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Background: Genetic variants in SCN10A , encoding the neuronal voltage-gated sodium channel Na V 1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A , encoding the major essential cardiac sodium channel Na V 1.5. Methods: The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. Results: We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene ( Scn10a-short ). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a , the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short –encoded Na V 1.8-short increased Na V 1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts Na V 1.5-mediated sodium current and heart rhythm. Conclusions: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of Na V 1.8 in cardiomyocytes that impacts on Na V 1.5 function, cardiac conduction velocities, and arrhythmia susceptibility. more...

Rationale: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Objective: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. Methods and Results: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell–derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell–specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. Conclusions: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2 , TBX3 , and ISL1 , and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3 . more...

International audience; The rate of contraction of the heart relies on proper development and function of the sinoatrial node, which consists of a small heterogeneous cell population, including Tbx3(+) pacemaker cells. Here, we have isolated and characterized the Tbx3(+) cells from Tbx(3+/Venus) knock-in mice. We studied electrophysiological parameters during development and found that Venus-labeled cells are genuine Tbx3(+) pacemaker cells. We analyzed the transcriptomes of late fetal FACS-purified Tbx3(+) sinoatrial nodal cells and Nppb-Katushka(+) atrial and ventricular chamber cardiomyocytes, and identified a sinoatrial node-enriched gene program, including key nodal transcription factors, BMP signaling and Smoc2, the disruption of which in mice did not affect heart rhythm. We also obtained the transcriptomes of the sinoatrial node region, including pacemaker and other cell types, and right atrium of human fetuses, and found a gene program including TBX3, SHOX2, ISL1 and HOX family members, and BMP and NOTCH signaling components conserved between human and mouse. We conclude that a conserved gene program characterizes the sinoatrial node region and that the Tbx(3+/Venus) allele provides a reliable tool for visualizing the sinoatrial node, and studying its development and function. more...

International audience; The components of the cardiac conduction system, responsible for coordinated activation of the heart chambers, are well defined and their cells differ in gene expression profile and phenotype from those of the surrounding working myocardium. Yet, when and on what basis the myocardium of each of the conduction system components become distinguishable from other myocardium during heart development has not been well established. To identify and assess cell type-specific expression profiles and differentiation markers, we performed transcriptome analysis on fluorescence activated cell sorted purified conduction system (Venus +) and chamber myocardial cells (Katushka +) of Tbx3 +/Venus ;TgNppb(Katushka) double transgenic mouse fetuses. We found that transcripts associated with nervous system development and ion channel activity were enriched in Tbx3 + conduction system cells, whereas transcripts associated with mitochondrial function, muscle contraction and fatty acid metabolism were enriched in the Nppb + working myocardium. We analyzed spatio-temporal expression patterns of several candidate markers (Cacna2d2, Cacna1g, Ephb3, Tnni1), reviewed those of established conduction system markers (Tbx3, Hcn4, Gja5, Cntn2), and placed the patterns in the context of conduction system development. The overview indicates that different properties of conduction system components develop gradually and at different developmental stages, and that chamber myocardium gradually differentiates and diverges from conduction system myocardium until after birth. more...