TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway.

RATIONALE: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored. OBJECTIVE: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action. METHODS AND RESULTS: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca2+]i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca2+]i and velocity of shortening as well as an acceleration in [Ca2+]i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1-/- mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14-22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) (1-5 min) that correlated with increases in CM [Ca2+]i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca2+]i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L). CONCLUSIONS: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca2+ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.

TRPA1 and TRPV1 contribute to propofol-mediated antagonism of U46619-induced constriction in murine coronary arteries.

BACKGROUND: Transient receptor potential (TRP) ion channels have emerged as key components contributing to vasoreactivity. Propofol, an anesthetic is associated with adverse side effects including hypotension and acute pain upon infusion. Our objective was to determine the extent to which TRPA1 and/or TRPV1 ion channels are involved in mediating propofol-induced vasorelaxation of mouse coronary arterioles in vitro and elucidate the potential cellular signal transduction pathway by which this occurs. METHODS: Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). Isolated microvessels were cannulated and secured in a temperature-controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in microvessels pre-constricted with U46619 to assess the dose-dependent relaxation effects of propofol on coronary microvascular tone. RESULTS: Propofol-induced relaxation was unaffected in vessels obtained from TRPV1-/- mice, markedly attenuated in pre-constricted vessels obtained from TRPA1-/- mice and abolished in vessels obtained from TRPAV-/- mice. Furthermore, NOS inhibition with L-NAME or endothelium denuding abolished the proporfol-induced depressor response in pre-constricted vessels obtained from all mice. In the absence of L-NAME, BKCa inhibition with penitrem A markedly attenuated propofol-mediated relaxation in vessels obtained from wild-type mice and to a lesser extent in vessels obtained from TRPV1-/-, mice with no effect in vessels obtained from TRPA1-/- or TRPAV-/- mice. CONCLUSIONS: TRPA1 and TRPV1 appear to contribute to the propofol-mediated antagonism of U46619-induced constriction in murine coronary microvessels that involves activation of NOS and BKCa.

4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling.

We demonstrated previously that TRPV1-dependent regulation of coronary blood flow (CBF) is disrupted in diabetes. Further, we have shown that endothelial TRPV1 is differentially regulated, ultimately leading to the inactivation of TRPV1, when exposed to a prolonged pathophysiological oxidative environment. This environment has been shown to increase lipid peroxidation byproducts including 4-Hydroxynonenal (4-HNE). 4-HNE is notorious for producing protein post-translation modification (PTM) via reactions with the amino acids: cysteine, histidine and lysine. Thus, we sought to determine if 4-HNE mediated post-translational modification of TRPV1 could account for dysfunctional TRPV1-mediated signaling observed in diabetes. Our initial studies demonstrate 4-HNE infusion decreases TRPV1-dependent coronary blood flow in C57BKS/J (WT) mice. Further, we found that TRPV1-dependent vasorelaxation was suppressed after 4-HNE treatment in isolated mouse coronary arterioles. Moreover, we demonstrate 4-HNE significantly inhibited TRPV1 currents and Ca2+ entry utilizing patch-clamp electrophysiology and calcium imaging respectively. Using molecular modeling, we identified potential pore cysteines residues that, when mutated, could restore TRPV1 function in the presence of 4-HNE. Specifically, complete rescue of capsaicin-mediated activation of TRPV1 was obtained following mutation of pore Cysteine 621. Finally, His tag pull-down of TRPV1 in HEK cells treated with 4-HNE demonstrated a significant increase in 4-HNE binding to TRPV1, which was reduced in the TRPV1 C621G mutant. Taken together these data suggest that 4-HNE decreases TRPV1-mediated responses, at both the in vivo and in vitro levels and this dysfunction can be rescued via mutation of the pore Cysteine 621. Our results show the first evidence of an amino acid specific modification of TRPV1 by 4-HNE suggesting this 4-HNE-dependent modification of TRPV1 may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

TRPA1 is functionally co-expressed with TRPV1 in cardiac muscle: Co-localization at z-discs, costameres and intercalated discs.

Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) and vanilloid subtype-1 (TRPV1) are structurally related, non-selective cation channels that show a high permeability to calcium. Previous studies indicate that TRP channels play a prominent role in the regulation of cardiovascular dynamics and homeostasis, but also contribute to the pathophysiology of many diseases and disorders within the cardiovascular system. However, no studies to date have identified the functional expression and/or intracellular localization of TRPA1 in primary adult mouse ventricular cardiomyocytes (CMs). Although TRPV1 has been implicated in the regulation of cardiac function, there is a paucity of information regarding functional expression and localization of TRPV1 in adult CMs. Our current studies demonstrate that TRPA1 and TRPV1 ion channels are co-expressed at the protein level in CMs and both channels are expressed throughout the endocardium, myocardium and epicardium. Moreover, immunocytochemical localization demonstrates that both channels predominantly colocalize at the Z-discs, costameres and intercalated discs. Furthermore, specific TRPA1 and TRPV1 agonists elicit dose-dependent, transient rises in intracellular free calcium concentration ([Ca2+]i) that are abolished in CMs obtained from TRPA1-/- and TRPV1-/- mice. Similarly, we observed a dose-dependent attenuation of the TRPA1 and TRPV1 agonist-induced increase in [Ca2+]i when WT CMs were pretreated with increasing concentrations of selective TRPA1 or TRPV1 channel antagonists. In summary, these findings demonstrate functional expression and the precise ultrastructural localization of TRPA1 and TRPV1 ion channels in freshly isolated mouse CMs. Crosstalk between TRPA1 and TRPV1 may be important in mediating cellular signaling events in cardiac muscle.

Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction.

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

Requisite Role of Kv1.5 Channels in Coronary Metabolic Dilation.

RATIONALE: In the working heart, coronary blood flow is linked to the production of metabolites, which modulate tone of smooth muscle in a redox-dependent manner. Voltage-gated potassium channels (Kv), which play a role in controlling membrane potential in vascular smooth muscle, have certain members that are redox-sensitive. OBJECTIVE: To determine the role of redox-sensitive Kv1.5 channels in coronary metabolic flow regulation. METHODS AND RESULTS: In mice (wild-type [WT], Kv1.5 null [Kv1.5(-/-)], and Kv1.5(-/-) and WT with inducible, smooth muscle-specific expression of Kv1.5 channels), we measured mean arterial pressure, myocardial blood flow, myocardial tissue oxygen tension, and ejection fraction before and after inducing cardiac stress with norepinephrine. Cardiac work was estimated as the product of mean arterial pressure and heart rate. Isolated arteries were studied to establish whether genetic alterations modified vascular reactivity. Despite higher levels of cardiac work in the Kv1.5(-/-) mice (versus WT mice at baseline and all doses of norepinephrine), myocardial blood flow was lower in Kv1.5(-/-) mice than in WT mice. At high levels of cardiac work, tissue oxygen tension dropped significantly along with ejection fraction. Expression of Kv1.5 channels in smooth muscle in the null background rescued this phenotype of impaired metabolic dilation. In isolated vessels from Kv1.5(-/-) mice, relaxation to H2O2 was impaired, but responses to adenosine and acetylcholine were normal compared with those from WT mice. CONCLUSIONS: Kv1.5 channels in vascular smooth muscle play a critical role in coupling myocardial blood flow to cardiac metabolism. Absence of these channels disassociates metabolism from flow, resulting in cardiac pump dysfunction and tissue hypoxia.

Propofol restores TRPV1 sensitivity via a TRPA1-, nitric oxide synthase-dependent activation of PKCε.

We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCε)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCε activation and to elucidate the cellular mechanism by which this occurs. F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca(2+) concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCε, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing both channels, PKCε inhibition prevented the propofol- and allyl isothiocyanate (AITC)-induced restoration of TRPV1 sensitivity to agonist stimulation as well as increased phosphorylation of PKCε and TRPV1. In cells containing TRPV1 only, neither agonist induced PKCε or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCε phosphorylation, and PKCε inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. These data indicate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCε.

Propofol causes vasodilation in vivo via TRPA1 ion channels: role of nitric oxide and BKCa channels.

