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Right arrow Myocardial protection

J Thorac Cardiovasc Surg 2001;122:103-112
© 2001 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology (CSP)

{alpha}1-Adrenoceptors during simulated ischemia and reoxygenation of the human myocardium: Effect of the dose and time of administration

Mahmoud Loubani, FRCSI, Manuel Galiñanes, MD, PhD, FRCS

From the Division of Cardiac Surgery/ Department of Surgery, University of Leicester, Glenfield Hospital, Leicester, United Kingdom.

Received for publication Oct 27, 2000. Revisions requested Jan 4, 2001; revisions received Jan 22, 2001. Accepted for publication Jan 29, 2001. Address for reprints: Professor Manuel Galiñanes, Division of Cardiac Surgery/Department of Surgery, University of Leicester, Glenfield Hospital, Leicester, LE3 9QP, United Kingdom (E-mail: mg50{at}le.ac.uk).

Abstract

Objective: We sought to investigate the effect of {alpha}1-adrenoceptor activity on the ischemic and reoxygenated human myocardium.
Methods: Right atrial appendages (n = 6 per group) obtained during elective cardiac operations were sliced and stabilized in normoxic normothermic buffer solution for 30 minutes and then subjected to 90 minutes of simulated ischemia, followed by 120 minutes of reoxygenation. In study 1 the dose responses to the {alpha}1-adrenoceptor agonist phenylephrine (0.01, 0.1, 1, 10, and 100 µmol/L) and to the {alpha}1-adrenoceptor antagonist prazosin (0.1, 1, 10, and 100 µmol/L) when administered for 10 minutes before ischemia, during ischemia, and during reoxygenation were examined. The influence of the time of administration (ie, before ischemia, during ischemia, or during reoxygenation) of phenylephrine (0.1 µmol/L) and prazosin (10 µmol/L) was then investigated in study 2. In study 3 the effect of the combined administration of phenylephrine given before ischemia and prazosin given during ischemia was investigated. In study 4 the protective effect of phenylephrine given before ischemia (for 10 minutes or for 5 minutes with a 5-minute washout period) was compared with that of ischemic preconditioning (5 minutes of ischemia and 5 minutes of reoxygenation). At the end of each protocol, the leakage of creatine kinase (in units per gram of wet weight) and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide to insoluble formazan dye (in millimoles per gram of wet weight) were measured.
Results: Phenylephrine is maximally beneficial at 0.1 and 1 µmol/L (creatinine kinase, 0.97 ± 0.06 and 0.95 ± 0.03 U/g, respectively; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 153.0 ± 7.8 and 156.2 ± 6.7 mmol/g, respectively) compared with ischemic control (creatine kinase, 1.87 ± 0.03 U/g; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 108.5 ± 6.8 mmol/g; P < .05) but prazosin is detrimental at concentrations above 10 µmol/L (creatine kinase, 5.22 ± 0.29 U/g; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 69.8 ± 2.9 mmol/g; P < .05 vs ischemic control). In addition, phenylephrine (0.1 µmol/L) is protective when given before ischemia (creatine kinase, 2.06 ± 0.21 U/g; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 148.5 ± 4.5 mmol/g; P < .05 vs ischemic control) but is detrimental when given during ischemia alone (creatine kinase, 4.49 ± 0.98 U/g; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 70.5 ± 6.1 mmol/g; P < .05 vs ischemic control) and has no significant effect during reoxygenation. In contrast, prazosin (10 µmol/L) is beneficial when given during ischemia alone (creatine kinase, 1.34 ± 0.10 U/g; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 148.5 ± 4.5 mmol/g; P < .05 vs ischemic control), is detrimental when given during reoxygenation alone (creatine kinase, 1.5 ± 0.16 U/g; 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, 85.0 ± 4.7 mmol/g; P < .05 vs ischemic control), and has no effect when given before ischemia. The use of phenylephrine before ischemia alone is as protective as prazosin given during ischemia alone, but the combination of the two drugs does not cause additional benefit. Interestingly, the protection afforded by phenylephrine when given before ischemia is similar to that obtained with ischemic preconditioning.
Conclusions: In the human myocardium activation of {alpha}1-adrenoceptors before ischemia is protective but is detrimental during ischemia, whereas blockade of {alpha}1-adrenoceptors is beneficial during ischemia but detrimental during reoxygenation. The degree of protection achieved by activation of the {alpha}1-adrenoceptors before ischemia is similar to that obtained with blockade of {alpha}1-adrenoceptors during ischemia and that of ischemic preconditioning.

