Regulation Of Cardiac Development Research Articles

Cardiac-specific overexpression of CREM-IbΔC-X via CRISPR/Cas9 in mice presents a new model of atrial cardiomyopathy with spontaneous atrial fibrillation

Atrial cardiomyopathy (ACM) forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. However, generating stable animal models that accurately replicate the entire progression of atrial lesions, particularly the onset of AF, presents significant challenges. In the present study, we found that the isoform of CRE-binding protein modulator (CREM-IbΔC-X), which is involved in the regulation of cardiac development and atrial rhythm, was highly expressed in atrial biopsies from patients with AF. Building upon this finding, we employed CRISPR/Cas9 technology to create a mouse model with cardiac-specific overexpression of CREM-IbΔC-X (referred to as CS-CREM mice). This animal model effectively illustrated the development of ACM through electrophysiological and structural remodelings over time. Proteomics and Chip-qPCR analysis of atrial samples revealed significant upregulation of cell-matrix adhesion and extracellular matrix structural components, alongside significant downregulation of genes related to atrial functions in the CS-CREM mice. Furthermore, the corresponding responses to anti-arrhythmia drugs, i.e., amiodarone and propafenone, suggested that CS-CREM mice could serve as an ideal in vivo model for drug testing. Our study introduced a novel ACM model with spontaneous AF by cardiac-specifically overexpressing CREM-IbΔC-X in mice, providing valuable insights into the mechanisms and therapeutic targets of ACM.

Hand2os1 Regulates the Secretion of Progesterone in Mice Corpus Luteum.

Simple SummaryThe corpus luteum plays a key role in pregnancy maintenance and estrous cycle regulation by secreting progesterone. In this study, we investigate the expression and regulation of lncRNA Hand2os1 in the ovaries. We found Hand2os1 was specifically detected in luteal cells during the proestrus and estrus phases, and strongly expressed in the corpus luteum on day 4 and day 18 of pregnancy. Moreover, Hand2os1 regulates the secretion of progesterone in the mouse corpus luteum by affecting the key rate-limiting enzyme StAR, which suggests it may have an impact on the maintenance of pregnancy.The corpus luteum plays a key role in pregnancy maintenance and estrous cycle regulation by secreting progesterone. Hand2os1 is an lncRNA located upstream of Hand2, with which a bidirectional promoter is shared and is involved in the regulation of cardiac development and embryo implantation in mice. The aim of this study was to investigate the expression and regulation of Hand2os1 in the ovaries. Here, we used RNAscope to detect differential expression of Hand2os1 in the ovaries of cycling and pregnant mice. Hand2os1 was specifically detected in luteal cells during the proestrus and estrus phases, showing its highest expression in the corpus luteum at estrus. Additionally, Hand2os1 was strongly expressed in the corpus luteum on day 4 of pregnancy, but the positive signal progressively disappeared after day 8, was detected again on day 18, and gradually decreased after delivery. Hand2os1 significantly promoted the synthesis of progesterone and the expression of StAR and Cyp11a1. The decreased progesterone levels caused by Hand2os1 interference were rescued by the overexpression of StAR. Our findings suggest that Hand2os1 may regulate the secretion of progesterone in the mouse corpus luteum by affecting the key rate-limiting enzyme StAR, which may have an impact on the maintenance of pregnancy.

Bmp Signaling Regulates Hand1 in a Dose-Dependent Manner during Heart Development.

The bone morphogenetic protein (Bmp) signaling pathway and the basic helix–loop–helix (bHLH) transcription factor Hand1 are known key regulators of cardiac development. In this study, we investigated the Bmp signaling regulation of Hand1 during cardiac outflow tract (OFT) development. In Bmp2 and Bmp4loss-of-function embryos with varying levels of Bmp in the heart, Hand1 is sensitively decreased in response to the dose of Bmp expression. In contrast, Hand1 in the heart is dramatically increased in Bmp4 gain-of-function embryos. We further identified and characterized the Bmp/Smad regulatory elements in Hand1. Combined transfection assays and chromatin immunoprecipitation (ChIP) experiments indicated that Hand1 is directly activated and bound by Smads. In addition, we found that upon the treatment of Bmp2 and Bmp4, P19 cells induced Hand1 expression and favored cardiac differentiation. Together, our data indicated that the Bmp signaling pathway directly regulates Hand1 expression in a dose-dependent manner during heart development.

