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Cardiovascular Research 2000 45(2):351-359; doi:10.1016/S0008-6363(99)00371-5
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Urodilatin limits acute reperfusion injury in the isolated rat heart

Javier Inserte, David Garcia-Dorado*, Luis Agulló, Amaya Paniagua and Jordi Soler-Soler

Servicio de Cardiologìa, Hospital General Universitari Vall d’Hebron, Passeig Vall d’Hebron, 119–129, 08035 Barcelona, Spain

* Corresponding author. +34-93-4894038; fax: +34-93-4894032 dgdorado{at}hg.vhebron.es

Received 2 June 1999; accepted 12 October 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: Hypercontracture is an important mechanism of myocyte death during reperfusion. cGMP modulates the sensitivity of contractile myofilaments to Ca2+, and increasing cGMP concentration during the last minutes of anoxia prevents reoxygenation-induced hypercontracture in isolated cardiomyocytes. The purpose of this study was to determine whether stimulation of particulate guanylyl cyclase with the natriuretic peptide urodilatin, given at the time of reperfusion, reduces myocardial necrosis in the rat heart submitted to transient ischemia. Methods: Isolated rat hearts (n=38) were submitted to either 40 or 60 min of no-flow ischemia and 2 h of reperfusion, and were allocated to receive or not receive 0.05 µM urodilatin during the first 15 min of reperfusion or non-reperfusion treatment. Results: A marked reduction in myocardial cGMP concentration was observed in control hearts during reperfusion after 40 or 60 min of ischemia. Urodilatin significantly attenuated cGMP depletion during initial reperfusion, markedly improved contractile recovery after 40 min of ischemia (P<0.0309), and reduced reperfusion-induced increase in left ventricular end-diastolic pressure (P=0.0139), LDH release (P=0.0263), and contraction band necrosis (P=0.0179) after 60 min of ischemia. The beneficial effect of urodilatin was reproduced by the membrane permeable cGMP analog 8-Bromo-cGMP. Conclusions: These results indicate that reduced cGMP concentration may impair myocyte survival during reperfusion. Stimulation of particulate guanylyl cyclase may appear as a new strategy to prevent immediate lethal reperfusion injury.

KEYWORDS Ischemia; Natriuretic peptide; Necrosis; Reperfusion; Second messengers


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Early reperfusion may prevent necrosis of ischemic myocardium. However, when reperfusion is delayed it is associated with the appearance of areas of contraction band necrosis composed of hypercontracted cardiomyocytes [1,2]. Myocyte hypercontracture is the consequence of excessive contractile activity due to re-energization in the presence of elevated cytosolic Ca2+ concentration [3–6]. In in situ myocytes, hypercontracture causes sarcolemmal disruption and cell death [7,8]. Transient contractile blockade with 2,3-butanedione monoxime (BDM) during the time required for recovery of Ca2+ control prevents hypercontracture [9,10], and reduces final infarct size [1,11]. However, the potential therapeutic value of BDM in patients with acute myocardial infarction receiving reperfusion therapy is limited by the need for selective intracoronary delivery of the blocker, and its toxicity.

Previous studies have shown that treatments increasing cGMP concentration during reoxygenation and cGMP analogs may prevent reoxygenation-induced hypercontracture in isolated cardiomyocytes [12]. In the isolated rat heart submitted to transient hypoxia, cGMP synthesis and release into the coronary circulation are reduced during oxygen deprivation and remain reduced during reoxygenation [13]. In this model, L-arginine supplementation before hypoxia increases cGMP concentration during reoxygenation, reduces enzyme release, and improves functional recovery, and all these effects are abolished by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase. Since hypercontracture occurs during the first few seconds after reoxygenation, treatments increasing cGMP need to be present during the last minutes of anoxia in order to prevent it [12,13]. However, in myocardium submitted to transient ischemia, reperfusion-induced hypercontracture occurs only after disappearance of intracellular acidosis and its negative inotropic effect [6]. This time window could allow treatments increasing cGMP to exert their protective effect against hypercontracture when administered at the time of reperfusion. On the other hand, the protective effect of cGMP against reoxygenation-induced hypercontracture is observed at concentrations that have little effect on systolic shortening of normoxic myocytes [12]. Thus, drugs modulating cGMP appear as potentially useful in the limitation of lethal reperfusion injury occurring in clinical conditions.

