© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Release of a stable cardiodepressant mediator after myocardial ischaemia during reperfusion1
Medizinische Klinik und Poliklinik I, Charité, Humboldt-Universität zu Berlin, Schumannstr. 20–21, D-10098 Berlin, Germany
* Corresponding author. Tel.: +49 (30) 2802-5885; fax.: +49 (30) 2802-3996.
Received 21 November 1996; accepted 9 April 1997
 |
Abstract |
|---|
 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Objective: The aim of this study was to investigate whether cardiodepressant mediators are released after myocardial ischaemia during reperfusion. Methods: Using a double heart model, the effect of the reoxygenated coronary effluent of an isolated guinea pig heart on a sequentially perfused second heart was studied under control conditions and after 10 min ischaemia of the first heart. Investigation of the modulating role of known autacoids took place by using free radical scavengers, an NO synthase inhibitor and adenosine receptor antagonists. In order to identify the chemical nature of cardiac metabolites, the coronary effluent was also subjected to different chemical treatment modes. Results: No haemodynamic changes were observed during sequential perfusion under control conditions. After 10 min of global ischaemia in heart I, a marked decrease in LVP (–22%), LVdP/dtmax (–43%), LVdP/dtmin (–41%) and coronary perfusion pressure (–25%) was measured in heart II during sequential perfusion. The negative inotropic effect was rapid in onset and reversible within 5 min; free radicals, nitric oxide and adenosine were not involved. Storage of the coronary effluent of the first heart up to 24 h, heating, or protease treatment did not modify its cardiodepressant effects on the second sequentially perfused heart. Conclusions: These results suggest the release—from an isolated heart after ischaemia during reperfusion—of a cardiodepressant mediator which induces a potent reversible negative inotropic effect on a sequentially perfused heart. The mediator is stable and in all probability not a protein.
KEYWORDS Myocardial ischemia; Reperfusion; Cardiodepression; Free radicals; Free radical scavengers; Guinea pig, heart
|
1 Introduction |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Myocardial ischaemia induces numerous metabolic events resulting in reversible and irreversible functional and structural changes. In particular, contractile dysfunction after myocardial ischaemia is a common phenomenon investigated in numerous studies [1–3]. Potential underlying mechanisms for the depression of myocardial contractile function are excessive cellular and mitochondrial calcium overload [4], decreased sensitivity of myofilaments to calcium [5], injury mediated by release of free radicals [6–8]or cytotoxic enzymes, changes in calcium availability [9], and dysfunction of the sarcoplasmic reticulum [10]. In addition, a release of autacoids such as nitric oxide (NO) and adenosine may also contribute to changes in myocardial circulation and contractile function after ischaemia [11–14]. Furthermore, myocardial ischaemia stimulates the calcium-independent phospholipase A2 [15–17]. Activation of this enzyme also contributes to myocardial ischaemic damage [15–17]. The question arises whether myocardial ischaemia induces the release of further still unknown cardioactive mediators which may contribute to cardiac malfunction after ischaemia. However, studies on the functional role of agents generated during ischaemia or after an ischaemic period are hampered by the fact that ischaemia itself causes severe myocardial damage due to energy depletion. We therefore established a model of sequential perfusion of two isolated hearts in which we characterized the effects of the coronary effluent of an isolated heart (heart I) after global stop flow ischaemia on a sequentially perfused non-ischaemic second heart (heart II). In this double heart model, the coronary effluent of heart I is reoxygenated and rapidly transported to heart II by a roller pump. This approach allows direct characterization of the effects of cardiac metabolites on myocardial contractility and coronary circulation, independent of direct cardiac damage caused by ischaemia. In this model, heart II acts as a bioassay to further characterize the haemodynamic effects of these putative mediators. In the present study, we demonstrate the technical performance of serial perfusion of two isolated hearts and the changes in the contractile parameters with and without preceding ischaemia of heart I. In addition, the relative contribution of known autacoids such as adenosine and NO released after ischaemia is investigated by using antagonists and/or inhibitors of synthesis. Finally, chemical treatment of the coronary effluent allows conclusions on the stability and chemical nature of cardiac metabolites which may influence myocardial contractility.
|
2 Methods |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
2.1 Perfusion of two isolated guinea-pig hearts—the ‘double heart model’
In our model two guinea pigs of either sex (200–300 g) were killed mechanically. The hearts were excised and mounted for perfusion by the Langendorff technique. The hearts were perfused separately with a modified Krebs-Henseleit solution (NaCl 127.1 mmol/l, KCl 4.7 mmol/l, MgSO4 1.1 mmol/l, KH2PO4 1.19 mmol/l, NaHCO3 24.9 mmol/l, CaCl2 1.26 mmol/l, glucose 8.93 mmol/l, HEPES 10 mmol/l), equilibrated with 95% O2/5%CO2, 37°C, pH 7.4. Left and right ventricular pressures were recorded via a fluid-filled latex balloon inserted through the mitral and tricuspid valve, respectively, and attached to a pressure transducer and chart recorder. The right and left ventricular end-diastolic pressures were maintained at 2.5 and 5 mmHg, respectively. Balloon pressures were electronically differentiated to yield dP/dt and heart rate. Coronary flow rates were monitored with an ultrasonic flow meter (Transsonic) connected to a flow probe which was built into the aortic arch. Coronary perfusion pressure (CPP) was monitored with a pressure transducer attached to the aortic perfusion cannula. All parameters were continuously displayed. Registration included heart rate (HR), left ventricular pressure (LVP), left ventricular peak dP/dt (LVdP/dtmax and min), right ventricular pressure (RVP), right ventricular peak dP/dt (RVdP/dtmax and min), coronary flow rates (CF), and coronary perfusion pressure.
