Vascular smooth muscle-specific knockdown of the noncardiac form of the L-type calcium channel by microRNA-based short hairpin RNA as a potential antihypertensive therapy

Abstract

In different rodent models of hypertension, vascular voltage-gated L-type calcium channel (Ca L) current and vascular tone is increased because of increased expression of the noncardiac form of the Ca L (Ca v1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Ca v1.2 to reduce its expression in vascular smooth muscle cells (VSMCs). siRNAs expressing plasmids and appropriate controls were constructed and first screened in human embryonic kidney (HEK) 293 cells cotrans-fected with a rat Ca v1.2 expression vector. The most effective gene silencing was achieved with a modified mir-30a-based short hairpin RNA (shRNAmir) driven by the cytomegalovirus promoter. In A7r5 cells, a vascular smooth muscle cell line, two copies of shRNAmir driven by a chimeric VSMC-specific en- hancer/promoter reduced endogenous Ca v1.2 expression by 61 % and decreased the Ca L current carried by barium by 47%. Moreover, the chimeric vascular smooth muscle-specific enhancer/promoter displayed almost no activity in non-VSMCs (PC-12 and HEK 293). Because the proposed siRNA was designed to only target the noncardiac form of Ca v1.2, it did not affect the Ca L expression and function in cultured cardiomyo-cytes, even when driven by a stronger cytomegalovirus promoter. In conclusion, vascular Ca v1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Ca v1.2 siRNA without similarly affecting cardiac Ca L expression and function. When coupled with a viral vector, this molecular intervention in vivo may provide a novel long-term vascular-specific gene therapy for hypertension. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.