The transcription factor Sox7 modulates endocardiac cushion formation contributed to atrioventricular septal defect through Wnt4/Bmp2 signaling.

Cardiac septum malformations account for the largest proportion in congenital heart defects. The transcription factor Sox7 has critical functions in the vascular development and angiogenesis. It is unclear whether Sox7 also contributes to cardiac septation development. We identified a de novo 8p23.1 deletion with Sox7 haploinsufficiency in an atrioventricular septal defect (AVSD) patient using whole exome sequencing in 100 AVSD patients. Then, multiple Sox7 conditional loss-of-function mice models were generated to explore the role of Sox7 in atrioventricular cushion development. Sox7 deficiency mice embryos exhibited partial AVSD and impaired endothelial to mesenchymal transition (EndMT). Transcriptome analysis revealed BMP signaling pathway was significantly downregulated in Sox7 deficiency atrioventricular cushions. Mechanistically, Sox7 deficiency reduced the expressions of Bmp2 in atrioventricular canal myocardium and Wnt4 in endocardium, and Sox7 binds to Wnt4 and Bmp2 directly. Furthermore, WNT4 or BMP2 protein could partially rescue the impaired EndMT process caused by Sox7 deficiency, and inhibition of BMP2 by Noggin could attenuate the effect of WNT4 protein. In summary, our findings identify Sox7 as a novel AVSD pathogenic candidate gene, and it can regulate the EndMT involved in atrioventricular cushion morphogenesis through Wnt4-Bmp2 signaling. This study contributes new strategies to the diagnosis and treatment of congenital heart defects.

Tmem2 restricts atrioventricular canal differentiation by regulating degradation of hyaluronic acid.

BACKGROUND: Atrioventricular valve development relies upon the precisely defined dimensions of the atrioventricular canal (AVC). Current models suggest that Wnt signaling plays an important role atop a pathway that promotes AVC development. The factors that confine AVC differentiation to the appropriate location, however, are less well understood. RESULTS: Transmembrane protein 2 (Tmem2) is a key player in restricting AVC differentiation: in zebrafish, tmem2 mutants display an expansion of AVC characteristics, but the molecular mechanism of Tmem2 function in this context remains unclear. Through structure-function analysis, we demonstrate that the extracellular portion of Tmem2 is crucial for its role in restricting AVC boundaries. Importantly, the Tmem2 ectodomain contains regions implicated in the depolymerization of hyaluronic acid (HA). We find that tmem2 mutant hearts exhibit excess HA deposition alongside broadened distribution of Wnt signaling. Moreover, addition of ectopic hyaluronidase can restore the restriction of AVC differentiation in tmem2 mutants. Finally, we show that alteration of a residue important for HA depolymerization impairs the efficacy of Tmem2 function during AVC development. CONCLUSIONS: Taken together, our data support a model in which HA degradation, regulated by Tmem2, limits the distribution of Wnt signaling and thereby confines the differentiation of the AVC.

Atrioventricular canal defect and genetic syndromes: The unifying role of sonic hedgehog.

The atrioventricular canal defect (AVCD) is a congenital heart defect (CHD) frequently associated with extracardiac anomalies (75%). Previous observations from a personal series of patients with AVCD and "polydactyly syndromes" showed that the distinct morphology and combination of AVCD features in some of these syndromes is reminiscent of the cardiac phenotype found in heterotaxy, a malformation complex previously associated with functional cilia abnormalities and aberrant Hedgehog (Hh) signaling. Hh signaling coordinates multiple aspects of left-right lateralization and cardiovascular growth. Being active at the venous pole the secondary heart field (SHF) is essential for normal development of dorsal mesenchymal protrusion and AVCD formation and septation. Experimental data show that perturbations of different components of the Hh pathway can lead to developmental errors presenting with partially overlapping manifestations and AVCD as a common denominator. We review the potential role of Hh signaling in the pathogenesis of AVCD in different genetic disorders. AVCD can be viewed as part of a "developmental field," according to the concept that malformations can be due to defects in signal transduction cascades or pathways, as morphogenetic units which may be altered by Mendelian mutations, aneuploidies, and environmental causes.

