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Changes in myocardial β-adrenergic receptors during acute rejection of heterotopically transplanted rat hearts
Cardiac and Pulmonary Transplantation
Changes in myocardial f)-adrenergic receptors during acute rejection of heterotopically transplanted rat hearts To evaluate changes of the myocardial {j-adrenergic receptors in acute cardiac graft rejection, the density and binding affinity value of the myocardial {j-adrenergic receptors in heterotopically transplanted rat isografts and allografts were analyzed. Hearts from Fisher rat donors were transplanted either to the Fisher rats (isografts) or to Lewis rats (allografts). Histologic examination of the allografts showed mild to moderate rejection on the seventh and fourteenth days and showed severe rejection on the twenty-first day after transplantation. The density values in the allografts and isografts similarly increased significantly (p < 0.05) above the normal level on the seventh and fourteenth days after transplantation. The density in allografts on the twenty-first day decreased significantly (p < 0.05) below the normal level, while that in isografts remained at the normal level. In contrast, the binding affinity value of myocardial {j-adrenergic receptors in both isografts and allografts did not change after transplantation. These results demonstrated that myocardial {j-adrenergic receptors presented upregulation in mild to moderate rejection, whereas these receptors presented downregulation in severe rejection. The data suggested that downregulation of myocardial {j-adrenergic receptors plays a major role in decreased cardiac contractility during severe rejection, but not during mild and moderate rejection. (J THORAC CARDIOVASC SURG 1992;104:1567-71)
Hitoshi Yokoyama, MD, Mikio Ohmi, MD, Atsusi Iguchi, MD, Sadayuki Murata, MD, Takahiko Nakame, MD, Koichi Tabayashi, MD, and Hitoshi Mohri, MD, Sendai, Japan
Since cardiac transplantation has become an effective modality of therapy for selected patients with end stage heart diseases, acute rejection of the cardiac graft is a serious problem for postoperative care. The Stanford group-? demonstrated that detectable deterioration of From the Department of Thoracic and Cardiovascular Surgery, Tohoku University School of Medicine, Sendai, Japan. Supported by a Grant-in-Aid for Specific Project Research (No. 03670650) from the Ministry of Education, Science and Culture, Japan. Received for publication Aug. 16, 1991. Accepted for publication March 25, 1992. Address for reprints: Hitoshi Yokoyama, MD, Department of Thoracic and Cardiovascular Surgery, Tohoku University School of Medicine, I-I Seiryo-machi, Sendai 980, Japan.
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cardiac contractility at rest was a relatively late event in the course of rejection, suggesting myocardial necrosis as a major factor for decreased cardiac contractility. However, changes of the cellar mechanism of positive inotropy during acute rejection are still unclear. Myocardial {j-adrenergic receptors located on cardiac sarcolemmal membrane are known to be a major pathway in producing positive inotropic effects.' These receptors are changed in their density and binding affinity, in some cases resulting in changes in cardiac contractility and in response to catecholamines-"? Therefore changes in the density and binding affinity of myocardial {j-adrenergic receptors in transplanted cardiac grafts during acute rejection could be a subject worth investigating. The purpose of the present study is to evaluate the relationship between cardiac rejection and changes in the 1567
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Yokoyama et al.
myocardial l3-adrenergic receptors. In this study, posttransplant changes in the maximal density (B-max) and binding affinity value (Kd) of myocardial d-adrenergic receptors were examined in the heterotopically transplanted allografts and isografts of the rat heart in comparison with their histologic gradings of acute rejection. Isografts represent sympathetic denervated hearts without acute rejection, while allografts represent sympathetic denervated hearts with acute rejection. Thus comparison of allografts with isografts would reflect the effects of acute rejection.
