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Tetrahydrobiopterin and antioxidants reverse the coronary endothelial dysfunction associated with left ventricular hypertrophy in a porcine model

Olivier Malo, Fanny Desjardins, Jean-François Tanguay, Jean-Claude Tardif, Michel Carrier, Louis P. Perrault
DOI: http://dx.doi.org/10.1016/S0008-6363(03)00390-0 501-511 First published online: 1 August 2003


Objective: Endothelium-dependent G-protein mediated relaxations of epicardial coronary arteries is impaired with left ventricular hypertrophy. The objective of this study was to assess the effect of l-arginine, BH4 and the combination of two antioxidants, superoxide dismutase and catalase, on endothelium-dependent relaxations in a swine left ventricular hypertrophy model. Methods: Aortic banding was performed 3 cm above the coronary ostia. Vascular reactivity studies were performed in standard organ chamber experiments to assess the NO pathway in the presence of methyltetrahydropterin (a BH4 analogue), l-arginine, superoxide dismutase and catalase. Results: There was a statistically significant increase in endothelium-dependent relaxation to serotonin and to bradykinin with methyltetrahydropterin and with superoxide dismutase plus catalase (P<0.05) but not with l-arginine compared to untreated coronary arteries from left ventricular hypertrophy animals. Plasma 3-nitrotyrosine level increased significantly from 918±122 to 1844±300 μM (P<0.05 vs. control) after 60 days of aortic banding. Endothelial dysfunction was not associated with a reduced expression of endothelial nitric oxide synthase 2 months after pressure overload left ventricular hypertrophy. Conclusions: Treatment with BH4 and antioxidants constitutes an interesting approach for the prevention of endothelial dysfunction in epicardial coronary arteries associated with left ventricular hypertrophy.

  • Antioxidants
  • Endothelial dysfunction
  • Left ventricular hypertrophy
  • Nitric oxide
  • Tetrahydrobiopterin

Time for primary review 31 days.

1 Introduction

Left ventricular hypertrophy (LVH) represents an adaptive mechanism through which the heart normalizes ventricular wall stress and preserves systolic function in the early stages [1]. However, decompensation often eventually occurs and leads to heart failure [2]. LVH increases the risk of cardiovascular disease (CV) nearly three-fold and has been found to be associated with adverse outcomes including stroke, sudden death, myocardial infarction, congestive heart failure and coronary heart disease (CHD) [3]. Impairment of the coronary vasodilator capacity also occurs in the presence of LVH [4]. The endothelium plays a key role in regulating vascular and organ function through diverse signaling pathways. Loss or reduction of endothelium-dependent vascular relaxation is due to reduced bioavailability and/or increased degradation of nitric oxide (NO), the major endothelium-dependent vasorelaxing factor [5].

A number of risk factors for CV are associated with reduced synthesis and/or increased degradation of vascular NO, including hypercholesterolemia, diabetes, hypertension, and smoking [6,7]. We recently demonstrated that endothelial dysfunction in LVH induced pressure overload was due to an alteration of the Gi- and Gq-protein mediated endothelium-dependent relaxation pathways in epicardial coronary arteries [8]. It was also associated with a decreased release and production of NO as evidenced by the decrease of basal cyclic GMP content at the vascular wall level [8].

