Multiplexed measurement of cell type-specific calcium kinetics using high-content image analysis combined with
Tomoka Tabata1, Yuki Masumura1, Shuichiro Higo2
1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan.
Insights
This study introduces a new CRISPR/Cas9 and high-content image analysis method to track individual cardiomyocyte calcium kinetics. This approach rapidly determines the pathogenicity of genetic mutations linked to dilated cardiomyopathy (DCM).
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Genetic Disease Mechanisms
Background:
- Assessing genetic mutation pathogenicity in dilated cardiomyopathy (DCM) often relies on cardiomyocyte calcium (Ca2+) kinetics.
- Conventional methods analyze whole cell populations, obscuring individual cell behavior and introducing bias due to cellular heterogeneity.
Purpose of the Study:
- To develop a novel analytical method combining CRISPR/Cas9 genome editing and high-content image analysis (HCIA).
- To enable simultaneous, cell-by-cell measurement of Ca2+ kinetics and immunofluorescence in thousands of cardiomyocytes.
- To rapidly determine the pathogenicity of genetic mutations in DCM.
Main Methods:
- Utilized CRISPR/Cas9 genome editing and HCIA to link Ca2+ kinetics with immunofluorescence imaging in cultured mouse cardiomyocytes.
- Introduced specific genetic modifications, including Serca2a depletion and a patient-derived PKD1 frameshift mutation.
- Analyzed Ca2+ kinetics and protein expression at the single-cell level in mixed cultures.
Main Results:
- Identified prolonged action potential duration in Serca2a-depleted ventricular cardiomyocytes.
- Demonstrated that a PKD1 mutation decreases PC1 protein expression, which co-localizes with Serca2a and calcium channels.
- Observed suppressed Ca2+ amplitude in ventricular cardiomyocytes with reduced PC1 expression.
Conclusions:
- The developed HCIA method provides comprehensive kinetic and static data for individual cardiomyocytes.
- This technique allows for rapid and accurate determination of the pathogenicity of genetic mutations associated with DCM.
- Facilitates a deeper understanding of cellular mechanisms underlying cardiovascular diseases.
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.
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