Generation of a homozygous CRYAB p.Arg120Gly mutant (UKEi001-A-1) from a human iPSC line

Variants in CRYAB can lead to desmin-related (cardio-)myopathy (DRM), a genetic muscle disorder with no curative treatment available. We introduced a homozygous CRYAB c.358G > A (p.Arg120Gly) mutation, which is established for the study of DRM in mice, into a donor human induced pluripotent stem cell (hiPSC) line. Control and mutant hiPSCs were tested for karyotype integrity and pluripotency marker expression. HiPSCs could be differentiated into endoderm, ectoderm and cardiomyocytes as a mesodermal derivative in vitro. CRYABhom hiPSC-derived cardiomyocytes developed intracellular CRYAB aggregates, which is a hallmark of DRM. This newly created mutant can be utilized to study DRM and cardiac proteinopathy in a human context

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies