Upregulation of utrophin improves the phenotype of Duchenne muscular dystrophy hiPSC-derived CMs

Kalina Andrysiak; Paweł E Ferdek; Anna M Sanetra; Gabriela Machaj; Luisa Schmidt; Izabela Kraszewska; Katarzyna Sarad; Katarzyna Palus-Chramiec; Olga Lis; Marta Targosz-Korecka; Marcus Krüger; Marian H Lewandowski; Guillem Ylla; Jacek Stępniewski; Józef Dulak

doi:10.1016/j.omtn.2024.102247

Upregulation of utrophin improves the phenotype of Duchenne muscular dystrophy hiPSC-derived CMs

Mol Ther Nucleic Acids. 2024 Jun 11;35(3):102247. doi: 10.1016/j.omtn.2024.102247. eCollection 2024 Sep 10.

Authors

Kalina Andrysiak¹, Paweł E Ferdek², Anna M Sanetra³, Gabriela Machaj⁴, Luisa Schmidt⁵, Izabela Kraszewska¹, Katarzyna Sarad^{1

6}, Katarzyna Palus-Chramiec³, Olga Lis⁷, Marta Targosz-Korecka⁷, Marcus Krüger⁵, Marian H Lewandowski³, Guillem Ylla⁴, Jacek Stępniewski¹, Józef Dulak¹

Affiliations

¹ Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
² Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
³ Department of Neurophysiology and Chronobiology, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, 30-387 Kraków, Poland.
⁴ Laboratory of Bioinformatics and Genome Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Kraków, Poland.
⁵ Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50931 Cologne, Germany.
⁶ Doctoral School of Exact and Natural Sciences, Jagiellonian University, 30-348 Kraków, Poland.
⁷ Department of Physics of Nanostructures and Nanotechnology, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, 30-348 Kraków, Poland.

Abstract

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease. Although it leads to muscle weakness, affected individuals predominantly die from cardiomyopathy, which remains uncurable. Accumulating evidence suggests that an overexpression of utrophin may counteract some of the pathophysiological outcomes of DMD. The aim of this study was to investigate the role of utrophin in dystrophin-deficient human cardiomyocytes (CMs) and to test whether an overexpression of utrophin, implemented via the CRISPR-deadCas9-VP64 system, can improve their phenotype. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) lacking either dystrophin (DMD) or both dystrophin and utrophin (DMD KO/UTRN^(+/-)). We carried out proteome analysis, which revealed considerable differences in the proteins related to muscle contraction, cell-cell adhesion, and extracellular matrix organization. Furthermore, we evaluated the role of utrophin in maintaining the physiological properties of DMD hiPSC-CMs using atomic force microscopy, patch-clamp, and Ca²⁺ oscillation analysis. Our results showed higher values of afterhyperpolarization and altered patterns of cytosolic Ca²⁺ oscillations in DMD; the latter was further disturbed in DMD KO/UTRN^(+/-) hiPSC-CMs. Utrophin upregulation improved both parameters. Our findings demonstrate for the first time that utrophin maintains the physiological functions of DMD hiPSC-CMs, and that its upregulation can compensate for the loss of dystrophin.

Keywords: AAV; CRISPR-dCas9; DMD; Duchenne muscular dystrophy; MT: RNA/DNA Editing; calcium; cardiomyocytes; cardiomyopathy; hiPSC; utrophin.