MicroRNA-423-5p Mediates Cocaine-Induced Smooth Muscle Cell Contraction by Targeting Cacna2d2

Abstract

Cocaine abuse increases the risk of atherosclerotic cardiovascular disease (CVD) and causes acute coronary syndromes (ACS) and hypertension (HTN). Significant research has explored the role of the sympathetic nervous system mediating the cocaine effects on the cardiovascular (CV) system. However, the response of the sympathetic nervous system alone is insufficient to completely account for the CV consequences seen in cocaine users. In this study, we examined the role of microRNAs (miRNAs) in mediating the effect of cocaine on the CV system. MiRNAs regulate many important biological processes and have been associated with both response to cocaine and CV disease development. Multiple miRNAs have altered expression in the CV system (CVS) upon cocaine exposure. To understand the molecular mechanisms underlying the cocaine response in the CV system, we studied the role of miRNA-423-5p and its target Cacna2d2 in the regulation of intracellular calcium concentration and SMC contractility, a critical factor in the modulation of blood pressure (BP). We used in vivo models to evaluate BP and aortic stiffness. In vitro, cocaine treatment decreased miR-423-5p expression and increased Cacna2d2 expression, which led to elevated intracellular calcium concentrations and increased SMC contractility. Overexpression of miR-423-5p, silencing of its target Cacna2d2, and treatment with a calcium channel blocker reversed the elevated SMC contractility caused by cocaine. In contrast, suppression of miR-423-5p increased the intracellular calcium concentration and SMC contractibility. In vivo, smooth muscle-specific overexpression of miR-423-5p ameliorated the increase in BP and aortic stiffness associated with cocaine use. Thus, miR-423-5p regulates SMC contraction by modulating Cacna2d2 expression increasing intracellular calcium concentrations. Modulation of the miR-423-5p-Cacna2d2-Calcium transport pathway may represent a novel therapeutic strategy to improve cocaine-induced HTN and aortic stiffness.

Keywords: blood pressure; cocaine; intracellular calcium; microRNA; smooth muscle cells.

Figure 1

Cocaine and CM suppress miR-423-5P expression and increase Cacna2d2 expression in the mouse aortas. (A) QRT-PCR analysis was performed using RNA extracted from aortas isolated from mice treated with saline, cocaine, or CM for 10 consecutive days. Cocaine and CM resulted in decreased miR-423-5p expression and increased Cacna2d2 expression compared to saline treatment. Black circles = saline treatment, gray squares = CM treatment, open triangle = cocaine treatment (* p < 0.05 vs. saline). (B) Immunohistochemical (IHC) analysis of Cacna2d2 levels in aortic sections showed cocaine and CM treatment induced increased levels of Cacna2d2 protein expression compared to saline. Scale bars = 100 µm, magnification 20× (PBS group), 50 µm magnification 40× (CM and Cocaine groups) (C) The intensity of Cacna2d2 positive staining was quantified using Image J software. Student’s two tailed t-test was used for all statistical analysis (* p < 0.05 vs. saline).

Figure 2

miR-423 has putative binding sites in Cacna2d2. (A) In silico analysis identified 3 putative miR-423-5p binding sites in the Cacna2d2 3′ UTR (site 1: 426–448; site 2: 812–834; site 3: 1098–1121 based on the NM_020263 transcript variant). Point mutations were introduced in these three sites to determine the potential binding of miR-423-5p to the Cacna2d2 3′UTR. Green lettering represents the seed sequence of miR-423-5p while red lettering represents the seed match of the miR-423-5p binding site on the Cacna2d2 mRNA. (B) Luciferase reporter assays were performed by co-transfection of HEK-293 cells with luciferase reporter constructs containing the WT or mutant Cacna2d2 3′ UTR and either the miR-423-5p or a non-specific control miRNA (miR-Ctrl) expression vector. Gaussia luciferase activities (GLuc), measured and normalized to secreted alkaline phosphatase (SeAP) activities, show that Cacna2d2 is a direct target of miR-423-5p. Student’s two tailed t-test was used for all statistical analysis (* p < 0.05 vs. miR-Ctrl). Black circle = Cacna2d2 wt 3′ UTR + miR-Ctrl, gray circles = Cacna2d2 wt 3′ UTR + miR-423-5p, black squares = Cacna2d2 mutant 3′ UTR + miR-Ctrl, gray squares = Cacna2d2 mutant 3′ UTR + miR-423-5p.