BACKGROUND: Transient receptor potential (TRP) ion channels of the A1 (TRPA1) and V1 (TRPV1) subtypes are key regulators of vasomotor tone. Propofol is an intravenous anesthetic known to cause vasorelaxation. Our objectives were to examine the extent to which TRPA1 and/or TRPV1 ion channels mediate propofol-induced depressor responses in vivo and to delineate the signaling pathway(s) involved. METHODS: Mice were subjected to surgery under 1.5-2.5% sevoflurane gas with supplemental oxygen. After a stable baseline in mean arterial pressure (MAP) was achieved propofol (2.5, 5.0, 10.0 mg/kg/min) was administered to assess the hemodynamic actions of the intravenous anesthetic. The effect of nitric oxide synthase (NOS) inhibition with L-NAME and/or calcium-gated K+ channel (BKCa) inhibition with Penetrim A (Pen A), alone and in combination, on propofol-induced decreases in mean arterial pressure were assessed in control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). RESULTS: Propofol decreased MAP in control mice and this effect was markedly attenuated in TRPA1-/- and TRPAV-/- mice but unaffected in TRPV1-/-mice. Moreover, pretreatment with L-NAME or Pen A attenuated the decrease in MAP in control and TRPV1-/- mice, and combined inhibition abolished the depressor response. In contrast, the markedly attenuated propofol-induced depressor response observed in TRPA1-/- and TRPAV-/- mice was unaffected by pre-treatment with Pen A or L-NAME when used either alone or in combination. CONCLUSION: These data demonstrate for the first time that propofol-induced depressor responses in vivo are predominantly mediated by TRPA1 ion channels with no involvement of TRPV1 ion channels and includes activation of both NOS and BKCa channels.

Penitrem A as a tool for understanding the role of large conductance Ca(2+)/voltage-sensitive K(+) channels in vascular function.

Large conductance, Ca(2+)/voltage-sensitive K(+) channels (BK channels) are well characterized, but their physiological roles, often determined through pharmacological manipulation, are less clear. Iberiotoxin is considered the "gold standard" antagonist, but cost and membrane-impermeability limit its usefulness. Economical and membrane-permeable alternatives could facilitate the study of BK channels. Thus, we characterized the effect of penitrem A, a tremorigenic mycotoxin, on BK channels and demonstrate its utility for studying vascular function in vitro and in vivo. Whole-cell currents from human embryonic kidney 293 cells transfected with hSlo α or α + ß1 were blocked >95% by penitrem A (IC(50) 6.4 versus 64.4 nM; p < 0.05). Furthermore, penitrem A inhibited BK channels in inside-out and cell-attached patches, whereas iberiotoxin could not. Inhibitory effects of penitrem A on whole-cell K(+) currents were equivalent to iberiotoxin in canine coronary smooth muscle cells. As for specificity, penitrem A had no effect on native delayed rectifier K(+) currents, cloned voltage-dependent Kv1.5 channels, or native ATP-dependent K(ATP) current. Penitrem A enhanced the sensitivity to K(+)-induced contraction in canine coronary arteries by 23 ± 5% (p < 0.05) and increased the blood pressure response to phenylephrine in anesthetized mice by 36 ± 11% (p < 0.05). Our data indicate that penitrem A is a useful tool for studying the role of BK channels in vascular function and is practical for cell and tissue (in vitro) studies as well as anesthetized animal (in vivo) experiments.

Disruption of TRPV1-mediated coupling of coronary blood flow to cardiac metabolism in diabetic mice: role of nitric oxide and BK channels.