The precise molecular basis of endothelial dysfunction in CV is not completely understood [9]. However, clinical and experimental studies have reported that a decrease in 5,6,7,8-tetrahydrobiopterin (BH4) bioavailability, could be implicated in multiple cardiovascular pathologies which suggests an important role for BH4 in the genesis of CV [9–11]. BH4 is an important cofactor of endothelial NO synthase (eNOS) and plays a major role in the liaison of l-arginine with the enzyme eNOS [5,12]. Under conditions where tetrahydrobiopterin is reduced, eNOS may produce superoxide anions (O2) instead of NO [10,13]. An increase production of O2 in the vascular wall then inhibits the physiological role of NO through scavenging [14]. Nitric oxide can also participate in the deterioration of endothelial function by reacting rapidly with O2 to form peroxynitrite (ONOO), a strong vasotoxic oxidant [6,7,14–16]. Recent investigators have demonstrated a close relationship between the bioavailability of BH4 and NO in endothelial cells. The supplementation with BH4 favors the production of NO from the endothelial NOS. The administration of BH4 improves endothelial dysfunction not only in animals models of hypertension and diabetes but also in patients with atherosclerosis and hypercholesterolemia [17]. BH4 supplementation could represent an effective therapeutic option for endothelial dysfunction [7,13]. The aim of the present study was to assess the role of an analogue of BH4, 6-methyltetrahydropterin, l-arginine and two antioxidants, superoxide dismutase (SOD) and catalase (CAT), on endothelium-dependent relaxation of epicardial coronary arteries and to measure the plasma level of ONOO as a marker of oxidative stress in a pressure overload model of LVH.

2 Methods

2.1 Animals

Twenty-three Landrace swine weighing 28.6±1.8 kg of either sex were maintained and tested in accordance 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 research protocol was approved by the local committee. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Two groups were studied: one control group included swine sacrificed by exsanguination without aortic banding and the other group underwent aortic banding for a duration of 60 days before sacrifice.

2.2 Surgical procedure

Pigs were anaesthetized with intramuscular injection mixture of ketamine (20 mg/kg; Rogarsetic, QC, Canada) and xylasine (2 mg/kg; Rompun, ON, Canada). Swine were artificially ventilated with an O2/air mixture. Respiratory control was maintained by frequent determinations of arterial blood gases. Acidosis was balanced with 8.4% sodium bicarbonate (Abbott Laboratories, QC, Canada). Light anaesthesia was supported by halothane 1% v/v (Halocarbon Laboratories, NJ, USA).

Hairs were shaved in the operative field, the skin was surgically disinfected and intramuscular antibiotics (trivetrin 0.07 ml/kg; Shering-Plough, QC, Canada) were administered. A catheter was placed in an auricular vein for intravenous infusion during the procedure. A left anterior thoracotomy was performed in the third intercostal space. The ascending aorta and the pulmonary artery were carefully separated and an umbilical tape was placed 3 cm above the coronary ostia of the aorta, gently constricted to obtain a systolic gradient of 15 mmHg and then tied. The pericardium was closed, leaving a small opening to ensure drainage of pericardial fluid and the thorax was closed in layers [8].

2.3 Echocardiographic studies

Transthoracic echocardiography was performed before surgery and after a follow-up of 1, 4 weeks and 2 months. Pigs were sedated (see above) for serial echocardiographic examinations. A 2.5 MHz phased-array transducer and a standard echocardiographic system (Sonos 1000 Hewlett-Packard, Andover, MA, USA) were used. A 2-D guided M-mode study of the left ventricle and two-dimensional apical four- and two-chamber views were performed and recorded on videotape for off-line measurements. The thickness of the interventricular septum (IVS) and left ventricular posterior wall (LVPW) were measured on M-mode at end-diastole during an average of three measurements cardiac cycle. Left ventricular mass was calculated using the following formula: LV mass=1.05[(D+LVPW+IVS)3D3]−14 g, where D represents left ventricular cavity end-diastolic diameter.

2.4 Hemodynamics studies

Hemodynamic measurements of the left ventricle were performed by insertion of a 6 or 7-Fr pigtail catheter via 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 in the left ventricle to measure left intraventricular pressures. All studies were performed at baseline and 1 and 2 months after surgery.

2.5 Sacrifice and coronary harvesting

2.5.1 Experimental groups Control hearts

Hearts from normal 4-month-old swine (40 kg) not submitted to aortic surgery or to left thoracotomy and which received the same tranquilizing agent were excised through a median sternotomy (n = 6). Hearts 60 days after aortic stenosis

Hearts from swine submitted to aortic banding and sacrificed after 60 days of aortic were also excised through a median sternotomy (n = 12). Six animals were excluded due to death from sepsis and infection.

