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NEW FINDINGS: What is the central question of this study? Is there a causal relationship between gonadotrophin-releasing hormone (GnRH) receptor-activating autoantibodies and polycystic ovary syndrome (PCOS)? What is the main finding and its importance? Induction of GnRH receptor-activating autoantibodies in rats resulted in increased luteinizing hormone pulsatility and testosterone concentrations, disrupted oestrous cycles, increased atretic follicles, and activation of insulin signalling in the pituitary and ovary. These changes replicate those seen in humans with PCOS, suggesting that GnRH receptor-activating autoantibodies might be involved in the pathogenesis of PCOS. ABSTRACT: Gonadotrophin-releasing hormone receptor-activating autoantibodies (GnRHR-AAb) are associated with polycystic ovary syndrome (PCOS). In the present study, we examined the impact of GnRHR-AAb on reproductive function in GnRHR-immunized female rats. All immunized rats produced high titres of GnRHR-AAb targeting a dominant epitope located in the central region of the second extracellular loop of the GnRHR. Increased pulsatile luteinizing hormone secretion and testosterone concentrations were found in immunized rats. These rats exhibited disrupted oestrous cycles, increased ovarian follicular atresia, and activation of insulin signalling in the pituitary and ovary, as indicated by increased mRNA expressions of insulin receptor substrate, phosphatidylinositol 3-kinase and glucose transporter 1. No significant changes in inflammatory cytokines were detected in the ovarian tissue. These features mimic those observed in humans with PCOS. Our findings support the concept that chronic stimulation of the GnRHR by GnRHR-AAb, with an associated increase in pituitary luteinizing hormone secretion and ovarian androgen overproduction, might represent a new aetiological mechanism for PCOS.
Assuntos
Síndrome do Ovário Policístico , Animais , Autoanticorpos , Feminino , Atresia Folicular , Hormônio Liberador de Gonadotropina , Hormônio Luteinizante , Síndrome do Ovário Policístico/metabolismo , Ratos , Receptores LHRHRESUMO
AIMS: Postural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and ß1/2-adrenergic receptors (ß1/2AR). METHODS AND RESULTS: Immunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and ß1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and ß1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, ß1AR and ß2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and ß1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the ß1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated ß1AR and α1AR activity but not with ß2AR activity. CONCLUSION: These data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and ß1AR responsiveness are important in the pathophysiology of postural tachycardia.
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Músculos Abdominais/irrigação sanguínea , Autoanticorpos/sangue , Autoimunidade , Imunoglobulina G/sangue , Síndrome da Taquicardia Postural Ortostática/imunologia , Receptores Adrenérgicos alfa 1/imunologia , Receptores Adrenérgicos beta 1/imunologia , Receptores Adrenérgicos beta 2/imunologia , Adolescente , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Agonistas de Receptores Adrenérgicos beta 1/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Adulto , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Células CHO , Estudos de Casos e Controles , Cricetulus , Relação Dose-Resposta a Droga , Feminino , Humanos , Técnicas In Vitro , Masculino , Síndrome da Taquicardia Postural Ortostática/sangue , Síndrome da Taquicardia Postural Ortostática/diagnóstico , Ratos , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Transfecção , Vasoconstrição/efeitos dos fármacos , Adulto JovemRESUMO
Functional autoantibodies to the autonomic receptors are increasingly recognized in the pathophysiology of cardiovascular diseases. To date, no human activating monoclonal autoantibodies to these receptors have been available. In this study, we describe for the first time a ß2-adrenergic receptor (ß2AR)-activating monoclonal autoantibody (C5F2) produced from the lymphocytes of a patient with idiopathic postural hypotension. C5F2, an IgG3 isotype, recognizes an epitope in the N terminus of the second extracellular loop (ECL2) of ß2AR. Surface plasmon resonance analysis revealed high binding affinity for the ß2AR ECL2 peptide. Immunoblotting and immunofluorescence demonstrated specific binding to ß2AR in H9c2 cardiomyocytes, CHO cells expressing human ß2AR, and rat aorta. C5F2 stimulated cyclic AMP production in ß2AR-transfected CHO cells and induced potent dilation of isolated rat cremaster arterioles, both of which were specifically blocked by the ß2AR-selective antagonist ICI-118551 and by the ß2AR ECL2 peptide. This monoclonal antibody demonstrated sufficient activity to produce postural hypotension in its host. Its availability provides a unique opportunity to identify previously unrecognized causes and new pharmacological management of postural hypotension and other cardiovascular diseases.
