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CARDIOVASCULAR
Laboratoire de Pharmacologie, Institut National de la Santé et de la Recherche Médicale E 00.01, Faculté de Médecine Paris Sud, Le Kremlin-Bicêtre, France
Received April 9, 2003; accepted April 24, 2003.
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Abstract |
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;
Marber et al., 1993). To date,
the window of cardioprotection, i.e., the time interval between the
preconditioning stimulus and the final sustained ischemia, has received
extensive attention (Kloner and Jennings,
2001). The mechanism of this cardioprotection has been extensively
investigated and seems to be both complex and highly multifactorial
(Bolli, 2000;
Napoli et al., 2000;
Schulz et al., 2001). However,
only few studies have demonstrated that the efficacy of early preconditioning
progressively disappears with increasing durations of ischemia, thus
characterizing a "ceiling of protection"
(Murry et al., 1986;
Gumina et al., 1999). To date,
this issue has never been investigated with delayed preconditioning.
Nitroglycerin and nitric oxide (NO)-donors such as
S-nitroso-N-acetylpenicillamine (SNAP) have demonstrated
their efficacy at inducing delayed preconditioning both experimentally
(Takano et al., 1998;
Hill et al., 2001) and
clinically (Leesar et al.,
2001). This cardioprotection has been reported to involve the
generation of oxidant species, the activation of protein kinase C and nuclear
factor-
B as well as the induction of inducible nitric-oxide synthase
activity (Bolli, 2000).
Accordingly and taking into account our background with SNAP
(Lellouche et al., 2002), we
decided to investigate the ceiling of protection with this NO donor.
Myocardial infarct size was measured after increasing durations of coronary
artery occlusion followed by a long duration of reperfusion.
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Materials and Methods |
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Animal Surgery. Male New Zealand rabbits (2–2.5 kg) were anesthetized with pentobarbital sodium (20–30 mg/kg i.v.). They were intubated and mechanically ventilated with 100% oxygen (ventilation rate, 25 breaths/min; tidal volume, 25 ml). A catheter was positioned in the rabbit's ear marginal artery for arterial pressure measurement (Statham P23ID strain gauge; Statham Instruments, Oxnard, CA). An external ECG was also recorded. A left thoracotomy was performed at the 4th intercostal space under sterile conditions. The pericardium was opened and a 4/0 Prolene suture was passed beneath a major branch of the left coronary artery. The ends of the ligature were passed through a short segment of propylene tubing to form a snare. Regional myocardial ischemia was induced by pulling the snare through the tubing. Ischemia was confirmed by the presence of regional modifications of the myocardial surface and by the occurrence of ST segment deviation of the ECG. In all animals, a coronary artery occlusion (CAO) was performed and the snare was released. The chest was then closed in layers and a small tube was left in the thorax to evacuate air and fluids after surgery. All rabbits underwent 72 h of coronary artery reperfusion.
Measurement of Risk Area and Infarct Size. After completion of reperfusion, animals received heparin and sodium pentobarbital (50 mg/kg i.v.). Potassium chloride was then administered i.v. to induce cardiac arrest. The hearts were excised. The ascending aorta was cannulated and perfused (120 mm Hg) retrogradely with saline followed by Evans blue (1%) after ligation of the previously occluded artery. The left ventricle was cut into 8 to 10 slices. These slices were weighed and incubated with 1% triphenyltetrazolium chloride (TTC; Sigma Chemical, Poole, Dorset, UK) in a pH 7.4 buffer during 15 min at 37°C. Slices were overnight fixed in 10% formaldehyde and then photographed with a digital camera. Using a computerized planimetric program (Scion Image; Scion Corporation, Frederick, MD), the area at risk and the infarcted zones were quantified. The area at risk was identified as the nonblue region and was expressed as a percentage of the left ventricle weight. Infarcted area was identified as the TTC-negative zone and was expressed as a percentage of the area at risk.
