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Departments of Preclinical and Clinical Pharmacology, Human Anatomy and Histology, Section of Histology, University of Florence, and Prosperius Institute, Florence, Italy
Address all correspondence and requests for reprints to: Prof. Tatiana Bani Sacchi, Dipartimento di Anatomia Umana e Istologia, Sezione di Istologia, V.le G.Pieraccini, 6, I-50139 Firenze, Italy. E-mail: histology{at}cesit1.unifi.it
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Abstract |
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Introduction |
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Increasing evidence has been accumulating that a deficient generation of NO is involved in many of the pathogenic events underlying ischemia-reperfusion-induced myocardial injury (13, 14, 15, 16, 17, 18). During ischemia, there is a damage of the coronary endothelium associated with an impaired production of NO and a failure of endothelium-dependent vasodilation, which initiate a cascade of events resulting in myocardial dysfunction and eventually in irreversible myocardial injury (13, 14, 19, 20, 21, 22, 23). During reperfusion, oxygen availability allows for large amounts of toxic oxygen-derived free radicals to be generated (24, 25, 26, 27). In turn, these highly reactive radicals cause peroxidation of myocardial cell membrane lipids and exacerbate tissue injury (28). Ultimately, a loss of calcium homeostasis with excess calcium influx occurs in damaged myocardial cells, leading to irreversible impairment of mechanical function and eventually cell necrosis (29, 30). Resident mast cells also contribute to myocardial dysfunction. These cells undergo activation during postischemic reoxygenation, thus releasing a series of powerful chemical mediators such as histamine (30, 31), which affects coronary vascular resistance and permeability, increases interstitial edema (32, 33) and promotes ventricular arrhythmias (34).
It can be argued that an impairment of some beneficial NO-mediated functions, such as coronary vasodilation (35), scavenging of oxygen-derived free radicals (36), and inhibition of mast cell activation and histamine release (37), takes place in the ischemic and reperfused heart. Based on the above reported properties of RLX on the cardiocirculatory system and especially on its capacity to stimulate the endogenous production of NO, we hypothesized that RLX could have a protective effect against myocardial injury due to ischemia and reperfusion. The current study was designed to test this hypothesis using the isolated and perfused guinea pig heart.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatments
The guinea pigs were anesthesized by ip injection of sodium
pentobarbital (Pentothal, Abbott, Latina, Italy, 30 mg/kg BW). A
cannula was introduced into the trachea, and the animals were
ventilated with air using a Palmer pump (U. Basile, Comerio, Italy). A
midline thoracotomy was performed, and the heart was exposed after
opening the pericardium. Two loose 00 braided silk sutures were placed
around the left anterior descending coronary artery about 2 mm below
its origin. To facilitate successive removal of the sutures, small
silicon rings were inserted in the silk threads below the knots. The
hearts were quickly removed, a cannula was introduced into the aorta
and then attached to a Langendorff apparatus. The hearts were perfused
at a constant pressure of 40 cm of water with a modified Tyrode
solution. In this way, the aortic semilunar valve remains closed and
the perfusion fluid enters directly the coronary arteries. The
perfusion solution was composed of (mM): Na+
149.3, K+ 2.7, Ca2+ 1.8, Mg2+ 1.05,
Cl- 145.4, HCO3- 11.9,
H2PO4- 0.3, and glucose 5.6. It
was maintained at 37 C and gassed with a mixture of 95% O2
and 5% CO2, giving a final pH of 7.48 (38). A strain-gauge
transducer was connected to a clip hooked to the heart apex and coupled
with a thermic writing oscillograph to record heart contractility.
Stabilization of cardiac activity usually occurred within 30 min.
The protocol included four groups of ten hearts each, treated as follows: Group 1: Sham-operated hearts in which no tightening of the coronary sutures was performed. Group 2: Hearts in which ischemia was induced by tightening the sutures and maintained for 20 min. After this period, the sutures were released to allow reperfusion of the ischemic tissue. Reperfusion was maintained for 20 min. Group 3: Hearts subjected to the same procedure as for group 2 and treated with RLX, added to the perfusion fluid (final concentration: 30 ng/ml) at the coronary occlusion. This RLX concentration was chosen because it corresponds to the intermediate one among those which have been previously shown to be effective in increasing coronary flow and NO production in isolated guinea pig hearts (9). Highly purified porcine RLX (2500–3000 U/mg), prepared according to Sherwood and O’Byrne (39), was used. Group 4: Ischemic and reperfused hearts treated with RLX as for group 3 in which the NO synthase inhibitor NG-momomethyl-L-arginine (L-NMMA, Ultrafine Chemicals, Manchester, UK) was added to the perfusion fluid (final concentration: 100 µM). L-NMMA was given 1 h before induction of ischemia and was maintained during both ischemia and reperfusion together with RLX. Pretreatment with L-NMMA is needed to allow tissue depletion of the authentic substrate L-arginine and hence to obtain effective NO synthase inhibition (9). This group of hearts was included in the study to verify an involvement of the L-arginine-NO pathway in the response of the ischemic-reperfused hearts to RLX.