Sequential perfusion of two isolated hearts was performed according to the technique described by Schrader et al. [18]. At the onset of serial perfusion, the coronary effluent of heart I was reoxygenated by carbogen gas (95%O2/5%CO2) in a microchamber (500 µl) and rapidly transported to heart II by a roller pump. Reduction of transit time to less than 3 s was achieved by miniaturization of the oxygenator and the connecting tubes (inner diameter = 0.5 mm). With a system of valves and pumps, different perfusion modes could be realized: separate perfusion at constant pressure (60 cmH20), separate perfusion at constant flow (10 ml/min), and sequential perfusion (Fig. 1). During sequential perfusion of the two isolated hearts, the ventricular contractile parameters of both hearts were stable for at least 2 h.
|
2.2 Experimental protocol
After completion of the surgical preparation, the hearts were separately perfused at constant pressure (60 cmH2O). Then, after an equilibration period, perfusion at constant flow (10 ml/min) was performed for 30 min. This was followed by sequential perfusion without (control) or with preceding ischaemia (10 min) of heart I as described in Fig. 2.
|
Two different modes of serial perfusion were performed: in perfusion mode A, the coronary effluent of heart I was immediately (<3 s) transported to heart II. In perfusion mode B, the coronary effluent was fractionally collected at 6-s intervals over a period of 1 min and delivered to heart II after different periods of storage and biochemical interventions (Fig. 2).
2.3 Chemical and biochemical analyses of the coronary effluent
To ascertain the efficacy of reoxygenation, pH and pO2 were continuously measured by micro flow-through electrodes (Microelectrodes Inc., Londonderry, USA) inserted immediately upstream from the second heart. In addition, the following metabolic parameters were analyzed in the coronary effluent of heart I after reoxygenation in the microchamber: osmolality, sodium, potassium, chloride, calcium, phosphate, magnesium, lactate, and glucose.
2.4 Role of known autacoids released after ischaemia
The release of adenosine and catecholamines into the coronary effluent was determined by high-performance liquid chromatography (HPLC) measurements.
In addition, to investigate the role of known mediators released after ischaemia on the observed negative inotropic effect in the double heart model, the following experiments were performed:
1. Role of catecholamines: Intracoronary infusion of the selective β1-receptor antagonist, metoprolol (2.8 µmol/l, n = 3), to heart II. The infusion was started 10 min prior to serial perfusion and continued until the end of the experiment (perfusion mode A).
2. Role of adenosine: Intracoronary infusion of a selective adenosine A1-receptor antagonist (1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 1 µmol/l, n = 4) and a selective adenosine A2-receptor antagonist (3,7-dimethyl-1-propargylxanthine (DMPX), 1 µmol/l, n = 4) to heart II. The infusions were started 10 min prior to serial perfusion and continued until the end of the experiment (perfusion mode A). Furthermore, the coronary effluent was incubated with adenosine deaminase (0.2 U/l, n = 5), which hydrolyses adenosine to inosine.
3. Role of nitric oxide: Intracoronary infusion of a nitric oxide (NO) synthase inhibitor (N
-nitro-L-arginine, 100 µmol/l, n = 4) to heart I and heart II. In heart I, the intracoronary infusion was started 20 min prior to ischaemia, stopped during ischaemia, and restarted at the onset of reperfusion. In heart II infusion of the NO synthase inhibitor was started 20 min prior to sequential perfusion and then continued until the end of the experiment (perfusion mode A).
4. Role of free radicals: Incubation of the coronary effluent of heart I with the free radical scavengers superoxide dismutase (SOD, perfusate concentration 100 U/ml) and catalase (CAT, perfusate concentration 100 U/ml) (n = 5) for 30 min prior to delivery to heart II (perfusion mode B).
5. Role of activation of phospholipase A2 and generation of arachidonic acid or its metabolites: Intracoronary infusion of (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Haloenol lactone suicide substrate, HELSS) (25 µmol/l, n = 3), a specific inhibitor of calcium-independent phospholipase A2, to heart I and heart II. In heart I, the intracoronary infusion was started 10 min prior to ischaemia, stopped during ischaemia, and restarted at the onset of reperfusion. In heart II infusion of the phospholipase A2 inhibitor was started 10 min prior sequential perfusion and then continued until the end of the experiment (perfusion mode A).
2.5 Investigations on stability and chemical structure
In different series of experiments, an attempt was made to obtain further information on the size, stability and chemical structure of the mediator(s) released from heart I after ischaemia. The coronary effluent—collected over a period of 1 min after 10 min of ischaemia in heart I—was therefore subjected to various treatment modes prior to delivery to heart II:
1. Storage at room temperature for 30 min (n = 5) and for 24 h (n = 3; perfusion mode B).
2. Heating to 56°C for 30 min (n = 5; perfusion mode B).
3. Incubation at room temperature for 24 h with the protease, chymotrypsin (perfusate concentration 25 mU/ml, n = 3; perfusion mode B), and with papain, pepsin, bromelain, endoproteinase Glu-C, and thermolysin (perfusate concentration 25 mU/ml, n = 3 each).