Experimental Protocol. The animals were randomized and the protocol was realized during two consecutive days, i.e., 24 h apart as illustrated in Fig. 1. On day 1, rabbits were divided into three groups receiving a 75-min intravenous infusion of either saline (control group), S-nitroso-N-acetylpenicillamine at 3 µg/kg/min (SNAP-3 group), or S-nitroso-N-acetylpenicillamine at 30 µg/kg/min (SNAP-30 group). These doses of SNAP were chosen on the basis of a preliminary study showing that 3 µg/kg/min did not induce any hemodynamic effect, whereas 30 µg/kg/min significantly decreased mean arterial pressure (–14% from 89 ± 3 mm Hg). On day 2, all rabbits underwent either a 15-, 20-, or a 30-min CAO followed by 72-h reperfusion.
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Statistical Analysis. Data are reported as mean ± S.E.M. Comparisons were made using analysis of variance followed by post hoc Fisher's protected least significant difference test, if necessary. Significant differences were determined as p < 0.05.
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Results |
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Hemodynamic. On day 1, baseline values of heart rate and mean arterial pressure were not significantly different between groups (heart rate, 202 ± 8, 196 ± 12, and 197 ± 5 beats/min; and mean arterial pressure, 99 ± 4, 95 ± 4, and 97 ± 3 mm Hg for all control, SNAP-3, and SNAP-30, respectively). Infusion of saline and SNAP at 3 µg/kg/min did not significantly affect these parameters (heart rate, 208 ± 9 beats/min, and mean arterial pressure, 98 ± 3 mm Hg, for SNAP-3). SNAP at 30 µg/kg/min reduced mean arterial pressure by 15% and increased heart rate by 25% (82 ± 4 mm Hg and 247 ± 7 beats/min, respectively; p < 0.05). On day 2, these parameters were not significantly different between all groups at baseline, during CAO and reperfusion (data not shown).
Infarct Sizes. Sizes of area at risk were similar among groups (Table 1). After 15-min CAO, infarct sizes were significantly reduced by SNAP-3 and by SNAP-30 compared with control (Fig. 2). After 20-min CAO, SNAP-3 failed to reduce infarct size whereas SNAP-30 still exerted beneficial effects. After 30-min CAO, infarct sizes were similar among groups. As illustrated in Fig. 3, the intensity of cardioprotection (i.e., reduction of mean infarct size versus control) was plotted against the duration of CAO. Interestingly, SNAP-30 provided a near maximal cardioprotection after 15-min CAO (–84%). However, a ceiling of protection was observed with SNAP-30 after 30-min CAO and also with SNAP-3 after 20-min CAO.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Tissier et al., 2002).
In literature, the evaluation of the limiting effect of any procedure on
infarct size is usually performed using a single duration of CAO. However,
Gumina et al. (1999)
demonstrated in dogs that two different cardioprotective procedures (i.e., an
Na+/H+ exchange inhibitor and early preconditioning)
might be equipotent to reduce infarct size after a 60-min CAO but not after
90-min CAO. Thus, the use of a single CAO duration does not provide complete
information about the cardioprotective potency of a drug or a preconditioning
maneuver. Accordingly, we investigated SNAP-induced delayed preconditioning
with three different durations of CAO in the present study. Although this
pharmacological strategy was able to exert a potent cardioprotective effect
after short periods of ischemia, the magnitude of cardioprotection
significantly diminished with increasing durations of CAO and hence with
subsequent increasing in infarct size. This study clearly demonstrates a
ceiling of cardioprotection with delayed preconditioning as it was
demonstrated with early ischemic preconditioning
(Gumina et al., 1999).
Interestingly, the time to reach this ceiling of protection with SNAP was
dose-dependent and therefore related to the intensity of the preconditioning
stimulus. One can argue that higher doses of SNAP would have further delay the
time to reach the ceiling point but regarding the potent and undesirable
hypotensive effect of higher doses than 30 µg/kg/min of SNAP, it was
difficult to test this hypothesis.
