Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Patients
2.2. Generation of iPSCs from Lymphoblastoid Cell Lines (LCLs)
2.3. Characterization of Reprogrammed Cells
2.3.1. Immunocytochemistry
2.3.2. Sequence and Karyotype Analyses
2.3.3. Teratoma Formation
2.4. Differentiation of Cardiomyocytes from iPSCs
2.5. Characterization of iPSC-CMs
2.5.1. Quantitative Real-Time PCR (qRT-PCR)
2.5.2. Field Potential Recording Using the MED64 System and Data Analysis
2.6. Construction of Expression Plasmids
2.7. Cell Culture and Generation of Nav1.5 Stable Expression Cell Lines
2.8. Automated Patch-Clamp Recording
2.9. Statistical Analysis
3. Results
3.1. Patient Information and Genetic Analysis
3.2. Generation and Characterization of iPSCs from a Patient Harboring R1623Q Mutation in Nav1.5
3.3. Differentiation of R1623Q Mutation-Harboring iPSCs into Cardiomyocytes
3.4. MED64 Analysis
3.5. Automated Patch-Clamp Analysis of Neonatal R1623Q Nav1.5 Variant
4. Discussion
Study Limitations
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Parameter | ‘Adult’ Nav1.5 | ‘Neonatal’ Nav1.5 | ‘R1623Q’ Nav1.5 | ‘Neonatal-R1623Q’ Nav1.5 | Two-Way ANOVA (p-Value) | Interaction (p-Value) | |
---|---|---|---|---|---|---|---|
Adult vs. Neonatal | WT vs. R1623Q | ||||||
Peak sodium current density (pA/pF) | −315.0 ± 44.9 | −139.5 ± 11.0 | −199.3 ± 17.3 | −172.6 ± 9.9 | * | * | * a, * b, * c |
Steady-state activation (V1/2, mV) | 55.4 ± 1.3 | −57.9 ± 1.0 | −52.6 ± 1.2 | −45.0 ± 0.8 | * | * | * c, * d, * e, * f |
Steady-state activation (k) | 4.4 ± 0.4 | 4.9 ± 0.3 | 4.7 ± 0.3 | 4.6 ± 0.2 | - | ||
Steady-state inactivation (V1/2, mV) | −70.1 ± 1.0 | −68.0 ± 1.1 | −70.7 ± 0.8 | −70.3 ± 0.6 | - | ||
Steady-state inactivation (k) | 4.4 ± 0.1 | 4.8 ± 0.1 | 6.0 ± 0.1 | 6.8 ± 0.2 | * | * | * b, * c, * d, * e, * f |
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Hayama, E.; Furutani, Y.; Kawaguchi, N.; Seki, A.; Nagashima, Y.; Okita, K.; Takeuchi, D.; Matsuoka, R.; Inai, K.; Hagiwara, N.; et al. Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes. Biology 2021, 10, 1062. https://doi.org/10.3390/biology10101062
Hayama E, Furutani Y, Kawaguchi N, Seki A, Nagashima Y, Okita K, Takeuchi D, Matsuoka R, Inai K, Hagiwara N, et al. Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes. Biology. 2021; 10(10):1062. https://doi.org/10.3390/biology10101062
Chicago/Turabian StyleHayama, Emiko, Yoshiyuki Furutani, Nanako Kawaguchi, Akiko Seki, Yoji Nagashima, Keisuke Okita, Daiji Takeuchi, Rumiko Matsuoka, Kei Inai, Nobuhisa Hagiwara, and et al. 2021. "Induced Pluripotent Stem Cell-Derived Cardiomyocytes with SCN5A R1623Q Mutation Associated with Severe Long QT Syndrome in Fetuses and Neonates Recapitulates Pathophysiological Phenotypes" Biology 10, no. 10: 1062. https://doi.org/10.3390/biology10101062