Figure 3

Cocaine and CM increase intracellular free calcium [Ca²⁺] and induce primary mouse aortic SMC contraction. (A) Fluorescence image analysis of free cytosolic Ca²⁺ in mouse aortic SMCs treated with PBS, CM, cocaine, or the calcium ionophore ionomycin (positive control) and stained with the calcium specific dye Fluo-3 AM show that cocaine and CM increase the level of intracellular free calcium in SMCs. Scale bars = 400 μm. (B) Quantification by Image J software analysis fluorescence positive cells. Black circles = saline treatment, gray squares = CM treatment, open triangles = cocaine treatment, closed triangles = Ionomycin treatment (* p < 0.05 vs. saline). (C) Mouse Aortic SMCs were exposed to saline, cocaine, CM or the vasoactive peptide ET-1 (positive control), and their contractility was measured using the collagen gel contraction assay. Representative images of SMC contractility in the collagen contraction assay show increased SMC contraction in response to cocaine and CM. (D) Quantification of collagen gel contraction show both cocaine and CM substantially increase SMC contraction relative to PBS Student’s two tailed t-test was used for all statistical analysis (** p < 0.05 vs. PBS). Black circles = saline treatment, gray squares = CM treatment, open triangles = cocaine treatment, closed triangles = Endothelin-1 treatment.

Figure 4

The miR-423-5p-Cacna2d2 axis mediates cocaine-induced SMC contraction. SMCs were transduced with lentiviral vectors encoding control miRNA (miR-Ctr), miR-423-5p, Control miRNA antagonist (miRZip-Ctr) or the miR-423-5p antagonist (miRZip-423-5p). (A) qRT-PCR analysis shows that miR-423-5p expression was increased in the miR-423-5p-transduced cells and decreased in miR-Zip-423-5p-transduced cells. Left panel, black circles = miR-Ctrl treatment, gray squares = miR-423-5p treatment; Right panel, black dots = miRZip-Ctrl treatment, gray squares = miRZip-423-5p treatment. (* p < 0.05 vs. miR-Ctrl; ** p < 0.05 vs. miRZip-Ctrl). (B) MiR-423-5p overexpression led to a significant decrease in Cacna2d2 expression both in the absence (PBS) or presence of cocaine (p < 0.05 compared to miR-Ctr). Conversely, silencing of miR-423-5p by miRZip-423-5p treatment led to increased Cacna2d2 expression in the presence or absence of cocaine. Black triangles = miR-Ctrl + saline, gray triangles = miR-Ctrl + cocaine, black circles = miR-423-5p + saline, gray circles = miR-423-5p + cocaine, black squares = miRZip-Ctrl + saline, gray squares = miRZip-Ctrl + cocaine, black diamonds = miRZip-423-5p + saline, gray diamonds = miRZip-423-5p + cocaine (* p < 0.05 vs. miR-Ctrl + saline; ** p < 0.05 vs. miR-Ctrl + cocaine; # p < 0.05 vs. miRZip-Ctrl + saline; ## p < 0.05 vs. miRZip-Ctrl + Cocaine). (C) Aortic SMCs were treated with lentiviral vectors encoding miR-423-5p, miR-Ctrl, miRZip-423-5p or miRZip-Ctrl, and the transduced SMCs were seeded on collagen gel with or without treatment of cocaine. Representative images of the collagen contractility assay show miR-423-5p abrogates, whereas miRZip-423-5p potentiates cocaine-induced SMC contraction. (D) Quantification of collagen gel contractility assays confirm that cocaine-induced SMC contraction is mediated, at least in part, by miR-423-5p. Student’s two tailed t-test was used for all statistical analysis (ns: not significant; * p < 0.05 vs. saline; ** p < 0.05 vs. miR-Ctrl; # p < 0.05 vs. miR-Ctrl + saline). Black triangles = miR-Ctrl + saline, gray triangles = miR-Ctrl + cocaine, black circles = miR-423-5p + saline, gray circles = miR-423-5p + cocaine, black squares = miRZip-523-5p + saline, gray squares = miRZip-423-5p = cocaine.

