Pathogenesis Of Congenital Heart Disease Research Articles

Getting to the heart of the matter: long non-coding RNAs in cardiac development and disease.

Cardiogenesis in mammals requires exquisite control of gene expression and faulty regulation of transcriptional programs underpins congenital heart disease (CHD), the most common defect among live births. Similarly, many adult cardiac diseases involve transcriptional changes and sometimes have a developmental basis. Long non-coding RNAs (lncRNAs) are a novel class of transcripts that regulate cellular processes by controlling gene expression; however, detailed insights into their biological and mechanistic functions are only beginning to emerge. Here, we discuss recent findings suggesting that lncRNAs are important factors in regulation of mammalian cardiogenesis and in the pathogenesis of CHD as well as adult cardiac disease. We also outline potential methodological and conceptual considerations for future studies of lncRNAs in the heart and other contexts.

Jun Is Required in Isl1-Expressing Progenitor Cells for Cardiovascular Development

Jun is a highly conserved member of the multimeric activator protein 1 transcription factor complex and plays an important role in human cancer where it is known to be critical for proliferation, cell cycle regulation, differentiation, and cell death. All of these biological functions are also crucial for embryonic development. Although all Jun null mouse embryos die at mid-gestation with persistent truncus arteriosus, a severe cardiac outflow tract defect also seen in human congenital heart disease, the developmental mechanisms are poorly understood. Here we show that murine Jun is expressed in a restricted pattern in several cell populations important for cardiovascular development, including the second heart field, pharyngeal endoderm, outflow tract and atrioventricular endocardial cushions and post-migratory neural crest derivatives. Several genes, including Isl1, molecularly mark the second heart field. Isl1 lineages include myocardium, smooth muscle, neural crest, endocardium, and endothelium. We demonstrate that conditional knockout mouse embryos lacking Jun in Isl1-expressing progenitors display ventricular septal defects, double outlet right ventricle, semilunar valve hyperplasia and aortic arch artery patterning defects. In contrast, we show that conditional deletion of Jun in Tie2-expressing endothelial and endocardial precursors does not result in aortic arch artery patterning defects or embryonic death, but does result in ventricular septal defects and a low incidence of semilunar valve defects, atrioventricular valve defects and double outlet right ventricle. Our results demonstrate that Jun is required in Isl1-expressing progenitors and, to a lesser extent, in endothelial cells and endothelial-derived endocardium for cardiovascular development but is dispensable in both cell types for embryonic survival. These data provide a cellular framework for understanding the role of Jun in the pathogenesis of congenital heart disease.

PITX2c Loss-of-Function Mutations Responsible for Congenital Atrial Septal Defects

Congenital heart disease (CHD) is the most common form of developmental anomaly and is the leading non-infectious cause of infant mortality. A growing body of evidence demonstrates that genetic risk factors are involved in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disease and the genetic determinants for CHD in most patients remain unclear. In the present study, the entire coding region and splice junction sites of the PITX2c gene, which encodes a homeobox transcription factor crucial for normal cardiovascular genesis, was sequenced in 150 unrelated patients with various CHDs. The 200 unrelated control individuals were subsequently genotyped. The functional characteristics of the mutations were explored using a dual-luciferase reporter assay system. As a result, two novel heterozygous PITX2c mutations, p.H98Q and p.M119T, were identified in 2 unrelated patients with atrial septal defects, respectively. The variations were absent in 400 control chromosomes and the affected amino acids were completely conserved evolutionarily. The two variants were both predicted to be disease-causing by MutationTaster and PolyPhen-2, and the functional analysis revealed that the PITX2c mutants were consistently associated with significantly reduced transcriptional activity compared with their wild-type counterpart. These findings firstly link PITX2c loss-of-function mutations to atrial septal defects in humans, which provide novel insight into the molecular mechanism responsible for CHD, suggesting potential implications for the early prophylaxis and allele-specific treatment of CHD.

