RESUMO
Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.
Assuntos
Miócitos Cardíacos , Canais de Potássio Shal , Ubiquitina-Proteína Ligases , Animais , Humanos , Coelhos , Potenciais de Ação/fisiologia , Estudo de Associação Genômica Ampla , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293RESUMO
The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.
Assuntos
Endossomos/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Potenciais de Ação , Animais , Estudo de Associação Genômica Ampla , Humanos , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Proteínas Nucleares/genética , Ligação Proteica , Transporte Proteico , Coelhos , Fatores de Transcrição/genética , Ubiquitinação , Peixe-ZebraRESUMO
[This corrects the article DOI: 10.3389/fphys.2021.672360.].
RESUMO
Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in larger animals, and the mechanisms are unknown. Specifically, age-associated changes in timecourse of senescence and related changes in inflammation and fibrosis are not well understood. Additionally, the cellular and systemic role of senescence and its inflammatory milieu in influencing arrhythmogenesis with age is not clear, particularly in large animal models with cardiac electrophysiology more similar to humans than previously studied animal models. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a 12-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that therapeutic interventions targeting senescent cells may mitigate arrhythmias post-MI with age.
Assuntos
Infarto do Miocárdio , Miofibroblastos , Animais , Coelhos , Humanos , Idoso , Miofibroblastos/patologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Arritmias Cardíacas , Fibrose , Inflamação/patologiaRESUMO
Transcranial direct current stimulation (tDCS) is a method for modulating cortical excitability by weak constant electrical current that is applied through scalp electrodes. Although often described in terms of anodal or cathodal stimulation, depending on which scalp electrode pole is proximal to the cortical region of interest, it is the orientation of neuronal structures relative to the direct current (DC) vector that determines the effect of tDCS. To investigate the contribution of neural pathway orientation, we studied DCS-mediated neuromodulation in an in vitro rat hippocampal slice preparation. We examined the contribution of dendritic orientation to the direct current stimulation (DCS) neuromodulatory effect by recording field excitatory postsynaptic potentials (fEPSPs) in apical and basal dendrites of CA1 neurons within a constant DC field. In addition, we assessed the contribution of axonal orientation by recording CA1 and CA3 apical fEPSPs generated by stimulation of oppositely oriented Schaffer collateral and mossy fiber axons, respectively, during DCS. Finally, nonsynaptic excitatory signal propagation was measured along antidromically stimulated CA1 axons at different DCS amplitudes and polarity. We find that modulation of both the fEPSP and population spike depends on axonal orientation relative to the electric field vector. Axonal orientation determines whether the DC field is excitatory or inhibitory and dendritic orientation affects the magnitude, but not the overall direction, of the DC effect. These data suggest that tDCS may oppositely affect neurons in a stimulated cortical volume if these neurons are excited by oppositely orientated axons in a constant electrical field.
Assuntos
Axônios/fisiologia , Fenômenos Biofísicos/fisiologia , Estimulação Elétrica , Hipocampo/citologia , Neurônios/fisiologia , Orientação , Análise de Variância , Animais , Animais Recém-Nascidos , Biofísica , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/fisiologia , Vias Neurais/fisiologia , Ratos , Ratos Long-Evans , Sinapses/fisiologiaRESUMO
Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (I Na), transient outward potassium current (I to), L-type calcium channel peak current (I Ca,L), rapid and slow components of the delayed rectifier potassium current (I Kr and I Ks), and inward rectifier (I K1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.
RESUMO
Glutamate released from synaptic vesicles mediates excitatory neurotransmission by stimulating glutamate receptors. Glutamate transporters maintain low synaptic glutamate levels critical for this process, a role primarily attributed to astrocytes. Recently, vesicular release of glutamate from unmyelinated axons in the rat corpus callosum has been shown to elicit AMPA receptor-mediated currents in glial progenitor cells. Glutamate transporters are the only mechanism of glutamate clearance, yet very little is known about the role of glutamate transporters in normal development of oligodendrocytes (OLs) or in excitotoxic injury to OLs. We found that OLs in culture are capable of sodium-dependent glutamate uptake with a K(m) of 10 +/- 2 microm and a V(max) of 2.6, 5.0, and 3.8 nmol x min(-1) x mg(-1) for preoligodendrocytes, immature, and mature OLs, respectively. Surprisingly, EAAC1, thought to be exclusively a neuronal transporter, contributes more to [(3)H]l-glutamate uptake in OLs than GLT1 or GLAST. These data suggest that glutamate transporters on oligodendrocytes may serve a critical role in maintaining glutamate homeostasis at a time when unmyelinated callosal axons are engaging in glutamatergic signaling with glial progenitors. Furthermore, GLT1 was significantly increased in cultured mature OLs contrary to in vivo data in which we have shown that, although GLT1 is present on developing OLs when unmyelinated axons are prevalent in the developing rat corpus callosum, after myelination, GLT1 is not expressed on mature OLs. The absence of GLT1 in mature OLs in the rat corpus callosum and its presence in mature rat cultured OLs may indicate that a signaling process in vivo is not activated in vitro.
Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Transportador 2 de Aminoácido Excitatório/fisiologia , Transportador 3 de Aminoácido Excitatório/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Ácido Glutâmico/metabolismo , Oligodendroglia/metabolismo , Animais , Animais Recém-Nascidos , Ácido Aspártico/farmacologia , Benzodiazepinas/farmacologia , Bicuculina/farmacologia , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Transportador 2 de Aminoácido Excitatório/antagonistas & inibidores , Transportador 3 de Aminoácido Excitatório/antagonistas & inibidores , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Antagonistas GABAérgicos/farmacologia , Gangliosídeos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ácido Caínico/análogos & derivados , Ácido Caínico/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/metabolismo , Antígenos O/metabolismo , Técnicas de Patch-Clamp/métodos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Gravidez , Quinoxalinas/farmacologia , Ratos , Ratos Long-Evans , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Trítio/metabolismoRESUMO
OBJECTIVE: To investigate coronary endothelial protection of a small-conductance calcium-activated potassium (SK) channel activator against a period of cardioplegic-hypoxia and reoxygenation (CP-H/R) injury in mice and patients with diabetes (DM) and those without diabetes (nondiabetic [ND]). METHODS: Mouse small coronary arteries/heart endothelial cells (MHECs) and human coronary arterial endothelial cells (HCAECs) were dissected from the harvested hearts of mice (n = 16/group) and from discarded right atrial tissue samples of patients with DM and without DM (n = 8/group). The SK current density of MHECs was measured. The in vitro small arteries/arterioles, MHECs, and HCAECs were subjected to 60 minutes of CP hypoxia, followed by 60 minutes of oxygenation. Vessels were treated with or without the selective SK activator NS309 for 5 minutes before and during CP hypoxia. RESULTS: DM and/or CP-H/R significantly inhibited the total SK currents of MHECs and HCAECs and significantly diminished the mouse coronary relaxation response to NS309. Administration of NS309 immediately before and during CP hypoxia significantly improved the recovery of coronary endothelial function, as demonstrated by increased relaxation responses to adenosine 5'-diphosphate and substance P compared with those seen in controls (P < .05). This protective effect was more pronounced in vessels from ND mice and patients compared with DM mice and patients (P < .05). Cell surface membrane SK3 expression was significantly reduced after hypoxia, whereas cytosolic SK3 expression was greater than that of the sham control group (P < .05). CONCLUSIONS: Application of NS309 immediately before and during CP hypoxia protects mouse and human coronary microvasculature against CP-H/R injury, but this effect is diminished in the diabetic coronary microvasculature. SK inhibition/inactivation and/or internalization/redistribution may contribute to CP-H/R-induced coronary endothelial and vascular relaxation dysfunction.
Assuntos
Doença da Artéria Coronariana/etiologia , Vasos Coronários/patologia , Diabetes Mellitus Tipo 2/complicações , Endotélio Vascular/metabolismo , Indóis/farmacologia , Oximas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Vasodilatação/efeitos dos fármacos , Idoso , Animais , Células Cultivadas , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Camundongos , Transdução de Sinais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacosRESUMO
BACKGROUND: Diabetic (DM) inactivation of small conductance calcium-activated potassium (SK) channels contributes to coronary endothelial dysfunction. However, the mechanisms responsible for this down-regulation of endothelial SK channels are poorly understood. Thus, we hypothesized that the altered metabolic signaling in diabetes regulates endothelial SK channels and human coronary microvascular function. METHODS: Human atrial tissue, coronary arterioles and coronary artery endothelial cells (HCAECs) obtained from DM and non-diabetic (ND) patients (n = 12/group) undergoing cardiac surgery were used to analyze metabolic alterations, endothelial SK channel function, coronary microvascular reactivity and SK gene/protein expression/localization. RESULTS: The relaxation response of DM coronary arterioles to the selective SK channel activator SKA-31 and calcium ionophore A23187 was significantly decreased compared to that of ND arterioles (p < 0.05). Diabetes increases the level of NADH and the NADH/NAD+ ratio in human myocardium and HCAECs (p < 0.05). Increase in intracellular NADH (100 µM) in the HCAECs caused a significant decrease in endothelial SK channel currents (p < 0.05), whereas, intracellular application of NAD+ (500 µM) increased the endothelial SK channel currents (p < 0.05). Mitochondrial reactive oxygen species (mROS) of HCAECs and NADPH oxidase (NOX) and PKC protein expression in the human myocardium and coronary microvasculature were increased respectively (p < 0.05). CONCLUSIONS: Diabetes is associated with metabolic changes in the human myocardium, coronary microvasculature and HCAECs. Endothelial SK channel function is regulated by the metabolite pyridine nucleotides, NADH and NAD+, suggesting that metabolic regulation of endothelial SK channels may contribute to coronary endothelial dysfunction in the DM patients with diabetes.
