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

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Abstract

<p id="d14007408e325">Loss-of-function mutations in <i>PKP2</i>, 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 <i>PKP2</i> 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 <i>DSG2</i> 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 <i>PKP2</i> 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. </p><div class="fig panel" id="undfig1"> <a class="named-anchor" id="undfig1">  </a> <div class="figure-container so-text-align-c"> <img alt="" class="figure" src="/document_file/bc9c57e1-b662-48d4-9005-143b1971fe96/PubMedCentral/image/fx1"/> </div> <div class="panel-content"/> </div><p id="d14007408e347"> <div class="list"> <a class="named-anchor" id="ulist0010">  </a> <ul class="so-custom-list" style="list-style-type: none"> <li id="u0010"> <div class="so-custom-list-label so-ol">•</div> <div class="so-custom-list-content so-ol"> <p id="p0010">Generation of isogenic iPSC-CMs with a precise dose of plakophilin-2</p> </div> </li> <li id="u0015"> <div class="so-custom-list-label so-ol">•</div> <div class="so-custom-list-content so-ol"> <p id="p0015">Modeling reduced contractility and impaired desmosome assembly using iPSC-CMs</p> </div> </li> <li id="u0020"> <div class="so-custom-list-label so-ol">•</div> <div class="so-custom-list-content so-ol"> <p id="p0020">Generation of isogenic iPSC-CMs for desmosome-imaging</p> </div> </li> <li id="u0025"> <div class="so-custom-list-label so-ol">•</div> <div class="so-custom-list-content so-ol"> <p id="p0025">Proof of concept of <i>PKP2</i> replacement using isogenic plakophilin-2-deficient iPSC-CMs </p> </div> </li> </ul> </div> </p><p class="first" id="d14007408e373">In this article, Higo and colleagues generate isogenic iPS cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of <i>PKP2</i>, modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency. <i>PKP2</i> gene replacement recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome imaging. The isogenic iPSC-CMs recapitulate the pathology of arrhythmogenic cardiomyopathy and provide a rapid and convenient cellular platform for therapeutic development. </p>

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Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes

Paul Burridge, Elena Matsa, Praveen Shukla … (2014)

Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification.

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Diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia: proposed modification of the task force criteria.

Frank I Marcus, William J. McKenna, Duane L Sherrill … (2010)

In 1994, an International Task Force proposed criteria for the clinical diagnosis of arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) that facilitated recognition and interpretation of the frequently nonspecific clinical features of ARVC/D. This enabled confirmatory clinical diagnosis in index cases through exclusion of phenocopies and provided a standard on which clinical research and genetic studies could be based. Structural, histological, electrocardiographic, arrhythmic, and familial features of the disease were incorporated into the criteria, subdivided into major and minor categories according to the specificity of their association with ARVC/D. At that time, clinical experience with ARVC/D was dominated by symptomatic index cases and sudden cardiac death victims-the overt or severe end of the disease spectrum. Consequently, the 1994 criteria were highly specific but lacked sensitivity for early and familial disease. Revision of the diagnostic criteria provides guidance on the role of emerging diagnostic modalities and advances in the genetics of ARVC/D. The criteria have been modified to incorporate new knowledge and technology to improve diagnostic sensitivity, but with the important requisite of maintaining diagnostic specificity. The approach of classifying structural, histological, electrocardiographic, arrhythmic, and genetic features of the disease as major and minor criteria has been maintained. In this modification of the Task Force criteria, quantitative criteria are proposed and abnormalities are defined on the basis of comparison with normal subject data. The present modifications of the Task Force Criteria represent a working framework to improve the diagnosis and management of this condition. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00024505.

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Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Daisuke Mashiko, Yoshitaka Fujihara, Yuhkoh Satouh … (2013)

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.