HOPX Exhibits Oncogenic Activity during Squamous Skin Carcinogenesis

Cutaneous squamous cell carcinomas (SCCs) are frequent heterogeneous tumors arising from sun-exposed regions of the skin and characterized by complex pathogenesis. HOPX is a member of the homeodomain-containing superfamily of proteins holding an atypical homeodomain unable to bind to DNA. First discovered in the heart as a regulator of cardiac development, in the skin, HOPX modulates the terminal differentiation of keratinocytes. There is a particular interest in studying HOPX in squamous skin carcinogenesis because it has the atypical structure and the functional duality as an oncogene and a tumor suppressor gene, reported in different malignancies. In this study, we analyzed the effects of HOPX knockdown and overexpression on SCC tumorigenicity invitro and invivo. Our data show that HOPX knockdown in SCC cells inhibits their proliferative and invasive activity through the acceleration of apoptosis. We established that methylation of two alternative HOPX promoters leads to differential expression of HOPX transcripts in normal keratinocytes and SCC cells. Importantly, we report that HOPX acts as an oncogene in the pathogenesis of SCC probably through the activation of the second alternative promoter and the modulation of apoptosis.

Aberrant expression of miR‐29b‐3p influences heart development and cardiomyocyte proliferation by targeting NOTCH2

ObjectivesmicroRNA‐29 (miR‐29) family have shown different expression patterns in cardiovascular diseases. Our study aims to explore the effect and mechanism of miR‐29 family on cardiac development.Materials and methodsA total of 13 patients with congenital heart disease (CHD) and 7 controls were included in our study. Tissues were obtained from the right ventricular outflow tract (RVOT) after surgical resection or autopsy. The next‐generation sequencing was applied to screen the microRNA expression profiles of CHD. Quantitative RT‐PCR and Western blot were employed to measure genes expression. Tg Cmlc2: GFP reporter zebrafish embryos were injected with microRNA (miRNA) to explore its role in cardiac development in vivo. Dual‐luciferase reporter assay was designed to validate the target gene of miRNAs. CCK‐8 and EdU incorporation assays were performed to evaluate cardiomyocyte proliferation.ResultsOur study showed miR‐29b‐3p expression was significantly increased in the RVOT of the CHD patients. Injection of miR‐29b‐3p into zebrafish embryos induced higher mortality and malformation rates, developmental delay, cardiac malformation and dysfunction. miR‐29b‐3p inhibited cardiomyocyte proliferation, and its inhibitor promoted cardiomyocyte proliferation in vitro and in vivo. Furthermore, we identified that miR‐29b‐3p influenced cardiomyocyte proliferation by targeting NOTCH2, which was down‐regulated in the RVOT of the CHD patients.ConclusionThis study reveals that miR‐29b‐3p functions as a novel regulator of cardiac development and inhibits cardiomyocyte proliferation via NOTCH2, which provides novel insights into the aetiology and potential treatment of CHD.

MiR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations.

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.

Role of WW domain E3 ubiquitin protein ligase 2 in modulating ubiquitination and Degradation of Septin4 in oxidative stress endothelial injury

