Cardiac hypertrophy, defined as an increase in myocardial mass, is an adaptive mechanism in response to chronic work overload and is a common clinical finding over the age of 59 years.1 Aortic stenosis imposes a pressure overload on the left ventricle, which leads to the development of LVH.2 Reduced coronary reserve, defined as the difference between basal flow and maximal flow during exercise or infusion of agents such as adenosine, occurs in LVH as a result of hypertension and aortic stenosis.1 The reduced coronary reserve limits the ability of hypertrophied hearts to meet blood flow and metabolic requirements under conditions of increased demand.1,3
The normal endothelium maintains local vasomotor tone regulation, offers a nonthrombogenic surface, acts as a selective barrier controlling permeability and transport of solutes and macromolecules, metabolizes circulating and locally generated factors, controls vascular smooth muscle cell proliferation, and inhibits platelet adhesion and aggregation. Furthermore, endothelial cells produce several substances that can cause relaxation or contraction of the underlying vascular smooth muscle cells (VSMC).4,5 The principal relaxing factors are nitric oxide (NO), prostacyclin (PGI2), and the endothelium-derived hyperpolarizing factor (EDHF). Opposed to them, the principal contracting factors identified to date include angiotensin II (AngII), superoxide anions, endoperoxides, thromboxane A2 (TXA2), and the peptide endothelin-1 (ET-1), which plays a role in long-term modulation of muscular tone.6
ET-1 is a potent vasoconstrictor, has positive inotropic and mitogenic properties, influences homeostasis of salt and water, and stimulates the renin-angiotensin-aldosterone axis and the sympathetic nervous system.7 There are 2 receptor subtypes for ET-1: ETA located on VSMC and ETB present on both VSMC and the endothelium. Both receptor subtypes present on VSMC cause a contraction, whereas the ETB subtype on the endothelium leads to a vasorelaxation via the release of NO and PGI2.8 Endogenous vascular production of ET-1 plays a role in the development of cardiac hypertrophy in in-vivo pressure overload models and is partially inhibited by selective antagonists for ETA receptors.9 Two distinct secretory pathways are now considered for ET-1 production: a constitutive pathway involving continuous release of ET-1 implicated in the control of normal vascular tone and a regulated pathway involving stimulated release of ET-1.10 ET-1 receptors are coupled to Gq proteins on smooth muscle cells and to Gi proteins on the endothelium. On VSMC, receptor activation leads to phospholipase C activation, which induces the formation of inositol trisphosphate (IP3) and diacylglycerol (DAG).11 IP3 stimulates Ca2+ release from sarcoplasmic reticulum and phosphorylates IP4, which in turn activates Ca2+ channels.12 DAG activates protein kinase C to stimulate extracellular release of Ca2+.13
Endothelial dysfunction occurs through a decreased secretion of vasodilator mediators, an increased production of endothelium-dependent vasoconstrictors, or an increased sensitivity of VSMC and/or resistance of vascular smooth muscle to endothelium-derived vasodilators.14 Responses may be altered by a modification of the expression of endothelial receptors, impairment of the signal transduction pathway (in particular, Gi proteins and second messengers), alterations in the activity or expression of enzymes involved in the formation of endothelial mediators (such as nitric oxide synthase, endothelin-converting enzyme, etc) and/or alteration in the response of target cells (ie, platelets and VSMC) to endothelial mediators.14 The endothelial dysfunction of epicardial coronary arteries in LVH secondary to 2 months of aortic banding in swine has been recently reported by our group and was characterized by decreased endothelium-dependent relaxations to serotonin and bradykinin, agonists whose receptors are respectively coupled to Gi and Gq proteins, resulting in decreases of both NO release (decreased basal coronary cGMP content) and production (decreased plasmatic nitrite/nitrate ratio).15 The present study was designed to assess the contribution of endothelium-dependent contracting factors to the coronary artery endothelial dysfunction in left ventricular hypertrophy caused by aortic banding in a swine model and to assess the role of endothelin receptors subtypes in this process.
MATERIALS AND METHODS
Forty-two Landrace swine of either gender, weighing 33.5 ± 2 kg and aged 8 weeks, were used. The experiments were performed in compliance with recommendations of the guidelines on the care and use of laboratory animals issued by the Canadian Council on Animal Research and the guidelines of the Animal Care, and the protocol was approved by the local committee.
