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In a first family (family#1), we identified 53 members of whom 17 present a syndromic cardiac disorder characterized by electrical disorders (sinus node dysfunction, atrial fibrillation...) and developmental defects (atrial septal defect, valvopathy, left ventricle non-compaction...) following an autosomal dominant model. Among the affected family members, 6 are implanted with a pacemaker and one experienced a sudden death at 43yo. Despite a strong linkage pointing to the 4q25 region, exome sequencing failed to identify causal variant. Interestingly, 6 additional non-related families presenting the same phenotype have been also identified. Our aims are to identity the causal mutation and the molecular mechanism underlying this complex cardiac syndrome. Genetic study has been performed using whole genome sequencing (WGS). Based on transgenic mouse strains, we assessed the impact of Family#1 mutation on the phenotype and on gene expression. Then, we generated human cardiomyocytes derived iPS cells (CM-iPS) isogenic models to evaluate the epigenome (CUT&RUN and ATAC-seq), transcriptome (RNA-seq) and topological associated domain (TAD) remodelling (Hi-C). By WGS we uncovered a deletion of 15kb in a gene desert area on 4q25, segregating in all affected relatives of Family#1. The 6 other families present overlapping deletions. Mouse model recapitulates the cardiac phenotype and exhibit a dysregulation of Pitx2 expression in cardiac specific compartments. Based on human CM-iPS models, epigenetic data highlight among the 15kb deletion a unique open region containing a CTCF binding site, crucial for delimiting TAD boundaries. Hi-C assay reveals the fusion of 2 TADs and highlights new interactions between PITX2 and atrial specific regulatory elements. We identified a deletion located within a gene desert area associated with a complex cardiac disorder. The CTCF binding site contained in the deletion seems key in the TAD border. The TAD remodelling leads to new (regulatory) interactions and expression dysregulation of PITX2. We describe a new molecular mechanism implying a yet unidentified non-coding regulatory element of PITX2 and responsible for a complex electrical and developmental cardiac syndrome. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Nantes UniversitéFrench national reserch agency (ANR)

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (−12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from −161 to +138) and enhancer (469 bp around −6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5′-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein–protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.

ChIP-seq is rapidly becoming a routine technique for the determination of the genome wide association of DNA binding proteins and histone modifications. Here we provide a protocol for the isolation, purification, and immunoprecipitation of DNA fragments associated with a target transcription factor of interest. Although the method makes use of adult mouse hearts, it can, with relative ease, be adapted for the in vivo ChIP isolation of DNA from other cell and tissue sources with the intention of massive parallel sequencing.

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.

Objective: In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. Design: Rats were treated with AM or Dron and the expression of TRα 1, TRα 2, TRβ 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. Methods: Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRα 1, TRα 2, TRβ 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. Results: AM and Dron affected TR expression in the RA similarly by decreasing TRα 1 and β 1 expression by about 50%. In the LVW, AM and Dron decreased TRβ 1 and, interestingly, AM increased TRα 1. In the apex, AM also increased TRα 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. Conclusion: Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.

Nuclear thyroid hormone (T3) receptors (TR) play a critical role in mediating the effects of T3 on development, differentiation and normal physiology of many organs. The heart is a major target organ of T3, and recent studies in knockout mice demonstrated distinct effects of the different TR isoforms on cardiac function, but the specific actions of TR isoforms and their specific localization in the heart remain unclear. We therefore studied the expression of TRα1, TRα2 and TRβ1 isoforms in the mouse heart at different stages of development, using monoclonal antibodies against TRα1, TRα2 and TRβ1. In order to identify distinct components of the embryonic heart, in situ hybridization for cardiac-specific markers was used with the expression pattern of sarcoplasmic reticulum calcium-ATPase 2a as a marker of myocardial structures, while the pattern of expression of connexin40 was used to indicate the developing chamber myocardium and peripheral ventricular conduction system. Here we show that in the ventricles of the adult heart the TRβ1 isoform is confined to the cells that form the peripheral ventricular conduction system. TRα1, on the other hand, is present in working myocardium as well as in the peripheral ventricular conduction system. In the atria and in the proximal conduction system (sinoatrial node, atrio-ventricular node), TRα1 and TRβ1 isoforms are co-expressed. We also found the heterogeneous expression of the TRα1, TRα2 and TRβ1 isoforms in the developing mouse heart, which, in the case of the TRβ1 isoform, gradually revealed a dynamic expression pattern. It was present in all cardiomyocytes at the early stages of cardiogenesis, but from embryonic day 11.5 and into adulthood, TRβ1 demonstrated a gradual confinement to the peripheral ventricular conduction system (PVCS), suggesting a specific role of this isoform in the formation of PVCS. Detailed knowledge of the distribution of TRα1 and TRβ1 in the heart is of importance for understanding not only their mechanism of action in the heart but also the design and (clinical) use of TR isoform-specific agonists and antagonists.