Urodilatin is a member of the natriuretic peptide family normally present in urine [14] but not detected in plasma [15] that stimulates particulate guanylyl cyclase in many cell types including myocytes [12,16,17]. Urodilatin has a half-life much longer than that of atrial natriuretic peptide (ANP), has low toxicity and may be safely administered intravenously to patients [18]. Our hypothesis was that urodilatin administered at the time of reperfusion may limit myocardial necrosis secondary to ischemia/reperfusion. Urodilatin was added during the initial phase of reperfusion in isolated rat hearts submitted to transient global ischemia of two different durations, and cGMP release, functional recovery, and myocardial necrosis were measured.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Isolated perfused rat heart
The care and use of animals conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the experimental procedures were approved by the Research Commission on Ethics of the Hospital of Vall d’Hebron. Adult male Sprague–Dawley rats, weighing 300–350 g, were deeply anaesthetized with intraperitoneal injection of thiopental sodium (150 mg kg–1). The hearts were removed from the thorax and immediately arrested in ice-cold saline solution. The aorta was quickly cannulated and the heart was retrogradely perfused with a Krebs–Henseleit bicarbonate buffer (KHB) at 37°C using a non-recirculating Langendorff apparatus, at a constant pressure of 60 mm Hg. The composition of KHB was as follows (in mM): NaCl, 140; NaHCO3, 24; KCl, 2.7; KH2PO4, 0.4; MgSO4, 1; CaCl2, 1.8; and, glucose, 11. KHB was filtered under vacuum through a 0.45-µm cellulose filter before use to remove any particulate matter, and continuously gassed with 95% O2 and 5% CO2.

Left ventricular pressure was monitored by means of a water-filled latex balloon placed in the left ventricle and connected to a pressure transducer 43600 F (Baxter, The Netherlands) through a Cordis 5F catheter (Cordis, Miami, FL). The left ventricular end-diastolic pressure (LVEDP) was set between 6 and 8 mm Hg by adjusting the filling of the balloon. The signal obtained was digitized and recorded continuously on a hard disk with the aid of an ad hoc developed software. The variables measured included heart rate (HR), LVEDP and left ventricular developed pressure (LVdevP), calculated as the difference between left ventricular peak systolic pressure and LVEDP. Coronary flow was measured by timed collection of effluent at regular intervals using a calibrated tube, and expressed in ml min–1.

2.2 Experimental protocol and groups of treatment
2.2.1 Normoxic studies
The effects of urodilatin on contractility and coronary flow under normoxic conditions were studied in 13 hearts perfused for 180 min. Following a 30-min stabilization period, hearts were exposed to 15 min of perfusion with urodilatin (n=4) followed by 135 min of perfusion without the drug. Five hearts perfused for 180 min without urodilatin were used as normoxic controls.

The correlation between cGMP released into the coronary effluent and myocardial cGMP content was studied in eight hearts. After 30 min of stabilization, hearts were perfused for 3 min with urodilatin at different concentrations (0, 0.005, 0.05 or 1 µM), and immediately frozen by dropping the hearts into liquid nitrogen for posterior measurement of myocardial cGMP content. cGMP release was measured in samples from the coronary effluent collected immediately before, and at 3 min into, urodilatin perfusion.

2.2.2 Ischemia-reperfusion studies
In a first set of experiments, after 30 min of normoxic perfusion, hearts were subjected to global ischemia for 40 min by clamping the aortic in-flow line, and reperfused for 120 min. Hearts were allocated to one of two groups, receiving, respectively, 0.05 µM urodilatin during the first 15 min of reperfusion (n=8) or no drug (control group, n=10). In a second set of experiments, hearts were allocated to the same two groups of treatment, but ischemia was extended to 60 min (n=6 per group).

