A standardised FACS assay based on native, receptor transfected cells for the clinical diagnosis and monitoring of ß1-adrenergic receptor autoantibodies in human heart disease.

BACKGROUND: Autoantibodies against ß1-adrenergic receptors (ß1AR) that stimulate cardiac cAMP-production play a causal role in the pathogenesis of human heart failure. Patients can be subjected to specific therapies, if the presence of potentially cardio-noxious ß1AR-autoantibodies is reliably diagnosed. This requires assessment of IgG-interactions with the native ß1AR because ß1AR-autoantibodies target a conformational epitope inadequately presented by denatured receptors or linear peptides. Here, we report on a standardised diagnostic procedure for the assessment of ß1AR-autoantibodies in heart failure patients, which is based on IgG-binding to native human ß1AR. METHODS: Good laboratory practice (GLP)-conform measurement of ß1AR-autoantibodies was based on flow-cytometric quantification of differential IgG-binding to native HT1080 cells overexpressing biofluorescent human ß1AR or not. Receptor-specific IgG-binding was derived from IgG-related median fluorescence of ß1AR-positive cells corrected for background staining of ß1AR-negative cells admixed to each measurement. The slope of IgG binding at two different concentrations was used as measure for the titre/avidity of ß1AR-autoantibodies. RESULTS: Sensitivity and specificity of the novel procedure for high ß1AR-autoantibody levels in dilated cardiomyopathy patients (n=40, NYHA class III-IV) relative to n=40 matched healthy subjects was >90%. It was similar to functional assays considered the gold standard and vastly superior to existing screening-procedures employing fixed cells or linear receptor-peptides as auto-antigenic targets. Inter-assay scatter was 7%-15% and linear dilution recovery was within ±10% of expected values throughout. CONCLUSIONS: The novel assay possibly provides a tool to determine true prevalence and clinical impact of ß1AR-autoantibodies. Furthermore, it may serve as companion diagnostic for therapies specifically directed at ß1AR-autoantibodies.

Negative regulation of mitochondrial transcription by mitochondrial topoisomerase I.

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 IIß 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.

Diagnostic and therapeutic aspects of ß1-adrenergic receptor autoantibodies in human heart disease.

Growing evidence indicates a cardio-pathogenic role of autoantibodies against ß1-adrenergic receptors (ß1AR). In particular autoantibodies stimulating ß1AR-mediated cAMP-production (i.e. agonistic ß1AR 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 ß1AR 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. ß1AR autoantibodies can be removed by extracorporeal absorption or neutralised by systemic administration of synthetic epitope mimics. Only patients bearing agonistic ß1AR autoantibodies in their bloodstream will benefit from these approaches. Therefore, reliable detection of agonistic ß1AR autoantibodies is a key pre-requisite for the future implementation of these strategies. ß1AR 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, ß1AR autoantibodies can reliably be detected only by assays utilising the native ß1AR as a test antigen. To provide a sufficient basis for diagnostic predictions or therapeutic decisions, one must also determine whether ß1AR 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 ß1AR-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.

Inadequate mito-biogenesis in primary dermal fibroblasts from old humans is associated with impairment of PGC1A-independent stimulation.

Extrinsic skin ageing converges on the dermis, a post-mitotic tissue compartment consisting of extracellular matrix and long-lived fibroblasts prone to damage accumulation and maladaptation. Aged human fibroblasts exhibit mitochondrial and nuclear dysfunctions, which may be a cause or consequence of ageing. We report on a systematic study of human dermal fibroblasts retrieved from female donors aged 20-67 years and analysed ex vivo at low population doubling precluding replicative senescence. According to gene set enrichment analysis of genome wide array data, the most prominent age-associated change of the transcriptome was decreased expression of mitochondrial genes. Consistent with that, mitochondrial content and cell proliferation declined with donor age. This was associated with upregulation of AMP-dependent protein kinase (AMPK), increased mRNA levels of PPARγ-coactivator 1α (PGC1A) and decreased levels of NAD(+)-dependent deacetylase sirtuin 1. In the old cells the PGC1A-mediated mito-biogenetic response to direct AMPK-stimulation by AICAR was undiminished, while the PGC1A-independent mito-biogenetic response to starvation was attenuated and accompanied by increased ROS-production. In summary, these observations suggest an age-associated decline in PGC1A-independent mito-biogenesis, which is insufficiently compensated by upregulation of the AMPK/PGC1A-axis leading under baseline conditions to decreased mitochondrial content and reductive overload of residual respiratory capacity.

Impact of human autoantibodies on ß1-adrenergic receptor conformation, activity, and internalization.

AIMS: Autoantibodies against second extracellular loops of ß(1)-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 RESULTS: IgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to ß(1)-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 ß(1)-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.