4. Passage through a 0.5 kDa filter: experiments (n = 3) were performed using a 0.5 kDa molecular weight cut-off ultrafiltration membrane (YC05 membrane Amicon INC) and a type 8050 stirred cell system (Amicon, Witten, Germany).
In addition, to guarantee that under control conditions (without preceding ischaemia of the first heart) no specific haemodynamic changes were induced by the different treatments, the coronary effluent of heart I was collected under basal conditions without preceding ischaemia and also subjected to identical storage and treatment modes.
To obtain further information on the duration of the negative inotropic effect, the coronary effluent was collected over 30 s, pooled from 10 postischaemic periods, reoxygenated in the microchamber, and then infused into the second heart over a period of 5 min.
2.6 Ethics
The investigation conforms with the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1985).
2.7 Statistical analysis
Results are expressed as mean () s.e.m. for n determinations if not otherwise indicated. Statistical analyses were performed by Kruskal-Wallis ANOVA, followed by Mann-Whitney U-test at a level of significance of P<0.05.
2.8 Drugs
1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) and 3,7-dimethyl-l-propargylxanthine (DMPX) were obtained from Research Biochemicals International (RBI, Köln, Germany); chymotrypsin, papain, pepsin, bromelain, endoproteinase Glu-C, thermolysin, catalase, superoxide dismutase, adenosine deaminase, metoprolol and N-nitro-L-arginine from Sigma Chemical (Deisenhofen, Germany), (E)-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Haloenol lactone suicide substrate (HELSS) from Biomol (Hamburg, Germany).
|
3 Results |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
3.1 Haemodynamic measurements
In both groups, left ventricular contractile parameters of heart II were similar at baseline. No relevant changes of these parameters were observed during serial perfusion in the control group (n = 7) (Fig. 3). On the other hand, after 10 min of global ischaemia of heart I (n = 13), LVdP/dtmax (–43%), LVdP/dtmin (–41%), LVP (–22%), and CPP (–25%) of heart II immediately decreased when reperfusion was started (Table 1, Fig. 3). These parameters increased again and reached a new baseline within 5 min. The heart rate of heart II remained stable. The pronounced decrease in LVdP/dtmax and LVdP/dtmin therefore primarily reflects direct negative inotropic and lusitropic effects of the coronary effluent of heart I. Similar data were obtained for the right ventricular contractile parameters (data not shown). Fig. 4 shows an original registration of the contractile parameters of heart I and heart II prior to and after the onset of serial perfusion after global ischaemia of heart I.
|
|
|
3.2 Chemical and biochemical analyses of the coronary effluent
The effectiveness of reoxygenation was assessed by gas analysis of the perfusate. Oxygen partial pressure in the coronary effluent after reoxygenation in the microchamber before delivery to heart II was stable at 661±11 mmHg. After 10 min of global ischaemia and reoxygenation in the microchamber, the pO2 of the coronary effluent of heart I only slightly decreased, to 610±10 mmHg within 0.5 min, and then increased again to 630±11 mmHg within 2 min (Table 2). These measurements demonstrate that the microchamber is able to effectively reoxygenate the coronary effluent of heart I and realizes an adequate oxygenation of heart II even after global ischaemia of heart I.
|
HEPES (10 mmol/l) was added to the perfusate to stabilize the pH of the coronary effluent. The addition of HEPES did not influence significantly the function of the isolated hearts. The pH of the perfusate was stable at 7.42 at baseline and did not change significantly in the coronary effluent of heart I in the control group. After 10 min of ischaemia of heart I, the pH moderately decreased to 7.20±0 within 0.5 min at the onset of serial perfusion. The pH then increased again to baseline levels within 2 min (Table 2).
The analysis of chemical parameters in the coronary effluent of heart I prior to and after a 10 min global ischaemia is shown in Table 3. The concentration of electrolytes was measured to exclude the possibility that ionic changes in the coronary effluent contribute to reperfusion-induced contractile malfunction of heart II. As shown in Table 3, the concentrations of sodium, chloride, calcium, and magnesium remained stable. In addition, potassium and phosphate increased only slightly to 5.8 and 1.63 mmol/l, respectively. Glucose decreased to a minimum of 8.18 mmol/l. These parameters again reached baseline values within 1 min. There were no relevant changes of perfusate osmolality. Lactate concentration in the coronary effluent slightly increased to a maximum of 3.22 mmol/l within 0.5 min and then progressively decreased during 2 min to baseline values.
|
3.3 Role of known cardioactive mediators
In the coronary effluent of heart I, catecholamines were not detectable by HPLC: neither prior to nor after 10 min stop flow ischaemia. Accordingly, the negative inotropic effect observed with heart II was not modulated by the β1-receptor antagonist, metoprolol (2.8 µmol/l, n = 3).