Anesthesia and Surgical Technique
Swine were anesthetized by intramuscular injection of ketamine (20 mg/kg; Rogarsetic, Toronto, ON) and xylazine (2 mg/kg; Rompun, Cambridge, ON) mixture. Swine were artificially ventilated with an O2/air mixture. Ventilation was maintained during surgical intervention with a respirator to maintain an arterial oxygen saturation of 95%, and a light anesthesia was ensured by isoflurane 1% vol/vol (Halocarbon Laboratories, NJ). Respiratory control was maintained by frequent determinations of arterial blood gases, and acidosis was balanced with 8.4% sodium bicarbonate (Abbott Laboratories, QC). Hair was shaved in the operative field, and the skin was disinfected with a 0.5% chlorhexidine solution. A catheter was placed in an auricular vein for intravenous infusion during the operation and for the administration of the antibiotic (Excenel 0.06 mL/kg; Pharmacia & Upjohn, Orangeville, ON). Arterial cannulation was performed through the right femoral artery for blood pressure analysis. A rectal probe was used for monitoring the temperature. The chest was entered through a left anterior thoracotomy in the third intercostal space and the aortic banding was done by tying an umbilical tape around the aorta, 3 cm above the coronary ostia, to obtain a minimal systolic peak gradient of 15 mm Hg. The gradient was measured by monitoring the systemic arterial pressure distally to the banding site. The pericardium was then closed, and the chest was closed in multiple layers.
The animals were treated with an antibiotic (Excenel 0.06 mL/kg; Pharmacia & Upjohn, Orangeville, ON) for 3 days following the surgical intervention and left to recover in temperature-controlled quarter and fed with a standard piglet chow. Swine were sacrificed 2 months after the operation.
Explantation Protocol and Experimental Groups
Hypertrophied Hearts (n = 24)
After 2 months of aortic banding, swine were anesthetized with the same mixture of ketamine and xylazine as used for the surgical intervention (see above). Sacrifice was performed by exsanguination, the thorax was opened through a sternotomy, and the heart was removed. The heart was then weighed for the measurement of heart-to-body weight ratio, which was used to assess left ventricular hypertrophy, and rapidly placed in a Krebs-bicarbonate solution (composition in mmol/L: NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, EDTA 0.026, dextrose 11.1; control solution).
Control Hearts (n = 18)
Control swine were sham animals, submitted to thoracotomy but not to aortic banding and kept in house for a 2-month period. Hearts were excised through a median sternotomy in the same fashion as for the aortic banding group.
After heart explantation, epicardial coronary arteries were dissected free from the adventitial tissue and divided into rings of 5 mm length. A total of 16 rings were obtained per animal; 4 from the left anterior descending, 4 from the left circumflex, and 8 from the right coronary arteries. Rings were then placed in organ chambers filled with a Krebs-bicarbonate solution (20 mL) at 37°C and oxygenated with a mixture of 95% O2/5% CO2 to study endothelial function. The rings were suspended between 2 metal stirrups, one of which was connected to an isometric force transducer. Data were recorded by data acquisition software (IOS3, Emka Inc, Paris, France).
After a 30-minute period of stabilization, tension on rings was increased progressively to an optimal tension (approximately 3.5 g), as determined by the construction of an active length-tension curve by measuring contractions to potassium chloride (KCl; 30 mM) at different levels of stretch. Then, 60 mM of KCl was added, and a maximal contraction was evaluated. After that, rings were washed, and blockers such as indomethacin (10−5 M; to prevent the endogenous production of prostanoids), daltroban (10−7 M; a TXA2 receptor antagonist), and propranolol (10−7 M; to prevent the activation of β-adrenergic receptors) were added. The rings were then stabilized for a 45-minute period.
Responses to endothelin-1 (ET-1; 10−11 to 10−6 M), with or without indomethacin and with or without daltroban, were compared between the control and the aortic banding groups. Responses to IRL-1620, an ETB receptor agonist (10−11 to 10−6 M), with and without endothelium, were compared with responses to ET-1 in the aortic banding and control groups. Responses to IRL-1620 (10−11 to 10−6 M) were also measured after a first contraction by prostaglandin F2 α (range, 2 × 10−6 to 3 × 10−5 M; to achieve a contraction averaging 50% of the maximal contraction obtained with KCl).