Prevalence and spectrum of GATA5 mutations associated with congenital heart disease

Congenital heart disease (CHD) is the most prevalent form of developmental abnormality with an incidence of approximately 1% in live newborns, and is the leading non-infectious cause of infant mortality with more than 29% of neonates who die of birth defects having cardiac anomalies [ [1] Roger V.L. Go A.S. Lloyd-Jones D.M. et al. Heart disease and stroke statistics—2012 update: a report from the American Heart Association. Circulation. 2012; 125: e2-e220 Crossref PubMed Scopus (0) Google Scholar ]. Despite its striking prevalence and important clinical significance, the etiology responsible for CHD remains largely unknown. Now it is generally understood that abnormal cardiovascular development during embryogenesis may be attributed to an aberrant biological process that is heterogeneous and complex, with both environmental and genetic risk factors involved [ [2] Bruneau B.G. The developmental genetics of congenital heart disease. Nature. 2008; 451: 943-948 Crossref PubMed Scopus (565) Google Scholar ]. Great advance in developmental biology has contributed to the discovery of numerous transcriptional regulators, signaling molecules and structural proteins that are crucial for normal cardiogenesis. This mechanistic understanding of cardiac morphogenesis results in the identification of multiple CHD related genes by using positional cloning or direct candidate gene strategies [ [3] Cecchetto A. Rampazzo A. Angelini A. et al. From molecular mechanisms of cardiac development to genetic substrate of congenital heart diseases. Future Cardiol. 2010; 6: 373-393 Crossref PubMed Scopus (16) Google Scholar ]. Aggregating evidence demonstrates that cardiac transcription factor genes GATA4 and NKX2-5 are most commonly causally implicated in the pathogenesis of CHD [ [4] McCulley D.J. Black B.L. Transcription factor pathways and congenital heart disease. Curr Top Dev Biol. 2012; 100: 253-277 Crossref PubMed Scopus (211) Google Scholar ]. Nevertheless, CHD is genetically heterogeneous and the genetic determinants underlying CHD in a vast majority of patients are still to be identified.

Novel GATA6 Mutations Associated with Congenital Ventricular Septal Defect or Tetralogy of Fallot

Congenital heart disease (CHD) is the most common form of developmental malformation and is the leading noninfectious cause of infant mortality. Emerging evidence indicates that genetic defects are involved in the pathogenesis of CHD. Nevertheless, CHD is genetically heterogeneous, and the molecular basis for CHD in a majority of patients remains unknown. In this study, the whole coding region of GATA6, a gene encoding a zinc-finger transcription factor crucial for normal cardiogenesis, was sequenced in 380 unrelated patients with CHD. The relatives of the index patients harboring the identified mutations and 200 unrelated control individuals were subsequently genotyped. The functional effect of the mutations was characterized using a luciferase reporter assay system. As a result, two novel heterozygous GATA6 mutations, p.D404Y and p.E460X, were identified in two families with ventricular septal defect and tetralogy of Fallot, respectively. The mutations co-segregated with CHD in the families with complete penetrance, and were absent in 400 control chromosomes. Functional analysis demonstrated that the mutated GATA6 proteins were associated with significantly decreased transactivational activity in comparison with their wild-type counterpart. These findings provide novel insight into the molecular mechanism implicated in CHD, suggesting potential implications for the early prophylaxis and personalized treatment of CHD.

Homeobox C9 Is Not Potentially Related to Congenital Heart Disease in Chinese Patients

Congenital heart disease (CHD) is one of the most common human birth defects. The etiology and pathogenesis of CHD are complex and involve several genes as well as multiple changes in signaling pathways. The aim of this study was to identify potential pathological mutations in the Homeobox C9 (Hoxc9) gene in 350 Chinese children with CHD to further understand the etiology of CHD. Sequence analysis of the Hoxc9 gene in 350 nonsyndromic patients with CHD Result: We did not identify any nonsynonymous variants in the coding regions of Hoxc9 in the patients with CHD. We found one synonymous variant c.C564T (p. his188his) in one ventricular septal defect patient. We also identified four previously reported polymorphisms (rs56368105, rs12817092, rs34079606, and rs2241820) in CHD. We did not find any diagnostic alterations in the coding regions of Hoxc9 among the patients with CHD. Nevertheless, to our knowledge, this is the first study of Hoxc9 in nonsyndromic CHD and has expanded our overall knowledge of the etiology of this disease.