Oxidative stress-associated endothelial injury is the initial event and major cause of multiple cardiovascular diseases such as atherosclerosis and hypertensive angiopathy. A protein homeostasis imbalance is a critical cause of endothelial injury, and homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligases are the core factors controlling protein homeostasis. Although HECT-type E3 ubiquitin ligases are involved in the regulation of cardiac development and diseases, their roles in endothelial injury remain largely unknown. This study aimed to identify which HECT-type E3 ubiquitin ligase is involved in endothelial injury and clarify the mechanisms at molecular, cellular, and organism levels. We revealed a novel role of the HECT-type E3 ubiquitin ligase WWP2 in regulating endothelial injury and vascular remodeling after endothelial injury. Endothelial/myeloid-specific WWP2 knockout in mice significantly aggravated angiotensin II/oxidative stress-induced endothelial injury and vascular remodeling after endothelial injury. The same results were obtained from in vitro experiments. Mechanistically, the endothelial injury factor Septin4 was identified as a novel physiological substrate of WWP2. In addition, WWP2 interacted with the GTPase domain of Septin4, ubiquitinating Septin4-K174 to degrade Septin4 through the ubiquitin-proteasome system, which inhibited the Septin4-PARP1 endothelial damage complex. These results identified the first endothelial injury-associated physiological pathway regulated by HECT-type E3 ubiquitin ligases in vivo as well as a unique proteolytic mechanism through which WWP2 controls endothelial injury and vascular remodeling after endothelial injury. These findings might provide a novel treatment strategy for oxidative stress-associated atherosclerosis and hypertensive vascular diseases.

Abstract 472: Smyd1 Variants Regulate Distinct Areas of Chromatin in the Cardiomyocyte

Heart failure is one of the most devastating conditions that contributes to 25% of all deaths in the USA every year. The development of heart failure, which is often preceded by cardiac hypertrophy resulting from an increased workload, is accompanied by global changes in gene expression that resemble a more fetal-like transcriptome. Although many epigenetic factors have been identified, which influence these underlying changes in transcription during disease, we still know very little about how this process is regulated on a molecular level. Smyd1 is a unique histone methyltransferase that modifies lysine residues to regulate gene expression in the cardiomyocyte. Originally, Smyd1 was identified as a necessary regulator of cardiac development, but recently we have shown that Smyd1 is differentially expressed during cardiac hypertrophy and failure and that loss of Smyd1 (in conditional, cardiomyocyte-specific knockout mice) leads to hypertrophic growth, metabolic dysfunction and ultimately heart failure. In mice, the smyd1 gene produces two variants, Smyd1a and Smyd1b, which differ only by a 13 amino acid deletion. In addition, we have shown that overexpression of Smyd1a (but not Smyd1b) can prevent phenylephrine-induced hypertrophy, however, both variants are capable of suppressing ANF expression. Despite this interesting data, the specific genomic regions bound by these variants and the unique genes they regulate are unknown. Therefore, to identify the genes regulated by Smyd1 variants we performed ChIP-Seq in cardiomyocytes under normal and hypertrophic conditions. Interestingly, our analyses show that Smyd1a binds DNA more frequently than Smyd1b under basal conditions (2,392 vs 1,004 binding events, respectively) but that Smyd1b exhibits greater binding in hypertrophic conditions (2,414 vs 5,045 binding events). Moreover, bioinformatics analysis of these sites confirmed both conserved and unique binding preferences and showed that while both variants are most abundant in distal intergenic regions (32-57%), Smyd1a is uniquely enriched (24%) at gene promoters (<1kb). These exciting results suggest novel and distinct regulatory functions of Smyd1 isoforms and begin to inform us how Smyd1 regulates cardiac morphology and physiology.

Abstract 894: Inhibition of Long Noncoding RNA lncExACT1 Induces Physiological Cardiac Hypertrophy and Protects Against Pathological Hypertrophy