Endothelium-dependent responses were studied by constructing concentration-response curves to serotonin (5-HT; 10−10 to 10−5 M) and bradykinin (BK; 10−12 to 10−6 M), with or without indomethacin, in control and aortic banding groups. In these experiments, rings were first contracted with prostaglandin F2 α (range, 2 × 10−6 to 3 × 10−5 M) to achieve a contraction averaging 50% of the maximal contraction obtained with KCl. These studies were performed in the presence of ketanserin (10−6 M, incubated 45 minutes before the addition of 5-HT to block the contraction induced by 5-HT2 receptors on VSMC).
Endothelium-independent responses were studied by constructing concentration-response curves to sodium nitroprusside (SNP; 10−10 to 10−5 M), with or without the addition of Nω-nitro-L-arginine (L-NA, 10−4 M; an inhibitor of endogenous NO), in control and aortic banding groups. In the same experiments, responses were evaluated after the removal of the endothelium by gently rubbing the interior surface of the vessel with a vascular clamp. In these experiments, rings were first contracted with prostaglandin F2 α (range 2 × 10−6 to 3 × 10−5 M) to achieve a contraction averaging 50% of the maximal contraction obtained with KCl.
ET-1 Plasma Measurement
Plasma levels of ET-1 were measured in control and aortic banding groups. Blood samples were drawn from the coronary sinus and centrifuged (4°C, 15 minutes, 2300 rpm) to collect plasma. Then, plasma was subsequently stored at −70°C until the measurement of ET-1. The peptide was measured by using an ELISA endothelin-1 assay (Biomedica, Montreal, QC) with a detection limit of 0.05 fmol/mL.
Confocal Microscopy Experiments
In each experiment, 3 rings of left anterior coronary arteries were frozen in 2-methylbutane. On the day of the experiment, arteries were cut in slices of 10 μm with a cryostat. Slices were fixed in 4% paraformaldehyde (pH 7.3) for 15 minutes at room temperature and then washed 3 times for 5 minutes in PBS. Slices were blocked and permeabilized in 10% normal donkey serum (NDS) and 0.5% Triton X-100 for 1 hour at room temperature. ETA (rabbit; Alomone Laboratory, Jerusalem, Israel) and ETB (rabbit; Alomone Laboratory, Jerusalem, Israel) antibodies were diluted 1:100 in 2% NDS and 0.1% Triton. The slices were incubated overnight at 4°C and washed for 5 minutes 3 times. On the next day, antirabbit (donkey)-TriTC diluted 1:500 in 2% NDS and 0.1% Triton were put on tissues and incubated 1 hour at room temperature in the dark and washed for 5 minutes 3 times in PBS. Then, 15 μL of 0.2% DABCO diluted 1:3 with glycerol was added to the tissues. Observations were made with a confocal microscope Zeiss LMS 510 (Jena, Germany) with NEHE (543 nm) laser to visualize TriTC.
Western Blot Experiments
Western blotting was conducted using fresh control and aortic banding epicardial coronary arteries. Briefly, proteins were extracted in liquid nitrogen using a mortar and pestle and placed in extraction buffer (20 mM β-glycerophosphate, 20 mM sodium fluoride, 2 mM EDTA pH 8, 5 mM EGTA pH 7.5, 25 mM TRIS pH 7.4, 1 mM Na3VO4, 10 mM benzamidine, 0.5 mM PMSF, 10 μg/mL leuptin, 5 mM DTT, 1 μM microcystin). Then, 4 μL of Triton (TX-100) was added to homogenates and then centrifuged at 49,000 rpm for 30 minutes at 4°C. The proteins thus obtained were used for immunoblotting. Proteins were separated in SDS-PAGE (2% to 10% gel) and transferred onto a PVDF membrane for ETB and nitrocellulose membrane for ETA. Blots were blocked with 5% powdered skim milk in TBS tween 0.1% for 2 hours at room temperature. The membranes were incubated with the first antibody (ETA and ETB; Alomone Laboratory, Jerusalem, Israel) diluted 1:200 overnight. The membranes were then washed for 7 minutes 3 times in TBS tween 0.1%. The second antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG antibody; Jackson ImmunoResearch Laboratory) was diluted 1:10,000, incubated 1 hour at room temperature, and washed for 7 minutes 3 times in TBS tween 0.1%. The optic density for each band was measured using Quantify one program (PDI company, PDR version 2.7.1, New York, NY).
