Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3^+/−) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3^+/− mutants. Notably, Tbx3^+/− atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

全基因组关联研究发现TBX3附近的非编码变异与 PR 间期和 QRS 持续时间相关，表明TBX3表达的细微变化会影响房室传导系统功能。为了探讨房室传导系统是否以及在多大程度上受到 Tbx3 剂量减少的影响，我们首先表征了杂合Tbx3突变体 ( Tbx3 ^+/- ) 小鼠心脏的电生理特性和形态。我们发现杂合子小鼠出生后 PR 间期缩短、QRS 时限延长以及房室束发育不全。房室结的大小不受影响。通过激光捕获显微切割分离的房室结的转录组分析揭示了Tbx3 ^+/-突变体中数百个失调的基因。值得注意的是， Tbx3 ^+/-房室结显示工作心肌基因程序（线粒体和代谢过程、肌肉收缩力）的表达增加，而起搏器基因程序（神经元、Wnt信号传导、钙/离子通道活性）的表达减少。通过整合房室组织的染色质可及性谱（ATAC 测序）和其他表观遗传数据，我们在全基因组范围内鉴定了 Tbx3 依赖性房室调节 DNA 元件 (RE)。我们使用转基因报告基因检测来确定Ryr2（一种上调的室富集基因）和Cacna1g（一种下调的传导系统特异性基因）附近候选 RE 的功能。 通过基因组编辑删除候选 RE，我们发现强内含子二分 RE 选择性地控制体内传导系统中Cacna1g 的表达。我们的数据提供了对调节心脏传导系统结构和功能的多因素 Tbx3 依赖性转录网络的见解，这可能是携带TBX3基因座变异的个体之间 PR 持续时间和 QRS 间期差异的基础。