A third set of experiments hearts were performed to investigate the effect of 8-Bromo-cGMP. In these experiments hearts (n=4 per group) were submitted to either 40 or 60 min of ischemia and perfused with 100 µM 8-Bromo-cGMP under hypoxic conditions for 2 min starting 5 min before reperfusion and, under normoxic conditions, during the first 15 min of reperfusion. The brief hypoxic infusion prior to reperfusion containing 8-Bromo-cGMP was performed to ensure an effective intracellular concentration of drug at the onset of reperfusion. Previous studies (unpubl. observations) have shown that this requires administering the drug at least 5 min before reperfusion. The infusion however was stopped after 2 min to minimize catabolite washout and modify as minimally as possible the reperfusion conditions. The composition of the hypoxic buffer was identical to that used for normoxic reperfusion except for the replacement of bicarbonate by HEPES 20 mM and for the absence of glucose. Its pH was adjusted to 6.3 to match the extracellular pH previously reported in the ischemic rat heart [19] and was bubbled with 100% N2. Finally, a fourth set of experiments was used to investigate the effect of urodilatin on contractile function during the initial minutes of reperfusion. These hearts were submitted to 40 min of ischemia and reperfused in the presence of urodilatin. After the first 7 min of reflow urodilatin was withdrawn from the perfusate for 3 min, and added again at between 10 and 15 min of reperfusion.

2.3 Lactate dehydrogenase release
Lactate dehydrogenase (LDH) activity was spectrophotometrically measured in samples collected from the coronary effluent at different times throughout the reperfusion period, as previously described [13].

2.4 cGMP release
Samples (9 ml) from the coronary effluent were collected at different time points throughout the perfusion period, and rapidly frozen in liquid nitrogen. Samples were boiled for 10 min, centrifuged at 1250g for 10 min and the supernatant lyophilized. cGMP was determined in concentrated samples by radioimmunoassay using acetylated [3H]-cGMP, as previously described [20].

2.5 Myocardial cGMP content
cGMP concentration was measured in eight normoxic hearts in which the correlation between myocardial cGMP concentration and release was analysed, and in an additional series of 24 hearts submitted to either 40 or 60 min of ischemia and to 10 or 120 min of reperfusion with or without urodilatin according to a 2x2x2 equilibrated design.

Frozen hearts were pulverized under liquid nitrogen. The powdered tissue was homogenized in cold trichloroacetic acid at 7.5% (weight/volume). After centrifugation at 14 000g for 15 min at 4°C, the supernatant was collected and washed five times with seven volumes of water-saturated diethylether. The residual ether was removed by placing tubes in a bath at 50°C for 30 min. cGMP was measured in the extracts using the radioimmunoassay method described above and results were expressed as fmols of cGMP per mg of protein. Total protein content from the powdered heart was determined according to Bradford [21].

2.6 Histological analysis
A 3-mm-thick, cross-sectional, midventricular slice was embedded in paraffin, and 4-µm sections were obtained (Leika RM2145 microtome, Leika, Germany) and stained with Masson's trichrome. The presence of contraction band necrosis was assessed as previously described [11] and its extent was quantified morphometrically. Serial microphotographs of adjacent optical fields (x400, Olympus IMT-2, Olympus Optical, Japan) were obtained according to four perpendicular lines irradiating from the center of the left ventricular cavity with one of the lines crossing the right ventricular cavity at its middle point. Microphotographs were digitized (Olympus DP10 camera, Olympus Optical, Japan) for subsequent analysis (Micro ImageTM, Olympus Optical, Japan). Microphotographs including ventricular cavities or blank extracardiac space were excluded. Microphotographs were classified into one of three scores: 0, no contraction band necrosis; 1, contraction band necrosis involving less than 50% of the photographed area; 2, contraction band necrosis involving more than 50% of the photographed area. The average score was calculated for each heart.

2.7 Data analysis and statistics
Statistical analysis was performed by using commercially available software (Instat, GraphPad Software). Differences between groups were assessed by means of one-way analysis of the variance. Individual comparisons between groups were performed by using the Student–Newman Keuls test. A critical P value of 0.05 was used. Values are expressed as mean±S.E.M.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Effects of urodilatin in normoxic hearts
3.1.1 Myocardial function
LVEDP and HR were stable throughout the 180 min of normoxic perfusion. A slight, non-statistically significant decrease in LVdevP and flow values was observed by the end of the experimental perfusion period, confirming the stability of the preparation. There were no significant differences between groups (Table 1).


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  		Table 1 Hemodynamic parameters and coronary flow in normoxic perfused heartsa

 
3.1.2 cGMP release
In normoxic hearts, cGMP release into the coronary effluent showed a non-significant decrease throughout the perfusion period (from 293±29 fmol min–1 at 30 min of perfusion to 283±28 fmol min–1 after 180 min of perfusion). Addition of urodilatin to the perfusion buffer for 15 min increased cGMP release since the first minute, reaching a maximum of 2710±102 fmol min–1 after 10 min of perfusion with urodilatin.