Materials and methods Animal preparations. Male Lewis rats and Fisher rats weighing 200 to 300 gm were used. Animal models of transplantation were produced by Fisher hearts transplanted to Lewis rats (allografts) or to Fisher rats (isografts). The surgical technique for heterotopic intraabdominal cardiac transplantation was a modification of the method described by Ono and Lindsey.8 Briefly, a pair of rats was anesthetized with an intraperitoneal injection of pentobarbital sodium (20 mg/kg of body weight). The donor rat was intubated, following which controlled ventilation was instituted using a respirator for small animals. After the intravenous injection of 1000 U of heparin sodium, the inferior vena cava and bilateral superior venae cavae were ligated and subsequently dissected. The ascending aorta and main pulmonary trunk were dissected, followed by mass ligation of the pulmonary veins and isolationof the donor heart. Twenty milliliters of ice-cold saline was injected into the ascending aorta and the isolated heart was kept in the same solution until transplantation. The ascending aorta and pulmonary artery of the donor heart were anastomosed to the abdominal aorta and inferiorvena cava of the recipient rat, respectively, in an end-to-side fashion with a continuous suture of 8-0 polypropylene.The ischemic time of the donor heart was within 30 minutes. In the preliminary experiments, allografts were completely rejected in 20 to 26 days tn = 9) when no immunosuppressive measures were taken. Then beating cardiac grafts were harvested on the seventh, fourteenth, or twenty-first day after transplantation and were submitted to both histologic and receptor-binding studies. Normal, untreated hearts of Fisher rats were also submitted to receptor-binding studies. All animals received humane care in accordance with the Guide for Animal Experimentation of Tohoku University and Tohoku University School of Medicine. Histologic analysis. Cross-sectional ventricular specimens of 1 mm in thickness were obtained from the midlevel of the longitudinal cardiac axis parallel to the horizontal axis. After fixation in buffered 10%formalin, the specimenswere examined with a light microscope using standard hematoxylin and eosin staining. Histologic gradings of the severity of cardiac rejection were classified by the method of Billingham?into mild, moderate, and severe rejection. Each histologic grading was given a score (rejection grade score) as no rejection = 0, mild rejection = 1, moderate rejection = 2, and severe rejection = 3. Receptor-binding studies. The remaining specimens ofventricular myocardium were immersed in ice-cold Tyrode's solution and were homogenized with three IS-second bursts at full
Surgery
speed on a Polytron PT-20 homogenizer (Brinkmann Instruments, Westbury, N.Y.). The homogenate was centrifuged at 1000 g for 10 minutes. The supernatant was centrifuged at 30,000 g for 15 minutes. The pellets containing sarcolemmal membranes were resuspended in ice-cold assay buffer (Tris 50 mmol/L, PMSF (phenylmethylsulfonylfluoride) 0.1 mmol/L, pH 7.4) by 10 strokes of a tight fitting Potter homogenizer (Wheaton Instruments, Millville, N.J.). The membrane preparations were stored at -80° C until binding assay was made. Protein concentrations of the membrane preparations were measured by the micro-Bradford method (Bio-Rad Laboratories, Cambridge, Mass.) with bovine serum albumin as the standard. IJ-Adrenergicdensity and binding affinity were assessedwith the IJ-adrenergic receptor ligand 3H-CGP 12117 (Amersham, Arlington Heights, IIl.). The membrane preparations (100 ~d) were added to tubes containing assay buffer and a variable concentration of 3H-CGP 12117 with or without 5 X 10- 5 mol/L propranolol in a final volume of 500 Ill. The assay mixture was incubated with shaking at 37° C for 40 minutes, and the reaction was discontinued by coolingthe mixture tubes to an ice-cold temperature. Then rapid filtering under a controlled vacuum through Watmann GF /C filters with a Brandel multiple port cell harvester (M-24R, Brandel Inc., Gaithersberg, Md.) was performed. The tubes were washed five times with 2 ml of icecold buffer. The glass filterswere dried under hot lightsand were packed in 20 ml scintillation vials containing 5 ml scintillation medium and were counted in a gamma counter. The B-max of 3H-CGP binding site and the Kd for 3H-CGP were calculated by Scatchard plots from the binding data. The regression lines in Scatchard plots were fitted by the method of least squares. Eight data points were determined for the Scatchard analysis. Each determination was done in triplicate. Statistical analysis. Values are expressed as means ± S.E. of n experiments, unless otherwise stated. The difference between mean values was analyzed by unpaired Student's t test or, when appropriate, by analysis of variance, and were judged to be significant when p values were less than 0.05.
Results Histologic findings. All isografts showed near normal histologic findings except for slight increases in interstitial fibrosis, which were seen only in the twenty-first day specimens. Posttransplant changes in the rejection grade scores of allografts showed near linear increases as time passed, registering scores of 1.3 ± 0.2 on the seventh day, 1.8 ± 0.4 on the fourteenth day and 2.8 ± OJ on the twenty-first day (Fig. I, A). Density of myocardial /3-adrenergic receptors. Posttransplant changes in the B-max of myocardial o-adrenergic receptors in the isografts and allografts are demonstrated in Fig. 1, B. B-max in the normal hearts of Fisher rats was 52.9 ± 4.5 fmol/rng protein (n = 6). B-max in the isografts was 90.0 ± 8.0,77.8 ± 2.3, and 51.9 ± 1.9 frnol/rng protein on the seventh, fourteenth, and twentyfirst day after transplantation, respectively. B-max values in the isografts on the seventh and fourteenth days after
Volume 104 Number 6 December 1992
Rejection changes in myocardial fJ-receptors
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Fig. 1. A, Posttransplant changes in histologic gradings of rejection in the heterotopically transplanted allografts of rat hearts. Rejection grade scoresare given in detail in the "Materialsand methods"section.Data points represent means ± SE (n = 6). B, Posttransplant changes in the density (B-max) of myocardial iJ-adrenergic receptors in the heterotopically transplanted rat isografts and allografts. Data points represent means ± SE (n = 6). *p < 0.05 against normalvalues(day 0); a represents a significant (p < 0.05) difference between isografts and allografts.