Many parameters such as anesthesia, the sedation protocol in conscious
preparations (e.g., use of diazepam), the size of area at risk
(Ytrehus et al., 1994), and
strains of animals (Bao et al.,
2000) may modify the myocardial oxygen balance, the severity of
ischemia, and hence the susceptibility to infarction. It is thus interesting
to speculate that different experimental conditions would lead to different
patterns of ceiling of protection. Indeed, after 30-min CAO, our present and
previous results (Lellouche et al.,
2002) failed to demonstrate any protective effect of SNAP, whereas
others reported a significant reduction in infarct size in rabbits
(Takano et al., 1998).
However, the investigation of increasing durations of CAO allows to avoid
discrepancies between studies and leads to the same final conclusion, i.e.,
delayed preconditioning with SNAP is effective at reducing infarct size, even
after 72 h of reperfusion. Therefore, the use of a single duration of CAO is
certainly one explanation to account for discrepancies observed between
studies regarding the cardioprotective effects of a drug or of a
preconditioning maneuver.
Importantly, it is well known that the prolongation of ischemia is
responsible for an increase in infarct size that will tend to a maximal value,
i.e., about 80 to 90% of the area at risk in rabbits
(Miura et al., 1989).
Preventive cardioprotective strategies tend to be inefficient when this
maximal value is reached, e.g., after 60- to 90-min CAO in native collateral
deficient species such as rabbit (Miura et
al., 1989). In contrast, in the present study, the loss of
protection conferred by delayed preconditioning with SNAP is different in its
mechanism. Indeed, the ceiling of protection occurs with infarct sizes
averaging 50% of the area at risk, i.e., far from the maximal possible infarct
size (Miura et al., 1989). In
other words, delayed preconditioning with SNAP cannot provide in all cases
additional salvage of myocardial tissue to the beneficial effects of
reperfusion, i.e., it will be beneficial only with early revascularization.
Consequently, it becomes important not only to define the ability of a drug to
reduce infarct size but also to characterize its ceiling of protection, i.e.,
the duration of ischemia and the infarct size at which protection is lost.
Although we did not specifically investigate this issue, it is tempting to
speculate that the concept of ceiling of protection could be extended to other
stimuli known to induce delayed preconditioning, e.g., brief ischemia or
adenosine, which partially share common signaling pathways with NO donors
(Bolli, 2000). Indeed, both
ischemic and pharmacological delayed preconditioning with an adenosine
A1-receptor agonist were unable to reduce infarct size after 30-min
CAO and 72-h reperfusion in rabbits (Miki
et al., 1999; Lellouche et al.,
2002; Tissier et al.,
2002). These data suggest once again that a ceiling of protection
was reached but additional studies are required to definitely extend this
concept to all kind of delayed preconditioning. Investigation of the
mechanisms responsible for a ceiling of protection were beyond the scope of
this study. One could speculate, however, that differential interactions exist
among the numerous mediators of cardioprotection and those of myocardial
injury with varying durations of ischemia.
In conclusion, our results demonstrate that delayed preconditioning induced by the NO donor SNAP reaches a ceiling of protection against myocardial infarction in rabbits. Moreover, our results support the necessary use of different durations of ischemia when investigating cardioprotective strategies. This finding also has potential important clinical implications supporting that delayed preconditioning might exert its optimal beneficial effect mainly with early revascularization.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: SNAP, S-nitroso-N-acetylpenicillamine; CAO, coronary artery occlusion; TTC, triphenyltetrazolium chloride.
Address correspondence to: Prof. A. Berdeaux, Laboratoire de Pharmacologie, INSERM E 00.01, Faculté de Médecine Paris-Sud, 63, rue Gabriel Péri, 94270 Le Kremlin-Bicêtre, France. E-mail: alain.berdeaux{at}kb.u-psud.fr
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