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 cotransfected 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 enhancer/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 cardiomyocytes, 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.