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1.
Int J Mol Sci ; 20(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614475

RESUMO

Dysfunction of the cardiac sodium channel Nav1.5 (encoded by the SCN5A gene) is associated with arrhythmias and sudden cardiac death. SCN5A mutations associated with long QT syndrome type 3 (LQT3) lead to enhanced late sodium current and consequent action potential (AP) prolongation. Internalization and degradation of Nav1.5 is regulated by ubiquitylation, a post-translational mechanism that involves binding of the ubiquitin ligase Nedd4-2 to a proline-proline-serine-tyrosine sequence of Nav1.5, designated the PY-motif. We investigated the biophysical properties of the LQT3-associated SCN5A-p.Y1977N mutation located in the Nav1.5 PY-motif, both in HEK293 cells as well as in newly generated mice harboring the mouse homolog mutation Scn5a-p.Y1981N. We found that in HEK293 cells, the SCN5A-p.Y1977N mutation abolished the interaction between Nav1.5 and Nedd4-2, suppressed PY-motif-dependent ubiquitylation of Nav1.5, and consequently abrogated Nedd4-2 induced sodium current (INa) decrease. Nevertheless, homozygous mice harboring the Scn5a-p.Y1981N mutation showed no electrophysiological alterations nor changes in AP or (late) INa properties, questioning the in vivo relevance of the PY-motif. Our findings suggest the presence of compensatory mechanisms, with additional, as yet unknown, factors likely required to reduce the "ubiquitylation reserve" of Nav1.5. Future identification of such modulatory factors may identify potential triggers for arrhythmias and sudden cardiac death in the setting of LQT3 mutations.


Assuntos
Substituição de Aminoácidos , Síndrome do QT Longo/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Motivos de Aminoácidos , Animais , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ligação Proteica , Ubiquitinação , Adulto Jovem
2.
Cardiol Young ; 29(10): 1257-1263, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31477192

RESUMO

INTRODUCTION: The SCN5A gene is implicated in many arrhythmogenic and cardiomyopathic processes. We identified a novel SCN5A variant in a family with significant segregation in individuals affected with progressive sinus and atrioventricular nodal disease, atrial arrhythmia, dilated cardiomyopathy, and early sudden cardiac arrest. METHODS: A patient pedigree was created following the clinical evaluation of three affected individuals, two monozygotic twins and a paternal half-brother, which lead to the evaluation of a paternal half-sister (four siblings with the same father and three mothers) all of whom experienced varying degrees of atrial arrhythmias, conduction disease, and dilated cardiomyopathy in addition to a paternal history of unexplained death in his 50s with similar autopsy findings. The index male underwent sequencing of 58 genes associated with cardiomyopathies. Sanger sequencing was used to provide data for bases with insufficient coverage and for bases in some known regions of genomic segmental duplications. All clinically significant and novel variants were confirmed by independent Sanger sequencing. RESULTS: All relatives tested were shown to have the same SCN5A variant of unknown significance (p. Asp197His) and the monozygotic twins shared a co-occurring NEXN (p. Glu575*). Segregation analysis demonstrates likely pathogenic trait for the SCN5A variant with an additional possible role for the NEXN variant in combination. CONCLUSIONS: There is compelling clinical evidence suggesting that the SCN5A variant p. Asp197His may be re-classified as likely pathogenic based on the segregation analysis of our family of interest. Molecular mechanism studies are pending.


Assuntos
Arritmias Cardíacas/genética , Cardiomiopatia Dilatada/genética , DNA/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adolescente , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/metabolismo , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , Ecocardiografia , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Linhagem , Fenótipo , Adulto Jovem
3.
Int Heart J ; 60(4): 979-982, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31257342

RESUMO

Congenital long QT syndrome (LQTS) is a cardiac channelopathy that leads to the prolongation of the QT interval. This prolongation can lead to ventricular tachyarrhythmia, syncope, and sudden cardiac death. There are various types of LQTS. Treatment of LQT1 and LQT2 is mainly based on antiadrenergic therapy. LQT3, on the other hand, is a result of a mutation of the SCN5A gene, which encodes the sodium channels. In this type, patients are sensitive to vagal stimuli and episodes tend to occur at rest. Sodium channel blocking compounds, such as ranolazine, mexiletine, and flecainide, have been found to be effective in selective mutations.In this case report, we report the case of a child with congenital LQT3 (V411M) who presented first with sudden cardiac death and three weeks later with an implantable cardioverter defibrillator storm. Knowing the specific mutation and understanding the mechanism at the molecular level through an in vitro study yielded a clinically meaningful result. The patient's arrhythmia burden was totally eliminated following successful treatment with flecainide.


