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European Journal of Heart Failure 2006 8(8):769-776; doi:10.1016/j.ejheart.2006.09.011
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© 2006 European Society of Cardiology

Functional alterations in NO, PGI2 and EDHF pathways in the aortic endothelium after myocardial infarction in rats

Gabor Csanyia,1,2, Michael Bauerb,1, Wolfgang Dietlb, Magdalena Lomnickaa, Tatiana Stepuroa, Bruno K. Podesserb and Stefan Chlopickia,*

a Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University Medical College Grzegorzecka Str. 16, 31-531 Krakow, Poland
b Ludwig Boltzmann Cluster for Cardiovascular Research c/o Institute for Biomedical Research, Allgemeines Krankenhaus Wien, Waehringer Guertel 18-20, A-1090 Vienna, Austria

* Corresponding author. Tel.: +48 12 421 11 68; fax: +48 12 421 72 17. E-mail address: s.chlopicki{at}cyfronet.krakow.pl


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Background: Previous work on endothelial dysfunction in post-MI heart failure has shown conflicting results.

Aim: To analyze gender related alterations in NO-, PGI2- and EDHF-dependent endothelial function in the thoracic aorta 7 and 42 days after myocardial infarction (MI).

Methods and results: MI was induced by coronary artery ligation in female and male Sprague–Dawley rats. There was no gender related difference in infarct-size or in the impairment of fractional shortening of the left ventricle 42 days after coronary ligation. Neither acetylcholine-induced (Ach) vasodilation nor basal PGI2 production in the aorta was modified by coronary ligation. Interestingly, 7 days after MI, basal NO production was impaired and the EDHF component of Ach-induced vasodilation was up-regulated, in both male and female rats. However, 42 days post-MI, basal NO was only impaired in male rats, while EDHF was only up-regulated in female rats.

Conclusion: MI induced impairment of functional activity of basal NO production and adaptive up-regulation of the EDHF component of Ach-induced relaxation. The above alterations in endothelial function in the aorta were gender-specific at 42 days but not 7 days after MI. Some of the previously reported discrepancies in the development of endothelial dysfunction in the post-MI period may be gender related differences.

Key Words: Heart failure • Endothelial dysfunction • NO • PGI2 • EDHF • Gender

Received February 8, 2006; Revised July 3, 2006; Accepted September 19, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Chronic heart failure (CHF) is characterized not only by alterations in cardiac function but also by increased peripheral resistance and impairment of peripheral blood flow that depends on neurohumoral activation [1]. Endothelial dysfunction (ED) contributes significantly to the increased peripheral resistance in CHF and therefore plays an important role in the pathophysiology of this disease. ED, diagnosed on the basis of decreased receptor-stimulated or basal vasodilator capacity is present in various types of peripheral vessels in patients with CHF of non-ischaemic or ischaemic origin [2,3]. Normalization of peripheral endothelial function e.g. by physical exercise, angiotensin converting enzyme inhibitors (ACE-I) or endothelin antagonists enhances cardiac output, arterial compliance and exercise capacity [4-6] and these changes are associated with prolonged survival in CHF.

The rat coronary ligation model has been extensively used as an experimental model to investigate the mechanisms of ED in CHF. Despite numerous studies, it is still not clear whether the endothelium-dependent relaxation in large conduit arteries and resistance vessels in this model is impaired or not. Indeed, some authors have demonstrated impaired [7,8], while others preserved [9] or augmented [10] NO-dependent relaxation in aortic rings following MI. Discordant results have also been reported in studies of mesenteric arteries [9,11].

It is well known that endothelial cells produce not only NO but a number of other vasodilators, such as PGI2 or EDHF, regulating vasomotor tone. Most previous studies however, have investigated the relative contribution of NO to endothelial function following MI and there are only a few reports concerning the role of PGI2 and EDHF [8,12,13] and again they are contradictory. Indeed, one study reported that diminished EDHF activity may be a major factor contributing to decreased endothelium-dependent relaxation in heart failure [8], while other studies have demonstrated increased activity of EDHF in CHF [13].