The techniques for hemodynamic measurements have already been published.15 Briefly, measurements of the pressures were performed by insertion of a 6- or 7-Fr pigtail catheter in the femoral artery, after which a guiding catheter was advanced into the ascending aorta and placed in each coronary ostium to measure intracoronary pressures. The guiding catheter was then advanced across the aortic valve into the left ventricle to measure left intraventricular pressures. All studies were performed at baseline, 1 and 2 months after surgery.
Epicardial coronary arteries from control and aortic banding groups were dissected with a myocardial block, fixed in formalin 10%, and cut transversely. The slices were stained with Verhoeff to identify the VSMC. Total vessel area and lumen area were determined in digital planimetry with a video microscope linked to a computer and customized software (Scion image 1.6, Frederick, MD). Wall area was determined by subtraction of the lumen area from the total area. To obtain comparable data at variable external diameters, the wall-to-lumen ratio was calculated by dividing the wall area by the lumen area.
All solutions were prepared on the day of the experiment. Bradykinin, 5-hydroxytryptamine creatinine sulfate (serotonin), indomethacin, daltroban, Nω-nitro-L-arginine, ketanserin, and sodium nitroprusside were purchased from Sigma Chemical Co (Oakville, ON). Propranolol was purchased from Biomol Research Laboratories Inc (Plymouth Meeting, PA) and prostaglandin F2 α from Cayman Chemical Co (Ann Arbor, MI). Endothelin-1 and IRL-1620 were purchased from American Peptide Company Inc (Sunnyvale, CA).
Contractions are expressed as a percentage of the maximal contraction obtained with KCl for each group, and relaxations are expressed as a percentage of contraction to prostaglandin F2 α or 5-HT (in the case of relaxations to BK) represented as mean ± SEM; n refers to the number of animals used. Half-maximum effective concentration (EC50) of each agonist (ET-1, IRL1620, 5-HT, BK and SNP) used for the dose-response curves was measured from each individual concentration-response curve using a logistic curve-fitting program (Allfit for Windows 2.12, Dr De Léan, University of Montreal, Montreal, QC). The EC50 is the concentration of the agonist used to obtain 50% of the maximal response to the agonist. The pD2 value, the negative log of the EC50, was obtained. ANOVA studies were performed to compare concentration-response curves, and the Fisher and Bonferroni tests were used for post-hoc test. A value of P < 0.05 was considered statistically significant.
Morphologic and Hemodynamic Studies
The heart-to-body ratio and the left ventricle-to-body ratio increased by 36% and 21%, respectively, after 2 months of aortic banding, confirming the presence of LVH (Table 2). The wall-to-lumen ratio of the epicardial coronary arteries increased in LVH with an increase in wall thickness. Hemodynamic results were described in a previous study.15 Intravascular pressures of coronary arteries were not altered in the aortic banding group. Left diastolic and end-diastolic intraventricular pressures were increased, and relaxation of the left ventricle was impaired. There were no significant changes in left ventricular ejection fraction between groups.
Contractions to potassium chloride (KCl) and to prostaglandin F2 α were significantly greater in the aortic banding group compared with the control group. There were no statistically significant differences in the concentration of prostaglandin F2 α needed to achieve the target level of contraction or in the ratio of prostaglandin F2 α contraction to KCl contraction in epicardial coronary artery rings from aortic banding and control groups (Table 1).
There was a statistically significant increase in contractions to ET-1 for the group submitted to 2 months of aortic banding compared with the control group (Fig. 1A). Indomethacin and daltroban decreased the maximal contraction obtained with ET-1 in the group submitted to aortic banding. In the control group, indomethacin and daltroban did not change the maximal contraction to ET-1 (data not shown). There was a statistically significant decrease in contractions to IRL-1620 for the aortic banding group compared with the control group in rings without endothelium (Fig. 1B). The maximal contraction to IRL-1620 is decreased in both groups in the presence of endothelium, but more significantly in the control group.