Adenosine is known to be released after ischaemia, and in our model HPLC analysis accordingly showed an increase in adenosine concentration in the coronary effluent of heart I after ischaemia (Fig. 5). The adenosine concentrations in the coronary effluent were measured after passage of the effluent through the micro-oxygenator. To exclude the possibility that this mediator may modulate the contractile effects observed in heart II, the second heart was pretreated with an A1 (DPCPX, 1 µmol/l, n = 4) or A2 (DMPX, 1 µmol/l, n = 4) receptor antagonist. During infusion of these antagonists under basal conditions, no significant changes in the contractile parameters were observed. At the onset of serial perfusion after 10 min global ischaemia of heart I, the adenosine receptor antagonists did not significantly influence the contractile response of heart II. In the presence of the A1-receptor antagonist, DPCPX, LVP, LVdP/dtmax and LVdP/dtmin decreased within 0.5 min by –22, –44 and –45%, respectively. In the presence of the A2-receptor antagonist, the decrease in the left ventricular contractile parameters was comparable. Furthermore, neither of the adenosine receptor antagonists significantly inhibited maximal decrease in coronary perfusion pressure in heart II. In addition, 30 min incubation of the coronary effluent of heart I with adenosine deaminase (which hydrolyses adenosine to inosine) did not modulate myocardial contractility of heart II during serial perfusion (data not shown).
|
As mentioned above, nitric oxide as well as free radicals may also be suspected of mediating the negative inotropic effects. To exclude this possibility, pretreatment with an NO synthase inhibitor and free radical scavengers was performed. Under basal conditions, infusion of the arginine analogue, N
-nitro-L-arginine (100 µmol/l, n = 4), induced a rapid increase of CPP—which stabilized on a new plateau (+20% above baseline)—as well as a concomitant decrease in contractility (LVdP/dtmax –16%). During sequential perfusion after 10 min ischaemia of heart I, the NO synthase inhibitor did not modulate the contractile response observed in heart II. In the presence of N-nitro-L-arginine the decreases of LVP, LVdP/dtmax, LVdP/dtmin, and CPP were comparable to those in the absence of the NO synthase inhibitor. Oxygen free radicals are considered to play a role in contractile dysfunction after myocardial ischaemia during reperfusion. To exclude the possibility that release of oxygen free radicals from heart I or generation of these autacoids in the micro-oxygenator contribute to contractile malfunction of heart II, the coronary effluent of heart I was incubated with free radical scavengers in additional series of experiments. Incubation of the coronary effluent collected from heart I during reperfusion after 10 min ischaemia with SOD (100 U/ml) and CAT (100 U/ml) did not modulate the negative inotropic effects in heart II (Table 4). Control experiments without preceding ischaemia of heart I showed that pretreatment of the coronary effluent of heart I with the free radical scavengers did not induce significant haemodynamic changes in heart II.
|
Myocardial ischaemia may induce a relevant activation of the calcium-independent phospholipase A2 (PLA2), which induces generation of arachidonic acid and its metabolites. A recent in vitro study on inhibition of canine myocardial cytosolic calcium-independent PLA2 has shown that haloenol lactone suicide substrate (HELSS) is a potent irreversible inhibitor of the calcium-independent PLA2 [19]. In smooth muscle cells HELSS blocks agonist-induced release of arachidonic acid release by specific and irreversible inhibition of the calcium-independent PLA2 [20]. In the present study the effects of an intracoronary infusion of 25 µmol/l HELSS (n = 3) were investigated. The inhibitor of the calcium-independent PLA2 did not modify the negative inotropic effects in heart II when serial perfusion was started. In the presence of HELSS LVP, LVdP/dtmax, LVdP/dtmin, and CPP decreased within 0.5 min by –20, –43, –45 and –28%, respectively.
Consequently adenosine, nitric oxide, free radicals and activation of the Ca2+-independent PLA2 do not play a major role in the observed changes in contractility in heart II.
3.4 Investigations of the stability and the chemical structure of the cardiodepressant mediator(s)
Control experiments have verified that under control conditions without preceding ischaemia of heart I, neither storage of the coronary effluent for 30 min or 24 h, nor its treatment with chymotrypsin or heating caused any changes in contractile parameters and coronary perfusion pressure when the coronary effluent was applied to the second heart.
After 10 min of ischaemia of heart I and storage of the coronary effluent for 30 min or 24 h, the cardiodepressant effect of the coronary effluent was still maintained (Tables 4 and 5
). Furthermore, heating of the coronary effluent and its incubation with the protease chymotrypsin did not modulate its cardiodepressant effect on heart II (Tables 4 and 5). Identical results were obtained using the proteases papain, pepsin, bromelain, endoproteinase Glu-C, and thermolysin. Furthermore, passage of the coronary effluent through a 0.5 kD filter (n = 3) likewise did not influence its cardiodepressive effects on heart II.
|
When the coronary effluent of 10 postischaemic periods was collected, pooled, and then infused into the second heart over 5 min, similar results were obtained to those with direct sequential perfusion. However, the negative inotropic effect was prolonged and continued until the end of perfusion (Fig. 6). The decrease of ventricular contractility was subsequently completely reversible and returned to baseline again.
|
A further series of experiments was performed to investigate whether the mediators released from heart I during reperfusion induce a decrease of the contractile parameters of heart II in a dose-dependent manner. As shown in Fig. 7, dilution of the coronary effluent collected from isolated hearts during reperfusion after 10 min of ischaemia caused a progressive decrease of its negative inotropic effect, depending on the extent of dilution.
