Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes

Abstract

Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.

Disease modeling of desmosome-related cardiomyopathy using induced pluripotent stem cell-derived cardiomyocytes

“…Among the desmosomal genes, mutations in PKP2 are most frequently identified in patients with AC[ 11 , 37 - 39 ], and have been extensively studied using patient-derived iPSC-CMs compared to other desmosomal genes ( DSG2 [ 30 , 40 , 41 ], DSP [ 42 , 43 ], and DSC2 [ 44 , 45 ]). Various clinical phenotypes and pathological characteristics observed in patients with AC harboring PKP2 mutations, downregulated desmosomal protein expression, upregulated lipogenesis, and increased apoptosis in heart tissues have been recapitulated using genetically engineered mouse models[ 11 ] and human cardiomyocytes differentiated from iPSCs[ 46 - 54 ] (Table 1 ). Most known mutations of PKP2 are heterozygous and are missense, nonsense, and frameshift mutations.…”

“…Studies on patient-derived iPSCs have identified that PKP2 variants are heterozygous missense[ 48 ], heterozygous frameshift[ 46 , 47 , 49 , 50 , 54 ], homozygous frameshift[ 47 , 51 ], compound heterozygous, and frameshift[ 52 ] mutations. Disease-specific iPSCs are generated from fibroblasts[ 46 - 48 , 51 ], keratinocytes[ 49 ], adipose tissue-derived stromal cells[ 52 ], and peripheral blood mononuclear cells[ 54 ], whereas control iPSCs are generated from healthy subjects[ 46 - 49 , 51 , 52 ], human embryonic stem cells[ 50 ], or isogenic cells engineered from patient-derived iPSCs using genome editing[ 54 ]. Genome editing allows disease modeling by introducing heterozygous and homozygous frameshift mutations in wild-type iPSC lines[ 53 ].…”

“…Decreased expression of desmosomal proteins, aberrant lipogenesis, and apoptosis of cardiomyocytes are observed in the heart tissues of patients with AC[ 9 , 55 , 56 ]. These pathological phenotypes are recapitulated in iPSC-CMs with PKP2 mutations as determined using immunostaining[ 46 - 54 ], lipid staining[ 47 - 50 ], electron microscopy[ 46 , 54 ], terminal transferase dUTP nick end labeling staining[ 46 , 47 ], and cleaved-CASP3 expression analysis[ 54 ]. Lethal arrhythmia is a hallmark of patients with AC.…”

“…We established iPSCs from a patient with AC harboring a heterozygous frameshift PKP2 mutation (c.1228dupG, p.D410fsX425) and generated an isogenic set of iPSC clones harboring three genotypes [heterozygous mutation (Hetero), homozygously corrected with homology-directed repair (HDR), and homozygously introduced frameshift mutations via non-homologous end joining (NHEJ)] using genome editing[ 54 ] (Figure 1 ). These isogenic sets of iPSCs comprise patient-derived Hetero-iPSCs, HDR-iPSCs with two-fold higher PKP2 expression relative to Hetero-iPSCs, and NHEJ-iPSCs, which do not express PKP2 , recapitulating both haploinsufficiency and complete loss of PKP2 .…”

“…In the isogenic background, the haploinsufficiency of PKP2 did not affect the localization or expression levels of desmosomal proteins in iPSC-CMs as evidenced by the results of immunostaining or western blotting analyses. However, the desmosome area represented by dot distribution on the cell periphery in Hetero-iPSC-CMs was significantly lower than that in HDR-iPSC-CMs[ 54 ], suggesting that desmosome assembly is impaired by PKP2 haploinsufficiency. The impaired assembly of desmosomal proteins in human iPSC-CMs is supported by another study using isogenic iPSC-CMs.…”