Contractions induced by stimulation of ETA receptors accounted for most of the contraction compared with stimulation of ETB receptor with IRL-1620 in both aortic banding (Fig. 2A) and control (Fig. 2B) groups, with and without endothelium. There were no statistically significant differences between the contractions in rings with and without endothelium for ET-1 in both the aortic banding and control groups. In the control group, statistically significant differences exist in the contractions induced by IRL-1620 between rings with and without endothelium; higher contractions were obtained in rings without endothelium. In rings contracted with PGF2 α, IRL-1620 induced a non-statistically significant relaxation at low concentrations in the banding and control groups in the presence of endothelium (data not shown).
There was a statistically significant increase in relaxations to 5-HT and BK in the presence of indomethacin for the group submitted to 2 months of aortic banding (Fig. 3A,B). Daltroban induces a similar increase in relaxations to 5-HT and BK in the aortic banding group (data not shown). There was no difference in the control group with or without indomethacin.
There was no statistically significant difference in maximal relaxation to the NO donor SNP between the control and the aortic banding groups, and there was no difference in the sensitivity between the 2 groups (Fig. 3C). There was a significant leftward shift of the relaxation-concentration curve to SNP in both groups when aortic rings were incubated for 45 minutes in the baths with the NO synthase inhibitor L-NA, as shown by their respective pD2 values (Table 3). There was also a significant leftward shift in rings without endothelium from both groups (Table 3).
ET-1 Plasma Measurement
There was a statistically significant increase of ET-1 plasma concentrations in the aortic banding group compared with the control group after 2 months of aortic banding (Fig. 4).
Confocal Microscopy Experiments
Qualitative evaluation obtained with confocal microscopy showed no statistically significant difference of ETA (in VSMC) and ETB (in both VSMC and endothelium) receptors density between groups (Fig. 5A).
Western Blot Experiments
There was a statistically significant decrease in ETB receptors expression in the aortic banding group compared with control group (Fig. 5B and Table 4).
LVH is an adaptive mechanism by which the heart normalizes wall stress and preserves left ventricular function.16 It represents a stronger risk factor for cardiovascular events than blood pressure elevation, smoking or cholesterol, and in multivariate analyses, only age and LVH remain independent prognostic factors.17 The major findings of the present study are: 1) ET-1-mediated contractions are increased in epicardial coronary arteries of swine with LVH due to 2 months of aortic stenosis, with a concomitant increase in ET-1 plasma concentration; 2) this increased propensity to contraction is due in part to the effect of cyclooxygenase products, such as TXA2; 3) the relative contribution of ETA receptors to the contractions of coronary arteries is more important than the one of ETB receptors in both aortic banding and control groups; 4) ETB receptors expression is decreased in coronary arteries in the presence of LVH; 5) with no alteration of the sensitivity of smooth muscle cells to exogenous NO.
The coronary vascular dysfunction in LVH renders the myocardium more susceptible to ischemia, since coronary reserve is reduced.18 Reduced coronary reserve in hypertrophied hearts is the hallmark of pathologic hypertrophy induced by pressure overload and is accentuated in the failing myocardium.19 The status of epicardial coronary arteries in LVH is controversial. Chilian et al found no enlargement of epicardial arteries in hypertrophy caused by pressure overload, whereas other investigators have reported that the diameter of epicardial coronary arteries is increased in proportion to the extent of LVH.20 Results from another study showed functional and morphologic abnormalities in the endothelium, intima, and medial VSMC of large epicardial coronary arteries in dogs with severe LVH and chronic coronary pressure overload as a result of aortic banding. On the other hand, intramyocardial small arteries and resistance vessels, in spite of medial thickening, retained normal functions and endothelial structure.18 In this model of LVH in swine, an increase in the wall-to-lumen ratio was observed in epicardial coronary arteries, which could partially explain the increase in vascular resistance in the coronary bed. However, intravascular pressures of coronary arteries were not altered in the aortic banding group.
