A Preliminary Zebrafish Model of ACTA2 Deficiency Reveals Increased Larval Phenotype Burden and Suggests Reduced Adult Mutant Survival.

ACTA2 encodes smooth muscle alpha-actin and is one of the most common genetic causes of inherited non-syndromic thoracic aortic aneurysm and dissection. Although murine models have provided important mechanistic insight, complementary vertebrate systems may enable more rapid in vivo phenotyping and future therapeutic screening. Utilizing a CRISPR/Cas9-based approach, we developed zebrafish acta2 mutant models and assessed phenotypes at larval and adult stages. At 3 days post-fertilization, larvae were screened by brightfield microscopy for edema, axis defects, hemorrhage, and a composite endpoint (any phenotype) under basal conditions and after exposure to 0.2 mM epinephrine. Phenotype occurrence was summarized as group-specific proportions across a 2 × 2 design defined by genotype (wild-type vs. acta2-deficient mutant) and treatment (DMSO control vs. epinephrine), with prespecified pairwise comparisons using Pearson's chi-squared tests. Pooled fish-level logistic regression models were also fit for each endpoint. Adult follow-up was performed by genotyping all available acta2 fish at the facility and tracking staggered tank-level cohorts longitudinally. Initial gross morphologic assessment did not reveal overt external phenotypic differences between heterozygous or homozygous acta2 mutant larvae and wild-type controls. In larval analyses, the mutant genotype was associated with a greater burden of adverse phenotypes than treatment exposure. For the composite endpoint, mutant larvae demonstrated higher proportions than normal larvae under both control (28.3% vs. 17.4%, p = 0.007) and epinephrine conditions (26.9% vs. 17.4%, p = 0.023), whereas epinephrine did not significantly alter composite phenotype frequency within either genotype. Similar genotype-associated trends were observed for axis defects and hemorrhage. In pooled logistic regression, acta2 deficiency was associated with increased odds of axis defects (OR 2.64, 95% CI 1.48-4.89, p = 0.001), hemorrhage (OR 2.40, 95% CI 1.09-5.68, p = 0.036), and the composite endpoint (OR 1.88, 95% CI 1.19-3.00, p = 0.008), whereas epinephrine exposure did not demonstrate a consistent independent effect across endpoints. Adult staggered follow-up showed greater attrition in homozygous mutant cohorts than in wild-type or heterozygous cohorts, with aggregated losses of 24%, 12%, and 3%, respectively. This preliminary zebrafish acta2 model demonstrated that acta2 deficiency is associated with increased adverse larval phenotype burden and suggested reduced long-term persistence of homozygous mutant fish in adulthood. The strongest signal in the current study was genotype-associated phenotype burden rather than a robust epinephrine-dependent effect. These findings support the feasibility of zebrafish-based ACTA2 phenotyping while highlighting the need for more specific vascular endpoints, refined longitudinal follow-up, and future variant-specific modeling.