transplantation were significantly (p < 0.05) greater than the normal value. B-max in the allografts was 77.6 ± 8.7, 79.6 ± 15.6, and 19.4 ± 9.0 fmol/rng protein on the seventh, fourteenth, and twenty-first days after transplantation, respectively. B-max values in the allografts on the seventh and fourteenth days after transplantation were significantly (p < 0.05) greater than the normal value. However, B-max values in the allografts on the twentyfirst day after transplantation were significantly (p < 0.05) smaller than the normal value and the value in the isografts. Relationship between the rejection grade scores and B-max of myocardial ,B-adrenergic receptors in the allografts. B-max of normal hearts of Fisher rats with no
rejection was 52.9 ± 4.5 frnol/rng protein (n = 6). B-max of allografts in rejection grade score 1 (mild rejection) was 65.5 ± 8.2 frnol/rng protein, which was not significantly greater than the normal value (p = 0.132). In hearts of rejection grade score 2 (moderate rejection), B-max increased significantly (p < 0.05) to higher levels (99.7 ± 9.5 frnol/rng protein) above the normal value. However, B-max of the hearts in rejection grade score 3 (severe rejection) decreased significantly (p> 0.05) below the normal value, to 19.4 ± 9.1 frnol/ mg protein (Fig. 2). Binding affinity value of myocardial ,B-adrenergic receptors. Posttransplant changes in the Kd of fJ-adrenergic receptors in the isografts and allografts are demonstrated in Fig. 3. Kd of fJ-adrenergic receptors in normal hearts of Fisher rats was 1.4 ± 0.3 nmol (n = 6). Kd values after transplantation were not significantly different from the normal value in both isografts and allografts. Discussion fJ-Adrenergic receptors on the myocardial sarcoplasmic membrane playa major role in producing positive inotropic effects in mammalian cardiac muscles.' Changes in B-max and Kd of fJ-adrenergic receptors have been investigated recently, especially in failing human hearts with dilated cardiomyopathy, ischemic cardiomyopathy, or mitral valve diseases.>? In these cases, decreased B-max of the myocardial fJ-adrenergic receptors was observed. Implications for the treatment of the failing human heart by modification of the fJ-adrenergic system have also been explored. 10, 11 Transplanted hearts
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The Journal of Thoracic and Cardiovascular Surgery
Yokoyama et al.
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Fig. 3. Posttransplant changes in the binding affinity value (Kd) of myocardial,B-adrenergic receptors in the heterotopically transplanted rat isograftsand allografts. Data points represent means ± SE (n = 6). All resultswerenot significantly different from normal values (day 0). during acute rejection show decreased cardiac contractility': 2 or impaired cardiac response to catecholamines.F Recently, changes in myocardial {3-adrenergic pathways in acute rejection have been suggested by assessing the cardiac response to {3-adrenergic receptor stimulation in animal experiments.'? However, the mode of changes in B-max and Kd of myocardial {3-adrenergic receptors during acute rejection has not been evaluated. The results of the current study showed increased B-max of myocardial {3-adrenergic receptors in the heterotopically transplanted rat heart allografts during mild to moderate rejection. In other words "upregulation" of {3-adrenergic receptors was observed. Isografts also presented the upregulation of {3-adrenergic receptors on the seventh and fourteenth days after transplantation. Cardiac denervation alone has been known to upregulate the myocardial {3-adrenergic receptors.l': 14 which promotes the sensitivity of cardiac responsiveness to adrenergic stimulation. 13, 15 Therefore a mechanism of the upregulation in the allografts is speculated to be mainly caused by sympathetic denervation, as in the case of the isografts. In addition, some compensatory mechanisms of the myocardium might be responsible for the upregulation when myocardial necrosis decreased the total amount of myocardium. This mechanism is likely because, in comparison with mild rejection, the level of upregulation was higher during moderate rejection when histologic myocardial necrosis starts. Another explanation may be an effect of myocardial ischemia. Upregulation of {3-adrenergic receptors was reported in some animal experiments in which myocardial ischemia was
