RESUMEN
Accumulating evidence suggests that liver injury can be induced by the over-expression of ß 1-adrenergic receptors (ß 1-ARs). High titers of autoantibodies specific to ß 1-adrenergic receptors (ß 1-AA) are detected in the sera of heart failure patients, potentially playing agonist-like roles. However, the role of ß 1-AA in liver function has not been characterized. In this study, we collect the sera of primary biliary cholangitis (PBC) patients, a condition which easily develops into liver fibrosis, and analyze the relationship between PBC and ß 1-AA. A passive immunization model is established to assess the effect of ß 1-AA on the liver. Subsequently, the effect of ß 1-AA on macrophages is investigated in vitro. Results show that PBC patients have a high titer and ratio of ß 1-AA, compared to controls. Liver injury and fibrosis are induced by ß 1-AA. In vitro experiments with ROS probe demonstrate that ß 1-AA induces macrophages to produce ROS and secrete TNFα. These effects can be partially reversed by metoprolol, a blocker for ß 1-AR. Results from the transwell and phagocytosis assays show that ß 1-AA promotes macrophage migration and phagocytosis. FCM tests suggest that ß 1-AA induces the alteration of M1 rather than M2 markers in macrophages. Finally, the Annexin V/PI assay indicates that macrophage culture supernatants stimulated by ß 1-AA cause hepatocyte apoptosis. Overall, these results suggest that ß 1-AA is involved in PBC. The ß 1-AA-induced activation, phagocytosis and phenotypic modification of macrophages may play an important role in the development of hepatic fibrosis and injury.
Asunto(s)
Metoprolol , Receptores Adrenérgicos beta 1/inmunología , Factor de Necrosis Tumoral alfa , Anexina A5 , Autoanticuerpos , Humanos , Cirrosis Hepática , Macrófagos/metabolismo , Metoprolol/farmacología , Especies Reactivas de Oxígeno/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
IMPORTANCE AND OBJECTIVES: There is unmet medical need to understand the pathogenic mechanism of the panoply of clinical manifestations associated with silicone breast implants (SBIs) such as severe fatigue, widespread pain, palpitations, dry mouth and eyes, depression, hearing loss etc. We aimed to determine whether autoantibodies against the autonomic nervous system receptors can explain the enigmatic and subjective clinical manifestation reported by women with SBIs. RESULTS: Circulating level of autoantibodies against G protein-coupled receptors (GPCRs) of the autonomic nervous system (adrenergic, muscarinic, endothelin and angiotensin receptors) have been evaluated in symptomatic women with SBIs using an ELISA method. These women with SBIs addressed our clinic due to various subjective and autonomic-related manifestations such as chronic severe fatigue, cognitive impairment, widespread pain, memory loss, sleep disorders, palpitations, depression, hearing abnormalities etc. We report for the first time, a significant reduction in the sera level of anti-ß1 adrenergic receptor (p < 0.001), anti-angiotensin II type 1 receptor (p < 0.001) and anti-endothelin receptor type A (p = 0.001) autoantibodies in women with SBIs (n = 93) as compared with aged matched healthy women (n = 36). Importantly, anti-ß1 adrenergic receptor autoantibody was found to significantly correlate with autonomic-related manifestations such as: sleep disorders and depression in women with SBIs. CONCLUSIONS: Chronic immune stimulation by silicone material may lead to an autoimmune dysautonomia in a subgroup of potentially genetically susceptible women with SBIs. The appearance of autoantibodies against GPCRs of the autonomic nervous system serve as an explanation for the subjective autonomic-related manifestations reported in women with SBIs.
