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 voltagegated 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 v 1.2). The objective of this study was to develop a small interfering RNA (siRNA) expression system against the noncardiac form of Ca v 1.2 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 v 1.2 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 v 1.2 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 v 1.2, 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 v 1.2 expression and function were effectively reduced by VSMC-specific delivery of the noncardiac form of Ca v 1.2 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 longterm vascular-specific gene therapy for hypertension.One hallmark finding in chronic hypertension is an anomalous constriction of small arteries and arterioles that is mediated by Ca 2ϩ influx through Ca L (Ca v 1.2). Ca v 1.2 expression is increased in the renal, mesenteric, and skeletal muscle circulations of different rodent models of hypertension, and the increased expression corresponds to a higher density of Ca L current and the development of anomalous vascular tone (Ohya et al., 1993;Lozinskaya and Cox, 1997;Pratt et al., 2002;Pesic et al., 2004). In smooth musclespecific Ca v 1.2 knockout mice, depolarization-induced contraction and myogenic tone were abolished, and the mean blood pressure was reduced by ϳ30 mm Hg (Moosmang et al., 2003). These studies demonstrate collectively a strong, positive correlation between blood pressure and the number of Ca L in the arterial circulation in vivo. Thus, high blood pressure may be amenable to control by therapeutic interventions that down-regulate Ca v 1.2 expression in the vasculature.Ca v 1.2 is expressed in many tissues including heart, brain, lung, uterus, the gastrointestinal system, and VSMCs (Koch et al., 1990). Several alternatively spliced isoforms have been identified. It is notable that alternative splicing of exon 1 results in two different N termini of Ca v 1.2 (isoforms A and B) that are conserved in many species. Isoform A (cardia...

Contraction by Ca2+ Influx via the L-Type Ca2+ Channel Voltage Window in Mouse Aortic Segments is Modulated by Nitric Oxide

“…L-type Ca 2+ channels play a dominant role in blood pressure regulation as suggested by the fall in mean arterial blood pressure in mice with selective ablation of the channel gene (CACN1C) in vascular smooth muscle cells (VSMCs) (Moosmang et al, 2003), the increased CACN1C gene expression in VSMCs in rodent models of hypertension (Pesic et al, 2004;Rhee et al, 2009), the anti-hypertensive effect of L-type Ca 2+ channel blockers (CaBs) (Mancia et al, 2007) and the relationship between an autoantibody against vascular L-type Ca 2+ channels and clinical characteristics of hypertensive patients (Zhou et al, 2008). Transcripts and protein expression of CACN1C are found widely in the cardiovascular system, where the ion channels serve the time-and voltage-dependent influx of Ca 2+ ions to initiate muscle contraction (Akata, 2007;Bers, 2002).…”

Ion channel remodeling in vascular smooth muscle during hypertension: Implications for novel therapeutic approaches

“…This is mainly due to low patient compliance to drug regimens, which require daily dosing and often multi-drug therapy [174,175]. Using gene therapy to control vascular tone by altering the expression and function of ion channels is an approach that could reduce the impact of patient adherence by avoiding the need for daily drug administration and reducing side effects associated with the non-specific effects of small molecule drugs [173,176]. This also avoids blood pressure fluctuations caused by short drug half-lives and inconsistent patient dosing intervals [176].…”

Modification of cardiovascular ion channels by gene therapy

“…Initial experiments demonstrate the feasibility of Ad-mediated gene delivery to the VSM of intact arteries ex vivo. Strong enhancer-promoters with high specificity for VSM gene expression have been developed [59].…”