Influence of Anti-beta-receptor Antibodies on Cardiac Adenylate Cyclase in Patients With Idiopathic Dilated Cardiomyopathy. AHJ 1990

Irvin Goldenberg Influence of Anti-beta-receptor Antibodies on Cardiac Adenylate Cyclase in Patients With Idiopathic Dilated Cardiomyopathy
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  Influence of anti beta receptor antibodies on cardiac adenylate cyclase in patients with idiopathic dilated cardiomyopathy Autoantibodies against the cardiac ~l-adrenoceptor are present in the sera of patients with idiopathic dilated cardiomyopathy and may modulate the responsiveness of cardiac/~-adrenergic pathways to agonists. The regulation of cardiac adenylate cyclase activity by autoantibodies was examined in 50 patients with dilated cardiomyopathy. Inhibition of isoproterenol-sensitive adenylate cyclase activity could be demonstrated by serum dilutions or IgG in 52 (26 of 50) of the patients; basal and NaF-stimulated activities, in contrast, were unaffected. In 14 patients, both ligand binding to ~-receptor and isoproterenol-sensitive adenylate cyclase activity were inhibited by lO0-fold serum dilutions. Pretreatment of cardiac membranes with pertussis toxin did not affect inhibition of adenylate cyclase indicating that the effect of sera does not depend on Gi. The immunogeneUc control of antireceptor antibodies was examined by comparing the distribution of HLA antigens in antibody-positive and antibody-negative patients. HLA-DR4 and HLA-DR1 were strongly associated with antibodies inhibiting ligand binding and adenylate cyclase activity (71 of patients with such antibodies typed as either DR4 or DR1). Conversely 58 of patients with HLA-DR4 and 71 of patients with HLA-DR1 antibodies showed inhibition of adenylate cyclase activity compared to 46 of those who lacked both HLA-DR4 and HLA-DR1 antibodies. These results strongly suggest that cardiac ~-adrenergic receptors and adenylate cyclase activity in dilated cardiomyopathy can be modulated by circulating autoantibodies, the presence of which is under the control of the major histocompatibility complex. (AM HEART J 1990;119:1322.) Constantinos J. Limas, MD, Irvin F. Goldenberg, MD, and Catherine Limas, MD. Minneapolis Minn. Reduced inotropic responsiveness to fi-agonists in patients with heart failure appears to occur as a re- sult of a decline in the number of cardiac/~-adrenergic receptors and the activity of isoproterenol-sensitive adenylate cyclase. 1, 2 Defects at the level of the gua- nine nucleotide binding proteins have also been pro- posed but have not been adequately documented. 3 It is likely that these multiple defects are receptor driven, that is, they result from altered f~-receptor numbers, receptor-agonist interactions, or both. The stimulation of the sympathetic nervous system that accompanies the development of heart failure 4 is one potential mechanism for downregulating cardiac/3- From the Departments of Medicine Cardiovascular Division) and Labora- tory Medicine and Pathology, University of Minnesota School of Medicine and the Minneapolis Heart Institute, Received for publication Dec. 22, 1989; accepted Jan. 30, 1990. Reprint requests: Constantinos J. Limas, MD, Box 19, UMHC, Department of Medicine, University of Minnesota School of Medicine, Minneapolis, MN 55455. 4/1/19811 receptors. Receptor changes may thus be an epiphe- nomenon, that is, a consequence of the neurohumoral adaptations to the syndrome of clinical heart failure independent of the underlying cause. We have been interested in the alternative possi- bility that the cause of the disease process leading to heart failure determines the pathogenesis of/3-re- ceptor alterations. Recently we reported the presence of autoantibodies directed against the cardiac /~- receptor in sera of patients with idiopathic dilated cardiomyopathy. 