ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

Antonia T L Zech; Maksymilian Prondzynski; Sonia R Singh; Niels Pietsch; Ellen Orthey; Erda Alizoti; Josefine Busch; Alexandra Madsen; Charlotta S Behrens; Moritz Meyer-Jens; Giulia Mearini; Marc D Lemoine; Elisabeth Krämer; Diogo Mosqueira; Sanamjeet Virdi; Daniela Indenbirken; Maren Depke; Manuela Gesell Salazar; Uwe Völker; Ingke Braren; William T Pu; Thomas Eschenhagen; Elke Hammer; Saskia Schlossarek; Lucie Carrier

doi:10.3390/cells11172745

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

Cells. 2022 Sep 2;11(17):2745. doi: 10.3390/cells11172745.

Authors

Antonia T L Zech^{1

2}, Maksymilian Prondzynski^{1

2

3}, Sonia R Singh^{1

2}, Niels Pietsch^{1

2}, Ellen Orthey^{1

2}, Erda Alizoti^{1

2}, Josefine Busch^{1

2}, Alexandra Madsen^{1

2}, Charlotta S Behrens^{1

2}, Moritz Meyer-Jens^{1

2}, Giulia Mearini^{1

2}, Marc D Lemoine^{1

2

4}, Elisabeth Krämer^{1

2}, Diogo Mosqueira⁵, Sanamjeet Virdi⁶, Daniela Indenbirken⁶, Maren Depke⁷, Manuela Gesell Salazar⁷, Uwe Völker^{7

8}, Ingke Braren⁹, William T Pu^{3

10}, Thomas Eschenhagen^{1

2}, Elke Hammer^{7

8}, Saskia Schlossarek^{1

2}, Lucie Carrier^{1

2}

Affiliations

¹ Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
² DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany.
³ Department of Cardiology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Cardiology, University Heart and Vascular Center, 20246 Hamburg, Germany.
⁵ Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK.
⁶ Heinrich-Pette-Institute, Leibniz Institute of Virology, 20246 Hamburg, Germany.
⁷ Department for Functional Genomics, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, 17475 Greifswald, Germany.
⁸ DZHK (German Centre for Cardiovascular Research), Partner Site Greifswald, 17475 Greifswald, Germany.
⁹ Vector Facility, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
¹⁰ Harvard Stem Cell Institute, Cambridge, MA 02138, USA.

Abstract

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.

Keywords: autophagy; human induced pluripotent stem cell-derived cardiomyocytes; inherited cardiomyopathy; protein aggregation; sarcomere; ubiquitin-proteasome system; α-actinin-2.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actinin* / genetics
Actinin* / metabolism
Cardiomyopathy, Hypertrophic* / genetics
Cardiomyopathy, Hypertrophic* / metabolism
Humans
Induced Pluripotent Stem Cells / metabolism
Myocytes, Cardiac / metabolism
Protein Aggregation, Pathological* / genetics
Protein Aggregation, Pathological* / metabolism
Sarcomeres / metabolism

Substances

ACTN2 protein, human
Actinin

Grants and funding

This work was supported fully or in part by the German Centre for Cardiovascular Research (DZHK) to M.P., M.D.L., E.H., U.V. and L.C., German Ministry of Research Education (BMBF) to M.P., M.D.L., E.H., U.V. and L.C., Deutsche Herzstiftung (F/51/17) to Sa.S., Helmut und Charlotte Kassau Stiftung to L.C., European Research Council Advanced Grant (IndivuHeart) to T.E., Research Promotion Fund of the Faculty of Medicine (Hamburg) to A.T.L.Z., M.P. and M.D.L. (“Clinician Scientist Program” and “Project funding for young scientists”), Leducq Foundation (20CVD01) to L.C., National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs: NC/S001808/1) to D.M. and Pro Exzellenzia 4.0 to S.R.S.