Physiologically relevant increases in transcription factor dosage and their role in development and disease remain largely unexplored. Genomic deletions upstream of the Paired-like homeodomain transcription factor gene ( PITX2 ), identified in patients with sinus node dysfunction and atrial fibrillation and modeled in mice ( delB ), rewire the local epigenetic landscape, increasing PITX2 expression. Here, we demonstrate that pacemaker cardiomyocytes in the embryonic delB sinus node ectopically express PITX2 at physiological dosages in a heterogeneous pattern. The prenatal delB sinus node forms discrete subdomains showing PITX2 dosage-dependent mild or severe loss of pacemaker cardiomyocyte identity. Respective subdomain sizes and severity of sinus node dysfunction and atrial arrhythmia susceptibility align with PITX2 dosage. Ectopic PITX2c expression in human induced pluripotent stem cell-derived pacemaker cardiomyocytes causes PITX2 dosage-dependent transcriptional and electrophysiological changes paralleling those in delB mice. Our findings provide a mechanistic link between genetic variation–driven ectopic PITX2 expression, sinus node dysfunction and atrial arrhythmogenesis, illustrating how spatiotemporally defined increases in transcription factor dosage can translate into developmental defects and disease predisposition.

The natriuretic peptides A and B, encoded by NPPA and NPPB, respectively, have complementary and redundant functions in cardiovascular homeostasis. To establish their coordinated roles, we analyzed the cardiac phenotype of a mouse line in which the Nppa–Nppb cluster was deleted from the genome. At 8 weeks of age, Nppa–Nppb^−/− mice (HOM) had significantly larger hearts and cardiomyocytic hypertrophy compared to wild-type and heterozygous mice. Electrocardiogram comparisons showed QRS prolongation in HOM mice. Hypertrophy was confirmed by echocardiography, which further indicated preservation of left ventricular systolic function. Bulk-transcriptomic analysis revealed moderate changes in gene expression of the left ventricle. Genes involved in fatty acid metabolism, ion handling and conductivity, including genes marking the ventricular conduction system, were down-regulated. Spatial transcriptomic analysis revealed the greatest changes in gene expression in the subendocardial wall, where the ventricular conduction system is located. Tbx5, the encoding dosage-sensitive T-box transcription factor Tbx5 that is essential for the expression of ventricular conduction system genes and for Nppa and Nppb, was down-regulated in the ventricles of HOM mice, indicating that a positive feedback loop normally maintains Tbx5 expression. We conclude that homozygous Nppa–Nppb deficiency in mice causes cardiac hypertrophy, including a likely perturbation of the ventricular conduction system.

Variation in transcription factor (TF) activity modulates traits and disease susceptibility, yet how such variation translates into cellular phenotype and organ function in vivo is not well established. We utilized AAV-mediated gene delivery to express the dosage-sensitive TF TBX5 across a physiologically plausible range in postnatal ventricular cardiomyocytes. Transcriptomic profiling revealed that TBX5 dosage-dependent cardiomyocyte states changed gradually and often non-monotonically across the TBX5 dosage spectrum. The lowest dosages induced cardiomyocyte hypertrophy and upregulated gene programs governing oxidative metabolism, calcium handling, and contractility. In contrast, mid-to-high dosages induced a ventricular conduction system-like transcriptional profile. Supraphysiological dosages triggered cardiomyocyte dedifferentiation, characterized by cardiomyocyte size reduction, cell cycle re-entry, metabolic reprogramming, and impaired ventricular ejection fraction. Furthermore, the ventricular state of an Nppa-Nppb deficiency model characterized by cardiac hypertrophy and reduced Tbx5 expression was partially normalized by TBX5 delivery. By defining the non-linear relationship of TBX5 activity level, cardiomyocyte state and cardiac function, our study reveals how TF dosage regulates the transitions from physiological maturation to specialized lineage acquisition and dedifferentiation at the cell and organ level in vivo .