2016 Fiscal Year Research-status Report
The investigation of Vitamin D system on muscle
| Project/Area Number |
16K19555
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| Research Institution | The University of Tokushima |
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Principal Investigator |
MARIA TSOUMPRA 徳島大学, 先端酵素学研究所(オープンイノベ), 特任研究員 (80756198)
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Keywords | vitamin D / vitamin D receptor / muscle |
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| Outline of Annual Research Achievements |
In vivo experiments
We have generated two different strains of muscle specific VDR knockout mice. In VDRfl/fl/MlcCre/+ strain, VDR floxed targeted ablation is driven by myosin light chain (Mlc) Cre recombinase in fast fibers only. In VDRfl/fl/HSACre/+ mice population, Cre recombinase is driven by the human skeletal actin promoter and targets VDR in striated muscle. We first investigated muscle power alterations in male mice aged 6-10 week (wk) via grip power test. Reduced grip power in VDRfl/fl/MlcCre/+ versus their control littermates was evident throughout our experimental period (5-10 wk) and more prominently observed at 6 and 8 wk of age.
In vitro experiments
Through microarray studies performed on tibialis anterior (TA) obtained from 8 wk mice we confirmed modification of a range of potential candidate genes of VDR direct action in muscle. We then evaluated selected candidate genes for follow up using qPCR studies. We performed qPCR experiments using muscle tissue from mice of different age in selected muscles such as gastrocnemius (Gast), quadriceps (Qua) and TA. We observed that MyoD and SERCA2A genes were downregulated in Gast of 10 wk and TA of 8 wk homozygote males. Of particular interest for follow up, WD Repeat and FYVE domain containing 1 (Wdfy1) and TOX high mobility group box family member 4 (Tox4) gene demonstrated an upregulation in 8 and 6 wk homozygotes. Furthermore biglycan (Bgn) and calcium/calmodulin-dependent protein kinase I (CamK1) genes were upregulated in both Gast and TA of 5 and 6 wk old homozygotes respectively.
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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
We believe that our project progresses rather smoothly. It takes a considerable amount of time to establish knockout mice population. In our case, once we had generated a pool of VDRfl/fl/MlcCre/+ mice and their control littermates we needed to evaluate the following, since this specific mouse strain is not widely used for VDR targeted ablation: 1) whether these mice were muscle specific VDR knockout- we have confirmed VDR reduction in muscle such as TA and Gast and less so Qua due to its poorer content in fast fibers 2) Whether VDR ablation was muscle specific only - we confirmed this by assessing VDR amount in several tissues (skin, intestine, bone) other than muscle 3) whether the grip power effect or the gene alteration effect was muscle specific and not systemic- we therefore measured serum calcium or phosphate to ascertain equal amounts in both homozygotes and controls. We then needed to select the best tissue in order to perform microarray studies. We therefore performed HE and NADH staining to assess the predominance of fast fibers in our selected muscles and also to eliminate possibility of necrosis or other morphological changes that might have interfered with our outcome. Finally we needed to evaluate the best time-point to collect samples for microarray and we used the grip power test data to work when grip power alteration was at the maximum (wk 6-8).
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| Strategy for Future Research Activity |
In vivo
We are planning to repeat the grip power test and microarray studies on our second VDR specific knockout mice population (VDRfl/fl/HSACre/+) and identify more genes that can be direct targets of VDR.
In vitro
We wish to assess any possible link of Wdfy1, Tox4, CamK1 and Bgn genes in myogenic differentiation and muscle physiology. We have confirmed the in vitro suppressive effect of 1,25(OH)2D3 on these genes by induction of myogenesis in C2C12 cells via daily supplementation of Ham’s-F12 medium and 1% horse serum with or without 1,25(OH)2D3. We clearly demonstrated that all four gene expression was dose-dependently downregulated upon administration of 1,25(OH)2D3 on day six where myotubes were clearly formed and maximal suppression was observed with 10 nM 1,25(OH)2D3. However we still do not know whether these genes are direct targets of VDR. We propose the following experiments: 1) overexpression of all four genes in C2C12 and selection of specific time points during myogenesis in vitro to apply 1,25(OH)2D3 and observe alterations in myogenic genes 2) cloning of promoter regions of all four genes and exploration of myogenic or inflammatory marker alteration using qPCR or luciferase assays 3) proliferation/cytotoxicity assays performed in C2C12 myotubes when these genes are overexpressed and 4) generation of transgenic mouse if time permits, using the most promising of our genes to assess phenotypic and grip power alterations.
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| Causes of Carryover |
This is a grant for two years and I am continuing the research as described. It took some time to create model mice and I will use the rest in this year.
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| Expenditure Plan for Carryover Budget |
I will use this amount for molecular biological chemicals, fetal bovine serum, culture media, microarray analysis, mice maintenance and western blotting reagents.
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Research Products
(2 results)
