DES encodes the muscle-specific intermediate filament protein desmin, which is highly relevant to the structural integrity of cardiomyocytes. Mutations in this gene cause different cardiomyopathies including dilated cardiomyopathy. Here, we functionally validate DES-p.L112Q using SW-13, H9c2 cells, and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy in combination with deconvolution analysis. These experiments reveal an aberrant cytoplasmic aggregation of mutant desmin. In conclusion, these functional analyses support the re-classification of DES-p.L112Q as a likely pathogenic variant leading to dilated cardiomyopathy.

Peroxisomes are small, highly dynamic organelles involved in a plethora of metabolic pathways. They are essential for the efficient exchange of metabolites and cellular messengers orchestrating intracellular signaling. Calcium (Ca ²⁺ ) is one of the most prominent physiological signaling elements and regulates a wide variety of processes in cellular homeostasis and function. Recently, we showed that peroxisomes participate in cellular Ca ²⁺ dynamics by taking up and releasing Ca ²⁺ following store-operated calcium entry (SOCE), however, the mechanism of peroxisomal Ca ²⁺ uptake and its modulators remained unknown. Using live cell imaging in combination with genetically encoded calcium indicators (GECI), we show that peroxisomal calcium dynamics are independent of PEX11β and the pore protein PXMP2. Instead, we find that the ACBD5-dependent membrane contact site between peroxisomes and the endoplasmic reticulum (ER) is necessary for efficient peroxisomal Ca ²⁺ uptake. Further, we identify the ACBD5-dependent peroxisome-ER contact site as the major factor restricting peroxisome motility within the cell. Microtubules and SOCE stimulation exert smaller and independent effects on peroxisome motility. This work expands the range of known functions of the peroxisome-ER contact site.

DES encodes the muscle specific intermediate filament protein desmin and mutations in this gene cause different cardiomyopathies. Here, we functionally validate DES-p.L112Q using SW-13, H9c2 cells and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy. These experiments reveal an aberrant cytoplasmic aggregation of mutant desmin. In conclusion, these functional analyses support the re-classification of DES-p.L112Q as a likely pathogenic variant leading to dilated cardiomyopathy.

BACKGROUND: The DES gene encodes the IF (intermediate filament) protein desmin, which connects different multiprotein complexes, such as the cardiac desmosomes, and is highly important for the structural integrity of cardiomyocytes. Pathogenic DES mutations cause filament assembly defects leading to cardiomyopathies. However, most DES variants listed in genetic disease databases are currently classified as variants of unknown significance. Here, we characterized 21 different DES variants of unknown significance and 18 additional proline variants, localized in a highly conserved stretch at the C terminus of the desmin coil-2 subdomain. METHODS: We inserted desmin variants via site-directed mutagenesis and investigated the filament assembly in transfected cell lines and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy. In addition, we purified recombinant wild-type and mutant desmin and analyzed the filament formation by atomic force microscopy. Coexpression with wild-type desmin delivered by adeno-associated virus was used to model the heterozygous status of cardiomyopathy patients. RESULTS: Twelve DES variants of unknown significance formed cytoplasmic aggregates, which were likewise verified by atomic force microscopy. Of note, these 12 variants disturb the filament assembly even when coexpressed with wild-type desmin. Using a proline screen, we showed that proline residues localized at nearly each of the positions in this stretch cause filament assembly defects. By modeling the tetrameric structure of desmin, we demonstrated that specific heptad positions, as well as positions of intramolecular and intermolecular ion bridge sites, are particularly susceptible to mutations that promote desmin aggregation. CONCLUSIONS: In summary, our study demonstrated that the highly conserved stretch at the C terminus of the coil-2 subdomain is a hotspot region, where several pathogenic DES mutations cause an aberrant desmin aggregation. Based on our molecular data, we suggest reclassifying the aggregate-forming variants as likely pathogenic mutations rather than variants of unknown significance. Our study may have relevance for the genetic counseling of cardiomyopathy patients with similar DES variants.