Assuntos
DNA/genética , Eletrocardiografia , Flecainida/uso terapêutico , Síndrome do QT Longo/tratamento farmacológico , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Criança , Análise Mutacional de DNA , Feminino , Humanos , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
4.
Cell Physiol Biochem ; 53(1): 36-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169990

RESUMO

BACKGROUND/AIMS: Ivabradine lowers the heart rate by inhibition of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels mediating the 'funny' pacemaker current If in the sinoatrial node. It is clinically approved for the treatment of heart failure and angina pectoris. Due to its proposed high selectivity for If administration of ivabradine is not associated with the side effects commonly observed following the application of other heart rate lowering agents. Recent evidence, however, has shown significant affinity of ivabradine towards Kv11.1 (ether-a-go-go related gene, ERG) potassium channels. Despite the inhibition of Kv11.1 channels by ivabradine, cardiac action potential (AP) duration and heart rate corrected QT interval (QTc) of the human electrocardiogram (ECG) were not prolonged. We thus surmised that compensatory mechanisms might counteract the drug's inhibitory action on Kv11.1. METHODS: The effects of ivabradine on human Kv11.1 and Kv7.1 potassium, Cav1.2 calcium, and Nav1.5 sodium channels, heterologously expressed in tsA-201 cells, were studied in the voltage-clamp mode of the whole cell patch clamp technique. In addition, changes in action potential parameters of human induced pluripotent stem cell (iPSC) derived cardiomyocytes upon application of ivabradine were studied with current-clamp experiments. RESULTS: Here we show that ivabradine exhibits significant affinity towards cardiac ion channels other than HCN. We demonstrate for the first time inhibition of human voltage-gated Nav1.5 sodium channels at therapeutically relevant concentrations. Within this study we also confirm recent findings of human Kv11.1 inhibition by low µM concentrations of ivabradine and observed no prolongation of ventricular-like APs in cardiomyocytes derived from iPSCs. CONCLUSION: Our results provide an explanation why ivabradine, despite its affinity for Kv11.1 channels, does not prolong the cardiac AP and QTc interval. Furthermore, our results suggest the inhibition of voltage-gated Nav1.5 sodium channels to underlie the recent observations of slowed atrioventricular conduction by increased atrial-His bundle intervals upon administration of ivabradine.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Canais Iônicos/metabolismo , Ivabradina/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Canais Iônicos/antagonistas & inibidores , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp
5.
Acta Biochim Biophys Sin (Shanghai) ; 51(6): 562-570, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31139826

RESUMO

The protein voltage-gated sodium channel Nav1.5 is highly upregulated in various types of cancer and, in general, promotes cancer cell invasiveness and metastatic progression. A previous study found that Nav1.5 was highly expressed in poorly differentiated oral squamous cell carcinoma (OSCC). However, whether Nav1.5 enhances invasiveness and metastasis of OSCC are still unknown. In this study, we found that Nav1.5 was highly expressed in OSCC cell lines compared with normal oral keratinocyte HOK cell line by using western blot analysis. CCK-8 assay results revealed that downregulation of Nav1.5 expression by its specific siRNA reduced proliferation of OSCC HSC-3 cells. Moreover, transwell assay results showed Nav1.5 knockdown significantly inhibited migration and invasion of HSC-3 cells. Meanwhile, qRT-PCR and western blot analysis results showed that epidermal growth factor (EGF) induced Nav1.5 expression in a time- and dose-dependent manner. In addition, EGF promoted proliferation, migration and invasion of HSC-3 cells. Importantly, the Nav1.5 inhibitor tetrodotoxin significantly inhibited the proliferation of HSC-3 cells and impeded the migration and invasion of HSC-3 cells. Furthermore, it was found that siRNA-mediated knockdown of Nav1.5 also lessened the proliferation of HSC-3 cells and blocked the migration and invasion of HSC-3 cells. Taken together, these results indicate that Nav1.5 is involved in the progression of OSCC and Nav1.5 promotes the proliferation, migration and invasion of OSCC cells.


