A brief period of ischemia followed by reperfusion has been shown to reduce subsequent ischemia/reperfusion-induced damage to the myocardium, which is recognized as "ischemic preconditioning" (1,2). Preconditioning effect is of interest because it is an endogenous adaptation of the heart against lethal ischemia/reperfusion injury. In a rat model, cardioprotective effects of preconditioning have been demonstrated in terms of improvement of postischemic mechanical dysfunction (3-6), minimization of arrhythmias (7-10), and limitation of myocardial infarction (11,12). Although similar improvements were observed in the preconditioned hearts, the mechanisms underlying preconditioning protection may be different in each cardiac injury (12-14).
Recent observations show that translocation from cytosolic fraction to membrane and subsequent activation of protein kinase C play an important role in intracellular signaling for the preconditioning effects (15-19). To activate protein kinase C-mediated signaling, stimulation of α1-adrenoceptor is one of the essential events. In fact, α1-adrenoceptor seems to contribute to the preconditioning cardioprotection (20,21). However, contribution of the β-adrenergic system to the mechanism for preconditioning cardioprotection against postischemic contractile dysfunction has not been fully understood.
Several lines of evidence have suggested that myocardial ischemia increases catecholamine levels in plasma (22,23), which leads to exacerbation of ischemia-induced myocardial damage (24,25). Treatment with β-adrenoceptor blocking agents has been shown to ameliorate myocardial ischemic damage (26), probably because of a reduction in myocardial oxygen consumption during ischemia. In contrast, stimulation of β-adrenoceptor for a long period increases cardiac work load, which leads to an imbalance of myocardial oxygen supply and demand, resulting in an induction of global ischemia in the myocardium (27-29). However, a transient increase in cardiac work load, similar to the insult of ischemic preconditioning, would be expected to exert the cardioprotective effect against subsequent prolonged ischemia and reperfusion injury without an interruption of blood flow. In fact, overdrive pacing of the heart has been shown to induce preconditioning effect in dogs (30) and rabbits (31). Therefore it is possible that pharmacologic stimulation of the myocardial β-adrenergic system also exerts the cardioprotective effect. The purpose of our study was to investigate whether β-adrenergic stimulation exerts a cardioprotective effect as does ischemic preconditioning.
MATERIALS AND METHODS
Male Sprague-Dawley rats weighing 280-360 g (SLC Co., Shizuoka, Japan) were used in these experiments. Rats were fed with a standard diet and acclimated in a quarantine room for ≥1 week before experiments. The animal protocol was designed according to the Guideline of Experimental Animal Care issued from the Prime Minister's Office of Japan.
Phenylephrine hydrochloride and timolol maleate were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Bunazosin hydrochloride was a generous gift from Eisai Co., Ltd. (Tokyo, Japan). Norepinephrine bitartrate and dl-isoproterenol hydrochloride were purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). Creatine kinase (CK) assay kit (CK NAC) was obtained from Boehringer Mannheim, Mannheim, Germany.
Perfusion of the isolated rat heart
Rats were anesthetized with diethylether, and their hearts were quickly isolated and perfused in a Langendorff mode at a constant flow rate of 9.0 ml/min with Krebs-Henseleit bicarbonate solution with the following composition (mM); NaCl, 120.0; KCl, 4.8; CaCl2, 1.25; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; and glucose, 11.0; pH 7.4. The solution was equilibrated with a gas mixture of 95% O2 and 5% CO2. A water-filled latex balloon with an uninflated diameter of 3.7 mm (Hirokawa Seisakujo, Co., Ltd., Niigata, Japan) was inserted into the left ventricle through the left atrium to measure left ventricular developed pressure (LVDP). Left ventricular end-diastolic pressure (LVEDP) was adjusted to 5 mm Hg. LVDP and heart rate (HR) were measured by means of a pressure amplifier (AP-621G; Nihon Kohden, Tokyo, Japan) and a heart-rate counter (AT-601G; Nihon Kohden), respectively. The rate-pressure product (RPP) was calculated by multiplying LVDP and HR. Coronary perfusion pressure (CPP) was monitored through a branch of an aortic cannula with an electric manometer (TP-400T; Nihon Kohden) connected to a carrier amplifier (AP-621G; Nihon Kohden). Experiments were begun after an initial 30-min stabilization.