We have previously shown transient receptor potential vanilloid subtype 1 (TRPV1) channel-dependent coronary function is compromised in pigs with metabolic syndrome (MetS). However, the mechanisms through which TRPV1 channels couple coronary blood flow to metabolism are not fully understood. We employed mice lacking TRPV1 [TRPV1((-/-))], db/db diabetic, and control C57BKS/J mice to determine the extent to which TRPV1 channels modulate coronary function and contribute to vascular dysfunction in diabetic cardiomyopathy. Animals were subjected to in vivo infusion of the TRPV1 agonist capsaicin to examine the hemodynamic actions of TRPV1 activation. Capsaicin (1-100 µg·kg(-1)·min(-1)) dose dependently increased coronary blood flow in control mice, which was inhibited by the TRPV1 antagonist capsazepine or the nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (L-NAME). In addition, the capsaicin-mediated increase in blood flow was attenuated in db/db mice. TRPV1((-/-)) mice exhibited no changes in coronary blood flow in response to capsaicin. Vasoreactivity studies in isolated pressurized mouse coronary microvessels revealed a capsaicin-dependent relaxation that was inhibited by the TRPV1 inhibitor SB366791 l-NAME and to the large conductance calcium-sensitive potassium channel (BK) inhibitors iberiotoxin and Penetrim A. Similar to in vivo responses, capsaicin-mediated relaxation was impaired in db/db mice compared with controls. Changes in pH (pH 7.4-6.0) relaxed coronary vessels contracted to the thromboxane mimetic U46619 in all three groups of mice; however, pH-mediated relaxation was blunted in vessels obtained from TRPV1((-/-)) and db/db mice compared with controls. Western blot analysis revealed decreased myocardial TRPV1 protein expression in db/db mice compared with controls. Our data reveal TRPV1 channels mediate coupling of myocardial blood flow to cardiac metabolism via a nitric oxide-dependent, BK channel-dependent pathway that is corrupted in diabetes.

PKCα mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells.

Transient receptor potential vanilloid channel 4 (TRPV4) is a polymodally activated nonselective cationic channel implicated in the regulation of vasodilation and hypertension. We and others have recently shown that cyclic stretch and shear stress activate TRPV4-mediated calcium influx in endothelial cells (EC). In addition to the mechanical forces, acetylcholine (ACh) was shown to activate TRPV4-mediated calcium influx in endothelial cells, which is important for nitric oxide-dependent vasodilation. However, the molecular mechanism through which ACh activates TRPV4 is not known. Here, we show that ACh-induced calcium influx and endothelial nitric oxide synthase (eNOS) phosphorylation but not calcium release from intracellular stores is inhibited by a specific TRPV4 antagonist, AB-159908. Importantly, activation of store-operated calcium influx was not altered in the TRPV4 null EC, suggesting that TRPV4-dependent calcium influx is mediated through a receptor-operated pathway. Furthermore, we found that ACh treatment activated protein kinase C (PKC) α, and inhibition of PKCα activity by the specific inhibitor Go-6976, or expression of a kinase-dead mutant of PKCα but not PKCε or downregulation of PKCα expression by chronic 12-O-tetradecanoylphorbol-13-acetate treatment, completely abolished ACh-induced calcium influx. Finally, we found that ACh-induced vasodilation was inhibited by the PKCα inhibitor Go-6976 in small mesenteric arteries from wild-type mice, but not in TRPV4 null mice. Taken together, these findings demonstrate, for the first time, that a specific isoform of PKC, PKCα, mediates agonist-induced receptor-mediated TRPV4 activation in endothelial cells.

Endothelin-mediated in vivo pressor responses following TRPV1 activation.