Assuntos
Carcinoma de Células Escamosas/genética , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Bucais/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Invasividade Neoplásica , Interferência de RNA , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
6.
Nat Commun ; 10(1): 2267, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118417

RESUMO

Mutations in LMNA, which encodes the nuclear proteins Lamin A/C, can cause cardiomyopathy and conduction disorders. Here, we employ induced pluripotent stem cells (iPSCs) generated from human cells carrying heterozygous K219T mutation on LMNA to develop a disease model. Cardiomyocytes differentiated from these iPSCs, and which thus carry K219T-LMNA, have altered action potential, reduced peak sodium current and diminished conduction velocity. Moreover, they have significantly downregulated Nav1.5 channel expression and increased binding of Lamin A/C to the promoter of SCN5A, the channel's gene. Coherently, binding of the Polycomb Repressive Complex 2 (PRC2) protein SUZ12 and deposition of the repressive histone mark H3K27me3 are increased at SCN5A. CRISPR/Cas9-mediated correction of the mutation re-establishes sodium current density and SCN5A expression. Thus, K219T-LMNA cooperates with PRC2 in downregulating SCN5A, leading to decreased sodium current density and slower conduction velocity. This mechanism may underlie the conduction abnormalities associated with LMNA-cardiomyopathy.


Assuntos
Cardiomiopatia Dilatada/genética , Sistema de Condução Cardíaco/patologia , Lamina Tipo A/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Adolescente , Adulto , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/cirurgia , Linhagem Celular , Regulação para Baixo , Epigênese Genética , Feminino , Transplante de Coração , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Pessoa de Meia-Idade , Mutação , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Complexo Repressor Polycomb 2/metabolismo
7.
Dokl Biochem Biophys ; 484(1): 9-12, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012002

RESUMO

An effective bacterial system for the production of ß-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of ß-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.


Assuntos
Venenos de Escorpião , Bloqueadores dos Canais de Sódio , Animais , Humanos , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Venenos de Escorpião/biossíntese , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/isolamento & purificação , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
8.
Nat Commun ; 10(1): 1514, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944319

RESUMO

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas
9.
Cardiovasc J Afr ; 30(2): 79-86, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30882133

RESUMO

AIM: We aimed to study the effect of allocryptopine (All) on the late sodium current (INa,Late) of atrial myocytes in spontaneously hypertensive rats (SHR). METHODS: The enzyme digestion method was used to separate single atrial myocytes from SHR and Wistar-Kyoto (WKY) rats. INa,Late was recorded using the patch-clamp technique, and the effect of All was evaluated on the current. RESULTS: Compared with WKY rat cells, an increase in the INa,Late current in SHR myocytes was found. After treatment with 30 µM All, the current densities were markedly decreased; the ratio of INa,Late/INa,peak of SHR was reduced by 30 µM All. All reduced INa,Late by alleviating inactivation of the channel and increasing the window current of the sodium channel. Furthermore, INa,Late densities of three SCN5A mutations declined substantially with 30 µM All in a concentration-dependent manner. CONCLUSIONS: The results clearly show that an increase in INa,Late in SHR atrial myocytes was inhibited by All derived from Chinese herbal medicine.