|
|
4 Discussion |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
We established a model of two isolated, sequentially perfused hearts. This approach makes it possible to investigate effects of mediators released from an isolated perfused heart after global ischaemia during reperfusion on a serially perfused non-ischaemic second heart. Using a buffer-perfused double heart model, we attempted to investigate whether release of mediators from myocardial tissue may contribute to contractile dysfunction during reperfusion independent of the effects of blood cells. This procedure may furthermore exclude the detrimental effects of ischaemia per se. Investigations on the functional role of mediators released from myocardial tissue during ischaemia and reperfusion in an isolated perfused heart are hampered by methodical difficulties: the cardiac effects of mediators are required to be distinguished from direct effects of ischaemia on myocardial tissue. Furthermore, ischaemia-induced regional changes in pH and in electrolyte concentrations may contribute to cardiac contraction abnormalities.
In this double heart model of two sequentially perfused isolated hearts, 3 essential technical features were implemented. First, flow rates of the two hearts were exactly synchronized by electronic processing. Second, reoxygenation and transit times were reduced to less than 3 s, so that the effects of unstable mediators could also be ascertained. In addition, gas analysis of the Krebs-Henseleit solution showed that the micro-oxygenator is able to effectively reoxygenate the coronary effluent of heart I within 1 s. Thirdly, the volume of the oxygenator was minimized (500 µl) to avoid dilution of potentially active mediators.
In our experiments, we have shown that serial perfusion of two isolated hearts is feasible under control conditions without relevant changes in the left ventricular contractile parameters, heart rate, and coronary perfusion pressure of heart II. These parameters remained stable for at least 2 h.
In contrast, after a 10 min stop flow ischaemia of heart I, serial perfusion induced a rapid decrease of left ventricular contractile parameters and coronary perfusion pressure of heart II (Figs. 3 and 4
, Table 1). Within 5 min, myocardial contractility and coronary perfusion pressure increased again to baseline. In this model of serial perfusion after myocardial ischaemia, the major events are a pronounced but reversible negative inotropic effect and simultaneous coronary vasodilation.
The reperfusion-induced contractile dysfunction of heart II cannot be explained by changes in electrolytes, glucose, osmolality, pH, or pO2 in the coronary effluent of heart I. During reperfusion, no relevant changes in the concentration of calcium, phosphate, sodium, chloride, magnesium, and glucose were measured (Table 2). The osmolality of the perfusate remained stable. After 0.5 min, potassium and phosphate in the coronary effluent of heart I increased moderately. Furthermore, the pH decreased from 7.4 to 7.2, and pO2 slightly decreased. Additional control experiments showed that an increase in perfusate potassium and phosphate—by external supply and a decrease in pH and pO2 comparable to the changes observed after ischaemia at the onset of reperfusion—did not induce significant haemodynamic changes. In addition, infusion of NaHCO3 during serial perfusion after ischaemia entirely prevented decrease in the pH, but did not inhibit the negative inotropic effect in heart II (data not shown). It may therefore be concluded that the observed changes in contractile function in our model are not caused by acidosis or by changes in the ionic concentration and oxygen pressure of the perfusate. To exclude the possibility of artefacts due to rapid reoxygenation in the micro-oxygenator, we also performed control experiments in which isolated hearts were perfused with Krebs-Henseleit solution which was initially deoxygenated by equilibration with nitrogen before reoxygenation in the micro-oxygenator. This perfusion mode, however, did not impair the contractile function of the isolated heart, which indicates that the micro-oxygenator is able to effectively oxygenate the perfusate without any detrimental effects.
Catecholamines may attenuate the negative inotropic effects in heart II. However, norepinephrine was not detected in the coronary effluent of heart I after 10 min stop-flow ischaemia. Furthermore, administration of β-receptor antagonists did not modulate the contractile response of heart II. This is in accordance with previous reports which demonstrate a release of catecholamines only after longer periods of ischaemia [21].
A role of adenosine, nitric oxide, or free radicals in modulating the cardiodepressive effect during serial perfusion could also be excluded by administration of specific receptor antagonists or synthesis inhibitors (Tables 1 and 4). Adenosine is known to be released after ischaemia. In addition, adenosine has been reported to attenuate contractile dysfunction after brief periods of ischaemia [13, 14]. Despite enhanced release of adenosine after ischaemia in our model (as confirmed by HPLC measurements, Fig. 5), this metabolite did not influence the negative inotropic effect observed in the second heart.
Recent studies with cardiomyocytes, papillary muscles, and isolated hearts indicate that NO influences myocardial inotropic responses [22–24]. Exogenous NO has been reported to attenuate cardiac myocyte contraction [22]. However, conflicting data exist concerning NO release after myocardial ischaemia. Reduced release of NO following ischaemia [11]and increased NO production [12]are both described. In an in vivo model (dog), it has been shown that enhanced NO release reduces myocardial contractility in the ischaemic heart [12]. In our model, it is unlikely that NO plays a role, since the coronary effluent is reoxygenated before reaching the second heart; NO would rapidly react to form NO2– and NO3–. Nevertheless, to exclude this possibility, we treated the two hearts with an NO synthase inhibitor during the entire experimental period. The cardiodepressive effect observed in the second heart was not modified. Recent in vivo studies have shown that the ability of NO synthase inhibitors to block NO-mediated effects depends on the exact timing of administration of these substances [25]. Therefore, we performed additional experiments (n = 4) in which the intracoronary infusion of N-nitro-L-arginine (100 µmol/l) was started 60 min prior to onset of ischaemia. However, the prolonged administration of the NO synthase inhibitor was also ineffective in modulating myocardial contractility of heart II during sequential perfusion as well, which suggests that in our model of sequential perfusion of two isolated hearts NO is not likely to be involved in the mechanism of action of the putative mediator(s).