The schematic protocol of this study is shown in Fig. 1. After equilibration periods, the hearts were randomly divided into the following groups: (group a) vehicle-treated group: hearts perfused for 2 min with vehicle followed by 10 min of perfusion with normal Krebs-Henseleit buffer (n = 7); (group b) norepinephrine-treated group: hearts perfused for 2 min with norepinephrine (0.25 μM) followed by 10 min of drug-free perfusion (n = 6); (group c) phenylephrine-treated group: hearts perfused for 2 min with phenylephrine (10 μM) followed by 10 min of drug-free perfusion (n = 5); and (group d) isoproterenol-treated group: hearts perfused for 2 min with isoproterenol (0.25 μM) followed by 10 min of isoproterenol-free perfusion (n = 6). Either norepinephrine, phenylephrine, or isoproterenol was applied directly to the heart for 2 min at an infusion rate of 0.2 ml/min through a syringe located just anterior to aortic cannula, by using an infusion pump (Syringepump STC-523; Terumo Co., Ltd., Tokyo, Japan). In a preliminary study, we examined RPP recovery of the ischemic/reperfused hearts pretreated with different concentrations (0.1, 0.25, and 1 μM) of either norepinephrine or isoproterenol. We found that norepinephrine and isoproterenol at a concentration of 0.25 μM produced the largest recovery of the RPP in the ischemic/reperfused hearts. The concentration of phenylephrine used (10 μM) was sufficient to activate myocardial α1-adrenoceptor (32,33). Agents were dissolved in the Krebs-Henseleit buffer, pH 7.0, just before use. Infusion of 0.2 ml/min vehicle did not alter the pH of the perfusion buffer.
After this treatment, the hearts were subjected to 40 min of ischemia. To achieve global ischemia in hearts, the perfusion was stopped and the hearts were submerged in a chamber filled with the Krebs-Henseleit buffer as previously described, except that it contained 11 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride (TRIS-HCl) to replace 11 mM glucose, and maintained at 37°C to prevent hypothermia-induced cardioprotection. Ischemic contracture was taken as a rise in LVEDP after the onset of ischemia. During ischemia, changes in LVEDP were measured to determine the time to onset of ischemic contracture and an increase in LVEDP. The time to contracture was defined as the time during ischemia for LVEDP to elevate >5 mm Hg (34,35). After 40 min of ischemia, the buffer in the chamber was drained and the hearts were reperfused for 30 min with an aerobic buffer. Effluent from the hearts was collected during reperfusion, and the CK activity in the effluent was determined according to the method of Bergmeyer et al. (36).
To examine the effect of β-adrenoceptor blocker, timolol (5 μM) was applied to the hearts 3 min before norepinephrine infusion until the onset of 40-min ischemia, and the hearts were subjected to 40 min of ischemia and 30 min of reperfusion (group e): norepinephrine/timolol-treated group (n = 5). Furthermore, to clarify a possible role in β-adrenoceptor stimulation as a trigger for exerting the ischemic preconditioning-like cardioprotective action in the norepinephrine-treated heart, timolol was applied to the heart only during the preischemic period. In our experiment, timolol, a nonselective β-adrenoceptor blocker that does not possess membrane-stabilizing action was used because β-adrenoceptor blockers with membrane-stabilizing action per se have been shown to exert a cardioprotective effect in the perfused rat heart (37,38). The effect of timolol alone on the ischemic/reperfused heart also was examined (group f): timolol-treated group (n = 4).
