Insights into CAD Pathogenesis: A Multi-Model Study of PHACTR1 and EDN1 at the 6p24 Risk Locus

Background:
Coronary artery disease (CAD) and myocardial infarction remain major contributors to global mortality, with the 6p24 locus — encompassing EDN1 and PHACTR1 — persistently associated with CAD risk in genome-wide association studies (GWAS). Intronic variants within PHACTR1 have been identified as major contributors to CAD risk, yet the direct link between these SNPs and disease pathogenesis remains under scrutiny. Recent findings suggest that the lead SNP, previously attributed to PHACTR1, as harboring gene, may act as a distal regulator of EDN1, a key player in endothelial function, challenging established SNP-gene associations and underscoring the complexity of genetic regulation in CAD.

Methods:
To elucidate the roles of PHACTR1 and EDN1 in CAD, we utilized zebrafish (Danio rerio), mouse, and cell culture models. Gene knockdown of edn1 in zebrafish was achieved using morpholino oligonucleotides, while CRISPR/Cas9 technology was employed to generate phactr1 knockouts, as well as a dual knockout for edn1 and phactr1. Phenotypic assessments in zebrafish included angiogenesis and hemodynamic measurements, with blood flow, heart rhythm, and contractility quantified using our custom PyHeart4Fish software in Tg(myl7) transgenic lines. Vascular development was analyzed via fluorescence imaging in Tg(fli1a) lines, and hemodynamic parameters were recorded. For lipid metabolism studies, phactr1 knockout zebrafish and mice were placed on high-cholesterol diets to assess plaque formation and lipid deposition, with comparisons made against wild-type and heterozygous controls.

Results:
Using edn1-specific morpholinos in zebrafish, we observed significant effects on angiogenesis and blood pressure surrogate parameters, underscoring edn1's essential role in vascular development and cardiovascular function. In our phactr1 knockout (KO) zebrafish, CRISPR/Cas9-induced phactr1 deletion led to a marked reduction in fatty deposits, similar findings were shown in the mouse model, suggesting PHACTR1’s involvement in lipid regulation and potential atheroprotection. Functional assessments via PyHeart4Fish revealed opposing effects of the two genes on ejection fraction and related parameters. Additionally, an endothelial cell (EC) tube formation assay with PHACTR1 knockdown via siRNA showed significant angiogenic effects.

Conclusions:
Our study highlights the complex interplay between PHACTR1 and EDN1 at the 6p24 locus, with both genes influencing distinct aspects of vascular biology relevant to CAD. By using a cross-species approach with zebrafish, mouse models as well human cell culture, we provide compelling evidence that PHACTR1 may play a role in lipid regulation, while EDN1 primarily affects vascular development and function. These findings underscore the importance of exploring multi-gene loci in CAD to better understand the genetic mechanisms driving disease and may inform future therapeutic strategies aimed at mitigating CAD risk through targeted molecular interventions.

Funding: This work was supported by the DZHK