TBX5 G125R induces profound transcriptional deregulation and atrial dysfunction in vivo [RNA-seq]

Rationale: Pathogenic variants in the gene for T-box transcription factor (TF) 5 (TBX5) cause Holt-Oram syndrome (HOS), characterized by congenital heart defects and limb abnormalities. A particular missense variant in TBX5 (G125R) in a Dutch family causes atypical HOS as well as early onset atrial fibrillation (AF). Understanding the effects of such an altered key cardiac TF on the transcription regulatory network and cardiac physiology provides a unique opportunity to gain insight into the mechanisms underlying diseases such as AF. Objective: To determine the in vivo TBX5-G125R-induced changes in the transcriptional regulatory network and epigenetic state underlying the atypical HOS with early onset AF found in an extended pedigree. Methods and Results: We modeled the TBX5-G125R pathogenic variant in vivo in the mouse and found severe cardiac defects and fetal lethality in Tbx5G125R/G125R mice, whereas Tbx5G125R/+ mice were viable and morphologically unaffected. Electrophysiological analysis of adult Tbx5G125R/+ mice revealed variable RR interval, atrial extra systole and susceptibility to AF upon pacing. These characteristics were also observed in the patient family. Additionally, calcium homeostasis as well as conduction were changed in the mutant atrial cardiomyocytes. Single-nucleus transcriptional profiling of right atria revealed the most profound changes in expression in the cardiomyocytes of Tbx5G125R/+ mice. Transcriptional profiling of atrial tissue identified differential expression of over a thousand genes, whereas expression levels of several genes known to respond to Tbx5 insufficiency did not change. Epigenetic profiling revealed shifts in sites associated with acetylated H3K27 as well as in chrom atin accessibility in Tbx5G125R/+ atrial cardiomyocytes indicating Tbx5-G125R has altered DNA binding and TF interaction properties. Whole tissue RNA-seq of left and right atria of Tbx5G125R/+ and control adult female mice (n=5)研究背景：T盒转录因子5（TBX5）编码基因的致病变异可引发霍尔特-奥拉姆综合征（HOS），该病以先天性心脏缺损与肢体畸形为典型特征。荷兰某家族中存在的TBX5特定错义变异（G125R）可导致非典型霍尔特-奥拉姆综合征，同时伴早发性心房颤动（AF）。解析此类关键心脏转录因子的变异对转录调控网络及心脏生理功能的影响，可为揭示心房颤动等疾病的发病机制提供独特研究视角。 研究目标：明确该扩展大家系中检出的伴早发性心房颤动的非典型霍尔特-奥拉姆综合征相关TBX5-G125R变异，在体内诱导的转录调控网络与表观遗传状态改变。 方法与结果：我们在小鼠体内构建了TBX5-G125R致病变异模型。纯合子Tbx5G125R/G125R小鼠出现严重心脏缺损并发生胚胎致死，而杂合子Tbx5G125R/+小鼠可正常存活且形态无异常。对成年杂合子小鼠的电生理分析显示，其RR间期存在波动、可出现房性期前收缩，且起搏时房颤易感性升高，这些表型与患者家族的临床特征一致。此外，突变型心房心肌细胞的钙稳态与传导功能均发生改变。对右心房开展的单细胞核转录组测序显示，杂合子小鼠的心肌细胞基因表达出现最显著的变化。心房组织转录组分析鉴定出超过一千个差异表达基因，而若干已知受Tbx5功能不足调控的基因表达水平未发生改变。表观遗传组分析显示，杂合子小鼠心房心肌细胞中，与乙酰化H3K27结合的位点以及染色质开放区域均出现偏移，提示TBX5-G125R变异改变了其DNA结合能力与转录因子互作特性。我们对杂合子Tbx5G125R/+小鼠与对照成年雌性小鼠（n=5）的左右心房开展了全组织RNA测序。