Altered Electrical, Biomolecular, and Immunologic Phenotypes in a Novel Patient-Derived Stem Cell Model of Desmoglein-2 Mutant ARVC

Figure 1. Family pedigree and electrocardiogram from hiPSC donor. (A) Family pedigree of patient JHU013 (dashed circle) harboring a pathogenic variant in desmoglein-2 (DSG2 c.2358delA). Arrow signifies proband. (B) 12-lead ECG of patient JHU013 showing T wave inversion in leads V1-V4 (red markers), a classic feature in ARVC patients. Dashed box inset, highlighting T wave inversion in lead V1.

Figure 2. Donor-derived hiPSC line harbors heterozygous DSG2 c.2358delA variant and expresses markers of pluripotency. (A) Schematic protein structure of desmoglein-2. EC1–4, extracellular cadherin domains; EA, extracellular anchor domain; TM, transmembrane domain; IA, intracellular anchor domain; ICD, intracellular cadherin-like domain; LD, linker domain; RUD, repeat unit domain with 6 repeats; TD, terminal domain; DUR, DSG unique region; red arrow indicates location of truncation caused by the DSG2 c.2358delA variant. (B) Chromosomal analysis of JHU013 (DSG2Mut) hiPSCs. (C) Sanger sequencing of DSG2 in control and DSG2Mut hiPSCs showing the c.2358delA mutation. Yellow oval highlights c.2358 nucleotide under investigation. ‘N’ indicates likely heterozygosity at that location. (D) Confocal images of DSG2Mut hiPSCs depicting immunopositivity for canonical markers of pluripotency: Sex determining region Y-box 2 (SOX2), homeobox NANOG, and Octamer binding transcription factor-3/4 (OCT3/4). (E) RT-qPCR analysis showed abundant expression of pluripotency-associated transcripts in DSG2Mut hiPSCs relative to terminally differentiated control cells (primary neonatal human cardiac fibroblasts,…

Figure 3. RT-qPCR, immunostaining and Western blot analyses of DSG2Mut hiPSC-CMs. (A) RT-qPCR analysis of normal and DSG2Mut hiPSC-CMs, normalized to Ctrl1. DSG2 primer targets a region upstream of the c.2358 deletion. n = 3 for all groups. (B–E) Representative control and DSG2Mut hiPSC-CMs immunostained for desmosomal and structural proteins. Yellow arrows highlight presence of desmosomal proteins at cell-cell boundaries; yellow arrowheads denote perinuclear localization of DSG2. White scale bars: 10 µm (B–D), 5 µm (E). (F) Whole lysate western immunoblots of DSG2Mut and control hiPSC-CMs, probed for desmosomal proteins and α-actinin (ACTN2). (G) Quantification of western blot signal intensity relative to GAPDH and normalized to Ctrl1. DSG2 was quantified using the full-length 130 kDa band. n = 3 for each line. (H) Z-line lengths quantified from confocal images of α-actinin immunostaining. Shorter Z-lines in DSG2Mut cells correspond to narrower myofibrils. Two regions were randomly selected for quantification from three unique images of each line. * p < 0.05; ** p < 0.01.

Figure 4. Cytokine expression in DSG2Mut hiPSC-CMs. (A,C,E,F,H,J) Representative cytokine immunoblots from wildtype control (Ctrl1) and DSG2Mut hiPSC-CMs, grouped based on their role in inflammation: (A) the innate immune response (purple); (E) chemoattractant and pleiotropic regulation (green); (F) interleukins that regulate immune cell differentiation and proliferation (orange); and (J) adipo-fibrokines involved in lipid accumulation and metabolism and fibrotic remodeling (blue). (B,D,G,I) Box and whisker plots of selected cytokines from Figure 4A,E,F,J. Data presented as mean ± standard deviation.; Blue dashed line represents normalized values for Ctrl1; Ctrl1 (n = 4); DSG2Mut (n = 6); For all values shown in (B,D,G,I) p < 0.05 comparing DSG2Mut vs Ctrl1.

Figure 5. Bulk RNA-Seq analysis reveals both canonical and non-canonical NFĸB pathways regulate immune signaling in DSG2Mut hiPSC-CMs. (A) RNA-Seq heatmap analysis and (B) KEGG NFĸB pathway schematic. For (A), we identified genes in the NFĸB pathway that were differentially expressed between Ctrl1 and DSG2Mut hiPSC-CMs (BH-adjusted p-value < 0.05). NFĸB pathway genes were identified using genes associated with the gene ontology term “NIK/NF-kappaB signaling” (GO:0038061). Color scale denotes row-scaled log expression values. For (B), purple lines and arrows indicate canonical NFĸB pathway; green lines and arrows indicate non-canonical NFĸB pathway; red shading indicates higher expression in DSG2Mut relative to control, blue indicates lower expression, and gray indicates no difference. (C) Schematic demonstrating canonical NFĸB signaling through RELA and non-canonical NFĸB signaling through RELB.

Figure 6. Action potential characteristics of hiPSC-CM monolayers measured via optical mapping. (A) Representative traces of normalized transmembrane potential (Vm). (B) Action Potential Duration (APD) measured at 30% and 80% repolarization. (C) Relative upstroke velocity measured at 1000ms pacing and spatial heterogeneity of upstroke velocity, calculated as the relative standard deviation (RSD) of all measurements within each sample. (D) Activation maps with isochrone lines spaced 5ms apart. (E) Mean conduction velocity and conduction heterogeneity measurements at 1000 ms pacing. (F) Representative normalized calcium traces. (G) Time-to-peak Ca2+. (H) Ca2+ decay rate, measured using normalized data. Vm mapping (B–E): Ctrl1 (n = 8), Ctrl2 (n = 13) DSG2Mut (n = 9). Ca2+ mapping (G,H): Ctrl1 (n = 10), Ctrl2 (n = 7), DSG2Mut (n = 9). * p < 0.05; ** p < 0.01, *** p < 0.001.

Figure 7. Effects of DSG2 suppression on Ctrl1 hiPSC-CMs. (A) Immunostaining for desmoglein-2 and plakoglobin in Ctrl1 hiPSC-CMs treated with anti-DSG2 and negative control siRNA. White scale bars, 20 µm. (B) RT-qPCR measurements of key transcripts in Ctrl1 hiPSC-CMs with and without DSG2 knockdown (C) Activation maps with 5 ms isochrones and (D) mean conduction velocities and conduction heterogeneity of Ctrl1 hiPSC-CMs with DSG2 knockdown (E) Representative Vm traces of Ctrl1 hiPSC-CMs with DSG2 knockdown and (F) mean action potential characteristics. (G) Representative Ca2+ traces for Ctrl1 hiPSC-CMs with DSG2 knockdown and (H) mean Ca2+ transient characteristics. Neg. Control (n = 9); DSG2-KD (n = 8). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = no significant difference.