The discrepancies between results obtained previously may be due to the use of different strains of experimental animals, or to assessment of endothelial function in different types of vessels at different time points during the post-MI period. Furthermore, even if comparable experimental conditions were used (rat aorta, 10 weeks post-MI) the assessment of peripheral endothelial function did not yield similar results [9,14] suggesting the involvement of additional factors such as infarct-size (small/large MI) [8] or the gender of the animals.

Interestingly, there is evidence that heart failure in females has a milder progression and is associated with a better outcome [15]. There is also increasing evidence that oestrogen improves endothelial function. Indeed, oestrogen up-regulates the expression of endothelial nitric oxide synthase (eNOS) [16], cyclooxygenase-2 dependent PGI2 production [17], as well as EDHF synthesis [18]. Thus, the endothelial response induced by post-MI remodelling may be gender-specific as regards the NO, PGI2 and EDHF mediated pathways.

Taking all the above into consideration, the aim of the present study was to assess NO-, PGI2- and EDHF-dependent endothelial function in the aortic endothelium following large MI, and to assess whether post-MI changes in endothelial function are dependent on the time period following MI and on the gender of the animals.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
The experiments were conducted according to the Guidelines for Animal Care and Treatment of the European Community and were approved by the animal ethics committees of the Medical Universities of Krakow and Vienna. The investigation conforms with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised 1985).

2.1. Animals
Adult male (n=25, weighing 300±20 g) and female (n=25, weighing 200±20 g) Sprague-Dawley rats were obtained from the Core Unit of Biomedical Research Himberg of the Medical University of Vienna. The rats were housed in an isolated room at controlled temperature with free access to food and drinking water.

2.2. Coronary artery ligation model
Coronary artery ligation was performed according to a technique described by Pfeffer et al. [19]. Briefly, rats were anesthetized with ketamine (1 mg/100 g bodyweight, ip) and xylazine (10 mg/100 g bodyweight, ip), intubated and ventilated with air supplemented with 1% isoflurane using a Bigler animal respirator (Bigler Instruments, Vienna, Austria). Lateral thoracotomy was performed in the fourth intercostal space and MI induced by ligation of the left anterior descending (LAD) coronary artery approximately 3 mm from its origin using a 6.0 prolene® suture. SHAM-operated rats underwent the same surgical procedure, except coronary ligation. Altogether 50 animals were used for the experiments. Fifteen rats (7 female and 8 male) served as SHAM controls, while 35 underwent coronary ligation. The surviving rats were randomly assigned to be sacrificed at day 7 (4 female and 4 male) or 42 days after MI (4 female and 5 male). Importantly, animals with an infarct-size≥30% of the left ventricle were used for the vascular experiments. Animals with an infarct-size<30% were rejected (approximately 5% of animals).

2.3. In vivo haemodynamic measurements
At baseline before the induction of MI and 42 days post-MI, in vivo haemodynamics were assessed by transthoracic echocardiography. Echocardiographic studies were carried out under light anaesthesia and spontaneous respiration with xylazin (1 mg/100 g bodyweight) and ketamin (10 mg/100 g bodyweight) using the same dosage in MI and SHAM rats. Imaging was performed by an ultrasonographer experienced in rodent imaging with a 7.5 MHz commercially available standard paediatric transducer connected to a echocardiographic computer console (VINGMEDSound, CFM 800, software version 1.0) [20]. Phased array technology with a spatial resolution of 0.2 mm was used. The interrogation depth was set at 4 cm. A parasternal long axis view was followed by a parasternal short axis view. After a good image quality of the mid-papillary muscle level of the left ventricle in 2-dimensional echocardiography had been obtained M-mode was added for measurement of left ventricular dimensions at end-systole and end-diastole. Fractional shortening was calculated. Measurements of 3 beats of the heart were performed online from the screen and averages were used for further analysis.

2.4. Determination of infarct-size
At day 7 or 42 post-MI, rats were anaesthetized with sodium thiopental (12 mg/100 g body weight, ip; Biochemie GmbH, Kundl-Rakusko, Austria) and sacrificed by cervical dislocation. The heart was rapidly removed, blotted dry and weighed to determine the total heart weight. The atria were removed from the ventricles, the right ventricle was separated from the left ventricle (LV), the infarcted region was dissected from the non-infarcted LV, and each part was weighed. Infarct-size was expressed as the ratio of the infarct region to total LV mass.