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Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Hipotensão Ortostática/imunologia , Hipotensão Ortostática/fisiopatologia , Imunoglobulina G/imunologia , Receptores Adrenérgicos beta 2/imunologia , Vasodilatadores/imunologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/farmacologia , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Arteríolas/metabolismo , Arteríolas/patologia , Arteríolas/fisiopatologia , Autoanticorpos/sangue , Autoanticorpos/farmacologia , Células CHO , Cricetinae , Cricetulus , Humanos , Hipotensão Ortostática/sangue , Hipotensão Ortostática/genética , Hipotensão Ortostática/patologia , Imunoglobulina G/sangue , Imunoglobulina G/farmacologia , Masculino , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Peptídeos/imunologia , Peptídeos/farmacologia , Propanolaminas/farmacologia , Ratos , Receptores Adrenérgicos beta 2/sangue , Receptores Adrenérgicos beta 2/genética , Ressonância de Plasmônio de Superfície , Vasodilatadores/sangue , Vasodilatadores/farmacologiaRESUMO
Previous studies demonstrated burst pacing and intravenous infusion of ACh induced sustained atrial tachycardia when rabbits were immunized to produce ß2-adrenergic receptor (ß2AR)-activating autoantibodies. The objective of this study was to examine the arrhythmogenic effect of ß1-adrenergic receptor (ß1AR)-activating autoantibodies in the rabbit. Eight New Zealand white rabbits were immunized with a ß1AR second extracellular loop peptide to raise ß1AR antibody titers. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization. Arrhythmia occurrence was determined in response to burst pacing before and after ACh infusion in incremental concentrations of 10 µM, 100 µM, and 1 mM. The baseline sinus heart rate before and after immunization averaged 149 ± 17 per min and 169 ± 16 per min, respectively (P < 0.05). In the preimmune studies, there were five sustained (≥10 s) arrhythmias in 32 induction attempts, which occurred in only four of eight rabbits. In the postimmune studies, there were 22 sustained arrhythmias in 32 induction attempts, which occurred in all eight rabbits (P < 0.0001 for the independent effect of immunization). Of the 22 sustained arrhythmias postimmunization, 15 were sinus tachycardia compared with only two before immunization (P < 0.01 for the independent effect of immunization). Postimmune (but not preimmune) rabbit sera demonstrated specific binding to ß1AR and induced significant ß1AR activation in transfected cells in vitro. No cross-reactivity with ß2AR was observed. In conclusion, in contrast with rabbits with ß2AR-activating autoantibodies that demonstrate predominantly atrial tachycardias, enhanced autoantibody activation of ß1AR in the rabbit leads to tachyarrhythmias mainly in the form of sustained sinus tachycardia.
Assuntos
Arritmias Cardíacas/imunologia , Autoanticorpos/metabolismo , Receptores Adrenérgicos beta 1/imunologia , Acetilcolina/farmacologia , Animais , Arritmias Cardíacas/metabolismo , Autoanticorpos/imunologia , Frequência Cardíaca , Coelhos , Receptores Adrenérgicos beta 1/metabolismoRESUMO
Sinus tachycardia (ST) is ubiquitous, but its presence outside of normal physiological triggers in otherwise healthy individuals remains a commonly encountered phenomenon in medical practice. In many cases, ST can be readily explained by a current medical condition that precipitates an increase in the sinus rate, but ST at rest without physiological triggers may also represent a spectrum of normal. In other cases, ST may not have an easily explainable cause but may represent serious underlying pathology and can be associated with intolerable symptoms. The classification of ST, consideration of possible etiologies, as well as the decisions of when and how to intervene can be difficult. ST can be classified as secondary to a specific, usually treatable, medical condition (eg, pulmonary embolism, anemia, infection, or hyperthyroidism) or be related to several incompletely defined conditions (eg, inappropriate ST, postural tachycardia syndrome, mast cell disorder, or post-COVID syndrome). While cardiologists and cardiac electrophysiologists often evaluate patients with symptoms associated with persistent or paroxysmal ST, an optimal approach remains uncertain. Due to the many possible conditions associated with ST, and an overlap in medical specialists who see these patients, the inclusion of experts in different fields is essential for a more comprehensive understanding. This article is unique in that it was composed by international experts in Neurology, Psychology, Autonomic Medicine, Allergy and Immunology, Exercise Physiology, Pulmonology and Critical Care Medicine, Endocrinology, Cardiology, and Cardiac Electrophysiology in the hope that it will facilitate a more complete understanding and thereby result in the better care of patients with ST.