Long noncoding-RNAs (lncRNAs) are critical regulators of cardiac development as well as pathological hypertrophy and heart failure (HF). However, their roles in exercise-induced physiological hypertrophy are unclear. Here, we used RNAseq to identify a novel class of cardiac lncRNAs that are dynamically regulated by exercise. We call these l ong n on c oding Ex ercise A ssociated C ardiac T ranscripts (lncExACTs). Among them, lncExACT1, a highly conserved lncRNA, is down-regulated in exercised hearts but upregulated in transverse aortic constriction (TAC)-induced pathological hypertrophy and HF. In primary neonatal cardiomyocytes (CMs), transfection of LNA antisense oligonucleotide complementary to lncExACT1 (GapmeR) was sufficient to inhibit lncExACT1 expression and increase CM size with a gene expression pattern consistent with physiological hypertrophy (increased PCG1α and α/βMHC ratio, all p <0.05 vs . control). In contrast, lentiviral overexpression of lncExACT1 in primary CMs induced a pathological gene expression pattern (decreased PCG1α and β/αMHC ratio, with increased ANP and BNP, all p <0.05 vs . control). In vivo, GapmeR treatment for two weeks reduced cardiac lncExACT1 expression (0.5-fold at 2 weeks), increased ventricular wall thickness and fractional shortening (FS, p =0.034 vs . control), increased heart weight to tibial length ratio (HW/TL) and α/βMHC. In contrast, injection of AAV9-lncExACT1 increased cardiac lncExACT1 (6-fold at 5 weeks) increased HW/TL as well as βMHC and ANP. Further, lncExACT1 acted as a sponge for microRNA-222 (a microRNA we previously reported is necessary for physiological cardiac growth) thereby increasing expression of microRNA-222 targets, including p27, a cell cycle inhibitor protein. Moreover, GapmeR injection reduced TAC-induced increase of interventricular septal end diastole (IVSd) and left Ventricular Posterior Wall Dimensions (LVPWd) without affecting FS. We conclude that inhibition of lncExACT1 is sufficient to induce physiological hypertrophy and protect against pathological hypertrophy, while induction of lncExACT1 promotes pathological hypertrophy. lncExACT1 appears to mediate these effects, at least in part, by acting as competing endogenous RNA for microRNA-222.

Abstract 785: Modeling Congenital Heart Disease-Associated Variants in GATA6 Using CRISPR/Cas9 and Human Induced Pluripotent Stem Cells

The discovery of damaging gene mutations in congenital heart disease (CHD) patients enables identification of regulators of cardiac development. Exome sequencing identified de novo heterozygous loss-of-function (LoF) and missense variants in GATA6 among CHD probands, most with outflow tract malformations. Other subjects with GATA6 LoF mutations developed pancreatic agenesis. To elucidate the molecular basis for the predominance of this heart defect, we modeled GATA6 mutations in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). GATA6 variants were introduced into isogenic hiPSCs using CRISPR/Cas9 genome editing. Genome-wide molecular profiles including chromatin accessibility (ATAC-Seq) and gene expression (single cell and bulk RNA-Seq) were evaluated during hiPSC-CM differentiation. Analyses of GATA6 mutant hiPSC-CMs showed deficits in hiPSC-CM differentiation, chromatin accessibility and transcriptional profiles. Heterozygous GATA6 LoF hiPSCs made hiPSC-CMs but exhibited reduced expression of second heart field genes. Homozygous GATA6 LoF hiPSCs failed to differentiate and adopted fibroblast expression profiles. hiPSCs carrying a homozygous GATA6 missense variant, R456G, which altered a DNA-binding domain residue, showed enhanced capacity to differentiate into neuroepithelial-like cells. Chromatin-accessibility studies confirmed that GATA6 normally binds to genes in the promoter region and other genes at distal enhancers. Human GATA6 haploinsufficiency disrupts developmental transcriptional responses driving cardiac morphogenesis. The HAND2 -dependent genetic program, operant during outflow tract development, is particularly sensitive to GATA6 dosage. The mixed differentiation patterns observed in mutation-carrying hiPSCs likely contributes to vascular phenotypes observed in CHD patients. GATA6 haploinsufficiency preferentially alters binding of distal enhancers to promoters in genes where GATA6 normally binds the enhancer rather than the promoter. We speculate that pathogenicity of GATA6 haploinsufficiency is mediated by weaker binding of GATA6 to distal enhancers than to promoter elements, altering expression of these genes in GATA6 haploinsufficient patients.

A polymorphism rs3746444 within the pre-miR-499 alters the maturation of miR-499-5p and its antiapoptotic function.