Injury from oxygen-derived free radicals has been shown to be an underlying mechanism of myocardial stunning after ischaemia [6, 26]. These free radicals are held responsible for reperfusion-mediated tissue injury, secondary to lipid peroxidation and other irreversible alterations of cell constituents [26, 27]. Free radicals have furthermore been shown to inactivate NO and alter membrane permeability to ions, all of which can lead to damage to the endothelium and myocardial tissue. According to these data, increased production of free radicals in heart I may account for the reversible decrease of myocardial contractility in heart II of this double heart model. Furthermore, oxygen free radicals may be generated in the micro-oxygenator. However, as indicated in Table 4, administration of free radical scavengers did not influence the decrease in contractility observed in heart II, which suggests that in the model of sequential perfusion of two isolated hearts, oxygen-derived free radicals are not likely to be involved in the reversible negative inotropic effect in heart II.
Previous reports have demonstrated that myocardial ischaemia stimulates the calcium-independent phospholipase A2 (PLA2), which induces the generation of arachidonic acid and its metabolites. Activation of this enzyme liberates arachidonic acid from the sarcolemma [15–17]. Therefore, arachidonic acid or its metabolites, leukotrienes and prostaglandins, may contribute to the reversible changes of myocardial contractile performance of heart II. However, the PLA2-inhibitor did not influence the reversible decrease of ventricular contractile parameters during reperfusion. It may therefore be concluded that in the double heart model, activation of the calcium-independent PLA2—which induces a generation of arachidonic acid or one of its metabolites—is most likely not the underlying mechanism of the cardiodepressant effects in the second heart at the onset of serial perfusion. On the other hand, activation of this enzyme has been implicated as an important mechanism of myocardial ischaemic damage itself. Myocardial ischaemia very rapidly activates the calcium-independent PLA2 in the ischaemic tissue, which accelerates hydrolysis of myocardial membrane constituents and results in ischaemic membrane dysfunction [15–17].
It is of particular interest whether a dose–effect relationship of the coronary effluent exists which would indicate that the cardiodepressant mediator(s) are soluble. The coronary effluent collected during reperfusion from heart I was accordingly diluted with fresh perfusate, which resulted in a decrease of its negative inotropic effects, as a function of the extent of dilution (Fig. 7). This result supports the hypothesis that soluble cardiodepressant agent(s) are released from heart I.
The observed effect is reversible and short-lasting in this model of serial perfusion. This may be due to rapid wash-out. When the effluent was collected from 10 postischaemic periods and then administered over 5 min to the second heart, the observed haemodynamic effects were prolonged and continued until the end of perfusion (Fig. 6). Further studies using a recirculating perfusion mode, papillary muscle preparations, or isolated myocytes could provide additional information on the duration of the negative inotropic effect.
The effects of a different cardiodepressant mediator have been previously described by others in several models of circulatory shock [28, 29]. A myocardial depressant factor (MDF) has been detected in the blood of experimental animals as well as of humans. This factor is generated in the splanchnic region primarily in the pancreas [29]. The critical step in MDF production is splanchnic hypoperfusion. MDF is a low-molecular-weight peptide which exerts a direct negative inotropic effect. In contrast, the cardiodepressant mediator characterized in the double heart model is most likely not a peptide or a protein, since administration of several proteases with different restriction sites of amino acid bindings do not prevent the negative inotropic effects of the coronary effluent. The initial chemical characterization indicates that the factor is stable, since after storage of the coronary effluent at room temperature up to 24 h before delivery to heart II, the effect was still present (Table 5). Even heating to 56°C did not prevent the effect of this putative mediator, which further supports the conclusion that the factor is not a protein. Passage of the coronary effluent through a 0.5 kDa filter likewise failed to influence the negative inotropic effect, suggesting that the factor(s) are small molecules.
Taken together, these results suggest that a new, still unknown cardiodepressant mediator is released after global ischaemia from an isolated heart and induces a pronounced reversible decrease in contractility in a sequentially perfused second heart.
Recent in vitro studies have shown that endocardial and coronary vascular endothelium modulate myocardial contractile function [30–34]. Removal of endocardial endothelium causes an abbreviation of twitch contraction and a decrease in peak isometric twitch tension of papillary muscles [30]. Endothelial cells have been shown to release unidentified mediators which upregulate or downregulate myocardial contractility [32]. In addition, in vitro experiments have demonstrated that endothelial cells release a cardiac myofilament desensitizing factor [33]. Release of endothelium-derived factors may also contribute to cardiac malfunction during ischaemia or reperfusion. It is of particular interest that hypoxic incubated endothelial cells release diffusible factors which decrease myocardial contractility [34]. No information is available from the data of the present study whether the endocardial or coronary vascular endothelium is the cellular source of the cardiodepressant factors.