5 Both the prevalence and the titer of these antibodies defined by a ligand binding inhi- bition assay) were higher in patients w.ith cardiomy- opathy than in control subjects with ischemic or valvular heart disease and comparable degrees of he- modynamic impairment. In this study we have ex- amined the effect of sera and IgG in patients with dilated cardiomyopathy on the cardiac adenylate cy- clase activity and identify possible immunogenetic factors controlling the development of antibodies that interact with cardiac fi-receptors and result in ihibition of adenylate cyclase activity. 1322   olume 119 Number 6 Antireceptor antibodies in cardiomyopathy 1323 r ~ E .9 Z C P 0 0 50 40 30 20 10 0 0 50 100 150 200 Serum Dilution Fig. 1. Typical ligand binding inhibition curve using se- rum dilutions from patient with dilated cardiomyopathy. Results are expressed as percentage of inhibition of [3HI dihydroalprenolol DHA) binding to Cardiac membranes compared to control subjects (incubated in absence of se- rum). . 40 O 0 g ~ 20 6O O 50 u r O ~z 40 r .2 9 30 m ._ m ==o 20 ~ 10 a 0 I I 0 50 100 150 200 250 Serum Dilution Fig. 2. Inhibitionofisoproterenol-sensitiveadenylatecy- clase activity by serum dilutions from patients with dilated cardiomyopathy. Results represent mean _+ standard error for eight patients and are expressed as percentage of inhi- bition of control enzymatic activity (incubated in absence of serum). METHODS Studies on anti-/3-recep~or antibodies were carried out with the use use of diluted serum on IgG from 50 patients with idiopathic dilated cardiomyopathy. Nine of these pa- tients were women ranging in age from 36 to 59 years (mean = 46). The degree of hemodynamic impairment was reflected in a low cardiac output (3.85 m 0.2 L/min), low ejection fraction (20.7 z 6~ ), and high pulmonary capil- lary wedge pressure (23.6 z 4 mm Hg). The presence of coronary artery disease had been excluded by means of coronary arteriography in all patients. Treatment at the time of the study included digitalis, diuretics, and vasodi- lators; none was taking fl-blockers. The presence of autoantibodies against the 8-receptor was assessed by means of the ligand binding inhibition as- say as previously described. 5 Rat cardiac membranes were prepared by homogenizing minced ventricular tissues in five volumes of cold 50 mmol/L tris-HC1-5 mmol/L EDTA, pH 7.5, with a Polytron PT-20 homogenizer at half- maximal speed for 30 seconds ã 3. The homogenates were centrifuged at 500 g for 10 minutes, and the supernates passed through gauze before centrifugation at 40,000 fl for 15 minutes at 4 ~ C. The resultant pellet was washed twice and used as the membrane fraction. For the [3HI dihydroalprenolol (DHA) binding inhibi- tion assay, 100/~l of serum (1:50 to 1:400 final dilution) or buffer was preincubated with 250 1 of the membrane sus- pension (0.1 to 0.2 mg protein determined by the method of Lowry et al. 6) in 50 mmol/L tris-HC1 (pH 7.5), 5 mmol/ L MgC12, 0.1 mmol/L phenylmethylsulfonylfluoride, 5/~g/ ml leupeptin, and 7 ~g/ml pepstatin for 60 minutes at 30 ~ C with DHA (Du Pont New England Nuclear Research '~ 10 0 -- ~'~ C Fig. 3. 0 0.0 I I I I I I I I 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 IgG (mg/ml) Concentration dependence of influence of IgG isolated from sera of patients with dilated cardiomyopathy on isoproterenol-sensitive adenylate cyclase activity of rat membrane preparations. Results are expressed as percent- age of inhibition of control enzymatic activity (incubated in absence of IgG) and represent mean -~ standard error for eight patients. Products, Boston, Mass., specific activity 105 Ci/mmol) and continued for 15 minutes. It was terminated by filtra- tion through Whatman GF/C filters as previously de- scribed. 