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Role Of Gamma‐Adducin Gene In Cerebral Vascular Myogenic Response
Author(s) -
Pabbidi Mallikarjuna,
Ge Ying,
Harder David R,
Roman Richard J,
Fan Fan
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.945.7
Subject(s) - transfection , congenic , vascular smooth muscle , microbiology and biotechnology , western blot , endocrinology , medicine , chemistry , biology , gene , biochemistry , smooth muscle
Previous studies suggest that middle cerebral arteries (MCA) isolated from FHH rat exhibit impaired myogenic response (MR) that is associated with elevation in large conductance potassium (BK) channel function. Impaired MR is restored in FHH.1 BN congenic strain in which 2.4‐Mbp region containing 15 genes including Gamma‐adducin (Add3) in chromosome 1 from BN rats is transferred onto FHH rat. The present study tested the hypothesis that a mutation in Add3 gene, is responsible for the elevation of BK channel function that contribute to impaired vascular MR in FHH rats. We used 27‐mer Dicer‐Substrate RNAi (DsiRNA) to silence Add3 gene in cerebral vessels and HEK293 cells that are permanently transfected with BKα subunit to measure vascular function and BK channel function respectively using arteriomyography, patch clamp and western blot methods. MCA transfected with Add3 DsiRNA and cultured for 36hrs reduced mRNA expression in a dose‐dependent manner (50±0.2% reduction; n=3). MCA transfected with Add3 DsiRNA exhibited impaired MR when luminal pressure is increased from 50 to 150 mmHg (112±1 to 121±2μm; n=3). In contrast, the MR is intact in vessels treated with scrambled DsiRNA (50mmHg: 131±16 to 150mmHg: 91±5 μm; n=3). Total outward K channel currents were ~3 fold higher in smooth muscle cells freshly isolated from MCA that were transfected with Add3 DsiRNA compared with non‐transfected cells (DsiRNA:141±18 pA/pF; non‐ transfected: 44±10 pA/pF; n=10–11). Similarly, BK channel current was ~3 fold higher in HEK293 cells expressing BKα that were transfected with Add3 DsiRNA (+120mV; DsiRNA: 334±8 pA/pF; non‐ transfected: 109±17 pA/pF; n=3–4). Finally, expression of BKα protein is ~2 fold greater in the membrane of cerebral vessels isolated from FHH compared to FHH.1 BN rats (FHH: 2.6±0.5; FHH.1 BN : 1.3±0.1; n=3). In conclusion, these results indicate that Add3 is a viable candidate gene that may play a causal role in impaired MR by elevation of BK channel activity in FHH rats. Support or Funding Information This research has received funding support from the American Heart Association, Scientist Development Grant (13SDG146) to Pabbidi and NIH 1R01DK104184‐01 to Richard J. Roman.