3.1.3 Correlation between cGMP release and myocardial cGMP content
In hearts exposed for 3 min to different concentrations of urodilatin, myocardial cGMP content ranged between 48 and 260 fmols/mg protein, and total cGMP release into the coronary effluent during the stimulation period ranged between 276 and 3856 fmol min–1. There was an excellent correlation between both variables (r2=0.96).

3.2 Effects of urodilatin during reperfusion
3.2.1 Myocardial function
In control hearts subjected to 40 min of ischemia, LVEDP and LVdevP were, respectively, 7.8±0.6 and 112.1±9.9 mm Hg at the end of the equilibration period. At this time HR was 270±15 beats min–1 and coronary flow 11.2±0.4 ml min–1. No-flow ischemia resulted in cessation of left ventricular contractile activity [Fig. 1(A)], and in a steep increase in LVEDP with a peak of 53.4±3.3 mm Hg 20 min after the onset of the ischemic period [Fig. 1(B)]. Reperfusion induced a further increase in LVEDP with a peak of 78.2±6.0 mm Hg 3 min after its onset. LVdevP recovered to 37% of its initial value after 30 min of reperfusion and 38% after 120 min [Fig. 1(A)]. Coronary flow during reperfusion did not reach pre-ischemic values. After 30 min of reperfusion it was 5.3±0.5 ml min–1 and at the end of reperfusion 4.4±0.4 ml min–1 (P<0.0001 with respect to pre-ischemic values).


Figure 1
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  		Fig. 1 Changes in left ventricular developed pressure (LVdevP) and left ventricular end-diastolic pressure (LVEDP) during equilibration (EQUIL), 40 min of ischemia (dashed area) and 120 min of reperfusion in control hearts (Control) and in hearts receiving 0.05 µM urodilatin (Urodilatin) during the first 15 min of reperfusion. The 15 min infusion period is denoted by INF. Data are presented as mean±S.E.M. *, P<0.05 vs. control group; {dagger}, P<0.05 urodilatin group vs. control group.

 
Addition of urodilatin to the perfusion buffer during the first 15 min of reperfusion was associated with a markedly improved contractile recovery of LVdevP (67% after 30 min of reperfusion and 55% after 120 min [Fig. 1(A)] but only after cessation of urodilatin infusion). The rapid increase in LVdevP after withdrawal of urodilatin suggested a negative inotropic effect that was confirmed in an additional series of hearts submitted to consecutive addition–withdrawal of the drug during the initial minutes of reflow (Fig. 2). These studies disclosed a marked effect of the urodilatin during initial reperfusion, as demonstrated by a 50% reduction in LVdevP, in sharp contrast with the absence of contractile effects in normoxic hearts not previously submitted to ischemia.


Figure 2
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  		Fig. 2 Changes in left ventricular developed pressure (LVdevP) and heart rate (HR) in hearts submitted to consecutive addition (+) withdrawal (–) of 0.05 µM urodilatin during normoxia without previous ischemia or during the first 30 min of reperfusion after 40 min of ischemia. Data are presented as mean±S.E.M. *, P<0.05 vs. control group.

 
Hearts subjected to 60 min of ischemia showed identical behaviour to those submitted to 40 min of occlusion during the equilibration period, as well as during the first 40 min of ischemia [Fig. 3(A) and (B)]. The time and magnitude of LVEDP rise during ischemia were identical in hearts submitted to both durations of ischemia. In control hearts, reperfusion was followed by an increase in LVEDP with a peak of 128.9±7.0 mm Hg 3 min after its onset. LVdevP recovered to 8% of its initial value after 30 min of reperfusion and 10% after 120 min. Addition of urodilatin to the perfusion buffer during the first 15 min of reperfusion did not produce significant changes in the recovery of LVdevP, HR or coronary flow compared to control hearts, but reduced significantly the peak of LVEDP during early reperfusion (105.6±3.5 mm Hg, P=0.0139).


Figure 3
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  		Fig. 3 Changes in left ventricular developed pressure (LVdevP) and left ventricular end-diastolic pressure (LVEDP) during equilibration (EQUIL), 60 min of ischemia (dashed area) and 120 min of reperfusion in control hearts (Control) and in hearts receiving 0.05 µmol/l urodilatin (Urodilatin) during the first 15 min of reperfusion. The 15 min infusion period is denoted by INF. Data are presented as mean±S.E.M. *, P<0.05 vs. control group; {dagger}, P<0.05 URO 0.05 µmol/l group vs. control group.

