cases. Moreover, new high-resolution methods such as comparative genomic hybridization enabled the discovery of a high number of different copy number variations, leading to gain or loss of chromosomal regions often containing multiple genes, in patients with VSD. In this chapter, we will describe the broad genetic heterogeneity observed in VSD patients considering recent advances in this field.

Molecular Pathways and Animal Models of Truncus Arteriosus.

In normal cardiovascular development in birds and mammals, the outflow tract of the heart is divided into two distinct channels to separate the oxygenated systemic blood flow from the deoxygenated pulmonary circulation. When the process of outflow tract septation fails, a single common outflow vessel persists resulting in a serious clinical condition known as persistent truncus arteriosus or common arterial trunk. In this chapter, we will review molecular pathways and the cells that are known to play a role in the formation and development of the outflow tract and how genetic manipulation of these pathways in animal models can result in common arterial trunk.

Molecular Pathways and Animal Models of Semilunar Valve and Aortic Arch Anomalies.

The great arteries of the vertebrate carry blood from the heart to the systemic circulation and are derived from the pharyngeal arch arteries. In higher vertebrates, the pharyngeal arch arteries are a symmetrical series of blood vessels that rapidly remodel during development to become the asymmetric aortic arch arteries carrying oxygenated blood from the left ventricle via the outflow tract. At the base of the aorta, as well as the pulmonary trunk, are the semilunar valves. These valves each have three leaflets and prevent the backflow of blood into the heart. During development, the process of aortic arch and valve formation may go wrong, resulting in cardiovascular defects, and these may, at least in part, be caused by genetic mutations. In this chapter, we will review models harboring genetic mutations that result in cardiovascular defects affecting the great arteries and the semilunar valves.

Cardiac Transcription Factors and Regulatory Networks.

Cardiac development is a fine-tuned process governed by complex transcriptional networks, in which transcription factors (TFs) interact with other regulatory layers. In this chapter, we introduce the core cardiac TFs including Gata, Hand, Nkx2, Mef2, Srf, and Tbx. These factors regulate each other's expression and can also act in a combinatorial manner on their downstream targets. Their disruption leads to various cardiac phenotypes in mice, and mutations in humans have been associated with congenital heart defects. In the second part of the chapter, we discuss different levels of regulation including cis-regulatory elements, chromatin structure, and microRNAs, which can interact with transcription factors, modulate their function, or are downstream targets. Finally, examples of disturbances of the cardiac regulatory network leading to congenital heart diseases in human are provided.

Human Genetics of Tetralogy of Fallot and Double-Outlet Right Ventricle.

Tetralogy of Fallot (TOF) and double-outlet right ventricle (DORV) are conotruncal defects resulting from disturbances of the second heart field and the neural crest, which can occur as isolated malformations or as part of multiorgan syndromes. Their etiology is multifactorial and characterized by overlapping genetic causes. In this chapter, we present the different genetic alterations underlying the two diseases, which range from chromosomal abnormalities like aneuploidies and structural mutations to rare single nucleotide variations affecting distinct genes. For example, mutations in the cardiac transcription factors NKX2-5, GATA4, and HAND2 have been identified in isolated TOF cases, while mutations of TBX5 and 22q11 deletion, leading to haploinsufficiency of TBX1, cause Holt-Oram and DiGeorge syndrome, respectively. Moreover, genes involved in signaling pathways, laterality determination, and epigenetic mechanisms have also been found mutated in TOF and/or DORV patients. Finally, genome-wide association studies identified common single nucleotide polymorphisms associated with the risk for TOF.

Human Genetics of Semilunar Valve and Aortic Arch Anomalies.

Lesions of the semilunar valve and the aortic arch can occur either in isolation or as part of well-described clinical syndromes. The polygenic cause of calcific aortic valve disease will be discussed including the key role of NOTCH1 mutations. In addition, the complex trait of bicuspid aortic valve disease will be outlined, both in sporadic/familial cases and in the context of associated syndromes, such as Alagille, Williams, and Kabuki syndromes. Aortic arch abnormalities particularly coarctation of the aorta and interrupted aortic arch, including their association with syndromes such as Turner and 22q11 deletion, respectively, are also discussed. Finally, the genetic basis of congenital pulmonary valve stenosis is summarized, with particular note to Ras-/mitogen-activated protein kinase (Ras/MAPK) pathway syndromes and other less common associations, such as Holt-Oram syndrome.

Human Genetics of Truncus Arteriosus.

