Abstract
The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.
Highlights
Pathogenic variants in T-box transcription factor (TF) TBX5 have been linked to Holt-Oram syndrome (HOS), which is typically characterized by varying degrees of limb and cardiac malformations and conduction defects (OMIM #142900).[1,2,3]
E14.5-E16.5 Tbx5G125R/G125R fetuses showed ventricular and atrial septal defects as well as enlarged right atria at E16.5 (Supplemental Figure 1), which were present in patients heterozygous for this pathogenic variant (Supplemental Table 1, Figure 1A)
As typical HOS is usually associated with loss of function variants leading to haploinsufficiency, the atypical HOS phenotype of TBX5-p.G125R carriers suggests the pathogenic missense variant causes TBX5 to gain specific function(s)
Summary
Pathogenic variants in T-box transcription factor (TF) TBX5 have been linked to Holt-Oram syndrome (HOS), which is typically characterized by varying degrees of limb and cardiac malformations and conduction defects (OMIM #142900).[1,2,3] Tbx[5] is dose-dependently required for heart development, specification and function of the conduction system and determines the working myocardial phenotype of the atrium.[3,4,5,6,7,8] Tbx[5] has been put forward as key regulator of ion handling protein-encoding gene expression and rhythm control in vivo.[9,10,11,12,13] Genome wide association studies uncovered several variants near TBX5 associated with atrial fibrillation (AF). We engineered the p.G125R variant in the highly conserved T-box of mouse Tbx[5], and used this model to gain insight into the molecular mechanisms underlying structural and electrophysiological changes predisposing to AF in TBX5 mutation carriers
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