The aims of this study were to determine whether 1) like ischemic preconditioning, transient exposure to norepinephrine before ischemia exacerbates contracture during ischemia and 2) protection afforded by norepinephrine is stereospecific (receptor mediated). Isolated perfused rat hearts were randomized into five groups (n = 6/group):1) ischemic preconditioning (3 min of ischemia + 3 min of reperfusion + 5 min of ischemia + 5 min of reperfusion), 2) untreated control, 3) vehicle control (ascorbic acid), 4) substitution of preconditioning ischemia by perfusion withd-norepinephrine, and5) substitution of preconditioning ischemia by perfusion withl-norepinephrine. This was followed by 40 min of zero-flow ischemia and 50 min of reperfusion. Ischemic preconditioning andl-norepinephrine exacerbated contracture (time to 50% contracture = 9.2 ± 1.1 and 9.0 ± 1.1 vs. 13.3 ± 0.3, 12.4 ± 0.5, and 13.2 ± 0.4 min for untreated control, vehicle control, andd-norepinephrine, respectively,P < 0.05). Postischemic left ventricular developed pressure was poor in untreated control (23.0 ± 2.2%), vehicle control (26.9 ± 2.3%), andd-norepinephrine (19.8 ± 2.8%) groups but good in preconditioned (52.4 ± 5.1%) andl-norepinephrine (52.5 ± 1.1%) groups (P < 0.05). Thus norepinephrine preconditioning, like ischemic preconditioning, causes a paradoxical exacerbation of contracture coupled with enhanced postischemic recovery; both effects are stereospecific.
it is widely accepted that it is possible to protect the heart against the detrimental effects of an extended period of ischemia by prior exposure to one or more brief periods of ischemia. This protection may be manifest as a limitation of infarct size, a reduction of arrhythmias, or an improvement of postischemic function (8, 15, 18, 20). It is also well established that similar protection can be achieved by substituting the preconditioning ischemia with similar periods of transient perfusion with catecholamines or other agents that are capable of activating a variety of cardiac membrane receptors. There is a substantial body of evidence supporting the hypothesis that adrenergic stimulation is an important component mechanism in the protection afforded by preconditioning. Thus adrenergic agonists, substituting for ischemic preconditioning, protect against postischemic myocardial dysfunction (1, 14, 17), reduce infarct size (2, 22, 26), and reduce the incidence of reperfusion-induced ventricular fibrillation and ventricular tachycardia (23). However, although it is frequently assumed that this catecholamine-induced protection is achieved via an adrenergic receptor-mediated mechanism, other than studies using α1-antagonists (1), no studies have sought to confirm this or determine whether some secondary (non-receptor-mediated) mechanism might be involved. One approach to test such a possibility would be to exploit the chiral nature of norepinephrine, which exists in two forms:d-norepinephrine (inactive at the receptor) and l-norepinephrine (active). Routine laboratory norepinephrine is supplied as a racemic mixture of d- andl-norepinephrine. However, the two optical isomers are available separately and, as used in the present study, are invaluable for distinguishing receptor-mediated from non-receptor-mediated events.
Although preconditioning with ischemia enhances postischemic functional recovery, it has a paradoxical effect of exacerbating contracture during ischemia (6, 9, 11). It is not known whether the mechanism of this unexpected consequence of ischemic preconditioning is an unrelated epiphenomenon or a necessary component of the preconditioning mechanism. One possibility is that the exacerbation of contracture is a consequence of the antecedent preconditioning ischemia, which, although unfavorable, can be overcome by the greater protective power of the phenomenon. One approach to the clarification of this issue would be to study the effect on ischemic contracture of pharmacological preconditioning procedures (such as the use of catecholamines) that do not involve antecedent ischemia. Therefore, in the present rat heart study we also compared the effects on ischemic contracture of preconditioning with ischemia vs. pharmacological preconditioning withd- orl-norepinephrine.
