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Effects of α-human atrial natriuretic peptide in guinea-pig isolated heart
Inte~tio~I Journal of Cardiology, 40 (1993) 21 I-220 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0167-5273/93/SO6.00
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Effects of a-human atria1 natriuretic peptide in guinea-pig isolated heart Paolo Di Nardo, Marco Pafi, Manuela Bartoli, Marilena Minieri, Fausta Bellegrandi, Gianfranco Raimondi, Giuseppe Peruzzi and Giuseppe Tallarida Laboratory of Cellular and Molecular Cardiology. Department of Internal Medicine. University qf Rome “Tor Yergata” Via 0. Raimondo. 00173 Rome. Italy
(Received 27 October 1992: revision accepted 24 March 1993)
The aim of the present investigation has been to ascertain whether or not atria1 natriuretic peptides (ANP) can exert a direct effect on myocardial contractility. Alpha-human ANP (a-hANP) concentrations ranging from 1 pM to 50 nM have been used to perfuse guinea-pig isolated hearts in a non-recirculating Langendorff apparatus. A dual concentration-related effect has been induced by a-hANP on myocardial function. A maximal increase of +LV dP/dt,,, (+56%; P < 0.001) has been observed when guinea-pig hearts were perfused with 100 pM ar-hANP, whereas a 25% decrease (P < 0.01) occurred with 50 nM cw-hANP. Similar effects have also been induced by cr-hANP on the coronary flow rate (CFR). A significant CFR increase (maximal at 10 pM cu-hANP) was induced ,by picomolar concentrations of a-hANP, whereas a progressive decrease, which was maximal (-28%; P < 0.01) at 50 nM o-hANP, was observed with nanomolar concentrations of the peptide. No effects have been observed on heart rate. These results suggest that ANP has direct effects on both vascular and myocardial muscle cells. Coronary vasoconstriction induced by nanomolar concentrations of ANP can contribute to the cardiodepression, whereas ANP in picomolar concentrations can induce a coronary vasodilation which is not coupled with the enhanced myocardial contractility. The latter is the likely expression of a direct effect of the peptide on myocardial function. Key words: Atria1 natriuretic peptide ; Isolated heart ; Inotropic state ; Coronary flow
1. In~~uetion Intravenous administration of alpha-human atria1 natriuretic peptide (ol-hANP) and other atria1 natriuretic factors lowers the arterial pressure in animals [l-5] and man [6,7] through a still undefined mechanism, The depressant activity of atria1 peptides (ANP) has been mainly related to a direct vasodilatory effect. In fact it has been Corresponding to: Paolo Di Nardo, Laboratory of Cellular and Molecular Cardiology, Department of Internal Medicine, University of Rome “Tor Vergata”. Via 0. Raimondo, 00173 Rome, Italy.
observed that ANP is able to counteract vessel precontraction induced in vitro by norepinephrine or angiotensin II [8,9]. Further investigations in rats [lo], rabbits [ll], dogs [12] and man [13-151 have confirmed that a-hANP can exert a direct vasodilating effect. However, systemic vasodilation has been observed only when ANP circulating levels were remarkably increased with respect to physiological concentrations [ 16- 181. The hypotensive effect can also be induced by ANP through a decrease in stroke volume and cardiac output. In fact, ANP can induce a fluid shift from the intravascular to the extravascular com-
212
partment, and a reduction in venous return leading to a decrease in cardiac filling pressure [ 19-261. It has been also hypothesized that the stimulation of receptors with sympathocardiopulmonary inhibitory effects might consistently contribute to the cardio-inhibitory and vasodilatory action of ANP [1,27-291. Recent experimental data from our laboratory do not support this view, providing evidence that a significant neurovegetative component is not involved in the mechanism of a-hANP depressor effect on the cardiovascular function [ 111.In keeping with these results, some studies indicate that ANP causes a reduction in the cardiac output through a direct negative inotropic effect [30,31]. However, no exhaustive results have been reported in previous studies, in which only a possible direct depressor effect of ANP on the myocardial function has been emphasized [2,4,11,3 1,321. The role of ANP-induced coronary vasoactivity in eliciting myocardial effects has also been investigated. In isolated guinea-pig hearts, Wangler et al. [3 1] observed a depression of the myocardial performance which was apparently caused by coronary vasoconstriction induced by atriopeptin II (23 amino acids). These findings were obtained with very high doses of ANP and were not confirmed by the same investigators [33] in rat isolated hearts exposed to a-hANP (28 amino acids). The present investigation in a guinea-pig isolated heart model has been undertaken to: (a) verify whether a-hANP exerts a direct effect on myocardial function and coronary flow; (b) ascertain whether concentrations of o-hANP in the physiological and pharmacological range might induce different responses in myocardial function and coronary flow, and (c) determine the relationship between the effects of ANP on myocardial function and coronary flow. Materials and methods Perfusion procedure Guinea-pigs (250-300 g bw) were stunned and hearts were rapidly excised and cooled in a bath of chilled perfusion medium (4°C). After removing the auricles, the heart was perfused by retrograde aorta perfusion in a non-recirculating Langendorff
apparatus at 37°C with a modified KrebsHenseleit buffer (KH) containing (mM) NaCl 127, KC1 4.7, KH2P04 1.1, MgS04 1.2, NaHC03 25, CaC12 2.5, glucose 5.5 and Na-pyruvate 2; pH 7.4. The KH buffer was gassed with 95% 02-5% CO*. The gas partial pressure and pH were periodically measured by a gas-analyzer (Radiometer ABL 330, Copenhagen, Denmark) in samples withdrawn proximally to the heart. The aortic PO, was approx. 80 kPa (600 mmHg). The hydrostatic perfusion pressure was measured in aortic cannula 1 cm above the heart with a Gould P23XL pressure transducer (Gould Inc., Valley View, Ohio) and was maintained at 10 kPa (100 cm H20). Coronary flow rate (CFR) was estimated by collecting coronary effluent in a funnel with a calibrated stem lumen and counting drops by a photoelectric device [34]. A fluid-filled latex balloon was passed through the left atrio-ventricular orifice and placed in the left ventricle. The balloon was connected to a Gould P23XL pressure transducer (Gould Inc., Valley View, Ohio) and inflated to maintain an end-diastolic pressure of approximately 0.67 kPa (5 mmHg). A second thin polyethylene tubing was inserted into the left ventricle before the balloon inflation to drain thebesian flow. Hearts were not electrically stimulated. An epicardial unipolar electrogram (EG) was obtained by a spring electrode positioned to the apex. Heart rate (HR) and LV dP/dt,,, were automatically computed. All electrical and mechanical signals were recorded on a Gould (Bioamplifiers: multichannel polygraph 13.4615.50, 13.4615.71 and Biotach 13.4615.65A). Isolated hearts were perfused with KH until CFR, electrical and mechanical activities were stabilized (approx 15 min), then the myogenic autoregulation of smooth muscles of coronary vessels (perfusion pressure: 9.33 kPa (70 mmHg) and 4 kPa (30 mmHg)) as well as the effect of increasing preloads (0.67 kPa (5 mmHg), 1.33 kPa (10 mmHg) and 2 kPa (15 mmHg)) on cardiac working musculature have been evaluated before each experimental test was started [34]. All studies have been in accordance with the Italian regulations for the care and use of laboratory animals.
