AimsAutoantibodies against second extracellular loops of β1-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function.Methods and resultsIgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to β1-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed.Conclusions(i) Autoantibodies trigger conformation changes in the β1-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

Mitochondrial topoisomerase I is a genetically distinct mitochondria-dedicated enzyme with a crucial but so far unknown role in the homeostasis of mitochondrial DNA metabolism. Here, we present data suggesting a negative regulatory function in mitochondrial transcription or transcript stability. Deficiency or depletion of mitochondrial topoisomerase I increased mitochondrial transcripts, whereas overexpression lowered mitochondrial transcripts, depleted respiratory complexes I, III and IV, decreased cell respiration and raised superoxide levels. Acute depletion of mitochondrial topoisomerase I triggered neither a nuclear mito-biogenic stress response nor compensatory topoisomerase IIβ upregulation, suggesting the concomitant increase in mitochondrial transcripts was due to release of a local inhibitory effect. Mitochondrial topoisomerase I was co-immunoprecipitated with mitochondrial RNA polymerase. It selectively accumulated and rapidly exchanged at a subset of nucleoids distinguished by the presence of newly synthesized RNA and/or mitochondrial RNA polymerase. The inactive Y559F-mutant behaved similarly without affecting mitochondrial transcripts. In conclusion, mitochondrial topoisomerase I dampens mitochondrial transcription and thereby alters respiratory capacity. The mechanism involves selective association of the active enzyme with transcriptionally active nucleoids and a direct interaction with mitochondrial RNA polymerase. The inhibitory role of topoisomerase I in mitochondrial transcription is strikingly different from the stimulatory role of topoisomerase I in nuclear transcription.

Growing evidence indicates a cardio-pathogenic role of autoantibodies against β1-adrenergic receptors (β1AR). In particular autoantibodies stimulating β1AR-mediated cAMP-production (i.e. agonistic β1AR autoantibodies) play a paramount role in chronic heart failure. When induced by immunisation, such autoantibodies cause heart failure in rodents; when present in patients they negatively affect survival in heart failure. However, the true prevalence and clinical impact of agonistic β1AR autoantibodies in human heart disease are still unclear, as are the events triggering their production, and the inter-relationship between autoantibody level and disease activity. β1AR autoantibodies can be removed by extracorporeal absorption or neutralised by systemic administration of synthetic epitope mimics. Only patients bearing agonistic β1AR autoantibodies in their bloodstream will benefit from these approaches. Therefore, reliable detection of agonistic β1AR autoantibodies is a key pre-requisite for the future implementation of these strategies. β1AR autoantibodies impact on conformation and down-stream signalling of the receptor by binding a conformational epitope, which is poorly represented by synthetic mimics and readily destroyed by fixation. Consequently, β1AR autoantibodies can reliably be detected only by assays utilising the native β1AR as a test antigen. To provide a sufficient basis for diagnostic predictions or therapeutic decisions, one must also determine whether β1AR autoantibodies stimulate the receptor, which again requires native, cell-based reporter systems. Translation of these procedures into versatile diagnostic tests fitting the requirements of general health care is a challenge for future development. Here, we will review the state of diagnostic and therapeutic efforts in the field of β1AR-directed autoimmunity, thereby aiming to furnish a conceptual frame for the further development of novel, more reliable diagnostic tools and more specific antibody-targeted therapeutic concepts.

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The novel assay possibly provides a tool to determine true prevalence and clinical impact of β1AR-autoantibodies. Furthermore, it may serve as companion diagnostic for therapies specifically directed at β1AR-autoantibodies.

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