Assuntos
Diabetes Mellitus , Células Endoteliais , Arteríolas , Vasos Coronários/diagnóstico por imagem , Coração , HumanosRESUMO
BACKGROUND: Long QT syndrome has been associated with sudden cardiac death likely caused by early afterdepolarizations (EADs) and polymorphic ventricular tachycardias (PVTs). Suppressing the late sodium current (INaL) may counterbalance the reduced repolarization reserve in long QT syndrome and prevent EADs and PVTs. METHODS: We tested the effects of the selective INaL blocker GS967 on PVT induction in a transgenic rabbit model of long QT syndrome type 2 using intact heart optical mapping, cellular electrophysiology and confocal Ca2+ imaging, and computer modeling. RESULTS: GS967 reduced ventricular fibrillation induction under a rapid pacing protocol (n=7/14 hearts in control versus 1/14 hearts at 100 nmol/L) without altering action potential duration or restitution and dispersion. GS967 suppressed PVT incidences by reducing Ca2+-mediated EADs and focal activity during isoproterenol perfusion (at 30 nmol/L, n=7/12 and 100 nmol/L n=8/12 hearts without EADs and PVTs). Confocal Ca2+ imaging of long QT syndrome type 2 myocytes revealed that GS967 shortened Ca2+ transient duration via accelerating Na+/Ca2+ exchanger (INCX)-mediated Ca2+ efflux from cytosol, thereby reducing EADs. Computer modeling revealed that INaL potentiates EADs in the long QT syndrome type 2 setting through (1) providing additional depolarizing currents during action potential plateau phase, (2) increasing intracellular Na+ (Nai) that decreases the depolarizing INCX thereby suppressing the action potential plateau and delaying the activation of slowly activating delayed rectifier K+ channels (IKs), suggesting important roles of INaL in regulating Nai. CONCLUSIONS: Selective INaL blockade by GS967 prevents EADs and abolishes PVT in long QT syndrome type 2 rabbits by counterbalancing the reduced repolarization reserve and normalizing Nai. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Antiarrítmicos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Síndrome do QT Longo/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Piridinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Taquicardia Ventricular/prevenção & controle , Triazóis/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Simulação por Computador , Canais de Potássio de Retificação Tardia/metabolismo , Modelos Animais de Doenças , Feminino , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Masculino , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Coelhos , Canais de Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia , Fatores de Tempo , Fibrilação Ventricular/genética , Fibrilação Ventricular/metabolismo , Fibrilação Ventricular/fisiopatologia , Fibrilação Ventricular/prevenção & controleRESUMO
Glutamate is the major excitatory neurotransmitter of the central nervous system and is toxic to neurons even at low concentrations. GLT1, the rodent analog of human EAAT2, is primarily responsible for glutamate clearance in the cerebrum. GLT1 was thought to be expressed exclusively in astrocytes in the mature brain. Recently, however, GLT1a was demonstrated in excitatory axon terminals where synaptic glutamate concentration rises above 1 mm during excitatory transmission. GLT1 function in neurons with accurate control of both intracellular and extracellular solutions mimicking synaptic concentration gradients has never been studied. Here we characterized the kinetics of coupled glutamate transporter current in whole-cell configuration and [(3)H]-l-glutamate uptake in cultured rat cerebral neurons across the entire range of synaptic glutamate concentrations. In both neurons and GLT1a-transfected COS-7 cells, the kinetics were similar and revealed two specific components: a high-affinity component with glutamate k(D) value around 15 mum and a low-affinity component with k(D) value around 0.2 mm. The specific low-affinity component was discovered as a result of significant deviation of the transporter current from Michaelis-Menten kinetics in the 100-300 mum concentration range. Activation of the specific low-affinity component led to a two-fold decrease in the current/flux ratio, implying a change in the transport coupling. Our data indicate that GLT1 endogenously expressed in cultured rat forebrain neurons displays high and low glutamate affinity uptake components that are different in current-flux coupling ratios. This property is intrinsic to the protein because it was also observed in GLT1a-transfected COS-7 cells.