At the end of reperfusion, the hearts were detached from the Langendorff apparatus and weighed. Fragments of ischemic-reperfused myocardium were collected for biochemical and morphological studies. They were taken 3–5 mm below the left coronary artery ligatures to exclude tissue areas damaged by the surgical procedure. Parallel experiments were carried out in guinea pig hearts to evaluate the extension of the ischemic area after ligation of the left descending coronary artery. The hearts were perfused with 1% nitro-blue tetrazolium dye (40) dissolved in the perfusion fluid soon after a 20-min ischemia. By direct examination of the left ventricle, the ischemic area appeared unstained. This area was measured by computer assisted morphometry and corresponded to 94 ± 8 mm2 downstream the ligature. For the current study, the tissue samples were taken from corresponding areas of the left ventricles.
Determination of coronary flow
Coronary flow was evaluated by collecting the perfusates of the
ischemic and reperfused hearts in graduated tubes over 10-min intervals
for the entire duration of the experiments. The perfusates collected
during the last 10 min of the stabilization period were assessed as
basal coronary flow. In the experiments with L-NMMA, the basal coronary
flow was measured during the last 10 min before addition of the drug to
the perfusion fluid.
Evaluation of NO production
NO was determined by measuring the amount of nitrites
(NO2-), the stable end products of NO
metabolism, in the perfusates of the ischemic and reperfused hearts.
Part of each perfusate was lyophilized and resuspended in 4 ml of water
just before use. NO2- were measured
spectrophotometrically using the Griess reagent (aqueous solution of
1% sulfanylamide and 0.1% naphthylethylendiamine dihydrochloride in
2.5% H3PO4, Ultrafine Chemicals Ltd.,
Manchester, UK), which forms a stable chromophore with
NO2-, absorbing at 546 nm wave length (41).
NO2- concentration was determined by
comparison with standard concentrations of sodium nitrite, dissolved in
the perfusion solution. The NO2- amounts in
the perfusates collected during the last 10 min of the stabilization
period were assessed as basal NO2- levels. In
the experiments with L-NMMA, the basal NO2-
levels were measured in the perfusates collected during the last 10 min
before addition of the drug to the perfusion fluid.
Determination of histamine
Histamine was measured fluorimetrically in the perfusates of the
ischemic and reperfused hearts from groups 2 and 3 using the method of
Shöre et al. (42) as modified by Lorenz et
al. (43). Part of each perfusate was lyophilized and resuspended
in 4 ml of 0.4 M HClO4. Two ml of each sample
were shaken with 1.5 g NaCl in 10 ml of n-butanol
(Merck, Darmstadt, Germany) in a glass tube and then centrifuged at
1800 x g for 5 min. The butanol phase was separated
and mixed with 5 ml of 0.1 M NaOH, saturated with NaCl, in
a glass tube. Histamine was redistributed to the aqueous phase by
shaking 8 ml of butanol phase together with 3 ml of 0.1 M
HCl and 15 ml of n-heptane (Merck) for at least 6 min. After
centrifugation as above, the organic phase was carefully removed and 2
ml of the aqueous phase were transferred into a glass tube and mixed
with 0.4 ml of 1 M NaOH and 0.1 ml of
o-phtaldialdehyde solution (Merck, 1% wt/vol in methanol).
After 4 min, 0.2 ml of 3 M HCl were added and the
fluorescence was measured in a spectrofluorimeter (RF 5000, Shimadzu,
Kyoto, Japan). The excitation wave length was set at 360 nm and the
emission wave length was read at 450 nm. The authenticity of histamine
in the samples was demonstrated by recording the excitation and
emission fluorescence spectra. Histamine concentration was determined
by comparison with standard concentrations of histamine. The histamine
amounts in the perfusates collected during the last 10 min of the
stabilization period were assessed as basal histamine levels.
