Abstract Body (Do not enter title and authors here): Background:
Cardiac fibrosis is a central pathological feature of heart failure, still a leading cause of cardiovascular death in the developed world. Despite its relevance, the transcriptional regulation of fibroblast activation remain poorly investigated. Novel approaches, such as single-nuclei RNA sequencing (snRNA-seq), might identify potential therapeutic targets. This study aimed to investigate the role of the transcription factor ATOH8 in fibroblast activation, based on single-nuclei transcriptomic analyses from a murine myocardial infarction (MI) model and validating its function in vitro in human cardiac fibroblasts.

Methods:
Single-nuclei ATAC sequencing (snATAC-seq) was performed using murine myocardial tissue at days 3 and 10 post-MI and compared to sham controls. Each subcohort consisted of 3 males and 3 females. Accessible chromatin sections were identified, thus describing the activity of profibrotic transcriptional regulators, including ATOH8.
Subsequent in vitro validation was performed using primary human cardiac fibroblasts (PromoCell, Cat. No. C-12375) stimulated with transforming growth factor-beta 1 (TGF-β1, 10 ng/ml) to induce myofibroblast activation. ATOH8 was silenced using siRNA. Fibroblast activation was assessed via qPCR (COL1A1, α-SMA, Periostin), α-SMA immunostaining, and bulk RNA sequencing of ATOH8-silenced vs. control cells. Statistical comparisons were made using unpaired t-tests or ANOVA, with significance defined as p < 0.05.

Results:
snATAC-seq data from the murine MI model showed increased activation of the ATOH8 gene locus in infarcted myocardial tissue compared to sham, suggesting a regulatory role in the early fibrotic response.
In further in vitro validation experiments ATOH8 knockdown in human cardiac fibroblasts significantly reduced TGF-β1-induced expression of COL1A1, α -SMA and Periostin and led to a decrease in α-SMA-positive fibroblasts. Bulk RNA-seq analysis of cell culture samples confirmed the downregulation of fibrosis-associated gene transcripts and TGF-β-responsive pathways upon ATOH8 silencing.

Conclusions:
Transcriptomic analyses in a murine MI model identified ATOH8 as a pivotal transcriptional regulator of cardiac fibrosis. Functional validation in primary human cardiac fibroblasts supports a relevant role for ATOH8 in promoting profibrotic gene expression and myofibroblast activation. These findings suggest the potential of ATOH8 as a therapeutic target in cardiac fibrosis that should be addressed.