Under normal conditions, basal ET-1 plasmatic levels are very low because most of the peptide is released abluminally toward the vascular smooth muscle, and potent inhibitory mechanisms prevent significant ET-1 production.22 In disease states such as atherosclerosis and congestive heart failure, circulating concentrations of ET-1 are elevated, and endothelium-dependent relaxation to acetylcholine is decreased.23 Elevation of plasmatic ET-1 is thought to be one of the major events in the development of the early atherosclerotic lesion, characterized by functional alterations of endothelial cells without significant morphologic changes.24
ET-1-mediated contractions are increased in epicardial coronary arteries from the aortic banding group when compared with the control group, and the level of contraction is reduced by indomethacin, an inhibitor of COX. These results suggest that ET-1 induces the release of COX products, as demonstrated by Plante et al, who showed that ET-1 induces the release of TXA2 and PGI2 via activation of ETB receptors.25 Taddei and Vanhoutte have also reported that ET-1-mediated contractions of rat aortic rings without endothelium are reduced by indomethacin; this fact has been demonstrated in rats with hypertension but not in normotensive rats.26 Furthermore, an inhibitor of TXA2 synthesis, dazoxiben, and a TXA2 receptor antagonist, SQ-29-548, both have an inhibitory effect on contractions similar to that of indomethacin, suggesting that COX-derived products are implicated in contraction, and more specifically TXA2,26 as also suggested by our results. Indeed, in the present model, contractions to ET-1 were reduced in the aortic banding group with daltroban, an inhibitor of TXA2 receptors. Furthermore, because contractions to ET-1 in the aortic banding group in the presence of indomethacin are not restored to the control level, other phenomena such as an increased state of oxidative stress or hypertrophy of the VSMC themselves could be implicated.
An increased state of oxidative stress from free radicals generated in great amount can cause an endothelial dysfunction27 and induce contractions of VSMC by stimulation of COX, with the subsequent release of TXA2 and its precursor, prostaglandin H2 (PGH2). We have previously reported that the nitrotyrosine content in plasma, as an indicator of peroxynitrite production (a marker of oxidative stress), is increased in the same swine model of LVH following 2 months of aortic banding. A treatment with 6-methyl tetrahydrobiopterin, a BH4 analogue, or with antioxidants superoxide dismutase and catalase significantly improved endothelium-dependent relaxations of epicardial coronary arteries in LVH. The contribution of COX-derived products to the decreased relaxation is also suggested by the concentration-response curve to 5-HT, and less importantly with BK, because indomethacin improved endothelium-dependent relaxations in epicardial coronary arteries from the aortic banding group but not in control. Similarly, in hypertensive rats, contractions of aortic rings by stimulation of TXA2/PGH2 receptors were greater than in normal rats.27 In this study, epicardial coronary arteries of LVH swine have an increased sensitivity to TXA2 and PGH2 because indomethacin improves relaxations only in the aortic banding group.
Endothelium-independent responses, evaluated by dose-response curves to sodium nitroprussiate (SNP), an exogenous NO donor, were not altered in the aortic banding group. This fact suggests that the dysfunction of epicardial coronary arteries is related to endothelial alterations and not to a decreased sensitivity of VSMC to NO or a mechanical dysfunction of VSMC.
ETA and ETB receptors mediate vasoconstriction in numerous vessel beds, but the contribution of each subtype is different according to the vascular bed and species.28 To assess the contribution of both receptor subtypes to contractions in the aortic banding group, concentration-response curves to ET-1 and to IRL-1620 (a selective agonist of ETB receptors) in epicardial coronary artery rings without endothelium were constructed. Under these conditions, in LVH secondary to aortic banding, ETA receptors contribute to the majority of the contraction (86% versus 14%) compared with ETB receptors. In rings with endothelium, the contraction mediated by ETB receptors is smaller and accounts for only 8% of total contraction because endothelial ETB receptors induce NO and PGI2 release. Thus, endothelial ETB receptors only partially inhibit the contraction induced by smooth muscle ETB, but this is not significant because the contraction is mainly evoked by ETA receptors. Elmoselhi et al have demonstrated that, in control porcine epicardial coronary arteries, ETA receptors contribute to 78% of the contraction compared with 22% for ETB receptors.29 In our experiments, the contraction induced by ETA and ETB receptor subtypes in control arteries are, respectively, 57% versus 43%. Thus, a change in the relative contribution of each receptor subtype in the contraction to ET-1 was observed in the endothelial dysfunction. Contrary to the increased contraction in the aortic banding group observed with ET-1, the contraction via ETB receptors on VSMC is decreased compared with the control group without endothelium, which could be due to the decreased receptor density observed in Western blot experiments (see below). Moreover, the relative contribution of endothelial ETB receptors is of lesser magnitude in the aortic banding group because the decreased contraction to IRL-1620 in the presence of endothelium is greater in the control group than in the aortic banding group (73% versus 44%). In human epicardial coronary arteries, the relative contribution of ETA and ETB receptors is approximately the same as in swine.30
Qualitative results obtained with confocal microscopy indicate a nonsignificant decrease in endothelial and smooth muscle ETB receptors and increase in ETA receptors in epicardial coronary arteries in the aortic banding group compared with controls. Western blots showed a statistically significant decrease of ETB receptors only. Two mechanisms may be involved in the decreased density of ET receptors: down-regulation and receptor occupancy. Indeed, after prolonged exposure to ligands, receptors are either internalized by endocytosis or associated with the ligand and are unable to induce a further response.31 Also, the total ET receptor number decreases without change in the binding affinity when ET-1 plasma concentrations are increased.31 In addition, the synthesis of ETB receptors is inhibited by ET-1, by inhibition of the ETB receptors' mRNA stabilizing factor.31 Thus, ETB receptors may be down-regulated more rapidly than ETA receptors and/or ETA receptors are synthesized more promptly after internalization,31 accounting for the selective decreased density of ETB receptors in this LVH model.