Determination of malonyldialdehyde
Malonyldialdehyde (MDA) is the end-product of lipid peroxidation
of cell membranes due to oxygen-derived free radicals, and is
considered a reliable marker of myocardial cell damage (44). It was
determined by measuring the chromogen obtained from the reaction of MDA
with 2-thiobarbituric acid, according to Aruoma et al. (45).
About 100 mg of left ventricular tissue was homogenized with 1 ml of 50
mM Tris-HCl buffer containing 180 mM KCl and 10
mM EDTA, final pH 7.4, using a tissue homogenizer (Ing.
Terzano, Milan, Italy). To 0.5 ml of sample were added 0.5 ml of
2-thiobarbituric acid (Merck, 1% wt/vol) in 0.05 M NaOH
and 0.5 ml of HCl (25% wt/vol in water). The mixture was placed in
glass tubes, sealed with screw caps, and heated in boiling water for 10
min. After cooling, the chromogen was extracted in 3 ml of
n-butanol (Merck) and the organic phase was separated by
centrifugation at 2000 x g for 10 min. The absorbance
of the organic phase was read spectrophotometrically at 532 nm wave
length. The values are expressed as nmol of thiobarbituric
acid-reactive substance (MDA equivalent)/mg of protein, using a
standard curve of 1,1,3,3-tetramethoxypropane. Protein concentration
was determined according to Bradford (46).
Evaluation of calcium content
Excessive calcium influx is a critical event accompanying
irreversible injury in myocardial ischemia-reperfusion (29, 30). The
calcium content of the myocardial tissue was measured by atomic
absorption spectrometry (47). Briefly, the tissue fragments were rinsed
thoroughly in calcium-free buffered solution. Then, 30 mg of tissue
were dried in an oven at 80 C and digested overnight with 65%
HNO3 (100 µl/10 mg of dry tissue). Upon addition of 32%
HCl (150 µl/10 mg of dry tissue), the samples were dried at 45 C
under nitrogen. At the moment of the assay the samples were suspended
in 50 µl of 32% HCl and added with lanthanum chloride
(LaCl3.7H2O) to a final concentration of 1%
lanthanum (wt/vol). The amounts of calcium in the samples were read in
an atomic absorption spectrophotometer (Perkin-Elmer 303,
Überlingen, Germany) at 422 nm wave length. The values were
determined by comparison with a standard curve obtained with increasing
concentrations of CaCl2 and expressed as ng of calcium/mg
of tissue (dry weight).
Computer-assisted densitometry of cardiac mast cells
Tissue samples from the hearts of groups 1 to 3 were fixed
by immersion in isotonic formaldehyde-acetic acid (IFAA), dehydrated in
graded ethanol, and embedded in paraffin wax. Sections 5-µm thick
were cut and stained with Astra blue which selectively binds heparin
contained in mast cell granules (48). Light transmittance across mast
cells, which is inversely related to their content in secretory
granules, was evaluated by a computer-assisted method, as described
previously (10). The mast cells were viewed by a CCTV television camera
(Sony, Tokyo, Japan) applied to a Reichert-Jung Microstar IV light
microscope (Cambridge Instruments Inc., Buffalo, NY) with a x100 oil
immersion objective, and interfaced with an Apple Macintosh LC III
personal computer through a Videospigot card (Supermac, Sunnyvale, CA).
The card allows for the light transmitted across the microscopical
slide to be determined within a range of 256 gray levels, which are
comprised between 0 (black level) and 255 (white level). The card also
allows for a digitized image of mast cells to be reproduced on the
basis of the values estimated. Measurements of transmittance were
carried out using an NIH 1.49 image analysis program. The transmittance
of 100 different mast cells, 10 from each animal of the different
groups, was analyzed and the mean transmittance value (±
SE) was then calculated.
Morphology
Tissue samples from the hearts of groups 1 to 3 were fixed in
cold 4% glutaraldehyde in 0.1 M sodium cacodylate buffer,
pH 7.4, for 3 h at room temperature, and postfixed in 1% osmium
tetroxide in 0.1 M phosphate buffer, pH 7.4, for 1 h
at 4 C. They were then dehydrated in graded acetone, passed through
propylene-oxide and embedded in Epon 812 (Fluka, Buchs, Switzerland).
Electron microscopic examination was carried out on ultrathin sections
stained with uranyl acetate and alkaline bismuth subnitrate (49) and
viewed under an Elmiskop 102 electron microscope (Siemens, Berlin,
Germany) at 80 kV.