Activation of {alpha}1-adrenoceptors induces positive inotropic, chronotropic, and dromotropic actions in the heart and vascular effectsGo Go 1-4 that are clinically exploited to optimize hemodynamic conditions. However, it is generally believed that activation of {alpha}1-adrenoceptors is detrimental to the ischemic heart, a thesis that would be supported by the increased release of cardiac and plasma catecholaminesGo 5 and the enhanced density of cardiac {alpha}1-adrenoceptors during ischemia.Go Go 6-8 However, it has recently been shown that {alpha}1-adrenoceptors mediate the protection induced by ischemic preconditioning in animals and in the human myocardiumGo Go 9-14 by means of phospholipase C and protein kinase C activation,Go 13 and this has given rise to the possibility that the effect of {alpha}1-adrenoceptors in ischemic injury may depend on the dose and the time of their activation (ie, before, during, or after ischemia). To investigate this, right atrial appendages were obtained from patients undergoing elective coronary bypass operations, and the muscles were subjected to various protocols in a validated in vitro model of simulated ischemia and reoxygenation.Go 15

Methods

Experimental preparation
Experiments were performed on myocardium obtained from the right atrial appendage of patients undergoing elective coronary artery operations or aortic valve replacement in a cell necrosis model that was developed in our laboratory and described previously.Go 15 Patients were excluded if they had enlarged atriums, atrial arrhythmias, poor left ventricular function (ejection fraction, <30%), and right ventricular failure or were taking oral hypoglycemic agents, opioid analgesics, KATP channel openers, or catecholamines. Local ethical committee approval was obtained for the harvesting technique. The specimens were collected in oxygenated N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid– buffered solution at 4°C to 5°C and immediately sectioned and prepared for study. The appendage was mounted onto a ground glass plate with the epicardial surface face down and then sliced freehand with surgical skin graft blades (Shwann-Morton) to a thickness of between 300 and 500 µm. The specimen and the slide were always kept moist throughout the procedure. The muscles (weight 30-50 mg) were then transferred to conical flasks (25-mL Erlenmeyer flasks, Schott Glaswerk) containing 10 mL of oxygenated buffered solution, and the flasks were placed in a shaking water bath maintained at 37°C. The oxygenation of the incubation medium was maintained by a continuous flow of 95% O2/5% CO2 gas mixture to obtain a PO2 of between 185 and 225 mm Hg and a PCO2 of between 45 and 50 mm Hg. The PO2, PCO2, and pH in the incubation medium were monitored by means of intermittent analyses of the effluent with an automated blood gas analyzer (model 855 Blood Gas System, Chiron Diagnostics), and the pH was kept between 7.36 and 7.45. For the induction of simulated ischemia, the medium was bubbled with 95% N2/5% CO2 (pH 6.80-7.00), and D-glucose was replaced with 2-deoxyglucose. In this preparation creatine kinase (CK) leakage (in units per gram of wet weight) was taken as a measure of tissue injury, and the reduction of 3-[4,5 dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) to an insoluble formazan dye (in millimoles per gram of wet weight) was used to assess tissue viability. The atrial tissue was not paced, and the force developed was not measured in these studies.

Solutions and drugs
The incubation medium was prepared daily with deionized distilled water and contained the following: NaCl2, 118 mmol/L; KCl, 4.8 mmol/L; NaHCO3, 27.2 mmol/L; MgCl2, 1.2 mmol/L; KH2PO4, 1.0 mmol/L; CaCl2, 1.25 mmol/L; D-glucose, 10 mmol/L; and N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 20 mmol/L. As mentioned above, during simulated ischemia, D-glucose was removed and substituted with 2-deoxyglucose (10 mmol/L) to maintain a constant osmolarity. Phenylephrine and prazosin were dissolved in deionized distilled water immediately before their use. All reagents were obtained from Sigma.