After 60 days of aortic banding, the swine were anaesthetized using the same approach as for the aortic banding. Sacrifice was performed by exsanguination and the thorax reopened to explant the heart. The total weight of the heart was determined and it was placed in a Krebs–bicarbonate solution for harvesting of coronary arteries. The left descending coronary artery, the left circumflex and right coronary arteries were dissected free from the surrounding myocardium, cleaned of adherent fat and connective tissue, and cut into 4–5 mm long rings. A total of 16 coronary arterial rings were prepared from each heart. Rings from the left anterior descending and circumflex coronary arteries were used randomly.

2.6 Organ chamber experiments

The vascular reactivity of coronary arteries was studied in organ chambers filled with control solution (20 ml) at 37°C. The rings were suspended between two metal stirrups, one being connected to an isometric force transducer. Data were collected with data acquisition software (IOS3, Emka, Paris, France). Except when stated otherwise, the studies were performed in the presence of indomethacin (10−5 M, to exclude the production of endogenous prostanoids) and propranolol (10−5 M, to prevent the activation of β-adrenergic receptors). Each preparation was stretched to the optimal point of its active length–tension curve (usually 3.5 g), as determined by measuring the contraction to potassium chloride (30 mM) at different levels of stretch and was then allowed to stabilize for 90 min. The maximal contraction was determined with potassium chloride (60 mM) and the baths were then washed. Rings were excluded if they failed to contract with potassium chloride (exclusion rate <5%). After washing and 30 min of stabilization, endothelium-dependent relaxations were studied in preparations contracted with prostaglandin F2α (range 2×10−6–10−5 M) to achieve a contraction averaging 50% of the maximal contraction to KCl (60 mM).

2.6.1 NO pathway

The NO mediated-relaxation pathway was studied by constructing concentration–response curves to serotonin [5-HT, an agonist that binds to 5-HT1D receptors coupled to Gi-proteins (10−10–10−5 M; in the presence of 10−6 M ketanserin, incubated 40 min before the addition of serotonin to block serotonin 5HT2 receptors)], and bradykinin [BK, an agonist that binds to B2 receptors coupled to Gq-proteins leading to the release of NO and the endothelium-derived hyperpolarizing factor (EDHF 10−12–10−6 M)].

In paired experiments, the preparations were incubated with a liposoluble analogue of tetrahydrobiopterin, 6-methyltetrahydropterin (10−4 M) for 45 min before exposure to PGF2α. In a separate series of experiments, rings were incubated with SOD (150 U/ml) and CAT 1200 U/ml) 5 min before exposure to PGF. In another set of experiments, the eNOS substrate l-arginine (10−4 M) was added in the organ chamber 45 min before exposure to PGF.

Endothelium-independent relaxations were studied by constructing concentration–response curves to sodium nitroprusside (SNP, 10−10–10−5 M, an exogenous NO donor). Except for potassium chloride, no rings were exposed to more than one agonist in the course of the experiments. Control and treated rings from the same animal were studied in parallel. Only one concentration–response curve was obtained in each vascular ring.

2.7 Measurement of total plasma nitrotyrosine levels

The plasma levels of ONOO were assessed using high-pressure liquid chromatography (HPLC) measuring nitrotyrosine (NTYR) content as an indicator of ONOO production [16]. Blood samples from the aortic banding and control groups were drawn from the coronary sinus at the sacrifice in EDTA to prevent coagulation and subsequently centrifuged. The isolated plasma was then frozen and kept at −70°C until analysis. The plasma samples were analyzed by HPLC 501 with a C18 reversed-phase column (Waters Corporation, Milford, MA, USA). The peak concentrations of NTYR were measured with an ultraviolet detector Waters 484 set at 274 nm. The peak was identified on the basis of the retention time of authentic NTYR [18]. ONOO production was assessed by measuring the production of nitrotyrosine from tyrosine which served as an index of ONOO produced from blood samples from the aortic banding and control groups.

2.8 Endothelial (eNOS) nitric oxide synthase expression

After fixation in Tissuetek, preservation at −70°C and subsequent fixation in formaldehyde 1%, a bovine antibody specific for eNOS was used for the ABC technique (avidin–biotin complex immunoperoxidase method) with nickel enhancement and the expression of eNOS was compared in control and treated coronary arteries and graded semiquantitatively by an independent evaluator.

