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Smooth muscle specific knockdown of L‐type calcium channel by exogenous microRNA (miRNA)
Author(s) -
Pang Li,
Stimers Joseph,
Rhee Sung,
Palade Philip
Publication year - 2008
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.22.1_supplement.912.32
Subject(s) - gene knockdown , transfection , hek 293 cells , small hairpin rna , microrna , cav1.2 , vascular smooth muscle , microbiology and biotechnology , gene silencing , western blot , downregulation and upregulation , gene isoform , green fluorescent protein , chemistry , calcium channel , rna interference , biology , calcium , endocrinology , gene , rna , biochemistry , organic chemistry , smooth muscle
In spontaneously hypertensive rats, vascular L‐type calcium channel pore forming α1 subunit (Cav1.2) protein expression, L‐type calcium current (Ca L ) and vascular tone are all increased, due to increased expression of the ubiquitously expressed isoform containing an exon 1b encoded N‐terminus. The objective of this study is to develop shRNA or miRNA constructs against the non‐cardiac form of Cav1.2 (exon1b isoform) to reverse the upregulation in a tissue‐specific manner. Cav1.2 exon 1b‐specific shRNA, miRNA, and appropriate controls were constructed and tested for silencing effects. In HEK cells, rat Cav1.2 expression vector (rCav1.2) was cotransfected with different constructs, CMV promoter‐driven miRNA (Cav1.2) had the strongest effect and decreased rCav1.2 protein ∼72%. More interestingly, miRNA(Cav1.2) driven by the vascular smooth muscle‐specific promoter SM22α reduced endogenous Cav1.2 expression (western blot) ∼51% and decreased Ca L (barium) current (patch clamp) ∼46% in transfected A7r5 cells. Moreover, the SM22‐GFP‐miRNA(Cav1.2) had no effect on cotransfected rCav1.2 expression in HEK cells and showed little or no GFP expression in PC12 or HL1 cells. Conclusion: SM22–GFP‐miRNA(Cav1.2) can effectively and smooth muscle‐specifically reduce Cav1.2 expression. Coupled to adeno‐associated virus, this may provide a novel long‐term therapy for hypertension. Supported by NIH HL63903.