To examine the effect of an α1-adrenoceptor blocker, bunazosin (1 μM) was applied to the hearts from 3 min before isoproterenol infusion until the end of 30 min of reperfusion, and the hearts were subjected to 40 min of ischemia and 30 min of reperfusion: (group g) isoproterenol/bunazosin-treated group (n = 4). Furthermore, to examine an involvement of α1-adrenoceptor activation in the cardioprotection during an entire period of perfusion, bunazosin was applied to the heart during both preischemic and postischemic periods. The effect of bunazosin alone on the ischemic/reperfused heart also was examined: (group h) bunazosin-treated group (n = 4).
In another series of experiments, hearts were preconditioned with 5 min of ischemia followed by 5-min reperfusion and then subjected to 40 min of ischemia and 30 min of reperfusion as described previously: (group i) preconditioned group (n = 7). Nonpreconditioned hearts without any treatment were subjected to 40 min of ischemia followed by 30 min of reperfusion: (group j) nonpreconditioned group (n = 6). Total perfusion time was matched with the appropriate preconditioned hearts.
Ischemic preconditioning used in this study consisted of one cycle of zero-flow global ischemia for 5 min followed by 5 min of reperfusion. A greater recovery of the postischemic function was obtained by this preconditioning. In the pharmacologically preconditioned heart, each drug was applied to the heart for 2 min and then the heart was subjected to drug-free perfusion for 10 min. Thus the time for pharmacologic preconditioning was not equal to that for ischemic preconditioning. We found that this procedure elicited a better recovery of the ischemic/reperfused hearts on reperfusion than did others.
All values are expressed as mean ± SEM. Statistical differences among groups were estimated by using one-way analysis of variance followed by either Dunnett's or Bonferroni's multiple-comparison test. Student's paired t test was used for intragroup comparison. Student's unpaired t test was used for comparisons between two groups. Values of p < 0.05% were considered statistically significant.
Effects of norepinephrine, phenylephrine, and isoproterenol on ischemic/reperfused hearts
In the first series of experiments, the effects of norepinephrine, phenylephrine, and isoproterenol on the hearts subjected to 40-min ischemia followed by 30-min reperfusion were studied. Changes in LVDP, LVEDP, HR, and CPP are shown in Figs. 2a-d, respectively. Baseline values of cardiac variables were not significantly different among groups. During ischemia, LVEDP of the hearts gradually increased, indicating the development of ischemic contracture. In the vehicle-treated hearts, despite a considerable recovery of HR (171 ± 28 beats/min at the end of reperfusion), there was little restoration of LVDP during reperfusion (14.3 ± 2.1 mm Hg at the end of reperfusion). During reperfusion, CPP and LVEDP of the vehicle-treated hearts were increased as compared with their initial values. In this model, there were very few arrhythmias except for sudden irregular contraction (one of 20).
Treatment with norepinephrine before a prolonged period of ischemia markedly increased LVDP and decreased CPP. Withdrawal of norepinephrine rapidly reduced the increased LVDP below the baseline level. On reperfusion, LVDP and HR recovered almost to their preischemic levels, and the increase in LVEDP was attenuated in the norepinephrine-treated heart. At the end of reperfusion, the values for LVDP, HR, and LVEDP were 71.2 ± 9.9 mm Hg, 242 ± 11 beats/min, and 44.3 ± 7.6 mm Hg, respectively.
LVDP was increased by treatment with phenylephrine, whereas HR and CPP remained unchanged in the preischemic heart. In contrast to that with norepinephrine, the increased LVDP returned to the initial baseline level at 10 min of phenylephrine-free perfusion. Phenylephrine neither enhanced the restoration of LVDP nor preserved the increase in LVEDP on reperfusion. In a preliminary study, a lower concentration of phenylephrine (0.25 μM) also failed to enhance the recovery of RPP on reperfusion (8.0 ± 2.7% recovery of its initial value; n = 4).
Treatment with isoproterenol alone markedly increased the LVDP and HR of the preischemic heart. During reperfusion after 40-min ischemia, the recovery of the LVDP and HR was enhanced, and the increases in LVEDP and CPP were attenuated in the isoproterenol-treated heart.
