Development of an Arrhythmogenic Cardiomyopathy Model Using CRISPR-Cas9 and Homology-Directed Repair

DOI: https://doi.org/10.63181/ujcvs.2025.33(3).62-74
Keywords: CRISPR-Cas9, gene editing, model of cardiomyopathy, iPSC, PKP2 mutation, homology-directed repair, desmosomal integrity, correction of cardiomyopathy

Abstract

Arrhythmogenic cardiomyopathy (ACM) frequently results from loss-of-function variants in PKP2, leading to desmosomal failure, electrical instability, and fibrofatty remodeling.

Aim: To create a human cellular ACM model by CRISPR–Cas9 knock-in of PKP2 c.2011delC in control induced pluripotent stem cells (iPSCs) and to evaluate allele-specific correction by homology-directed repair (HDR) in patient-derived iPSCs.

Methods: Two complementary iPSC systems were engineered: (i) pathogenic PKP2 c.2011delC knock-in (exon 10; p.Lys672Argfs*12) in control iPSCs and (ii) CRISPR HDR correction in patient iPSCs. Clonal edits were confirmed by Sanger/TIDE and long-range PCR (~2 kb); karyotypes were normal and off-targeting was below method thresholds (TIDE ≈2%, amplicon-seq ≤1%). iPSC-derived cardiomyocytes were assessed for PKP2 expression/localization (IF/Western), desmosomal organization (PKP2/DSP/Cx43), electrophysiology (whole-cell patch clamp: APD90, arrhythmic events), Ca²⁺ handling (Fluo-4; unit of analysis = differentiation; 5 cells × 3 differentiations/group), and fibrofatty remodeling (Oil Red O, Picrosirius Red). From patient edits, 12 single-cell clones were isolated; 9 were fully corrected, 6 advanced to functional testing.

Results: Mutant cardiomyocytes recapitulated ACM: PKP2 protein ~34.2% of control; desmosomal score 0.83±0.27 (vs 2.91±0.17), prolonged APD90 275±18 ms (vs 224±15 ms), and arrhythmias in 78% (Healthy 5%). Ca²⁺ transients showed reduced ΔF/F₀ 0.704±0.034 (vs 1.000±0.039) and frequency shifts (Healthy 1.009±0.024 Hz, ACM 0.964±0.120 Hz, corrected 1.401±0.069 Hz; ANOVA p=0.0167). CRISPR correction restored PKP2 to 92.1% of control, improved desmosomal organization to 2.68±0.19, shortened APD90 to 225±13 ms, reduced arrhythmias to 12%, increased Ca²⁺ amplitude to 1.161±0.023, and normalized collagen (4.8±0.6%) and lipid (8.2±1.2%) burdens.

Conclusions: Dual-direction editing–pathogenic knock-in for modeling and isogenic HDR correction for rescue – provides a robust human platform for ACM. Correction of PKP2 c.2011delC reverses desmosomal, electrical, Ca²⁺-handling, and fibrofatty defects, supporting translational development of gene-editing therapies for ACM.