induced by transient occlusion of the coronary arteries. 16, 17 A decrease in coronary blood flow in the early phase of acute rejection has been reported in animal experiments on dogs" and rats.!" DiSesa and associates I 2 reported impaired myocardial contractile reserve during mild and moderate rejection. However, the results in the present study did not reveal downregulation of the myocardial {3-adrenergicreceptors. Thus the impaired myocardial contractile reserve found by DiSesa and colleagues may not be caused by {3-adrenergic receptor downregulation. They also reported that chronotropic responses to isoproterenol in rat allografts were preserved nearly equally to those in isografts when the allografts showed moderate rejection.U Preserved chronotropic response during moderate rejection is consistent with the results in the present study, because positive chronotropy is mainly modulated by myocardial {3-adrenergic receptors. 3 The present study demonstrated that the allografts showed significant downregulation of the myocardial {3-adrenergic receptors in the terminal stage of acute rejection. Besides myocardial necrosis, this downregulation may be a reason for deterioration of left ventricular contractility in the late period of acute rejection.': 2, 20 Decrease in myocardial high-energy phosphate content in the terminal stage of acute rejection has been reported recently." This finding suggested impaired synthesis of cellular proteins such as {3-adrenergic receptors in severe rejection, accompanied by the receptor downregulation. The presence of anti-{3-adrenergic receptor antibodies was recognized recently in a substantial proportion of patients with idiopathic dilated cardiomyopathy.F However, whether myocardial {3-adrenergic receptors are one of the direct immunologic targets or not is still unknown. The sarcolemmal membrane of lymphocytes also have {3-adrenergic receptors.P Therefore whether the observed {3-adrenergic receptor changes were caused by the changes in myocardium or by infiltrated lymphocytes must be determined. Since the amount of infiltrated lymphocytes assessed by histologic findings increased steadily despite downregulation of B-max during severe rejection, the changes in B-max of {3-adrenergic receptors obtained in this study were likely caused by the changes in the myocardium but not by the lymphocytes. The Kd of {3-adrenergic receptors is determined by the three-dimensional conformation of their binding site.' Since the present study showed that the Kd values did not change in both isografts and allografts after transplantation, conformational changes of the myocardial {3-adrenergic receptor proteins, at least their binding sites, may not have occurred in acute cardiac graft rejection.
Volume 104 Number 6 December 1992
We thank Dr. Kazuo N unoki of the Second Department of Pharmacology, Tohoku University School of Medicine, for his useful advice in the receptor-binding studies, and Dr. Nobuaki Tamahashi of the Japan Pathology Institute, Sendai, for his help in histologic evaluation of cardiac rejection.
REFERENCES
I. Stinson EB, Griepp RB, Bieber CP, Shumway NE. Hemodynamic observations after orthotopic transplantation of the canine heart. J THORAC CARDIOVASC SURG 1972; 63:344-52. 2. Stinson EB, Tecklenberg PL, Hollingsworth JF, Jones KW, Sloane R, Rahmoeller G. Changes in left ventricular mechanical and hemodynamic function during acute rejection of orthotopically transplanted hearts in dogs. J THORAC CARDIOVASC SURG 1974;68:783-91. 3. Stiles GL, Caron MG, Lefkowitz RJ. ,B-adrenergic receptors: biochemical mechanisms of physiological regulation. Physiol Rev 1984;64:661-743. 4. Sibley DR, Lefkowitz RJ. Molecular mechanisms of receptor desensitization using the ,B-adrenergic receptor coupled adenylatecyclase system as a model. Nature 1985;317: 1249. 5. Bristow MR, Ginsburg R, Umans Y, et al. (31- and (3yadrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective ,Bl-receptor down-regulation in heart failure. Circ Res 1986;59:297-309. 6. Bohm M, Diet F, Feiler G, et al. Subsensitivity of the failing human heart to isoprenaline and milrinone is related to ,B-adrenoceptor downregulation. J Cardiovasc Pharmacol 1988;12:726-32. 7. Murphree SS, Saffitz JE. Distribution of ,B-adrenergic receptors in failing human myocardium: implications for mechanism of down-regulation. Circulation 1989;79: 121425. 8. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J THORAC CARDIOVASC SURG 1969; 57:225-9. 9. Billingham ME. Diagnosis of cardiac rejection by endomyocardial biopsy. J Heart Transplant 1982; I:25-30. 10. Bristow MR, Port JD, Hershberger RE, Gilbert EM, Feldman AM. The ,B-adrenergic receptor-adenylate cyclase complex as a target for therapeutic intervention in heart failure. Eur Heart J I989;IO(suppl 8):45-54. II. Horn EM, Corwin SJ, Steinberg SF, et al. Reduced lymphocyte stimulatory guanine nucleotide regulatory protein
Rejection changes in myocardial (3-receptors
12.
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15.
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20.
21.
22.
23.
