2021 Fiscal Year Research-status Report
Elucidation of the pathology in ARVC caused by Japanese-specific DSG2 mutations using knock-in mice models: searching for the therapeutic targets
Project/Area Number |
21K08119
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Research Institution | National Cardiovascular Center Research Institute |
Principal Investigator |
ZANKOV DimitarP 国立研究開発法人国立循環器病研究センター, 研究所, 室長 (20631295)
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Co-Investigator(Kenkyū-buntansha) |
大野 聖子 国立研究開発法人国立循環器病研究センター, 研究所, 部長 (20610025)
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Project Period (FY) |
2021-04-01 – 2024-03-31
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Keywords | Cardiomyopathy / Arrhythmia / Desmosome |
Outline of Annual Research Achievements |
We generated knock-in mice carrying the two most frequent mutations in the Japanese cohort of arrhythmogenic right ventricular cardiomyopathy (ARVC): desmoglein 2 R297C and D499A using CRISPR/Cas9 genome editing. The phenotype of the mice shows significant similarity to the clinical appearance of human patients. There are also inequality in phenotype between the two kind of mice. Only 297C carriers (both hetero- and homozygous) die suddenly starting from the age of 9 weeks. Dissection of the hearts show enlargement of cavities predominantly on the right side. In homozygous 297C mice we also observed pale zones with increased density scattered allover the heart. Histology confirmed that these areas are fibrotic replacement of myocardium. Telemetry experiment in 297C homozygous mice uncovered cardiac conduction and rhythm defects: ventricular tachycardia, premature complexes, AV block. MRI showed enlargement of the cavities of both left and right ventricles but predominantly right in 297C homozygous mice at the age of 19 weeks. Confocal images stained for TUNEL reaction confirmed fragmented DNA, the hallmark of apoptosis, in all mice groups. Treadmill experiment of 11 weeks old mice of all groups produced cardiac dysfunction significantly faster (19 weeks old) than spontaneous development (after 25 weeks of age). We performed RNA sequencing of 11 weeks old 297C mice separating right and left ventricles. Initial analysis shows that most differentially expressed genes are related to immune system and inflammation.
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Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
All planned experiments are conducted in time and sufficient experimental data are collected.
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Strategy for Future Research Activity |
We plan further experiments to continue phenotype description, uncover mechanisms underlying the phenotype and discover putative target for curative treatment: - Western blot and immunohistochemistry to evaluate how intercalated proteins are affected by the desmoglein 2 mutations - Patch clamp recordings, particularly of sodium current, which is known to be located in the intercalated disks and affected by desmosomal mutations - RNA sequencing to evaluate transcriptome in very young mice (5 weeks old) and compare to the already available RNA sequencing data from 11 weeks old mice in order to follow the progression and initiation of molecular mechanisms - According to the results of RNA sequencing analysis, molecular and imaging experiments will be designed to verify bioinformatic data and identify therapeutic target
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Causes of Carryover |
実験で使用する予定のマウスの繁殖が遅れ、次年度の解析に回したため。現在繁殖は順調に進んでおり、次年度に使用する予定である。
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Research Products
(1 results)