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COVID-19 , Síndrome da Taquicardia Postural Ortostática , Humanos , Taquicardia Sinusal/diagnóstico , Taquicardia Sinusal/terapiaRESUMO
Polycystic ovary syndrome (PCOS), a metabolic and reproductive disease, is frequently associated with type 2 diabetes. We have demonstrated activating autoantibodies (AAb) directed toward the second extracellular loop (ECL2) of the gonadotropin-releasing hormone receptor (GnRHR) are present in a significant subgroup of PCOS patients. It is unclear whether GnRHR-AAb can induce peripheral tissue insulin resistance (IR) in animal models. Sixteen rats were divided equally into a GnRHR ECL2 peptide-immunized group (IMM group) and a control group (CON group). Sera GnRHR-AAb titer, luteinizing hormone (LH), and testosterone (T) were higher in IMM rats compared with CON rats. No significant difference in fasting blood glucose was observed between the two groups. However, the plasma glucose level at other time points of the IMM group was higher than that of the CON group during an intraperitoneal glucose tolerance test (IPGTT) and an insulin tolerance test (ITT) (p < 0.01). These data support the likelihood of the GnRHR-AAb induction of glucose intolerance and IR. Compared with the CON group, the IMM group showed a significant increase in insulin-stimulated phosphorylation of IRS-1 (p-IRS-1 S636/639) and a decrease in insulin-stimulated phosphorylation of Akt (p-AKT S473). Expression of the glucose transport genes including GLUT-2 in liver and GLUT-4 in white adipose tissue and skeletal muscle was significantly decreased in IMM rats compared with the CON rats. Serum levels of proinflammatory cytokines (TNF-α, IL-1α, and IL-18) were increased, while anti-inflammatory cytokines (IL-4 and IL-10) were decreased in the IMM group. Taken together, elevated GnRHR-AAb enhanced LH, hyperandrogenism, and inflammation. These changes are likely related to the observed peripheral tissue IR through the downregulation of the insulin-stimulated IRS/PI3K/Akt/Glut signaling pathway.
Assuntos
Autoanticorpos/imunologia , Resistência à Insulina , Obesidade/metabolismo , Síndrome do Ovário Policístico/metabolismo , Receptores LHRH/imunologia , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Citocinas/sangue , Feminino , Transportador de Glucose Tipo 2/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The ubiquitin-proteasome system (UPS) plays a central role in intracellular protein degradation and regulates many cellular processes, including cell proliferation, inflammation, adaptation to stress, cell death, and the removal of damaged or misfolded proteins. Numerous studies have demonstrated that altered UPS function is involved in the pathogenesis of a wide range of cardiac diseases including hypertrophy and failure, myocardial ischemia, atherosclerosis, and diabetic cardiovascular disease. Impairment of proteasome function is a common feature of cardiac disease; however several studies have also demonstrated increased proteasome activity in models similar but not identical with those having decreased function. Recent studies have shown that use of proteasome inhibitors before or following production of the model of cardiac disease may confer cardioprotection under certain conditions.
Assuntos
Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Complexo de Endopeptidases do Proteassoma/uso terapêutico , Inibidores de Proteassoma , Animais , Sistemas de Liberação de Medicamentos , Quinase 2 de Receptor Acoplado a Proteína G/fisiologia , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Ubiquitina/fisiologiaRESUMO
OBJECTIVES: 1) To confirm the correlation of GnRH receptor (GnRHR) activating autoantibody (AAb) activity with polycystic ovary syndrome (PCOS) diagnosis in large well defined cohorts; and 2) to evaluate suppression of AAb activity with GnRH antagonist medication in transfected GnRHR cells exposed to serum of PCOS patients. DESIGN: Cross-sectional matched case-control study. SETTING: University-based research facility. PATIENTS: Sera from 200 patients with PCOS from the Pregnancy in Polycystic Ovary Syndrome II (PPCOS II) trial and from 200 race, parity-, age-, and body mass index (BMI)-matched ovulatory unexplained infertile control patients from the Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) trial were obtained and used for this study. INTERVENTIONS: GnRHR AAb activity was determined with the use of the GeneBlazer cell-based fluorescence resonance energy transfer assay with and without cetrorelix, a GnRH antagonist. MAIN OUTCOME MEASURES: 1) GnRHR AAb activity in PCOS patients compared with control subjects; and 2) effectiveness of GnRH antagonist in suppressing GnRHR AAb activity. RESULTS: GnRHR AAb activity levels in the PCOS group were significantly higher than in the control group. With cetrorelix, GnRHR AAb activity was largely suppressed in the PCOS group but not in the control group. These differences remained significant after adjusting for within-pair differences in age, BMI, and antimüllerian hormone (AMH) levels. CONCLUSIONS: We confirmed higher GnRHR AAb activity levels in the sera of a large cohort of PCOS patients compared with unexplained infertile control subjects. Addition of cetrorelix resulted in significant suppression of AAb activity levels in PCOS patients as a group whereas control subjects were unaffected. GnRHR AAb, along with patient age and AMH level, may provide a promising future diagnostic test for PCOS.