2.8.1 Drugs

All solutions were prepared daily. Bradykinin, dl-6-methyl-5,6,7,8-tetrahydropterin, 5-hydroxytryptamine creatinine sulfate (serotonin), indomethacin, ketanserin, l-arginine, SOD, CAT and SNP were purchased from Sigma (ON, Canada). Propranolol was purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA) and prostaglandin F2α from Cayman Chemicals (Ann Arbor, MI, USA).

2.9 Statistical analysis

Relaxation and contraction are expressed as a percentage of the maximal contraction to prostaglandin F2α for each group and expressed as mean±standard error of the mean (S.E.M.); n refers to the number of animals from which blood vessels were taken. One-way analysis of variance studies was performed to compare concentration–response curves. The Newman–Keuls test was used as the post-hoc test. Student's t-test for paired/unpaired observations was used for the comparison of the plasma 3-nitrotyrosine concentrations between groups. Differences were considered to be statistically significant when P<0.05.

3 Results

3.1 Morphological data, echocardiographic measurements and hemodynamic studies

Echocardiographic and hemodynamic studies were associated with statistically significant changes in cardiovascular parameters. Morphological, echocardiographic and hemodynamic data are presented in Tables 1–3.

View this table:
Table 3

Hemodynamic data

Basal1 Month2 Months
LV systolic pressure (mmHg)110±7108±8107±10
LV end-diastolic pressure (mmHg)17±422±333±2*
LAD systolic pressure (mmHg)85±1293±1084±10
LAD diastolic pressure (mmHg)52±1251±635±5
LCx systolic pressure (mmHg)76±992±1090±6
LCx diastolic pressure (mmHg)49±851±442±2
RC systolic pressure (mmHg)91±990±1291±5
RC diastolic pressure (mmHg)63±950±848±11
LV+dP/dT (mmHg/s)1910±3041324±3741077±91
LV−dT/dT (mmHg/s)1980±251266±208*1187±151*
  • Data are presented as mean±S.E.M.

    *P<0.05 versus basal group.

    LV, left ventricle; LAD, left anterior descending; LCx, left circumflex; RC, right coronary.

View this table:
Table 2

Echocardiographic data

ControlSham24-h banding2-month banding
Left atrium FAS (cm2)32±134±134±229±2
Left atrium FS (%)22±221.7±0.422±215±1*
Left ventricle FS (%)40±242±245±337±3
Left ventricle EF (%)57±258±163.2±0.459±3
Left ventricle IVS (g)0.82±0.040.85±0.050.98±0.041.01±0.03*
Left ventricle PW (g)0.83±0.060.80±0.030.88±0.030.95±0.03*
  • Data are presented as mean±S.E.M.

    *P<0.05 versus all groups.

    FAS, fractional area shortening; FS, fractional shortening; EF, ejection fraction; IVS, interventricular septum; PW, posterior wall.

View this table:
Table 1

Morphological data

NBody weightHeart weightLV weightLV weight/bodyHeart weight/body
(kg)(g)(g)weight (g/kg)weight (g/kg)
2-Month banding931±2194±12*141±1*4.59±0.25*6.21±0.86*
  • Data are presented as mean±S.E.M. LV, left ventricular.

    *P<0.05 vs. control.

3.2 Vascular reactivity in hypertrophied heart

3.2.1 Contraction

There were no differences in the amplitude of the contraction to potassium chloride (60 mM) between the groups. There were no differences in the amplitude of the contractions to prostaglandin F2α between groups. There were no differences in the concentration of prostaglandin F2α needed to achieve the target level of contraction in coronary rings from swine submitted to aortic banding compared to the control group (data not shown).