Assuntos
Transportador 2 de Aminoácido Excitatório/biossíntese , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Prosencéfalo/metabolismo , Potenciais Sinápticos/fisiologia , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/administração & dosagem , Neurônios/efeitos dos fármacos , Prosencéfalo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Potenciais Sinápticos/efeitos dos fármacosRESUMO
BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Proteínas de Ligação a DNA/genética , Coração/embriologia , Humanos , Proteínas de Membrana/genética , Miócitos Cardíacos/enzimologia , Ubiquitina-Proteína Ligases Nedd4/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coelhos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinação , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Sudden death in long-QT syndrome type 1 (LQT1), an inherited disease caused by loss-of-function mutations in KCNQ1, is triggered by early afterdepolarizations (EADs) that initiate polymorphic ventricular tachycardia (pVT). We investigated ionic mechanisms that underlie pVT in LQT1 using a transgenic rabbit model of LQT1. METHODS: Optical mapping, cellular patch clamping, and computer modeling were used to elucidate the mechanisms of EADs in transgenic LQT1 rabbits. RESULTS: The results showed that shorter action potential duration in the right ventricle (RV) was associated with focal activity during pVT initiation. RV cardiomyocytes demonstrated higher incidence of EADs under 50 nmol/L isoproterenol. Voltage-clamp studies revealed that the transient outward potassium current (Ito) magnitude was 28% greater in RV associated with KChiP2 but with no differences in terms of calcium-cycling kinetics and other sarcolemmal currents. Perfusing with the Ito blocker 4-aminopyridine changed the initial focal sites of pVT from the RV to the left ventricle, corroborating the role of Ito in pVT initiation. Computer modeling showed that EADs occur preferentially in the RV because of the larger conductance of the slow-inactivating component of Ito, which repolarizes the membrane potential sufficiently rapidly to allow reactivation of ICa,L before IKr has had sufficient time to activate. CONCLUSIONS: Ito heterogeneity creates both triggers and an arrhythmogenic substrate in LQT1. In the absence of IKs, Ito interactions with ICa,L and IKr promote EADs in the RV while prolonging action potential duration in the left ventricle. This heterogeneity of action potential enhances dispersion of refractoriness and facilitates conduction blocks that initiate pVTs.
Assuntos
Frequência Cardíaca , Ventrículos do Coração/metabolismo , Canal de Potássio KCNQ1/metabolismo , Potássio/metabolismo , Síndrome de Romano-Ward/metabolismo , Taquicardia Ventricular/metabolismo , Função Ventricular Direita , Potenciais de Ação , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , Simulação por Computador , Modelos Animais de Doenças , Feminino , Ventrículos do Coração/fisiopatologia , Canal de Potássio KCNQ1/genética , Masculino , Modelos Cardiovasculares , Mutação , Miócitos Cardíacos/metabolismo , Coelhos , Síndrome de Romano-Ward/genética , Síndrome de Romano-Ward/fisiopatologia , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatologia , Fatores de TempoRESUMO
During long-term potentiation (LTP) of excitatory synapses, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by Ca(2+) influx through NMDA receptors that potentiate AMPA receptor currents by insertion of additional GluR1-containing receptors at the synapse and by increasing AMPA channel conductance, as well as by stimulating structural changes. CaMKII is also involved in the maintenance of LTP and contributes to maintenance of behavioral sensitization by cocaine or amphetamine. Recent studies show that transient expression of catalytically dead αCaMKII K42M mutant after exposure to amphetamine persistently reverses the behavioral effects of the addiction. A suggested interpretation is that this mutant acts as a dominant negative in the control of synaptic strength, but this interpretation has not been physiologically tested. Here we investigate the effect of αCaMKII K42M mutant expressed in single CA1 pyramidal neurons on basal excitatory neurotransmission in cultured rat hippocampal organotypic slices. The mutant caused nearly 50% reduction in the basal CA3-CA1 transmission, while overexpression of the wild-type αCaMKII had no effect. This result is consistent with the dominant negative hypothesis, but there are complexities. We found that the decrease in basal transmission did not occur when activity in the slices was suppressed after transfection by TTX or when NMDA receptors were blocked by APV. Thus, the dominant negative effect requires neural activity for its expression.