Combined Mutation Screening of NKX2-5, GATA4, and TBX5 in Congenital Heart Disease: Multiple Heterozygosity and Novel Mutations

BackgroundVariants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt–Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development.DesignIn order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes.ResultsTwo cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M).ConclusionsIn addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts.

Novel NKX2-5 mutations responsible for congenital heart disease

Congenital heart disease (CHD) is the most common birth defect and is the leading cause of infant morbidity and mortality resulting from birth defects. Increasing evidence demonstrates that genetic variation in the NKX2-5 gene, which encodes a homeobox-containing transcription factor crucial to cardiogenesis, is an important molecular determinant for CHD. Nevertheless, the genetic components underlying CHD remain largely unknown. We screened NKX2-5 for potential molecular defects in patients with CHD. The entire coding region of NKX2-5 was initially sequenced in a cohort of 268 unrelated patients with CHD. The relatives of the patients carrying identified mutations and 200 unrelated control individuals were subsequently genotyped. Three novel heterozygous missense NKX2-5 mutations, p.Q22K, p.R36S, and p.E54K, were identified in three families with autosomal dominantly inherited atrial septal defect, ventricular septal defect, and tetralogy of Fallot, respectively. These mutations, absent in 200 control individuals, appear to be highly conserved evolutionarily and co-segregated with CHD in the families, with complete penetrance. These findings expand the spectrum of mutations in NKX2-5 associated with CHD and provide new insight into the molecular etiology involved in the pathogenesis of CHD, which signifies potential implications for genetic diagnosis and gene-specific therapy for this common disease in newborns.

Planar Cell Polarity Signaling Pathway in Congenital Heart Diseases

Congenital heart disease (CHD) is a common cardiac disorder in humans. Despite many advances in the understanding of CHD and the identification of many associated genes, the fundamental etiology for the majority of cases remains unclear. The planar cell polarity (PCP) signaling pathway, responsible for tissue polarity in Drosophila and gastrulation movements and cardiogenesis in vertebrates, has been shown to play multiple roles during cardiac differentiation and development. The disrupted function of PCP signaling is connected to some CHDs. Here, we summarize our current understanding of how PCP factors affect the pathogenesis of CHD.

A novel GATA6 mutation in patients with tetralogy of Fallot or atrial septal defect

GATA6 is a member of the GATA family of transcription factors, and its expression and functions overlap with those of GATA4 during heart development. Mutations in GATA4 have been related to human congenital heart diseases (CHDs) in several studies, whereas mutations in GATA6 have only recently been reported in patients with persistent truncus arteriosus. Animal experiments have revealed critical roles for GATA6 in the development of the myocardium and cardiac morphogenesis, thereby highlighting the potential involvement of GATA6 defects in the pathogenesis of CHDs. Here, we screened the GATA6 in 270 individuals with sporadic CHDs by direct sequencing. After identification of the mutation, a luciferase reporter assay and real-time quantitative polymerase chain reaction were performed to detect functional changes in the mutant transcription factor. The same heterozygous missense mutation (Ser184Asn) was identified in three patients, including one with tetralogy of Fallot and two with atrial septal defects. This mutation was not found in 500 unrelated ethnically matched healthy subjects. Direct sequencing of this region in the parents of these three patients revealed the same mutation in one of the parents for each patient, and one of the parent carriers presented with a bicuspid aortic valve. Biological analysis revealed clearly decreased transcriptional activity of GATA6 Ser184Asn in vitro. All these data suggest that GATA6 Ser184Asn is a novel mutation associated with CHDs and has an important role in disease pathogenesis.