Animals and Chemicals
Male Wistar rats (200–300 g) were obtained from B & K Universal. All animals received humane care in compliance with thePrinciples of Laboratory Animal Careformulated by the National Society for Medical Research and theGuide for the Care and Use of Laboratory Animals [DHHS Publ. No. (NIH) 86-23]. Ascorbic acid (to prevent oxidation of norepinephrine) was obtained from Sigma Chemical, and l- andd-norepinephrine were obtained from Fluka.
Buffer-Perfused Rat Heart Preparation
Rats were anesthetized with pentobarbital sodium (60 mg/kg ip) and anticoagulated with heparin (1,000 IU/kg iv). Hearts were immediately excised and immersed in cold (4.0°C) perfusion buffer (for composition see below). The aorta was rapidly cannulated, and each heart was perfused in the Langendorff mode (constant pressure equivalent to 100 cmH2O) with bicarbonate buffer at 37.0°C. The buffer contained (in mM) 118.5 NaCl, 25.0 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 11.0 glucose, and 1.4 CaCl2 and was gassed with 95% O2-5% CO2 (pH 7.4).
After removal of the left atrial appendage, a fluid-filled balloon catheter, attached to a pressure transducer, was introduced into the left ventricle via the mitral valve. The balloon was inflated with water until a stable left ventricular end-diastolic pressure of 4–6 mmHg was obtained, and balloon volume was not altered thereafter. The pressure transducer was connected to a four-channel chart recorder, which was run continuously. Each heart was surrounded by a thermostatically controlled water-jacketed chamber to maintain its temperature at 37.0 ± 0.2°C throughout the experiment.
Hearts were aerobically perfused for 10 min at 37.0 ± 0.2°C, during which time left ventricular developed pressure (LVDP), heart rate, and coronary flow were measured. Heart rate was measured by running the recorder at a speed that enabled the individual heartbeats to be counted. Coronary flow was measured by the timed collection of the coronary effluent. Hearts were randomly assigned to five groups (n = 6/group):1) untreated controls subjected to a further 16 min of aerobic perfusion, i.e., no intervention before ischemia; 2) ischemic preconditioned hearts subjected to 3 min of ischemia + 3 min of reperfusion followed by 5 min of ischemia + 5 min of reperfusion; 3) vehicle control hearts perfused with buffer containing 0.1 mM ascorbic acid, an antioxidant that was included in alld- andl-norepinephrine solutions to prevent their oxidation (i.e., 3 min of perfusion + 3 min of washout followed by 5 min of perfusion + 5 min of washout);4)d-norepinephrine-treated hearts perfused with buffer containingd-norepinephrine (2.5 nmol/min), rather than ischemic preconditioning (i.e., 3 min of perfusion + 3 min of washout followed by 5 min of perfusion + 5 min of washout); and5)l-norepinephrine-treated hearts perfused with buffer containingl-norepinephrine (2.5 nmol/min), rather than ischemic preconditioning (i.e., 3 min of perfusion + 3 min of washout followed by 5 min of perfusion + 5 min of washout). LVDP, heart rate, and coronary flow were recorded during all treatment periods. Hearts were then subjected to 40 min of normothermic (37.0°C), global ischemia, during which time each heart was immersed in buffer. The pre- and postischemic LVDP and development of contracture during ischemia were recorded by means of the intraventricular balloon. The time to onset of contracture was taken as the time at which the left ventricular end-diastolic pressure increased by 4 mmHg from the value recorded after 2 min of ischemia, and the time to 50% contracture was taken as the time taken to achieve one-half of its peak reading. Finally, hearts were reperfused for 50 min, during which time LVDP, heart rate, and coronary flow were monitored (Fig. 1).
Drug Preparation and Delivery
Norepinephrine was delivered via a peristaltic pump (Gilson Miniplus 3) attached to a sidearm of the aortic cannula. To deliverl- andd-norepinephrine at the required rate of 2.5 nmol/min [which had been shown previously (1) to mimic ischemic preconditioning], stock solutions (6.25 μM) were prepared daily in 0.1 mM ascorbic acid in bicarbonate buffer and infused into the aortic inflow line at 0.4 ml/min. Untreated controls received buffer and vehicle controls received ascorbic acid via the sidearm at an identical flow rate.