 
3.2.2 LDH release
No measurable LDH activity was detected in the coronary effluent of hearts submitted to 40 min of ischemia independently of the presence or absence of urodilatin. However, in hearts subjected to 60 min of ischemia reperfusion was associated with important LDH release with an early peak 4 min after the onset of reoxygenation followed by a rapid decay. Addition of urodilatin during the first 15 min of reperfusion reduced significantly the total LDH release during the reperfusion period compared to the control group (164.3±9.7 U/g dry weight/120 min vs. 131.5±8.0 U/g dry weight/120 min, P=0.0263) without modifying its time course (Fig. 4).


Figure 4
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  		Fig. 4 LDH release during reperfusion following 60 min of ischemia in control hearts (Control) and in hearts receiving 0.05 µmol/l urodilatin (Urodilatin) during the first 15 min of reperfusion. Data are presented as mean±S.E.M. *, P<0.05 vs. control group.

 
3.2.3 cGMP release
In hearts subjected to 40 min of ischemia, cGMP release at the end of the stabilization period was 197±18 fmol min–1. After 10 min of reperfusion, cGMP release reached 32±11% of its initial value in the control group and 94±22% in hearts receiving urodilatin (P<0.001 with respect to controls). After cessation of urodilatin infusion, cGMP release decreased progressively [Fig. 5(A)]. In hearts submitted to 60 min of ischemia the time course and magnitude of cGMP release were similar to those observed in the corresponding groups submitted to 40 min of ischemia [Fig. 5(B)].


Figure 5
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  		Fig. 5 cGMP release into the coronary effluent during equilibration (EQUIL) and reperfusion in control hearts (Control), and in hearts receiving 0.05 µmol/l urodilatin (Urodilatin) during the first 15 min of reperfusion. The 15 min of infusion period is denoted by INF. Panels A and B correspond, respectively, to hearts submitted to 40 min and 60 min of ischemia. The dashed area corresponds to the ischemic period. Data are presented as mean±S.E.M. *, P<0.05 vs. control group.

 
3.2.4 Myocardial cGMP concentration
cGMP content was severely reduced in myocardium reperfused after 40 or 60 min of ischemia (Fig. 6). Addition of urodilatin to the perfusate during the first 15 min of reflow prevented this reduction in hearts reperfused after 40 min of ischemia, and markedly attenuated it in hearts reperfused after 60 min of ischemia. These effects were lost after 120 min of reperfusion (0.2±0.1 fmols/mg protein in the control group and 1.7±1.5 fmols/mg protein in the urodilatin group subjected to 40 min of ischemia and 1.91±1.81 fmols/mg protein in the control group and 0.2±0.1 fmols/mg protein in the urodilatin group subjected to 60 min of ischemia).


Figure 6
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  		Fig. 6 Myocardial cGMP after 30 min of equilibration (Normoxia) and after 10 min of reperfusion in control hearts (Control), and in hearts receiving 0.05 µmol/l urodilatin (Urodilatin) during the first minutes of reperfusion following either 40 min of ischemia (Isch40/Rep10) or 60 min of ischemia (Isch60/Rep10). Data are presented as mean±S.E.M. *, P<0.05 vs. 10 min reperfusion control group.

 
3.2.5 Effect of 8-Bromo-cGMP
Hearts receiving 8-Bromo-cGMP, a soluble analog of cGMP, during initial reperfusion after 40 min of ischemia showed a significantly better functional recovery than those not receiving it (Table 2). Hearts receiving 8-Bromo-cGMP after 60 min of ischemia did not differ from their controls in their functional behaviour, although a trend towards a better postischemic recovery of LVdevP was observed (Table 2), but showed a marked reduction of reperfusion-induced LDH release compared to the control group (111.4±24.7 U/g dry weight/120 min vs. 182.6±19.1 U/g dry weight/120 min, P=0.0365).


