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 Ca(2+)-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 approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 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.

Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.

Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise ␣ and ␤ subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype. Smooth muscle cells (SMCs)2 are of crucial importance in maintaining normal structural and contractile integrity of various organs and tissues throughout the vertebrate body. Unlike skeletal and cardiac muscle cells, which largely undergo irreversible terminal differentiation, SMCs have the inherent ability to alter their differentiated state in response to diverse stimulatory cues. This process of SMC phenotypic modulation is a hallmark of several human pathological conditions, including vascular occlusive disease, asthma, intestinal and bladder obstruction, and Alzheimer disease. SMC phenotypic modulation is often defined in terms of unique molecular signatures of gene expression involved with contraction and cytoskeletal architecture as well as extracellular matrix remodeling (1, 2). For example, vascular SMCs display reduced levels of contractile filaments following injury to the vessel wall, adopting the so-called synthetic phenotype characterized by an overabundance of rough endoplasmic reticulum (3). On the other hand, recent studies have shown an exaggerated SMC contractile phenotype thought to contribute to disease progression (4, 5).The majority of SMC-restricted genes contain one or more conserved CArG elements that bind the widely express...

. Hypercholesterolemia abolishes voltage-dependent K ϩ channel contribution to adenosine-mediated relaxation in porcine coronary arterioles. Am J Physiol Heart Circ Physiol 288: H568 -H576, 2005. First published September 30, 2004; doi:10.1152/ajpheart.00157.2004.-Hypercholesterolemic patients display reduced coronary flow reserve in response to adenosine infusion. We previously reported that voltagedependent K ϩ (Kv) 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 (ϳ150 m 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 ϩ 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. Kv currents were significantly reduced (ϳ15%) in smooth muscle cells from hypercholesterolemic compared with control animals. Furthermore, K v current sensitive to low concentrations of TEA was reduced (ϳ45%) 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. microcirculation; smooth muscle; K ϩ current; voltage clamp HYPERCHOLESTEROLEMIA IS RECOGNIZED as a primary independent risk factor for coronary artery disease (3, 31) and is associated with altered vascular reactivity and ion channel function of the coronary microcirculation (20,21,27). Both hypercholesterolemic patients and animal models display impaired vasodilatory responses of the coronary microcirculation, which are generally attributed to endothelium dysfunction (16,20,27). However, numerous studies (11,12,24,32) have documented reduced coronary flow reserve in hypercholesterolemic patients in response to intravenous infusion of adenosine or dipyridamole, which also act directly on smooth muscle. These reports indicate that changes in vascular reactivity of the coronary microcirculation in response to hypercholesterolemia may a...

Hypercholesterolemia impairs endothelial function [e.g., the nitric oxide (NO)-cyclic GMP-phosphodiesterase 5 (PDE5) pathway], limits shear stress-induced vasodilation, and is therefore expected to reduce exercise-induced vasodilation. To assess the actual effects of hypercholesterolemia on endothelial function and exercise-induced vasodilation, we compared the effects of endothelial NO synthase (eNOS) and PDE5 inhibition in chronically instrumented Yucatan (Control) and Rapacz familial hypercholesterolemic (FH) swine, at rest and during treadmill exercise. The increases in systemic vascular conductance produced by ATP (relative to nitroprusside) and exercise were blunted in FH compared with Control swine. The vasoconstrictor response to eNOS inhibition, with nitro-l-arginine (NLA), was attenuated in FH compared with Control swine, both at rest and during exercise. Furthermore, whereas the vasodilator response to nitroprusside was enhanced slightly, the vasodilator response to PDE5 inhibition, with EMD360527, was reduced in FH compared with Control swine. Finally, in the pulmonary circulation, FH resulted in attenuated vasodilator responses to ATP, while maintaining the responses to both NLA and EMD360527. In conclusion, hypercholesterolemia reduces exercise-induced vasodilation in the systemic but not the pulmonary circulation. This reduction appears to be the principal result of a decrease in NO bioavailability, which is mitigated by a lower PDE5 activity.

The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) was first described by Gardos in erythrocytes and later confirmed to play a significant role in T-cell activation and the immune response. More recently, K(Ca)3.1 has been characterized in numerous cell types which contribute to the development of vascular disease, such as T-cells, B-cells, endothelial cells, fibroblasts, macrophages, and dedifferentiated smooth muscle cells (SMCs). Physiologically, K(Ca)3.1 has been demonstrated to play a role in acetylcholine and endothelium-derived hyperpolarizing factor (EDHF) induced hyperpolarization, and thus control of blood pressure. Pathophysiologically, K(Ca)3.1 contributes to proliferation of T-cells, B-cells, fibroblasts, and vascular SMCs, as well as the migration of SMCs and macrophages and platelet coagulation. Recent studies have indicated that blockade of K(Ca)3.1, by specific blockers such as TRAM-34, could prove to be an effective treatment for vascular disease by inhibiting T-cell activation as well as preventing proliferation and migration of macrophages, endothelial cells, and SMCs. This vasculoprotective potential of K(Ca)3.1 inhibition has been confirmed in both rodent and swine models of restenosis. In this review, we will discuss the physiological and pathophysiological role of K(Ca)3.1 in cells closely associated with vascular biology, and the effect of K(Ca)3.1 blockers on the initiation and progression of vascular disease.

Emter CA, Tharp DL, Ivey JR, Ganjam VK, Bowles DK. Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca 2ϩ -sensitive K ϩ current in miniature swine with LV hypertrophy. Am J Physiol Heart Circ Physiol 301: H1687-H1694, 2011. First published August 12, 2011 doi:10.1152/ajpheart.00610.2011.-Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P Ͻ 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ETA) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K ϩ currents (IKϩ) in coronary smooth muscle cells. Raising internal Ca 2ϩ from 200 to 500 nM increased Ca 2ϩ -sensitive K ϩ current in HF-TR and control, but not HF animals. In conclusion, an ETA-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca 2ϩ -sensitive IKϩ was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca 2ϩ -sensitive IKϩ, illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy. coronary circulation; vascular smooth muscle; potassium channels; heart failure CORONARY VASCULAR DYSFUNCTION is a hallmark feature of the progression to heart failure (HF). Altered responsiveness to mediators of vascular tone, such as endothelin-1 (ET-1), have been observed and may contribute to this phenomena (8,21,32,33,46,49). Impaired coronary vascular function can have profound effects on myocardial oxidative capacity and function and may play a significant role in the inability of the failing heart to respond to situations of increasing stress. The mechanism by which this occurs remains unclear. Considerable evidence exists indicating that profound modulation of both cardiomyocyte and smooth muscle cell electrophysiological phenotype are involved in cardiovascular disease (36, 47, 51). Calcium-activated K ϩ (K Ca...