Transient receptor potential vanilliod 1 (TRPV1) channels have recently been postulated to play a role in the vascular complications/consequences associated with diabetes despite the fact that the mechanisms through which TRPV1 regulates vascular function are not fully known. Accordingly, our goal was to define the mechanisms by which TRPV1 channels modulate vascular function and contribute to vascular dysfunction in diabetes. We subjected mice lacking TRPV1 [TRPV1((-/-))], db/db, and control C57BLKS/J mice to in vivo infusion of the TRPV1 agonist capsaicin or the α-adrenergic agonist phenylephrine (PE) to examine the integrated circulatory actions of TRPV1. Capsaicin (1, 10, 20, and 100 µg/kg) dose dependently increased MAP in control mice (5.7 ± 1.6, 11.7 ± 2.1, 25.4 ± 3.4, and 51.6 ± 3.9%), which was attenuated in db/db mice (3.4 ± 2.1, 3.9 ± 2.1, 7.0 ± 3.3, and 17.9 ± 6.2%). TRPV1((-/-)) mice exhibited no changes in MAP in response to capsaicin, suggesting the actions of this agonist are specific to TRPV1 activation. Immunoblot analysis revealed decreased aortic TRPV1 protein expression in db/db compared with control mice. Capsaicin-induced responses were recorded following inhibition of endothelin A and B receptors (ET(A) /ET(B)). Inhibition of ET(A) receptors abolished the capsaicin-mediated increases in MAP. Combined antagonism of ET(A) and ET(B) receptors did not further inhibit the capsaicin response. Cultured endothelial cell exposure to capsaicin increased endothelin production as shown by an endothelin ELISA assay, which was attenuated by inhibition of TRPV1 or endothelin-converting enzyme. TRPV1 channels contribute to the regulation of vascular reactivity and MAP via production of endothelin and subsequent activation of vascular ET(A) receptors. Impairment of TRPV1 channel function may contribute to vascular dysfunction in diabetes.

Propofol restores transient receptor potential vanilloid receptor subtype-1 sensitivity via activation of transient receptor potential ankyrin receptor subtype-1 in sensory neurons.

BACKGROUND: Cross talk between peripheral nociceptors belonging to the transient receptor potential vanilloid receptor subtype-1 (TRPV1) and ankyrin subtype-1 (TRPA1) family has been demonstrated recently. Moreover, the intravenous anesthetic propofol has directly activates TRPA1 receptors and indirectly restores sensitivity of TRPV1 receptors in dorsal root ganglion (DRG) sensory neurons. Our objective was to determine the extent to which TRPA1 activation is involved in mediating the propofol-induced restoration of TRPV1 sensitivity. METHODS: Mouse DRG neurons were isolated by enzymatic dissociation and grown for 24 h. F-11 cells were transfected with complementary DNA for both TRPV1 and TRPA1 or TRPV1 only. The intracellular Ca concentration was measured in individual cells via fluorescence microscopy. After TRPV1 desensitization with capsaicin (100 nM), cells were treated with propofol (1, 5, and 10 µM) alone or with propofol in the presence of the TRPA1 antagonist, HC-030031 (0.5 µM), or the TRPA1 agonist, allyl isothiocyanate (AITC; 100 µM); capsaicin was then reapplied. RESULTS: In DRG neurons that contain both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in DRG neurons containing only TRPV1 receptors, exposure to propofol or AITC after desensitization did not restore capsaicin-induced TRPV1 sensitivity. Similarly, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in F-11 cells transfected with TRPV1 only, neither propofol nor AITC was capable of restoring TRPV1 sensitivity. CONCLUSIONS: These data demonstrate that propofol restores TRPV1 sensitivity in primary DRG neurons and in cultured F-11 cells transfected with both the TRPV1 and TRPA1 receptors via a TRPA1-dependent process. Propofol's effects on sensory neurons may be clinically important and may contribute to peripheral sensitization to nociceptive stimuli in traumatized tissue.

Short-term exercise training prevents micro- and macrovascular disease following coronary stenting.

The purpose of this study was to determine the effects of exercise on coronary blood flow and macrovascular atherosclerosis in response to stent deployment. Male Yucatan swine were placed on a control diet (C); on a high-fat/cholesterol diet (hypercholesterolemic; H); or on a high-fat/cholesterol diet and aerobically exercise trained (HX) starting after 36 wk on the diet. All pigs underwent coronary angiography and intravascular ultrasound (IVUS) guided placement of a bare metal stent in the circumflex coronary artery after 40 wk on diets and 3 wk later pigs underwent repeat angiography and IVUS and coronary blood flow (CBF) measurement. Average peak velocity (APV) was measured under basal conditions and in response to intracoronary application of the endothelium-independent vasodilator adenosine and the endothelium-dependent vasodilator bradykinin. There was a similar approximately 8-fold increase in total cholesterol in H and HX compared with control. Baseline CBF was increased above control and H in HX (P<0.05). At all doses adenosine-induced CBF was impaired in H, but preserved in HX. Similarly, bradykinin-induced CBF was impaired in H vs. control, yet was potentiated in HX. Microvessel density was decreased in H and preserved in HX vs. control. Native atheroma in HX was lower relative to H and control, while in-stent stenosis in HX was not different from H. Hyperlipidemia-induced microvascular dysfunction after stent deployment may be a result of reduction in microvessel density. This is the first report that short-term exercise training near the time of stenting prevents stent-induced microvascular dysfunction and attenuates native atheroma independent of changes in plasma cholesterol in this porcine model.