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OBJECTIVE: Is polycystic ovary syndrome (PCOS) associated with activating autoantibodies (AAb) to the second extracellular loop (ECL2) of gonadotropin-releasing hormone receptor (GnRHR)? DESIGN AND METHODS: We retrospectively screened sera from 40 patients with PCOS and 14 normal controls (NCs) with regular menses using enzyme-linked immunosorbent assay (ELISA) for the presence of GnRHR-ECL2-AAb. We obtained similar data from 40 non-PCOS ovulatory but infertile patients as a control group (OIC) of interest. We analyzed GnRHR-ECL2-AAb activity in purified immunoglobulin (Ig)G using a cell-based GnRHR bioassay. RESULTS: The mean ELISA value in the PCOS group was markedly higher than the NC (Pâ =â .000036) and the OIC (Pâ =â .0028) groups. IgG from a sample of 5 PCOS subjects, in contrast to a sample of 5 OIC subjects, demonstrated a dose-dependent increase in GnRHR-stimulating activity qualitatively similar to the acute action of the natural ligand GnRH and the synthetic agonist leuprolide. The GnRHR antagonist cetrorelix significantly suppressed (Pâ <â .01) the elevated GnRHR activity induced by IgG from 7 PCOS patients while the IgG activity level from 7 OIC subjects was unchanged. Five other OIC subjects had relatively high ELISA values at or above the 95% confidence limits. On further study, 3 had normal or low activity while 2 had elevated IgG-induced GnRHR activity. One suppressed with cetrorelix while the other did not. The copresence of PCOS IgG increased the responsiveness to GnRH and shifted the dosage response curve to the left (Pâ <â .01). CONCLUSIONS: GnRHR-ECL2-AAb are significantly elevated in patients with PCOS compared with NCs. Their presence raises important etiological, diagnostic, and therapeutic implications.
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Background Previous studies have demonstrated that functional autoantibodies to adrenergic receptors may be involved in the pathogenesis of postural tachycardia syndrome. The objective of this study was to examine the impact of these autoantibodies on cardiovascular responses to postural changes and adrenergic orthosteric ligand infusions in immunized rabbits. Methods and Results Eight New Zealand white rabbits were coimmunized with peptides from the α1-adrenergic receptor and ß1-adrenergic receptor (ß1AR). Tilt test and separate adrenergic agonist infusion studies were performed on conscious animals before and after immunization and subsequent treatment with epitope-mimetic peptide inhibitors. At 6 weeks after immunization, there was a greater percent increase in heart rate upon tilting compared with preimmune baseline. No significant difference in blood pressure response to tilting was observed. The heart rate response to infusion of the ß-adrenoceptor agonist isoproterenol was significantly enhanced in immunized animals, suggesting a positive allosteric effect of ß1AR antibodies. In contrast, the blood pressure response to infusion of the α1-adrenergic receptor agonist phenylephrine was attenuated in immunized animals, indicating a negative allosteric effect of α1-adrenergic receptor antibodies. Injections of antibody-neutralizing peptides suppressed the postural tachycardia and reversed the altered heart rate and blood pressure responses to orthosteric ligand infusions in immunized animals at 6 and 30 weeks. Antibody production and suppression were confirmed with in vitro bioassays. Conclusions The differential allosteric effect of α1-adrenergic receptor and ß1AR autoantibodies would lead to a hyperadrenergic state and overstimulation of cardiac ß1AR. These data support evidence for an autoimmune basis for postural tachycardia syndrome.
