2018 Fiscal Year Research-status Report
心室細動発症におけるTMEM168遺伝子変異解析とトラスレーショナル研究への応用
| Project/Area Number |
18K08033
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Brugada syndrome |
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| Outline of Annual Research Achievements |
Exome-wide analysis in a family with inherited Brugada syndrome reveal heterozygous point mutation in TMEM168, a gene with unknown function. Cloning of the human variant and expression in HL-1 cardiomyocytes found that TMEM168 is localized in the nuclear membrane and endogenous Na+ current of HL-1 cells transfected with TMEM168 variant had reduced density and Nav1.5 protein (cardiac Na+ channel alpha subunit). We generated knock-in mice (CRISPR/Cas9 genome editing) that incorporated similar to the studied human family point mutation. Like in HL-1 cells, TMEM168 is localized in the nuclear membrane and co-localized with lamin A (nuclear membrane protein). Nav1.5 protein and Na+ current density were reduced in a similar pattern. ECG recordings in the presence of Ajmaline (Na+ channel blocker) detected ST segment elevation in right precordial leads, conduction disturbances and ventricular arrhythmias selectively in the knock-in animals. There was no transcriptional dysregulation of Na+ channel subunits. We identified increased ubiquitination of Nav1.5 in the knock-in hearts. There was also increased interaction between mutated TMEM168 and alphaB-crystallin, a chaperon protein that is part of Nav1.5 molecular complex and is physiological suppressor of Nedd4 - ubiquitine ligase also associated with Nav1.5. We propose the following mechanism of Nav1.5 regulation: increased affinity of variant TMEM168 protein to alphaB-crystallin deplete its association with Nav1.5 and release inhibitory effect on Nedd4. This results in enhanced ubiquitination and degradation of Nav1.5.
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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
1. Exome sequencing identified successfully genomic variant and it was verified by Sanger method.
2. We managed to confirm the association between TMEM168 variant and electrophysiological properties of HL-1 cardiomyocytes and in vivo in TMEM168 knock-in mouse model of genetic defect.
3. Detection of unexpected mechanism of Nav1.5 regulation by nuclear membrane protein.
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| Strategy for Future Research Activity |
1. Sreening Brugada syndrome patients with unknown genomic defect for TMEM168 mutations.
2. Further experiments to study mechanistic effects of TMEM168 mutants on cardiac ion channels in addition to Nav1.5 e.g., other Brugada syndrome-related channels like Ito (transient outward K+ current).
3. Attempt to rescue the phenotype of knock-in mice by interfering with mechanisms that regulate Nav1.5 trafficking or ubiquitination.
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Research Products
(1 results)
