Abstract Body: Introduction. Intercalated discs (ICDs) mechanically, electrically, and chemically couple cardiomyocytes, preserving tissue integrity and coordinating signaling. Their disruption underlies arrhythmogenic cardiomyopathy (ACM), characterized by ventricular arrhythmias and progressive remodeling, frequently caused by mutations in desmosomal proteins such as Desmoplakin (DSP). Although gene therapy holds promise for monogenic cardiac diseases, the large size of the DSP gene exceeds the packaging capacity of standard viral vectors.
Research Question. We hypothesized that previously unrecognized ICD components regulate disc organization and signaling, and that their dysfunction contributes to ACM pathogenesis.
Methods. Proximity proteomics was used to map the ICD interactome in wild-type and ACM mouse models. Cardiomyocyte-specific tamoxifen-inducible DSP knockout (DspcKO) mice served as an ACM model. The candidate gene was investigated in vivo using CasAAV-mediated cardiomyocyte-specific deletion and a knockout mouse model.
Results. Proximity proteomics identified EPS15L1 (EGFR pathway substrate 15–like 1) as a previously unrecognized ICD-associated protein. Pediatric ACM hearts showed reduced EPS15L1 at the ICD by immunostaining, confirmed by Western blot and single-cell RNA analysis. Endocytic trafficking emerged as one of the most dysregulated pathways in the single-cell data. Cardiomyocyte-specific deletion induced ventricular arrhythmias, fibrosis, immune infiltration, and ICD disorganization. EPS15L1 knockout mice exhibited postnatal lethality, conduction defects, and marked ICD remodeling confirmed by super-resolution microscopy. ICD proteomics revealed reduced transmembrane proteins, including DSP, Nav1.5, and the β1-adrenergic receptor, with enrichment of scaffold and signaling proteins. EPS15L1 localized to early endosomes, and KO hearts showed reduced endosomal proteins and impaired receptor internalization, consistent with defective trafficking and recycling. Bulk RNA-seq revealed activation of adipogenic and pro-fibrotic transcriptional programs.
Conclusion. EPS15L1 is a previously unrecognized regulator of ICD integrity and cardiac homeostasis. Its loss recapitulates key features of ACM, linking membrane trafficking defects to disease pathogenesis and identifying a mutation-independent therapeutic target.