Assuntos
Autoanticorpos/sangue , Frequência Cardíaca , Fragmentos de Peptídeos/imunologia , Síndrome da Taquicardia Postural Ortostática/imunologia , Postura , Receptores Adrenérgicos beta 1/imunologia , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Imunização , Masculino , Fragmentos de Peptídeos/administração & dosagem , Síndrome da Taquicardia Postural Ortostática/sangue , Síndrome da Taquicardia Postural Ortostática/fisiopatologia , Coelhos , Receptores Adrenérgicos beta 1/administração & dosagemRESUMO
BACKGROUND: Activating autoantibodies (AAb) to adrenergic receptors (AR) have previously been reported in patients with postural tachycardia syndrome (POTS). These AAb may contribute to a final common pathway for overlapping disease processes, reflecting a possible autoimmune contribution to POTS pathophysiology. In prior studies, measurement of AAb activity was inferred from costly, low-throughput, and laborious physiological assays. In the present study, we developed and validated an alternative cell-based bioassay for measuring AAb activity in serum by means of pre-treatment with monoamine oxidase (MAO). METHODS: A total of 37 POTS patients and 61 sex-matched healthy control participants were included. Serum was pre-treated with MAO to remove endogenous catecholamines that could falsely inflate AR activation by AAb. A receptor-transfected cell-based bioassay was used to detect presence of α1AR-AAb and ß1AR-AAb in serum. RESULTS: MAO effectively degraded catecholamines as demonstrated by suppression of norepinephrine-induced α1AR activation in POTS (6.4 â± â0.7 vs. 5.5 â± â0.9; P â= â0.044) and in controls (4.1 â± â0.5 vs. 3.9 â± â0.6; P â= â0.001). Mean activity values were greater in the POTS vs. Controls for α1AR-AAb (6.2 â± â1.2 vs. 5.3 â± â1.0; P â< â0.001) and ß1AR-AAb (5.7 â± â1.8 vs. 4.1 â± â0.9; P â< â0.001). Compared to controls, more POTS patients were positive for α1AR-AAb activity (22% vs 4%; P â= â0.007) and ß1AR-AAb activity (52% vs. 2%; P â< â0.001). CONCLUSIONS: The co-presence of norepinephrine in serum samples can artifactually elevate α1AR and ß1AR activity, which can be avoided by serum pre-treatment with MAO. Using this novel bioassay, we show that POTS patients have increased α1AR-AAb and ß1AR-AAb activity compared to healthy controls in the largest POTS cohort reported to-date.
RESUMO
BACKGROUND: Both the adrenergic and renin-angiotensin systems contribute to orthostatic circulatory homeostasis, which is impaired in postural orthostatic tachycardia syndrome (POTS). Activating autoantibodies to the α1-adrenergic and ß1/2-adrenergic receptors have previously been found in sera from patients with POTS. We hypothesized that patients with POTS might also harbor activating autoantibodies to the angiotensin II type 1 receptor (AT1R) independently of antiadrenergic autoimmunity. This study examines a possible pathophysiological role for AT1R autoantibodies in POTS. METHODS AND RESULTS: Serum immunoglobulin G from 17 patients with POTS, 6 patients with recurrent vasovagal syncope, and 10 normal controls was analyzed for the ability to activate AT1R and alter AT1R ligand responsiveness in transfected cells in vitro. Of 17 subjects with POTS, 12 demonstrated significant AT1R antibody activity in immunoglobulin G purified from their serum. No significant AT1R antibody activity was found in the subjects with vasovagal syncope or healthy subjects. AT1R activation by POTS immunoglobulin G was specifically blocked by the AT1R blocker losartan. Moreover, POTS immunoglobulin G significantly shifted the angiotensin II dosage response curve to the right, consistent with an inhibitory effect. All subjects with POTS were positive for one or both autoantibodies to the AT1R and α1-adrenergic receptor. CONCLUSIONS: Most patients with POTS harbor AT1R antibody activity. This supports the concept that AT1R autoantibodies and antiadrenergic autoantibodies, acting separately or together, may exert a significant impact on the cardiovascular pathophysiological characteristics in POTS.
Assuntos
Autoanticorpos/sangue , Autoimunidade , Síndrome da Taquicardia Postural Ortostática/fisiopatologia , Receptor Tipo 1 de Angiotensina/imunologia , Adolescente , Adulto , Autoanticorpos/imunologia , Feminino , Humanos , Masculino , Síndrome da Taquicardia Postural Ortostática/sangue , Síndrome da Taquicardia Postural Ortostática/imunologia , Receptor Tipo 1 de Angiotensina/sangue , Vasoconstrição/fisiologia , Adulto JovemRESUMO
BACKGROUND: Diabetes is associated with a cardiomyopathy that is independent of coronary artery disease or hypertension. In the present study we used in vivo magnetic resonance imaging (MRI) and echocardiographic techniques to examine and characterize early changes in myocardial function in a mouse model of type 1 diabetes. METHODS: Diabetes was induced in 8-week old C57BL/6 mice with two intraperitoneal injections of streptozotocin. The blood glucose levels were maintained at 19-25 mmol/l using intermittent low dosages of long acting insulin glargine. MRI and echocardiography were performed at 4 weeks of diabetes (age of 12 weeks) in diabetic mice and age-matched controls. RESULTS: After 4 weeks of hyperglycemia one marker of mitochondrial function, NADH oxidase activity, was decreased to 50% of control animals. MRI studies of diabetic mice at 4 weeks demonstrated significant deficits in myocardial morphology and functionality including: a decreased left ventricular (LV) wall thickness, an increased LV end-systolic diameter and volume, a diminished LV ejection fraction and cardiac output, a decreased LV circumferential shortening, and decreased LV peak ejection and filling rates. M-mode echocardiographic and Doppler flow studies of diabetic mice at 4 weeks showed a decreased wall thickening and increased E/A ratio, supporting both systolic and diastolic dysfunction. CONCLUSION: Our study demonstrates that MRI interrogation can identify the onset of diabetic cardiomyopathy in mice with its impaired functional capacity and altered morphology. The MRI technique will lend itself to repetitive study of early changes in cardiac function in small animal models of diabetic cardiomyopathy.
