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Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells
Published on: February 12, 2021
Phenotypic recapitulation and correction of desmoglein-2-deficient cardiomyopathy using human-induced pluripotent
Mikio Shiba1, Shuichiro Higo1,2, Takumi Kondo1
1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
Insights
A novel homozygous mutation in Desmoglein-2 (DSG2) caused severe heart failure due to desmoglein-2 deficiency. Correcting the DSG2 mutation in patient-derived cardiomyocytes restored normal heart tissue function, proving gene replacement therapy potential.
Area of Science:
- Cardiovascular Biology
- Genetics
- Stem Cell Biology
Background:
- Desmoglein-2 (DSG2) is crucial for cardiac tissue integrity.
- DSG2 mutations are linked to arrhythmogenic cardiomyopathy.
- Severe biventricular heart failure can stem from genetic defects in cardiac proteins.
Purpose of the Study:
- To investigate the role of DSG2 deficiency in a patient with severe biventricular heart failure.
- To model the disease using patient-derived induced pluripotent stem cells (iPSCs) and cardiomyocytes (iPSC-CMs).
- To evaluate the therapeutic potential of gene correction and replacement for DSG2-deficient cardiomyopathy.
Main Methods:
- Identified homozygous stop-gain mutation (c.C355T, p.R119X) in DSG2.
- Generated patient-derived iPSCs and corrected the DSG2 mutation via homology-directed repair (HDR).
- Differentiated iPSCs into iPSC-CMs for functional and structural analysis using multielectrode arrays, micro-force testing, and electron microscopy.
Main Results:
- Complete desmoglein-2 deficiency led to disrupted myocardial desmosomes and intercalated disks.
- Patient-derived iPSC-CMs exhibited abnormal electrical activity, tissue fragility, and weak contraction force.
- Gene correction in HDR-iPSC-CMs significantly rescued these disease phenotypes.
- DSG2 replacement therapy via adeno-associated virus restored cardiac contraction force.
Conclusions:
- Confirms desmoglein-2-deficient cardiomyopathy as a distinct clinical entity.
- Patient-derived iPSC-CMs effectively recapitulate and allow correction of the disease phenotype.
- Demonstrates the potential for precision medicine and gene replacement therapy in treating DSG2-related heart conditions.
Abstract:
Desmoglein-2, encoded by DSG2, is one of the desmosome proteins that maintain the structural integrity of tissues, including heart. Genetic mutations in DSG2 cause arrhythmogenic cardiomyopathy, mainly in an autosomal dominant manner. Here, we identified a homozygous stop-gain mutations in DSG2 (c.C355T, p.R119X) that led to complete desmoglein-2 deficiency in a patient with severe biventricular heart failure. Histological analysis revealed abnormal deposition of desmosome proteins, disrupted intercalated disk structures in the myocardium. Induced pluripotent stem cells (iPSCs) were generated from the patient (R119X-iPSC), and the mutated DSG2 gene locus was heterozygously corrected to a normal allele via homology-directed repair (HDR-iPSC). Both isogenic iPSCs were differentiated into cardiomyocytes [induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs)]. Multielectrode array analysis detected abnormal excitation in R119X-iPSC-CMs but not in HDR-iPSC-CMs. Micro-force testing of three-dimensional self-organized tissue rings (SOTRs) revealed tissue fragility and a weak maximum force in SOTRs from R119X-iPSC-CMs. Notably, these phenotypes were significantly recovered in HDR-iPSC-CMs. Myocardial fiber structures in R119X-iPSC-CMs were severely aberrant, and electron microscopic analysis confirmed that desmosomes were disrupted in these cells. Unexpectedly, the absence of desmoglein-2 in R119X-iPSC-CMs led to decreased expression of desmocollin-2 but no other desmosome proteins. Adeno-associated virus-mediated replacement of DSG2 significantly recovered the contraction force in SOTRs generated from R119X-iPSC-CMs. Our findings confirm the presence of a desmoglein-2-deficient cardiomyopathy among clinically diagnosed dilated cardiomyopathies. Recapitulation and correction of the disease phenotype using iPSC-CMs provide evidence to support the development of precision medicine and the proof of concept for gene replacement therapy for this cardiomyopathy.
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