3.2.2 Endothelium-dependent relaxations Effect of aortic banding

There was a decrease in endothelium-dependent relaxations to 5-HT and bradykinin in coronary rings after 2 months of aortic banding compared with controls (data not shown). Effect of methyltetrahydropterin on vascular reactivity

There were improvements in endothelium-dependent relaxations to 5-HT (Fig. 1A) and to BK (Fig. 1B) when coronary rings after 2 months of aortic banding were incubated with the liposoluble analogue of tetrahydrobiopterin (10−4 M).

Fig. 1

Cumulative concentration–responses curves to serotonin (5-HT); (a) bradykinin (BK); (b) in rings of porcine coronary arteries after 2 months of left ventricular pressure overload (□) and after preincubation with l-arginine (▴). Responses are given as the percent of relaxation to the contraction induced by prostaglandin F2α. Results are presented as the mean±S.E.M. The symbols indicate statistically significant differences between the groups for the indicated concentration: *P<0.05. Effect of l-arginine on vascular reactivity

Incubation with l-arginine (10−4 M) did not improve the endothelium-dependent relaxations to serotonin (Fig. 2A) and to bradykinin (Fig. 2B) after 2 months of banding.

Fig. 2

Cumulative concentration–responses curves to serotonin (5-HT); (a) bradykinin (BK); (b) in rings of porcine coronary arteries after 2 months of left ventricular pressure overload (□) and after preincubation with methyltetrahydropterin (▴). Responses are given as the percent of relaxation to the contraction induced by prostaglandin F2α. Results are presented as the mean±S.E.M. The symbols indicate statistically significant differences between the groups for the indicated concentration: *P<0.05. Effect of SOD and CAT on vascular reactivity

Increases in endothelium-dependent relaxations to serotonin (Fig. 3A) and to bradykinin (Fig. 3B) were observed when rings from the bandings group were pretreated with SOD and CAT.

Fig. 3

Cumulative concentration–responses curves to serotonin (5-HT); (a) bradykinin (BK); (b) in rings of porcine coronary arteries after 2 months of left ventricular pressure overload (□) and after preincubation with superoxide dismutase and catalase (▴). Responses are given as the percent of relaxation to the contraction induced by prostaglandin F2α. Results are presented as the mean±S.E.M. The symbols indicate statistically significant differences between the groups for the indicated concentration: *P<0.05.

3.2.3 Endothelium-independent relaxations

No differences in endothelium-independent relaxations to the NO donor SNP were observed in coronary rings 60 days of banding with ad without endothelium compared with control (Fig. 4).

Fig. 4

Cumulative concentration–response curves to sodium nitroprussiate (SNP) in rings of porcine coronary arteries with (▴) and without (●) endothelium 2 months after aortic banding. Responses are given as the percent of relaxation to the contraction induced by prostaglandin F2a. Results are presented as the mean±S.E.M. The symbols indicate statistically significant differences between the groups for the indicated concentration: *P<0.05.

3.2.4 Plasma levels of nitrotyrosine

There was a statistically significant increase of 3-NT plasma levels after 60 days of banding from 918±122 to 1844±300 μM (Fig. 5).

Fig. 5

Total plasmatic 3-nitrotyrosine after 2 months of aortic banding. Results are presented as the mean±S.E.M. The asterisk indicate statistically significant differences between the 2 groups: *P<0.05.

3.2.5 eNOS expression

Immunohistochemistry showed preservation of the expression of eNOS compared to control rings after 2 months of aortic banding (Fig. 6).

Fig. 6

Representative photomicrograph of eNOS expression in (a) control coronary endothelial cells and (b) after 2 months of pressure overloads hypertrophy (magnification: ×250). eNOS is present 2 months after aortic banding.

4 Discussion

The major findings of the present study are that (1) LVH is associated with increased nitrotyrosine formation in the plasma after 60 days of pressure overload. (2) eNOS expression is preserved in the coronary arterial wall. (3) Acute in vitro treatment with a tetrahydrobiopterin analogue and with SOD and CAT significantly improves vascular reactivity of epicardial coronary arteries in LVH.