microRNAs (miRNAs) are non‐coding RNAs that function as post‐transcriptional regulators of cardiac development and cardiovascular diseases. Single nucleotide polymorphisms (SNPs) in miRNA genes are a novel class of genetic variations in the human genome that confer the risk of cardiovascular diseases. Here, we identified a polymorphism A→G (rs3746444) in miR‐499 precursor (pre‐miR‐499) that affects the maturation of miR‐499‐5p and alters its antiapoptotic function by converting stable A‐U base pair to wobble G‐U base pair in pre‐miR‐499 secondary structure. Furthermore, our results showed that the concentrations of plasma miR‐499‐5p could be correlated with myocardial infarction (MI) and heart failure (HF) patients in comparison with control subjects and polymorphism rs3746444 in miR‐499 could influence its abundance in plasma. Finally, our results also showed that the variant of polymorphism in miR‐499 influenced the severity of the myocardial infarction significantly. This is the first report to highlight the biological significance of this polymorphism on the maturation of miR‐499‐5p and its antiapoptotic role during MI. These findings may pave a way to better understand the individual variability based on miRNA SNPs in heart diseases and may contribute to better treatment for disease severity on a personalized level.

Approaches to augment vascularisation and regeneration of the adult heart via the reactivated epicardium.

Survival rates following myocardial infarction have increased in recent years but current treatments for post-infarction recovery are inadequate and cannot induce regeneration of damaged hearts. Regenerative medicine could provide disease-reversing treatments by harnessing modern concepts in cell and developmental biology. A recently-established paradigm in regenerative medicine is that regeneration of a tissue can be achieved by reactivation of the coordinated developmental processes that originally formed the tissue. In the heart, the epicardium has emerged as an important regulator of cardiac development and reactivation of epicardial developmental processes may provide a means to enable cardiac regeneration. Indeed, in adult mouse hearts, treatment with thymosin β4 and other drug-like molecules reactivates the epicardium and improves outcomes after myocardial infarction by inducing regenerative paracrine signalling, neovascularisation and de novo cardiomyocyte production. However, there are considerable limitations to current methods of epicardial reactivation that prevent direct translation into clinical practice. Here, we describe the rationale for targeting the epicardium and the successes and limitations of this approach. We consider how several recent advances in epicardial biology could be used to overcome these limitations. These advances include insight into epicardial signalling and heterogeneity, epicardial modulation of inflammation and epicardial remodelling of extracellular matrix.

Loss of Mouse P2Y6 Nucleotide Receptor Is Associated with Physiological Macrocardia and Amplified Pathological Cardiac Hypertrophy

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy.

Common miR-590 Variant rs6971711 Present Only in African Americans Reduces miR-590 Biogenesis.

MicroRNAs (miRNAs) are recognized as important regulators of cardiac development, hypertrophy and fibrosis. Recent studies have demonstrated that genetic variations which cause alterations in miRNA:target interactions can lead to disease. We hypothesized that genetic variations in miRNAs that regulate cardiac hypertrophy/fibrosis might be involved in generation of the cardiac phenotype in patients diagnosed with hypertrophic cardiomyopathy (HCM). To investigate this question, we Sanger sequenced 18 miRNA genes previously implicated in myocyte hypertrophy/fibrosis and apoptosis, using genomic DNA isolated from the leukocytes of 199 HCM patients. We identified a single nucleotide polymorphism (rs6971711, C57T SNP) at the 17th position of mature miR-590-3p (= 57th position of pre-miR-590) that is common in individuals of African ancestry. SNP frequency was higher in African American HCM patients (n = 55) than ethnically-matched controls (n = 100), but the difference was not statistically significant (8.2% vs. 6.5%; p = 0.5). Using a cell culture system, we discovered that presence of this SNP resulted in markedly lower levels of mature miR-590-5p (39 ± 16%, p<0.003) and miR-590-3p (20 ± 2%, p<0.003), when compared with wild-type (WT) miR-590, without affecting levels of pri-miR-590 and pre-miR-590. Consistent with this finding, the SNP resulted in reduced target suppression when compared to WT miR-590 (71% suppression by WT vs 60% suppression by SNP, p<0.03). Since miR-590 can regulate TGF-β, Activin A and Akt signaling, SNP-induced reduction in miR-590 biogenesis could influence cardiac phenotype by de-repression of these signaling pathways. Since the SNP is only present in African Americans, population studies in this patient population would be valuable to investigate effects of this SNP on myocyte function and cardiac physiology.