Assuntos
Cardiomiopatias/etiologia , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Animais , Glicemia/metabolismo , Peso Corporal , Cardiomiopatias/diagnóstico , Angiopatias Diabéticas/diagnóstico , Feminino , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/enzimologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Estreptozocina , Disfunção Ventricular Esquerda/etiologiaRESUMO
Activating autoantibodies to the ß1-adrenergic and M2 muscarinic receptors are present in a very high percentage of patients with Graves' disease and atrial fibrillation (AF). The objective of this study was to develop a reproducible animal model and thereby to examine the impact of these endocrine-like autoantibodies alone and with thyroid hormone on induction of thyroid-associated atrial tachyarrhythmias. Five New Zealand white rabbits were coimmunized with peptides from the second extracellular loops of the ß1-adrenergic and M2 muscarinic receptors to produce both sympathomimetic and parasympathomimetic antibodies. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization and subsequent treatment with thyroid hormone. Antibody expression facilitated the induction of sustained sinus, junctional and atrial tachycardias, but not AF. Addition of excessive thyroid hormone resulted in induced sustained AF in all animals. AF induction was blocked acutely by the neutralization of these antibodies with immunogenic peptides despite continued hyperthyroidism. The measured atrial effective refractory period as one parameter of AF propensity shortened significantly after immunization and was acutely reversed by peptide neutralization. No further decrease in the effective refractory period was observed after the addition of thyroid hormone, suggesting other cardiac effects of thyroid hormone may contribute to its role in AF induction. This study demonstrates autonomic autoantibodies and thyroid hormone potentiate the vulnerability of the heart to AF, which can be reversed by decoy peptide therapy. These data help fulfill Witebsky's postulates for an increased autoimmune/endocrine basis for Graves' hyperthyroidism and AF.
Assuntos
Fibrilação Atrial/etiologia , Modelos Animais de Doenças , Doença de Graves/fisiopatologia , Receptor Muscarínico M2/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Taquicardia/etiologia , Tiroxina/metabolismo , Agonistas de Receptores Adrenérgicos beta 1/sangue , Agonistas de Receptores Adrenérgicos beta 1/química , Agonistas de Receptores Adrenérgicos beta 1/metabolismo , Animais , Antígenos/farmacologia , Antígenos/uso terapêutico , Antígenos/toxicidade , Fibrilação Atrial/induzido quimicamente , Fibrilação Atrial/imunologia , Fibrilação Atrial/prevenção & controle , Autoanticorpos/análise , Autoanticorpos/biossíntese , Autoanticorpos/química , Seio Coronário/efeitos dos fármacos , Seio Coronário/imunologia , Seio Coronário/fisiopatologia , Doença de Graves/sangue , Doença de Graves/imunologia , Doença de Graves/metabolismo , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/imunologia , Átrios do Coração/fisiopatologia , Sistema de Condução Cardíaco/efeitos dos fármacos , Sistema de Condução Cardíaco/imunologia , Sistema de Condução Cardíaco/fisiopatologia , Masculino , Agonistas Muscarínicos/sangue , Agonistas Muscarínicos/química , Agonistas Muscarínicos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Fragmentos de Peptídeos/toxicidade , Coelhos , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 1/química , Período Refratário Eletrofisiológico/efeitos dos fármacos , Taquicardia/induzido quimicamente , Tiroxina/sangue , Tiroxina/farmacologia , Tiroxina/intoxicação , Regulação para Cima/efeitos dos fármacosRESUMO
In human myocarditis and its sequela dilated cardiomyopathy (DCM), the mechanisms and immune phenotype governing disease and subsequent heart failure are not known. Here, we identified a Th17 cell immunophenotype of human myocarditis/DCM with elevated CD4+IL17+ T cells and Th17-promoting cytokines IL-6, TGF-ß, and IL-23 as well as GM-CSF-secreting CD4+ T cells. The Th17 phenotype was linked with the effects of cardiac myosin on CD14+ monocytes, TLR2, and heart failure. Persistent heart failure was associated with high percentages of IL-17-producing T cells and IL-17-promoting cytokines, and the myocarditis/DCM phenotype included significantly low percentages of FOXP3+ Tregs, which may contribute to disease severity. We demonstrate a potentially novel mechanism in human myocarditis/DCM in which TLR2 peptide ligands from human cardiac myosin stimulated exaggerated Th17-related cytokines including TGF-ß, IL-6, and IL-23 from myocarditic CD14+ monocytes in vitro, and an anti-TLR2 antibody abrogated the cytokine response. Our translational study explains how an immune phenotype may be initiated by cardiac myosin TLR ligand stimulation of monocytes to generate Th17-promoting cytokines and development of pathogenic Th17 cells in human myocarditis and heart failure, and provides a rationale for targeting IL-17A as a therapeutic option.
