Tbx3 governs a transcriptional program to maintain atrioventricular conduction system form and function [ATAC-seq]

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. Overall design: ATAC-seq on microdissected and FACS-sorted E13.5 Tbx3-Venus+ atrioventricular junction cardiomyocytes

研究依据：房室传导系统（atrioventricular conduction system）负责调控心室激活，其界定以Tbx3的表达为标志。全基因组关联研究已鉴定出TBX3基因邻近区域的遗传变异与传导速度相关指标（PR间期与QRS时限）相关，提示TBX3剂量的微小变化即可影响传导系统功能。
研究目的：本研究旨在评估Tbx3剂量降低是否以及如何影响房室传导系统的结构完整性。
方法与结果：心电图检测结果显示，与野生型小鼠相比，杂合子Tbx3突变小鼠的PR间期缩短，QT间期及QRS时限延长。我们观察到，出生后Tbx3+/-小鼠的房室束（atrioventricular bundle）及其近端束支出现发育不全，而房室结（atrioventricular node）的大小未受影响。本研究利用BAC-Tbx3-Egfp小鼠模型，通过激光捕获显微切割技术特异性分离房室结，随后进行RNA-sequencing（RNA测序），以此分析野生型与Tbx3+/-小鼠房室结的转录组。结果显示数百个基因出现程度较轻但持续一致的表达失调。将本研究数据与源自Tbx3+/Venus;BAC-Nppb-Katushka心脏的传导系统心肌细胞及腔室心肌细胞的转录组数据进行交叉比对后发现，Tbx3+/-小鼠房室结中一组富集于腔室心肌的基因（包括Kcne1（MinK）、Ryr2及Scn5a）表达上调，而富集于传导系统的基因（包括Hcn4与Cacna2d2）表达下调。我们对纯化后的胎鼠Tbx3+房室传导系统心肌细胞进行ATAC-sequencing（转座酶可及性测序），以在全基因组范围内筛选潜在的房室特异性调控DNA元件，并鉴定出介导Tbx3对房室结内Ryr2及其他靶基因进行调控的调控元件。
结论：Tbx3剂量降低会导致大量影响房室结电生理特性的基因表达失调，并无法维持房室束的结构完整性。本研究数据为携带TBX3基因座相关变异的个体中PR间期与QRS时限的个体差异提供了潜在机制。
整体实验设计：对经显微切割及FACS（荧光激活细胞分选，Fluorescence-Activated Cell Sorting）分选的胚胎龄E13.5 Tbx3-Venus+房室交界区心肌细胞开展ATAC测序