Predefined exclusion criteria stated that hearts would be excluded from the study if the 10-min control value did not fulfill the following criteria: LVDP ≥100 mmHg, heart rate ≥280 beats/min, and coronary flow ≥10 ml/min. Only one heart (in the vehicle control group) was excluded, because of a low heart rate; this heart was immediately replaced by another.
Values are means ± SE. To account for interanimal variability, the functional indexes measured during treatment periods and the 50-min reperfusion period were expressed as a percentage of the control value recorded for each heart before any test intervention was made (i.e., 10 min after the start of the experiment). To compare the effects of the various treatments on function over the 16-min intervention period preceding the induction of 40 min of ischemia and also during reperfusion, trapezoid integration was used to calculate the area under the time-response curve for each variable, for each heart, and these individual values were then employed for statistical comparison of the various groups (13). Individual comparisons of summary variables, final function data points, and the contraction data were carried out by one-way ANOVA, and, if this revealed significant differences, Bonferroni’s test was used (multiway comparison). A difference was considered to be statistically significant whenP < 0.05.
Control Function Before Interventions
As shown in Table 1, there were no significant differences in the control values for LVDP, coronary flow, and heart rate in any of the five groups after the initial 10 min of aerobic perfusion.
Function During Interventions
Figure 2 shows LVDP, coronary flow, and heart rate recorded at various times during the 16-min treatment period preceding the 40-min period of ischemia in each of the study groups. As expected, in the untreated control and vehicle control groups, all indexes remained constant throughout this period. Also as expected, in the ischemic preconditioning group the functional indexes fell during the brief periods of ischemia, recovering to near the control values during the subsequent periods of reperfusion. Although administration ofl-norepinephrine caused a large positive inotropic effect (LVDP increased by ∼40% during both periods of infusion) and a small positive chronotropic effect (an ∼15% increase in heart rate during both periods of infusion), it affected coronary flow only during the second period of administration, with a small increase (∼4%) in coronary flow.d-Norepinephrine had a very small inotropic effect (∼10%) but had no effect on the heart rate or coronary flow.
Ischemic Contracture During 40 Min of Ischemia
The temporal profiles for the development of ischemic contracture are shown in Fig. 3. There were no substantial differences between the two control groups (untreated control and vehicle control) and thed-norepinephrine group at any time point on the contracture curve. As shown in Fig. 3, compared with these three groups, ischemic preconditioning andl-norepinephrine infusion exacerbated the development of contracture, accelerating the time to onset of contracture (6.0 ± 1.0 and 6.8 ± 0.7 vs. 11.5 ± 0.3, 10.2 ± 0.7, and 10.9 ± 0.5 min for untreated controls, vehicle control, and d-norepinephrine, respectively, P < 0.05) and time to 50% contracture (9.2 ± 1.1 min for ischemic preconditioning and 9.0 ± 1.1 min for l-norepinephrine vs. 13.3 ± 0.3, 12.4 ± 0.5, and 13.2 ± 0.4 min for untreated control, vehicle control, andd-norepinephrine, respectively,P < 0.05). Although ischemic preconditioning and l-norepinephrine greatly exacerbated the rate of development of contracture, after ∼15 min of ischemia, the extent of contracture was similar in all groups and remained this way for the rest of the ischemic period. Once peak contracture was attained, there was a gradual decline in all groups to a similar value at the end of ischemia.
Postischemic Recovery Profiles
Figure 4 shows the profiles of the recovery of LVDP (expressed as a percentage of its preintervention basal value) in each study group. The postischemic recovery of LVDP was poor in the untreated control (23.0 ± 2.2%), vehicle control (26.9 ± 2.3%), and d-norepinephrine (19.8 ± 2.8%) groups, whereas substantial, similar, and significant improvements were observed in the ischemic preconditioning andl-norepinephrine groups (52.4 ± 5.1 and 52.5 ± 1.1%, respectively,P < 0.05). It is evident that LVDP in the ischemic preconditioning andl-norepinephrine groups recovered significantly better than in the other groups, where the recovery profiles were poor and nearly identical.
