Abstract Body (Do not enter title and authors here): Introduction. Intercalated discs (ICDs) are specialized membrane domains that physically, electrically, and chemically connect adjacent cardiomyocytes (CMs). They preserve structural integrity under mechanical stress and serve as critical hubs for signal integration. The importance of ICDs is underscored by arrhythmogenic cardiomyopathy (ACM), a genetic disorder marked by ventricular arrhythmias and progressive cardiac remodeling, often due to mutations in desmosomal proteins. While gene therapy holds promise for monogenic cardiac diseases, desmoplakin large gene size exceeds the packaging limits of standard viral vectors, making direct gene replacement challenging.
Research Question. We hypothesized that unexplored components of the ICD may regulate its structural and signaling functions and that their dysfunction contributes to ACM. Identifying such regulators could offer new, broadly applicable therapeutic targets beyond individual gene correction.
Methods. We performed proximity proteomics to define the ICD interactome in wild-type and ACM model mice. A cardiomyocyte-specific, tamoxifen-inducible knockout of Desmoplakin (DspcKO) served as a novel ACM mouse model. Candidate genes were screened in vivo using CasAAV-mediated cardiomyocyte-specific deletion.
Results. Proximity proteomics revealed two previously uncharacterized ICD-associated proteins: Cobll1 (Cordon-bleu WH2 repeat protein-like 1) and Eps15l1 (Epidermal growth factor receptor pathway substrate 15-like 1). ICD immunostaining in ACM pediatric patient's heart biopsies showed reduced expression of both proteins, confirmed by Western blot and single-cell analysis. Endocytic trafficking emerged as a top dysregulated pathway in the single-cell data, prompting investigation of endosomal involvement. Cardiomyocyte-specific depletion of both proteins in mice induced ACM-like phenotypes, including arrhythmias, fibrosis, immune infiltration, and ICD disorganization. Western blotting showed enrichment in early endosome fractions. Super-resolution microscopy confirmed colocalization with EEA1+ but not RAB7+ compartments, supporting a role in early endosomal trafficking.
Conclusion. Cobll1 and Eps15l1 are newly identified regulators of ICD integrity and cardiac homeostasis. Their loss mimics core features of ACM, linking membrane trafficking defects to disease pathogenesis. These findings highlight new therapeutic entry points and advance efforts to develop mutation-independent treatments for ACM.