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Cardiovascular Research 2001 51(1):100-107; doi:10.1016/S0008-6363(01)00280-2
© 2001 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Short-acting calcium antagonist clevidipine protects against reperfusion injury via local nitric oxide-related mechanisms in the jeopardised myocardium

Andrey Gourine*, Adrian Gonon, Per-Ove Sjöquist and John Pernow

Department of Cardiology, Karolinska Hospital, S-171 76 Stockholm, Sweden

* Corresponding author. Tel.: +46-8-5177-3560; fax: +46-8-311-044

Received 16 October 2000; accepted 1 March 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Background: Calcium antagonists may, in addition to their classical actions, release nitric oxide (NO) from coronary arteries. The aim of this study was to elucidate the possible interaction between the cardioprotective effect of a short-acting calcium antagonist and NO during myocardial ischaemia and reperfusion. Methods: Anaesthetised pigs were subjected to 45 min ligation of the left anterior descending coronary artery (LAD) followed by 4 h of reperfusion. Five groups were given vehicle (n=9), clevidipine (n=8), the NO synthase inhibitor L-NMMA (n=6), clevidipine in combination with L-NMMA (n=6) or clevidipine in combination with L-NMMA and NO precursor L-arginine (n=6) into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. Results: There were no significant differences in LAD blood flow, mean arterial pressure, rate–pressure product or dP/dt between the groups before ischaemia or during reperfusion. The infarct size (IS) was 86±2% of the area at risk in the vehicle group. Clevidipine reduced the IS to 59±3% (P<0.001). When clevidipine was administered together with L-NMMA, the protective effect of clevidipine was abolished (IS, 87±3%; P<0.001 vs. clevidipine), whereas addition of L-arginine restored its cardioprotective effect (IS 60±3%; P<0.001 vs. vehicle). L-NMMA did not affect IS per se (88±5%). Endothelium-dependent coronary vasodilation induced by substance P was significantly larger in the clevidipine group than in the other groups. Conclusion: Local administration of a calcium antagonist during the late ischaemia and early reperfusion reduces IS and preserves coronary endothelial function. The cardioprotective effect of clevidipine is suggested to be dependent on maintained local bioavailability of NO.

KEYWORDS Coronary circulation; Endothelial function; Ischaemia; Nitric oxide; Reperfusion; Vasoconstriction/dilation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Interruption of coronary blood flow causes myocardial ischaemia and, after a critical time period, myocardial cell death. Despite that restitution of flow is mandatory for reducing the ultimate damage, restoration of arterial blood flow per se will increase functional and structural injury in the jeopardised myocardium. This phenomenon has been referred to as reperfusion injury [1,2]. The ultimate mechanism underlying this seemingly paradoxical outcome is still unknown. However, the involvement of different pathophysiological pathways has been proposed including e.g. cytosolic calcium overloading, formation of free radicals, accumulation and activation of neutrophils [3,4].

Recently, much attention has been focused on the role of the coronary vascular endothelium during ischaemia and reperfusion [4]. The endothelium produces several different relaxing and contracting factors such as nitric oxide (NO), prostacyclin, endothelium-derived hyperpolarizing factor, endothelin-1 (ET-1) and angiotensin II [5]. Endothelial NO is formed from L-arginine by the constitutive form of the enzyme NO synthase. The enzyme can be inhibited competitively by L-arginine analogs such as N-monomethyl-L-arginine (L-NMMA). NO is an important regulator of vascular tone, prevents platelet and leukocyte adherence and is a scavenger of superoxide [6,7]. Endothelial dysfunction is an early event in various pathological cardiovascular conditions. This dysfunction is characterised by an impairment of endothelium-dependent relaxation, due to reduced bioavailability of endothelial NO. The reduced bioavailability may be the result of attenuated production/release or enhanced inactivation of NO [5]. During myocardial ischaemia and reperfusion, endothelial dysfunction already occurs during early reperfusion and is considered to be an important event for the development of ischaemia–reperfusion injury [8].

Several studies indicate that calcium antagonists can protect against ischaemia–reperfusion injury [9–11]. Suggested mechanisms are related to amelioration of ischaemia-induced endothelial cell permeability [12], activation of KATP channels [11], protective effect against oxygen free radicals and attenuated neutrophil accumulation [13]. More recent studies have also demonstrated that calcium antagonists can release NO from coronary microvessels [14] and attenuate the severity of low flow myocardial ischaemia via an NO-dependent mechanism [15,16].