Experimental protocols
After sectioning the atrium, the preparations were allowed to stabilize for 30 minutes and then randomly allocated to various protocols. In all the studies, simulated ischemia was induced for a period of 90 minutes, followed by 120 minutes of reoxygenation. Some of the preparations were not made ischemic and instead were aerobically perfused for 240 minutes to serve as aerobic matched controls.

Study 1: Dose response to phenylephrine and prazosin
In this study various concentrations of the {alpha}1-adrenoceptor agonist phenylephrine (0.01, 0.1, 1, 10, and 100 µmol/L) and the {alpha}1-adrenoceptor antagonist prazosin (0.1, 1, 10, and 100 µmol/L) were added to the incubation media for 10 minutes before ischemia, during ischemia, and during reoxygenation (n = 6 preparations per group).

Study 2: Influence of the time of administration of phenylephrine and prazosin
Influence of phenylephrine (0.1 µmol/L) and prazosin (10 µmol/L) was investigated by the exposure of the myocardial tissue to the optimal concentrations of the drugs shown in study 1 for 10 minutes before ischemia, during ischemia, and during reoxygenation alone and in combination (n = 6 preparations per group).

Study 3: Potency of phenylephrine- and prazosin-induced protection
The most beneficial dose and time of administration for phenylephrine and prazosin seen in the 2 previous studies were used alone and in combination (n = 6 preparations per group) to study the potency of the protection offered by the drugs.

Study 4: Phenylephrine and ischemic preconditioning
To investigate the potency of protection of the {alpha}1-agonist phenylephrine compared with that of ischemic preconditioning, right atrial specimens (n = 6 per group) were subjected to a protocol of ischemia-reoxygenation identical to the one used in the previous studies. Phenylephrine at a concentration of 0.1 µmol/L was used before ischemia for 10 minutes or for 5 minutes followed by 5-minute washout period. Ischemic preconditioning was induced by 5 minutes of ischemia and 5 minutes of reoxygenation immediately before the 90-minute ischemia, a protocol shown to afford maximal protection in this preparation.Go 16

Assessment of tissue injury and viability
Tissue injury was determined by measuring the leakage of CK into the incubation medium during the 120-minute reoxygenation period. This was assayed by means of a kinetic UV method based on the formation of NAD (Sigma Catalogue No. 1340-K), and the results were expressed as units per gram of wet weight.

Tissue viability was assessed by the reduction of MTT to a blue formazan product at the end of the experimental period. The tissue was loaded into a Falcon conical tube (15 mL, Becton Dickinson Labware) into which 2 mL of phosphate buffer solution (0.05 mol/L) containing MTT (1.25 mg/mL; 3 mmol/L at final concentration) was added and then incubated for 30 minutes at 37°C. After this, the tissue was homogenized in 2 mL of dimethylsulfoxide (Homogenizer Ultra-Turrax T25, dispersing tool G8, IKA-Labortechnic) at 9500 rpm for 1 minute. The homogenate was then centrifuged at 1000g for 10 minutes, and 0.2 mL of the supernatant was dispensed into a 98-well flat-bottom microtiter plate (Nunc Brand Products). After this, the absorbance of the blue formazan formed was measured on a plate reader (Benchmark, Bio-Rad Laboratories) at 550 nm, and the results were expressed as millimoles per gram of wet weight.

Statistical analysis
All data are presented as means ± SEM. Analysis of variance was used for multiple comparisons, with application of a post hoc Tukey test.

Results

Study 1: Dose response to phenylephrine and prazosin
The results shown in Figure 1, A and B, on CK leakage and MTT reduction demonstrate that phenylephrine induces a dose-response curve with maximal protection at 0.1 and 1 µmol/L and a loss of protection at doses of 10 µmol/L and greater. It is important to note that although protection was lost at the highest concentrations of phenylephrine, higher doses did not exert a detrimental effect, and therefore CK leakage and MTT mean values were similar to those seen in the phenylephrine-free group.