Genetics of Congenital Heart Disease

Cardiovascular malformations are the most common type of birth defect and result in significant mortality worldwide. The etiology for the majority of these anomalies remains unknown but genetic factors are being recognized as playing an increasingly important role. Advances in our molecular understanding of normal heart development have led to the identification of numerous genes necessary for cardiac morphogenesis. This work has aided the discovery of an increasing number of monogenic causes of human cardiovascular malformations. More recently, studies have identified single nucleotide polymorphisms and submicroscopic copy number abnormalities as having a role in the pathogenesis of congenital heart disease. This review discusses these discoveries and summarizes our increasing understanding of the genetic basis of congenital heart disease.

The role of HIRA gene in the pathogenesis of congenital heart disease

Conotruncal defects(CTD) is a kind of severe cardiovascular abnormality, which is the major cause of death in rinatal pelriod. Some researches suggest that CTD and 22q11 microdeletion had close relationship. In addition, HIRA gene is located in 22q11, which was speculated as a candidate gene in CTD. Key words: HIRA; Conotruncal defect; 22q11 microdeletion

Role of genomics in cardiovascular medicine.

As all branches of science grow and new experimental techniques become readily accessible, our knowledge of medicine is likely to increase exponentially in the coming years. Recently developed technologies have revolutionized our analytical capacities, leading to vast knowledge of many genes or genomic regions involved in the pathogenesis of congenital heart diseases, which are often associated with other genetic syndromes, coronary artery disease and non-ischemic cardiomyopathies and channelopathies. The knowledge-base of the genesis of cardiovascular diseases is likely going to be further revolutionized in this new era of genomic medicine. Here, we review the advances that have been made over the last several years in this field and discuss different genetic mechanisms that have been shown to underlie a variety of cardiovascular diseases.

Myocardin-Related Transcription Factors

The association of transcriptional coactivators with DNA-binding proteins provides an efficient mechanism to expand and modulate genetic information encoded within the genome. Myocardin-related transcription factors (MRTFs), including myocardin, MRTF-A/MKL1/MAL, and MRTF-B/MKL2, comprise a family of related transcriptional coactivators that physically associate with the MADS box transcription factor, serum response factor, and synergistically activate transcription. MRTFs transduce cytoskeletal signals to the nucleus, activating a subset of serum response factor-dependent genes promoting myogenic differentiation and cytoskeletal organization. MRTFs are multifunctional proteins that share evolutionarily conserved domains required for actin-binding, homo- and heterodimerization, high-order chromatin organization, and transcriptional activation. Mice harboring loss-of-function mutations in myocardin, MRTF-A, and MRTF-B, respectively, display distinct phenotypes, including cell autonomous defects in vascular smooth muscle cell and myoepithelial cell differentiation and function. This article reviews the molecular basis of MRTF function with particular focus on the role MRTFs play in regulating cardiovascular patterning, vascular smooth muscle cell and cardiomyocyte differentiation and in the pathogenesis of congenital heart disease and vascular proliferative syndromes.

Familial congenital heart disease, progressive atrioventricular block and the cardiac homeobox transcription factor gene NKX2.5:

D. Woodrow Benson Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA Sirs: Congenital heart disease has the highest incidence of all birth defects affecting about 6–8/1000 live births. These heart malformations are believed to arise during cardiac development, but there is little known about the underlying mechanisms of disease [12]. Cardiac development is a complex process, and in recent years, the role of transcription factors as possible navigators of development has been frequently investigated [7, 10, 19]. The cardiac transcription factor NKX2.5 was identified as the first genetic cause of nonsyndromic congenital heart disease [22]. A total of 33 heterozygous mutations in NKX2.5 have been reported in individuals with a variety of congenital heart malformations, including atrial septal defect, ventricular septal defect, tetralogy of Fallot and abnormalities of the tricuspid valve [2, 6, 8, 9, 11, 13, 14, 16, 18, 21–23]. Specific types of mutations result in progressive atrioventricular (AV) conduction disturbance, requiring pacemaker implantation. These studies have provided important insight into the genetic origins of cardiac malformations and, by providing tools for the developmental biologist, have contributed to improved insights into the pathogenesis of congenital heart disease [3, 5]. As such, these results are important to pediatricians, cardiologists and surgeons who diagnose, treat and provide longterm follow-up to these patients. Here we report a novel NKX2.5 mutation in a small family where progressive AV block and congenital heart disease was identified in two generations.

GATA-6 regulates semaphorin 3C and is required in cardiac neural crest for cardiovascular morphogenesis

GATA transcription factors play critical roles in restricting cell lineage differentiation during development. Here, we show that conditional inactivation of GATA-6 in VSMCs results in perinatal mortality from a spectrum of cardiovascular defects, including interrupted aortic arch and persistent truncus arteriosus. Inactivation of GATA-6 in neural crest recapitulates these abnormalities, demonstrating a cell-autonomous requirement for GATA-6 in neural crest-derived SMCs. Surprisingly, the observed defects do not result from impaired SMC differentiation but rather are associated with severely attenuated expression of semaphorin 3C, a signaling molecule critical for both neuronal and vascular patterning. Thus, the primary function of GATA-6 during cardiovascular development is to regulate morphogenetic patterning of the cardiac outflow tract and aortic arch. These findings provide new insights into the conserved functions of the GATA-4, -5, and -6 subfamily members and identify GATA-6 and GATA-6-regulated genes as candidates involved in the pathogenesis of congenital heart disease.

Developmental transitions in electrical activation patterns in chick embryonic heart.

The specialized conduction tissue network mediates coordinated propagation of electrical activity through the adult vertebrate heart. Following activation of the atria, the activation wave is slowed down in the atrioventricular canal or node, then spreads rapidly into the left and right ventricles via the His-Purkinje system (HPS). This results in the ventricle being activated from the apex toward the base and is thought to represent HPS function. The development of mature HPS function in embryogenesis follows significant phases of cardiac morphogenesis. Initially, cardiac impulse propagates in a slow, linear, and isotropic fashion from the sinus venosus at the most caudal portion of the tubular heart. Although the speed of impulse propagation gradually increases, ventricular activation in the looped heart still follows the direction of blood flow. Eventually, the immature base-to-apex sequence of ventricular activation undergoes an apparent reversal, maturing to apex-to-base pattern. The embryonic chick heart has been studied intensively by both electrophysiological and morphological techniques, and the morphology of its conduction system (which is similar to mammals) is well characterized. One interesting but seldom studied feature is the anterior septal branch (ASB), which came sharply to focus (together with the rest of the ventricular conduction system) in our birthdating studies. Using an optical mapping approach, we show that ASB serves to activate ventricular surface between stages 16 and 25, predating the functionality of the His bundle/bundle branches. Heart morphogenesis and conduction system formation are thus linked, and studying the abnormal activation patterns could further our understanding of pathogenesis of congenital heart disease.

Developmental changes in ventricular diastolic function correlate with changes in ventricular myoarchitecture in normal mouse embryos.