RESUMO
Activating autoantibodies to the angiotensin II type 1 receptor (AT1R) have been implicated in hypertensive disorders. We investigated whether AT1R antibodies produced in immunized rabbits will activate AT1R and contribute to hypertension by a direct contractile effect on the vasculature and whether they can be blocked by a novel decoy peptide. A multiple antigenic peptide containing the AT1R epitope AFHYESQ, which is the receptor-binding epitope of AT1R-activating autoantibodies, was used to immunize 6 rabbits. AT1R antibody activity was analyzed in AT1R-transfected cells, and their contractile effects were assayed using isolated perfused rat cremaster resistance arterioles. A retro-inverso D-amino acid epitope-mimetic peptide was tested for AT1R antibody inhibition in vitro and in vivo. All immunized animals produced high AT1R antibody titers and developed elevated blood pressure. No changes in measured blood chemistry values were observed after immunization. Rabbit anti-AT1R sera induced significant AT1R activation in transfected cells and vasoconstriction in the arteriole assay, both of which were blocked by losartan and the retro-inverso D-amino acid peptide. A single intravenous bolus injection of the retro-inverso d-amino acid peptide (1 mg/kg) into immunized rabbits dropped the mean arterial pressure from 122±11 to 82±6 mm Hg. Rabbit anti-AT1R sera partially suppressed angiotensin II-induced contraction of isolated rat cremaster arterioles, and the pressor response to angiotensin II infusion was attenuated in immunized animals. In conclusion, AT1R-activating autoantibodies and the retro-inverso d-amino acid peptide, respectively, have important etiologic and therapeutic implications in hypertensive subjects who harbor these autoantibodies.
Assuntos
Autoanticorpos/imunologia , Pressão Sanguínea/fisiologia , Hipertensão/imunologia , Receptor Tipo 1 de Angiotensina/imunologia , Vasoconstrição/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hipertensão/fisiopatologia , CoelhosRESUMO
PURPOSE: Previous studies demonstrated that burst pacing and subthreshold infusion of acetylcholine in ß1-adrenergic receptor (ß1AR)-immunized rabbits induced sustained sinus tachycardia. The aim of this study was to examine the anti-arrhythmogenic effect of a newly designed retro-inverso (RI) peptidomimetic inhibitor that specifically targets the ß1AR antibodies in the rabbit. METHODS: Six New Zealand white rabbits were immunized with a ß1AR second extracellular loop peptide to produce sympathomimetic ß1AR antibodies. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization and subsequent treatment with the RI peptide inhibitor. Each rabbit served as its own control. RESULTS: No sustained arrhythmias were induced at preimmune baseline. At 6 weeks after immunization, there was a marked increase in induced sustained tachyarrhythmias, predominantly sinus tachycardia, which was largely suppressed by the RI peptide. The atrial effective refractory period was shortened significantly in immunized rabbits compared to their preimmune state. The RI peptide reversed and prolonged this shortening. ß1AR antibody levels were negatively correlated with the atrial effective refractory period. Postimmune sera-induced ß1AR activation in transfected cells in vitro was also blocked by the RI peptide. CONCLUSIONS: ß1AR-activating autoantibodies are associated with reduction of the atrial effective refractory period and facilitate arrhythmia induction in this model. The RI peptide reversal may have important therapeutic implications in subjects who harbor these autoantibodies.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/administração & dosagem , Arritmias Cardíacas/imunologia , Arritmias Cardíacas/prevenção & controle , Autoanticorpos/imunologia , Peptídeos/administração & dosagem , Receptores Adrenérgicos beta 1/imunologia , Animais , Materiais Biomiméticos/administração & dosagem , Peptídeos/química , Coelhos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Resultado do TratamentoRESUMO
Autoantibodies to the angiotensin II type 1 receptor (AT1R) have been reported in patients with primary aldosteronism, including aldosterone producing adenoma (APA) and idiopathic adrenal hyperplasia (IAH). Sera from 25 primary aldosteronism subjects (12 with IAH and 13 with APA) and 15 normotensive control subjects were assayed for AT1R autoantibodies by enzyme-linked immunosorbent assay and an AT1R-transfected cell-based bioassay. Nine of 12 IAH subjects (75%) and six of 13 APA subjects (46%) were positive for AT1R autoantibodies in the bioactivity assay. The mean AT1R autoantibody activity for the IAH and APA subjects was significantly greater than controls (P < .001 and P < .01, respectively), and this in vitro activity was suppressed by the AT1R blocker losartan. None of the controls had significant AT1R autoantibody activity. Enzyme-linked immunosorbent assay values were less sensitive but were positive in some subjects with IAH and APA. The mean arterial pressure of these primary aldosteronism subjects correlated modestly with AT1R autoantibody activity. These data confirm the presence of active AT1R autoantibodies in a high percentage of subjects with primary aldosteronism irrespective of their underlying etiology. These observations have both pathophysiological and clinical implications.