Evaluation of heart contractility
To test heart contractility, amplitude of cardiac contractions
was measured on the oscillographic recordings at the end of the
stabilization period (basal amplitude), at the end of ischemia, and at
the end of reperfusion. In the experiments with L-NMMA, the basal
amplitude of cardiac contractions was measured just before addition of
the drug to the perfusion fluid.
Statistical analysis
The values of the parameters determined in the perfusates and of
the amplitude of heart contractions are reported as percent changes
over the basal values, to normalize individual differences between
basal values within each group. All data are expressed as means ±
SEM. The distribution of the measured values in the
different experimental groups was assessed to be Gaussian. Statistical
analysis was performed by either two-way ANOVA test or one-way ANOVA
test followed by Student-Newman-Keuls multiple comparison test.
Calculations were carried out using a GraphPad Prism 2.0 statistical
program (GraphPad Software, San Diego, CA). P < 0.05
was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) in the
ischemic-reperfused hearts was under the basal level for the entire
experimental period. In contrast, in the ischemic-reperfused hearts
treated with RLX, the coronary flow remained over the basal level
during the ischemic period and was even more elevated during
reperfusion. Treatment with L-NMMA significantly reduced the effect of
RLX on the coronary flow.
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) in the perfusates of the
ischemic-reperfused hearts remained nearly unchanged as compared with
the basal level, whereas it was significantly elevated in the
perfusates of the ischemic-reperfused hearts treated with RLX.
Treatment with L-NMMA significantly reduced the effect of RLX on
NO2- levels.
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) in the
perfusates of the ischemic-reperfused hearts was nearly unchanged
during ischemia and increased significantly during reperfusion as
compared with the basal level. Conversely, in the perfusates of the
ischemic-reperfused hearts treated with RLX the amount of histamine
remained nearly unchanged for the entire experimental period.
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) in myocardial
tissue of the ischemic-reperfused hearts increased significantly when
compared with the sham-operated hearts. The increase in MDA production
was significantly lower in the ischemic-reperfused hearts treated with
RLX. Treatment with L-NMMA together with RLX resulted in a significant
increase in MDA production as compared with RLX alone.
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) of myocardial
tissue of the ischemic-reperfused hearts increased significantly when
compared with the sham-operated hearts. In the ischemic-reperfused
hearts treated with RLX, the calcium content was significantly reduced,
thus reaching levels similar to the sham-operated hearts. Treatment
with L-NMMA together with RLX resulted in a significant increase in
calcium content as compared with RLX alone.
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) showed
that the light transmittance across mast cells, which is inversely
related to their content in secretory granules, was markedly increased
in the myocardium of the ischemic-reperfused hearts compared with the
sham-operated ones. Conversely, in the ischemic-reperfused hearts
treated with RLX, the light transmittance was markedly reduced, thus
dropping to levels even lower than those of the sham-operated
hearts.
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). These abnormalities were
never found in the ischemic-reperfused hearts treated with RLX, in
which the ultrastructural features of myocardial cells were similar to
those of the sham-operated hearts (Fig. 7B).
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) showed that, in the ischemic and
reperfused hearts, the mean amplitude of contractions was significantly
reduced during both ischemia and reperfusion. In the ischemic and
reperfused hearts treated with RLX the mean amplitude of contractions
remained virtually unchanged as compared with the basal mean value. On
the other hand, in the ischemic and reperfused hearts treated with RLX
in the presence of L-NMMA the mean amplitude of contractions was
significantly reduced in both ischemia and reperfusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RLX, added to the perfusion fluid, causes a marked reduction of biochemical and morphological markers of myocardial injury as well as of mechanical heart dysfunction. In fact, RLX strongly decreases the production of MDA, the end-product of lipid peroxidation of cell membranes due to oxygen-derived free radicals, which is considered a reliable marker of myocardial cell damage (44). This indicates that RLX can reduce peroxidation of cell membrane lipids due to harmful oxygen-derived free radicals that are known to be generated during ischemia and reperfusion (24, 25, 26, 27, 28). Moreover, RLX prevents the overload of calcium, which is a critical event accompanying irreversible injury of the myocardial tissue (29, 30). Of note, a similar effect of RLX in inhibiting intracellular calcium rise has also been observed in other target cells, such as vascular smooth muscle cells (3), mast cells (10), and platelets (11). The protective effect of RLX against myocardial injury induced by ischemia and reperfusion is further strengthened by the lack of ultrastructural signs of myocyte alterations - such as hypercontraction of myofibrils, mitochondrial swelling and accumulation of dense granules in the mitochondrial matrix - in the ischemic-reperfused hearts treated with the peptide. The above findings fit well with the functional data showing that RLX prevents the reduction of contractile activity found in the ischemic-reperfused hearts not treated with the hormone.