References

  1. Bosman LP, Te Riele ASJM. Arrhythmogenic right ventricular cardiomyopathy: a focused update on diagnosis and risk stratification. Heart. 2022 Jan;108(2):90-97. https://doi.org/10.1136/heartjnl-2021-319113.
  2. Laurita KR, Vasireddi SK, Mackall JA. Elucidating arrhythmogenic right ventricular cardiomyopathy with stem cells. Birth Defects Res. 2022 Oct 1;114(16):948-958. https://doi.org/10.1002/bdr2.2010.
  3. Khedro T, Duran JM, Adler ED. Modeling Nonischemic Genetic Cardiomyopathies Using Induced Pluripotent Stem Cells. Curr Cardiol Rep. 2022 Jun 3;24(6):631-644. https://doi.org/10.1007/s11886-022-01683-8
  4. Raad N, Bittihn P, Cacheux M, Jeong D, Ilkan Z, Ceholski D, Kohlbrenner E, Zhang L, Cai CL, Kranias EG, Hajjar RJ, Stillitano F, Akar FG. Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation. Circulation. 2021 Aug 10;144(6):441-454. https://doi.org/10.1161/CIRCULATIONAHA.119.043502.
  5. Madonna R, Moscato S, Cufaro MC, Pieragostino D, Mattii L, Del Boccio P, Ghelardoni S, Zucchi R, De Caterina R. Empagliflozin inhibits excessive autophagy through the AMPK/GSK3beta signalling pathway in diabetic cardiomyopathy. Cardiovasc Res. 2023 May 22;119(5):1175-1189. https://doi.org/10.1093/cvr/cvad009.
  6. Stava TT, Leren TP, Bogsrud MP. Molecular genetics in 4408 cardiomyopathy probands and 3008 relatives in Norway: 17 years of genetic testing in a national laboratory. Eur J Prev Cardiol. 2022 Oct 18;29(13):1789-1799. https://doi.org/10.1093/eurjpc/zwac102.
  7. Vučković S, Dinani R, Nollet EE, Kuster DWD, Buikema JW, Houtkooper RH, Nabben M, van der Velden J, Goversen B. Characterization of cardiac metabolism in iPSC-derived cardiomyocytes: lessons from maturation and disease modeling. Stem Cell Res Ther. 2022 Jul 23;13(1):332. https://doi.org/10.1186/s13287-022-03021-9.
  8. Amin G, Castañeda SL, Zabalegui F, et al. Generation of two edited iPSCs lines by CRISPR/Cas9 with point mutations in PKP2 gene for arrhythmogenic cardiomyopathy in vitro modeling. Stem Cell Research. 2023;71:103157. https://doi.org/10.1016/j.scr.2023.103157
  9. Janz A, Zink M, Cirnu A, et al. CRISPR/Cas9-edited PKP2 knock-out (JMUi001-A-2) and DSG2 knock-out (JMUi001-A-3) iPSC lines as an isogenic human model system for arrhythmogenic cardiomyopathy (ACM). Stem Cell Research. 2021;53:102256. https://doi.org/10.1016/j.scr.2021.102256
  10. Prondzynski M, Lemoine MD, Zech AT, et al. Disease modeling of a mutation in α-actinin 2 guides clinical therapy in hypertrophic cardiomyopathy. EMBO Molecular Medicine. 2019;11(7):e11115. https://doi.org/10.15252/emmm.201911115
  11. Li J, Wiesinger A, Fokkert L, Bakker P, de Vries DK, Tijsen A.J, Pinto Y.M, Verkerk A.O, Christoffels V.M, Boink GJJ, Devalla H.D. Modeling the atrioventricular conduction axis using human pluripotent stem cell-derived cardiac assembloids. Cell Stem Cell. 2024 Nov 7;31(11):1667-1684.e6. https://doi.org/10.1016/j.stem.2024.08.008.
  12. Wang H, La Russa M, Qi LS. CRISPR/Cas9 in genome editing and beyond. Annual Review of Biochemistry. 2017;86:227–264. https://doi.org/10.1146/annurev-biochem-060815-014607
  13. Bruna O S Câmara, Bruno M Bertassoli, Natália M Ocarino, Rogéria Serakides. Differentiation of Mesenchymal Stem Cells from Humans and Animals into Insulin-producing Cells: An Overview In Vitro Induction Forms. Curr Stem Cell Res Ther 2021;16(6):695-709. https://doi.org/10.2174/1574888X16666201229124429.
  14. Gharanei M, Shafaattalab S, Sangha S, Gunawan M, Laksman Z, Hove-Madsen L, Tibbits G.F. Atrial-specific hiPSC-derived cardiomyocytes in drug discovery and disease modeling. Methods. 2022 Jul;203:364-377. https://doi.org/10.1016/j.ymeth.2021.06.009.
  15. Hill M, Andrews-Pfannkoch C, Atherton E, Knudsen T, Trncic E, Marmorstein AD. Detection of Residual iPSCs Following Differentiation of iPSC-Derived Retinal Pigment Epithelial Cells. J Ocul Pharmacol Ther. 2024 Dec;40(10):680-687. https://doi.org/10.1089/jop.2024.0130.