The hypothesis that we tested in the present study is that calcium antagonists preserve endothelial function and protect against myocardial injury by local NO-mediated mechanisms during late ischaemia and early reperfusion. This is possible to test in vivo by administration of a newly developed ultra-short acting calcium antagonist clevidipine [17,18], which when given into a coronary artery is devoid of systemic effects, together with an NO synthase inhibitor and the NO precursor L-arginine.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animal preparation
The study was approved by the regional ethical committee for laboratory animal experiments and conforms 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).

Pigs of either sex were premedicated with ketamine hydrochloride (20 mg/kg i.m.) and atropine sulphate (0.1 mg/kg i.m.). Anaesthesia was induced by sodium pentobarbital (20 mg/kg. i.v.) and maintained by a continuous infusion (2–4 mg/kg/h i.v.). The animals were intubated and mechanically ventilated with air and oxygen. Respiratory rate and tidal volume were adjusted to keep arterial blood pH, pO2 and pCO2 within the physiological range. Rectal temperature was kept at 38.5–39.0°C by means of a heated operating table. A 7French catheter was positioned in the superior caval vein through the internal jugular vein for drug and fluid administration. Another 7French catheter was positioned in the descending aorta via the left femoral artery for sampling of blood and for measurement of mean arterial pressure (MAP) via a Statham P23Db transducer. Heart rate (HR) was determined from the arterial pressure curve. The rate–pressure product (RPP) was calculated as systolic blood pressure multiplied by HR. A catheter-tip manometer (Millar instruments, Houston, TX, USA) for the determination of left ventricular pressures and the first derivative of left ventricular pressure development (dP/dt) was inserted in the left ventricular cavity via the carotid artery. All variables were continuously recorded on a Grass polygraph (model 7). The heart was exposed via a sternotomy. A ligature was placed around the left anterior descending coronary artery (LAD) at a position from which the distal third of the artery would be occluded by tightening the ligature. An ultrasonic flow probe (Transonic Systems, New York, USA) for measurement of blood flow was placed around the artery just proximal to the snare. The flow probe was connected to a Transonic 208 blood flow meter. A needle (0.4 mm O.D.) connected to a catheter was introduced into the LAD just distal to the snare.

2.2 Experimental protocol
Following 30 min stabilization after the preparation, the pigs were subjected to 45 min of coronary artery ligation followed by 4 h reperfusion. Four groups were randomised to receive either vehicle (n=10), clevidipine (0.3 nmol/kg/min, n=9), the NO synthase inhibitor L-NMMA (0.2 mg/kg, n=7) or clevidipine in combination with L-NMMA (n=6) into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. An additional fifth group was given clevidipine in combination with L-NMMA and the NO precursor L-arginine (2 mg/kg, n=6) as above. This group was included in order to reverse the inhibitory effect of L-NMMA on NO synthesis and it was randomised with two pigs included in the vehicle group. The dose of clevidipine was based on a previous study by Segawa et al. [19]. All infusions were given at a rate of 3 ml/min. Endothelium-dependent responses were evaluated at the end of reperfusion by intracoronary infusion of substance P (0.02 and 0.2 µg/min at a rate of 2 ml/min) immediately before the animals were sacrificed.

2.3 Determination of infarct size
At the end of the experiment LAD was reoccluded and 2% Evans blue was injected into the left atrium to outline the ischaemic myocardium, after which the pigs were killed by injection of a high dose of potassium chloride into the left atrium. The heart was rapidly extirpated. The atria and the right ventricle were removed. The left ventricle was cut into 1 cm thick slices perpendicular to the heart base–apex axis. The slices were then incubated in 0.8% triphenyl tetrazolium chloride at 37°C which stained the viable myocardium red in order to measure the extent of myocardial necrosis [20]. The extent of myocardial necrosis and the area at risk was determined by planimetry.