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and ,B-adrenergic receptors in congestive heart failure and reversal with angiotensin converting enzyme inhibitor therapy. Circulation 1988;78:1373-9. DiSesa YJ, Masetti P, Diaco M, Schoen FJ, Marsh JD, Cohn LH. The mechanism of heart failure caused by cardiac allograft rejection. J THORAC CARDIOVASC SURG 1991;101:446-9. Yatner DE, Lavallee M, Amano J, Finizola A, Homey CJ, Yatner SF. Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog. Circ Res 1985;57:55-64. Lurie KG, Bristow MR, Reitz BA. Increased ,B-adrenergic receptor density in an experimental model of cardiac transplantation. J THORAC CARDIOVASC SURG 1983;86:195201. Yusef S, Theodoropoulos S, Mathias CJ, et al. Increased sensitivity of the denervated transplanted human heart to isoprenaline both before and after ,B-adrenergic blockade. Circulation 1987;75:696-704. Mukherjee A, WongTM, Buja LM, Lefkowitz RJ, Willerson JT. Beta-adrenergic and muscarinic cholinergic receptors in canine myocardium: effects of ischemia. J Clin Invest 1979;64: 1423-8. Mukherjee A, Bush LR, McCoy KE, et al. Relationship between ,B-adrenergic receptor numbers and physiological responses during experimental canine myocardial ischemia. Circ Res 1982;50:735-41. Salomon NW, Stinson EB, Griepp RB, Shumway NE. Alterations in total and regional myocardial blood flow during acute rejection of orthotopic canine cardiac allografts. J THORAC CARDIOVASC SURG 1978;75:542-7. Hamano K, Ohmi M, Esato K, Mohri H. Myocardial tissue blood flow in allotransplanted rat heart with a special reference to acute rejection. J Heart Transplant 1989;8:4852. Hansen DE, Daughters GT II, Alderman EL, et al. Effect of acute human cardiac allograft rejection on left ventricular systolic torsion and diastolic recoil measured by intramyocardial markers. Circulation 1987;76:998-1008. Canby RC, Evanochko WT, Barrett LY, et al. Monitoring the bioenergetics of cardiac allograft rejection using in vivo P-31 nuclear magnetic resonance spectroscopy. J Am Coil Cardiol 1987;9:1067-74. Limas CJ, Goldberg IF, Limas C. Autoantibodies against ,B-adrenoceptors in human idiopathic dilated cardiomyopathy. Circ Res 1989;64:97-103. Williams LT, Snyderman R, Lefkowitz RL. Identification of ,B-adrenergic receptors in human lymphocytes by (-)[3H]alprenolol binding. J Clin Invest 1976;57:149-55.
Changes in myocardial f)-adrenergic receptors during acute rejection of heterotopically transplanted rat hearts To evaluate changes of the myocardial {j-adrenergic receptors in acute cardiac graft rejection, the density and binding affinity value of the myocardial {j-adrenergic receptors in heterotopically transplanted rat isografts and allografts were analyzed. Hearts from Fisher rat donors were transplanted either to the Fisher rats (isografts) or to Lewis rats (allografts). Histologic examination of the allografts showed mild to moderate rejection on the seventh and fourteenth days and showed severe rejection on the twenty-first day after transplantation. The density values in the allografts and isografts similarly increased significantly (p < 0.05) above the normal level on the seventh and fourteenth days after transplantation. The density in allografts on the twenty-first day decreased significantly (p < 0.05) below the normal level, while that in isografts remained at the normal level. In contrast, the binding affinity value of myocardial {j-adrenergic receptors in both isografts and allografts did not change after transplantation. These results demonstrated that myocardial {j-adrenergic receptors presented upregulation in mild to moderate rejection, whereas these receptors presented downregulation in severe rejection. The data suggested that downregulation of myocardial {j-adrenergic receptors plays a major role in decreased cardiac contractility during severe rejection, but not during mild and moderate rejection. (J THORAC CARDIOVASC SURG 1992;104:1567-71)
Hitoshi Yokoyama, MD, Mikio Ohmi, MD, Atsusi Iguchi, MD, Sadayuki Murata, MD, Takahiko Nakame, MD, Koichi Tabayashi, MD, and Hitoshi Mohri, MD, Sendai, Japan
Since cardiac transplantation has become an effective modality of therapy for selected patients with end stage heart diseases, acute rejection of the cardiac graft is a serious problem for postoperative care. The Stanford group-? demonstrated that detectable deterioration of From the Department of Thoracic and Cardiovascular Surgery, Tohoku University School of Medicine, Sendai, Japan. Supported by a Grant-in-Aid for Specific Project Research (No. 03670650) from the Ministry of Education, Science and Culture, Japan. Received for publication Aug. 16, 1991. Accepted for publication March 25, 1992. Address for reprints: Hitoshi Yokoyama, MD, Department of Thoracic and Cardiovascular Surgery, Tohoku University School of Medicine, I-I Seiryo-machi, Sendai 980, Japan.
