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Mutations and variations in and around SCN5A, encoding the major cardiac sodium channel, influence impulse conduction and are associated with a broad spectrum of arrhythmia disorders. Here, we identify an evolutionary conserved regulatory cluster with super enhancer characteristics downstream of SCN5A, which drives localized cardiac expression and contains conduction velocity-associated variants. We use genome editing to create a series of deletions in the mouse genome and show that the enhancer cluster controls the conformation of a >0.5 Mb genomic region harboring multiple interacting gene promoters and enhancers. We find that this cluster and its individual components are selectively required for cardiac Scn5a expression, normal cardiac conduction and normal embryonic development. Our studies reveal physiological roles of an enhancer cluster in the SCN5A-SCN10A locus, show that it controls the chromatin architecture of the locus and Scn5a expression, and suggest that genetic variants affecting its activity may influence cardiac function., Mutations associated with SCN5A, which encodes the major cardiac sodium channel, influence impulse conduction and are associated with arrhythmia disorders. Here, the authors identify an evolutionary conserved, super enhancer-like, regulatory cluster downstream of SCN5A and show that it controls the chromatin architecture of the locus and Scn5a expression.

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10(-68); rs9388451, P = 5.1 × 10(-17)) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10(-14)). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10(-81)). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Nav1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Nav1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Nav1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Nav1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean ± SEM 309 ± 32 V/s; n = 14) compared to subendocardial myocytes (394 ± 32 V/s; n = 11; P

Cardiac expression of a transgene is a common approach for determining the role of gene products in the processes underlying cardiomyopathy and heart failure (HF). We have generated transgenic mice that express the ‘harmless’ yeast transcription factor Gal4 in the heart under control of the α-myosin heavy chain promoter and found that expression of this gene causes cardiomyopathy and HF, the severity of which correlated with the number of copies of the transgene integrated into the genome and with the expression level. A line with a single copy of the transgene targeted to the hprt locus correctly expressed the transgene but did not develop cardiomyopathy. Our results indicate that expression of a transgene in the heart may non-specifically cause HF in a dose-dependent manner.

The heart of higher vertebrates is a structurally complicated multi-chambered pump that contracts synchronously. For its proper function a number of distinct integrated components have to be generated, including force-generating compartments, unidirectional valves, septa and a system in charge of the initiation and coordinated propagation of the depolarizing impulse over the heart. Not surprisingly, a large number of regulating factors are involved in these processes that act in complex and intertwined pathways to regulate the activity of target genes responsible for morphogenesis and function. The finding that mutations in T-box transcription factor-encoding genes in humans lead to congenital heart defects has focused attention on the importance of this family of regulators in heart development. Functional and genetic analyses in a variety of divergent species has demonstrated the critical roles of multiple T-box factor gene family members, including Tbx11, -2, -3, -5, -18 and -20, in the patterning, recruitment, specification, differentiation and growth processes underlying formation and integration of the heart components. Insight into the roles of T-box factors in these processes will enhance our understanding of heart formation and the underlying molecular regulatory pathways.

s ingediend voor het Amsterdam Kindersymposium 2013 19 A novel fluorescent marker for molecular, structural and functional analysis of the cardiac conduction system Martijn L. Ba kker, Bas J. Boukens, Vincent Wakker, Arie O. Verkerk, Corrie de Gier-de Vries, Malou van den Boogaard, Antoon F. Moorman, Vincent M. Christoff els Department of anatomy, embryology and fysiology, AMC INTRODUCTION The function of the conduction system depends on structure, function and molecular composition of its components. Studying the relation between these aspects would greatly benefi t from a mouse model in which the cells of the conduction system can be easily recognized in situ. METHODS We generated a transgenic mouse model that expresses yellow fl uorescent protein Venus under control of the Tbx3 locus (Tbx3Venus). RESULTS The expression pattern of Venus precisely recapitulates that of Tbx3, and specifi cally marks the conduction system components, including the sinus node, but excluding the Purkinje fi ber network. Combined optical mapping and fl uorescence microscopy demonstrated that the cardiac impulse always originated from the fl uorescently labeled sinus node region. Furthermore, patch-clamp of fl uorescently labeled cells isolated from embryos, fetuses and adults revealed that these cells display sinus node action potentials and spontaneous depolarization. These data indicate that the fl uorescent cells represent the sinus node. From embryonic day 12.5 onwards, the pacemaker current If, was present and sodium current INa was absent from sinus node cardiomyocytes. An interactive three-dimensional model of the atria including the sinus node was generated and revealed novel aspects of the complex structure of the sinus node in the adult mouse heart. CONCLUSION We conclude that Tbx3Venus is a reliable tool to visualize the conduction system cells in situ, to isolate pacemaker cells and to facilitate studies integrating structure, function and molecular composition of the conduction system during development and disease.