As shown in Fig. 5, recovery of coronary flow was significantly better in the ischemic preconditioning andl-norepinephrine groups than in the other groups. This higher rate of flow was evident immediately (5 min) before any functional recovery occurred and persisted throughout the recovery period. At the end of the 50-min reperfusion period, coronary flow had recovered to 73.2 ± 2.9 and 75.9 ± 4.9%, respectively, in these two groups; by contrast, recovery of coronary flow in untreated control,d-norepinephrine, and vehicle control groups was poor (42.3 ± 3.5, 46.2 ± 3.9, and 56.1 ± 3.4%, respectively, P < 0.05).
At the end of the 50-min reperfusion period, there were no significant differences between any of the groups.
The present studies have confirmed other investigations (1, 2, 14) showing that transient exposure to norepinephrine is able to substitute for ischemia as an effective means of preconditioning the rat heart and protecting it against damage during ischemia and reperfusion. Furthermore, we have shown that the degree of protection afforded is identical to that achieved with ischemic preconditioning, with the postischemic functional recovery being more than doubled in both instances. Our studies have also confirmed our earlier findings (9, 11) that ischemic preconditioning causes a paradoxical exacerbation of contracture during the extended period of ischemia. A new finding of the present investigation is that preconditioning with norepinephrine also exacerbates contracture to an extent similar to that seen with ischemic preconditioning (see below). Another new finding arising from the present study is that the ability of norepinephrine to precondition the heart and accelerate contracture is stereospecific and is restricted to thel-form.d-Norepinephrine was unable to improve postischemic recovery or influence the development of postischemic contracture.
Significance of Stereospecificity of Preconditioning by Norepinephrine
Although adrenergic receptor activation is the most obvious and likely pathway by which norepinephrine would exert a profound effect on the myocardium and its vulnerability to ischemic injury, other mechanisms cannot be automatically ruled out. For example, there is a substantial literature on the potent cardiovascular effects of the oxidation (spontaneous and enzyme-mediated) products of catecholamines, such as adrenochrome (4, 27). Catecholamine oxidation is associated with free radical production, and free radical production has been considered a possible mechanism underlying preconditioning (16, 24, 25). However, the present study strongly suggests that the potent protective effects observed in the present study involve receptor activation and are fully consistent with the “Downey hypothesis” that effective preconditioning involves receptor-activated, G protein-mediated events (5). This conclusion is based on our observation that the ability of norepinephrine to precondition the heart is exclusively restricted to the l-isomer, thed-isomer being without effect. If catecholamine oxidation and free radical production were involved, then efficacy would also be expected with thed-isomer. This conclusion is further supported by our observation thatl-norepinephrine infusion provoked substantial inotropic and chronotropic effects during its infusion, whereas the infusion of an identical dose ofd-norepinephrine was generally without effect. Our results therefore fully support a receptor-mediated mechanism and support the important and original contributions made to this field by Banerjee and colleagues (1) and Mitchell et al. (14). Furthermore, the observation thatl-norepinephrine was as effective as transient ischemia in preconditioning the heart against injury surely argues strongly for a major role for adrenergic receptor stimulation in the mechanism of ischemic preconditioning, at least in the rat.