213
ANF
a- human
HPLC
of the perfusion fluid used for each heart was 400-450 ml. The purity of o-hANP has been preliminarily tested by HPLC (Fig. 1). Before and during o-hANP perfusion CFR. LVP (left ventricular pressure), LV dP/dt,,,, EG and HR were countinuously recorded. Statistical analysis During ANP perfusion, all parameters although continuously recorded, have been sampled every 60 s and CFR every 8 s. The mean of ten consecutive signals for each time point has been calculated. The baseline value of each parameter was represented by the means of values 3 min and 1 min before ANP perfusion, if their difference was lower than 5%. Data reported in the present paper are expressed as percent difference from control values ( 100%). Correlation coefficients among parameters have been evaluated by the non-parametric Spearman’s test. Significant differences from the baseline have been tested by Student’s t-test.
A chart recording from a typical experiment is shown in Figure 2. Basal values for HR, LVP, I
1
1
I
0
10
20
30
min.
guinea-pig
Fig. 1. HPLC analysis of synthetic a-human ANP used in the present investigation. The graph shows a single peak at 19 min retention time. Column: Vydac C18, 5 pm, 300 A pore, 250 x 64.6 mm. Mobile phase: 0.25 N HsPO, +TEA pH 2.25 (Solvent A); TEAP pH 2.25/CH,CN 4060 (Solvent B). Linear gradient from 30 to 50% B in 40 min. Flow: I.5 ml/mm. HPLC apparatus: Varian Vista 5500. Detector (Varian Polychrom 9060): 215 nm.
Experimental protocol A total of 28 hearts were tested. After heart stabilization and responsiveness testing, each heart was continuously perfused once for 16 min with one of the following concentrations of a single lot of 28-amino acid synthetic o-human ANP (CYhANP) (Novabiochem, Laufelfingen, Switzerland) in KH medium: 1, 10 and 100 pM, and 1, 10 and 50 nM. Depending on heart size, the final volume
isolated
heart
$=-01 mn
0
2
4
6
a
10 15
Fig. 2. Original chart recording from a typical experiment of guinea-pig isolated heart perfused with 10 pM o-hANP. LVP. left ventricular pressure (arbitrary units); dP/dt (arbitrary units); CFR. coronary flow rate (ml/mitt); HR, heart rate (beats/min x 100).
214
HEART RATE (guinea pig isolated heart)
120
100 80 0
5
15
10
ml.
Fig. 3. Percent variation of heart rate (HR) during guinea-pig isolated heart perfusion with different cu-hANP concentrations (baseline: 100%).
+LV dP/dt,,,, -LV dP/dt,,, and CFR were 199 f 15.9 beatslmin, 103.2 f 8.9 mmHg, 1391 f 158.4 mmHg/s, 1028 f 123.8 mmHg/s, and 13.7 f 3.1 ml/min, respectively.
Heart rate (HR) was not affected by perfusion with a-hANP at any concentration tested (Fig. 3). Only during 100 pM a-hANP perfusion, LVP did increase (+39%; P < 0.05), but it appeared unchanged when either lower or higher peptide concentrations were used (Fig. 4). A composite time-course representing the effects of different concentrations of a-hANP on both +LV dP/dt,,, and -LV dP/dt,,, is shown in Fig. 5. Alpha-hANP displays a concentration-related biphasic effect on +LV dP/dt,,,. In fact, myocardial perfusion with 1, 10, and 100 pM cr-hANP was associated with a transient +LV dP/dt,,, increase of approx 25% (P < O.Ol), 21% (P < 0.05) and 56% (P < O.OOl), respectively. Within a few seconds after starting perfusion, +LV dP/dt,, gradually increased for 4-6 min before returning to baseline levels. At higher o-hANP concentrations (1 and 10 nM) there was a slight increase in +LV dP/dt,,, followed by a sustained depression of approx 13%, whereas 50 nM o-hANP resulted in a continuous decrease reaching -25% after 15 min perfusion (P < 0.01). Myocardial reperfusion
+LV dP/dtm, (guinea LEFT
and -LV
pig isolated
dP/dtm, heart)
VENTRICULAR PRESSURE (guinea pig isolated heart)
150
0
5
10
15 “I”
Fig. 4. Percent variation of left ventricular pressure (LVP) during guinea-pig isolated heart perfusion with different cr-hANP concentrations (baseline: 100%). *P < 0.05 vs. control (time 0).