Metabolic syndrome reduces the contribution of K+ channels to ischemic coronary vasodilation.

This investigation tested the hypothesis that metabolic syndrome decreases the relative contribution of specific K(+) channels to coronary reactive hyperemia. Ca(2+)-activated (BK(Ca)), voltage-activated (K(V)), and ATP-dependent (K(ATP)) K(+) channels were investigated. Studies were conducted in anesthetized miniature Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) for 20 wk. The latter diet induces metabolic syndrome, increasing body weight, fasting glucose, total cholesterol, and triglyceride levels. Ischemic vasodilation was determined by the coronary flow response to a 15-s occlusion before and after cumulative administration of antagonists for BK(Ca) (penitrem A; 10 microg/kg iv), K(V) (4-aminopyridine; 0.3 mg/kg iv) and K(ATP) (glibenclamide; 1 mg/kg iv) channels. Coronary reactive hyperemia was diminished by metabolic syndrome as the repayment of flow debt was reduced approximately 30% compared with lean swine. Inhibition of BK(Ca) channels had no effect on reactive hyperemia in either lean or metabolic syndrome swine. Subsequent inhibition of K(V) channels significantly reduced the repayment of flow debt ( approximately 25%) in both lean and metabolic syndrome swine. Additional blockade of K(ATP) channels further diminished ( approximately 45%) the repayment of flow debt in lean but not metabolic syndrome swine. These data indicate that the metabolic syndrome impairs coronary vasodilation in response to cardiac ischemia via reductions in the contribution of K(+) channels to reactive hyperemia.

Contribution of BK(Ca) channels to local metabolic coronary vasodilation: Effects of metabolic syndrome.

This investigation was designed to examine the hypothesis that impaired function of coronary microvascular large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in metabolic syndrome (MetS) significantly attenuates the balance between myocardial oxygen delivery and metabolism at rest and during exercise-induced increases in myocardial oxygen consumption (MVo(2)). Studies were conducted in conscious, chronically instrumented Ossabaw swine fed a normal maintenance diet (11% kcal from fat) or an excess calorie atherogenic diet (43% kcal from fat, 2% cholesterol, 20% kcal from fructose) that induces many common features of MetS. Data were collected under baseline/resting conditions and during graded treadmill exercise before and after selective blockade of BK(Ca) channels with penitrem A (10 microg/kg iv). We found that the exercise-induced increases in blood pressure were significantly elevated in MetS swine. No differences in baseline cardiac function or heart rate were noted. Induction of MetS produced a parallel downward shift in the relationship between coronary venous Po(2) and MVo(2) (P < 0.001) that was accompanied by a marked release of lactate (negative lactate uptake) as MVo(2) was increased with exercise (P < 0.005). Inhibition of BK(Ca) channels with penitrem A did not significantly affect blood pressure, heart rate, or the relationship between coronary venous Po(2) and MVo(2) in lean or MetS swine. These data indicate that BK(Ca) channels are not required for local metabolic control of coronary blood flow under physiological (lean) or pathophysiological (MetS) conditions. Therefore, diminished function of BK(Ca) channels does not contribute to the impairment of myocardial oxygen-supply demand balance in MetS.

Exercise training decreases store-operated Ca2+entry associated with metabolic syndrome and coronary atherosclerosis.