2.5. Protocol of experiments in isolated aortic rings
Following sacrifice as described above, the descending thoracic aorta was quickly removed, and after washing with ice-cold saline it was placed in cold, freshly-prepared Krebs-Heinseleit buffer of the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 10, pyruvic acid 2 and EDTA 0.5. After removal of connective tissue each aorta was cut into 6 rings, each of 3-4 mm length. Vascular rings were then transferred to organ chambers filled with 5 ml of Krebs-Heinseleit solution maintained at 37 °C, pH 7.4 and gassed with 95% O2 and 5% CO2. Rings were mounted between 2 hooks attached to an isometric force transducer (Biegastab K30 type 351; Hugo Sachs March-Fr, Germany) connected to a recorder (Graphtec WR3320, UK) for continuous recording of tension. After an equilibration period of 30 min resting tension was increased stepwise to reach a final value of 4 g. Then rings were incubated further for 30 min. Stretching of the aortic rings to a resting tension of 4 g was chosen on the basis of preliminary experiments in which it was found to result in optimal and sustained vascular responses.

Viability of the vessels was documented by a contractile response to potassium chloride (KCl, 60 mM). The aortic rings were then precontracted with phenylephrine (Phe, 5x10–8-10–7 M) and after obtaining a stable plateau phase, cumulative concentration-dependent response to Ach (10–9 to 10–5 M) was induced. Phenylephrine-induced contraction did not differ between the experimental groups since the phenylephrine concentration was adapted to achieve a comparable level of precontraction (80-90% of KCl-induced contraction). After Ach-induced response, the aortic rings were mildly precontracted by Phe to reach 10-20% of KCl-induced maximal vasoconstriction, and basal NO activity in the aortic rings was determined on the basis of the magnitude of contraction induced by nitric oxide synthase (NOS) inhibitor L-NG-nitroarginine methyl ester (L-NAME, 300 µM). The endothelium-independent vasorelaxation was evoked by sodium nitroprusside (NaNP, 10–9 to 10–5 M). Since the NO-independent component of the Ach-induced response was not modified by indomethacin, EDHF-mediated relaxation was analyzed after combined incubation with L-NAME and indomethacin. In some experiments COX-1 or COX-2 inhibitors (indomethacin 1 µM, rofecoxib 1 µM, respectively) were used to test the involvement of COX in Ach-induced vasorelaxation. The EDHF component of Ach response was analyzed by pre-treatment (10 min) with the K+ channel blocker tetraethylammonium chloride (TEA, 10 mM) or epoxygenase synthesis inhibitor miconazole (MICO, 10 µM).

2.6. Determination of basal prostacyclin production in aortic rings
Basal PGI2 production by the aortic rings was analyzed by measuring its stable hydrolysis product 6-keto-PGF1P in an organ bath medium. A commercially available EIA kit from Cayman Chemical Company (MI, USA) was used. The aortic rings were equilibrated in the fresh Krebs-Henseleit solution as for the functional experiments, and samples of supernatant were collected after 10 and 30 min of incubation. PGI2 production was expressed as pg/mg of dry weight of the aortic rings. The enzymatic source of PGI2 was assessed using non-selective COX or selective COX-2 inhibitors such as indomethacin or rofecoxib, respectively.

2.7. Statistical analysis
Vasodilator responses are shown as a percentage of dilatation relative to phenylephrine-induced preconstriction. All results are expressed as mean±SEM. The significance of differences between two groups was established by Student's t-test. Significance of the differences between more than two groups was assessed by ANOVA followed by Scheffe test or Kruskal-Wallis test, for normally and non-normally distributed data, respectively. A p value<0.05 was considered statistically significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1. Post-MI remodelling with LV dysfunction after coronary ligation
3.1.1. Animal characteristics and morphometric results
As summarized in Tables 1A and 1B, body weight of male rats used for MI and SHAM experiments was greater than of female rats. There was no significant difference in infarct-size between female and male animals. As expected, in the late phase (42 days) but not in the early phase (7 days) after MI, LV weight/body weight ratio significantly increased in animals with MI as compared to respective SHAM animals. This increase was more pronounced in female than male rats with MI (3.6±0.3 vs. 2.8±0.1; p<0.05).