HIRA directly targets the enhancers of selected cardiac transcription factors during in vitro differentiation of mouse embryonic stem cells.

HIRA is a histone chaperone known to modulate gene expression through the deposition of H3.3. Conditional knockout of Hira in embryonic mouse hearts leads to cardiac septal defects. Loss of function mutation in HIRA, together with other chromatin modifiers, was found in patients with congenital heart diseases. However, the effects of HIRA on gene expression at earlier stages of cardiogenic mesoderm differentiation have not yet been studied. Differentiation of mouse embryonic stem cells (mESCs) towards cardiomyocytes mimics some of these early events and is an accepted model of these early stages. We performed RNA-Seq and H3.3-HA ChIP-seq on both WT and Hira-null mESCs and early cardiomyocyte progenitors of both genotypes. Analysis of RNA-seq data showed differential down regulation of cardiovascular development-related genes in Hira-null cardiomyocytes compared to WT cardiomyocytes. We found HIRA-dependent H3.3 deposition at these genes. In particular, we observed that HIRA influenced directly the expression of the transcription factors Gata6, Meis1 and Tbx2, essential for cardiac septation, through H3.3 deposition. We therefore identified new direct targets of HIRA during cardiac differentiation.

T-box genes and retinoic acid signaling regulate the segregation of arterial and venous pole progenitor cells in the murine second heart field.

The arterial and venous poles of the mammalian heart are hotspots of congenital heart defects (CHD) such as those observed in 22q11.2 deletion (or DiGeorge) and Holt-Oram syndromes. These regions of the heart are derived from late differentiating cardiac progenitor cells of the Second Heart Field (SHF) located in pharyngeal mesoderm contiguous with the elongating heart tube. The T-box transcription factor Tbx1, encoded by the major 22q11.2 deletion syndrome gene, regulates SHF addition to both cardiac poles from a common progenitor population. Despite the significance of this cellular addition the mechanisms regulating the deployment of common progenitor cells to alternate cardiac poles remain poorly understood. Here we demonstrate that Tbx5, mutated in Holt-Oram syndrome and essential for venous pole development, is activated in Tbx1 expressing cells in the posterior region of the SHF at early stages of heart tube elongation. A subset of the SHF transcriptional program, including Tbx1 expression, is subsequently downregulated in Tbx5 expressing cells, generating a transcriptional boundary between Tbx1-positive arterial pole and Tbx5-positive venous pole progenitor cell populations. We show that normal downregulation of the definitive arterial pole progenitor cell program in the posterior SHF is dependent on both Tbx1 and Tbx5. Furthermore, retinoic acid (RA) signaling is required for Tbx5 activation in Tbx1-positive cells and blocking RA signaling at the time of Tbx5 activation results in atrioventricular septal defects at fetal stages. Our results reveal sequential steps of cardiac progenitor cell patterning and provide mechanistic insights into the origin of common forms of CHD.

Novel mutation of GATA4 gene in Kurdish population of Iran with nonsyndromic congenital heart septals defects.

BACKGROUND: The mutations in GATA4 gene induce inherited atrial and ventricular septation defects, which is the most frequent forms of congenital heart defects (CHDs) constituting about half of all cases. METHOD: We have performed High resolution melting (HRM) mutation scanning of GATA4 coding exons of nonsyndrome 100 patients as a case group including 39 atrial septal defects (ASD), 57 ventricular septal defects (VSD) and four patients with both above defects and 50 healthy individuals as a control group. Our samples are categorized according to their HRM graph. The genome sequencing has been done for 15 control samples and 25 samples of patients whose HRM analysis were similar to healthy subjects for each exon. The PolyPhen-2 and MUpro have been used to determine the causative possibility and structural stability prediction of GATA4 sequence variation. RESULTS: The HRM curve analysis exhibit that 21 patients and 3 normal samples have deviated curves for GATA4 coding exons. Sequencing analysis has revealed 12 nonsynonymous mutations while all of them resulted in stability structure of protein 10 of them are pathogenic and 2 of them are benign. Also we found two nucleotide deletions which one of them was novel and one new indel mutation resulting in frame shift mutation, and 4 synonymous variations or polymorphism in 6 of patients and 3 of normal individuals. Six or about 50% of these nonsynonymous mutations have not been previously reported. CONCLUSION: Our results show that there is a spectrum of GATA4 mutations resulting in septal defects.