Both genetic and epigenetic factors, such as abnormal hemodynamics, affect cardiac morphogenesis and the pathogenesis of congenital heart disease. Diastolic function is an important determinant of cardiac function, and tools for evaluating diastolic function in the embryo would be very valuable for assessment of cardiac performance. Using histological measurements of ventricular myoarchitecture, Doppler assessment of ventricular inflow velocities, and direct measurement of ventricular pressure, we investigated developmental changes of ventricular diastolic function in the mouse embryos from embryonic days 9.5 to 19.5. Regression analysis showed that peak velocity of A wave (an index of passive compliance) correlated with the area of trabecular myocardium in right ventricle (RV) (r2=0.92, P<0.0001) and left ventricle (LV) (r2=0.93, P<0.0001). Peak velocity of E wave (an index of active relaxation) exponentially correlated with the area of compact myocardium in RV (r2=0.98, P<0.0001) and LV (r2=0.97, P<0.0001). We used these techniques to analyze FOG-2 null embryos. FOG-2 null embryos had thin compact myocardium, higher EDP and E/A ratio, smaller -dP/dt, and diminished sucking pressure than wild-type littermates, indicating that decreased ventricular diastolic function might be the primary cause of embryonic lethality. In conclusion, during embryogenesis the development of compact myocardium tightly regulates the development of ventricular distensibility. Our study in normal mice forms the basis for future studies of embryonic cardiac function in genetically manipulated mice with abnormalities of the cardiovascular system.

Serotonin is a novel survival factor of cardiomyocytes: mitochondria as a target of 5-HT2B receptor signaling.

Identification of factors regulating cardiomyocyte survival and growth is important to understand the pathogenesis of congenital heart diseases. Little is known about the molecular mechanism of cardiac functions triggered by serotonin. The link between signaling circuitry of external stimuli and the mitochondrial apoptotic machinery is of wide interest in cardiac diseases. Using cultured cardiomyocytes and 5-hydroxytryptamine (5-HT)2B-receptor knockout mice as an animal model of dilated cardiomyopathy, for the first time we show that serotonin via the Gq-coupled 5-HT2B-receptor protect cardiomyocytes against serum deprivation-induced apoptosis as manifested by DNA fragmentation, nuclear chromatin condensation, and TUNEL labeling. Serotonin prevents cytochrome c release and caspase-9 and -3 activation after serum deprivation via cross-talks between phosphatidylinositol-3 kinase/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Serotonin binding to 5-HT2B-receptor activates ERK kinases to inhibit Bax expression induced by serum deprivation. Serotonin via phosphatidylinositol-3 kinase/Akt can activate NF-kappaB that is required for the regulation of the mitochondrial adenine nucleotide translocator (ANT-1). Parallel to these observations, ultrastructural analysis in the 5-HT2B-receptor knockout mice heart revealed pronounced mitochondrial defects in addition to altered mitochondrial enzyme activities (cytochrome oxidase and succinate dehydrogenase) and ANT-1 and Bax expressions. These findings identify 5-HT as a novel survival factor targeting mitochondria in cardiomyocytes.

Sérotonine et système cardio-vasculaire : rôle du récepteur sérotoninergique 5-HT2B

Identification of factors controlling myocardial differentiation and proliferation is of great importance for understanding the pathogenesis of congenital heart diseases. The molecular mechanism by which serotonin (5-hydroxytryptamine, 5-HT) regulates embryonic development of heart and cardiovascular functions remained unknown until recently. Inactivation of the 5-HT2B receptor (5-HT2BR) gene leads to embryonic and neonatal death due to the following defects in the heart: (i) 5-HT2BR mutant embryos exhibit a lack of trabeculae in the heart and a reduction in the expression levels of a tyrosine kinase receptor, ErbB-2, leading to mid-gestation lethality. These in vivo data suggest that the 5-HT2BR coupled to the heterotrimeric G protein Gq uses the signaling pathway of ErbB-2 for cardiac differentiation. (ii) Newborn 5-HT2BR-ko mutant mice exhibit cardiac dilation resulting from contractility and structural deficits at the intercellular junctions between cardiomyocytes. (iii) In adult 5-HT2BR mutant mice, echocardiography and electrocardiography confirm the presence of left ventricular dilation and decreased systolic function. These results constitute the first genetic evidence that 5-HT, via the 5-HT2BR, regulates differentiation and proliferation during development as well as cardiac structure and function in adults.