Fig. 5. Percent variation of left ventricular dP/dt (+LV dP/dt,,,; -LV dP/dt,,,) during guinea-pig isolated heart perfusion with different cr-hANP concentrations (baseline: loo%). ‘P < 0.05. **P < 0.01. ***P < 0.001 vs. control (time 0).
215
with KH buffer only partially restored the baseline values. The time-courses of -LV dP/dt,,, and +LV dP/dt,,, were similar, and the modifications induced by a-hANP were not significantly different from their basal values. A 30% increase (P < 0.05) was observed only when guinea-pig hearts were perfused with 100 pM cr-hANP, whereas a sustained decrease (more than 20% after 1.5 min; P < 0.05) was observed with 50 nM a-hANP. Non-significant enhancements of the contractility index (+LV dP/dt,,,/LVP) were observed at all the lower cr-hANP concentrations (1 pM, 11%; 10 pM, 18%; 100 pM. 14%), whereas 25% decrement (P < 0.01) was detected only with 50 nM o-hANP (Fig. 6). Concentration-dependent effects were also induced by o-hANP on coronary flow rate (CFR). In fact, CFR appeared unmodifled with respect to basal values during heart perfusion with 1 pM (YhANP, while 10 pM a-hANP determined a 45% increase (P < 0.01) after 2 min perfusion. This was followed by a smooth, continuous decline until the 15th min (+25% with respect to basal values) of ANP perfusion (P < 0.05). A modest transient
CONTRACTILITY (guinea pig isolated
increase of CFR (approx. + 15%) was also seen at 100 pM a-hANP, whereas a progressive decrease was observed at higher ANP concentrations. After 15 min perfusion with 1, 10 and 50 nM cr-hANP, CFR was reduced by 15% (NS), 25% (NS) and 28% (P < O.Ol), respectively (Fig. 7). Statistical analysis (Table 1) showed high correlation coefficients between LVP and +LV dP/dt,,, at 100 pM, 1, 10 and 50 nM cr-hANP,
CORONARY (guinea-pig
FLOW isolated
RATE heart 1
INDEX heart)
0
Fig 6. Percent variation of contractility index (Cl = +LV dP/dt,,,/LVP) during guinea-pig isolated heart perfusion with different o-hANP concentrations (baseline: 100%). *P < 0.01 vs. control (time 0).
5
to
l5
min.
Fig. 7. Percent variation of coronary flow rate (CFR) during guinea pig isolated heart perfusion with different o-hANP concentrations (baseline: 100%). Standard deviations are not reported, however they did not exceed 10% of the related CFR mean value. *P < 0.05and **P < 0.01 vs. control (time 0).
216 TABLE 1 Correlation coefilcients evaluated by non-parametric [ANPI
+dP/dt vs. LVP
1 PM 10 pM 100 pM 1 nM 10 nM 50 nM
0.80 0.84 0.99 0.90 0.95 0.91
Spearman’s test.
CFR vs. +dP/dt,,,
CFR vs. -dP/dt,,,
CFR vs. LVP
CFR vs Cl
0.66 0.91
0.69
0.76 0.78
0.79 0.82
0.90 0.81 0.83
0.91 0.75 0.90
[ANP], concentration of atria1 natriuretic peptide; LVP, left ventricular pressure; CFR, coronary flow rate; CI, contractility index.
and to a lesser extent, at 1 and 10 pM cr-hANP. CFR, however, appeared to be highly correlated to LVP, +LV dP/dL,, -LV dP/dt,,, and contractility index only at 100 pM, 10 and 50 nM ar-hANP. Discussion Myocardial contractile function
Atria1 peptide binding sites have been found in the ventricular myocardium [35-371 suggesting that ANP could exert direct effects on the myocardial function. Several in vitro models have been used to evaluate the ANP effects on myocardial function. A marked impairment of myocardial function attributed to coronary vasoconstriction has been observed in guinea-pig isolated hearts after 1- 100 nmoles atriopeptin II (23 amino acids) bolus injections [31]. These results have not been confirmed in the same experimental model using both injected boli (approx. 20 nmoles/each) or infusion of 0.03-300 nM ANP [33,38]. Moreover, ANP did not modify the myocardial performance in cat [39] and guinea-pig papillary muscles [40] or atria [41]. Nevertheless, investigations on isolated cardiomyocytes have shown that ANP concentrations in the range l-50 nM could induce a direct negative inotropic effect possibly mediated by a reduction in intracellular calcium concentration [42-441. Studies performed in vivo on animal models and
man generated conflicting results. ANP administration provoked no or mild depressor effects on myocardial function in rat [45,46], dog [47,48] and sheep [49]. In dog, a cardiodepressor effect has been reported after intravenous 3 rglkg bolus + 0.3 pg/min per kg infusion of 24-amino acid auriculin A [30]. No or modest effects have been also observed on the myocardium of human subjects after ANP infusion [50,51]. A positive effect on myocardial performance has been observed in humans after intravenous or intraventricular administration of 0.5-2.5 pg/kg boli of ANP [52,53]. The increase in ventricular performance has not been related to a direct ANP effect, but to an increased cardiac sympathetic activity [5 1,531. Only a few studies reported ANP plasma concentrations after administration of natriuretic peptides. However, in these studies the positive inotropic effects seemed to be associated to ANP plasma concentrations not exceeding 300 pM [52,53]. The present study provides evidence that (YhANP can influence the myocardial function of guinea-pig isolated heart by inducing two dosedependent effects. In fact, in our experiments, ANP concentrations in the picomolar range (l-100 PM) provoked a significant increase in the inotropic state of the myocardium (P < O.OOl), whereas a myocardial perfusion with higher concentrations (nanomolar range) of cz-hANP provoked a progressively increasing negative inotropic effect statistically significant at 50 nM (P < 0.01). These observations are consistent with
217
