Basic/Translational Science -> Cell Physiology, Pharmacology, and Signaling D-PO01 - Featured Poster Session (ID 11) Poster

D-PO01-205 - Desmoplakin Mutations Impair Autophagy Via Beclin-1 Dysregulation In Mesenchymal Stromal Cells (mscs), Leading To Excessive Fibrosis In ARVC Hearts (ID 963)

Abstract

Background: Among desmosome gene mutations, desmoplakin (DSP) mutations in Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) involve left ventricle more frequently and appear to have more fibrosis than fatty infiltrations in ARVC hearts. MSCs are the progenitor cells for adipocytes and myofibroblasts responsible for adipogenesis and fibrogenesis, respectively.
Objective: We used induced pluripotent stem cell (iPSC)-derived MSCs to elucidate the pathogenic mechanisms by which DSP mutations in MSCs preferentially cause fibrosis in ARVC hearts, which results may lead to new therapy.
Methods: We generated iPSC-derived MSCs from normal individuals and 2 unrelated ARVC patients with DSP c.478C>T (p. Arg160X) or DSP c.3474_3475insA (p. Glu1159ArgfsX3) mutations. We studied the fibrogenic responses of these MSCs to 3 days of TGF-β1 treatment using standard Western/Co-IP, autophagy flux assay, immunostaining, and qPCR.
Results: Both DSP mutations result in no detected mutant DSP proteins (Dsp) and ~50% of wild-type (WT) Dsp (a haplo-insufficiency phenotype). Compared to normal MSCs, mutant DSP MSCs showed exaggerated collagen accumulation and secretion after TGF-β1, consistent with enhanced fibrogenic responses. Dsp normally binds to intermediate filaments, vimentin (VIM) in MSCs, and this interaction was decreased while beclin-1 (BECN1) binding to VIM was increased in DSP mutant MSCs at baseline and after TGF-β1. VIM-bound BECN1 was not available for initiating autophagy, which led to decreased autophagosomes and autolysosomes in DSP mutant MSCs (40-60% of normal MSCs) by autophagy flux assays as well as lower LC3B-II to LC3B-I ratios (60-70% of normal MSCs) by Western blots after TGF-β1. Decreased autophagy after TGF-β1 permitted collagen accumulation, secretion and complex formation in mutant MSCs, which fibrogenic features could be reversed by rapamycin, an autophagy activator.
Conclusion: Our study shows for the first time that DSP mutations lead to decreased WT Dsp levels and increased non-Dsp bound VIM in mutant MSCs. Unbound VIM sequester BECN1 from initiating autophagy, causing accumulation of collagen after TGF-β1, which likely accounts for the aggressive fibrosis seen in DSP mutant hearts.
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