Gladka, M. M. | bioRxiv Science

bioRxiv2026

TBX5 dosage governs ventricular cardiomyocyte maturation, specialization and dedifferentiation in vivo

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.

developmental biology↗

bioRxiv2026

Advancing Nuclei Isolation from Frozen Human Heart for Single-Nucleus RNA Sequencing Applications

While single-cell RNA sequencing (scRNA-seq) has been the first widely adopted single-cell transcriptomic approach, its reliance on fresh tissue samples has substantially limited its applicability to clinically relevant specimen. Single-nucleus RNA sequencing (snRNA-seq) overcomes this constrain by enabling transcriptomic profiling from frozen material. However, isolating high-quality nuclei from frozen cardiac tissue remains technically challenging due to the dense extracellular matrix, complex tissue architecture, and heterogeneous cellular composition of the heart. To address these challenges, numerous nuclei isolation protocols have been adapted and optimized, resulting in substantial methodological heterogeneity across studies. Despite the widespread use of snRNA-seq in cardiac research, a robust and standardized nuclei isolation protocol that consistently yields high-quality nuclei from frozen human heart tissue is still lacking. Here, we present a comprehensive, end-to-end protocol for nuclei isolation from frozen human left ventricle, along with a detailed downstream pipeline for snRNA-seq data analysis. Our hybrid nuclei isolation strategy integrates multiple sequential clean-up steps designed to preserve nuclear integrity and RNA quality prior to sequencing. Compared with commonly used nuclei isolation protocols, this approach yields substantially higher number of nuclei while maintaining comparable numbers of detected genes and counts, even at lower sequencing depth. Adoption of this protocol may reduce technical variability across studies and facilitate more reproducible snRNA-seq analyses of human cardiac tissue.

molecular biology↗