Asunto(s)
Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/etiología , Implantes de Mama/efectos adversos , Disautonomías Primarias/diagnóstico , Disautonomías Primarias/etiología , Siliconas/efectos adversos , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Biomarcadores , Estudios de Casos y Controles , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Femenino , Humanos , Receptores Adrenérgicos beta 1/inmunología , Receptores Acoplados a Proteínas G/inmunologíaRESUMEN
Dysregulation of immune responses has been linked to the generation of immunoglobulin G (IgG) autoantibodies that target human ß1ARs and contribute to deleterious cardiac outcomes. Given the benefits of ß-blockers observed in patients harboring the IgG3 subclass of autoantibodies, we investigated the role of these autoantibodies in human ß1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using human embryonic kidney (HEK) 293 cells expressing human ß1ARs. Unexpectedly, pretreatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) generation while enhancing biased ß-arrestin recruitment and Extracellular Regulated Kinase (ERK) activation. In contrast, the ß-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Because IgG3(+) autoantibodies are specific to human ß1ARs, non-failing human hearts were used as an endogenous system to determine their ability to bias ß1AR signaling. Consistently, metoprolol increased AC activity, reflecting the ability of the IgG3(+) autoantibodies to bias ß-blocker toward G-protein coupling. Importantly, IgG3(+) autoantibodies are specific toward ß1AR as they did not alter ß2AR signaling. Thus, IgG3(+) autoantibody biases ß-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein-independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) ß1AR autoantibodies.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Receptores Adrenérgicos beta 1/inmunología , Autoanticuerpos/sangre , Cardiomiopatías/inmunología , Cardiomiopatías/fisiopatología , AMP Cíclico , Células HEK293 , Corazón/fisiología , Humanos , Inmunoglobulina G/metabolismo , Receptores Adrenérgicos/inmunología , Receptores Adrenérgicos beta 1/metabolismo , Transducción de Señal , beta-ArrestinasRESUMEN
OBJECTIVE: To determine the association between autoantibodies to G-protein-coupled receptors with effect on the cardiovascular system and the cardiac biomarker N-terminal pro-brain natriuretic peptide reflecting heart function in Graves' disease. DESIGN AND METHODS: Sixty premenopausal women with Graves' disease were analyzed for IgG autoantibodies against ß1-adrenergic, muscarinic acetylcholine type 2 and angiotensin II type 1 receptors using enzyme-linked immunosorbent assays based on cell membranes overexpressing receptors in their native conformations. N-terminal pro-brain natriuretic peptide and heart symptoms were analyzed in hyperthyroidism and after 7.5 months of antithyroid treatment. Matched thyroid healthy controls were also assessed. RESULTS: Serum levels of antibodies against the ß1-adrenergic and the muscarinic acetylcholine type 2 receptors were higher in hyperthyroid patients than in controls (median ß1-adrenergic receptor antibodies 1.9 [IQR 1.3-2.7] vs. 1.1 [0.8-1.7] µg/mL, P<0.0001; muscarinic acetylcholine type 2 receptor 20.5 [14.0-38.3] vs. 6.0 [3.2-9.9] U/mL, P<0.0001). These antibodies decreased in euthyroidism (P<0.01), but were still higher than in controls (P<0.01). Angiotensin II type 1 receptor levels did not differ. N-terminal pro-brain natriuretic peptide was higher in hyperthyroidism (240 [134-372] vs. <35 [<35-67] ng/L, P<0.0001), normalized after treatment and did not correlate with autoantibodies. CONCLUSION: Autoantibodies against the ß1-adrenergic and the muscarinic acetylcholine type 2 receptors were increased in Graves' patients, decreased with treatment, but did not correlate with cardiac function. However, an autoimmune effect on the heart cannot be excluded in subpopulations, as the functional properties of the analyzed antibodies remain to be determined.
Asunto(s)
Antitiroideos/farmacología , Autoanticuerpos/sangre , Autoanticuerpos/efectos de los fármacos , Enfermedad de Graves/sangre , Enfermedad de Graves/tratamiento farmacológico , Receptor de Angiotensina Tipo 1/inmunología , Receptor Muscarínico M2/inmunología , Receptores Adrenérgicos beta 1/inmunología , Adulto , Biomarcadores/sangre , Femenino , Humanos , Resultado del TratamientoRESUMEN
OBJECTIVES: Peripartum cardiomyopathy (PPCM) is a pregnancy-associated and life-threatening cardiac disease. However, the causes and pathogenesis are not fully understood. Accumulating studies show that cardiomyopathy often appears to be associated with elevated levels of ß1-adrenoceptor (ß1AR) antibodies, indicating a possible involvement of ß1AR antibodies in the development of PPCM. DESIGN: We injected the antigen peptide segment of the ß1AR into the postpartum Wistar rats to make the immune models and their cardiac function was detected by echocardiography. Also, the concentration of ß1AR antibodies and apoptosis rate of left ventricular myocytes was tested by SA-ELISA, TUNEL, HE staining, qRT-PCR and western blot methods. Finally, the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its related proteins were examined by qRT-PCR and western blot methods. RESULTS: We found that the level of ß1AR antibodies in the serum was significantly increased and the postpartum rats exhibited symptoms of PPCM after autoimmunity. Moreover, the expression of peroxisome PGC-1α, which was a master regulator of mitochondrial metabolism, and its downstream transcript vascular endothelial growth factor (VEGF), was decreased in autoimmune perinatal rats. In addition, the expression of the apoptosis factor caspase 3 as well as the apoptosis rate of left ventricular myocytes was significantly increased. CONCLUSIONS: The results suggested that the symptoms of PPCM that appeared in autoimmune perinatal rats may be due to the increase of ß1AR antibodies, which inhibited the pathway associated with peroxisome PGC-1α.