2.4 Chemicals
Ketamin hydrochloride was purchased from Parke-Davis (USA), sodium pentobarbital from Apotekebolaget (Sweden), atropine sulphate and sodium heparin from Lovens (Denmark) and L-NMMA and substance P from Sigma (USA). Clevidipine was supplied by AstraZeneca (Sweden). Clevidipine was initially dissolved in 98% ethanol and thereafter diluted in 20% lipid emulsion (Intralipid, Pharmacia, Sweden). Further dilutions were made in saline giving a final concentration of ethanol and intralipid of 0.01% and 0.05%, respectively. Substance P was dissolved in saline. L-NMMA was dissolved in distilled water and diluted in saline.

2.5 Calculations and statistical analysis
All values are presented as means±S.E.M. Statistically significant differences were calculated using Friedman's test or Kruskal–Wallis analyses of variance for multiple paired and unpaired observations, respectively, followed by Dunnett's posthoc test. A P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Mortality and exclusions from the study
Of thirty-eight initially included pigs sixteen developed ventricular fibrillation during ischaemia and two during reperfusion. Of these animals, fifteen were successfully converted by DC shocks (10–20 J), whereas three developed irreversible ventricular fibrillation during ischaemia and were therefore excluded from the study. One animal was excluded in the vehicle group, one animal in the clevidipine group and one animal in the L-NMMA treated group. The remaining thirty-five pigs were included in the final analysis of the study.

3.2 Haemodynamics
MAP, HR, dP/dt max and RPP, before ischaemia, before drug administration, at the end of ischaemia and during reperfusion are presented in Table 1. There were no significant differences in haemodynamics before drug administration and during ischaemia between the groups. MAP decreased significantly during reperfusion in all groups. The reduction in MAP tended to be more pronounced in the in L-NMMA group but there were no significant differences in MAP between the groups during reperfusion. HR increased in all groups at the end of reperfusion but the changes were only statistically significant in the vehicle, L-NMMA and clevidipine+L-NMMA+L-arginine treated groups. Due to the opposite changes in HR and MAP, the RPP did not differ between the five groups during ischaemia and reperfusion. Left ventricular dP/dt did not change significantly in the experimental groups during ischaemia–reperfusion and there were no significant differences between the groups. LAD blood flow increased at the onset of reperfusion in all groups (Fig. 1). The degree of hyperemia varied between 200 and 250% of basal flow and there were no significant differences between the groups. At the end of reperfusion LAD blood flow had returned to pre-ischaemic levels (Fig. 1).


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  		Table 1 Haemodynamic data before ischaemia, before drug infusion, at the end of ischaemia and during reperfusiona

 

Figure 1
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  		Fig. 1 LAD blood flow before ischaemia (–45 min) and during reperfusion in animals subjected to 45 min of ischaemia and 4 h of reperfusion. The animals were given either vehicle (n=9), clevidipine (Clev, n=8), the NO synthase inhibitor L-NMMA (n=6), Clev in combination with L-NMMA (n=6) or Clev in combination with L-NMMA and the NO precursor L-arginine (L-Arg, n=6) into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. Data are presented as the mean±S.E.M.

 
3.3 Infarct size
Fig. 2a shows the infarct size expressed as a percentage of the area at risk in all experimental groups. The infarct size was 86±2% of the area at risk in the vehicle group. The infarct size was reduced to 59±3% in the group given clevidipine (P<0.001 vs. vehicle). When clevidipine was administered together with L-NMMA, the infarct size (87±3%) was not different from that of the vehicle group, but significantly larger than that of the group given clevidipine alone (P<0.001). Addition of L-arginine restored the cardioprotective effect of clevidipine (IS 60±3%; P<0.001 vs. vehicle). Administration of L-NMMA alone did not affect infarct size in comparison to the vehicle group. No significant differences in the areas at risk were observed between the groups (Fig. 2b).


Figure 2
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  		Fig. 2 Infarct size expressed as percent of the area at risk (a) and the area at risk as percent of the left ventricle (b) after 45 of ischaemia followed by 4 h of reperfusion. The animals were given either vehicle (n=9), clevidipine (Clev, n=8), the NO synthase inhibitor L-NMMA (n=6), Clev plus L-NMMA (n=6) or Clev in combination with L-NMMA and the NO precursor L-arginine (L-Arg, n=6) into the LAD during the last 10 min of ischaemia and the first 5 min of reperfusion. Data are presented as the mean±S.E.M. Significant differences between the groups are shown; ***, P<0.001.