It is conceivable that RLX produces its cardioprotective effect in the ischemic-reperfused myocardium through a vasodilation of the coronary vessels. Vasodilation and increase in the coronary flow are typical effects of this hormone, which were previously recognized in several target organs (4, 5, 6, 7, 8) including isolated and perfused rat and guinea pig hearts (9). In the current study, the values of the coronary flow in the guinea pig hearts perfused with RLX were above the basal values during the ischemic period and became even more elevated at reperfusion. It is conceivable that RLX had induced a dilation of collateral coronary vessels, which are well developed in the guinea pig heart (50), thus affording an increased supply of perfusion fluid to the ischemic myocardium. An improved myocardial tissue perfusion could also allow the removal of dangerous mediators and metabolites, generated during the ischemic period and in the acute phase of reperfusion, thus facilitating myocardial cell salvage in the area at risk.
The results of the current study show that RLX inhibits ischemia-reperfusion-induced mast cell degranulation and histamine release, thus providing a further contribution to myocardial salvage. In fact, cardiac mast cells are known to undergo degranulation during postischemic reoxygenation (30, 31). These cells are thought to mediate a significant component of the myocardial injury because they release powerful mediators, including peroxidase, histamine, serotonin and leukotrienes. These substances affect coronary vascular resistance and permeability and may enhance myocardial damage by increasing tissue edema (32, 51, 52), which in turn contributes to hinder restoration of a normal coronary blood flow. Our findings on cardiac mast cells fit well with the results of our previous studies on serosal mast cells, showing that RLX inhibits granule exocytosis and histamine release (10).
The cardioprotective effect of RLX against ischemia-reperfusion-induced injury probably relies on the ability of the peptide to stimulate endogenous NO production by cells in the heart, as inferred by the marked increase in the amount of NO2-, the end-product of NO metabolism, in the perfusates of the RLX-treated hearts, as well as by the abrogation of the protective effects of RLX by the NO synthase inhibitor L-NMMA. Enhanced generation of NO by RLX may account for the coronary vasodilation and the inhibition of mast cell activation and histamine release, in keeping with previous findings in isolated hearts (9) and mast cells (10), as well as for the reduced membrane lipoperoxydation of cardiac cells, NO being a quenching agent for oxygen-derived free radicals (36).
In view of a possible therapeutic use of RLX for the management of myocardial ischemic diseases, one might ask whether the conclusion of this study could be applied to humans. We favor an affirmative answer. In fact, the human heart somewhat resembles the guinea pig heart because it is also provided with coronary collaterals, even if at a lesser extent. Noteworthy, in patients with a clinical history of coronary atherosclerosis and/or angina pectoris, there is a growth of collateral vessels due to prolonged ischemia (50), which render the collateral circulation even more similar to that of the guinea pig heart. RLX might be particularly effective in this kind of patients, being able to favor myocardial perfusion through the coronary collaterals and hence to reduce the area at infarct risk.
It is known that the incidence of coronary heart disease is very low in cycling and pregnant women (53), whereas it becomes markedly elevated after menopause (54). Until now, estrogens have been held responsible for protection against ischemia-derived heart disease (55, 56, 57). The current findings indicate that RLX is able to lessen the consequences of ischemic heart disease by increasing blood supply to the area at risk. This raises the possibility that RLX, by favoring patency of coronary blood vessels as shown in previous studies in rats and guinea pigs (9) and as can be inferred from the current findings, could also prevent the occurrence of ischemic heart disease in women during nonconceptive cycles and pregnancy, when this hormone is released in blood by the corpus luteum (58, 59, 60). If this possibility will be validated, RLX could be included among the endogenous cardioprotective factors.
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Acknowledgments |
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Footnotes |
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Received March 10, 1997.
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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) or presence (
) of RLX, or in the presence of
L-NMMA plus RLX (
). The values are reported as mean percentage (±
SEM) of the basal coronary flow. Significance of
differences between curves (two-way ANOVA, each point: N = 10):
P < 0.0001.