Integrated human genetics and molecular/developmental biology studies have revealed that truncus arteriosus is highly associated with 22q11.2 deletion syndrome. Other congenital malformation syndromes and variants in genes encoding TBX, GATA, and NKX transcription factors and some signaling proteins have also been reported as its etiology.

Significant improvement of cardiac outflow tract septation defects in a DiGeorge syndrome model after minoxidil treatment.

The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

Multiwalled Carbon Nanotubes-Reprogrammed Macrophages Facilitate Breast Cancer Metastasis via NBR2/TBX1 Axis.

In recent years, carbon nanotubes have emerged as a widely used nanomaterial, but their human exposure has become a significant concern. In our former study, we reported that pulmonary exposure of multiwalled carbon nanotubes (MWCNTs) promoted tumor metastasis of breast cancer; macrophages were key effectors of MWCNTs and contributed to the metastasis-promoting procedure in breast cancer, but the underlying molecular mechanisms remain to be explored. As a follow-up study, we herein demonstrated that MWCNT exposure in breast cancer cells and macrophage coculture systems promoted metastasis of breast cancer cells both in vitro and in vivo; macrophages were skewed into M2 polarization by MWCNT exposure. LncRNA NBR2 was screened out to be significantly decreased in MWCNTs-stimulated macrophages through RNA-seq; depletion of NBR2 led to the acquisition of M2 phenotypes in macrophages by activating multiple M2-related pathways. Specifically, NBR2 was found to positively regulate the downstream gene TBX1 through H3k27ac activation. TBX1 silence rescued NBR2-induced impairment of M2 polarization in IL-4 & IL-13-stimulated macrophages. Moreover, NBR2 overexpression mitigated the enhancing effects of MWCNT-exposed macrophages on breast cancer metastasis. This study uncovered the molecular mechanisms underlying breast cancer metastasis induced by MWCNT exposure.

Variants in FOXC1 and FOXC2 identified in patients with conotruncal heart defects.

Conotruncal heart defects (CTD), subtypes of congenital heart disease, result from abnormal cardiac outflow tract development (OFT). FOXC1 and FOXC2 are closely related members of the forkhead transcription factor family and play essential roles in the development of OFT. We confirmed their expression pattern in mouse and human embryos, identifying four variants in FOXC1 and three in FOXC2 by screening these two genes in 605 patients with sporadic CTD. Western blot demonstrated expression levels, while Dual-luciferase reporter assay revealed affected transcriptional abilities for TBX1 enhancer in two FOXC1 variants and three FOXC2 variants. This might result from the altered DNA-binding abilities of mutant proteins. These results indicate that functionally impaired FOXC1 and FOXC2 variants may contribute to the occurrence of CTD.

Lymphatic endothelial transcription factor Tbx1 promotes an immunosuppressive microenvironment to facilitate post-myocardial infarction repair.

The heart is an autoimmune-prone organ. It is crucial for the heart to keep injury-induced autoimmunity in check to avoid autoimmune-mediated inflammatory disease. However, little is known about how injury-induced autoimmunity is constrained in hearts. Here, we reveal an unknown intramyocardial immunosuppressive program driven by Tbx1, a DiGeorge syndrome disease gene that encodes a T-box transcription factor (TF). We found induced profound lymphangiogenic and immunomodulatory gene expression changes in lymphatic endothelial cells (LECs) after myocardial infarction (MI). The activated LECs penetrated the infarcted area and functioned as intramyocardial immune hubs to increase the numbers of tolerogenic dendritic cells (tDCs) and regulatory T (Treg) cells through the chemokine Ccl21 and integrin Icam1, thereby inhibiting the expansion of autoreactive CD8+ T cells and promoting reparative macrophage expansion to facilitate post-MI repair. Mimicking its timing and implementation may be an additional approach to treating autoimmunity-mediated cardiac diseases.

Structural alterations in the amygdala and impaired social incentive learning in a mouse model of a genetic variant associated with neurodevelopmental disorders.