0177 : Deciphering multiple roles of Nkx2-5 during ventricular non-compaction

Left ventricular non-compaction (LVNC), characterized by prominent trabeculations and deep trabecular recesses in the ventricles, is the most common cardiomyopathy with a spectrum ranging from extreme normal variants to a pathological phenotype. In all forms, heart failure and sudden cardiac death represent the most severe complications related with noncompaction and arrhythmias. The presence of ventricular trabeculae is a normal component of ventricular myocardium during embryonic development. Trabeculae are also progenitors of the ventricular conduction system (VCS), which is composed of a complex network of Purkinje fibers controlling the rapid propagation of electrical activity in the ventricles. The transcription factor Nkx2.5 is a key regulator of cardiac development. Conduction disturbances and ventricular non-compaction are traits that have been observed in patients and mutant mice carrying NKX2-5 mutations. However, the relationship between Purkinje fiber differentiation and trabecular compaction is unknown, despite recent evidence that abnormal conduction system morphology underlies conduction defects and arrhythmias. In order to dissect the role of this gene in the apparition of the pathological outcomes of LVNC, we specifically inactivate Nkx2-5 in a time and tissue specific manner. Nkx2-5 floxed mice were crossed with Cx40-creERT2 mice and tamoxifen injections, to induce gene deletion, were performed at different time of trabecular development to detect the impact of this gene in ventricular trabeculation, compaction and in the VCS. Morphological and functional cardiac phenotypes in these different mutant mice were analysed by oupling non-invasive imaging techniques and immunofluorescent studies. Preliminary results showed that the degree of non-compaction and the VCS morphology differ according to the timing of Nkx2-5 inactivation, suggesting multiple roles for this transcription factor during ventricular trabeculae development. The author hereby declares no conflict of interest

Mitochondrial Function during and Regulation of Cardiac Development

The roles played by mitochondria during cardiac development are poorly understood. However, recent data has suggested that mitochondria are vitally important for normal formation and function of the heart in the embryo, the fetus, and the neonate. As the heart develops, mitochondrial ATP production becomes increasingly important, while mitochondrial regulation of the cellular redox state is important for cardiac myocyte differentiation. The mechanisms that control mitochondrial function during this period are becoming clearer and may include changes in protein acetylation, permeability transition pore activity, assembly of the electron transport chain, and mitochondrial dynamics and biogenesis.

Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation

Vertebrate heart formation is a spatiotemporally regulated morphogenic process that initiates with bilaterally symmetric cardiac primordial cells migrating toward the midline to form a linear heart tube. The heart tube then elongates and undergoes a series of looping morphogenesis, followed by expansions of regions that are destined to become primitive heart chambers. During the cardiac morphogenesis, cells derived from the first heart field contribute to the primary heart tube, and cells from the secondary heart field, cardiac neural crest, and pro-epicardial organ are added to the heart tube in a precise spatiotemporal manner. The coordinated addition of these cells and the accompanying endocardial cushion morphogenesis yield the atrial, ventricular, and valvular septa, resulting in the formation of a four-chambered heart. Perturbation of progenitor cells' deployment and differentiation leads to a spectrum of congenital heart diseases. Two of the genes that were recently discovered to be involved in cardiac morphogenesis are Numb and Numblike. Numb, an intracellular adaptor protein, distinguishes sibling cell fates by its asymmetric distribution between the two daughter cells and its ability to inhibit Notch signaling. Numb regulates cardiac progenitor cell differentiation in Drosophila and controls heart tube laterality in Zebrafish. In mice, Numb and Numblike, the Numb family proteins (NFPs), function redundantly and have been shown to be essential for epicardial development, cardiac progenitor cell differentiation, outflow tract alignment, atrioventricular septum morphogenesis, myocardial trabeculation, and compaction. In this review, we will summarize the functions of NFPs in cardiac development and discuss potential mechanisms of NFPs in the regulation of cardiac development.

Abstract 304: Crystal Structure Of Fak/mef2 Complex Reveals The Role Of Fak In The Regulation Of Mef2 Transcription Factor.