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  		Fig. 1. CK leakage (A) and MTT reduction (B) of human right atrial myocardium subjected to 90 minutes of ischemia and 120 minutes of reoxygenation and incubated with various concentrations of phenylephrine. The drug was present in the incubation media for 10 minutes before the induction of ischemia, during ischemia, and during reoxygenation. Columns represent the mean of 6 experiments, and bars represent the SEM. *P < .05 versus phenylephrine-free group.

 
As seen in Figure 2, A and B, CK leakage and MTT reduction were not significantly affected by prazosin when present in the incubation media throughout the experiment at the lowest concentrations of 0.1 and 1 µmol/L. However, in contrast with the results for phenylephrine, prazosin was detrimental at the highest concentrations (10 and 100 µmol/L), as shown by the significant increase in CK leakage and a decrease in MTT reduction.



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  		Fig. 2. CK leakage (A) and MTT reduction (B) of human right atrial myocardium subjected to 90 minutes of ischemia and 120 minutes of reoxygenation and incubated with various concentrations of prazosin. The drug was present in the incubation media for 10 minutes before the induction of ischemia, during ischemia, and during reoxygenation. Columns represent the mean of 6 experiments, and bars represent the SEM. *P < .05 versus prazosin-free group.

 
Study 2: Influence of the time of administration of phenylephrine and prazosin
Figure 3, A and B, shows the results of phenylephrine when given at different times at a chosen concentration of 0.1 µmol/L, which was shown to be the most effective dose in study 1. These results demonstrate, in terms of both CK leakage and MTT reduction, that maximal protection was obtained when phenylephrine was administered before ischemia alone, an effect that was equivalent to the one obtained with phenylephrine throughout the entire experimental period. Phenylephrine given during the reoxygenation period alone was less protective than when given before ischemia, and this was reflected by a modest but significant decrease in CK leakage without affecting MTT reduction. Interestingly, the administration of phenylephrine during ischemia alone was detrimental, as seen by the significant decrease in MTT reduction. It is worth noting that the adverse effect of phenylephrine during ischemia was completely counteracted when the drug was also present before ischemia and during reoxygenation.



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  		Fig. 3. CK leakage (A) and MTT reduction (B) of human right atrial myocardium subjected to 90 minutes of ischemia and 120 minutes of reoxygenation and incubated with 0.1 µmol/L phenylephrine for different times. Columns represent the mean of 6 experiments, and bars represent the SEM. *P < .05 versus the phenylephrine-free group.

 
Figure 4, A and B, shows the results on CK leakage and MTT reduction for prazosin given at various times at a concentration of 10 µmol/L. These results show that prazosin did not have a significant effect when given before ischemia alone, that it was detrimental when given during reoxygenation alone to a degree similar to the administration of prazosin during the entire experimental period, and that it was protective when given during ischemia alone. Importantly, the beneficial action of prazosin during ischemia was abolished when the drug was present before ischemia.



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  		Fig. 4. CK leakage (A) and MTT reduction (B) of human right atrial myocardium subjected to 90 minutes of ischemia and 120 minutes of reoxygenation and incubated with 10 µmol/L prazosin for different times. Columns represent the mean of 6 experiments, and bars represent the SEM. *P < .05 versus the prazosin-free group.

 
Study 3: Potency of phenylephrine- and prazosin-induced protection
As indicated by the results on CK leakage and MTT reduction shown in Figure 5, A and B, phenylephrine given before ischemia alone and prazosin given during ischemia alone afforded similar protection, and the combination of the two did not result in greater protection than that obtained with each drug given alone.



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  		Fig. 5. CK leakage (A) and MTT reduction (B) of human right atrial myocardium subjected to 90 minutes of ischemia and 120 minutes of reoxygenation and incubated with phenylephrine (0.1 µmol/L) for 10 minutes before ischemia, with prazosin (10 µmol/L) during ischemia, and with the 2 drugs in combination. Columns represent the mean of 6 experiments, and bars represent the SEM. *P < .05 versus the group with ischemia-reoxygenation (I/R) alone.