Asunto(s)
Cardiomiopatías , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores Adrenérgicos beta 1 , Transducción de Señal , Animales , Cardiomiopatías/epidemiología , Femenino , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/antagonistas & inhibidores , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 1/inmunologíaRESUMEN
Autoantibodies against muscarinic and beta1-adrenergic receptors are considered a potential cause and/or risk factor for chronic heart failure. Association of periodontitis with such autoantibodies and with impaired heart function has been observed in patients exposed to endemic Chagas' disease, which triggers by itself cardiomyopathy and receptor immunization.Here we studied the association between periodontitis, markers of cardiac injury and receptor autoimmunization in periodontitis patients (n = 147) not exposed to Chagas' disease. The autoantibodies were determined by IgG binding to native intact muscarinic and beta1-adrenergic receptors or to a cyclic peptide mimicking the disease-relevant conformational autoepitope presented by the active beta1-adrenergic receptor. Possible cardiac injury and inflammatory status were judged by serum levels of proBNP/Troponin I and CRP/IL-6, respectively. These parameters were analysed in healthy and periodontally diseased individuals as well as before and after periodontal therapy.Patients with periodontitis had significantly (p < 0.001) higher levels of autoantibodies against M5-muscarinic and beta1-adrenergic receptors, which further increased following periodontal therapy. Receptor autoantibodies were associated with increased inflammatory status but not with increased markers of cardiac injury. Thus, our data indicate that periodontitis triggers systemic inflammation, which is associated with receptor autoimmunization, and, independently thereof, with cardiac injury.
Asunto(s)
Autoanticuerpos/sangre , Periodontitis/inmunología , Receptor Muscarínico M5/inmunología , Receptores Adrenérgicos beta 1/inmunología , Adolescente , Adulto , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Femenino , Humanos , Mediadores de Inflamación/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Péptido Natriurético Encefálico/sangre , Periodontitis/sangre , Periodontitis/diagnóstico , Periodontitis/terapia , Estudios Prospectivos , Resultado del Tratamiento , Troponina I/sangre , Adulto JovenRESUMEN
BACKGROUND: Peripartum cardiomyopathy (PPCM) is life-threatening heart disease. However, the causes and pathogenesis of PPCM remain unclear. Previous studies found that ß1 adrenoceptor antibodies (ß1AA) had possible involvement in the development of PPCM. In the present study, we determined the potential relationship between PPCM and ß1AA, including the mechanism of ß1AA leading to PPCM. METHODS: We extracted the ß1AA from the postpartum Wistar rats that were injected by the antigen peptide segment of the ß1 adrenoceptor to produce PPCM. We tested the effects of ß1AA on H9C2 cell line by CCK-8, LDH, TUNEL, SA-ELISA, qRT-PCR, and western blot methods. Furthermore, PGC-1α was overexpressed to rescue the effect of ß1AA on H9C2 cells. RESULTS: We found that the extracted ß1AA induced apoptosis of cardiac myocytes of H9C2 cell line. Moreover, the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is a master regulator of mitochondrial metabolism, and its downstream transcript vascular endothelial growth factor (VEGF) got decreased in H9C2 cells after ß1AA treatment. In addition, the effect of ß1AA could be inhibited by atenolol, the antagonist of ß1 adrenoceptors (ß1AR) and imitated by isoprenaline, the agonist of ß1AR. Furthermore, overexpression of PGC-1α in the H9C2 cells rescued the apoptosis of cells and inhibitory expression of VEGF induced by ß1AA. CONCLUSIONS: Our results suggest that the symptoms of PPCM due to myocardial cell apoptosis induced by ß1AA inhibiting the PGC-1α-related pathway impairs mitochondrial energy metabolism. Therefore, our results uncover a previously unknown role of the ß1AA pathway in the etiology of PPCM and provide a novel potential target for the treatment of PPCM.
Asunto(s)
Anticuerpos/inmunología , Apoptosis , Cardiomiopatías/inmunología , Miocitos Cardíacos/inmunología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Complicaciones Cardiovasculares del Embarazo/inmunología , Receptores Adrenérgicos beta 1/inmunología , Animales , Anticuerpos/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Línea Celular , Femenino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Periodo Periparto , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Embarazo , Complicaciones Cardiovasculares del Embarazo/genética , Complicaciones Cardiovasculares del Embarazo/metabolismo , Complicaciones Cardiovasculares del Embarazo/patología , Ratas Wistar , Receptores Adrenérgicos beta 1/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Antibodies that activate the ß1-AR (ß1-adrenoreceptor) can induce heart failure in animal models. These antibodies are often found in patients with heart failure secondary to varying etiologies. Their binding to the ß1 receptor leads to prolonged receptor activation with subsequent induction of cellular dysfunction, apoptosis, and arrhythmias. ß-blocker therapy while highly effective for heart failure, may not be sufficient treatment for patients who have ß1 receptor autoantibodies. Removal of these autoantibodies by immunoadsorption has been shown to improve heart failure in small studies. However, immunoadsorption is costly, time consuming, and carries potential risks. An alternative to immunoadsorption is neutralization of autoantibodies through the intravenous application of small soluble molecules, such as peptides or aptamers, which specifically target and neutralize ß1-AR autoantibodies. Peptides may induce immunogenicity. Animal as well as early phase human studies with aptamers have not shown safety concerns to date and have demonstrated effectiveness in reducing autoantibody levels. Novel aptamers have the potential advantage of having a wide spectrum of action, neutralizing a variety of known circulating G-protein coupled receptor autoantibodies. These aptamers, therefore, have the potential to be novel therapeutic option for patients with heart failure who have positive for ß1-AR autoantibodies. However, clinical outcomes trials are needed to assess the clinical utility of this novel approach to treat heart failure.
