Development of an arrhythmogenic cardiomyopathy model using CRISPR-Cas9 and homology-directed repair

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. Materials and 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 rescueprovides 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.