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  		Table 1A Animal characteristics and morphometric results 7 days post-MI

 


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  		Table 1B Animal characteristics and morphometric results 42 days post-MI

 
3.1.2. Analysis of heart function in vivo after coronary ligation
There were slight increases in the size of MI hearts 7 days after coronary ligation (data not shown) which further increased 42 days after coronary ligation. Moreover, there was a significant increase in both systolic and diastolic LV diameters compared to SHAM rats in females and males 42 days after coronary ligation. Consequently fractional shortening (FS) was equally reduced in female and male MI hearts compared to SHAM (Table 1B).

3.2. Vascular function in the aorta after coronary ligation
3.2.1. Effects of coronary ligation on NO-dependent endothelial responses in the aorta
There was no impairment of acetylcholine (Ach)-induced vasodilation either 7 or 42 days after coronary ligation irrespective of the gender of the animals (Fig. 1A and B). Maximal relaxation induced by Ach (10–5 M) and EC50 values were similar in all experimental groups. Seven days after coronary ligation maximal Ach-induced relaxation in the aorta was: 94.07±2.25%, 89.14±2.32%, 98.27±1.47% and 96.81±2.88% and EC50 values (log M) were: –7.25±0.1, –6.98±0.1, –7.54±0.04 and –7.26±0.1 for female-MI, female-SHAM, male-MI and male-SHAM, respectively. After 42 days the maximal Ach-induced relaxation was: 96.01±3.22%, 92.01±2.23%, 99.03±3.36% and 98.28±3.9% and EC50 values (log M) were: –6.97±0.1, –7.13±0.1,–6.91±0.1 and–7.21±0.1 for female-MI, female-SHAM, male-MI and male-SHAM, respectively. It is however worth noting, that response to acetylcholine at a concentration of 10–7 to 10–6 was augmented in female and male MI vs. SHAM-operated rats 7 days after the coronary ligation, though this difference only reached statistical significance in males. This augmentation of Ach response was not seen 42 days after coronary ligation.


Figure 1
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  		Fig. 1 Lack of impairment of endothelium-dependent response in the aorta in the early (A) and late (B) phases of the post-MI period in female and male rats. 1A. Concentration-response curves for acetylcholine-induced relaxation in aortic rings taken from female ({square}), male ({blacksquare}) MI rats and female ({circ}), male (bullet) SHAM-operated rats 7 days after coronary ligation. Data represent mean±SEM for aortic rings, n=11-23. ## indicates p<0.01 when male-MI was compared with male-SHAM, ¤¤ indicates p<0.01 when female-MI was compared with male-MI, ¤¤¤ indicates p<0.001 when female-MI was compared with male-MI. 1B. Concentration-response curves for acetylcholine-induced relaxation in aortic rings taken from female ({square}), male ({blacksquare}) MI rats and female ({circ}), male (bullet) SHAM-operated rats 42 days after coronary ligation. Data represent mean±SEM for aortic rings, n=19-24.

 
Endothelium-independent relaxation was assessed by cumulative concentration of NaNP (from 10–9 to 10–5). Relaxation curves were nearly identical in MI and SHAM-operated rats, irrespective of gender. The magnitude of endothelium-independent relaxation induced by NaNP (10–6 M) was not different between the female and male MI groups and SHAM-operated rats. Forty two days after coronary ligation, NaNP (10–6 M) induced relaxation was: 101.55±2.71%, 101.44±2.87%, 100.97±4.46% and 105.1±1.8% in female-MI, female-SHAM, male-MI and male-SHAM, respectively. EC50 values for relaxation to NaNP were –8.43±0.02, –8.38±0.04, –8.31±0.03 and –8.24±0.05 for female-MI, female-SHAM, male-MI and male-SHAM, respectively.