 
3.4 Endothelial function
Endothelium-dependent responses were evaluated by intracoronary administration of substance P (0.02 and 0.2 µg/min) at the end of reperfusion. There was no differences in basal flow between the different groups when the infusions were performed. Substance P only increased LAD blood flow significantly in the group given clevidipine, but did not affect LAD flow in the other four groups (Fig. 3a). Both doses of substance P reduced coronary vascular resistance more in the clevidipine treated group than in the other groups (Fig. 3b).


Figure 3
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  		Fig. 3 Increase in LAD blood flow (a) and decrease in LAD vascular resistance (b) evoked by administration of substance P (0.02 and 0.2 µg/min) at the end of reperfusion to pigs subjected to 45 min ischaemia followed by 4 h of reperfusion. The animals were given either vehicle, clevidipine (Clev), the NO synthase inhibitor L-NMMA, Clev plus L-NMMA, Clev in combination with L-NMMA and NO precursor L-arginine (L-Arg, n=4–6). Data are presented as the mean±S.E.M. Significant differences between the Clev group and all other groups are shown; *, P<0.05; **, P<0.01.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The aim of the present study was to investigate whether the cardioprotective effect of a short-acting dihydropyridine calcium antagonist administered at the end of severe ischaemia and during early reperfusion was dependent on local production of NO. Intracoronary administration of clevidipine during the last 10 min of ischaemia and the first 5 min of reperfusion significantly reduced infarct size compared to the vehicle treated group following 45 min ischaemia and 4 h reperfusion and preserved the endothelium-dependent vasodilator response to substance P. The most important finding of the present study is that inhibition of NO synthase by L-NMMA abolished the protective effect of clevidipine whereas addition of L-arginine restored its cardioprotective effect. These observations indicate that the cardioprotective action of the dihydropyridine calcium antagonist is dependent on local bioavailability of NO.

The present finding that clevidipine protects against ischaemia–reperfusion injury is in accordance with previous studies of various experimental models in vivo which have demonstrated that different calcium antagonists protect the myocardium, both when administered before ischaemia [11,21,22], and at the end of ischaemia immediately before reperfusion [10]. Several different mechanisms of action have been proposed to explain the cardioprotection afforded by calcium antagonists administered during ischaemia–reperfusion. These include amelioration of ischaemia-induced endothelial cell injury [12], activation of KATP channels [11], increase in blood flow to the jeopardised myocardium during reperfusion [23], inactivation of oxygen free radicals [24] and attenuated neutrophil accumulation [13]. Recent studies have shown that certain calcium antagonists can mediate their vasodilator effects via an NO-dependent mechanism. Zhang and Hintze [14] demonstrated that the dihydropyridine calcium antagonist amlodipine releases NO from coronary microvessels via a kinin2-dependent mechanism. Furthermore, Kitakaze and coworkers [15,16] demonstrated that both benidipine and nifedipine increased coronary blood flow during low flow ischaemia, attenuated the severity of myocardial ischaemia and increased nitrate/nitrite level in the coronary sinus blood. All these effects were blunted by the NO synthase inhibitor L-NAME, suggesting that their effects were mediated via an NO-dependent mechanism. Long-term administration of amlodipine significantly increased NO synthase activity and endothelial NO synthase mRNA in the left ventricle of L-NAME-induced hypertension in rats [25]. Our results demonstrating that the infarct limiting effect of the calcium antagonist clevidipine was abolished following blockade of NO synthase, which is in accordance with and extends the above-mentioned data, and they suggest that the cardioprotection is related to maintained bioavailability of NO.

It is well documented that early reperfusion results in impaired endothelium-dependent vasodilatation which is the result of reduced NO formation or rapid inactivation of NO by oxygen-derived free radicals [26]. Several studies have reported that augmentation of NO levels during reperfusion by administration of L-arginine or NO donors preserves endothelial function, reduces neutrophil accumulation and decreases myocardial injury following ischaemia and reperfusion [27,28]. More recent studies indicate that endothelial NO synthase also exists in rabbit sarcoplasmic reticulum [29] and guinea pig mitochondria [30] and NO may protect against reperfusion injury after ischaemia by inhibiting Ca2+ influx into mitochondria which are otherwise damaged by superoxide anion [30]. Thus, NO appears to be one of the crucial factors mediating protection against ischemia–reperfusion injury.