In posing a teleologically oriented explanation of this pathophysiological process, it may be discussed whether the observed decrease in myocardial contractility is beneficial in endowing the cardiac myocyte with an endogenous protective mechanism, or deleterious to cardiac performance after myocardial ischaemia. In terms of cardiac energy balance, the reversible decrease of myocardial contractility reflects a salutary effect, allowing the heart to recover better metabolically before full contractility is restored. Furthermore, the question arises whether a release of still unknown cardiodepressant factors from myocardial tissue also contributes to the phenomenon of myocardial stunning after ischaemia.
In summary, the data of the present study strongly suggest that a stable cardiodepressant mediator is released from myocardial tissue after ischaemia. This mediator is obviously not a protein and induces a reversible negative inotropic effect.
Time for primary review 23 days.
|
Acknowledgements |
|---|
The present study was supported in part by the Deutsche Forschungsgemeinschaft (Fe 250/3.1). The technical assistance of Adelheid Gatzke, Christiane Gögelein, Angelika Westphal, is gratefully acknowledged.
|
Notes |
|---|
1 Part of this work was presented at the Joint meeting of the European Society of Cardiology, in Birmingham, on August 25–29, 1996.
|
References |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
- Braunwald E., Kloner R.A. The stunned myocardium: prolonged, postischemic ventricular dysfunction. Circulation (1982) 66:1146–1149.
[Abstract/Free Full Text] - Hearse D.J., Bolli R. Reperfusion induced injury: manifestations, mechanisms and clinical relevance. Cardiovasc Res (1992) 26:101–108.
[Abstract/Free Full Text] - Jennings RB, Yellon DM. Reperfusion injury. Definitions and historical background. In: Yellon DM, Jennings RB, editors. Myocardial protection: the pathophysiology of reperfusion and reperfusion injury. New York: Raven Press, 1992:1–11.
- Opie L.H. Role of calcium and other ions in reperfusion injury. Cardiovasc Drugs Ther (1991) 5:237–248.[CrossRef][Web of Science][Medline]
- Kusuoka H., Porterfield J.K., Weisman H.F., Weisfeldt M.L., Marban E. Pathophysiology and pathogenesis of stunned myocardium. Depressed Ca2+-activation of contraction as a consequence of reperfusion-induced cellular calcium overload in ferret hearts. J Clin Invest (1987) 79:950–961.[Web of Science][Medline]
- Bolli R. Postischemic myocardial stunning. Pathogenesis, pathophysiology and clinical relevance. In: Yellon, DM Jennings RB, editors. Myocardial protection: the pathophysiology of reperfusion and reperfusion injury. New York: Raven Press, 1992:105–149.
- Bolli R. Mechanism of myocardial ‘stunning’. Circulation (1990) 82:723–738.
[Abstract/Free Full Text] - Reimer K.A., Tanaka M., Murray C.E., Richard V.J., Jennings R.B. Evaluation of free radical injury in myocardium. Toxicol Pathol (1990) 18:470–480.[Web of Science][Medline]
- Ito B.R., Tate H., Kobayashi M., Schaper W. Reversibly injured, postischemic canine myocardium retains normal contractile reserve. Circ Res (1987) 61:834–846.
[Abstract/Free Full Text] - Krause S.M., Jacobus W.E., Becker L.C. Alterations in cardiac sarcoplasmic reticulum calcium transport in the postischemic ‘stunned’ myocardium. Circ Res. (1989) 65:526–530.
[Abstract/Free Full Text] - Maulik N., Engelman D.T., Watanabe M., et al. Nitric oxide signaling in ischemic heart. Cardiovasc Res (1995) 30:593–601.
[Abstract/Free Full Text] - Node K., Kitakaze M., Kosaka H., et al. Increased release of NO during ischemia reduces myocardial contractility and improves metabolic dysfunction. Circulation (1996) 93:356–364.
[Abstract/Free Full Text] - Kitakaze M., Hori M., Kamada T. Role of adenosine and its interaction with alpha adrenoceptor activity in ischaemic and reperfusion injury of the myocardium. Cardiovasc Res (1993) 27:18–27.[Web of Science][Medline]
- Rynning S.E., Hexeberg E., Birkeland S., Westby J., Grong K. Blockade of adenosine receptors during ischaemia increases systolic dysfunction but does not affect diastolic creep in stunned myocardium. Eur Heart J (1994) 15:1705–1711.
[Abstract/Free Full Text] - Hazen S.L., Stuppy R.J., Gross R.W. Purification and characterization of canine myocardial cytosolic phospholipase A2. A calcium-independent phospholipase with absolute sn-2 regiospecificity for diradyl glycerophospholipids. J Biol Chem (1990) 265:10622–10630.
[Abstract/Free Full Text] - Hazen S.L., Wolf M.J., Ford D.A., Gross R.W. The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia. FEBS Lett (1994) 339:213–216.[CrossRef][Web of Science][Medline]
- Ford D.A., Hazen S.L., Saffitz J.E., Gross R.W. The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A2 during myocardial ischemia. J Clin Invest (1991) 88:331–335.[Web of Science][Medline]
- Schrader J., Bardenheuer H. Assessment of vasoactive metabolites released from the isolated guinea pig during heart hypoxia and β-adrenergic stimulation. Basic Res Cardiol (1981) 6:365–368.