Asunto(s)
Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Autoanticuerpos/inmunología , Insuficiencia Cardíaca/tratamiento farmacológico , Receptores Adrenérgicos beta 1/inmunología , Animales , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/inmunología , Insuficiencia Cardíaca/fisiopatología , HumanosRESUMEN
The arrhythmogenic potential of ß1-adrenoceptor autoantibodies (ß1-AA), as well as antiarrhythmic properties of omega-3 in heart diseases, have been reported while underlying mechanisms are poorly understood. We aimed to test our hypothesis that omega-3 (eicosapentaenoic acid-EPA, docosahexaenoic acid-DHA) may inhibit matrix metalloproteinase (MMP-2) activity to prevent cleavage of ß1-AR and formation of ß1-AA resulting in attenuation of pro-arrhythmic connexin-43 (Cx43) and protein kinase C (PKC) signaling in the diseased heart. We have demonstrated that the appearance and increase of ß1-AA in blood serum of male and female 12-month-old spontaneously hypertensive rats (SHR) was associated with an increase of inducible ventricular fibrillation (VF) comparing to normotensive controls. In contrast, supplementation of hypertensive rats with omega-3 for two months suppressed ß1-AA levels and reduced incidence of VF. Suppression of ß1-AA was accompanied by a decrease of elevated myocardial MMP-2 activity, preservation of cardiac cell membrane integrity and Cx43 topology. Moreover, omega-3 abrogated decline in expression of total Cx43 as well as its phosphorylated forms at serine 368 along with PKC-ε, while decreased pro-fibrotic PKC-δ levels in hypertensive rat heart regardless the sex. The implication of MMP-2 in the action of omega-3 was also demonstrated in cultured cardiomyocytes in which desensitization of ß1-AR due to permanent activation of ß1-AR with isoproterenol was prevented by MMP-2 inhibitor or EPA. Collectively, these data support the notion that omega-3 via suppression of ß1-AA mechanistically controlled by MMP-2 may attenuate abnormal of Cx43 and PKC-ε signaling; thus, abolish arrhythmia substrate and protect rats with an advanced stage of hypertension from malignant arrhythmias.
Asunto(s)
Antiarrítmicos/farmacología , Arritmias Cardíacas/etiología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Ácidos Grasos Omega-3/farmacología , Hipertensión/complicaciones , Receptores Adrenérgicos beta 1/inmunología , Animales , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Biomarcadores , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Conexina 43/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Ácidos Grasos Omega-3/metabolismo , Femenino , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/ultraestructura , Proteína Quinasa C-epsilon/metabolismo , Ratas , Ratas Endogámicas SHR , Sarcolema/metabolismo , Sarcolema/ultraestructura , Fibrilación Ventricular/tratamiento farmacológico , Fibrilación Ventricular/etiología , Fibrilación Ventricular/fisiopatologíaRESUMEN
Although several risk factors exist for acute coronary syndrome (ACS) no biomarkers for survival or risk of re-infarction have been validated. Previously, reduced serum concentrations of anti-ß1AR Ab have been implicated in poorer ACS outcomes. This study further evaluates the prognostic implications of anti-ß1AR-Ab levels at the time of ACS onset. Serum anti-ß1AR Ab concentrations were measured in randomly selected patients from within the PLATO cohort. Stratification was performed according to ACS event: ST-elevation myocardial infarct (STEMI) vs. non-ST elevation myocardial infarct (NSTEMI). Antibody concentrations at ACS presentation were compared to 12-month all-cause and cardiovascular mortality, as well as 12-month re-infarction. Sub-analysis, stratifying for age and the correlation between antibody concentration and conventional cardiac risk-factors was subsequently performed. Serum anti-ß1AR Ab concentrations were measured in 400/799 (50%) STEMI patients and 399 NSTEMI patients. Increasing anti-ß1AR Ab concentrations were associated with STEMI (p = 0.001). Across all ACS patients, no associations between anti-ß1AR Ab concentration and either all-cause cardiovascular death or myocardial re-infarction (p = 0.14) were evident. However among STEMI patients ≤60 years with anti-ß1AR Ab concentration
Asunto(s)
