technical-applications-microelectrode-array-patch-clamp-recordings-on

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

Stanford University School of Medicine

Multiple strategies have been developed to efficiently differentiate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we describe a protocol for measuring three key functional parameters of hiPSC-CMs, including contractile function, calcium (Ca) handling, and action potential. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.

Hypertrophic cardiomyopathy (HCM) is a common inherited heart disease with a prevalence of about 0.2%. HCM is typically caused by mutations in genes encoding sarcomere or sarcomere-associated proteins. Here, we characterized induced pluripotent stem cell (iPSC) lines generated from the peripheral blood mononuclear cells of three HCM patients each carrying c.433C > T, c.610C > T, or c.235C > T mutation in the TNNI3 gene by non-integrated Sendai virus. All of the three lines exhibited normal morphology, expression of pluripotent markers, stable karyotype, and the potential of trilineage differentiation. The cardiomyocytes differentiated from these iPSC lines can serve as useful tools to model HCM in vitro.

Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying TNNI3 mutations.

 truncating mutations () are prevalent causes of inherited dilated cardiomyopathy (DCM), with a high risk of developing arrhythmogenic cardiomyopathy. We investigated the molecular mechanisms of mutant FLNC in the pathogenesis of arrhythmogenic DCM (a-DCM) using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We demonstrated that iPSC-CMs from two patients with different  mutations displayed arrhythmias and impaired contraction. FLNC ablation induced a similar phenotype, suggesting that  are loss-of-function mutations. Coimmunoprecipitation and proteomic analysis identified β-catenin (CTNNB1) as a downstream target. FLNC deficiency induced nuclear translocation of CTNNB1 and subsequently activated the platelet-derived growth factor receptor alpha (PDGFRA) pathway, which were also observed in human hearts with a-DCM and . Treatment with the PDGFRA inhibitor, crenolanib, improved contractile function of patient iPSC-CMs. Collectively, our findings suggest that PDGFRA signaling is implicated in the pathogenesis, and inhibition of this pathway is a potential therapeutic strategy in FLNC-related cardiomyopathies.

Article	Year
Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes. STAR protocols\| PubMed ID: 34746854	2021
Generation of three induced pluripotent stem cell lines from hypertrophic cardiomyopathy patients carrying TNNI3 mutations. Stem cell research\| PubMed ID: 34798544	2021
Activation of PDGFRA signaling contributes to filamin C-related arrhythmogenic cardiomyopathy. Science advances\| PubMed ID: 35196083	2022

Shane Rui Zhao

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