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  		Table 2 Hemodynamic parameters from hearts subjected to ischemia and receiving or not receiving 8-Bromo-cGMP during initial reperfusiona

 
3.2.6 Histology
No contraction band necrosis was observed in hearts subjected to 3 h of normoxic perfusion. Hearts submitted to 40 min of ischemia did not show areas of contraction band necrosis. After 60 min of ischemia all hearts showed extensive areas of contraction band necrosis. Treatment with urodilatin reduced significantly the presence of contraction band necrosis (mean score (0–2) of 1.17±0.17 vs. 1.83±0.17 in the control group, P=0.0179). A similar reduction in the extent of contraction band necrosis was observed in hearts receiving 8-Bromo-cGMP (1.05±0.21, P=0.0214).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
This study shows that, in the isolated rat heart, myocardial cGMP is severely depleted during reperfusion following prolonged ischemia, and that stimulation of particulate guanylyl cyclase with urodilatin during the first 15 min of reperfusion attenuates this depletion, markedly improves functional recovery, and protects cardiomyocytes against hypercontracture and necrosis induced by reperfusion after 60 min of ischemia. The effects on functional recovery were evident after 40 min of ischemia, a duration that resulted in minimal necrosis in both treated and control hearts. At the concentration used, urodilatin did not modify coronary flow either during normoxic conditions or during reperfusion and lacks effects on myocardial contraction in normoxic, control hearts, but had a marked, rapidly reversible negative inotropic effect on reperfused myocardium. These results indicate that reduced cGMP levels may have a detrimental influence on cell survival during myocardial reperfusion, and identify stimulation of membrane-bound guanylyl cyclase as a potentially useful pharmacological approach to enhance myocardial salvage during coronary reperfusion.

4.1 cGMP in reperfused myocardium
Although many studies have investigated the changes in NO synthesis and availability in reperfused myocardium [22–24], little is known about the modifications in cGMP concentration induced by ischemia/reperfusion. Previous studies have described either a reduction [25,26] or an absence of changes [27] in myocardial cGMP content in reperfused myocardium. In a recent study on the isolated rat heart [13] we reported a marked reduction in cGMP release into the coronary circulation during hypoxia and reoxygenation. The results of the present study demonstrate a marked reduction in myocardial cGMP concentration and coronary cGMP release in reperfused myocardium. Moreover, the response of myocardial cGMP to stimulation with urodilatin is markedly reduced in the reperfused myocardium: myocardial cGMP increased from 19.4±3.0 fmols/mg protein to 107.2±14.4 fmols/mg protein in response to urodilatin in normoxic hearts, but only to 19.4±6.4 fmols/mg protein in response to urodilatin during the initial 10 min of reperfusion following 40 min of ischemia. This blunted response to urodilatin was even more pronounced in myocardium reperfused after 60 min of ischemia, and suggests that ischemia/reperfusion may damage the enzymatic system controlling cGMP concentration.

The effect of manoeuvres increasing the activity of soluble guanylyl cyclase (such as the addition of NO donors or L-arginine supplementation) on myocardial injury secondary to ischemia/reperfusion or hypoxia/reoxygenation has been investigated in many studies, most of them with positive results [22–24]. This protective effect can be abolished by selective inhibitors of guanylyl cyclase [13]. However, NO is a free radical with potentially toxic actions at high concentrations that could counterbalance the beneficial effects of increased cGMP synthesis [28]. An alternative approach to increasing cGMP synthesis is stimulation of particulate guanylyl cyclase. In a recent study, addition of urodilatin to the incubation media during the last 15 min of anoxia and the first 15 min of reoxygenation prevented reoxygenation-induced hypercontracture in isolated cardiomyocytes [12], and the soluble cGMP analog 8-Bromo-cGMP mimicked this effect. The present study supports and extends these previous observations showing the beneficial effect of increasing cGMP in reoxygenated or reperfused myocardium, and demonstrates the feasibility of achieving this effect by the addition of urodilatin only at the time of reperfusion.

4.2 Protection mechanism
That the protective effect of urodilatin against myocardial necrosis secondary to ischemia/reperfusion is mediated through its effect on myocardial cGMP is supported by the ability of the soluble cGMP analog 8-Bromo-cGMP to reproduce it. This effect of cGMP against reperfusion injury can be explained by its ability to desensitize contractile myofilaments to Ca2+ during the initial minutes of reperfusion. Desensitization by BDM during the initial phase of reoxygenation or reperfusion prevents hypercontracture and limits myocardial necrosis in different models [1,9–11]. In this study urodilatin lacked contractile effects in normoxic hearts, but had a marked negative inotropic effect during the first minutes of reperfusion, a situation in which Ca2+ sensitivity of myofilaments and contractility are already depressed (stunning).