Síndrome Coronario Agudo/diagnóstico , Anticuerpos/sangre , Receptores Adrenérgicos beta 1/inmunología , Infarto del Miocardio con Elevación del ST/diagnóstico , Síndrome Coronario Agudo/sangre , Anciano , Anciano de 80 o más Años , Biomarcadores , Epítopos , Femenino , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/diagnóstico , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Infarto del Miocardio , Pronóstico , Factores de Riesgo , Infarto del Miocardio con Elevación del ST/sangre , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/diagnósticoRESUMEN
BACKGROUND: Dilated cardiomyopathy (DCM) is a common cause of heart failure with high morbidity and mortality rates. The association of anti-ß1 adrenergic receptor (ß1AR) autoantibodies with disease progression was shown by various studies and in vivo animal experiments. The prevalence of these disease-driving autoantibodies was estimated as 25% to 75% in DCM. The removal of autoantibodies or the interruption of their action leads to a prolonged improvement of heart function. However, presence and impact of the autoimmune aspect in DCM patients must be examined for targeted treatment. METHODS: We developed a heterogeneous immunoassay to support the diagnosis of anti-ß1AR autoantibody-induced DCM. The presentation of the native conformational epitope was enabled by reconstitution of human ß1AR into lipid bilayer nanodiscs, which stabilize the incorporated receptor in aqueous solution for measurements with standard immunological techniques. RESULTS: The incorporation of ß1AR into nanodiscs was verified by chromatographic, spectroscopic, and immunological methods. The functionality was shown by interaction assays with appropriate binding partners. Furthermore, ß1AR nanodiscs were applied to immunoassays for the detection of anti-ß1AR in human sera. Surface plasmon resonance spectroscopy and ELISA were developed, optimized, and validated. The optimized ß1AR nanodisc ELISA enabled a simultaneous measurement of 40 samples in duplicate. An interassay variance of 24% and an intraassay variance of 5% were determined. The limit of detection and the limit of quantification were determined as 0.64 ng/mL and 1.26 ng/mL, respectively (related to a monoclonal anti-ß1AR). CONCLUSIONS: Nanodisc technology is suitable as a novel biomimetic membrane system to stabilize and present ß1AR for detection of autoantibodies with immunological methods in DCM patients.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos , Cardiomiopatía Dilatada/sangre , Cardiomiopatía Dilatada/diagnóstico , Inmunoensayo/métodos , Receptores Adrenérgicos beta 1 , Adulto , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Cardiomiopatía Dilatada/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Humanos , Masculino , Nanotecnología , Receptores Adrenérgicos beta 1/inmunología , Sensibilidad y EspecificidadRESUMEN
Autophagy reduction has been confirmed as an important mechanism in apoptosis induction. Our previous study showed that decreased autophagy induced by ß1-adrenoceptor autoantibodies (ß1-AAs) enhanced cardiomyocyte apoptosis and contributed to heart failure progression. Endoplasmic reticulum stress (ERS) is known to be an important mechanism in intracellular homeostasis and is closely related to autophagy. However, ERS in ß1-AA-induced autophagy dysfunction of cardiomyocytes remains unclear. In this study, we used an active immunization rat model and H9c2 cardiomyocytes to study the role of ERS in ß1-AA-induced autophagy. Results showed that prolonged action of ß1-AAs significantly reduced the autophagy of myocardial tissues and H9c2 cardiomyocytes, and ERS and its related apoptotic pathways were significantly activated. Moreover, mRFP-GFP-LC3 double-labeled adenoviruses were used to detect cardiomyocyte autophagic flux to confirm that ß1-AAs caused a significant decrease in autophagic flux in H9c2 cardiomyocytes. The ERS inhibitor, 4-phenylbutyrate (4-PBA), partially attenuated the ß1-AA-induced reduction of cardiomyocyte autophagy, consistent with the effect of the mammalian target of rapamycin inhibitor rapamycin (Rapa). Compared to the pretreatment with 4-PBA or Rapa alone, pretreatment with the combination of 4-PBA and Rapa had a greater effect on attenuating the ß1-AA-induced decrease in autophagy and ß1-AA-induced apoptosis in cardiomyocytes. This study provides an experimental basis for the role of ß1-AAs in the homeostatic maintenance of cardiomyocytes in patients with heart failure with respect to autophagy and ERS.
