The N-Terminal Part of the 1A Domain of Desmin Is a Hot Spot Region for Putative Pathogenic DES Mutations Affecting Filament Assembly

Abstract

Desmin is the major intermediate filament protein of all three muscle cell types, and connects different cell organelles and multi-protein complexes such as the cardiac desmosomes. Several pathogenic mutations in the DES gene cause different skeletal and cardiac myopathies. However, the significance of the majority of DES missense variants is currently unknown, since functional data are lacking. To determine whether desmin missense mutations within the highly conserved 1A coil domain cause a filament assembly defect, we generated a set of variants with unknown significance and systematically analyzed the filament assembly using confocal microscopy in transfected SW-13, H9c2 cells and cardiomyocytes derived from induced pluripotent stem cells. We found that mutations in the N-terminal part of the 1A coil domain affect filament assembly, leading to cytoplasmic desmin aggregation. In contrast, mutant desmin in the C-terminal part of the 1A coil domain forms filamentous structures comparable to wild-type desmin. Our findings suggest that the N-terminal part of the 1A coil domain is a hot spot for pathogenic desmin mutations, which affect desmin filament assembly. This study may have relevance for the genetic counselling of patients carrying variants in the 1A coil domain of the DES gene.

Atlas ofDES(desmin) variants: Impact of variants located within the head domain on filament assembly

“…However, the variants DES-p.S13P, -p.N107D,p.E108G and -p.K109E cause a cytoplasmic desmin aggregation. Of note, p.N107D, p.E108G and p.K109E are localized in close proximity to the 1A domain, which was recently recognized as a mutation hotspot for pathogenic desmin mutations [41]. These data were verified at the single molecular level by atomic force microscopy (AFM).…”

“…Recently, we described a genetic hotspot in the 1A subdomain, where several likely pathogenic mutations affect the desmin filament formation [41]. Desmin consists of an Nterminal non-helical head domain [62], a central rod domain and a C-terminal tail domain [63].…”

“…Phalloidin conjugated with the fluorescence dye Texas Red (1:400, Thermo Fisher Scientific) was used for staining of F-actin (40 min, RT), 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL) was used for staining of the nuclei (5 min, RT). α-Actinin was stained by using antibodies as recently described [41]. Finally, two washing steps with PBS were performed and the cells were stored at 4 °C until confocal microscopy analyses.…”

“…Of note, desmin mutations interfere at different stages within the complex filament formation [23]. We have recently identified a genetic hotspot in the N-terminal part of the 1A subdomain where several likely pathogenic mutations affect the desmin filament assembly [41]. Up to now, some desmin head mutations have been previously described [15,[42][43][44].…”

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation

“…However, this model would not be optimal for further analysis of pathological cell mechanisms. In parallel, Brodelh and collaborators published a series of studies where healthy hiPSC-CMs were transiently transfected with different mutants of desmin(Brodehl et al , 2019, 2022; Kubánek et al , 2020; Kulikova et al , 2021; Protonotarios et al , 2021). Unfortunately, the mitochondrial function and/or phenotype was not characterized in these cardiomyocytes, probably because the efficiency of transient transfection was not sufficient to allow such analyses.…”