Seven days after coronary ligation, basal NO production was blunted in the MI groups irrespective of gender (21.76±2.89%, 54.32±5.11%, 37.61±4.81% and 51.98±5.05% for female-MI, female-SHAM, male-MI and male-SHAM, respectively). Forty two days after coronary ligation the attenuation of basal NO production in MI group vs. SHAM group was present in male (21.42±2.6% vs. 33.85±4.1%, respectively) but not in female animals (24.49±2.78% vs. 27.01±2.56%, respectively).

3.2.2. Up-regulation of NO- and COX-independent endothelial response in the aorta
The most striking gender-related difference induced by coronary ligation was seen in the magnitude of the NOS- and COX-independent component of the Ach response. The magnitude of the NOS- and COX-independent component of Ach-induced vasodilation was substantially up-regulated at the early phase of post-MI period, irrespective of gender (Fig. 2A). In the late phase of the post-MI period, up-regulation of the NOS- and COX-independent component of Ach-induced vasodilation was seen in female-MI rats only (Fig. 2B).


Figure 2
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  		Fig. 2 Up-regulation of the EDHF-component of acetylcholine-induced vasodilation in the early (A) and late (B) phases of the post-MI period in aorta from female and male rats. 2A. Acetylcholine-induced vasodilation in the presence of L-NAME (300 µM) and indomethacin (1 µM) in aortic rings taken from female ({square}), male ({blacksquare}) MI rats and female ({circ}), male (bullet) SHAM-operated rats 7 days after coronary ligation. Data represent mean±SEM for aortic rings, n=9-21. ** indicates p<0.01 when female-MI was compared with female-SHAM, ## indicates p<0.01 when male-MI was compared with male-SHAM. 2B. Acetylcholine-induced vasodilation in the presence of L-NAME (300 µM) and indomethacin (1 µM) in aortic rings taken from female ({square}), male ({blacksquare}) MI rats and female ({circ}), male (bullet) SHAM-operated rats 42 days after coronary ligation. Data represent mean±SEM for aortic rings, n=17-21., ** indicates p<0.01 when female-MI was compared with female-SHAM, *** indicates p<0.001 when female-MI was compared with female-SHAM, ¤¤ indicates p<0.01 when female-MI was compared with male-MI, ¤¤¤ indicates p<0.001 when female-MI was compared with male-MI.

 
Indomethacin alone did not modify the NO-independent component of Ach-induced vasodilation remaining in the presence of L-NAME. Moreover, basal PGI2 production by the aortic rings, as assessed by levels of 6-keto-PGF1P, did not differ significantly between MI and SHAM rats at either 7 or 42 days after coronary ligation (in pg/mg: 66.28±13.2, 101.79±41.9, 85.43±34.96 and 81.5±24.74 for female-MI, female-SHAM, male-MI and male-SHAM 42 days after MI, respectively).

The comparison of the magnitude of the NOS- and COX-independent component of vasodilation induced by 10–6 M Ach in relation to gender in the early (36.24±5.62% and 41.8±5.9% in female and male-MI, respectively) and in the late phase (25.99±4.02% and 3.79±1.09% in female and male-MI, respectively) of the post-MI period is shown in Fig. 3.


Figure 3
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  		Fig. 3 Comparison of the magnitude of the EDHF-component of vasodilation induced by 10–6 M acetylcholine in the early and late phases of the post-MI period in aorta from female and male rats. Data represent mean±SEM for aortic rings, n=9-21. * indicates p<0.05 when female-MI was compared with female-SHAM, *** indicates p<0.001 when female-MI was compared with female-SHAM, ##indicates p<0.01 when male-MI was compared with male-SHAM, ¤¤¤ indicates p<0.001 when female-MI was compared with male-MI.

 
3.2.3. Role of cytP450 metabolites and potassium channels in EDHF-mediated relaxation
If KCl (60 mM) was used to preconstrict the vessels instead of phenylephrine, the NOS- and COX-independent component of vasodilation induced by Ach was abrogated in all experimental groups (<1% in female-MI, female-SHAM, male-MI and male-SHAM animals both 7 and 42 days after coronary ligation).