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Autoanticuerpos/inmunología , Autofagia , Estrés del Retículo Endoplásmico , Miocitos Cardíacos/citología , Receptores Adrenérgicos beta 1/inmunología , Animales , Apoptosis , Línea Celular , Células Cultivadas , Masculino , Miocitos Cardíacos/inmunología , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Autoanticuerpos/sangre , Frecuencia Cardíaca , Fragmentos de Péptidos/inmunología , Síndrome de Taquicardia Postural Ortostática/inmunología , Postura , Receptores Adrenérgicos beta 1/inmunología , Animales , Presión Sanguínea , Modelos Animales de Enfermedad , Inmunización , Masculino , Fragmentos de Péptidos/administración & dosificación , Síndrome de Taquicardia Postural Ortostática/sangre , Síndrome de Taquicardia Postural Ortostática/fisiopatología , Conejos , Receptores Adrenérgicos beta 1/administración & dosificaciónRESUMEN
BACKGROUND: Autoimmunity associated with autoantibodies against the ß1-adrenergic receptor (ß1-AAB) is increasingly accepted as the driver of human dilated cardiomyopathy (DCM). Unfortunately, there is a lack of animal models to extend the knowledge about ß1-AAB autoimmunity in DCM and to develop appropriate treatment strategies. OBJECTIVES: To introduce an animal model, we investigated the ß1-AAB associated autoimmunity in Doberman Pinscher (DP) with dilated cardiomyopathy, which has similarities to human DCM. MATERIALS AND METHODS: Eighty-seven DP with cardiomyopathy in terms of pathological ECG and echocardiography (DoCM) and 31 dogs (at enrollment) without DoCM (controls) were analyzed for serum activity of ß1-AAB with a bioassay that records the chronotropic response of spontaneously beating cultured neonatal rat cardiomyocytes to the DP's IgG. To locate the receptor binding site of ß1-AAB and the autoantibody's sensitivity to inhibition, competing experiments with related blockers were performed with the bioassay. In controls that developed DoCM during follow-up, ß1-AAB were analyzed during progress. RESULTS: Fifty-nine (67.8%) DoCM dogs and 19 (61.3%) controls were ß1-AAB positive. Of the controls that developed DoCM, 8 were ß1-AAB positive (p = 0.044 vs. dogs remaining in the control group); their ß1-AAB activity increased with the cardiomyopathy progress (p<0.02). To supplement DoCM group with the 9 animals which developed cardiomyopathy in the follow up, a more pronounced ß1-AAB positivity became visible in the DoCM group (p = 0.066). Total and cardiac mortality were higher in ß1-AAB positive DP (p = 0.002; p = 0037). The dogs' ß1-AAB recognized a specific epitope on the second extracellular receptor and were sensitive to inhibition by drugs already successfully tested to inhibit the corresponding human autoantibody. CONCLUSIONS: Doberman Pinschers presented ß1-AAB associated autoimmunity, similar as in the pathogenesis of human DCM. Consequently, DP could compensate the lack of animal models for the investigation of ß1-AAB autoimmunity in human DCM.
Asunto(s)
Autoanticuerpos/inmunología , Autoinmunidad , Cardiomiopatía Dilatada/inmunología , Enfermedades de los Perros/inmunología , Receptores Adrenérgicos beta 1/inmunología , Animales , Cardiomiopatía Dilatada/veterinaria , Modelos Animales de Enfermedad , Perros , Femenino , Humanos , MasculinoRESUMEN
G protein-coupled receptors (GPCRs) in the G protein-coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the ß1-adrenoceptor (ß1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of ß1AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.
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Agonistas de Receptores Adrenérgicos beta 1/química , Diseño de Fármacos , Receptores Acoplados a Proteínas G/agonistas , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Sitio Alostérico/inmunología , Dominio Catalítico/inmunología , Enlace de Hidrógeno , Ligandos , Unión Proteica , Estructura Secundaria de Proteína , Receptores Adrenérgicos beta 1/química , Receptores Adrenérgicos beta 1/inmunología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/inmunología , Anticuerpos de Dominio Único/inmunologíaRESUMEN
OBJECTIVES: The pathogenesis of hypertensive heart disease (HHD) remains unclear, which might include autoimmunity. The aim of the present study was to determine whether a relationship exists between the presence of autoantibodies against ß1, ß2, α1 adrenoreceptors, M2-muscarinic receptors, angiotensin II type1 receptors and HHD. METHODS: In the present study, 44 patients diagnosed with HHD, 36 patients with hypertension, and 40 controls were also enrolled. The measurement of these 5 autoantibodies was performed by enzyme-linked immunosorbent assay. RESULTS: The frequencies of autoantibodies against ß1, ß2, α1 adrenoreceptors, autoantibodies against M2-muscarinic receptors and autoantibodies against angiotensin II type1 receptors were significantly higher in patients with HHD, when compared to patients with hypertension and normal controls (all p < 0.001). In addition, the titers of these 5 autoantibodies significantly increased in patients with HHD. Patients who were positive for all 5 autoantibodies had larger left ventricular end-diastolic diameter (60.5 ± 4.9 vs. 57.8 ± 5.0 vs. 52.5 ± 5.3 mm) and worse left ventricular ejection fraction (45.0 ± 11.0 vs. 56.6 ± 10.4 vs. 57.8 ± 5.3%), when compared to patients not positive for all the 5 autoantibodies and patients negative for all the 5 autoantibodies (χ2 = 9.524, p = 0.009 and χ2 = 7.689, p = 0.021). Furthermore, a significant positive correlation was observed between each 2 autoantibodies of these 5 autoantibodies (all p < 0.001). CONCLUSION: Multiple autoantibodies of cardiovascular receptors may be involved in the pathogenesis and may be predictive factors of HHD.