some previous in vivo and in vitro findings and suggest that physiological concentrations (3-25 pmol/l) of ANP [54] have a positive inotropic effect, while higher ANP concentrations result in a negative inotropic effect. Coronary flow In vitro and in vivo ANP effects on coronary arteries have been recently investigated. Most investigators have observed that ANP provokes a vasodilating effect on coronary arteries, whereas a few others have found that the peptide induces a coronary vasoconstriction. However, a comparative analysis of the results is not easily undertaken owing to different ANP doses and experimental models as well as techniques of peptide handling. In rats, synthetic ANP~101_126)administered through an intraventricular cannula (1 kg) resulted in a significant increase in coronary blood flow measured with radioactive microspheres [55]. In dog hearts perfused in situ at constant flow rate, Bathe et al. [47] reported that bolus injections of ANP (0.2 pg/kg-2 pg/kg) into the left coronary circulation induced a dose-related coronary vasodilation. A significant vasodilation has also been observed in anesthetized dogs after intracoronary [56], intrarterial [57], and intravenous [58] injection of 0.1-100 @kg of ANP in both normal [56,57] and embolized coronary arteries [%I. In contrast, no coronary effects induced by an intravenous administration of ANP (5 pg/kg) were observed in conscious chronically instrumented dogs [59]. In man, intraventricular administration of ANP (2.5 &kg) provoked a sustained proximal coronary dilation lasting more than 30 min, as quantitated by digital angiography [52,60]. More accurate experiments in which ANP was infused directly into the left coronary artery demonstrated that the atria1 peptide (1.75-84 &min) dilates the epicardial coronary arteries in normal subjects [50] and increases coronary flow similarly to nitroglycerin in patients with coronary artery disease [61]. Coronary vasoconstriction has been observed by a few investigators after ANP administration [31,62]. Intravenous injection of atria1 extracts (540 pg protein/kg) in conscious Wistar-Kyoto
and spontaneously hypertensive rats provoked a significant increase in the heart vascular resistance index [62]. Wangler et al. [31] observed a doserelated reduction of coronary flow in isolated guinea-pig heart after bolus injection (1- 100 nmoles) of atriopeptin II (23 amino acids), whereas no effect on the coronary blood flow was found in anesthetized rats in which synthetic ANP (28 amino acids) was intravenously infused (95 pmohmin) for 20 min [46]. Unfortunately, only in very few studies have plasma ANP levels been assayed after exogenous administration of the peptide. After administration of a 2.5 &kg intraventricular bolus of ANP in man, the immunoreactive ANP plasma level was in the picomolar range [52,60]. Since coronary vasodilation has been observed upon injection of ANP in doses lower than 2.5 &kg and also considering the results of the present study, it can reasonably be assumed that a relaxing effect is induced on coronary artery wall by ANP plasma concentrations in the picomolar range, whereas coronary vasoconstriction is provoked by ANP plasma concentrations in the nanomolar range. In conclusion, the results obtained in the present study and in the previous investigations support the possibility that ANP can induce enhancement or depression of myocardium and coronary artery function depending on its concentration in the perfusion medium or blood stream. These results are also consistent with the finding that ANP can bind receptor populations with affinity constants in the physiological as well as pharmacological range [63] and activate distinct intracellular responses
[W. Myucardial function vs. coronary flow relationship Present findings also answer the question of whether any relationship exists between the effects produced by ANP on the myocardial function and those concurrently induced on the coronary flow. In fact, maximal positive inotropic effect was induced by ol-hANP at a concentration of 100 pM, whereas the most pronounced increase in coronary flow rate occurred at 10 pM. This uncoupling of coronary and myocardial effects in our experimental model was also confirmed by the lack of any
218
statistical correlation between CFR and both LV dP/dt,,, and the contractility index at 10 pM. These data clearly support the possibility that (1) the positive inotropic effect caused by ANP at low concentrations is not strictly dependent on the increase in coronary flow and (2) ANP is able to induce direct effects on the myocardial function. In fact, coronary vasodilation should not be entirely dependent on myocardial oxygen requirements, otherwise it would have reached its maximal value at 100 pM when the maximal increase in myocardial inotropism had been observed. Thus, coronary dilation caused by (YhANP can be ascribed, at least in part, to a direct relaxing effect of the peptide on the coronary smooth muscles. The high correlation between CFR and left ventricular function indexes observed at nanomolar concentrations of ANP, could be related to a simultaneous effect of the natriuretic peptide on coronary vessels (vasoconstriction) and myocardial muscle cells (negative inotropic effect). However, it cannot be excluded that ANP-induced coronary vasoconstriction could contribute to produce the depression of myocardial function observed at the higher ANP concentrations. Present results on guinea-pig isolated heart provide a further insight into cardiac effects of ANP. Further attempts are needed to verify if ANP can induce direct in vivo effects on both myocardial and coronary function and to evaluate the relevance of these effects on hemodynamic control mechanisms in physiological as well as pathological conditions.
Acknowledgements This work has been supported in part by the Consiglio Nazionale delle Ricerche, Italy, Grants n. 90.02016.11 and n. 91.02125.11 and by the Minister0 dell’universita’ e della Ricerca Scientitica e Tecnologica, funds 400/o (Progetto Nazionale Insufficienza Cardiaca). The authors are grateful to Marco Pallante and Gianluca Ciarlantini for their technical assistance and to Anna Maria Maccari for helping with statistical analysis.
