Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease.

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action.

Human antibodies with muscarinic activity modulate ventricular repolarization: basis for electrical disturbance.

INTRODUCTION: In chronic chagasic patients sudden death has been reported when QT interval dispersion is increased and antibodies with muscarinic-like activity have been demonstrated to trigger arrhythmias. The aims were to investigate, in vivo and in vitro, relation between these antibodies and heterogeneity of ventricular repolarization and to identify predictors of cardiac death in chronic chagasic patients. METHODS AND RESULTS: Clinical, electrocardiograph and echocardiograph variables from 32 chronic chagasic patients with moderate to severe left ventricular dysfunction, followed-up for 10 years were analyzed. Sera from chronic chagasic patients with or without muscarinic activity were tested in isolated rabbit hearts to study ventricular repolarization. Stepwise multivariate logistic analysis was applied to identify independent predictors of cardiac death. QT interval dispersion of patients with muscarinic activity (75.9+/-5.5 ms) was larger than that of patients without muscarinic activity (51.3+/-4.0 ms, p<0.001). Maximum uncorrected and corrected QT intervals were not significantly different between groups of patients. Sera from patients with muscarinic activity significantly and reversibly increased QT interval in isolated rabbit hearts (p=0.002). This effect was abolished in the presence of the muscarinic antagonist atropine. Multivariate analysis identified maximum corrected QT intervals and left ventricular end diastolic index as independent predictors of cardiac death (p=0.03 and p=0.02, respectively). CONCLUSIONS: Sera with muscarinic activity from chagasic patients have a strong contribution to evoke ventricular repolarization rhythm disorder. In these patients, ventricular repolarization heterogeneity is increased significantly. In vitro, muscarinic sera reversibly increased repolarization duration. Maximum corrected QT intervals and left ventricular end diastolic index are independent predictors of cardiac death.

DNA immunizations with M2 muscarinic and beta1 adrenergic receptor coding plasmids impair cardiac function in mice.

Autoimmune mediated myocardial damage is likely to be a pathogenic mechanism for acquired dilated cardiomyopathies. Evidence confirms that autoantibodies that bind to M(2) muscarinic (M(2)AChR) and beta(1) adrenergic receptors (beta(1)AR) are present in idiopathic dilated cardiomyopathy and Chagasic patients' sera. To elucidate the role of these antibodies in cardiac functional impairment, we used a murine model immunized with plasmids encoding the M(2)AChR or beta(1)AR via gene-gun bombardment. Anti-M(2)AChR and beta(1)AR antibodies were detected over the course of 37 weeks. These antibodies were directed to the second extracellular loop (el2) of both receptors and the third intracellular loop (il3) of the M(2)AChR. Peak antibody titers from weeks 2 to 5 against M(2)AChR-el2 and beta(1)AR-el2 as well as elevated titers against M(2)AChR-il3 were detected. Anti-M(2)AChR-il3 and anti-beta(1)AR-el2 antibodies were predominant in IgG1 subclass immunoglobulins, suggesting a T-helper-2 biased lymphocyte response. Heart morphology and function was assessed by echocardiography over the course of 42 weeks. Data showed progressive decrease in left ventricular (LV) wall thickness and LV mass that was mostly evident for beta(1)AR-immunized mice albeit a small change in LV dimensions. Fractional shortening was altered and values of 41%, 37% and 48% were observed at week 42 for the M(2)AChR, beta(1)AR and control groups respectively. In support of autonomic deregulation, a twofold increase in M(2)AChR and a similar decrease in beta(1)AR density were observed in radioligand saturation assays for both experimental groups. Histological analysis revealed myofibril disarray and fibrosis, pointing towards remodeling as a consequence of the long-term presence of anti-receptor antibodies.