ACTN2 missense variant causes proteopathy in human iPSC-derived cardiomyocytes

Orthey, E.; Zech, A. T.; Prondzynski, M.; Singh, S. R.; Alizoti, E.; Busch, J.; Madsen, A.; Behrens, C. S.; Mearini, G.; Lemoine, M. D.; Kraemer, E.; Mosqueira, D.; Virdi, S.; Inderbirken, D.; Depke, M.; Salazar, M. G.; Voelker, U.; Braren, I.; Pu, W. T.; Eschenhagen, T.; Hammer, E.; Schlossarek, S.; Carrier, L.

doi:10.1101/2021.10.28.466251

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bioRxiv · 10.1101/2021.10.28.466251

ACTN2 missense variant causes proteopathy in human iPSC-derived cardiomyocytes

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Abstract

Genetic variants in -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 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-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.

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Orthey, E., Zech, A. T., Prondzynski, M., Singh, S. R., Alizoti, E., Busch, J., Madsen, A., Behrens, C. S., Mearini, G., Lemoine, M. D., Kraemer, E., Mosqueira, D., Virdi, S., Inderbirken, D., Depke, M., Salazar, M. G., Voelker, U., Braren, I., Pu, W. T., Eschenhagen, T., Hammer, E., Schlossarek, S., Carrier, L.. 2021-10-28. ACTN2 missense variant causes proteopathy in human iPSC-derived cardiomyocytes. https://doi.org/10.1101/2021.10.28.466251

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