In addition, the up-regulated NOS- and COX-independent component of Ach-induced vasodilation was completely blocked by pretreatment with tetraethylammonium chloride (TEA, 10 mM), an antagonist of Ca2+-activated K+ channels and miconazole (MICO, 10 µM), a selective inhibitor of epoxyeicosatrienoic acids (EETs) synthesis (Fig. 4).


Figure 4
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  		Fig. 4 The mechanism of the EDHF-component of acetylcholine-induced vasodilation in the post-MI period in aorta from female rats. Aortic rings were treated with L-NAME and indomethacin (white bars), L-NAME, indomethacin and TEA (grey bars) or L-NAME, indomethacin and MICO (black bars). Data represent mean±SEM for aortic rings, n=12. xx indicates p<0.01 when L-NAME treatment was compared with the combination of L-NAME+TEA, bulletbulletbullet indicates p<0.001 when L-NAME treatment was compared with the combination of L-NAME+MICO.

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
Results of the present study indicate that functional changes in the aortic endothelium occurring after myocardial infarction in rats are gender-dependent in the late but not in the early post-MI period. Indeed, in the early phase of post-MI remodelling the impairment of basal NO activity and up-regulation of EDHF activity were seen in the aorta of both female and male rats. However, in the late phase of the post-MI period characterized by typical features of severe LV dysfunction, the aortic endothelium displayed a gender-specific response. In males, we observed an impairment of basal NO without concomitant up-regulation of EDHF, while in females, up-regulation of EDHF without impairment in basal NO production. It could well be, that this preserved NO-dependent function and increased adaptive EDHF synthesis in the post-MI period, contributes to the better outcome in female patients with HF as compared to males reported in clinical trials [15]. Interestingly, gender-specific response of the endothelium could not be linked to differential LV dysfunction that developed after MI. Indeed, despite a more pronounced hypertrophic response of the female rat heart to coronary ligation, subsequent impairment of cardiac function (FS) was identical in rats of both genders.

Previous studies investigating the mechanism of peripheral endothelial dysfunction have shown quite controversial results. In an attempt to clarify the reasons for these discordant results, we comprehensively analyzed alterations in the NO, PGI2 and EDHF pathways in the thoracic aorta of female and male Sprague-Dawley rats in the early (7 days) and late (42 days after coronary ligation) phases of the post-MI period. Importantly, only animals with a large infarct-size (32-42%) were included and the development of post-MI LV dysfunction was validated by in vivo haemodynamic measurements. We showed that there was no impairment of Ach-stimulated relaxation in the aortic rings taken from female and male MI animals, either in the early or in the late phase post-MI. However, the functional activity of basal NO production was impaired in both genders in the early phase, but in the late phase it was present only in male rats.

These results indicate that assessment of endothelium-dependent vasodilation induced by acetylcholine is not sensitive enough to detect functional changes in the aortic endothelium following MI, as compared to the assessment of functional activity of basal NO. This notion is in line with the results of another previous study [21] and could explain the lack of endothelial dysfunction after coronary artery ligation reported by some authors using only the measurement of acetylcholine-induced response [22].

We did not investigate the possible mechanisms that could explain why basal NO production was impaired in a gender-unspecific manner in the early post-MI period and a gender-specific manner in late post-MI period, while Ach-induced vasodilation was preserved. However, it is worth noting that in other experimental settings, basal NO synthesis was more sensitive to superoxide anion-dependent inactivation than NO stimulated by acetylcholine [23], suggesting that decreased basal NO activity could be due to increased generation of superoxide anions [24]. Obviously in the early post-MI period, endothelial oxidant stress would be related to neurohormonal activation not to HF itself, while in the late post-MI period both factors could be involved [25]. On the other hand, in the late post-MI period oestrogen-dependent up-regulation of eNOS expression [26] may contribute to preservation of basal NO production in female rats.

The important finding of this study was that the NO-independent component of Ach-induced vasodilation was up-regulated both in female and male rats in the early phase of the post-MI period, while in the late phase it was only seen in females. The identity of the NO-independent component could be attributed to EDHF but not to COX-derived products. Indeed, in our experiments the cyclooxygenase (COX) inhibitor, indomethacin did not affect the up-regulated NO-independent vasodilation. Moreover, basal PGI2 synthesis did not differ significantly between the experimental groups.