Assuntos
Biocatálise , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sinapses/fisiologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Hipocampo/enzimologia , Hipocampo/fisiologia , Técnicas In Vitro , Masculino , Ratos , Ratos Long-Evans , Sinapses/enzimologiaRESUMO
Glutamate is the major excitatory neurotransmitter, but prolonged exposure even at micromolar concentrations causes neuronal death. Extracellular glutamate is maintained at nanomolar level by glutamate transporters, which, however, may reverse transport and release glutamate. If and when the reverse occurs depends on glutamate transport stoichiometry (GTS). Previously we found that in the presence of chloride, the coupled GLT-1 glutamate transporter current and its relationship to radiolabeled glutamate flux significantly decreased when extracellular glutamate concentration increased above 0.2 mM, which implies a change in GTS. Such high concentrations are feasible near GLT-1 expressed close to synaptic release site during excitatory neurotransmission. The aim of this study was to determine GLT-1 GTS at both low (19-75 µM) and high (300-1200 µM) glutamate concentration ranges. GTS experiments were conducted in the absence of chloride to avoid contributions by the GLT-1 uncoupled chloride conductance. Mathematical analysis of the transporter thermodynamic equilibrium allowed us to derive equations revealing the number of a particular type of ion transported per elementary charge based on the measurements of the transporter reversal potential. We found that GLT-1a expressed in COS-7 cells co-transports 1.5 Na+, 0.5 Glu-, 0.5 H+ and counter-transports 0.6 K+ per elementary charge in both glutamate concentration ranges, and at both 37°C and 26°C temperatures. The thermodynamic parameter Q10 = 2.4 for GLT-1 turnover rate of 19 s-1 (37°C, -50 mV) remained constant in the 10 µM-10 mM glutamate concentration range. Importantly, the previously reported decrease in the current/flux ratio at high glutamate concentration was not seen in the absence of chloride in both COS-7 cells and cultured rat neurons. Therefore, only in the absence of chloride, GLT-1 GTS remains constant at all glutamate concentrations. Possible explanations for why apparent GTS might vary in the presence of chloride are discussed.
Assuntos
Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Transmissão Sináptica/genética , Animais , Células COS , Cloretos/metabolismo , Chlorocebus aethiops , Transportador 2 de Aminoácido Excitatório/biossíntese , Gluconatos/metabolismo , Ácido Glutâmico/química , Neurônios/patologia , Técnicas de Patch-Clamp , Ratos , Sódio/metabolismoRESUMO
Excessive extracellular glutamate after traumatic brain injury (TBI) contributes to excitotoxic cell death and likely to post-traumatic epilepsy. Glutamate transport is the only known mechanism of extracellular glutamate clearance, and glutamate transporter 1 (GLT-1) is the major glutamate transporter of the mammalian brain. We tested, by immunoblot, in the rat lateral fluid percussion injury TBI model whether GLT-1 expression is depressed in the cortex after TBI, and whether GLT-1 expression after TBI is restored after treatment with ceftriaxone, a well-tolerated ß-lactam antibiotic previously shown to enhance GLT-1 expression in noninjured animals. We then tested whether treatment with ceftriaxone mitigates the associated regional astrogliosis, as reflected by glial fibrillary acid protein (GFAP) expression, and also whether ceftriaxone treatment mitigates the severity of post-traumatic epilepsy. We found that 7 days after TBI, GLT-1 expression in the ipsilesional cortex was reduced by 29% (n=7/group; p<0.01), relative to the contralesional cortex. However, the loss of GLT-1 expression was reversed by treatment with ceftriaxone (200 mg/kg, daily, intraperitoneally). We found that ceftriaxone treatment also decreased the level of regional GFAP expression by 43% in the lesioned cortex, relative to control treatment with saline (n=7 per group; p<0.05), and, 12 weeks after injury, reduced cumulative post-traumatic seizure duration (n=6 rats in the ceftriaxone treatment group and n=5 rats in the saline control group; p<0.001). We cautiously conclude that our data suggest a potential role for ceftriaxone in treatment of epileptogenic TBI.