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Effects of a-human atria1 natriuretic peptide in guinea-pig isolated heart Paolo Di Nardo, Marco Pafi, Manuela Bartoli, Marilena Minieri, Fausta Bellegrandi, Gianfranco Raimondi, Giuseppe Peruzzi and Giuseppe Tallarida Laboratory of Cellular and Molecular Cardiology. Department of Internal Medicine. University qf Rome “Tor Yergata” Via 0. Raimondo. 00173 Rome. Italy
(Received 27 October 1992: revision accepted 24 March 1993)
The aim of the present investigation has been to ascertain whether or not atria1 natriuretic peptides (ANP) can exert a direct effect on myocardial contractility. Alpha-human ANP (a-hANP) concentrations ranging from 1 pM to 50 nM have been used to perfuse guinea-pig isolated hearts in a non-recirculating Langendorff apparatus. A dual concentration-related effect has been induced by a-hANP on myocardial function. A maximal increase of +LV dP/dt,,, (+56%; P < 0.001) has been observed when guinea-pig hearts were perfused with 100 pM ar-hANP, whereas a 25% decrease (P < 0.01) occurred with 50 nM cw-hANP. Similar effects have also been induced by cr-hANP on the coronary flow rate (CFR). A significant CFR increase (maximal at 10 pM cu-hANP) was induced ,by picomolar concentrations of a-hANP, whereas a progressive decrease, which was maximal (-28%; P < 0.01) at 50 nM o-hANP, was observed with nanomolar concentrations of the peptide. No effects have been observed on heart rate. These results suggest that ANP has direct effects on both vascular and myocardial muscle cells. Coronary vasoconstriction induced by nanomolar concentrations of ANP can contribute to the cardiodepression, whereas ANP in picomolar concentrations can induce a coronary vasodilation which is not coupled with the enhanced myocardial contractility. The latter is the likely expression of a direct effect of the peptide on myocardial function. Key words: Atria1 natriuretic peptide ; Isolated heart ; Inotropic state ; Coronary flow
1. In~~uetion Intravenous administration of alpha-human atria1 natriuretic peptide (ol-hANP) and other atria1 natriuretic factors lowers the arterial pressure in animals [l-5] and man [6,7] through a still undefined mechanism, The depressant activity of atria1 peptides (ANP) has been mainly related to a direct vasodilatory effect. In fact it has been Corresponding to: Paolo Di Nardo, Laboratory of Cellular and Molecular Cardiology, Department of Internal Medicine, University of Rome “Tor Vergata”. Via 0. Raimondo, 00173 Rome, Italy.
observed that ANP is able to counteract vessel precontraction induced in vitro by norepinephrine or angiotensin II [8,9]. Further investigations in rats [lo], rabbits [ll], dogs [12] and man [13-151 have confirmed that a-hANP can exert a direct vasodilating effect. However, systemic vasodilation has been observed only when ANP circulating levels were remarkably increased with respect to physiological concentrations [ 16- 181. The hypotensive effect can also be induced by ANP through a decrease in stroke volume and cardiac output. In fact, ANP can induce a fluid shift from the intravascular to the extravascular com-
212
partment, and a reduction in venous return leading to a decrease in cardiac filling pressure [ 19-261. It has been also hypothesized that the stimulation of receptors with sympathocardiopulmonary inhibitory effects might consistently contribute to the cardio-inhibitory and vasodilatory action of ANP [1,27-291. Recent experimental data from our laboratory do not support this view, providing evidence that a significant neurovegetative component is not involved in the mechanism of a-hANP depressor effect on the cardiovascular function [ 111.In keeping with these results, some studies indicate that ANP causes a reduction in the cardiac output through a direct negative inotropic effect [30,31]. However, no exhaustive results have been reported in previous studies, in which only a possible direct depressor effect of ANP on the myocardial function has been emphasized [2,4,11,3 1,321. The role of ANP-induced coronary vasoactivity in eliciting myocardial effects has also been investigated. In isolated guinea-pig hearts, Wangler et al. [3 1] observed a depression of the myocardial performance which was apparently caused by coronary vasoconstriction induced by atriopeptin II (23 amino acids). These findings were obtained with very high doses of ANP and were not confirmed by the same investigators [33] in rat isolated hearts exposed to a-hANP (28 amino acids). The present investigation in a guinea-pig isolated heart model has been undertaken to: (a) verify whether a-hANP exerts a direct effect on myocardial function and coronary flow; (b) ascertain whether concentrations of o-hANP in the physiological and pharmacological range might induce different responses in myocardial function and coronary flow, and (c) determine the relationship between the effects of ANP on myocardial function and coronary flow. Materials and methods Perfusion procedure Guinea-pigs (250-300 g bw) were stunned and hearts were rapidly excised and cooled in a bath of chilled perfusion medium (4°C). After removing the auricles, the heart was perfused by retrograde aorta perfusion in a non-recirculating Langendorff
apparatus at 37°C with a modified KrebsHenseleit buffer (KH) containing (mM) NaCl 127, KC1 4.7, KH2P04 1.1, MgS04 1.2, NaHC03 25, CaC12 2.5, glucose 5.5 and Na-pyruvate 2; pH 7.4. The KH buffer was gassed with 95% 02-5% CO*. The gas partial pressure and pH were periodically measured by a gas-analyzer (Radiometer ABL 330, Copenhagen, Denmark) in samples withdrawn proximally to the heart. The aortic PO, was approx. 80 kPa (600 mmHg). The hydrostatic perfusion pressure was measured in aortic cannula 1 cm above the heart with a Gould P23XL pressure transducer (Gould Inc., Valley View, Ohio) and was maintained at 10 kPa (100 cm H20). Coronary flow rate (CFR) was estimated by collecting coronary effluent in a funnel with a calibrated stem lumen and counting drops by a photoelectric device [34]. A fluid-filled latex balloon was passed through the left atrio-ventricular orifice and placed in the left ventricle. The balloon was connected to a Gould P23XL pressure transducer (Gould Inc., Valley View, Ohio) and inflated to maintain an end-diastolic pressure of approximately 0.67 kPa (5 mmHg). A second thin polyethylene tubing was inserted into the left ventricle before the balloon inflation to drain thebesian flow. Hearts were not electrically stimulated. An epicardial unipolar electrogram (EG) was obtained by a spring electrode positioned to the apex. Heart rate (HR) and LV dP/dt,,, were automatically computed. All electrical and mechanical signals were recorded on a Gould (Bioamplifiers: multichannel polygraph 13.4615.50, 13.4615.71 and Biotach 13.4615.65A). Isolated hearts were perfused with KH until CFR, electrical and mechanical activities were stabilized (approx 15 min), then the myogenic autoregulation of smooth muscles of coronary vessels (perfusion pressure: 9.33 kPa (70 mmHg) and 4 kPa (30 mmHg)) as well as the effect of increasing preloads (0.67 kPa (5 mmHg), 1.33 kPa (10 mmHg) and 2 kPa (15 mmHg)) on cardiac working musculature have been evaluated before each experimental test was started [34]. All studies have been in accordance with the Italian regulations for the care and use of laboratory animals.