Significance of Improving Postischemic Functional Recovery
The primary end point for injury and protection in the present study was left ventricular function. Preconditioning by ischemia and catecholamines was able to exert a profound protective effect, such that the recovery of postischemic LVDP improved from <25% in controls to >50% in the ischemic orl-norepinephrine preconditioning groups. In considering the mechanism underlying this protection, it is possible that the improved postischemic function might arise indirectly as a consequence of a limitation of necrosis (infarct size limitation) or possibly a reduction in the incidence of arrhythmias. Alternatively, it might be that the improved function is a direct consequence of a preconditioning-mediated attenuation of myocardial stunning. In this connection, it has been argued (19, 21) that preconditioning cannot attenuate stunning and that any improvements in postischemic contractile function are most likely to be secondary to a limitation of infarct size. Because we did not measure infarct size (and arrhythmias were minimal), the present results provide no additional information on this issue. However, they do provide further support for the ability of preconditioning to achieve a major improvement in the rate (and the extent) of postischemic functional recovery.
Significance of Improving Postischemic Coronary Flow
Postischemic coronary flow was substantially higher in the groups subjected to ischemia- andl-norepinephrine-mediated preconditioning than in the other groups. This higher flow was present immediately (5 min) before any functional recovery occurred and persisted throughout the recovery period irrespective of the method of preconditioning. In this connection, we and others have demonstrated that ischemic preconditioning protects vascular function (3, 7, 10,12). Thus, although we have no direct evidence from this study, we would hypothesize that this might be the mechanism underlying the improved coronary flow in the preconditioned groups.
Significance of Change in Ischemia-Induced Contracture
A striking aspect of this study is the concordance between the enhanced functional recovery and accelerated contracture with both preconditioning procedures. We were the first to systematically characterize the paradoxical ability of ischemic preconditioning to exacerbate ischemic contracture (9, 11). Despite the profound protective effects of preconditioning in relation to the recovery of contractile function during reperfusion, it is now well established that preconditioning causes an exacerbation in the early phases (rate of development) of contracture during ischemia (6, 9, 11). This finding might appear to challenge some of the fundamental concepts underlying cardioprotection, since, until now, it has been generally assumed that severe ischemic contracture was associated with irreversible injury and that interventions that reduced contracture (e.g., cardioplegia) improved tissue survival and functional recovery on reperfusion, whereas interventions that accelerated contracture had the reverse effect.
Irrespective of the significance of ischemic contracture as a predictor of tissue injury, the present study sheds some light on the question as to whether the ability of preconditioning to exacerbate ischemic contracture is an epiphenomenon arising as a necessary adverse consequence of the antecedent preconditioning ischemia (which although detrimental is overcome by the powerful overall protective effects of preconditioning) or whether it is a necessary component of the preconditioning mechanism. Our observation that preconditioning with l-norepinephrine also results in an exacerbation of contracture would argue for a close relationship between associated mechanisms. It would also argue against any suggestion that the exacerbation of contracture is merely a consequence of the antecedent ischemia that is necessary to achieve preconditioning. Of course, the argument could be advanced that transient increases in heart rate and inotropic state that characterize preconditioning by norepinephrine might result in the induction of moderate “ischemia” during the periods of catecholamine infusion. However, this possibility can probably be discounted by our observation that there were no reductions in coronary flow during catecholamine infusion, with coronary flow remaining constant or even increasing. However, definitive resolution of this question would require detailed metabolic analysis of the tissue (e.g., lactate and high-energy phosphate content) during the preconditioning episodes.
Alternatively, it may well be that preconditioning is so protective that there is an enhanced recovery of postischemic function, despite the detrimental effect of contracture. If this were the case, it would raise the intriguing possibility that if the accelerated contracture present in preconditioning could be abolished, cardioprotection might be enhanced.
The present study sheds further light on ischemic preconditioning and the ability of adrenergic agonists to mimic this phenomenon, the exact mechanism of which remains obscure. The unexpected ability of pharmacological preconditioning to mimic the effects of ischemic preconditioning on ischemic contracture might prove a valuable tool for further investigating the mechanism(s) underlying this phenomenon and its relationship to cardioprotection.
The advice and discussion of Drs. M. Avkiran and A. Shipolini are gratefully acknowledged.
Address for reprint requests: F. J. Sutherland, Cardiovascular Research, The King’s Center for Cardiovascular Biology and Medicine, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK.
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- Copyright © 1999 the American Physiological Society