RESUMO
BACKGROUND: Glutamate and its specific ionotropic receptors, including N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, are supposed to play an important role in neurodegeneration as well as neuronal regeneration. Although autoantibodies (aab) to glutamate receptors (GluR) have been identified in several neurologic diseases, including paraneoplastic encephalitis and Rasmussen's encephalitis (RE) with an increasing prevalence, the presence and role of anti-GluR aab in multiple sclerosis (MS) have not been studied yet. OBJECTIVES AND METHODS: In this study, we tested the serum samples of 56 subjects, including patients with relapsing-remitting MS (n = 25), patients with RE (n = 8), and healthy donors (HD; n = 23), for anti-GluR aab by immunoblot analysis of a panel of recombinantly expressed GluR proteins, including GluN1, GluN2C, GluA3, GluK2, and GluD2. RESULTS: aab were mainly found directed against GluN1 and, except for one aab positive to GluK2 in 1 MS patient and 2 HD controls positive for GluA3, no other anti-GluR aab were detected. In the sera of RE patients, no anti-GluR aab were found. In patients with MS, 8 of the 25 sera (32%) tested positive for GluN1. Compared to the HD (6/23; 26%), this difference was not statistically significant (p = 0.28). CONCLUSIONS: Our study showed that if anti-GluR aab were detectable in HD and MS patients, they were mainly directed against GluN1 (in particular to oligomeric protein complexes) and were not found in RE. Those antibodies may have low titers and low affinities and might be considered an immune epiphenomenon. Hence, further studies will have to clarify their potential role as a surrogate marker for the extent of neuronal destruction or regeneration, respectively.
Assuntos
Autoanticorpos/sangue , Encefalite/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Receptores de Glutamato/imunologia , Adulto , Autoantígenos/imunologia , Encefalite/sangue , Feminino , Humanos , Immunoblotting , Masculino , Esclerose Múltipla Recidivante-Remitente/sangueRESUMO
Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.
Assuntos
Enterovirus Humano B/classificação , Enterovirus Humano B/fisiologia , Regulação da Expressão Gênica/fisiologia , Canais Iônicos/metabolismo , Miócitos Cardíacos/metabolismo , Transporte Proteico/fisiologia , Animais , Simulação por Computador , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Miócitos Cardíacos/virologia , Oócitos , Polimorfismo Genético , XenopusRESUMO
The mortality rate of neonatal piglets after heart surgery is high. Searching for a possible explanation for the death of neonatal piglets early after cardiopulmonary bypass, we analyzed hemodynamic parameters regarding survival and non-survival. Initially, 10 neonatal piglets (younger than 7 days) were connected to cardiopulmonary bypass (CPB). The mean body weight was 2.98 ± 0.44 kg. Exposure of the heart was performed through a median sternotomy. After connection to the CPB, the piglets were cooled to 32°C core temperature before the ascending aorta was cross-clamped and the heart arrested (90 min). Thereafter, piglets were re-warmed to 37°C and separated from CPB. During follow-up, the piglets did not receive inotropic support or vasopressors. Piglets who survived at least 2 h after termination of CPB were included in the study for further data analysis (n = 9). Five piglets died 2.5 to 4.0 h (median: 3.5 h) after CPB; these piglets formed the non-survivors group. Four animals survived the complete follow-up of 6 h after CPB and formed the survivors group. Regarding contractility (dP/dt(max) , dP/dt(max) /P, and wall thickening) there were not statistically significant differences between the groups. Non-survivors showed prolonged decrease of mean arterial pressure of more than 20% of baseline values, corresponding with a value of below 30 mm Hg. In conclusion, the death of neonatal piglets early after cardiopulmonary bypass was not determined by low output.
Assuntos
Baixo Débito Cardíaco , Ponte Cardiopulmonar/mortalidade , Causas de Morte , Animais , Animais Recém-Nascidos , Hemodinâmica , SuínosRESUMO
BACKGROUND: Cardiac action potential repolarisation is determined by K(+) currents including I(Ks). I(Ks) channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype. METHODS: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation. RESULTS: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits. CONCLUSION: The mutation acted in a E-subunit dependent manner, suggesting I(Ks) function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.
Assuntos
Canal de Potássio KCNQ1/genética , Mutação , Adulto , Sequência de Aminoácidos , Animais , Criança , Eletrocardiografia , Feminino , Humanos , Lactente , Recém-Nascido , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/fisiologia , Masculino , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Linhagem , Fenótipo , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Cooling before circulatory arrest or ischemic arrest has been reported to influence myocardial performance in isolated neonatal hearts. The aim of the present study was to analyze indices of myocardial contractility and relaxation in an in vivo neonatal model after deep hypothermic circulatory arrest (DHCA). DHCA (18°C; DHCA group; n = 8) or mild hypothermic cardiopulmonary bypass ([MH-CPB] 32°C; MH-CPB group; n = 10) was applied in newborn piglets. After reperfusion (60 and 120 min), left ventricular dP/dt(max) increased in DHCA and MH-CPB, while-dP/dt(max) decreased slightly in DHCA and increased in MH-CPB. Nevertheless, the differences between the two groups did not reach statistical significance. In conclusion, left ventricular contractility remained stable after reperfusion following DHCA, to some degree at the expense of the diastolic function.
