Abstract Thu149: Pathogenic desmoplakin variants induce long cell-membrane caveolin-1 (CAV1) cluster formation and decrease CAV1-mediated TGFβ1 receptor endocytosis via beclin-1 deficiency

Introduction: Among arrhythmogenic cardiomyopathy (AC), desmoplakin (DSP) mutations cause a distinctive cardiomyopathy with excessive cardiac fibrosis that could precede ventricular dysfunction. We previously reported that pathogenic DSP variants led to no mutant but ~50% decreased wild-type (WT) DSP proteins (a common DSP haplo-insufficient phenotype). In DSP -mutant fibroblasts, cytosolic vimentins (usually bind to WT DSP) become unbound to DSP and sequester beclin-1 (BECN1) from activating autophagy, leading to collagen accumulations via decreased autophagic degradation. Research Question: DSP variants also hyper-activate p38 and downstream fibrotic genes via unknown mechanisms. Goal: Further elucidation of pathogenic mechanisms by which DSP mutations trigger p38 overactivation may enable novel antifibrotic therapies. Methods: We generated human induced pluripotent stem cell (iPSC) -derived cardiac mesenchymal stomal/stem cells (MSCs, fibroblasts progenitors) from normal individuals and 2 unrelated AC patients with DSP c. 478C>T (p. Arg160X) or DSP c. 3474₃475insA (p. Glu1159Argfs*3) mutations. We studied the fibrogenic responses of these MSCs to 1 ng/ml TGFβ1 using standard Western/Co-IP, immunostaining, over-expression vectors, and qPCR. Results: Mutant DSP MSCs showed abnormally long CAV1 clusters on cell membrane (34-36 in mutant vs. 5. 54 µm/cell in normal MSCs) and knocking down DSP in normal MSCs with siRNA induced long CAV1 cluster. Knocking down CAV1 in mutant MSCs reduced p38 activation by TGFβ1. BECN1 over-expression in mutant MSCs decreased CAV1 cluster lengths and TGFβ1-indcued activation of p38 and fibrotic genes. TGFβ receptor 1 (TGFβR1) accumulated in CAV1 long clusters at baseline and further increased after 2-hour TGFβ1. Cardiac fibroblasts directly isolated from mouse hearts with periostin-Cre mediated, heterozygous deletion of DSP showed the same phenomena. Thus, we show here that hyperactivation of fibrotic genes and p38 pathways by TGFβ1 in DSP mutant MSCs is mediated by down-regulated CAV1- TGFβR1 endocytosis due to BECN1 deficiency, which could be reversed by BECN1 overexpression. Conclusions: We show for the first time that pathogenic DSP variants downregulate BECN1-mediated CAV1-TGFβR1 endocytosis and induce abnormally long CAV1 clusters that over-activate p38 signaling and fibrogenic genes after TGFβ1.