Effects of adrenergic blocking agents on the cardioprotection elicited by either norepinephrine or isoproterenol treatment
Effects of timolol on norepinephrine-induced changes in LVDP, HR, and CPP of the preischemic heart are shown in Table 1. Treatment with timolol per se did not alter cardiac parameters in the preischemic heart. Timolol attenuated increases in LVDP and HR elicited by norepinephrine infusion. CPP was markedly increased by norepinephrine infusion in the presence of timolol. Treatment with timolol alone during preischemia neither reduced cardiac function nor stimulated the recovery of contractile function during reperfusion: the recovery of RPP at the end of reperfusion was 14.3 ± 6.9%. In the presence of bunazosin, there were no appreciable changes in cardiac parameters induced by isoproterenol infusion (data not shown). Treatment with bunazosin alone during both preischemic and postischemic periods did not affect the recovery of RPP on reperfusion: the recovery of RPP at the end of reperfusion was 2.3 ± 1.1%.
Percentage recovery of the RPP of these treated animals at the end of reperfusion is shown in Fig. 3. After reperfusion, restoration of the RPP afforded by norepinephrine was blocked by pretreatment with timolol. Treatment with bunazosin did not affect isoproterenol-induced enhancement of recovery of the RPP.
Effects of ischemic preconditioning on cardiac parameters
Changes in LVDP, LVEDP, HR, and CPP of nonpreconditioned and ischemic preconditioned hearts are shown in Fig. 4. Five minutes of ischemia resulted in decreases of LVDP, HR, and CPP without any change in LVEDP. The decreased LVDP did not completely recover to the baseline level after 5 min of reperfusion. In both nonpreconditioned and preconditioned hearts, LVDP, HR, and CPP decreased to the lowest levels during 40 min of ischemia. The recovery of LVDP was enhanced in the ischemic preconditioned heart as compared with that in the nonpreconditioned heart. An increase of LVEDP during reperfusion was attenuated in the preconditioned heart. The effects of ischemic preconditioning on changes in LVDP, LVEDP, and HR during reperfusion were comparable with those of either norepinephrine or isoproterenol treatment.
Time to onset of ischemic contracture
The results on time to onset of ischemic contracture are shown in Fig. 5. In both norepinephrine- and isoproterenol-treated hearts, time to the onset of ischemic contracture was significantly shortened as compared with that in the vehicle-treated hearts. The time to contracture was significantly shorter in the preconditioned heart than in the nonpreconditioned heart. Shortening of the time to onset of ischemic contracture in both norepinephrine- and isoproterenol-treated hearts was similar to that observed in the preconditioned heart.
Release of creatine kinase
There was no appreciable release of CK [<0.5 nmol reduced nicotinamide adenine dinucleotide phosphate (NADPH)/min/g wet wt] in the effluent of the heart perfused under normoxic conditions for 90 min. Any pharmacologic intervention per se did not affect the amount of basal release of CK (data not shown). Release of CK during reperfusion is shown in Fig. 6. The release of CK was markedly increased during reperfusion after a prolonged period of ischemia (54.1 ± 5.2 nmol NADPH/min/g wet wt for vehicle-treated group). The values for CK release in norepinephrine-, isoproterenol-, and phenylephrine-treated hearts were 29.9 ± 1.0, 31.3 ± 4.4, and 46.0 ± 5.9 nmol NADPH/min/g wet wt, respectively. Treatment with either norepinephrine or isoproterenol, but not phenylephrine, reduced the release of CK into the effluent. Pretreatment with timolol abolished the reduction of CK release afforded by norepinephrine treatment, whereas that with bunazosin did not affect the decrease in CK release during reperfusion afforded by isoproterenol treatment. The release of CK also was reduced in the preconditioned heart (28.8 ± 1.5 nmol NADPH/min/g wet wt) as compared with that in the nonpreconditioned heart (54.1 ± 5.2 nmol NADPH/min/g wet wt).