Assuntos
Parada Circulatória Induzida por Hipotermia Profunda/efeitos adversos , Parada Cardíaca Induzida/efeitos adversos , Hipotermia Induzida/efeitos adversos , Contração Miocárdica/fisiologia , Traumatismo por Reperfusão Miocárdica/etiologia , Suínos/fisiologia , Animais , Animais Recém-Nascidos , Ponte Cardiopulmonar/métodos , Modelos Animais de Doenças , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Função Ventricular Esquerda/fisiologiaRESUMO
The stress reaction includes the release of stress hormones such as cortisol via the HPA axis4. One of the genes regulated by cortisol is the serum- and glucocorticoid-inducible kinase 1 (SGK1) a stimulator of the slow outward potassium channel KCNQ1/KCNE1-one of the major mediators of cardiac repolarization. Apart from KCNE1, several other KCNE beta subunits including KCNE3 and KCNE5 have been detected at the mRNA level in cardiac tissue as well as in the inner ear and the gastro-intestinal tract. Here, we extend our previous investigations to KCNQ1/KCNE3 channels and their modulation by SGKs. We show that these channels are not stimulated by any of the three SGK isoforms when expressed in a heterologous expression system. 3D docking simulations suggest that crucial residues within KCNQ1 and KCNE1 are co-localized to a region close to the putative inner phase of the membrane, suggesting a key region important for channel complex sorting into vesicles. Identification of the KCNQ1/KCNE recycling pathway and its modulation by SGK provides a mechanistic insight into stress-induced modulation of KCNQ1/KCNE channels.
Assuntos
Proteínas Imediatamente Precoces/fisiologia , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Transporte Biológico , Membrana Celular/metabolismo , Simulação por Computador , Modelos Moleculares , Isoformas de Proteínas , Ratos , Transdução GenéticaRESUMO
Cardiac function and hemodynamics are frequently decreased during the first hours after heart surgery, resulting in inotropic support for treatment and prevention of further hemodynamic deterioration. The aim of this study was analysis of hemodynamics of neonatal piglets who survived early postoperative course after cardiopulmonary bypass (CPB) and cardioplegic arrest without the use of inotropic drugs. Newborn piglets (younger than 7 days) were placed on mild hypothermic CPB (32 degrees C) for 180 minutes, including 90 minutes of cardioplegic arrest. Hemodynamics were examined after termination of CPB and none of the animals received any inotropic support. After 6 hours, survived animals were euthanized (CPB group, n=4). For control, neonatal piglets were examined for the same time interval after surgery without CPB (control group, n=3). Systolic left-ventricular pressure increased after CPB, mean arterial blood pressure and amplitude of left ventricular wall thickness decreased. Compared with control group, systolic left-ventricular pressure in CPB group was higher (p<0.05). Present data demonstrated hemodynamic depression after cardiac procedures in survived neonatal animals. Although the effects may not be solely attributed to CPB and myocardial ischemia effects may be potentiate by CPB.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Hemodinâmica/fisiologia , Animais , Animais Recém-Nascidos , Oxigenação por Membrana Extracorpórea/efeitos adversos , Período Pós-Operatório , SuínosRESUMO
Physical and emotional stress is accompanied by release of stress hormones such as the glucocorticoid cortisol. This hormone upregulates the serum- and glucocorticoid-inducible kinase (SGK)1, which in turn stimulates I(Ks), a slow delayed rectifier potassium current that mediates cardiac action potential repolarization. Mutations in I(Ks) channel alpha (KCNQ1, KvLQT1, Kv7.1) or beta (KCNE1, IsK, minK) subunits cause long QT syndrome (LQTS), an inherited cardiac arrhythmia associated with increased risk of sudden death. Together with the GTPases RAB5 and RAB11, SGK1 facilitates membrane recycling of KCNQ1 channels. Here, we show altered SGK1-dependent regulation of LQTS-associated mutant I(Ks) channels. Whereas some mutant KCNQ1 channels had reduced basal activity but were still activated by SGK1, currents mediated by KCNQ1(Y111C) or KCNQ1(L114P) were paradoxically reduced by SGK1. Heteromeric channels coassembled of wild-type KCNQ1 and the LQTS-associated KCNE1(D76N) mutant were similarly downregulated by SGK1 because of a disrupted RAB11-dependent recycling. Mutagenesis experiments indicate that stimulation of I(Ks) channels by SGK1 depends on residues H73, N75, D76, and P77 in KCNE1. Identification of the I(Ks) recycling pathway and its modulation by stress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered by physical or psychological stress.
Assuntos
Endossomos/genética , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteínas de Xenopus/genética , Animais , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Feminino , Canal de Potássio KCNQ1/fisiologia , Oócitos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevisRESUMO
We evaluated the newly developed miniaturized HIA microdiagonal blood pump (MDP) as a continuous flow left ventricular assist device. In a sheep model (n = 6), the MDP was implanted through left lateral thoracotomy and placed paracorporeally with inflow conduit to left atrium and outflow conduit to descending aorta. The sheep were pumped at a mean flow rate of 2.5 L/min for 7 days. Anticoagulation was applied by intravenous heparin administration. Postoperatively, activated clotting time was held stable with values of 200 seconds. During follow-up, blood samples (creatinine kinase, creatinine, glutamic-oxaloacetic transaminase (aspartate aminotransferase) (GOT), glutamate dehydrogenase (GLDH), gamma-GT, plasma-free hemoglobin, and hemoglobine) were taken daily. After 7 days, the sheep were killed for macroscopic examination. Systemic artery pressures remained stable during the whole test period. Because of operative reasons, the hemoglobin value (7.5 +/- 0.61 g/dl) decreased perioperatively, but recovered within the test period, whereas creatinine kinase increased initially after thoracotomy, but decreased to normal within days. Renal and liver functions were slightly impaired perioperatively, indicated by temporarily enhanced values of GOT, gamma-GT, GLDH, and creatinine. The MDP did not produce significant hemolysis as measured by plasma-free hemoglobin levels. Wound infections did not occur. We conclude that the MDP ran successfully as an left ventricular assist device for 7 days in sheep has potential for long-term support, and may serve as an alternative to current technologies. Presented data were not obtained in a clinical trial; however, the results are promising enough to proceed with longer duration animal studies.