5 Nonspecific binding was determined in the pres- ence of I mol/L propranolol. The number of fl-receptors was determined from Scatchard plots 7 of the binding data. The effects of serum or IgG (prepared by protein  June 1990 1324 Limas Goldenberg and Limas merican Heart Journal .> E 0 U ) <~- O~ ,r 0.8 [] Control [] Serum 0.6 0.4 0.2 0.0 ~~ Basal Isoproteranol stimulated m NaF stimulated Fig 4. Effect of sera from patients with dilated cardiomyopathy on basal, isoproterenol-, and NaF-stim- ulated adenylate cyclase activities of rat cardiac membrane preparations. Experiments were carried out in presence or absence of 100-fold serum dilutions as described in text. Results represent mean _+ standard error for 26 comparisons. *p < 0.01 compared to control subjects. Table I. Effect of pertussis toxin pretreatment of cardiac membranes on the ability of sera (100-fold dilution) from patients with cardiomyopathy to inhibit isoproterenol- sensitive adenylate cyclase activity Pertussis toxin Control treated (-) (+) (-) (+) Additions Serum Serum Serum Serum None 111 _+ 7 114 _+ 8 130 _+ 9 132 _+ 10 Isoproterenol 198 -+ 11 162 _+ 9* 252 _+ 12 203 -+ 11 (0.1 mmol/L) NaF(Smmol/L) 538_+ 16 594_+ 15 550_+ 18 548_+ 17 Results represent mean _+ standard error for six experiments. p < 0.01 compared to enzymatic activities in the absence of serum. A-Sepharose CL-4B column chromatography) on cardiac adenylate cyclase activities were assayed in a reaction mix- ture containing 40 mmol/L tris-HC1, pH 7.5, 0.5 mmol/L, [~_32p] adenosine triphosphate (Amersham/Searle, Arling- ton Heights, Ill. specific activity 30 Ci/mmol), 15 mmol/L MgC12, 8.0 mmol/L theophylline, 20 mmol/L phospho- enolpyruvate, 130 ~g/mg phosphoenolpyruvate kinase, 5.5 mmol/L KC1, 30 to 50 #g membrane protein, and when in- dicated 0.1 mmol/L (-) isoproterenol or 8.0 mmol/L NaF. Reactions were carried out at 30 ~ C for 15 minutes, and [32p] cyclic adenosine monophopshate formed was deter- mined according to the method of Salomon et al. s For study of the effects of pertussis toxin on the adenylate cyclase activity, cardiac membranes (40 to 50 ~g protein) were pretreated with or without the toxin (20 /~g/ml) in a medium containing 25 mmol/L glycylgtycine buffer, pH 7.5, 1 mmol/L NAD, 0.4 mmol/L adenosine triphosphate, 0.4 mmol/L guanosine 5'-triphosphate, 15 mmol/L thymi- dine, 10 mmol/L dithiothreitol, and 0.1 mg/ml ovalbumin at 30 ~ C for 30 minutes. The membranes were washed twice with 10 mmol/L tris-1 mmol/L EDTA buffer, pH 7.5, and resuspended in the assay buffer for determination of ade- nylate cyclase after preincubation with or without 100-fold dilutions of cardiomyopathic serum, as previously de- scribed. Lymphocytes for HLA antigen typing were isolated from 20 to 30 ml of heparinized blood by means of Ficoll- Hypaque density gradient centrifugation as previously described. 9 Purified B lymphocytes for HLA-DR typing were prepared by the method of Danilovs et al. 1~ HLA-A, B, and C antigens were assayed by means of lymphocyte suspensions with the microcytoxicity method, whereas HLA-DR typing was performed in microlymphocytotoxic- ity assays with prolonged incubation times using isolated B lymphocytes. The statistical evaluation of the results followed the method of Svejgaard et al. 1~ Chi-square 2 x 2 contingency tables were used for comparisons between pa- tient and control groups. Associations involving small numbers were tested with Fisher's exact test. p Values were corrected for the number of antigens tested, and associa- tions were considered significant if p < 0.05. RESULTS Sera of patients with idiopathic dilated cardiomy- opathy inhibit [3H] DHA binding to cardiac mem- branes in a concentration-dependent manner (Fig. 1). A 100-fold serum dilution induced 38 _+ 7% inhi- bition of [3H] DHA binding in a series of 78 consec- utive patients with dilated cardiomyopathy (data not shown); a few patients showed significant inhibition  Volume 119 Number 6 ntireceptor antibodies in cardiomyopathy 1325 70 ~ 60 '~ 50 o< 40 ~o .r._~ s 30 o Q lO o 9 2z 10 20 30 40 50 60 70 Percent Ligand Binding Inhibition Fig. 5. Correlation between extent of ligand binding and isoproterenol-sensitive adenylate cyclase inhibition by 100- fold serum dilutions from 14 patients with dilated cardio- myopathy. Percentage of inhibition is expressed in refer- ence to [Sill dihydroalprenolol binding or adenylate cyclase activity in control cardiac membranes (preincubated in absence of serum). 