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

Abstract

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. 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, utilizing CQ-treated C2C12 cells and muscle biopsies derived from CQ-myopathy patients, we performed different proteomic approaches and CARS (Coherent Anti-Stokes Raman Scattering) microscopy, in addition to immunohistochemical studies. 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. 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.