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 result 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 guanine nucleotide binding proteins have also been proposed 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 failure4 is one potential mechanism for downregulating cardiac/3-

From the Departments of Medicine (Cardiovascular Division) and Laboratory 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

1322

receptors. Receptor changes may thus be an epiphenomenon, 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 possibility that the cause of the disease process leading to heart failure determines the pathogenesis of/3-receptor 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 inhibition assay) were higher in patients w.ith cardiomyopathy than in control subjects with ischemic or valvular heart disease and comparable degrees of hemodynamic impairment. In this study we have examined the effect of sera and IgG in patients with dilated cardiomyopathy on the cardiac adenylate cyclase 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.

Volume

119

Antireceptor antibodies in cardiomyopathy

Number 6

1323

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rum dilutions from p a t i e n t 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 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 patients were women ranging in age from 36 to 59 years (mean = 46). T h e degree of hemodynamic i m p a i r m e n t was reflected in a low cardiac output (3.85 m 0.2 L/min), low ejection fraction (20.7 z 6 ~ ), and high pulmonary capillary 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. T r e a t m e n t at the time of the study included digitalis, diuretics, and vasodilators; none was taking fl-blockers. The presence of autoantibodies against the 8-receptor was assessed by means of the ligand binding inhibition assay as previously described. 5 R a t cardiac membranes were prepared by homogenizing minced ventricular tissues in five volumes of cold 50 m m o l / L tris-HC1-5 mmol/L EDTA, p H 7.5, with a Polytron PT-20 homogenizer at halfmaximal speed for 30 seconds • 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. T h e resultant pellet was washed twice and used as the m e m b r a n e fraction. For the [3HI dihydroalprenolol (DHA) binding inhibition assay, 100/~l of serum (1:50 to 1:400 final dilution) or buffer was p r e i n c u b a t e d with 250 #1 of the membrane suspension (0.1 to 0.2 mg protein determined by the method of Lowry et al. 6) in 50 m m o l / L tris-HC1 (pH 7.5), 5 m m o l / L MgC12, 0.1 m m o l / L phenylmethylsulfonylfluoride, 5/~g/ ml leupeptin, and 7 ~g/ml pepstatin for 60 minutes at 30 ~ C with D H A (Du P o n t New England Nuclear Research

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Fig. 2. I n h i b i t i o n o f i s o p r o t e r e n o l - s e n s i t i v e a d e n y l a t e c y clase activity by serum dilutions from patients with dilated cardiomyopathy. Results represent mean _+ standard error for eight patients and are expressed as percentage of inhibition of control enzymatic activity (incubated in absence of serum).

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isolated from sera of patients with dilated cardiomyopathy on isoproterenol-sensitive adenylate cyclase activity of r a t membrane preparations. Results are expressed as percentage 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 t e r m i n a t e d by filtration through W h a t m a n G F / C filters as previously described. 5 Nonspecific binding was determined in the presence of I #mol/L propranolol. The number of fl-receptors was d e t e r m i n e d from Scatchard plots 7 of the binding data. T h e effects of serum or IgG (prepared by protein

1324

June 1990 American Heart Journal

Limas, Goldenberg, and Limas

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