Long telomeres protect against age-dependent cardiac disease caused by NOTCH1 haploinsufficiency.

Diseases caused by gene haploinsufficiency in humans commonly lack a phenotype in mice that are heterozygous for the orthologous factor, impeding the study of complex phenotypes and critically limiting the discovery of therapeutics. Laboratory mice have longer telomeres relative to humans, potentially protecting against age-related disease caused by haploinsufficiency. Here, we demonstrate that telomere shortening in NOTCH1-haploinsufficient mice is sufficient to elicit age-dependent cardiovascular disease involving premature calcification of the aortic valve, a phenotype that closely mimics human disease caused by NOTCH1 haploinsufficiency. Furthermore, progressive telomere shortening correlated with severity of disease, causing cardiac valve and septal disease in the neonate that was similar to the range of valve disease observed within human families. Genes that were dysregulated due to NOTCH1 haploinsufficiency in mice with shortened telomeres were concordant with proosteoblast and proinflammatory gene network alterations in human NOTCH1 heterozygous endothelial cells. These dysregulated genes were enriched for telomere-contacting promoters, suggesting a potential mechanism for telomere-dependent regulation of homeostatic gene expression. These findings reveal a critical role for telomere length in a mouse model of age-dependent human disease and provide an in vivo model in which to test therapeutic candidates targeting the progression of aortic valve disease.

Dysregulated endocardial TGFß signaling and mesenchymal transformation result in heart outflow tract septation failure.

Heart outflow tract septation in mouse embryos carrying mutations in retinoic acid receptor genes fails with complete penetrance. In this mutant background, ectopic TGFß signaling in the distal outflow tract is responsible for septation failure, but it was uncertain what tissue was responsive to ectopic TGFß and why this response interfered with septation. By combining RAR gene mutation with tissue-specific Cre drivers and a conditional type II TGFß receptor (Tgfbr2) allele, we determined that ectopic activation of TGFß signaling in the endocardium is responsible for septation defects. Ectopic TGFß signaling results in ectopic mesenchymal transformation of the endocardium and thereby in improperly constituted distal OFT cushions. Our analysis highlights the interactions between myocardium, endocardium, and neural crest cells in outflow tract morphogenesis, and demonstrates the requirement for proper TGFß signaling in outflow tract cushion organization and septation.

[Potential role of CRELD1 gene in the pathogenesis of atrioventricular septal defect].

OBJECTIVE: To screen potential mutation of the CRELD1 gene in congenital atrioventricular septal defect (AVSD) and explore its functional implications. METHODS: Fragments encompassing the 11 coding exons of CRELD1 gene, including at least 50 bp of flanking intronic regions, were amplified with PCR and subjected to DNA sequencing. Results of sequencing were compared with predicted sequence from the GenBank database. Eukaryotic expression vector pcDNA3.1CRELD1 containing the mutational sequence was constructed. Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ RT-PCR) was applied to examine the expression of CRELD1, Tenascin C and Aggrecan. RESULTS: C857G was identified in a girl with an isolated partial AVSD. The mutation has resulted in a substitution of Alanine for Proline at amino acid 286 in the first cbEGF domain. Western blotting and FQ RT-PCR confirmed that the P286R missense mutation has been a gain-of-function mutation. Compared with the unloaded control, the Aggrecan mRNA expression was downregulated for both wild-type and mutant type samples (t=140.27 vs. 26.36, P < 0.01). The downregulation was more significant in mutant type (t=25.69, P=0.002). There was no significant difference of the Tenascin C expression between wild-type and the unload control (t=1.167, P> 0.05), whilst the Tenascin C expression was up-regulated in mutant type (t=6.66, P=0.022). CONCLUSION: Mutation of the CRELD1 gene may increase the risk for AVSD rather than being directly causative. The P286R mutation of CRELD1 can downregulate the expression of Aggrecan and upregulates the expression of Tenascin C protein, both of which are crucial to extracellular matrix in the formation of the atrioventricular septum. The P286R mutation of CRELD1 may be correlated to the occurrence of AVSD.