Previous studies demonstrate that under normoxic conditions cGMP may desensitize myofilaments to Ca2+ without altering Ca2+ kinetics. The molecular mechanism of this effect has not been established. It has been described that cGMP may directly reduce the relative myofilament response to Ca2+, probably via a cGMP-dependent protein kinase [29]. More recently, atrial natriuretic peptide has been shown to indirectly decrease myofilament Ca2+ sensitivity via cytosolic acidification secondary to modulation of the sarcolemmal Na+–H+ exchanger and/or other sarcolemmal transport systems by cGMP-dependent protein kinase [30,31]. In reperfused myocardium intracellular acidosis is rapidly corrected upon restoration of coronary blood flow by the combined actions of the Na+–H+ exchanger and the Na+–HCO3– symporter [32], and prolongation of acidosis during the initial phase of reperfusion has been shown to have a protective effect against hypercontracture in isolated myocytes [33,34] and in intact animals [35]. However, there is ample evidence from whole-cell voltage clamp studies that cGMP may have an inhibitory effect on L-type Ca2+ channels [36] and the possibility has not been excluded that changes in cGMP concentration may modulate Ca2+ kinetics in reperfused myocytes. Either improved Ca2+ kinetics or enhanced and/or prolonged acidosis could explain the striking beneficial effect of urodilatin at 0.05 µM on contractile recovery after 40 min of ischemia.

4.3 Methodological considerations
In the present study two different durations of ischemia (40 and 60 min) were used. The 40-min experiments allowed us to detect the beneficial effect of urodilatin against post-ischemic dysfunction in the absence of significant necrosis, as assessed by LDH release and histology, while the 60-min experiments allowed us to detect the protective effect of urodilatin against necrosis. The failure of urodilatin to significantly improve the extremely severe contractile dysfunction of myocardium surviving 60 min of ischemia may be reflected in the limited efficiency of the drug against stunning.

Changes in cGMP release was used as an index of myocardial cGMP content. Certainly, factors other than myocardial cGMP concentration can influence release of cGMP into the coronary circulation. However cGMP has been successfully used by previous authors to assess the effect of treatments stimulating cGMP synthesis [37,38], and in the present study an excellent correlation was observed between both variables at least under normoxic conditions. In addition, the results of cGMP measured in the coronary effluent were fully correlated with the analysis of myocardial cGMP content at a single time-point early during reperfusion.

4.4 Implications
The isolated perfused rat heart model presents many differences with in vivo situations. In fact, urodilatin has important non-cardiac effects when administered to intact animals [18,39,40], that are not considered in this model. However, the present results identify a new strategy to prevent immediate lethal reperfusion injury of myocardium based on stimulation of particulate guanylyl cyclase. The observation that urodilatin may protect reperfused myocardium from hypercontracture and necrosis when applied at the reflow time at concentrations lacking any detectable effect on normoxic myocardium not previously submitted to ischemia has potential therapeutic relevance, since urodilatin has been safely administered to normal volunteers [40,41] and to patients with heart failure [18]. Further research is clearly needed to define its therapeutic value during in vivo myocardial reperfusion.

Time for primary review 31 days.


   Acknowledgements
 
The authors wish to thank Yolanda Puigfel for her excellent technical assistance and Dr. Marisol Ruiz-Meana and Dr. Juan Cinca for their contribution in reviewing the manuscript. Urodilatin was kindly provided by Prof. Dr. Wolf-Georg Forssmann and PD Dr. Markus Meyer from the Niedersächsisches Institute für Peptid-Forschung, Hannover, Germany. This work was partially supported by the European Union (BMH1-PL95/1254), and by the Spanish Ministry of Health (FIS 97/0948).


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

  1. Schlack W, Uebing A, Schafer M, et al. Regional contractile blockade at the onset of reperfusion reduces infarct size in the dog heart. Pflugers Arch (1994) 428:134–141.[CrossRef][ISI][Medline]
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A. Kuno, N. V. Solenkova, V. Solodushko, T. Dost, Y. Liu, X.-M. Yang, M. V. Cohen, and J. M. Downey
Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1288 - H1295.
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