| 研究課題/領域番号 |
21K08119
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| 研究機関 | 国立研究開発法人国立循環器病研究センター |
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研究代表者 |
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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| 研究分担者 |
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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| 研究期間 (年度) |
2021-04-01 – 2024-03-31
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| キーワード | Cardiomyopathy / Arrhythmia / Desmosome |
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| 研究実績の概要 |
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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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
All planned experiments are conducted in time and sufficient experimental data are collected.
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| 今後の研究の推進方策 |
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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| 次年度使用額が生じた理由 |
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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