Oculofaciocardiodental syndrome: novel BCOR mutations and expression in dental cells.

Oculofaciocardiodental (OFCD) syndrome is a rare X-linked dominant condition. Mutations in BCOR have been described as causal in OFCD syndrome. Almost all BCOR mutations result in premature termination codons (PTCs); therefore, nonsense-mediated mRNA decay (NMD) might have an important role in pathogenesis. The purpose of this study was to identify BCOR mutations in two OFCD patients, if it present, and to clarify the pathogenesis of radiculomegaly using one OFCD patient's pulp and periodontal ligament (PDL) cells. In our genetic analysis, two novel BCOR mutations were found. We also examined the transcript levels and the effects of NMD using cultured pulp and PDL cells from one affected patient. BCOR expression was normal in pulp but reduced in PDL cells, which is consistent with the higher rates of NMD in PDL cells. The mutant PDL cells had unstable mutant transcripts and proliferated faster than did wild-type cells, but mutant pulp cells appeared normal by these measures. In summary, the nonsense and frameshift mutations, which introduce PTCs, were found to contribute to OFCD syndrome in our two patients. Furthermore, in PDL cells, the mutation resulting in a PTC corresponded to greater NMD, unstable mutant transcripts and increased cell proliferation, which may contribute to hyperactive root formation.

Imbalanced expression of Vcan mRNA splice form proteins alters heart morphology and cellular protein profiles.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.

Expression of the BMP receptor Alk3 in the second heart field is essential for development of the dorsal mesenchymal protrusion and atrioventricular septation.

RATIONALE: The dorsal mesenchymal protrusion (DMP) is a prong of mesenchyme derived from the second heart field (SHF) located at the venous pole of the developing heart. Recent studies have shown that perturbation of its development is associated with the pathogenesis of atrioventricular (AV) septal defect. Although the importance of the DMP to AV septation is now established, the molecular and cellular mechanisms underlying its development are far from fully understood. Prior studies have demonstrated that bone morphogenetic protein (BMP) signaling is essential for proper formation of the AV endocardial cushions and the cardiac outflow tract. A role for BMP signaling in regulation of DMP development remained to be elucidated. OBJECTIVE: To determine the role of BMP signaling in DMP development. METHODS AND RESULTS: Conditional deletion of the BMP receptor Alk3 from venous pole SHF cells leads to impaired formation of the DMP and a completely penetrant phenotype of ostium primum defect, a hallmark feature of AV septal defects. Analysis of mutants revealed decreased proliferative index of SHF cells and, consequently, reduced number of SHF cells at the cardiac venous pole. In contrast, volume and expression of markers associated with proliferation and active BMP/transforming growth factor ß signaling were not significantly altered in the AV cushions of SHF-Alk3 mutants. CONCLUSIONS: BMP signaling is required for expansion of the SHF-derived DMP progenitor population at the cardiac venous pole. Perturbation of Alk3-mediated BMP signaling from the SHF results in impaired development of the DMP and ostium primum defects.

Molecular signatures of cardiac defects in Down syndrome lymphoblastoid cell lines suggest altered ciliome and Hedgehog pathways.

Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD) and less frequently a ventricular septal defect (VSD) or atrial septal defect (ASD). Lymphoblastoid cell lines (LCLs) were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD(-); n = 22) were compared with those of LCLs from patients with cardiac malformations (CHD(+); n = 21). After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD) carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD(-) and AVSD and CHD(-) and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset). Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21.

Functional variant in methionine synthase reductase intron-1 significantly increases the risk of congenital heart disease in the Han Chinese population.

BACKGROUND: Homocysteine is known to be an independent risk factor for congenital heart disease (CHD). Methionine synthase reductase (MTRR) is essential for the adequate remethylation of homocysteine, which is the dominant pathway for homocysteine removal during early embryonic development. METHODS AND RESULTS: Here, we report that the c.56+781 A>C (rs326119) variant of intron-1 of MTRR significantly increases the risk of CHD in the Han Chinese population. In 3 independent case-control studies involving a total of 2340 CHD patients and 2270 healthy control participants from different geographic areas, we observed that patients carrying the heterozygous AC and homozygous CC genotype had a 1.40-fold (odds ratio=1.40; P=2.32×10(-7)) and 1.84-fold (odds ratio=1.84; P=2.3×10(-11)) increased risk, respectively, of developing CHD than those carrying the wild-type AA genotype. Both in vivo quantitative real-time polymerase chain reaction analysis of MTRR mRNA in cardiac tissue samples from CHD patients and in vitro luciferase assays in transfected cells demonstrated that the c.56+781 C allele profoundly decreased MTRR transcription. Further analysis demonstrated that the c.56+781 C allele manifested reduced CCAAT/enhancer binding protein-α binding affinity. In addition, healthy individuals with the homozygous CC genotype had significantly elevated levels of plasma homocysteine compared with the wild-type AA carriers. CONCLUSIONS: We have demonstrated that the MTRR c.56+781 A>C variant is an important genetic marker for increased CHD risk because this variant results in functionally reduced MTRR expression at the transcriptional level. Our results accentuate the significance of functional single-nucleotide polymorphisms in noncoding regions of the homocysteine/folate metabolism pathway core genes for their potential contributions to the origin of CHD.

Effects of regulator of G protein signaling 19 (RGS19) on heart development and function.

Wnt/Wg genes play a critical role in the development of various organisms. For example, the Wnt/beta-catenin signal promotes heart formation and cardiomyocyte differentiation in mice. Previous studies have shown that RGS19 (regulator of G protein signaling 19), which has Galpha subunits with GTPase activity, inhibits the Wnt/beta-catenin signal through inactivation of Galpha(o). In the present study, the effects of RGS19 on mouse cardiac development were observed. In P19 teratocarcinoma cells with RGS19 overexpression, RGS19 inhibited cardiomyocyte differentiation by blocking the Wnt signal. Additionally, several genes targeted by Wnt were down-regulated. For the in vivo study, we generated RGS19-overexpressing transgenic (RGS19 TG) mice. In these transgenic mice, septal defects and thin-walled ventricles were observed during the embryonic phase of development, and the expression of cardiogenesis-related genes, BMP4 and Mef2C, was reduced significantly. RGS19 TG mice showed increased expression levels of brain natriuretic peptide and beta-MHC, which are markers of heart failure, increase of cell proliferation, and electrocardiogram analysis shows abnormal ventricle repolarization. These data provide in vitro and in vivo evidence that RGS19 influenced cardiac development and had negative effects on heart function.

A functional genetic study identifies HAND1 mutations in septation defects of the human heart.

