Project description:Rationale: The atrioventricular conduction system controls ventricular activation and is delineated by expression of Tbx3. Genome-wide association studies identified genetic variants near TBX3 associated with conduction velocities (PR interval and QRS duration), suggesting minor changes in TBX3 dose affect conduction system function. Objective: To assess whether and how Tbx3 dose reduction affects the integrity of the atrioventricular conduction system. Methods and Results: Electrocardiograms revealed a PR interval shortening and prolonged QT interval and QRS duration in heterozygous Tbx3 mutants compared to wild-types. We observed that the atrioventricular bundle and proximal bundle branches of Tbx3+/- mice after birth became hypoplastic, whereas the size of the atrioventricular node was not affected. The transcriptomes of wild-type and Tbx3+/- atrioventricular nodes were analyzed using BAC-Tbx3-Egfp mice enabling specific isolation of the atrioventricular node by laser capture microdissection followed by RNA-sequencing. Hundreds of genes were slightly but consistently deregulated. Cross-referencing with transcriptome data of isolated cardiomyocytes of the conduction system and chamber myocardium derived from Tbx3+/Venus;BAC-Nppb-Katushka hearts revealed that a set of chamber-enriched genes, including Kcne1 (MinK), Ryr2, and Scn5a, were upregulated in Tbx3+/- atrioventricular nodes, whereas conduction system-enriched genes, including Hcn4 and Cacna2d2, were downregulated. We performed ATAC-sequencing on purified fetal Tbx3+ atrioventricular cardiomyocytes to identify potential atrioventricular-specific regulatory DNA elements on a genome-wide scale, and identified regulatory elements mediating the Tbx3-dependent regulation of Ryr2 and other target genes in the atrioventricular node. Conclusions: Tbx3 dose reduction results in deregulation of a large number of genes affecting the electrical properties of the atrioventricular node and causes failure to maintain the structural integrity of the atrioventricular bundle. These data provide a mechanism underlying differences in PR interval and QRS duration in individuals carrying associated variants in the TBX3 locus.

Project description:Rationale: A critical step in heart development is the coordinated formation of nodal myocardium and cushion tissue from the atrioventricular canal (AVC). After its specification, the myocardium of the AVC aligns the chambers, forms the AV node, and induces the formation of the mesenchymal AV cushions, primordia of valves and septa, while it resists working myocardial differentiation. Objective: To assess what roles Tbx2 and Tbx3, two closely related T-box transcription factors expressed in the AVC, play in these processes. Methods and Results: We analyzed mice ectopically expressing Tbx3 in the atrial myocardium by genome-wide microarray and expression analysis. We found a prominent role for Tbx3 in defining the nodal phenotype by repressing working myocardial genes (sarcomeric, mitochondrial, fast conduction) and cell proliferation regulators, and in inducing node-associated genes. Moreover, there was a striking induction of genes associated with endocardial cushions and mesenchyme. Using gain-of-function models, we found that in the developing heart both Tbx2 and Tbx3 induce ectopic Bmp2 and Tgfb2 expression and endocardial cushion formation. Analysis of compound Tbx2/Tbx3 mutant embryos revealed that upon loss of more than two functional alleles, expansion of the AV myocardium does not occur and AV cushions fail to form. Conclusions: Tbx2 and Tbx3 locally stimulate development of the AVC myocardium, induce the AV nodal phenotype therein and trigger AV cushion formation from the overlying AV endocardium, providing a mechanism for the colocalization and coordination of these two important processes in heart development. Nppa-Cre4 (Cre4) mice were crossed with CT mice to obtain efficient activation of Tbx3 in atria of double transgenic Cre4-CT mice, as previously described (Hoogaars et al., 2007). To investigate the gene expression profile of atria of Cre4-CT mice, we performed whole genome microarray analysis using Sentrix Mouse-6 oligonucleotide beadchips. We compared the atrial gene expression profiles of six male double transgenic Cre4-CT mice and six male Cre4 control mice.

Project description:to study the effect of induced expression of TBX3 within the atrium of the heart Background: Treatment of congenital or acquired disorders of the sinus node or atrioventricular node requires insight into the molecular mechanisms for the development and homeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogram terminally differentiated working cardiomyocytes into pacemaker cells. This would be an attractive approach in biological pacemaker formation. Methods and results: TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na+ channel (NaV1.5; INa) and inwardly rectifying K+ ion channels (Kir-genes; IK1). Concordantly, we observed conduction slowing in these hearts, and reductions in INa and IK1 in cardiomyocytes isolated from these hearts. The reduction in IK1 resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current, If, were observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes. Conclusions: TBX3 partially reprograms terminally differentiated working cardiomyocytes into pacemaker-like cells and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce IK1 density to enable diastolic depolarization, are very promising TBX3 characteristics for biological pacemaker formation strategies. 5 TBX3 expressing left atrial appendage samples (Tamoxifen-treated Myh6MCM;CTBX3 adult male mice) and 6 controls ((Tamoxifen-treated Myh6MCM adult male mice)

Project description:to study the effect of induced expression of TBX3 within the atrium of the heart Background: Treatment of congenital or acquired disorders of the sinus node or atrioventricular node requires insight into the molecular mechanisms for the development and homeostasis of these pacemaker tissues. In the developing heart, transcription factor TBX3 is required for pacemaker and conduction system development. Here, we explore the role of TBX3 in the adult heart and investigate whether TBX3 is able to reprogram terminally differentiated working cardiomyocytes into pacemaker cells. This would be an attractive approach in biological pacemaker formation. Methods and results: TBX3 expression was ectopically induced in cardiomyocytes of adult transgenic mice. Expression analysis revealed an efficient switch from the working myocardial expression profile to that of the pacemaker myocardium. This included suppression of genes encoding gap junction subunits (Cx40, Cx43), the cardiac Na+ channel (NaV1.5; INa) and inwardly rectifying K+ ion channels (Kir-genes; IK1). Concordantly, we observed conduction slowing in these hearts, and reductions in INa and IK1 in cardiomyocytes isolated from these hearts. The reduction in IK1 resulted in a more depolarized maximum diastolic potential, thus enabling spontaneous diastolic depolarization. Neither ectopic pacemaker activity nor pacemaker current, If, were observed. Lentiviral expression of TBX3 in ventricular cardiomyocytes resulted in conduction slowing and development of heterogeneous phenotypes, including depolarized and spontaneously active cardiomyocytes. Conclusions: TBX3 partially reprograms terminally differentiated working cardiomyocytes into pacemaker-like cells and induces important pacemaker properties. The ability of TBX3 to reduce intercellular coupling to overcome current-to-load mismatch and the ability to reduce IK1 density to enable diastolic depolarization, are very promising TBX3 characteristics for biological pacemaker formation strategies.

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