  16. Varzideh F, Gambardella J, Kansakar U, Jankauskas S.S., Santulli G. Molecular Mechanisms Underlying Pluripotency and Self-Renewal of Embryonic Stem Cells. Int J Mol Sci. 2023 May 7;24(9):8386. https://doi.org/10.3390/ijms24098386.
  17. Bo Zhang, Jinying Lin, Vanja Perčulija, Yu Li, Qiuhua Lu, Jing Chen, Songying Ouyang. Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system. Nucleic Acids Res. 2022 Nov 11;50(20):11820-11833. https://doi.org/10.1093/nar/gkac987.
  18. Joshi J, Albers C, Smole N, Guo S and Smith SA (2024) Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) for modeling cardiac arrhythmias: strengths, challenges and potential solutions. Front. Physiol. 15:1475152. https://doi.org/ 10.3389/fphys.2024.1475152
  19. Janz K, Tucker NR, Courtney SM, et al. Generation of human iPSC lines with PKP2 and DSG2 knockouts using CRISPR/Cas9: a novel in vitro model for arrhythmogenic cardiomyopathy. Cell Stem Cell. 2021;29(3):594–606.e8. https://doi.org/10.1016/j.scr.2023.103157
  20. Loiben A, Friedman CE, Chien WM, et al. Generation of human iPSC line from an arrhythmogenic cardiomyopathy patient with a DSP protein-truncating variant. Stem Cell Res. 2022 Nov;66:102987. https://doi.org/10.1016/j.scr.2022.102987
  21. Wu P, Li Y, Cai M, Ye B, Geng B, Li F, Zhu H, Liu J, &Wang X (). Ubiquitin carboxyl-terminal hydrolaseL1 of Cardiomyocytes promotes macroautophagy andProteostasis and protects against post-myocardial infarctioncardiac remodeling and heart failure. Frontiers in Cardiovas-cular Medicine. 2022. 9. 866901. https://doi.org/10.3389/fcvm.2022.866901
  22. Saffitz JE. Molecular mechanisms in the pathogenesis of arrhythmogenic cardiomyopathy. Cardiovasc Pathol. Author manuscript. 2017 Feb 27;28:51-58. https://doi.org/10.1016/j.carpath.2017.02.005
  23. Wu I, Zeng A, Greer-Short A, Aycinena JA, Tefera AE, Shenwai R, Farshidfar F. AAV9:PKP2 improves heart function and survival in a Pkp2-deficient mouse model of arrhythmogenic right ventricular cardiomyopathy. Commun Med (Lond). 2024 Mar 18;4(1):38. https://doi.org/10.1038/s43856-024-00450-w.
  24. Yanlin Zhu 1, Weili Fu. Peripheral Blood-Derived Stem Cells for the Treatment of Cartilage Injuries: A Systematic Review. Front Bioeng Biotechnol. 2022 Jul 22;10:956614. https://doi.org/10.3389/fbioe.2022.956614.
  25. Zhu K, Bao X, Wang Y, Lu T, Zhan L. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte modelling of cardiovascular diseases for natural compound discovery. Biomedicine & Pharmacotherapy. 2023;157:113970. https://doi.org/10.1016/j.biopha.2022.113970.
  26. Ou M, Zhao M, Li C, Tang D, Xu Y, Dai W, Sui W, Zhang Y, Xiang Z, Mo C, Lin H, Dai Y. Single-cell sequencing reveals the potential oncogenic expression atlas of human iPSC-derived cardiomyocytes. Biol Open. 2021 Feb 15;10(2):bio053348. https://doi.org/10.1242/bio.053348.
  27. Røsand Ø, Wang J, Scrimgeour N, Marwarha G, Høydal MA. Exosomal Preconditioning of Human iPSC-Derived Cardiomyocytes Beneficially Alters Cardiac Electrophysiology and Micro RNA Expression. Int J Mol Sci. 2024 Aug 2;25(15):8460. https://doi.org/10.3390/ijms25158460.
  28. Hanna P, Hoover DB, Kirkland LG, Smith EH, Poston MD, Peirce SG, Garbe CG, Cha S, Mori S, Brennan JA, Armour JA, Rytkin E, Efimov IR, Ajijola OA, Ardell JL, Shivkumar K. Noradrenergic and cholinergic innervation of the normal human heart and changes associated with cardiomyopathy. Anat Rec (Hoboken). 2025 May 14:10.1002/ar.25686. doi: 10.1002/ar.25686
  29. Pascual-Gilabert M, Artero R, López-Castel A. The myotonic dystrophy type 1 drug development pipeline: 2022 edition. Drug Discov Today. 2023 Mar;28(3):103489. https://doi.org/10.1016/j.drudis.2023.103489.
Published
2025-09-25
How to Cite
1.
Gramatiuk SM, Estrin SI, Ivanova YV, Kravchenko TV, Hubbard E, Sargsyan K. Development of an Arrhythmogenic Cardiomyopathy Model Using CRISPR-Cas9 and Homology-Directed Repair. ujcvs [Internet]. 2025Sep.25 [cited 2025Oct.9];33(3):62-4. Available from: https://www.cvs.org.ua/index.php/ujcvs/article/view/757
Issue
Ukrainian Journal of Cardiovascular Surgery Vol 33 No 3 2025
Section
MYOCARDIAL PATHOLOGY AND HEART FAILURE

Most read articles by the same author(s)