Heart and neural crest derivatives expressed 1 (HAND1) is a basic helix-loop-helix (bHLH) transcription factor essential for mammalian heart development. Absence of Hand1 in mice results in embryonal lethality, as well as in a wide spectrum of cardiac abnormalities including failed cardiac looping, defective chamber septation and impaired ventricular development. Therefore, Hand1 is a strong candidate for the many cardiac malformations observed in human congenital heart disease (CHD). Recently, we identified a loss-of-function frameshift mutation (p.A126fs) in the bHLH domain of HAND1 frequent in hypoplastic hearts. This finding prompted us to continue our search for HAND1 gene mutations in a different cohort of malformed hearts affected primarily by septation defects. Indeed, in tissue samples of septal defects, we detected 32 sequence alterations leading to amino acid change, of which 12 are in the bHLH domain of HAND1. Interestingly, 10 sequence alterations, such as p.L28H and p.L138P, had been identified earlier in hypoplastic hearts, but the frequent p.A126fs mutation was absent except in one aborted case with ventricular septal defect and outflow tract abnormalities. Functional studies in yeast and mammalian cells enabled translation of sequence alterations to HAND1 transcriptional activity, which was reduced or abolished by certain mutations, notably p.L138P. Our results suggest that HAND1 may also be affected in septation defects of the human hearts, and thus has a broader role in human heart development and CHD.

Dominant-negative ALK2 allele associates with congenital heart defects.

BACKGROUND: Serious congenital heart defects occur as a result of improper atrioventricular septum (AVS) development during embryogenesis. Despite extensive knowledge of the genetic control of AVS development, few genetic lesions have been identified that are responsible for AVS-associated congenital heart defects. METHODS AND RESULTS: We sequenced 32 genes known to be important in AVS development in patients with AVS defects and identified 11 novel coding single-nucleotide polymorphisms that are predicted to impair protein function. We focused on variants identified in the bone morphogenetic protein receptor, ALK2, and subjected 2 identified variants to functional analysis. The coding single-nucleotide polymorphisms R307L and L343P are heterozygous missense substitutions and were each identified in single individuals. The L343P allele had impaired functional activity as measured by in vitro kinase and bone morphogenetic protein-specific transcriptional response assays and dominant-interfering activity in vivo. In vivo analysis of zebrafish embryos injected with ALK2 L343P RNA revealed improper atrioventricular canal formation. CONCLUSIONS: These data identify the dominant-negative allele ALK2 L343P in a patient with AVS defects.

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development.

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth.

A critical role for the EphA3 receptor tyrosine kinase in heart development.

Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3(-/-) mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3(-/-) atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3(-/-) endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.

Blood cardioplegia provides superior protection in infant cardiac surgery.

BACKGROUND: We hypothesized that blood cardioplegia preserves myocardial metabolism and function more effectively than St Thomas' crystalloid cardioplegia in infant cardiac surgery. METHODS: Thirty infants with atrioventricular septal defects were randomly allocated to either blood or crystalloid intermittent cold (4 degrees C) cardioplegia. Arterial and coronary sinus blood was analyzed for lactate and oxygen. Cardiac output (thermodilution) and left ventricular function (echocardiography) were evaluated. RESULTS: The lactate concentration in coronary sinus blood early after bypass was significantly higher after crystalloid cardioplegia than after blood cardioplegia (2.1 +/- 0.3 vs 1.3 +/- 0.1 mmol/L, p = 0.006), with a significant myocardial release of lactate after crystalloid but not after blood cardioplegia. Oxygen extraction (arterial-coronary sinus O2 content) was higher early after crystalloid cardioplegia (3.02 +/- 0.13 vs 2.35 +/- 0.22 mmol/L, p = 0.01), possibly reflecting a difference in oxygen debt. The cardiac index was higher after blood cardioplegia (4.9 +/- 0.3 vs 4.0 +/- 0.3 L/min(-1)/m(-2), p = 0.04) and echocardiographic grading of left ventricular function was better (4.1 +/- 0.17 vs 3.5 +/- 0.22 arbitrary units, p = 0.046). CONCLUSIONS: This study indicates that blood cardioplegia preserves myocardial metabolism and function more effectively than crystalloid cardioplegia in infant cardiac surgery. The clinical significance of this finding is uncertain, but the more than 20% increase in cardiac index in the critical phase during weaning from bypass may be advantageous.