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Autoanticuerpos/sangre , Cardiopatías/inmunología , Hipertensión/inmunología , Receptor Muscarínico M2/inmunología , Receptores Adrenérgicos beta 1/inmunología , Anciano , Biomarcadores/sangre , Presión Sanguínea , Estudios de Casos y Controles , Ecocardiografía Doppler , Ensayo de Inmunoadsorción Enzimática , Femenino , Cardiopatías/etiología , Humanos , Hipertensión/complicaciones , Masculino , Persona de Mediana Edad , Función Ventricular IzquierdaRESUMEN
An autoimmune reaction directed against the cardiac b1-adrenergic receptor (beta1-ADR) leading to the generation of autoantibodies (AA) against this G-coupled receptor has been described in patients with heart failure (HF). Agonist-like beta1-ADR-AA are associated with morbidity in HF patients and even predict mortality. Standardised and valid diagnostic tools to detect beta 1-ADR-AA in clinical routine are lacking. We used a novel ELISA approach to investigate beta 1-ADR-AA in a cohort of 574 HF patients of the CIBIS-ELD trial with follow up. The CIBIS-ELD trial compared the titration of bisoprolol and carvedilol to recommended target doses in regard to BB tolerability in patients aged 65 years and older. Patient with left ventricular (LV) ejection fraction (EF) less than 50% or LV diameter end diastolic (DED) more than 55 cm showed significantly higher levels of beta1-ADR-AA. Although not yet fully validated, this ELISA allowed for a negative correlation of beta1-ADR-AA with the EF at baseline and at the follow up, beta1-ADR-AA further correlated positively with basal heart rate at follow up 12 weeks later. beta1-ADR-AA levels thus determined significantly increased under titration with beta-blockers (pless than 0.01). Changes in beta1-ADR-AA between F-Up and baseline were significantly higher in patients who used beta blockers (p=0.016) before study inclusion. The type of beta-blocker titrated in this study did not affect log beta1-ADR-AA levels at baseline (p=0.132), follow-up (p=0.058), nor the change (p=0.426). beta1-ADR-AA levels were estimated using a novel, commercially available ELISA. Although not yet fully validated, this ELISA allowed for pathophysiological insights: beta1-ADR-AA levels thus determined significantly increased under titration with beta-blockers (pless than 0.01), irrespective of type of BB. Higher levels of beta1-ADR-AA at baseline are associated with higher heart rates, lower ejection fraction and enlarged left ventricles. The relevance of the beta1-ADR-AA biomarker should be further evaluated.
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Autoanticuerpos/sangre , Insuficiencia Cardíaca/inmunología , Receptores Adrenérgicos beta 1/inmunología , Antagonistas Adrenérgicos beta/uso terapéutico , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Bisoprolol/uso terapéutico , Carvedilol/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Tasa de Filtración Glomerular , Insuficiencia Cardíaca/tratamiento farmacológico , Frecuencia Cardíaca , Humanos , Masculino , Resultado del Tratamiento , Función Ventricular IzquierdaRESUMEN
Therapeutic adoptive transfer of natural regulatory T cells (nTreg, CD4+ CD25+ Foxp3+ T cells) or in vivo selective expansion of nTreg cells has been demonstrated to improve the cardiac function in various cardiovascular disease models. The differentiation of nTreg cells is mediated by catecholamines via ß1-adrenergic receptor (ß1-AR) activation. Autoantibody against ß1-adrenoceptor (ß1-AA) as a ß1-AR agonist is closely associated with the occurrence and deterioration of cardiac dysfunction. However, whether ß1-AA has any impact on nTreg cells has not been reported. The aim of the present study was intended to assess the potential impact of ß1-AA on nTreg cell differentiation and explore the underlying mechanism. It was found that the expression of multiple proteins involved in nTreg cell differentiation, immunosuppressive function, and migration was up-regulated in mice after ß1-AA administration, suggesting that ß1-AA may promote nTreg cell activation. In vitro, ß1-AA promoted nTreg cell differentiation by up-regulating mitochondrial fatty acid oxidation (FAO) in activated CD4+ T cells via AMP-activated protein kinase (AMPK) activation and mitochondrial membrane potential reduction. In addition, the AMPK agonist facilitated ß1-AA-mediated FAO and nTreg cell differentiation. To further confirm the role of AMPK in ß1-AA-mediated nTreg cell differentiation, ß1-AA was acted on the CD4+ T cells isolated from AMPK-deficient (AMPK-/-) mice. The result showed that the effect of ß1-AA on nTreg cell differentiation was attenuated markedly after AMPK knockout. In conclusion, AMPK-mediated metabolic regulation targeting for nTreg cell restoration may be a promising therapeutic target for ß1-AA-positive patients with cardiac dysfunction.