In the present study we used clevidipine as a pharmacological tool for investigating the cardioprotective mechanism of calcium antagonists, since it is short-acting with a half-life of 0.27, 0.36 and 1.5 min in rat, dog and human, respectively, and the blood clearance in healthy volunteers is 0.14 l/min/kg [31–33]. Thus it seems appropriate to assume that its cardioprotective effect in the present study was mediated for a limited time period in connection with its administration [19].

It may be argued that L-NMMA abolished the cardioprotective effect of clevidipine secondarily to the haemodynamic effects of L-NMMA. Since NO synthase inhibition evokes vasoconstriction it may increase myocardial oxygen demand, which will increase myocardial injury during ischaemia. However, in the present study L-NMMA was administered locally in order to avoid systemic haemodynamic effects. Accordingly, MAP and RPP were not increased by L-NMMA, which indicates that myocardial oxygen demand was not increased in the present study. Inhibition of NO synthase did not affect infarct size per se which is in accordance with previous observations in pigs in vivo [34]. These findings indicate that it is not likely that altered systemic haemodynamics by L-NMMA have contributed to the present results. A coronary vasoconstrictory effect of L-NMMA may counteract the protective effect of clevidipine. However, NO synthase blockade evokes little or no coronary constriction in vivo [35,36] and any coronary constriction by NO synthase inhibition is attenuated following ischaemia [37]. In addition, it has been demonstrated that the vasodilator effect of clevidipine in the human internal mammary artery is independent of endothelial function [38]. Thus it seems less likely that changes in coronary flow explain the present results.

The endothelial function at the end of reperfusion was evaluated by local administration of substance P. It has been demonstrated that the increase in coronary flow evoked by substance P in pigs is blocked by NO synthase inhibition [39]. Substance P increased LAD flow in the group given clevidipine but did not affect LAD flow in the other groups. The increase in LAD blood flow and the reduction in LAD vascular resistance in response to both doses of substance P were significantly larger in the clevidipine group than in all other groups. These results indicate that endothelium-dependent vasodilatation to substance P measured at the end of reperfusion was significantly impaired by ischaemia–reperfusion but was maintained by clevidipine which further supports the view that clevidipine preserved NO production. We did not observe any significant increase in LAD flow in the group given clevidipine in combination with L-NMMA and L-arginine which may be somewhat surprising considering that the infarct size was reduced and NO production may be expected to be restored in this group. However, since the effect of L-NMMA may be more long lasting than that of L-arginine [40] it is possible, that L-arginine restored NO production in connection with its administration and that NO synthase was inhibited by L-NMMA at the time of substance P administration. We did not evaluate endothelium-independent vasodilator responses in these experiments. Several previous studies have clearly demonstrated that only endothelium-dependent, but not endothelium-independent coronary dilator responses are attenuated by ischaemia–reperfusion [41,42].

A limitation of the present study is that quantitative measurement of NO formation was not performed and related to the effect on infarct size. NO is rapidly oxidized to nitrite/nitrate [43]. Since total nitrite/nitrate originates from several sources other than endothelial NO, determination of plasma nitrite/nitrate poorly reflects NO production in vivo. Furthermore, since the plasma half-life of nitrate is 7–8 h [44], a reduction in plasma nitrate due to attenuated NO production during ischaemia–reperfusion in the present experiments is unlikely to occur. In addition, it has been demonstrated that nitrite is a source rather than a product of NO in ischaemic tissues [45].

Another possibility would be to determine cGMP levels. However myocardial cGMP levels have been reported not to be reduced by administration of NO synthase inhibitors [46]. In addition, other substances like atrial natriuretic peptide can also increase cGMP levels [47]. Thus, methodological problems limit the possibilities to quantitate NO production under the present experimental conditions.

In conclusion, local administration of clevidipine, during the last 10 min of severe ischaemia and early reperfusion reduces the infarct size and preserves endothelial function. Inhibition of NO synthase abolishes, whereas addition of L-arginine restored the protective effect of clevidipine suggesting that it is dependent on maintained local bioavailability of NO.

Time for primary review 28 days.


   Acknowledgements
 
The study was supported by grants from the Swedish Medical Research Council (10 857), the Swedish Heart and Lung foundation. We are grateful to Mrs. Marita Wallin for technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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