The major finding of our study is that 2-min perfusion with either norepinephrine or isoproterenol, but not phenylephrine, followed by 10-min drug-free perfusion before prolonged ischemia/reperfusion restored postischemic contractile function and attenuated CK release similar to ischemic preconditioning. In addition, the norepinephrine-induced protective effect was completely abolished by β-adrenoceptor blocker timolol, whereas the isoproterenol-induced effect was not blocked by the α1-adrenoceptor blocker bunazosin. The results suggest that preceding stimulation of myocardial β-adrenoceptor, but not α1-adrenoceptor, induces a cardioprotective effect against subsequent prolonged ischemia/reperfusion-induced injury in the isolated rat heart. This effect is similar to that of ischemic preconditioning with respect to following: (a) the time to ischemic contracture was shortened, (b) the LVDP was recovered almost to the preischemic level on reperfusion, (c) the elevation of LVEDP during reperfusion was attenuated, and (d) the CK release was attenuated.
Although the onset of ischemic contracture has been suggested to be a sign of irreversible cell damage (39), time to contracture during ischemia was decreased by preceding β-adrenoceptor stimulation as well as ischemic preconditioning. This phenomenon may be attributed to an early reduction in the content of glycolytic substrates because ischemic contracture was considered to begin when glycolytic adenosine triphosphate (ATP) production was reduced (40,41). In addition, depletion of glycogen content before prolonged ischemia plays an important role in the postischemic contractile recovery (42) and the protective effect of preconditioning (43). Shortening of the time to ischemic contracture during prolonged ischemia seems to be a typical phenomenon of the hearts afforded by preconditioning insults (11,44,45). It is suggested, therefore, that transient stimulation of β-adrenoceptor renders signaling to the myocardium similar to that of ischemic preconditioning.
As is well known, β-adrenoceptor blocking agents exert cardioprotective effects in ischemic/reperfused hearts. In contrast, a prolonged stimulation of myocardial β-adrenoceptor with isoproterenol results in the myocardial ischemic damage because of an increased cardiac work load (27,28). In both norepinephrine- and isoproterenol-treated hearts, the RPP once increased markedly during drug treatment and then decreased below the initial level after drug-free perfusion, suggesting that the heart was susceptible to global ischemia by an increase in the cardiac work load caused by transient activation of myocardial β-adrenoceptor. Consequently, a short period of infusion of either norepinephrine or isoproterenol followed by the drug-free perfusion, which may be similar to an ischemic preconditioning episode, appears to be beneficial against ischemia/reperfusion injury.
Several possible mechanisms underlying such a phenomenon are considered. One possible mechanism is metabolic alteration of the myocardium caused by β-adrenoceptor stimulation. Depletion of glycogen and reduced ATP content before prolonged ischemia have been shown to attenuate the ischemic damage because they reduce intracellular acidosis (42,46). An increase in cardiac work, accompanied by a marked elevation of myocardial oxygen consumption, may result in a decrease of endogenous energy substrates such as glycogen and high-energy phosphates in the preischemic heart, because supply of energy substrates and oxygen did not alter under the constant flow perfusion in our study even when cardiac work was markedly increased by the stimulation of β-adrenoceptor. In addition, in the presence of timolol, norepinephrine-induced increase in cardiac work was reduced, and then its protective effect disappeared, probably because of the lack of β-adrenoceptor stimulation-induced metabolic alterations such as glycogen depletion. These metabolic changes may play a key role in producing the β-adrenoceptor-mediating, preconditioning-mimetic protective effect in the ischemic/reperfused heart.
Another possibility is that transient stimulation of β-adrenoceptor may change a receptor function. β-Adrenoceptor function is regulated by phosphorylation via β-adrenoceptor kinase, and the receptor phosphorylation leads to its desensitization (47). Catecholamine release is increased in the ischemic myocardium, and the resulting stimulation of cardiac β-adrenoceptor exacerbates ischemic damage. If desensitization of receptor occurs before prolonged ischemia, ischemic damage would be expected to be lessened.