50 20 [] Antibody-negative n=24) [] Antibody-positive n=26) 11) 40 8) 8) :30 0 6) 7) 15 -- (5) DR1 DR2 DR3 DR4 DR5DRw6DR7 DR8 Fig. 6. Distribution of HLA-DR antigens in antibody- positive and negative patients with dilated cardiomyopa- thy. Presence of antibody was defined by >--20 inhibition of isoproterenol-sensitive adenylate cyclase activity in presence of a 100-fold serum dilution. Numbers in paren- theses represent number of patients typing for each HLA- DR antigen. (>--20 ) even at a 400-fold dilution. In contrast only 15 of patients with ischemic or valvular disease and none of the normal control subjects showed inhibi- tion at 100-fold serum dilution. In all instances inhi- bition of ligand binding can be prevented by human anti-IgG, suggesting that it is due to the presence of autoantibodies. In the present patient population, 14 of the 50 patients (30 ) showed 20 or higher inhi- bition of [3H] DHA binding at 100-fold serum dilu- tion, suggesting the presence of anti-/~-receptor an- tibodies. We sought to examine whether adenylate cyclase activities were also affected by these sera. As shown in Figs. 2 and 3, isoproterenol-stimulated adenylate cyclase is inhibited by diluted sera or IgG in a con- centration-dependent manner. Furthermore, the basal and NaF-stimulated activities, in contrast to hormone-sensitive adenylate cyclase, are not affected by sera (Fig. 4). Overall 52 (26 of 50) of patients had adenylate cyclase activity that was inhibitied by more than 20 . Of these, 14 had sera that inhibited both adenylate cyclase and ligand binding to /~- receptors, whereas 12 had inhibited isoproterenol- sensitive adenylate cyclase only. In those patients n whom both ligand binding and adenylate cyclase ac- tivities were inhibited, a good correlation (r = 0.72) between the extent of inhibition of the two processes could be demonstrated (Fig. 5). To determine whether the inhibitory effect of cardiomyopathic sera on iso- proterenol-sensitive adenylate cyclase is dependent on Gi, the effects of pretreatment with pertussis toxin were examined (Table I). Isoproterenol stimulated adenylate cyclase activity by 78 and 46 in the absence and presence of 100-fold serum dilutions, respectively. Pretreatment of membranes with per- tussis toxin resulted in enhanced isoproterenol effect (94 stimulation) in accordance with previous observations12,13; however, the inhibitory effect of serum was still detectable (54 stimulation by iso- proterenol). In contrast, NaF-stimulated adenylate cyclase activity was unaffected by pertussis toxin and the cardiomyopathic sera. The immunogenetic influences on ~-receptor and adenylate cyclase modulation by patient sera were investigated by comparing the distribution of HLA antigens in antibody-positive and negative patients (as defined by either ligand binding or adenylate cy- clase inhibition). We had previously observed that the frequency of HLA-DR4 and DR1 phenotypes is disproportionately high in anti-/~-receptor antibody- positive patients. 14 This observation was confirmed n the current series: all but four of the patients who were positive according to results of the ligand inhi- bition assay were typed as either HLA-DR4 or DR1 (six as DR4 and four as DR1). When the effect on isoproterenol-stimulated adenylate cyclase activity
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