 
Study 4: Phenylephrine and ischemic preconditioning
Figure 6, A and B, shows that ischemic preconditioning induces a significant decrease in CK leakage and better preserves MTT reduction when compared with the effects seen in muscles subjected to ischemia-reoxygenation alone. The results confirm previous findings from our laboratory using an identical preparation and protocol.Go 16 They also show that the {alpha}1-agonist phenylephrine given for 10 minutes before ischemia or for 5 minutes with a 5-minute washout period before ischemia, thus mimicking the ischemic preconditioning protocol, results in similar protection to that seen with ischemic preconditioning.



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  		Fig. 6. CK leakage (A) and MTT reduction (B) of human right atrial myocardium subjected to 90 minutes of ischemia and 120 minutes of reoxygenation after ischemic preconditioning (5 minutes of ischemia and 5 minute of reoxygenation) or incubation with phenylephrine (0.1 µmol/L) for 10 minutes or for 5 minutes with a 5-minute washout period. Columns represent the mean of 6 experiments, and bars represent the SEM. *P < .05 versus the group with ischemia-reoxygenation (I/R) alone.

 
Discussion

The present study has demonstrated that {alpha}1-adrenoceptors play an important role in the ischemia-reoxygenation–induced injury of the human atrial myocardium. Thus they show that stimulation of {alpha}1-adrenoceptors with phenylephrine protects against injury, whereas {alpha}1-adrenoceptor blockade with prazosin is detrimental; both effects are obtained in a dose-dependent manner. They have also shown that the effect of the stimulation or blockade of {alpha}1-adrenoceptors depends on the time of administration so that {alpha}1-adrenoceptor stimulation is protective when given before ischemia but detrimental when given during ischemia. On the contrary, {alpha}1-adrenoceptor blockade is beneficial during ischemia and detrimental during reoxygenation and has no significant effect before ischemia. It appears that similar maximal protection can be obtained with {alpha}1-adrenoceptor stimulation before ischemia and with {alpha}1-adrenoceptor blockade during ischemia, although the combination of the two does not induce additional protection. Furthermore, the protective effect of {alpha}1-adrenoceptor stimulation before ischemia is as potent as ischemic preconditioning. These studies are the first in dissecting the role of {alpha}1-adrenoceptors during ischemia and reoxygenation of the human myocardium, and the results have important mechanistic and clinical implications that warrant further discussion.

Our results showing that {alpha}1-adrenoceptor activation before ischemia is cardioprotective are in agreement with the observation of other investigators that {alpha}1-adrenoceptors participate in the protection induced by ischemic preconditioning in the rat heartGo Go 9,17 and in the human myocardium.Go 13 However, it is worth noting that although {alpha}1-adrenoceptor stimulation mimicked the protection of ischemic preconditioning in our studies, protection was less and did not replicate that obtained with ischemic preconditioning in the study of Cleveland and colleagues,Go 13 who also used the human myocardium. Certainly, there are differences in the experimental model and the doses of the {alpha}1-adrenoceptor agonist phenylephrine used in the 2 studies that may explain the differing results. Thus our studies were carried out in a model of necrosis in which muscles were not electrically stimulated and were subjected to 90 minutes of simulated ischemia, whereas in the study of Cleveland and colleagues,Go 13 the muscles were stimulated, and functional recovery, as opposed to necrosis, was assessed after only 45 minutes of ischemia. Furthermore, our dose-response study with phenylephrine showed that there is a bell-shaped response, with maximal protection at concentrations of 0.1 and 1 µmol/L and loss of protection at concentrations of 10 µmol/L or greater, as used in their study.Go 13 It is of interest that similar protection was obtained with phenylephrine administered for 10 minutes immediately before ischemia or for only 5 minutes with a 5-minute washout period before ischemia, thus suggesting that a short period of stimulation of {alpha}1-adrenoceptors is sufficient to attain maximal benefit.

