Elucidating cardiomyocyte state transitions in zebrafish heart development using transcriptome dynamics.

Congenital heart disease (CHD), the most prevalent congenital anomaly, remains genetically incompletely understood. To address this knowledge gap, we employed a zebrafish model to investigate transcriptional dynamics during cardiac morphogenesis. Bulk RNA sequencing (Bulk RNA-seq) analysis of embryonic hearts at 24, 30, 36, and 48 h post-fertilization (hpf) linked the gene expression dynamics to four morphogenetic events: heart tube formation, chamber formation, heart tube looping, and valve formation. Weighted gene co-expression network analysis identified the M5 module, enriched for cardiac muscle development and representing a crucial transition state. Single-cell RNA sequencing (scRNA-seq) analysis revealed the M5 module's association with cardiomyocyte state transitions at 30 hpf. Pseudotime analysis confirmed this association and established the module's regulatory role in cardiomyocyte development. Notably, smoothelin like 1 (smtnl1) emerged as a hub gene within the M5 module, and its knockout induced cardiac dysfunction, including abnormal heart rate and stroke volume. Collectively, we delineate stage-specific molecular drivers of heart development and identify candidate genes for understanding CHD pathogenesis.