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cardiac contractility at rest was a relatively late event in the course of rejection, suggesting myocardial necrosis as a major factor for decreased cardiac contractility. However, changes of the cellar mechanism of positive inotropy during acute rejection are still unclear. Myocardial {j-adrenergic receptors located on cardiac sarcolemmal membrane are known to be a major pathway in producing positive inotropic effects.' These receptors are changed in their density and binding affinity, in some cases resulting in changes in cardiac contractility and in response to catecholamines-"? Therefore changes in the density and binding affinity of myocardial {j-adrenergic receptors in transplanted cardiac grafts during acute rejection could be a subject worth investigating. The purpose of the present study is to evaluate the relationship between cardiac rejection and changes in the 1567
I 568
The Journal of Thoracic and Cardiovascular
Yokoyama et al.
myocardial l3-adrenergic receptors. In this study, posttransplant changes in the maximal density (B-max) and binding affinity value (Kd) of myocardial d-adrenergic receptors were examined in the heterotopically transplanted allografts and isografts of the rat heart in comparison with their histologic gradings of acute rejection. Isografts represent sympathetic denervated hearts without acute rejection, while allografts represent sympathetic denervated hearts with acute rejection. Thus comparison of allografts with isografts would reflect the effects of acute rejection.
Materials and methods Animal preparations. Male Lewis rats and Fisher rats weighing 200 to 300 gm were used. Animal models of transplantation were produced by Fisher hearts transplanted to Lewis rats (allografts) or to Fisher rats (isografts). The surgical technique for heterotopic intraabdominal cardiac transplantation was a modification of the method described by Ono and Lindsey.8 Briefly, a pair of rats was anesthetized with an intraperitoneal injection of pentobarbital sodium (20 mg/kg of body weight). The donor rat was intubated, following which controlled ventilation was instituted using a respirator for small animals. After the intravenous injection of 1000 U of heparin sodium, the inferior vena cava and bilateral superior venae cavae were ligated and subsequently dissected. The ascending aorta and main pulmonary trunk were dissected, followed by mass ligation of the pulmonary veins and isolationof the donor heart. Twenty milliliters of ice-cold saline was injected into the ascending aorta and the isolated heart was kept in the same solution until transplantation. The ascending aorta and pulmonary artery of the donor heart were anastomosed to the abdominal aorta and inferiorvena cava of the recipient rat, respectively, in an end-to-side fashion with a continuous suture of 8-0 polypropylene.The ischemic time of the donor heart was within 30 minutes. In the preliminary experiments, allografts were completely rejected in 20 to 26 days tn = 9) when no immunosuppressive measures were taken. Then beating cardiac grafts were harvested on the seventh, fourteenth, or twenty-first day after transplantation and were submitted to both histologic and receptor-binding studies. Normal, untreated hearts of Fisher rats were also submitted to receptor-binding studies. All animals received humane care in accordance with the Guide for Animal Experimentation of Tohoku University and Tohoku University School of Medicine. Histologic analysis. Cross-sectional ventricular specimens of 1 mm in thickness were obtained from the midlevel of the longitudinal cardiac axis parallel to the horizontal axis. After fixation in buffered 10%formalin, the specimenswere examined with a light microscope using standard hematoxylin and eosin staining. Histologic gradings of the severity of cardiac rejection were classified by the method of Billingham?into mild, moderate, and severe rejection. Each histologic grading was given a score (rejection grade score) as no rejection = 0, mild rejection = 1, moderate rejection = 2, and severe rejection = 3. Receptor-binding studies. The remaining specimens ofventricular myocardium were immersed in ice-cold Tyrode's solution and were homogenized with three IS-second bursts at full
Surgery
speed on a Polytron PT-20 homogenizer (Brinkmann Instruments, Westbury, N.Y.). The homogenate was centrifuged at 1000 g for 10 minutes. The supernatant was centrifuged at 30,000 g for 15 minutes. The pellets containing sarcolemmal membranes were resuspended in ice-cold assay buffer (Tris 50 mmol/L, PMSF (phenylmethylsulfonylfluoride) 0.1 mmol/L, pH 7.4) by 10 strokes of a tight fitting Potter homogenizer (Wheaton Instruments, Millville, N.J.). The membrane preparations were stored at -80° C until binding assay was made. Protein concentrations of the membrane preparations were measured by the micro-Bradford method (Bio-Rad Laboratories, Cambridge, Mass.) with bovine serum albumin as the standard. IJ-Adrenergicdensity and binding affinity were assessedwith the IJ-adrenergic receptor ligand 3H-CGP 12117 (Amersham, Arlington Heights, IIl.). The membrane preparations (100 ~d) were added to tubes containing assay buffer and a variable concentration of 3H-CGP 12117 with or without 5 X 10- 5 mol/L propranolol in a final volume of 500 Ill. The assay mixture was incubated with shaking at 37° C for 40 minutes, and the reaction was discontinued by coolingthe mixture tubes to an ice-cold temperature. Then rapid filtering under a controlled vacuum through Watmann GF /C filters with a Brandel multiple port cell harvester (M-24R, Brandel Inc., Gaithersberg, Md.) was performed. The tubes were washed five times with 2 ml of icecold buffer. The glass filterswere dried under hot lightsand were packed in 20 ml scintillation vials containing 5 ml scintillation medium and were counted in a gamma counter. The B-max of 3H-CGP binding site and the Kd for 3H-CGP were calculated by Scatchard plots from the binding data. The regression lines in Scatchard plots were fitted by the method of least squares. Eight data points were determined for the Scatchard analysis. Each determination was done in triplicate. Statistical analysis. Values are expressed as means ± S.E. of n experiments, unless otherwise stated. The difference between mean values was analyzed by unpaired Student's t test or, when appropriate, by analysis of variance, and were judged to be significant when p values were less than 0.05.