213
ANF
a- human
HPLC
of the perfusion fluid used for each heart was 400-450 ml. The purity of o-hANP has been preliminarily tested by HPLC (Fig. 1). Before and during o-hANP perfusion CFR. LVP (left ventricular pressure), LV dP/dt,,,, EG and HR were countinuously recorded. Statistical analysis During ANP perfusion, all parameters although continuously recorded, have been sampled every 60 s and CFR every 8 s. The mean of ten consecutive signals for each time point has been calculated. The baseline value of each parameter was represented by the means of values 3 min and 1 min before ANP perfusion, if their difference was lower than 5%. Data reported in the present paper are expressed as percent difference from control values ( 100%). Correlation coefficients among parameters have been evaluated by the non-parametric Spearman’s test. Significant differences from the baseline have been tested by Student’s t-test.
A chart recording from a typical experiment is shown in Figure 2. Basal values for HR, LVP, I
1
1
I
0
10
20
30
min.
guinea-pig
Fig. 1. HPLC analysis of synthetic a-human ANP used in the present investigation. The graph shows a single peak at 19 min retention time. Column: Vydac C18, 5 pm, 300 A pore, 250 x 64.6 mm. Mobile phase: 0.25 N HsPO, +TEA pH 2.25 (Solvent A); TEAP pH 2.25/CH,CN 4060 (Solvent B). Linear gradient from 30 to 50% B in 40 min. Flow: I.5 ml/mm. HPLC apparatus: Varian Vista 5500. Detector (Varian Polychrom 9060): 215 nm.
Experimental protocol A total of 28 hearts were tested. After heart stabilization and responsiveness testing, each heart was continuously perfused once for 16 min with one of the following concentrations of a single lot of 28-amino acid synthetic o-human ANP (CYhANP) (Novabiochem, Laufelfingen, Switzerland) in KH medium: 1, 10 and 100 pM, and 1, 10 and 50 nM. Depending on heart size, the final volume
isolated
heart
$=-01 mn
0
2
4
6
a
10 15
Fig. 2. Original chart recording from a typical experiment of guinea-pig isolated heart perfused with 10 pM o-hANP. LVP. left ventricular pressure (arbitrary units); dP/dt (arbitrary units); CFR. coronary flow rate (ml/mitt); HR, heart rate (beats/min x 100).
214
HEART RATE (guinea pig isolated heart)
120
100 80 0
5
15
10
ml.
Fig. 3. Percent variation of heart rate (HR) during guinea-pig isolated heart perfusion with different cu-hANP concentrations (baseline: 100%).
+LV dP/dt,,,, -LV dP/dt,,, and CFR were 199 f 15.9 beatslmin, 103.2 f 8.9 mmHg, 1391 f 158.4 mmHg/s, 1028 f 123.8 mmHg/s, and 13.7 f 3.1 ml/min, respectively.
Heart rate (HR) was not affected by perfusion with a-hANP at any concentration tested (Fig. 3). Only during 100 pM a-hANP perfusion, LVP did increase (+39%; P < 0.05), but it appeared unchanged when either lower or higher peptide concentrations were used (Fig. 4). A composite time-course representing the effects of different concentrations of a-hANP on both +LV dP/dt,,, and -LV dP/dt,,, is shown in Fig. 5. Alpha-hANP displays a concentration-related biphasic effect on +LV dP/dt,,,. In fact, myocardial perfusion with 1, 10, and 100 pM cr-hANP was associated with a transient +LV dP/dt,,, increase of approx 25% (P < O.Ol), 21% (P < 0.05) and 56% (P < O.OOl), respectively. Within a few seconds after starting perfusion, +LV dP/dt,, gradually increased for 4-6 min before returning to baseline levels. At higher o-hANP concentrations (1 and 10 nM) there was a slight increase in +LV dP/dt,,, followed by a sustained depression of approx 13%, whereas 50 nM o-hANP resulted in a continuous decrease reaching -25% after 15 min perfusion (P < 0.01). Myocardial reperfusion
+LV dP/dtm, (guinea LEFT
and -LV
pig isolated
dP/dtm, heart)
VENTRICULAR PRESSURE (guinea pig isolated heart)
150
0
5
10
15 “I”
Fig. 4. Percent variation of left ventricular pressure (LVP) during guinea-pig isolated heart perfusion with different cr-hANP concentrations (baseline: 100%). *P < 0.05 vs. control (time 0).