- Hazen S.L., Zupan L.A., Weiss R.H., Getman D.P., Gross R.W. Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and-independent phospholipase A2. J Biol Chem (1991) 266:7227–7232.
[Abstract/Free Full Text] - Lehmann J.J., Brown K.A., Ramanadham S., Turk J., Gross R.W. Arachidonic acid release from aortic smooth muscle cells induced by [Arg8]vasopressin is largely mediated by calcium-independent PLA2. J Biol Chem (1993) 268:20713–20716.
[Abstract/Free Full Text] - Schömig A., Fischer S., Kurz T., Richardt G., Schömig E. Nonexocytotic release of endogenous noradrenaline in the ischemic and anoxic rat heart: Mechanism and metabolic requirements. Circ Res (1987) 60:194–205.
[Abstract/Free Full Text] - Brady A.J., Warren J.B., Poole-Wilson P.A., Williams T.J., Harding S.E. Nitric oxide attenuates cardiac myocyte contraction. Am J Physiol (1993) 265:H176–H182.[Web of Science][Medline]
- Mohan P., Brutsaert D.L., Sys S.U. Myocardial performance is modulated by interaction of cardiac endothelium derived nitric oxide and prostaglandins. Cardiovasc Res (1995) 29:637–640.
[Abstract/Free Full Text] - Shah A.M., Prendergast B.D., Grocott-Mason R., Lewis M.J., Paulus W.J. The influence of endothelium-derived nitric oxide on myocardial contractile function. Int J Cardiol (1995) 50:225–231.[CrossRef][Web of Science][Medline]
- Tietjen C.S., Kirsch J.R., Clavier N., Traystman R.J. Time-dependent inhibition of oxotremorine-induced cerebral hyperemia by N-nitro-L-arginine in cats. Stroke (1995) 26:2160–2165.
[Abstract/Free Full Text] - Ambrosio G., Flaherty J.T., Duilio C., et al. Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts. J Clin Invest (1991) 87:2056–2066.[Web of Science][Medline]
- McCord J.M. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med (1985) 312:159–163.[Abstract]
- Brand E.D., Cowgill R., Lefer A.M. Further characterisation of a myocardial depressant factor present in hemorrhagic shock. J Trauma (1969) 9:216–226.[Web of Science][Medline]
- Lefer A.M. Interaction between myocardial depressant factor and vasoactive mediators with ischemia and schock. Am J Physiol (1987) 252:R193–R205.[Web of Science][Medline]
- Brutsaert D.L., Meulemans A.L., Sipido K.R., Sys S.U. Effects of damaging the endocardial surface on the mechanical performance of isolated cardiac muscle. Circ Res (1988) 62:358–366.
[Abstract/Free Full Text] - Henderson A.H., Lewis M.J., Shah A.M., Smith J.A. Endothelium, endocardium, and cardiac contraction. Cardiovasc Res (1992) 26:305–308.
[Free Full Text] - Ramaciotti C., McClellan G., Sharkey A., Rose D., Weisberg A., Winegrad S. Cardiac endothelial cells modulate contractility of rat heart in response to oxygen tension and coronary flow. Circ Res (1993) 72:1044–1064.
[Abstract/Free Full Text] - Shah A.M., Mebazaa A., Wetzel R.C., Lakatta E.G. Novel cardiac myofilament desensitizing factor released by endocardial and vascular endothelial cells. Circulation (1994) 89:2492–2497.
[Abstract/Free Full Text] - Shah A.M., Mebazaa A., Cuda G., et al. Ca2+-independent inhibition of actomyosin crossbridge cycling in cardiac myocytes by endothelial cell factors in response to hypoxia. J Physiol (1994) 80P:475.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Lorenz, N. Hellige, P. Rieder, H.-T. Kinkel, C. Trimpert, A. Staudt, S. B. Felix, G. Baumann, K. Stangl, and V. Stangl Positive inotropic effects of epigallocatechin-3-gallate (EGCG) involve activation of Na+/H+ and Na+/Ca2+ exchangers Eur J Heart Fail, May 1, 2008; 10(5): 439 - 445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Birkenmeier, A. Staudt, W.-H. Schunck, I. Janke, C. Labitzke, T. Prange, C. Trimpert, T. Krieg, M. Landsberger, V. Stangl, et al. COX-2-dependent and potentially cardioprotective effects of negative inotropic substances released after ischemia Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2148 - H2154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Stangl, G. Baumann, K. Stangl, and S. B Felix Negative inotropic mediators released from the heart after myocardial ischaemia-reperfusion Cardiovasc Res, January 1, 2002; 53(1): 12 - 30. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Felix, V. Stangl, P. Pietsch, P. Bramlage, A. Staudt, S. Bartel, E.-G. Krause, J.o.-U. Borschke, K.-D. Wernecke, G. Isenberg, et al. Soluble substances released from postischemic reperfused rat hearts reduce calcium transient and contractility by blocking the L-type calcium channel J. Am. Coll. Cardiol., February 1, 2001; 37(2): 668 - 675. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-K. Yang, N. J. Draper, and A. M. Shah Ca2+-independent inhibition of myocardial contraction by coronary effluent of hypoxic rat hearts Am J Physiol Heart Circ Physiol, February 1, 1999; 276(2): H623 - H632. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||