Loss of nono impairs cardiac morphogenesis and excitation–contraction coupling via the PI3K/Akt pathway in zebrafish

Pathogenic variants in NONO are clinically linked to congenital heart disease (CHD), but in vivo mechanisms and gene-targeted interventions remain poorly explored. Using CRISPR/Cas9 gene editing and morpholino silencing, we established knockout (KO) and knockdown (KD) zebrafish models to study nono in cardiogenesis. Adult KO hearts displayed severe structural remodeling, characterized by ventricular distortion, disorganized sarcomeres, and myocardial fibrosis. High-speed optical mapping and calcium imaging revealed reduced conduction velocity, increased dispersion, and spatial heterogeneity in action potential and Ca2+ transients, disrupting excitation–contraction coupling. In larvae, nono deficiency downregulated core transcription factors (nkx2.5, gata4), suppressed the PI3K/Akt pathway, and triggered myocardial apoptosis. Exogenous delivery of nono mRNA (Rescue) restored PI3K/Akt signaling, mitigating embryonic cardiovascular defects and functional failure. Collectively, our dual-model system demonstrates that NONO is required for structural integrity and electrophysiological homeostasis. This study provides vital in vivo functional validation of nono as a molecular target for myocardial dysfunction, offering a proof-of-concept for gene-based therapeutic restoration in NONO-associated CHD.