Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca ²⁺ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca ²⁺ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM α-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM α-actin but had no effect on c-fos expression. Conversely, the Ca ²⁺ /calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM α-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM α-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM α-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM α-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM α-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca ²⁺ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca²⁺-activated K⁺channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K⁺current 20-fold; increased IKCa1 promoter histone acetylation and c- jun binding; increased IKCa1 mRNA ∼4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle α-actin (SMαA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMαA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by ∼50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.

Objective— We previously demonstrated that upregulation of intermediate-conductance Ca ²⁺ -activated K ⁺ channels (K _Ca 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K _Ca 3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results— Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K _Ca 3.1 blocker TRAM-34. Expression of K _Ca 3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K _Ca 3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K _Ca 3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion— Blockade of K _Ca 3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.

Hypercholesterolemia (HC) is a mary risk factor for the development of coronary heart disease. Coronary ion regulation, especially calcium, is thought to be important in coronary heart disease development; however, the influence of high dietary fat and cholesterol on coronary arterial smooth muscle (CASM) ion channels is unknown. The purpose of this study was to determine the effect of diet-induced HC on CASM voltage-gated calcium current ( I_Ca). Male miniature swine were fed a high-fat, high-cholesterol diet (40% kcal fat, 2% wt cholesterol) for 20–24 wk, resulting in elevated serum total and low-density lipoprotein cholesterol. Histochemistry indicated early atherosclerosis in large coronary arteries. CASM were isolated from the right coronary artery (>1.0 mm ID), small arteries (∼200 μm), and large arterioles (∼100 μm). I_Cawas determined by whole cell voltage clamp. L-type I_Cawas reduced ∼30% by HC compared with controls in the right coronary artery (-5.29 ± 0.42 vs. -7.59 ± 0.41 pA/pF) but not the microcirculation (small artery, -8.39 ± 0.80 vs. -10.13 ± 0.60; arterioles, -10.78 ± 0.93 vs. -11.31 ± 0.95 pA/pF). Voltage-dependent activation was unaffected by HC in both the macro- and microcirculation. L-type voltage-gated calcium channel (Ca_v1.2) mRNA and membrane protein levels were unaffected by HC. Inhibition of I_Caby HC was reversed in vitro by the cholesterol scavenger methyl-β-cyclodextrin and mimicked in control CASM by incubation with the cholesterol donor cholesterol:methyl-β-cyclodextrin. These data indicate that CASM L-type I_Cais decreased in large coronary arteries in early stages of atherosclerosis, whereas I_Cain the microcirculation is unaffected. The inhibition of calcium channel activity in CASM of large coronary arteries is likely due to increases in membrane free cholesterol.

Hypercholesterolemic patients display reduced coronary flow reserve in response to adenosine infusion. We previously reported that voltage-dependent K⁺ (K_v) channels contribute to adenosine-mediated relaxation of coronary arterioles isolated from male miniature swine. For this study, we hypothesized that hypercholesterolemia attenuates K_v channel contribution to adenosine-induced vasodilatation. Pigs were randomly assigned to a control or high fat/high cholesterol diet for 20–24 wk, and then killed. After completion of the experimental treatment, arterioles (∼150 μm luminal diameter) were isolated from the left-ventricular free wall near the apical region of the heart, cannulated, and pressurized at 40 mmHg. Adenosine-mediated relaxation was significantly attenuated in both endothelium-intact and -denuded arterioles from hypercholesterolemic compared with control animals. The classic K_v channel blocker, 4-aminopyridine (1 mM), significantly attenuated adenosine-mediated relaxation in arterioles isolated from control but not hypercholesterolemic animals. Furthermore, the nonselective K⁺ channel blocker, tetraethylammonium (TEA; 1 mM) significantly attenuated adenosine-mediated relaxation in arterioles from control but not hypercholesterolemic animals. In additional experiments, coronary arteriolar smooth muscle cells were isolated, and whole cell K _v currents were measured. K_v currents were significantly reduced (∼15%) in smooth muscle cells from hypercholesterolemic compared with control animals. Furthermore, K_v current sensitive to low concentrations of TEA was reduced (∼45%) in smooth muscle cells from hypercholesterolemic compared with control animals. Our data indicate that hypercholesterolemia abolishes K_v channel contribution to adenosine-mediated relaxation in coronary arterioles, which may be attributable to a reduced contribution of TEA-sensitive K _v channels in smooth muscle of hypercholesterolemic animals.

The present study examined whether regulation of coronary tone in conduit arteries (>1.0 mm ID) is altered by exercise training. Yucatan miniature swine were treadmill trained for 16–20 wk (Ex) and compared with sedentary counterparts (Sed). Endothelium-denuded arterial rings were stretched to optimal length and allowed to equilibrate for 60 min. Inhibition of either Ca²⁺-activated channels [1 mM tetraethylammonium (TEA) or 10 nM iberiotoxin (IBTX)] or voltage-dependent K⁺channels [1 mM 4-aminopyridine (4-AP)] significantly increased resting tension in both groups; however, the effect of all K⁺-channel blockers was greater in Ex. Addition of 1 mM sodium nitroprusside reduced resting tension in both groups, confirming the presence of active basal tone; however, sodium nitroprusside-sensitive tone was increased approximately twofold in Ex compared with Sed group. Perforated patch-clamp experiments on isolated smooth muscle cells demonstrated no effect of exercise training on whole cell TEA-sensitive, 4-AP-sensitive, or basal K⁺current. Similarly, whereas TEA, 4-AP, and IBTX all decreased resting membrane potential, there was no difference in depolarization between groups. The greater effect of TEA on resting tension in Ex could be mimicked in Sed by addition of the Ca²⁺-channel agonist BAY K 8644. In conclusion, the greater response to K⁺-channel blockers after exercise training is consistent with an increased contribution of K⁺channels to regulation of basal tone in conduit coronary arteries. The lack of an effect of training on K⁺current characteristics or membrane potential responses in isolated cells suggests that a requisite factor for enhanced K⁺-channel activation in arteries from Ex, possibly stretch, is absent in isolated cells.