Assuntos
Autoanticorpos/imunologia , Hiperaldosteronismo/imunologia , Receptor Tipo 1 de Angiotensina/metabolismo , Adulto , Idoso , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Losartan/farmacologia , Masculino , Pessoa de Meia-Idade , PrevalênciaRESUMO
We have previously demonstrated that activating autoantibodies to ß1-adrenergic receptor (ß1AR) and M2 muscarinic receptor (M2R) facilitate atrial fibrillation (AF) in patients with Graves' disease (GD). The objectives of this expanded study were to examine the prevalence of ß1AR, ß2AR, and M2R autoantibodies in hyperthyroidism subjects. Sera from 81 patients including 31 with GD and AF, 36 with GD and sinus rhythm, 9 with toxic multinodular goiter, 5 with subacute thyroiditis, and 10 control subjects were examined for these autoantibodies by ELISA. Sera from 20 ELISA-positive GD subjects, 10 with AF and 10 with sinus rhythm, were assayed for autoantibody bioactivity using cell-based bioassays. In patients with GD and AF, 45, 65, and 77 % were ELISA positive for ß1AR, M2R, and ß2AR autoantibodies, respectively. In patients with GD and sinus rhythm, 17, 39, and 75 % were ELISA positive for ß1AR, M2R, and ß2AR autoantibodies, respectively. ß1AR and M2R autoantibodies were co-present in 39 % of patients with GD and AF compared to 14 % in GD with sinus rhythm (p = 0.026). Patients with toxic multinodular goiter or subacute thyroiditis had a low prevalence of autoantibodies. The mean ß1AR and M2R autoantibody activity was elevated in both GD groups but higher in those with AF than those with sinus rhythm. ß2AR autoantibody activity was also increased in both groups. In conclusion, ß1AR, ß2AR, and M2R autoantibodies were elevated in GD. ß1AR and M2R autoantibodies appear to be related to concurrent AF, while ß2AR autoantibodies were equally prevalent in those with a sinus tachycardia and those with AF.
Assuntos
Fibrilação Atrial/sangue , Autoanticorpos/sangue , Doença de Graves/sangue , Receptor Muscarínico M2/imunologia , Receptores Adrenérgicos beta 1/imunologia , Receptores Adrenérgicos beta 2/imunologia , Taquicardia/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/etiologia , Feminino , Doença de Graves/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Taquicardia/etiologiaRESUMO
Activating autoantibodies to the angiotensin type 1 receptor (AT1R) are associated with hypertensive disorders. The angiotensin type 2 receptor (AT2R) is known to counter-regulate the actions of AT1R. We investigated whether AT2R autoantibodies produced in immunized rabbits will activate AT2R and suppress the vasopressor responses to angiotensin II and AT1R-activating autoantibodies. Five rabbits immunized with a peptide corresponding to the second extracellular loop of AT2R developed high AT2R antibody titers. Rabbit anti-AT2R sera failed to directly dilate isolated rat cremaster arterioles; however, when co-perfused with angiotensin II or AT1R-activating autoantibodies, the anti-AT2R sera significantly inhibited their contractile effects. Rabbit anti-AT2R sera recognized a predominant sequence near the N-terminus of the AT2R second extracellular loop. A decoy peptide based on this sequence effectively reversed the opposing effect of the anti-AT2R sera on angiotensin II-induced contraction of rat cremaster arterioles. A similar blockade of the anti-AT2R sera effect was observed with the AT2R antagonist PD 123319 and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Rabbit anti-AT2R sera reacted specifically with AT2R. No cross-reactivity with AT1R was observed. Blood pressure did not change in immunized animals. However, the pressor responses to incremental angiotensin II infusions were blunted in immunized animals. Thirteen subjects with primary aldosteronism demonstrated increased AT2R autoantibody levels compared with normal controls. In conclusion, AT2R autoantibodies produced in immunized rabbits have the ability to activate AT2R and counteract the AT1R-mediated vasoconstriction. These autoantibodies provide useful and selective tools for the study of their roles in blood pressure regulation and possible therapeutic intervention.