Fig. 5. Percent variation of left ventricular dP/dt (+LV dP/dt,,,; -LV dP/dt,,,) during guinea-pig isolated heart perfusion with different cr-hANP concentrations (baseline: loo%). ‘P < 0.05. **P < 0.01. ***P < 0.001 vs. control (time 0).
215
with KH buffer only partially restored the baseline values. The time-courses of -LV dP/dt,,, and +LV dP/dt,,, were similar, and the modifications induced by a-hANP were not significantly different from their basal values. A 30% increase (P < 0.05) was observed only when guinea-pig hearts were perfused with 100 pM cr-hANP, whereas a sustained decrease (more than 20% after 1.5 min; P < 0.05) was observed with 50 nM a-hANP. Non-significant enhancements of the contractility index (+LV dP/dt,,,/LVP) were observed at all the lower cr-hANP concentrations (1 pM, 11%; 10 pM, 18%; 100 pM. 14%), whereas 25% decrement (P < 0.01) was detected only with 50 nM o-hANP (Fig. 6). Concentration-dependent effects were also induced by o-hANP on coronary flow rate (CFR). In fact, CFR appeared unmodifled with respect to basal values during heart perfusion with 1 pM (YhANP, while 10 pM a-hANP determined a 45% increase (P < 0.01) after 2 min perfusion. This was followed by a smooth, continuous decline until the 15th min (+25% with respect to basal values) of ANP perfusion (P < 0.05). A modest transient
CONTRACTILITY (guinea pig isolated
increase of CFR (approx. + 15%) was also seen at 100 pM a-hANP, whereas a progressive decrease was observed at higher ANP concentrations. After 15 min perfusion with 1, 10 and 50 nM cr-hANP, CFR was reduced by 15% (NS), 25% (NS) and 28% (P < O.Ol), respectively (Fig. 7). Statistical analysis (Table 1) showed high correlation coefficients between LVP and +LV dP/dt,,, at 100 pM, 1, 10 and 50 nM cr-hANP,
CORONARY (guinea-pig
FLOW isolated
RATE heart 1
INDEX heart)
0
Fig 6. Percent variation of contractility index (Cl = +LV dP/dt,,,/LVP) during guinea-pig isolated heart perfusion with different o-hANP concentrations (baseline: 100%). *P < 0.01 vs. control (time 0).
5
to
l5
min.
Fig. 7. Percent variation of coronary flow rate (CFR) during guinea pig isolated heart perfusion with different o-hANP concentrations (baseline: 100%). Standard deviations are not reported, however they did not exceed 10% of the related CFR mean value. *P < 0.05and **P < 0.01 vs. control (time 0).
216 TABLE 1 Correlation coefilcients evaluated by non-parametric [ANPI
+dP/dt vs. LVP
1 PM 10 pM 100 pM 1 nM 10 nM 50 nM
0.80 0.84 0.99 0.90 0.95 0.91
Spearman’s test.
CFR vs. +dP/dt,,,
CFR vs. -dP/dt,,,
CFR vs. LVP
CFR vs Cl
0.66 0.91
0.69
0.76 0.78
0.79 0.82
0.90 0.81 0.83
0.91 0.75 0.90
[ANP], concentration of atria1 natriuretic peptide; LVP, left ventricular pressure; CFR, coronary flow rate; CI, contractility index.
and to a lesser extent, at 1 and 10 pM cr-hANP. CFR, however, appeared to be highly correlated to LVP, +LV dP/dL,, -LV dP/dt,,, and contractility index only at 100 pM, 10 and 50 nM ar-hANP. Discussion Myocardial contractile function
Atria1 peptide binding sites have been found in the ventricular myocardium [35-371 suggesting that ANP could exert direct effects on the myocardial function. Several in vitro models have been used to evaluate the ANP effects on myocardial function. A marked impairment of myocardial function attributed to coronary vasoconstriction has been observed in guinea-pig isolated hearts after 1- 100 nmoles atriopeptin II (23 amino acids) bolus injections [31]. These results have not been confirmed in the same experimental model using both injected boli (approx. 20 nmoles/each) or infusion of 0.03-300 nM ANP [33,38]. Moreover, ANP did not modify the myocardial performance in cat [39] and guinea-pig papillary muscles [40] or atria [41]. Nevertheless, investigations on isolated cardiomyocytes have shown that ANP concentrations in the range l-50 nM could induce a direct negative inotropic effect possibly mediated by a reduction in intracellular calcium concentration [42-441. Studies performed in vivo on animal models and
man generated conflicting results. ANP administration provoked no or mild depressor effects on myocardial function in rat [45,46], dog [47,48] and sheep [49]. In dog, a cardiodepressor effect has been reported after intravenous 3 rglkg bolus + 0.3 pg/min per kg infusion of 24-amino acid auriculin A [30]. No or modest effects have been also observed on the myocardium of human subjects after ANP infusion [50,51]. A positive effect on myocardial performance has been observed in humans after intravenous or intraventricular administration of 0.5-2.5 pg/kg boli of ANP [52,53]. The increase in ventricular performance has not been related to a direct ANP effect, but to an increased cardiac sympathetic activity [5 1,531. Only a few studies reported ANP plasma concentrations after administration of natriuretic peptides. However, in these studies the positive inotropic effects seemed to be associated to ANP plasma concentrations not exceeding 300 pM [52,53]. The present study provides evidence that (YhANP can influence the myocardial function of guinea-pig isolated heart by inducing two dosedependent effects. In fact, in our experiments, ANP concentrations in the picomolar range (l-100 PM) provoked a significant increase in the inotropic state of the myocardium (P < O.OOl), whereas a myocardial perfusion with higher concentrations (nanomolar range) of cz-hANP provoked a progressively increasing negative inotropic effect statistically significant at 50 nM (P < 0.01). These observations are consistent with
217
