Autoantibodies in vitro modulating the M2 acetylcholine receptor (M2ACh-R) were observed in patients with idiopathic dilated cardiomyopathy (IDC) or Chagas' cardiomyopathy (ChC). We investigated the in vivo consequences on heart rate of such antibodies in mice immunized with a peptide derived from the second extracellular loop of the M2ACh-R compared with mice immunized with an irrelevant peptide. Sera of mice immunized with the M2ACh-R-derived peptide recognized the M2ACh-R on immunoblots and enhanced agonist activity of carbachol toward the M2AChR transfected in CHO cells. In vivo, no difference could be shown in heart rate or heart rate variability between the two groups of mice. The decrease in heart rate induced by carbachol was more pronounced, however, in the M2ACh-R immunized mice. The increase in heart rate induced by atropine, gallamine, and isoproterenol was significantly attenuated in the M2ACh-R immunized mice. Analysis of heart rate variability further argued for an increased parasympathetic response to different drugs in the M2ACh-R immunized mice. Antibodies raised against the M2AChR can behave as positive M2AChR allosteric modulators in vivo. They might be protective in boosting the activity of the parasympathetic drive to the heart, especially in patients with a high sympathetic tone.
Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.The G protein-coupled receptor family is one of the main targets of currently used drugs (1). The M2 muscarinic acetylcholine receptor (M2Ach-R) 1 belongs to this receptor family. This receptor is an integral membrane protein consisting of seven membrane spanning ␣-helices linked together by extraand intracellular loops that form a pharmacophore pocket. One of the pharmacological challenges posed by this family of receptors is the presence of multiple subtypes, all recognizing the same endogenous ligands. This suggests a high conservation of the pharmacophore for a particular family of receptors, thus explaining the difficulty to develop drugs (agonists or antagonists) specific for one of these subtypes. Autoantibodies directed against cardiovascular G protein-coupled receptors functionally interfering with the target have been described in several cardiovascular diseases (2-6). Most of these autoantibodies are directed against the second extracellular loop and are exquisitely specific for one of the receptor subtypes in view of the highly variable sequence of this domain (7). Elies et al. (8) have produced a monoclonal antibody directed against a peptide derived from the N-terminal part of the second extracellular loop of the M2Ach-R. This monoclonal antibody (IgG 2a) displays a partial agonist activity on its target receptor because of its natural bivalency. It is able to stabilize constitutive active receptor dimers and paradoxically induces a small decrease in carbachol affinity for the M2Ach-R.In this study, this antibody was used to construct an scFv fragment (single chain variable fragment), which was cloned, sequenced and expressed in Escherichia coli. We describe the immunochemical, pharmacological, and physiological properties of this scFv fragment. As reported in a previous study with an inverse agonist scFv-directed against the  2 adrenergic receptor (9) this may open the way to the design of allosteric inverse agonist reagents that are specific for one subtype of receptor. EXPERIMENTAL ...
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