4.1 BH4 improves endothelial function in LVH

LVH induced by pressure overload is associated with significant decreases in endothelium-dependent relaxations involving Gi and Gq-protein mediated pathways suggesting the presence of endothelial dysfunction [8]. In the present study, preincubation with a liposoluble analogue of tetrahydrobiopterin, 6-methyltetrahydropterin, restored endothelial function in coronary arteries exposed to 60 days of left ventricular pressure overload. This endothelial dysfunction is associated with reduced basal cyclic GMP in coronary arteries after 60 days of aortic banding which confirms decreased bioavailability of NO [8]. The presence of endothelial dysfunction in patients with LVH could reduce perfusion during extracorporeal circulation and hamper myocardial recovery at the time of reperfusion after cardiac surgery. Although outcomes have greatly improved due to advances in myocardial protection strategies, revascularization and aortic valve surgery is still associated with substantial morbidity in patients with LVH [19]. Recently, the identification of tetrahydrobiopterin (BH4) as a crucial cofactor for optimal activity of eNOS has provided a direct link between cellular bioavailability and NO synthesis in endothelial cells [10]. Studies on isolated aorta, coronary and cerebral arteries demonstrated that inhibition of BH4 causes impairment of endothelial function [9]. Recent experimental and clinical studies demonstrated that administration of BH4 improves endothelial dysfunction in diseases associated with increased vascular superoxide production such as hypercholesterolemia and diabetes mellitus [11]. Hong et al. [13] also demonstrated that a relative deficiency of tetrahydrobiopterin may be responsible, at least in part, for the development of hypertension in SHR. At the present time, it is generally accepted that reduced availability of BH4 causes reduction in NO production [9]. However, measurements of endogenous BH4 have not been performed in our model to confirm the reduced bioavailability of tetrahydrobiopterin. In the absence of BH4, eNOS is uncoupled and produces mainly O2 instead of NO. The know antihypertrophic effect of NO on the myocardium and on vascular smooth muscle cells [20], suggests that the impaired NO bioavailability documented in this model may promote the progression of LVH. The exact mechanism by which tetrahydrobiopterin participates in the biosynthesis of NO is still not understood [21] but most likely involves increased enzymatic activity of eNOS and/or antioxidant activity of tetrahydrobiopterin [9,13], providing strong evidence for the key role of BH4 in the pathogenesis of endothelial dysfunction [9].

4.2 SOD and CAT improve endothelial function

Oxidative stress may cause a functional uncoupling of the receptor–G protein complex leading to impairment of the normal signal transduction within endothelial cells [22]. The present study demonstrated an increase in the production of ONOO in swine after 60 days of aortic banding as shown by the increased formation of NTYR in the plasma. Peroxynitrites and related species aggressively nitrate protein tyrosine residues, leading to the chemically stable biomarker 3-NT. Recent investigations have demonstrated the value of this marker as evidence of NO dysregulation in disease [6]. Taken together, these observations suggest that the increase of ONOO and oxidative stress may be partially responsible for the endothelial dysfunction in LVH because treatment with the antioxidants SOD and CAT, improves endothelium-dependent relaxations. In light of our results with BH4 and normal expression of eNOS, limited bioavailability of tetrahydrobiopterin may be a major cause of increased production of O2 and a decreased bioavailability of NO by eNOS in LVH. MacCarthy et al. [23] demonstrated an increase in reactive oxygen species (ROS) within guinea pigs hypertrophied hearts. Increased formation of O2 increases the concentration of the potent oxidant ONOO, formed by the extremely rapid reaction between NO and superoxide [15], which inhibits the physiological role of NO as seen in this LVH model. Preliminary experiments indicate that peroxynitrite (ONOO) avidly oxidizes BH4 to dihydrobiopterin leading to impairment of endothelial function as observed in this model [11]. An increased oxidation of BH4 by ONOO may cause BH4 deficiency, which favors NADPH oxidase activity of eNOS leading to the formation of O2 instead of NO [24].

