Molecular mechanisms in chloroquine‐exposed muscle cells elucidated by combined proteomic and microscopic studies

• Published in: Neuropathology and applied neurobiology Vol. 49; no. 1; pp. e12877 - n/a
• Main Authors: Phan, Vietxuan, Hathazi, Denisa, Preuße, Corinna, Czech, Artur, Freier, Erik, Shema, Gerta, Zahedi, René P., Roos, Andreas
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01-02-2023
• Subjects: acquired myopathy; Biopsy; C2C12; Cardiac muscle; CARS microscopy; ChaFRADIC; Chloroquine; Chloroquine - pharmacology; COVID-19; COVID-19 Drug Treatment; Cytoskeleton; Extracellular matrix; Homeostasis; Humans; MeCP2; MeCP2 protein; Methyl-CpG binding protein; Molecular modelling; Muscle Cells; muscle proteomics; Muscular Diseases; Myopathy; Neuromuscular diseases; Protein folding; Proteins; Proteolysis; Proteomics; Rett syndrome; Skeletal muscle; Vacuoles; ChaFRADIC; acquired myopathy; MeCP2; C2C12; muscle proteomics; CARS microscopy; proteolysis
• ISSN: 0305-1846; 1365-2990
• DOI: 10.1111/nan.12877

Objectives
Chloroquine (CQ) is an antimalarial drug with a growing number of applications as recently demonstrated in attempts to treat Covid‐19. For decades, it has been well known that skeletal and cardiac muscle cells might display vulnerability against CQ exposure resulting in the clinical manifestation of a CQ‐induced myopathy. In line with the known effect of CQ on inhibition of the lysosomal function and thus cellular protein clearance, the build‐up of autophagic vacuoles along with protein aggregates is a histological hallmark of the disease. Given that protein targets of the perturbed proteostasis are still not fully discovered, we applied different proteomic and immunological‐based studies to improve the current understanding of the biochemical nature of CQ‐myopathy.
Methods
To gain a comprehensive understanding of the molecular pathogenesis of this acquired myopathy and to define proteins targets as well as pathophysiological processes beyond impaired proteolysis, utilising CQ‐treated C2C12 cells and muscle biopsies derived from CQ‐myopathy patients, we performed different proteomic approaches and Coherent Anti‐Stokes Raman Scattering (CARS) microscopy, in addition to immunohistochemical studies.
Results
Our combined studies confirmed an impact of CQ‐exposure on proper protein processing/folding and clearance, highlighted changes in the interactome of p62, a known aggregation marker and hereby identified the Rett syndrome protein MeCP2 as being affected. Moreover, our approach revealed—among others—a vulnerability of the extracellular matrix, cytoskeleton and lipid homeostasis.
Conclusion
We demonstrated that CQ exposure (secondarily) impacts biological processes beyond lysosomal function and linked a variety of proteins with known roles in the manifestation of other neuromuscular diseases.
CQ treatment may result in a muscle pathology associated with the build‐up of vacuoles in turn representing impaired proteolysis. By combining different proteomic and microscopic strategies, we here deciphered proteins affected by CQ‐exposure in muscle cells and thus provide comprehensive insights into targets of CQ‐myopathy. Hence, our studies significantly increased the current understanding of the etiology of this acquired muscle disorder.