Variation in a Left Ventricle–Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function

Rationale The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. Genome-wide association studies (GWAS) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5’ to the gene encoding the bHLH transcription factor HAND1. Objective Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS, and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify three additional SNPs, located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays (EMSA), disrupts GATA4 DNA-binding. Modeling two of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in EMSA and this enhancer in total, is required for VCS development and function in mice and perhaps humans

Pioneering disadvantage : consumer reactions to marketing mix strategies / M. Sadiq Sohail and Mohamad Farid Mahmood

Prior research has focused on the early entrant advantage, although a growing body of knowledgde suggests suggests that late movers have been outselling pioneers. In this research, the authors examine the issue of a pay phone service provider in Malaysia. In 1990, the company launches its product and creates an industry, but by the turn of the century, it has a meager 17 per cent of the market share. The Paper examines the specific issue of how a market pioneer has been eclipsed. It is hypothesized that the main cause of the declining sales is the in appropriate marketing mix strategies. The major findings based on a survey include consumers' acceptance level of the product, price, place, promotion, people, process and physical evidence strategies of the company

The human phrenic nerve serves as a morphological conduit for autonomic nerves and innervates the caval body of the diaphragm

Communicating fibres between the phrenic nerve and sympathetic nervous system may exist, but have not been characterized histologically and immunohistochemically, even though increased sympathetic activity due to phrenic nerve stimulation for central sleep apnoea may entail morbidity and mortality. We, therefore, conducted a histological study of the phrenic nerve to establish the presence of catecholaminergic fibres throughout their course. The entire phrenic nerves of 35 formalin-fixed human cadavers were analysed morphometrically and immunohistochemically. Furthermore, the right abdominal phrenic nerve was serially sectioned and reconstructed. The phrenic nerve contained 3 ± 2 fascicles in the neck that merged to form a single fascicle in the thorax and split again into 3 ± 3 fascicles above the diaphragm. All phrenic nerves contained catecholaminergic fibres, which were distributed homogenously or present as distinct areas within a fascicle or as separate fascicles. The phrenicoabdominal branch of the right phrenic nerve is a branch of the celiac plexus and, therefore, better termed the “phrenic branch of the celiac plexus”. The wall of the inferior caval vein in the diaphragm contained longitudinal strands of myocardium and atrial natriuretic peptide-positive paraganglia (“caval bodies”) that where innervated by the right phrenic nerve

Reduced sodium channel function unmasks residual embryonic slow conduction in the adult right ventricular outflow tract

In patients with Brugada syndrome, arrhythmias typically originate in the right ventricular outflow tract (RVOT). The RVOT develops from the slowly conducting embryonic outflow tract. We hypothesize that this embryonic phenotype is maintained in the fetal and adult RVOT and leads to conduction slowing, especially after sodium current reduction. We determined expression patterns in the embryonic myocardium and performed activation mapping in fetal and adult hearts, including hearts from adult mice heterozygous for a mutation associated with Brugada syndrome (Scn5a1798insD/+). The embryonic RVOT was characterized by expression of Tbx2, a repressor of differentiation, and absence of expression of both Hey2, a ventricular transcription factor, and Gja1, encoding the principal gap-junction subunit for ventricular fast conduction. Also, conduction velocity was lower in the RVOT than in the right ventricular free wall. Later in the development, Gja1 and Scn5a expression remained lower in the subepicardial myocardium of the RVOT than in RV myocardium. Nevertheless, conduction velocity in the adult RVOT was similar to that of the right ventricular free wall. However, in hearts of Scn5a1798insD/+ mice and in normal hearts treated with ajmaline, conduction was slower in the RVOT than in the right ventricular wall. The slowly conducting embryonic phenotype is maintained in the fetal and adult RVOT and is unmasked when cardiac sodium channel function is reduce

Embryonic Tbx3 + cardiomyocytes form the mature cardiac conduction system by progressive fate restriction

International audienceA small network of spontaneously active Tbx3+ cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3+ cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3+ cells was restricted to the definitive CCS. We identified a Tbx3+ population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3+ cardiomyocytes also contributed to the adjacent Gja5+ atrial and ventricular chamber myocardium, embryonic Gja5+ chamber cardiomyocytes did not contribute to the Tbx3+ sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3+ cell population in the early developing heart, which implicates Tbx3 as a useful tool for developing strategies to study and treat CCS diseases

Developmental Origin, Growth, and Three-Dimensional Architecture of the Atrioventricular Conduction Axis of the Mouse Heart

Rationale: The clinically important atrioventricular conduction axis is structurally complex and heterogeneous, and its molecular composition and developmental origin are uncertain. Objective: To assess the molecular composition and 3D architecture of the atrioventricular conduction axis in the postnatal mouse heart and to define the developmental origin of its component parts. Methods and Results: We generated an interactive 3D model of the atrioventricular junctions in the mouse heart using the patterns of expression of Tbx3, Hcn4, Cx40, Cx43, Cx45, and Nav1.5, which are important for conduction system function. We found extensive figure-of-eight rings of nodal and transitional cells around the mitral and tricuspid junctions and in the base of the atrial septum. The rings included the compact node and nodal extensions. We then used genetic lineage labeling tools (Tbx2(+Cre), Mef2c-AHF-Cre, Tbx18(+/Cre)), along with morphometric analyses, to assess the developmental origin of the specific components of the axis. The majority of the atrial components, including the atrioventricular rings and compact node, are derived from the embryonic atrioventricular canal. The atrioventricular bundle, including the lower cells of the atrioventricular node, in contrast, is derived from the ventricular myocardium. No contributions to the conduction system myocardium were identified from the sinus venosus, the epicardium, or the dorsal mesenchymal protrusion. Conclusions: The atrioventricular conduction axis comprises multiple domains with distinctive molecular signatures. The atrial part proliferates from the embryonic atrioventricular canal, along with myocytes derived from the developing atrial septum. The atrioventricular bundle and lower nodal cells are derived from ventricular myocardium. (Circ Res. 2010;107:728-736.