ACTN2 mutant causes proteopathy in human iPSC-derived cardiomyocytes

Genetic variants in a-actinin 2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we compared a mutant-expressing (ACTN2mut) and a wild-type-expressing (ACTN2wt) hiPSC lines, both created with CRISPR/Cas9 genetic tools, on CM structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, mass-spectrometry. This study showed that ACTN2mut present 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 culture of hiPSC-CMs. It was associated with a marked a marked reduction of sarcomere-associated protein levels and force impairment in engineered heart tissues. In conclusion, our study highlights the upregulation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs to proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.