Members of MEF2 (Myocyte Enhancer Factor 2) family of transcription factors are major regulators of cardiac development and homeostasis. Their functions are regulated at several levels, including the association with a variety of protein partners. We have previously shown that FAK (Focal Adhesion Kinase) regulates the stretch-induced activation of MEF2 in cardiomyocytes. But, the molecular mechanisms, involved in this process, are unclear. Here, we integrated biochemical, imaging and structural analyses to characterize a novel interaction between MEF2 and FAK. An association between MEF2 and FAK was detected by co-immunoprecipitation in the extracts of stretched cardiomyocytes (10%, 60Hz, 2 hours). MEF2 and FAK staining were co-localized in the nuclei of stretched cells. Pull down assays indicated that the Focal Adhesion Targeting (FAT) domain is sufficient to confer FAK interaction with MEF2. Gene reporter assays indicated that the interaction with FAK enhances the MEF2C transcriptional activity in cultured cardiomyocytes. Also, we present a 2.9-Å X-ray crystal structure for the FAK_FAT domain bound to MEF2C (1-95), comprised by the MADS box/MEF2 domain. The structural information, when used in combination with biochemical studies, small-angle X-ray scattering (SAXS) data and reporter gene assay, lead to a mechanistic model describing how FAK binds to MEF2C and stimulates its transcription function in cardiomyocytes. We further validated this model by showing that the binding of FAK to MEF2C is essential for the hypertrophy of cardiomyocyte in response to mechanical stress. Our results present FAK as a new positive regulator of MEF2, implicated in the fine control of the signal transduction between focal adhesions and the nucleus of cardiac myocytes during mechanical stress.

Effect of NRG-1/ErbB Signaling Intervention on the Differentiation of Bone Marrow Stromal Cells Into Sinus Node–like Cells

The neuregulin-1 (NRG-1)/ErbB signaling pathway is a crucial regulator of cardiac development and plays an important role in the formation of the cardiac special conduction system. To establish a rat bone marrow stromal cell (BMSC) cardiomyocyte (CM)-like differentiation model, BMSCs were treated with 5-azacytidine and fibroblast growth factor basic (FGF-basic) for 24 hours and then cocultured with neonatal rat CMs in a Transwell culture system. The feasibility of regulating the differentiation of BMSCs into sinoatrial node cells by manipulating the NRG-1/ErbB pathway was investigated. Three weeks after induction, reverse transcription-polymerase chain reaction analysis revealed that inhibition of NRG-1/ErbB signaling (using AG1478) greatly enhanced the expression of HCN4, Tbx3, and Tbx2. Additionally, Tbx3 protein levels were higher than in the control group and even produced distinct nodal-type action potentials. The expression of Nkx2.5 in the NRG-1 group (treated with exogenous NRG-1) was higher than the other 2 groups. The expression of phospho-Akt was also increased in the NRG-1 group but decreased in the AG1478 group. Together, these data demonstrate that inhibiting the endogenous NRG-1/ErbB signaling pathway when rat BMSCs differentiate into CMs can greatly enhance the pacemaker phenotype. Akt signaling may be one of the underlying molecular mechanisms responsible for these results.

Functional cooperation of spns2 and fibronectin in cardiac and lower jaw development

SummaryThe lipid mediator sphingosine-1-phosphate (S1P) is a regulator of cardiac development in zebrafish, as disruption of its receptor s1pr2 or transporter spns2 causes migration defects in cardiac progenitors. To examine the genetic interaction of S1P signaling and the cell adhesion molecule fibronectin, we have established a fn;spns2 double mutant. Cardiac migration defects in fn;spns2 mutants were more severe than those in fn or spns2 mutants. We further found that the lower jaw morphology was disorganized in the fn;spns2 mutant, while it had a slightly shortened anterior–posterior distance in the ventral pharyngeal arch in fn and spns2 mutants relative to wild type. Knockdown of fn in the s1pr2 mutant, but not in the s1pr1 mutant, resulted in severe defects in cardiac migration and ventral pharyngeal arch arrangement. Further, in the background of the fn mutant, knockdown of endothelin receptor A (ednra), which was downregulated in the spns2 mutant, caused pharyngeal defects resembling those in the fn;spns2 mutant. These results strongly suggest that Spns2-S1PR2 signaling and fibronectin cooperatively regulate both cardiac and lower jaw development in zebrafish.