polr2i is Required for Zebrafish Early Cardiac Development

Background: Congenital heart disease (CHD) is characterized by structural and functional anomalies of the heart and major blood vessels present at birth. It is recognized as the most common congenital defect. Epidemiological studies highlight the substantial contribution of genetic factors to CHD pathogenesis. In our previous study, RNA polymerase II subunit I (POLR2I protein) was identified as a candidate genetic contributor to CHD. However, its functional role remains largely unexplored. Methods: First, we performed bioinformatics analyses to evaluate the evolutionary conservation of the POLR2I protein across vertebrate species. The amino acid sequence similarity of the POLR2I protein exceeds 90% in different vertebrates, suggesting a correlation between their species. Quantitative real-time PCR (qRT-PCR) revealed significantly elevated polr2i gene expression during early embryonic stages and in adult zebrafish organs, including the heart, eyes, and brain. Morpholino oligonucleotide (MO)-mediated gene editing was used to downregulate the polr2i gene in zebrafish, and rescue experiments were performed by co-injecting capped polr2i gene mRNA. Transgenic zebrafish labeled with specific fluorescent protein facilitated detailed studies of cardiac and vascular development, myocardial mitochondrial quality, and embryonic asymmetry, respectively. Hemoglobin staining with o-Dianisidine assessed red blood cell accumulation. Results: Knocking down the polr2i gene through MO significantly disrupted developmental trajectories, as evidenced by reduced body size, axial curvature, enlarged yolk sacs, and elevated malformation and mortality rates. Rescue experiments confirmed the specificity of these phenotypes to polr2i gene loss. Affected embryos displayed elongated heart tubes with reduced overlap between chambers and significant pericardial edema, indicating severe cardiac malformations or functional impairments. Measured volume per beat, ejection fraction, and cardiac output decreased substantially. Furthermore, expression levels of critical cardiovascular markers were markedly reduced. Angiogenic processes were also disrupted, as evidenced by the reduced formation of intersegmental vessels and the caudal vein plexus. Impaired mitochondrial quality in myocardial cells was observed post-knockdown, along with notable defects in the left-right asymmetry of the heart, liver, and pancreas. Conclusion: Knockdown of the polr2i gene not only impairs cardiac structure and function but also disrupts the normal developmental asymmetry of multiple organs. These findings enhance our understanding of polr2i gene’s role in CHD and underscore its potential as a therapeutic target.