Characterization of a human cellular model of desmin-related cardiomyopathy

Abstract

Desmin-related (cardio-)myopathy (DRM) serves as a class of protein-misfolding diseases, characterized by conduction defects, skeletal muscle weakness and cardiomyopathy, which is caused by variants in desmin (DES) or ααB-crystallin (CRYAB) and there is no specific therapy available. Typical biomarkers of DRM are irregular cell structure, the formation of protein aggregates and impairment of ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP). The aim of this study was to characterize a novel human induced pluripotent stem cell (hiPSC)-derived DRM model with the CRYAB c.358A>G (p.R120G) variant, which was created in the institute with the CRISPR/Cas9 gene editing tools. The c.358A>G variant was produced in the CRYAB gene of a hiPSC control line (ERC001) by using the CRISPR/Cas9 technique (CRYABhom). And then both hiPSC lines were differentiated into cardiomyocytes (CMs). A qPCR assay was performed to determine the number of alleles in the CRYABhom cell line using genomic DNA (gDNA). The alterations of CRYAB and DES in CRYABhom and ERC001 hiPSC-CMs (at 30 days) were assessed through immunofluorescence analysis. The RT-qPCR assay was performed to evaluate the mRNA levels of CRYAB and DES in CRYABhom and ERC001 hiPSC-CMs at different time points. Western blot assay was conducted to assess the UPS function in CRYABhom and ERC001 hiPSC-CMs (60 days old) which were transduced using a GFPu adenovirus as well as treated by using the inhibitor of UPS epoxomicin (EPO) or dimethylsulfoxide (DMSO). Western blot assays were performed to evaluate the function of ALP by autophagy hallmark LC3-II in CRYABhom and ERC001 hiPSC-CMs (30 and 60 days old) with the treatment of the ALP inhibitor bafilomycin (BAF) A1 or DMSO. A homozygous clone with CRYAB knock-in was identified in which two alleles in the CRYABhom hiPSC line was determined. The protein levels of CRYAB and DES were markedly lower in CRYABhom than in ERC001 hiPSC-CMs at 30 days, whereas the mRNA levels of CRYAB and DES did not differ between the two cell lines. In addition, intracellular protein aggregates of CRYAB and DES were found in CRYABhom hiPSC-CMs but not in ERC001 hiPSC-CMs. Additionally, the levels of GFPu protein were much higher in CRYABhom hiPSC-CMs than in the control group with UPS inhibitor, suggesting that UPS in CRYABhom hiPSC-CMs was impaired. Furthermore, the levels of LC3-II protein were significantly reduced in CRYABhom hiPSC-CMs in comparison to ERC001 hiPSC-CMs at 30 days with DMSO, but not with ALP inhibitor, indicating that ALP was activated in CRYABhom hiPSC-CMs. We successfully established a novel hiPSC-derived DRM model, which may serve as a useful tool for future studies.