some previous in vivo and in vitro findings and suggest that physiological concentrations (3-25 pmol/l) of ANP [54] have a positive inotropic effect, while higher ANP concentrations result in a negative inotropic effect. Coronary flow In vitro and in vivo ANP effects on coronary arteries have been recently investigated. Most investigators have observed that ANP provokes a vasodilating effect on coronary arteries, whereas a few others have found that the peptide induces a coronary vasoconstriction. However, a comparative analysis of the results is not easily undertaken owing to different ANP doses and experimental models as well as techniques of peptide handling. In rats, synthetic ANP~101_126)administered through an intraventricular cannula (1 kg) resulted in a significant increase in coronary blood flow measured with radioactive microspheres [55]. In dog hearts perfused in situ at constant flow rate, Bathe et al. [47] reported that bolus injections of ANP (0.2 pg/kg-2 pg/kg) into the left coronary circulation induced a dose-related coronary vasodilation. A significant vasodilation has also been observed in anesthetized dogs after intracoronary [56], intrarterial [57], and intravenous [58] injection of 0.1-100 @kg of ANP in both normal [56,57] and embolized coronary arteries [%I. In contrast, no coronary effects induced by an intravenous administration of ANP (5 pg/kg) were observed in conscious chronically instrumented dogs [59]. In man, intraventricular administration of ANP (2.5 &kg) provoked a sustained proximal coronary dilation lasting more than 30 min, as quantitated by digital angiography [52,60]. More accurate experiments in which ANP was infused directly into the left coronary artery demonstrated that the atria1 peptide (1.75-84 &min) dilates the epicardial coronary arteries in normal subjects [50] and increases coronary flow similarly to nitroglycerin in patients with coronary artery disease [61]. Coronary vasoconstriction has been observed by a few investigators after ANP administration [31,62]. Intravenous injection of atria1 extracts (540 pg protein/kg) in conscious Wistar-Kyoto
and spontaneously hypertensive rats provoked a significant increase in the heart vascular resistance index [62]. Wangler et al. [31] observed a doserelated reduction of coronary flow in isolated guinea-pig heart after bolus injection (1- 100 nmoles) of atriopeptin II (23 amino acids), whereas no effect on the coronary blood flow was found in anesthetized rats in which synthetic ANP (28 amino acids) was intravenously infused (95 pmohmin) for 20 min [46]. Unfortunately, only in very few studies have plasma ANP levels been assayed after exogenous administration of the peptide. After administration of a 2.5 &kg intraventricular bolus of ANP in man, the immunoreactive ANP plasma level was in the picomolar range [52,60]. Since coronary vasodilation has been observed upon injection of ANP in doses lower than 2.5 &kg and also considering the results of the present study, it can reasonably be assumed that a relaxing effect is induced on coronary artery wall by ANP plasma concentrations in the picomolar range, whereas coronary vasoconstriction is provoked by ANP plasma concentrations in the nanomolar range. In conclusion, the results obtained in the present study and in the previous investigations support the possibility that ANP can induce enhancement or depression of myocardium and coronary artery function depending on its concentration in the perfusion medium or blood stream. These results are also consistent with the finding that ANP can bind receptor populations with affinity constants in the physiological as well as pharmacological range [63] and activate distinct intracellular responses
[W. Myucardial function vs. coronary flow relationship Present findings also answer the question of whether any relationship exists between the effects produced by ANP on the myocardial function and those concurrently induced on the coronary flow. In fact, maximal positive inotropic effect was induced by ol-hANP at a concentration of 100 pM, whereas the most pronounced increase in coronary flow rate occurred at 10 pM. This uncoupling of coronary and myocardial effects in our experimental model was also confirmed by the lack of any
218
statistical correlation between CFR and both LV dP/dt,,, and the contractility index at 10 pM. These data clearly support the possibility that (1) the positive inotropic effect caused by ANP at low concentrations is not strictly dependent on the increase in coronary flow and (2) ANP is able to induce direct effects on the myocardial function. In fact, coronary vasodilation should not be entirely dependent on myocardial oxygen requirements, otherwise it would have reached its maximal value at 100 pM when the maximal increase in myocardial inotropism had been observed. Thus, coronary dilation caused by (YhANP can be ascribed, at least in part, to a direct relaxing effect of the peptide on the coronary smooth muscles. The high correlation between CFR and left ventricular function indexes observed at nanomolar concentrations of ANP, could be related to a simultaneous effect of the natriuretic peptide on coronary vessels (vasoconstriction) and myocardial muscle cells (negative inotropic effect). However, it cannot be excluded that ANP-induced coronary vasoconstriction could contribute to produce the depression of myocardial function observed at the higher ANP concentrations. Present results on guinea-pig isolated heart provide a further insight into cardiac effects of ANP. Further attempts are needed to verify if ANP can induce direct in vivo effects on both myocardial and coronary function and to evaluate the relevance of these effects on hemodynamic control mechanisms in physiological as well as pathological conditions.
Acknowledgements This work has been supported in part by the Consiglio Nazionale delle Ricerche, Italy, Grants n. 90.02016.11 and n. 91.02125.11 and by the Minister0 dell’universita’ e della Ricerca Scientitica e Tecnologica, funds 400/o (Progetto Nazionale Insufficienza Cardiaca). The authors are grateful to Marco Pallante and Gianluca Ciarlantini for their technical assistance and to Anna Maria Maccari for helping with statistical analysis.
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