Abstract 12450: Identifying Differential Proteins of the Arrhythmogenic Right Ventricular Cardiomyopathy Type 5 Associated Mutation in Transmembrane Protein 43 Ser358Leu by Proteomics and Interaction Studies Using TurboID

Abstract

Introduction: Transmembrane protein 43 (TMEM43) is localized in the nuclear envelope and endoplasmic reticulum (ER). TMEM43- c.1073C>T, p.S358L leads to a rare fully penetrant type 5 arrhythmogenic right ventricular cardiomyopathy (ARVC5). The molecular pathomechanism of ARVC5 is not well understood. Methods: TurboID cloned to N- and C-terminal of TMEM43 WT and p.S358L were transfected in HEK293 cells. Biotinylation assay was performed and biotinylated proteins were immunoprecipitated using streptavidin-beads. After on beads-digestion, peptide fragments were analyzed by Orbitrap mass-spectrometry (MS). Furthermore, we cultivated primary dermal fibroblasts of three TMEM43 p.S358L carriers. Proteins were isolated from cell cultures and analyzed by MS. Additionally, TMEM43-p.S358L and control fibroblasts were investigated by transmission electron microscopy (TEM). Results: TurboID proteome data shows differences in the interacting proteome of TMEM43 WT and p.S358L. VDAC2, which is involved in metabolic regulation of lipids and part of the ER-mitochondrion contact site, was one of the prominent interacting proteins less represented in TMEM43 p.S358L. The proteome of fibroblasts show 11 proteins upregulated in TMEM43-p.S358L (p<0.003). Of note, proteins involved in fatty acid metabolism, such as acyl-coenzym A thioesterase 1 encoded by the gene ACOT1, and Malonate-CoA ligase encoded by ACSF3 were strongly upregulated. ACOT1 and ACSF3 regulate intracellular levels of free fatty acids and are expressed in human cardiomyocytes. TEM images of TMEM43 p.S358L carriers show accumulation of multilamellar bodies, which may support the dysregulation of the lipid metabolism as a prominent pathomechanism. Conclusions: The altered interaction of TMEM43 p.S358L with VDAC2 is a plausible mechanism for miscommunication of ER and mitochondria perturbing the lipid homeostasis. Additionally, the differentially regulated proteins in dermal fibroblasts and the accumulation of multilamellar bodies also suggest a dysregulation of the lipid metabolism by TMEM43 p.S358L, which may explain the development of myocardial fibrofatty replacement contributing to ARVC5.