Two majors ROS sources considered to be important in the genesis of vascular endothelial dysfunction are (1) dysfunctional eNOS deficient in BH4 or l-arginine and (2) NADPH oxidase [23]. Addition of exogenous BH4 usually inhibits O2 production by dysfunctional eNOS, restores NO production, and corrects endothelial dysfunction. In the present study, preincubation with a BH4 analogue corrected the coronary endothelial dysfunction. NADPH oxidases are expressed in several nonphagocytic cells, including the endothelium, vascular smooth muscle, fibroblasts and cardiac myocytes [23]. NADPH oxidase generates great amounts of ROS which are implicated in vascular pathophysiology, for example, angiotensin II-induced smooth muscle hypertrophy and hypertension, and the endothelial dysfunction associated with hypercholesterolemia [6]. Many stimuli could increase NADPH oxidase expression and/or activity include angiotensin II, tumor necrosis factor-α, and increased mechanical forces. These factors are all likely to be relevant in LVH [23]. Thus, BH4 is a prime target of oxidative stress which may lead to decrease NO bioavailability [15].

4.3 Effect of l-arginine on vascular reactivity

Other abnormalities of the endothelial signal transduction pathway could contribute to the pathogenesis of endothelial dysfunction in LVH. The substrate of the eNOS for the formation of NO, l-arginine, can restore normal dilator response to acetylcholine in the coronary microcirculation of hypercholesterolemic patients but not in epicardial coronary arteries [25]. In this study, preincubation with l-arginine did not affect the endothelial response to serotonin and to bradykinin of coronary arteries after 2 months of aortic banding. This indicates that the decreased availability of this substrate for the formation of NO is not the major cause of endothelial dysfunction in this model and maybe related to defective extracellular transport of l-arginine.

4.4 Limitations

The short-time frame covered in this study is convenient for characterization of the alterations of the signaling transduction pathways of endothelial cells but relates only to the situation of compensated LVH. Longer term experiments would be of interest to characterize the time course and progression of the coronary endothelial dysfunction observed in the present study with an increasing duration of pressure overload and development of congestive heart failure. Use of microswine for long-term studies would be necessary since the rapid increase in weight of the domestic swine poses a logistic problem. Use of mature animals would also enable to perform the studies in an adult habitus, more relevant to adult cardiac surgery and clinical cardiology. Another drawback of this model concerns the use of an experimental supracoronary aortic banding, which exposes the coronary arteries to a different hemodynamic pattern than the ones in systemic hypertension or valvular aortic stenosis. There was, however, no evidence of acute endothelial denudation due to modification of coronary artery perfusion pressure secondary to the experimental supraostial stenosis [8]. The absence of significant changes in coronary perfusion pressures in the presence of this experimental supravalvular aortic banding rules out the effect of pathological changes in shear stress, flow pattern as well as low cardiac output as the cause of the epicardial coronary dysfunction [8]. Another limitation of this model is the technique used for the measurement of total plasma nitrotyrosine plasma levels. 3-Nitro-l-tyrosin (nitrotyrosine), generated by the nitration of l-tyrosine (tyrosine) residues by ONOO induced tissue damage, is relatively stable and can be measured readily. Partially because ONOO has a very short half-life in vivo, its plasma fraction is difficult to measure. Thus, we have measured nitrotyrosine formation as an indicator of ONOO production.

5 Conclusion

The present study demonstrates that pressure overload-induced LVH is associated with an impairment of endothelium-dependent relaxation in epicardial coronary arteries and an increased in 3-NT in the plasma, a marker of production of peroxynitrite. Endothelial dysfunction of coronary arteries is reversed by a tetrahydrobiopterin analogue and the antioxidants SOD and CAT. This suggests that BH4 deficiency associated with increased ONOO production is responsible for the coronary endothelial dysfunction in LVH. Treatment with BH4 and antioxidants may be a promising therapy to favor the production of NO from eNOS. The acute administration of BH4 and antioxidants correct endothelial dysfunction in this animal model of LVH.


This work was supported through grant from Fonds de la Recherche de l’Institut de Cardiologie de Montréal. Dr Louis P. Perrault and Dr Jean-Claude Tardif are scholars from Fonds de la Recherche en Santé du Québec (FRSQ). We wish to thank Marie-Pierre Mathieu for her technical assistance.


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View Abstract