We also showed that elevated extracellular K+ concentration (60 mM), pre-treatment with the Ca2+-activated K+ channel antagonist, tetraethylammonium chloride (10 mM) and the epoxyeicosatrienoic acid (EETs) synthesis inhibitor, miconazol (10 µM), abolished the NO-independent vasodilation. In contrast, catalase, did not affect the EDHF-mediated vasodilation (data not shown). These results indicate the contribution of cytochrome P-450 metabolites of arachidonic acid, most likely an EET [27] but not H2O2, to the NO-independent component of Ach response in the aorta. On the basis of our pharmacological analysis we therefore suggest that the NO- and COX-independent pathway of vasorelaxation up-regulated by myocardial infarction is compatible with the nature of EDHF. Again, similar to changes in basal NO activity, up-regulation of EDHF was gender-specific only in the late phase of post-MI.

Here, we have demonstrated for the first time that EDHF up-regulation is gender-unspecific in the early and gender-specific in the late phase of post-MI period. It is tempting to speculate that long-lasting up-regulation of EDHF in females plays an important role in maintaining endothelial function in CHF. Indeed, the function of EDHF is not limited to the regulation of vasomotor tone. EDHF also displays potent anti-inflammatory action [28] which could be important in CHF.

Obviously, functional alterations in the aortic endothelium induced by myocardial infarction differ from those occurring at the same time in other vessel types, and this could also help explain discordant results obtained previously. Indeed in a previous study, endothelium-dependent relaxation was not uniformly impaired in pulmonary artery, abdominal aorta and mesenteric artery in rats with MI [9]. In our experiments, acetylcholine-induced vasodilation was not impaired in the aorta, but we observed the attenuation of acetylcholine-induced response in mesenteric resistance arteries (data not shown). Apparently, under physiological conditions in the aorta, the functional role of NO is more prominent than EDHF, whereas in the mesenteric resistance arteries, EDHF is more prominent than NO [29]. After myocardial infarction, large vessels such as the aorta still have the capacity for compensatory synthesis of EDHF, while this does not occur in the mesenteric artery displaying impairment of endothelium-dependent vasodilation in the post-MI period.

In summary, large myocardial infarction in rats did not modify the magnitude of acetylcholine-induced vasodilation and PGI2 production in the thoracic aorta, but induced impaired functional activity of basal NO production and adaptive up-regulation of the EDHF component of Ach-induced relaxation. The mechanisms of the latter involved cytochrome p450-dependent metabolites, most likely epoxyeicosatrienoic acids and opening of Ca2+-dependent K+ channels. The alterations in endothelial function in the aorta were gender-specific in the late but not in the early phase of the post-MI period. Neurohormonal activation induced by MI (e.g. AT-II, ET-1) is most likely to be involved in adaptive gender-unspecific alterations in endothelial function early after coronary ligation. On the other hand, the gender-specific adaptation of the aortic endothelium to the haemodynamic sequalae of coronary ligation seem to be apparent in the late phase of post-MI remodelling, at which stage severe LV dysfunction is present. Our findings may be relevant to the apparent protection of females against the progression of heart failure.


   Acknowledgment
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
This work was supported by the Polish Ministry of Science and Information Society Technologies (MNiI) (grants No P05A 003 25, PBZ-KBN-101/T09/2003/6 and scientific Austro-Polish exchange program (2004-12). Gabor Csanyi (Department of Pathophysiology, Albert Szent-Gyorgyi Medical and Pharmaceutical Center, University of Szeged) and Tatiana Stepuro (Department of Physiology, University of Grodno) were recipients of Jagiellonian University fellowships, J. Dietl's and from Queen Jadwiga fund, respectively. Professor Stefan Chlopicki is the recipient of a Professorial grant from the Foundation for Polish Science (SP/04/04).


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
1 The authors equally contributed to this work. Back

2 Current address: Department of Pathophysiology, Albert Szent-Gyorgyi Medical and Pharmaceutical Center, University of Szeged, Semmelweis u. 1., Pf., 427 H6701 Szeged, Hungary. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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