Results Histologic findings. All isografts showed near normal histologic findings except for slight increases in interstitial fibrosis, which were seen only in the twenty-first day specimens. Posttransplant changes in the rejection grade scores of allografts showed near linear increases as time passed, registering scores of 1.3 ± 0.2 on the seventh day, 1.8 ± 0.4 on the fourteenth day and 2.8 ± OJ on the twenty-first day (Fig. I, A). Density of myocardial /3-adrenergic receptors. Posttransplant changes in the B-max of myocardial o-adrenergic receptors in the isografts and allografts are demonstrated in Fig. 1, B. B-max in the normal hearts of Fisher rats was 52.9 ± 4.5 fmol/rng protein (n = 6). B-max in the isografts was 90.0 ± 8.0,77.8 ± 2.3, and 51.9 ± 1.9 frnol/rng protein on the seventh, fourteenth, and twentyfirst day after transplantation, respectively. B-max values in the isografts on the seventh and fourteenth days after
Volume 104 Number 6 December 1992
Rejection changes in myocardial fJ-receptors
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Fig. 2. Relationshipbetweenthe histologic grades of rejection and the density (B-max) of myocardial iJ-adrenergicreceptors in the heterotopically transplanted rat isografts and allografts. Rejectiongrade scoresare givenin detail in the "Materials and methods" section. Data points represent means ± SE (n = 6). *p < 0.05 against normal values (day 0).
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21
Time after transplantation (days)
Fig. 1. A, Posttransplant changes in histologic gradings of rejection in the heterotopically transplanted allografts of rat hearts. Rejection grade scoresare given in detail in the "Materialsand methods"section.Data points represent means ± SE (n = 6). B, Posttransplant changes in the density (B-max) of myocardial iJ-adrenergic receptors in the heterotopically transplanted rat isografts and allografts. Data points represent means ± SE (n = 6). *p < 0.05 against normalvalues(day 0); a represents a significant (p < 0.05) difference between isografts and allografts.
transplantation were significantly (p < 0.05) greater than the normal value. B-max in the allografts was 77.6 ± 8.7, 79.6 ± 15.6, and 19.4 ± 9.0 fmol/rng protein on the seventh, fourteenth, and twenty-first days after transplantation, respectively. B-max values in the allografts on the seventh and fourteenth days after transplantation were significantly (p < 0.05) greater than the normal value. However, B-max values in the allografts on the twentyfirst day after transplantation were significantly (p < 0.05) smaller than the normal value and the value in the isografts. Relationship between the rejection grade scores and B-max of myocardial ,B-adrenergic receptors in the allografts. B-max of normal hearts of Fisher rats with no
rejection was 52.9 ± 4.5 frnol/rng protein (n = 6). B-max of allografts in rejection grade score 1 (mild rejection) was 65.5 ± 8.2 frnol/rng protein, which was not significantly greater than the normal value (p = 0.132). In hearts of rejection grade score 2 (moderate rejection), B-max increased significantly (p < 0.05) to higher levels (99.7 ± 9.5 frnol/rng protein) above the normal value. However, B-max of the hearts in rejection grade score 3 (severe rejection) decreased significantly (p> 0.05) below the normal value, to 19.4 ± 9.1 frnol/ mg protein (Fig. 2). Binding affinity value of myocardial ,B-adrenergic receptors. Posttransplant changes in the Kd of fJ-adrenergic receptors in the isografts and allografts are demonstrated in Fig. 3. Kd of fJ-adrenergic receptors in normal hearts of Fisher rats was 1.4 ± 0.3 nmol (n = 6). Kd values after transplantation were not significantly different from the normal value in both isografts and allografts. Discussion fJ-Adrenergic receptors on the myocardial sarcoplasmic membrane playa major role in producing positive inotropic effects in mammalian cardiac muscles.' Changes in B-max and Kd of fJ-adrenergic receptors have been investigated recently, especially in failing human hearts with dilated cardiomyopathy, ischemic cardiomyopathy, or mitral valve diseases.>? In these cases, decreased B-max of the myocardial fJ-adrenergic receptors was observed. Implications for the treatment of the failing human heart by modification of the fJ-adrenergic system have also been explored. 10, 11 Transplanted hearts
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The Journal of Thoracic and Cardiovascular Surgery
Yokoyama et al.