A notable finding in our experiment is the lack of involvement of α1-adrenoceptor in the adrenoceptor-mediated cardioprotection, including the lack of the effects of phenylephrine on restoration of the RPP during reperfusion, the cardioprotective effect of isoproterenol in the presence of α1-adrenoceptor blocker, and the abolishment of the protective effect of norepinephrine by β-adrenoceptor blocker. Activation of myocardial α1-adrenoceptor has been shown to play a role in protective effects of ischemic preconditioning in dogs (21) and rabbits (48). Banerjee et al. (20) found that in the isolated rat heart, α1-adrenoceptor blockade with phentolamine eliminated the preconditioning effect, and they suggested that the beneficial effects of ischemic preconditioning were mediated by release of norepinephrine and stimulation of α1-adrenoceptors. In addition, Tosaki et al. (33) suggested the "pharmacologic" preconditioning occurred by phenylephrine infusion. These observations, particularly those in isolated rat hearts, are in conflict with our findings.
Several possible explanations for the discrepancy are considered. According to the report by Banerjee et al. (20), they used 20 min of ischemia and found no difference between preconditioned and nonpreconditioned hearts with regard to the time to ischemic contracture during ischemia and CK release into effluent on reperfusion. Recovery of LVDP after reperfusion in the nonpreconditioned heart was 57% of its initial value (20). Furthermore, Tosaki et al. (33) reported ∼70% recovery of LVDP on reperfusion after 30 min of ischemia in the drug-untreated working rat heart. These results indicate that the degree of their ischemic damage may be relatively weak compared with ours, because we used an ischemic period of 40 min, which resulted in only ≤15% recovery of postischemic LVDP and a marked increase in CK release during reperfusion. It should be noted that Suzuki et al. (49) suggested that β-adrenoceptor activation-induced cardioprotection was necessary for a relatively long ischemic period. Therefore our experimental conditions of a 40-min ischemic period, which produced more severe ischemic injury, may be too severe to exert α1-adrenoceptor-mediated cardioprotective effects. Different protective mechanisms may be induced by different preconditioning episodes even in the same animal species. It is likely that β-adrenoceptor-mediated signaling can offer the heart greater tolerance against ischemic/reperfusion injury than can α1-adrenoceptor stimulation in rats.
In addition, Banerjee et al. (20) used the perfused rat heart under constant pressure, and Tosaki et al. (33) used working heart perfusion; thereby coronary flow is changeable in response to interventions such as preconditioning insult and pharmacologic stimulation. Our results showed that phenylephrine or norepinephrine in the presence of timolol exerted coronary vasoconstriction by coronary α1-adrenoceptor stimulation during its infusion. Subsequent reduction in CPP, coronary vasodilation after washout of phenylephrine, may be an overcompensation of the myocardium after vasoconstriction. However, under perfusion with constant flow, oxygen delivery to myocardium would not be increased to meet the oxygen demand of the heart. Thus the lack of an increase in oxygen delivery may be another mechanism by which α1-adrenoceptor stimulation failed to induce cardioprotection under our experimental conditions. Tsuchida et al. (48) suggested that although α1-adrenoceptor stimulation could precondition the myocardium, receptors themselves did not participate in the preconditioning phenomenon, supporting our previous view.
Our study demonstrates that activation of myocardial β-adrenoceptor, but not α1-adrenoceptor, induces the preconditioning-mimetic protection in the ischemic/reperfused rat hearts. More recently, Asimakis et al. (50) reported that transient stimulation of β-adrenoceptor with dobutamine, a selective β1-adrenoceptor stimulant, could precondition the perfused rat heart. It is suggested, therefore, that an activation of myocardial β-adrenoceptor, possibly β1-receptor, plays an important role in the mechanism of such pharmacologic preconditioning effects.
Acknowledgment: This work was supported in part by a grant from The Vehicle Racing Commemorative Foundation of Japan.
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