Figure 5

Cacna2d2 and intracellular calcium concentration mediate cocaine-induced SMC contraction. (A) Cacna2d2 siRNA decreased Cacna2d2 mRNA expression in SMCs compared with Ctr siRNA in the absence or presence of cocaine. Black circle = Ctrl siRNA + saline, gray circle = Ctrl siRNA + cocaine, black triangle = Cacna2d2 siRNA + saline, gray triangle = Cacna2d2 siRNA + cocaine (ns: not significant; * p < 0.01 vs. Ctrl siRNA + saline; ** p < 0.01 vs. Ctrl siRNA + saline; # p < 0.01 vs. Ctrl siRNA + cocaine). (B) Representative images of the collagen contractility assay in SMCs in which Cacna2d2 was silenced in the absence or presence of cocaine show that the silencing of Cacna2d2 in SMCs leads to a decrease in SMC contractility compared to Ctr siRNA. (C) The effects of Cacna2d2 silencing on SMC contractility was measured by image J analysis of the collagen gel contractility assay. Black circle = Ctrl siRNA + saline, gray circle = Ctrl siRNA + cocaine, black triangle = Cacna2d2 siRNA + saline, gray triangle = Cacna2d2 siRNA + cocaine (* p < 0.05 vs. saline; ** p < 0.01 vs. Ctrl siRNA). (D) Treatment of SMCs with the L-type calcium channel antagonist Nimodipine (NM) reduces SMC contractility (compared to saline treatment) and abrogates the effect of cocaine on SMC contractility. (E) Quantification of collagen gel contraction images show NM inhibited SMC contraction compared with PBS treatment and abrogated SMC contraction induced by cocaine. Student’s two tailed t-test was used for all statistical analysis (* p < 0.05 vs. Ctrl siRNA). Black circles = SMC + saline, gray circles = SMC + cocaine, black squares = SMC + Nimodipine + saline, gray squares = SMC + Nimodipine + cocaine.

Figure 6

Treatment with miR-423-5p vector leads to increased expression of miR-423 and decreased expression of Cacna2d2 in mouse aortas. (A) Quantitative real-time PCR (qRT-PCR) analysis shows that the systemic treatment of mice with lentivirus expressing tmiR-423-5p from the smooth muscle- specific promoter Sm22a leads to an elevation of miR-423-5p compared to the control mice (vector control treatment) when measured in the aortas harvested from the cocaine (open circles) and saline (closed circles) treated mice. (ns: not significant; * p < 0.05 vs. miR-Ctrl; ** p < 0.05 vs. Saline). (B) qRT-PCR analysis also shows that miR-423-5p overexpression decreases Cacna2d2 expression in aortas compared with vector control treated mice in the absence or presence of cocaine. Student’s two tailed t-test was used for all statistical analysis (* p < 0.05 vs. saline; ** p < 0.05 vs. miR-Ctrl).

Figure 7

SMC-specific miR-423-5p ameliorated cocaine-induced increase BP and aortic stiffness in mice. Mice were injected with lentivirus encoding miR-423-5p from the smooth muscle-specific promoter (Sm22) or vector control before exposure to cocaine or PBS for 10 consecutive days. Pretreatment with miR-423-5p resulted in a partially reduction in the cocaine-induced (A) systolic and (B) Diastolic BP elevation. (* p < 0.05 vs. miR-Ctrl + cocaine). (C) Aortic stiffness was measured by pulse wave velocity (PWV) two days following the final treatment with cocaine. Pretreatment of the mice with the miR-423-5p expressing vector abrogates cocaine-induced elevation in aortic stiffness. Student’s two tailed t-test was used for all statistical analysis (ns: not significant; * p < 0.05 vs. miR-Ctrl; ** p < 0.01 vs. miR-Ctrl + cocaine). Black circles = empty vector + saline, gray circles = empty vector + cocaine, black triangle = miR-423-5p + saline, gray triangles = miR-423-5p + cocaine.