Copy number variants (CNVs) are robustly associated with psychiatric disorders and their dimensions and changes in brain structures and behavior. However, as CNVs contain many genes, the precise gene-phenotype relationship remains unclear. Although various volumetric alterations in the brains of 22q11.2 CNV carriers have been identified in humans and mouse models, it is unknown how the genes in the 22q11.2 region individually contribute to structural alterations and associated mental illnesses and their dimensions. Our previous studies have identified Tbx1, a T-box family transcription factor encoded in 22q11.2 CNV, as a driver gene for social interaction and communication, spatial and working memory, and cognitive flexibility. However, it remains unclear how TBX1 impacts the volumes of various brain regions and their functionally linked behavioral dimensions. In this study, we used volumetric magnetic resonance imaging analysis to comprehensively evaluate brain region volumes in congenic Tbx1 heterozygous mice. Our data show that the volumes of anterior and posterior portions of the amygdaloid complex and its surrounding cortical regions were reduced in Tbx1 heterozygous mice. Moreover, we examined the behavioral consequences of an altered volume of the amygdala. Tbx1 heterozygous mice were impaired for their ability to detect the incentive value of a social partner in a task that depends on the amygdala. Our findings identify the structural basis for a specific social dimension associated with loss-of-function variants of TBX1 and 22q11.2 CNV.

Genetic Screening of Targeted Region on the Chromosome 22q11.2 in Patients with Microtia and Congenital Heart Defect.

Microtia is a congenital malformation characterized by a small, abnormally shaped auricle (pinna) ranging in severity. Congenital heart defect (CHD) is one of the comorbid anomalies with microtia. However, the genetic basis of the co-existence of microtia and CHD remains unclear. Copy number variations (CNVs) of 22q11.2 contribute significantly to microtia and CHD, respectively, thus suggesting a possible shared genetic cause embedded in this genomic region. In this study, 19 sporadic patients with microtia and CHD, as well as a nuclear family, were enrolled for genetic screening of single nucleotide variations (SNVs) and CNVs in 22q11.2 by target capture sequencing. We detected a total of 105 potential deleterious variations, which were enriched in ear- or heart-development-related genes, including TBX1 and DGCR8. The gene burden analysis also suggested that these genes carry more deleterious mutations in the patients, as well as several other genes associated with cardiac development, such as CLTCL1. Additionally, a microduplication harboring SUSD2 was validated in an independent cohort. This study provides new insights into the underlying mechanisms for the comorbidity of microtia and CHD focusing on chromosome 22q11.2, and suggests that a combination of genetic variations, including SNVs and CNVs, may play a crucial role instead of single gene mutation.

TBX1 regulates myogenic differentiation by activating the TGFß-Smad2/3 pathway in myoblasts.

TBX1 is systematically conserved in the T-box transcription factor family and regulates craniofacial muscle development during various stages of myogenesis, including commitment, proliferation, terminal differentiation, and survival. However, the role and mechanism by which TBX1 regulates the myogenic development of myoblasts remains unclear. In our study, we overexpressed TBX1 in mouse C2C12 myoblasts using a lentivirus method. We found that TBX1 inhibited cell proliferation and muscle differentiation, which had no effect on apoptosis. During myogenic differentiation, we also found that TBX1 overexpressing cells regulate myogenic differentiation by upregulating the expression levels of Smad2 and Smad3 and downregulating the expression level of MEF2C. After treatment with a specific inhibitor of Smad3 (SIS3), the myogenic differentiation of wild-type and TBX1 overexpressing cells increased. Thus, TBX1 may regulate myoblast muscle differentiation by enhancing the expression of Smad2 and Smad3. TBX1 may be a therapeutic target for muscular dystrophy.

Congenital Heart Diseases: Genetic Risk Variants and Their Methylation Status.

(1) Background: The interaction between single nucleotide variants (SNVs) associated with congenital heart diseases (CHDs) and their gene methylation status has not been well researched. The aim of the present study was to determine if there is a relationship between the methy lation status (MS) of genes and the allelic variants associated with CHDs. (2) Methods: Seven SNVs of the genes AXIN1, TBX1, TBX20, and MTHFR were selected from the literature. DNA extraction, genotyping, and a methylation analysis were performed on healthy subjects and subjects with CHDs. (3) Results: Twenty-two subjects with CHDs were selected as the case group (15 with ventricular septal defects (VSDs) and 7 with atrial septal defects (ASDs)), and 44 healthy subjects comprised the control group. The MTHFR and AXIN1 genes were hypermethylated in the control group when compared to the case group. When analyzed separately, those with atrial septum defects exhibited greater methylation, except for the gene MTHFR where there were no differences. Only the alternate alleles of MTHFR showed a significantly different methylation status in those without cardiopathy. (4) Conclusions: The MTHFR and AXIN genes were hypermethylated in the control group; however, only the alternate alleles of MTHFR (rs1801133 and rs1801131) showed a significantly different methylation status.

A phenotypic rescue approach identifies lineage regionalization defects in a mouse model of DiGeorge syndrome.

TBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 (vB12) treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here, we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At the molecular level, in vivo vB12 treatment enabled us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SNAI2, also known as SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with, but distinct from, ISL1+ and TBX1+ populations. In addition, SNAI2+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos, and vB12 treatment restored cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in the core mesoderm and the arrangement of multiple lineages within the PhAp.

Multiple Recurrent Copy Number Variations (CNVs) in Chromosome 22 Including 22q11.2 Associated with Autism Spectrum Disorder.

Introduction: Autism spectrum disorder (ASD) is a developmental disorder that can cause substantial social, communication, and behavioral challenges. Genetic factors play a significant role in ASD, where the risk of ASD has been increased for unclear reasons. Twin studies have shown important evidence of both genetic and environmental contributions in ASD, where the level of contribution of these factors has not been proven yet. It has been suggested that copy number variation (CNV) duplication and the deletion of many genes in chromosome 22 (Ch22) may have a strong association with ASD. This study screened the CNVs in Ch22 in autistic Saudi children and assessed the candidate gene in the CNVs region of Ch22 that is most associated with ASD. Methods: This study included 15 autistic Saudi children as well as 4 healthy children as controls; DNA was extracted from samples and analyzed using array comparative genomic hybridization (aCGH) and DNA sequencing. Results: The aCGH detected (in only 6 autistic samples) deletion and duplication in many regions of Ch22, including some critical genes. Moreover, DNA sequencing determined a genetic mutation in the TBX1 gene sequence in autistic samples. This study, carried out using aCGH, found that six autistic patients had CNVs in Ch22, and DNA sequencing revealed mutations in the TBX1 gene in autistic samples but none in the control. Conclusion: CNV deletion and the duplication of the TBX1 gene could be related to ASD; therefore, this gene needs more analysis in terms of expression levels.

Morphogenesis of the Mammalian Aortic Arch Arteries.

The major vessels in mammals that take blood away from the heart and deliver it to the arms and the head take their origin from the aortic arch and are derived from the arteries formed within the embryonic pharyngeal arches. These pharyngeal arch arteries, initially symmetrical, form in a cranial to caudal sequence within the pharyngeal mesenchyme. They then undergo a complex process of remodeling to produce the asymmetrical brachiocephalic arteries as seen in the adult. A complex interaction between the tissues of the pharyngeal arches and the genes they express is required to ensure that arterial formation and remodeling is able to proceed normally. If this process is disrupted, life-threatening congenital cardiovascular malformations can occur, such as interruption of the aortic arch, isolation of individual arteries, or so-called vascular rings. Here, using state-of-the-art imaging techniques, we describe the morphogenesis of the arteries in humans and mice and the cardiovascular defects in the Tbx1 mutant mouse model. We provide details of the process of remodeling, clarifying also the morphogenesis of the external carotid artery and the so-called "migration" of the left subclavian artery.

MicroRNA-451a attenuates angiotensin II-induced cardiac fibrosis and inflammation by directly targeting T-box1.

Hypertension or angiotensin II (Ang II) induces cardiac inflammation and fibrosis, thus contributing to cardiac remodeling. MicroRNAs (miRNAs) are considered crucial regulators of cardiac homeostasis and remodeling in response to various types of stress. It has been reported that miR-451a is involved in regulating ischemic heart injury. However, its role in Ang II-induced cardiac fibrosis remains unknown. Cardiac remodeling was induced in mice by infusion of low-dose Ang II (490 ng/kg/min) with a minipump for 2 weeks. Echocardiography and histological examinations were performed to evaluate cardiac function and pathological changes. We observed that miR-451a expression was the most significantly downregulated in the hearts of Ang II-infused mice and in both primary cardiac myocytes and fibroblasts. Overexpression of miR-451a in mice significantly attenuated Ang II-induced cardiac fibrosis and inflammation. Conversely, knockdown of miR-451a in mice aggravated this effect. Bioinformatics analysis and a luciferase reporter assay revealed that TBX1 was a direct target of miR-451a. Mechanistically, miR-451a directly targeted TBX1 expression, which inhibited TGF-ß1 production in both cardiac myocytes and fibroblasts, inactivating of TGF-ß1/SMAD2/3 signaling, inhibiting myofibroblast differentiation and proinflammatory cytokine expression, and leading to attenuation of cardiac fibrosis and inflammation. In conclusion, these results indicate that miR-451a acts as a novel regulator of Ang II-induced cardiac fibrosis and inflammation by directly targeting TBX1, and may be a promising therapeutic target for treating hypertensive cardiac diseases.