The harmful effect seen when {alpha}1-adrenoceptors were activated only during ischemia and the protection obtained with their blockade were not unexpected and are supported by the reported literature.Go Go 18,19 However, an important contribution of the present study is that the activation of {alpha}1-adrenoceptors during ischemia does not diminish the protection induced by the activation of these receptors before ischemia. It was also important that the protection seen with {alpha}1-adrenoceptor blockade during ischemia is lost when the blockade is continued during reoxygenation. These results contradict the never-confirmed assumption that activation of {alpha}1-adrenoceptors during reoxygenation may extend reperfusion injury.Go 20 In fact, they show that activation of {alpha}1-adrenoceptors during reoxygenation does not significantly influence myocardial injury and that, on the contrary, {alpha}1-adrenoceptor blockade augments injury.

Although the signal transduction pathways that follow the activation of {alpha}1-adrenoceptors are well described,Go Go 21-33 the precise mechanisms responsible for their opposing actions in ischemia-reoxygenation remain unclear. It is possible that the increase in cytosolic calcium levels induced by {alpha}1-adrenoceptor agonists through cyclic adenosine monophosphateGo 19 may mediate the effects discussed above. Indeed, calcium overload can be harmful when occurring during ischemia,Go 34 and it may precondition the heart and be protectiveGo Go 35,36 when it occurs before ischemia. The demonstration by Miyawaki and AshrafGo 37 that a transient increase in cytosolic calcium levels during ischemic preconditioning is an important trigger for the activation and translocation of the protein kinase C isoforms {alpha} and {delta} further supports this hypothesis.

The binding of agonists to {alpha}1-adrenoceptors also causes the activation of phospholipase C, and this hydrolyzes phosphatidylinositol-4,5-biphosphate, resulting in the production of inositol-1,4,5-triphosphate and 1,2-diacylglycerol.Go Go 21,22 Inositol-1,4,5-triphosphate acts on the sarcoplasmic reticulum, increasing intracellular calcium levels,Go Go 23,24 whereas 1,2-diacylglycerol activates protein kinase C,Go 25 which in turn activates the transsarcolemal voltage-dependent calcium channelsGo Go 26-29 and the Na+/H+ channelsGo 30 and the opening of mitochondrial KATP channels.Go 31 There is evidence that stimulation of {alpha}1-adrenoceptors through activation of protein kinase C also enhances 5'-nucleotidase activity and hence adenosine formation,Go 32 which has been shown to influence the outcome of myocardial ischemia-reperfusion in a number of experimental models and species.Go Go Go 33,38,39 To what extent each of these mechanisms is participating in the action of {alpha}1-adrenoceptors during ischemia and reoxygenation is unclear; however, from our studies, it is evident that the result of the use of agents that activate or blockade these receptors may vary widely depending on time of initiation and termination of administration, and it is possible that more than one mechanism may be involved. Clearly, more studies are needed to elucidate the underlying mechanism of these actions.

A possible limitation of the present study is the use of atrial tissue as opposed to ventricular tissue, and therefore any extrapolation should be made with caution. Another possible limitation might be that right atrial appendages were obtained from patients taking antianginal medication, and this potentially may have had some influence on the ischemiareoxygenation injury. Furthermore, our studies were performed in an in vitro preparation, and therefore the clinical application of the results should also be made with caution.

Despite the above potential shortcomings, the findings of our study may have important therapeutic implications for myocardial protection during cardiac operations, cardiac transplantation, angioplasty, and acute myocardial infarction, for which agents acting on {alpha}1-adrenoceptors are frequently used. Particular attention must be paid during cardiac operations in which phenylephrine is used routinely to elevate the mean arterial blood pressure during cardiopulmonary bypass, and in doing so, one may be unwittingly exacerbating myocardial injury during cardiac ischemia, when collateral blood flow enters the ischemic myocardium. However, these studies have shown that such undesirable effects can be fully counteracted by the administration of phenylephrine before the induction of cardiac ischemia.

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S. Ghosh and M. Galinanes
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