Increases in ET-1 contractions can occur even with a decrease of ETA receptor density because an increased affinity of these receptors may evoke an increased response. Furthermore, because the density of ET receptors is high relatively to the dissociation constant (K d), ET-1 is immediately trapped by receptors, and fractional saturation builds up in the first cells, hence initiating a contraction.32 Finally, alterations in postreceptor events such as an increased release of second messengers IP3 and DAG could also explain the increased responsiveness to ET-1.
Different studies have suggested that angiotensin II may play a crucial role in the pathophysiology of cardiac hypertrophy. Moreover, in vivo studies have shown that inhibitors of the renin-angiotensin system (RAS) regress or prevent cardiac hypertrophy induced by hypertension, myocardial infarction, and aortic coarctation in an experimental rat model.33 It also has been shown that, in hypertrophy secondary to pressure overload, angiotensin II is released from the cardiac myocytes and induces the overexpression of angiotensinogen and preproendothelin-1 via AT1 receptors.34 However, the renin-angiotensin and endothelin systems appear generally to be parallel ones but can function in series only under specific and very particular conditions. In the present study, we focused on the role of ET-1 in cardiac hypertrophy, but the interaction of ET-1 with the RAS was not evaluated. Because the endothelium of the coronary circulation may be particularly sensitive to the stimulation by angiotensin II, which has been associated with an enhanced expression of ET-1,35 RAS might be implicated in the progression of LVH in our model.
The short-time frame covered in this study is convenient for characterization of the alterations in the transduction pathways of endothelial cells but is relevant only to compensated LVH. Longer-term experiments to characterize the time course of the endothelial dysfunction with progression to congestive heart failure are also of interest. It would necessitate the use of microswine for logistic reasons and to obtain an adult habitus more relevant to adult pathology. Although this model of supracoronary aortic banding exposes coronary arteries to hemodynamic patterns different from those in systemic hypertension or valvular aortic stenosis, no acute modifications of coronary artery perfusion patterns were documented in developing this model. Effects of angiotensin, as discussed, were not addressed in this study but could also contribute to the endothelial dysfunction observed.
Aortic banding imposes a pressure overload to the left ventricle, which leads to the development of LVH, which initially normalized wall stress to preserve left ventricular function.16 However, if the pressure overload is severe and longstanding, the hypertrophy may have adverse effects. The coronary blood flow reserve is diminished, and contractile and diastolic dysfunctions develop.3 Following LVH development, there is an endothelial dysfunction of swine epicardial coronary arteries from an imbalance between relaxing and contracting endothelium-derived factors.
Alterations in the coronary circulation play a major role in the enhanced susceptibility of the myocardium in LVH to ischemic injury.19 Endothelial dysfunction of swine epicardial coronary arteries in LVH secondary to 2 months of aortic banding involves both endothelial relaxing and contracting factors. ETA receptors are preferentially implicated in the increased contraction to ET-1, in association with COX-derived products. The treatment of the endothelial dysfunction associated to LVH with ET-1 antagonists selective for ETA receptors or with COX synthesis inhibitors represents an interesting strategy to improve the prognosis of patients with this condition.
The authors would like to thank Marie-Pierre Mathieu and Louis Villeneuve for their technical assistance and Dr. Eric Thorin, PhD for his collaboration.
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