Coronary artery disease (CAD), myocardial infarction (MI), and its sequelae, primarily ischemic heart disease, remain the leading causes of death worldwide. Recently, genome-wide association studies (GWAS) identified the 6p24 locus covering the genes encoding endothelin-1 (EDN1) and Phosphatase and actin regulator 1 (PHACTR1) to be associated with CAD. 

Although the lead single-nucleotide polymorphism (SNP) in 6p24, an intronic variant within PHACTR1, has been shown to affect PHACTR1 in most publications, this conclusion has recently been challenged. The upstream gene EDN1 has been described as a possible (co)causative gene as a distal regulator of PHACTR1. Therefore, it remains unclear which gene plays a driving role in disease progression and whether EDN1 and PHACTR1 regulate each other or have synergistic effects. Thus, identifying the causative gene and its molecular and functional effects is important for further progress in preventing and treating CAD.

The suitability of zebrafish (Danio rerio) in genetic cardiovascular research, is not only shown by the high degree of orthology but also by the physiological similarity of the zebrafish heart, whose functionality is in many respects closer to that of humans than, for example, that of murine models. 

While one first study has been conducted on the functions of phactr1 in zebrafish, possible mRNA transcripts have yet to be confirmed. Multiple mRNA transcript variants with differential expression of PHACTR1 have been described in both humans and mice, but it´s assumed, that zebrafish only have one transcript variant. Therefore, we designed primers to determine the presence of transcript variants via RT-PCR, before they will be confirmed in a tissue-specific RT-qPCR for the brain, eyes, gonads, heart, intestine, kidney, liver, muscle, skin, and spleen.

In a two-phase project approach, we achieved knockdown (KD) of the target genes phactr1 and edn1 (individually and in combination) using morpholino oligonucleotides. In wild-type fish, blood flow was measured as a surrogate parameter for blood pressure in 72 hpf larvae, here a significant reduction (P = .0306) in arterial pulse could be observed in edn1 KD. Effects on rhythm and contractility of the larval heart could be measured using the in-house developed software "PyHeart4Fish" in Tg(Myl7:eGFP)^twu34/+ fluorescent fish. The overall morphology of the heart and vasculature were assessed via fluorescent imaging using Tg(Myl7:eGFP)^twu34/+ resp. Tg(fli1a:nEGFP)^y7 at 72 hpf. In edn1 KD, with no change in the diameter of the cardinal vein (ortholog to the human venae cavae) (P = .2015), a significant increase in the diameter of the dorsal aorta (P = .0018), with a concomitant decrease in the diameter of the intersegmental vessels (P < .0001), was observed. Strongly indicative of a hemodynamically relevant vascular impact of this gene.

In the next phase, knockouts of the target genes will be generated in different lines using CRISPR/Cas9. To analyze plaque formation under a High Cholesterol Diet (HCD), endothelial colocalization of immune cellules, oxLDL, and ECG studies.

A better understanding of the two genes at 6p24 and their contribution to CAD risk may lead to the development of new targeted therapies.