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Time after transplantation (days)
Fig. 3. Posttransplant changes in the binding affinity value (Kd) of myocardial,B-adrenergic receptors in the heterotopically transplanted rat isograftsand allografts. Data points represent means ± SE (n = 6). All resultswerenot significantly different from normal values (day 0). during acute rejection show decreased cardiac contractility': 2 or impaired cardiac response to catecholamines.F Recently, changes in myocardial {3-adrenergic pathways in acute rejection have been suggested by assessing the cardiac response to {3-adrenergic receptor stimulation in animal experiments.'? However, the mode of changes in B-max and Kd of myocardial {3-adrenergic receptors during acute rejection has not been evaluated. The results of the current study showed increased B-max of myocardial {3-adrenergic receptors in the heterotopically transplanted rat heart allografts during mild to moderate rejection. In other words "upregulation" of {3-adrenergic receptors was observed. Isografts also presented the upregulation of {3-adrenergic receptors on the seventh and fourteenth days after transplantation. Cardiac denervation alone has been known to upregulate the myocardial {3-adrenergic receptors.l': 14 which promotes the sensitivity of cardiac responsiveness to adrenergic stimulation. 13, 15 Therefore a mechanism of the upregulation in the allografts is speculated to be mainly caused by sympathetic denervation, as in the case of the isografts. In addition, some compensatory mechanisms of the myocardium might be responsible for the upregulation when myocardial necrosis decreased the total amount of myocardium. This mechanism is likely because, in comparison with mild rejection, the level of upregulation was higher during moderate rejection when histologic myocardial necrosis starts. Another explanation may be an effect of myocardial ischemia. Upregulation of {3-adrenergic receptors was reported in some animal experiments in which myocardial ischemia was
induced by transient occlusion of the coronary arteries. 16, 17 A decrease in coronary blood flow in the early phase of acute rejection has been reported in animal experiments on dogs" and rats.!" DiSesa and associates I 2 reported impaired myocardial contractile reserve during mild and moderate rejection. However, the results in the present study did not reveal downregulation of the myocardial {3-adrenergicreceptors. Thus the impaired myocardial contractile reserve found by DiSesa and colleagues may not be caused by {3-adrenergic receptor downregulation. They also reported that chronotropic responses to isoproterenol in rat allografts were preserved nearly equally to those in isografts when the allografts showed moderate rejection.U Preserved chronotropic response during moderate rejection is consistent with the results in the present study, because positive chronotropy is mainly modulated by myocardial {3-adrenergic receptors. 3 The present study demonstrated that the allografts showed significant downregulation of the myocardial {3-adrenergic receptors in the terminal stage of acute rejection. Besides myocardial necrosis, this downregulation may be a reason for deterioration of left ventricular contractility in the late period of acute rejection.': 2, 20 Decrease in myocardial high-energy phosphate content in the terminal stage of acute rejection has been reported recently." This finding suggested impaired synthesis of cellular proteins such as {3-adrenergic receptors in severe rejection, accompanied by the receptor downregulation. The presence of anti-{3-adrenergic receptor antibodies was recognized recently in a substantial proportion of patients with idiopathic dilated cardiomyopathy.F However, whether myocardial {3-adrenergic receptors are one of the direct immunologic targets or not is still unknown. The sarcolemmal membrane of lymphocytes also have {3-adrenergic receptors.P Therefore whether the observed {3-adrenergic receptor changes were caused by the changes in myocardium or by infiltrated lymphocytes must be determined. Since the amount of infiltrated lymphocytes assessed by histologic findings increased steadily despite downregulation of B-max during severe rejection, the changes in B-max of {3-adrenergic receptors obtained in this study were likely caused by the changes in the myocardium but not by the lymphocytes. The Kd of {3-adrenergic receptors is determined by the three-dimensional conformation of their binding site.' Since the present study showed that the Kd values did not change in both isografts and allografts after transplantation, conformational changes of the myocardial {3-adrenergic receptor proteins, at least their binding sites, may not have occurred in acute cardiac graft rejection.
Volume 104 Number 6 December 1992
We thank Dr. Kazuo N unoki of the Second Department of Pharmacology, Tohoku University School of Medicine, for his useful advice in the receptor-binding studies, and Dr. Nobuaki Tamahashi of the Japan Pathology Institute, Sendai, for his help in histologic evaluation of cardiac rejection.
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