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• W4308045319 startingPage "40" @default.
• W4308045319 abstract "Kinetic analysis of intracellular calcium (Ca2+) in cardiomyocytes is commonly used to determine the pathogenicity of genetic mutations identified in patients with dilated cardiomyopathy (DCM). Conventional methods for measuring Ca2+ kinetics target whole-well cultured cardiomyocytes and therefore lack information concerning individual cells. Results are also affected by heterogeneity in cell populations. Here, we developed an analytical method using CRISPR/Cas9 genome editing combined with high-content image analysis (HCIA) that links cell-by-cell Ca2+ kinetics and immunofluorescence images in thousands of cardiomyocytes at a time. After transfecting cultured mouse cardiomyocytes that constitutively express Cas9 with gRNAs, we detected a prolonged action potential duration specifically in Serca2a-depleted ventricular cardiomyocytes in mixed culture. To determine the phenotypic effect of a frameshift mutation in PKD1 in a patient with DCM, we introduced the mutation into Cas9-expressing cardiomyocytes by gRNA transfection and found that it decreases the expression of PKD1-encoded PC1 protein that co-localizes specifically with Serca2a and L-type voltage-gated calcium channels. We also detected the suppression of Ca2+ amplitude in ventricular cardiomyocytes with decreased PC1 expression in mixed culture. Our HCIA method provides comprehensive kinetic and static information on individual cardiomyocytes and allows the pathogenicity of mutations to be determined rapidly." @default.
• W4308045319 created "2022-11-07" @default.
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• W4308045319 date "2022-12-01" @default.
• W4308045319 modified "2023-10-18" @default.
• W4308045319 title "Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with targeted gene disruption" @default.
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• W4308045319 doi "https://doi.org/10.1016/j.bbrc.2022.10.088" @default.
• W4308045319 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/36375249" @default.
• W4308045319 hasPublicationYear "2022" @default.
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