Impaired autophagic flux in skeletal muscle of plectin-related epidermolysis bullosa simplex with muscular dystrophy
Authors: Zrelski Michaela M. , Eckhard Margret , Fichtinger Petra , Hösele Sabrina , Sombke Andy , Mill Leonid , Kustermann Monika , Schmidt Wolfgang M. , Norwood Fiona , Schlötzer-Schrehardt Ursula , Wiche Gerhard , Schröder Rolf , Winter Lilli 
Published: February 2025

Background

Plectin, a multi-functional cytolinker and intermediate filament (IF) stabilizing protein, is essential for muscle fiber integrity and function. Mutations in the human plectin gene (PLEC) cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). The disorganization and aggregation of desmin IFs in conjunction with degenerative changes of the myofibrillar apparatus are key features in the skeletal muscle pathology of EBS-MD. We performed a comprehensive analysis addressing protein homeostasis in this rare protein aggregation disease by using human EBS-MD tissue, plectin knock-out mice, and plectin-deficient cells.

Methods

Protein degradation pathways were analyzed in muscles from EBS-MD patients, muscle-specific conditional plectin knockout (MCK-Cre/cKO) mice, as well as immortalized plectin-deficient (Plec-/-) myoblasts by electron and immunofluorescence microscopy. To obtain a comprehensive picture of autophagic processes, we evaluated the transcriptional regulation and expression levels of autophagic markers in plectin-deficient muscles and myoblasts (RNA-Seq, qRT-PCR, immunoblotting). Autophagic turnover was dynamically assessed by measuring baseline autophagy as well as specific inhibition and activation in mCherry-EGFP-LC3B-expressingPlec+/+andPlec-/-myoblasts, and by monitoring primary wild-type (WT) and plectin-deficient (P0) myoblasts using organelle-specific dyes. Analyses of chloroquine (CQ)-treated MCK-Cre/cKO mice corroborated that loss of plectin coincides with impaired autophagic clearance.

Results

Our study identified massive accumulation of degradative vacuoles as well as LC3 and SQSTM1-positive patches in EBS-MD patient and MCK-Cre/cKO mouse muscles andPlec-/-myoblasts. While the transcriptional regulation of autophagy-related proteins remained largely unaltered, protein levels of downstream targets of the autophagosomal degradation machinery were elevated in MCK-Cre/cKO muscle lysates (e.g. LAMP2, BAG3, and SQSTM1 to ∼160, ∼150, and ∼140% of control samples, respectively;P<0.05). Autophagosome turnover was compromised in mCherry-EGFP-LC3B-expressingPlec-/-myoblasts compared toPlec+/+cells (∼40% reduction in median red:green ratio, reduced puncta number, smaller puncta;P<0.01). By labelling autophagic compartments with CYTO-ID dye or lysosomes with LYSO-ID, we found reduced signal intensities in P0 cells (P<0.001). Treatment of primary myoblasts with CQ led to drastic swelling of autophagic vacuoles in WT myoblasts, while the swelling in P0 cells was moderate, establishing a defect in their autophagic clearance. Finally, CQ-treatment of MCK-Cre/cKO mice reassured in vivo the concept that autophagic flux is impaired in plectin-deficient muscles.

Conclusions

Our work demonstrates that the characteristic protein aggregation pathology in EBS-MD is linked to an impaired autophagic flux. The obtained results open a new perspective on the understanding of the protein aggregation pathology in plectin-related disorders and provides a basis for further pharmacological intervention.