AIMS: Stenting attenuates restenosis, but accelerated coronary artery disease (CAD) adjacent to the stent (peri-stent CAD) remains a concern in metabolic syndrome (MetS). Smooth muscle cell proliferation, a major mechanism of CAD, is mediated partly by myoplasmic Ca2+ dysregulation and store-operated Ca2+ entry (SOCE) via canonical transient receptor potential 1 (TRPC1) channels is proposed to play a key role. Exercise is known to prevent Ca2+ dysregulation in CAD. We tested the hypothesis that MetS increases SOCE and peri-stent CAD and exercise attenuates these events. METHODS AND RESULTS: Groups (n = 9 pigs each) were (i) healthy lean Ossabaw swine fed standard chow, (ii) excess calorie atherogenic diet fed (MetS), and (iii) aerobically exercise trained starting after 50 weeks of development of MetS (XMetS). Bare metal stents were placed after 54 weeks on diets, and CAD and SOCE were assessed 4 weeks later. Coronary cells were dispersed proximal to the stent (peri-stent) and from non-stent segments, and fura-2 fluorescence was used to assess SOCE, which was verified by Ni2+ blockade and insensitivity to nifedipine. XMetS pigs had increased physical work capacity and decreased LDL/HDL (P < 0.05), but no attenuation of robust insulin resistance, glucose intolerance, hypertriglyceridaemia, or hypertension. CAD was greater in peri-stented vs. non-stented artery segments. MetS had the greatest CAD, SOCE, and TRPC1 and STIM1 mRNA and protein expression, which were all attenuated in XMetS. CONCLUSION: This is the first report of the protective effect of exercise on native CAD, peri-stent CAD, SOCE, and molecular expression of TRPC1, STIM1, and Orai1 in MetS.

Impaired function of coronary BK(Ca) channels in metabolic syndrome.

The role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in regulation of coronary microvascular function is widely appreciated, but molecular and functional changes underlying the deleterious influence of metabolic syndrome (MetS) have not been determined. Male Ossabaw miniature swine consumed for 3-6 mo a normal diet (11% kcal from fat) or an excess-calorie atherogenic diet that induces MetS (45% kcal from fat, 2% cholesterol, 20% kcal from fructose). MetS significantly impaired coronary vasodilation to the BK(Ca) opener NS-1619 in vivo (30-100 microg) and reduced the contribution of these channels to adenosine-induced microvascular vasodilation in vitro (1-100 microM). MetS reduced whole cell penitrem A (1 microM)-sensitive K(+) current and NS-1619-activated (10 microM) current in isolated coronary vascular smooth muscle cells. MetS increased the concentration of free intracellular Ca(2+) and augmented coronary vasoconstriction to the L-type Ca(2+) channel agonist BAY K 8644 (10 pM-10 nM). BK(Ca) channel alpha and beta(1) protein expression was increased in coronary arteries from MetS swine. Coronary vascular dysfunction in MetS is related to impaired BK(Ca) channel function and is accompanied by significant increases in L-type Ca(2+) channel-mediated coronary vasoconstriction.

Endogenous adipose-derived factors diminish coronary endothelial function via inhibition of nitric oxide synthase.

Adipocytokines may be the molecular link between obesity and vascular disease. However, the effects of these factors on coronary vascular function have not been discerned. Accordingly, the goal of this investigation was to delineate the mechanisms by which endogenous adipose-derived factors affect coronary vascular endothelial function. Both isolated canine coronary arteries and coronary blood flow in anesthetized dogs were studied with and without exposure to adipose tissue. Infusion of adipose-conditioned buffer directly into the coronary circulation did not change baseline hemodynamics; however, endothelial-dependent vasodilation to bradykinin was impaired both in vitro and in vivo. Coronary vasodilation to sodium nitroprusside was unaltered by adipose tissue. Oxygen radical formation did not cause the impairment because quantified dihydroethidium staining was decreased by adipose tissue and neither a superoxide dismutase mimetic nor catalase improved endothelial function. Inhibition of nitric oxide (NO) synthase with L-NAME diminished bradykinin-mediated relaxations and eliminated the subsequent vascular effects of adipose tissue. In vitro measurement of NO demonstrated that adipose tissue exposure quickly lowered baseline NO and abolished bradykinin-induced NO production. The results indicate that adipose tissue releases factor(s) that selectively impair endothelial-dependent dilation via inhibition of NO synthase-mediated NO production.

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation.

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.