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Proteínas Quinasas Activadas por AMP/metabolismo , Autoanticuerpos/inmunología , Diferenciación Celular/inmunología , Ácidos Grasos/metabolismo , Activación de Linfocitos/inmunología , Receptores Adrenérgicos beta 1/inmunología , Linfocitos T Reguladores/inmunología , Proteínas Quinasas Activadas por AMP/genética , Traslado Adoptivo , Animales , Técnicas de Inactivación de Genes , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Oxidación-Reducción , Regulación hacia Arriba/inmunologíaRESUMEN
Background Autoantibodies against the second extracellular loop of the ß1-adrenoceptor (ß1- AA ) act similarly to agonist of ß1-adrenergic receptor, which plays an important role in the pathophysiological characteristics of ventricular remodeling. Recently, considerable lines of evidence have suggested that CTRP9 (C1q tumor necrosis factor-related protein 9) is a potent cardioprotective cardiokine and protects the heart from ventricular remodeling. The aim of this study was to determine the role of CTRP 9 in ventricular remodeling induced by ß1- AA . Methods and Results Blood samples were collected from 131 patients with coronary heart disease and 131 healthy subjects. The serum levels of ß1- AA and CTRP 9 were detected using ELISA . The results revealed that CTRP 9 levels in ß1- AA -positive patients were lower than those in ß1- AA -negative patients, and serum CTRP 9 concentrations were inversely correlated with ß1- AA . ß1- AA monoclonal antibodies (ß1- AA mAbs) were administered in mice with and without rAAV 9- cTnT -Full Ctrp9- FLAG virus for 8 weeks. Reverse transcription-polymerase chain reaction/Western analysis showed that cardiomyocyte CTRP 9 expression was significantly reduced in ß1- AA mAb-treated mice. Moreover, compared with the ß1- AA mAb alone group, cardiac-specific CTRP 9 overexpression improved cardiac function, attenuated adverse remodeling, and ameliorated cardiomyocyte apoptosis and fibrosis. Mechanistic studies demonstrated that CTRP 9 overexpression decreased the levels of G-protein-coupled receptor kinase 2 and promoted the activation of AMP-dependent kinase pathway. However, cardiac-specific overexpression of CTRP 9 had no effect on the levels of cAMP and protein kinase A activity elevated by ß1-AAmAb. Conclusions This study provides the first evidence that the long-term existence of ß1- AA mAb suppresses cardiac CTRP 9 expression and exaggerates cardiac remodeling, suggesting that CTRP 9 may be a novel therapeutic target against pathologic remodeling in ß1- AA -positive patients with coronary heart disease.
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Adiponectina/genética , Autoanticuerpos/inmunología , Enfermedad Coronaria/metabolismo , Regulación de la Expresión Génica , Ventrículos Cardíacos/fisiopatología , Receptores Adrenérgicos beta 1/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Remodelación Ventricular , Adiponectina/biosíntesis , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Enfermedad Coronaria/diagnóstico , Enfermedad Coronaria/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/biosíntesisRESUMEN
BACKGROUND: Numerous studies have reported significantly elevated titers of serum autoantibody against the second extracellular loop of ß1-adrenoceptor (ß1-AA), a catecholamine-like substance with ß1-adrenergic activity, in patients with heart failure. Although evidence demonstrates that this autoantibody may alter T cell proliferation and secretion, the role of T lymphocytes in heart failure induced by ß1-AA remains unclear. The current study was designed to determine whether T cell disorder contributes to heart failure induced by ß1-AA. METHODS AND RESULTS: ß1-AA monoclonal antibodies (ß1-AAmAb) produced using the hybridoma technique were administered in wild-type mice or T lymphocyte deficiency nudes for 12 weeks. T lymphocytes from heart failure patients and neonatal cardiomyocytes were utilized in vitro. Mouse protein antibody array analysis was employed to detect the cytokines responsible for ß1-AAmAb-induced heart failure. Compared to wild-type mice, T lymphocyte deficiency mice prevented cardiac function from getting worse, attenuated adverse remodeling, and ameliorated cardiomyocyte apoptosis and fibrosis. As shown by protein array, the serum level of interleukin (IL)-6 was significantly lower in the nude group as compared to wild-type after ß1-AAmAb treatment. Mechanistic studies in vitro demonstrated that T lymphocyte culture supernatants stimulated by ß1-AAmAb caused direct damage in the cardiomyocytes, and ß1-AAmAb promoted proliferation of T lymphocytes isolated from patients with heart failure and increased IL-6 release. IL-6-specific siRNA virtually abolished cardiomyocyte apoptosis, suggesting that IL-6 may be a key cytokine released by T lymphocytes and responsible for ß1-AAmAb-induced cardiac remodeling. CONCLUSIONS: Collectively, we demonstrate that ß1-AAmAb-induced cardiac remodeling via mediating T lymphocyte disorder and releasing a variety of IL-6.