Assuntos
Antiarrítmicos/farmacologia , Fibrilação Atrial/prevenção & controle , Alcaloides de Berberina/farmacologia , Átrios do Coração/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Sódio/metabolismo , Potenciais de Ação , Animais , Fibrilação Atrial/etiologia , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células HEK293 , Átrios do Coração/metabolismo , Frequência Cardíaca , Humanos , Hipertensão/complicações , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Mutação , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Fatores de Tempo
10.
Cell Mol Biol (Noisy-le-grand) ; 65(2): 58-62, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30860472

RESUMO

To investigate the expressions of Nav1.5 mRNA at different time points in a rat model of temporal lobe epilepsy (TLE), and to assess the potential contribution of Nav1.5 to epileptogenesis. Male Sprague-Dawley rats (72) weighing 230 to 250 g were used for this study. They were randomly assigned to six groups (12 rats/group): control and five TLE groups. The TLE groups were day 1 (acute period), days 7 and 14 (latent period), and days 30 and 60 (chronic period). With the exception of control, epilepsy was induced in the rats with an intraperitoneal (i.p.) injection of aqueous solution of lithium chloride 18 h prior to pilocarpine injection (i.p.) at a dose of 125 mg/kg body weight (b.wt). Rats in the control group were injected i.p. with 0.9 % sodium chloride (125 mg/kg b.wt.) in place of pilocarpine. A total of 84 out of 112 rats developed status epilepticus (SE). The expression of Nav1.5 in the brains of rats was assessed using quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry and Western blot analysis. The expressions of Scn5a mRNA in the hippocampus during the latent and chronic periods were significantly higher than in the control group (p < 0.05), but there were no significant differences in the corresponding expressions between the two different time points in the latent and chronic period groups (p > 0.05). The expression peaked 30 days post-SE, and was sustained for 60 days. There was no significant difference in the expression of Scn5a mRNA in the acute group, when compared to control. Immunohistochemical staining showed that expression levels of Nav1.5 in the CA3 region during latent and chronic periods were significantly higher than those in control group (p < 0.05), and the expressions peaked at day 30. However, there was no significant difference in the expression of Nav1.5 in the latent group, relative to the chronic period group. These results show that Nav1.5 might be involved in the pathogenesis of TLE.


Assuntos
Epilepsia do Lobo Temporal/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Epilepsia do Lobo Temporal/genética , Regulação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo
11.
FASEB J ; 33(4): 5366-5376, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30759345

RESUMO

The voltage-gated cardiac sodium channel, Nav1.5, is the key component that controls cardiac excitative electrical impulse and propagation. However, the dynamic alterations of Nav1.5 during cardiac ischemia and reperfusion (I/R) are seldom reported. We found that the protein levels of rat cardiac Nav1.5 were significantly decreased in response to cardiac I/R injury. By simulating I/R injury in cells through activating AMPK by glucose deprivation, AMPK activator treatment, or hypoxia and reoxygenation (H/R), we found that Nav1.5 was down-regulated by AMPK-mediated autophagic degradation. Furthermore, AMPK was found to phosphorylate Nav1.5 at threonine (T) 101, which then regulates the interaction between Nav1.5 and the autophagic adaptor protein, microtubule-associated protein 1 light chain 3 (LC3), by exposing the LC3-interacting region adjacent to T101 in Nav1.5. This study highlights an instrumental role of AMPK in mediating the autophagic degradation of Nav1.5 during cardiac I/R injury.-Liu, X., Chen, Z., Han, Z., Liu, Y., Wu, X., Peng, Y., Di, W., Lan, R., Sun, B., Xu, B., Xu, W. AMPK-mediated degradation of Nav1.5 through autophagy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Animais Recém-Nascidos , Autofagia/fisiologia , Imunoprecipitação , Masculino , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
12.
Circ Res ; 124(5): 737-746, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30602331

RESUMO

RATIONALE: Voltage-gated Na+ channel ( INa) function is critical for normal cardiac excitability. However, the Na+ channel late component ( INa,L) is directly associated with potentially fatal forms of congenital and acquired human arrhythmia. CaMKII (Ca2+/calmodulin-dependent kinase II) enhances INa,L in response to increased adrenergic tone. However, the pathways that negatively regulate the CaMKII/Nav1.5 axis are unknown and essential for the design of new therapies to regulate the pathogenic INa,L. OBJECTIVE: To define phosphatase pathways that regulate INa,L in vivo. METHODS AND RESULTS: A mouse model lacking a key regulatory subunit (B56α) of the PP (protein phosphatase) 2A holoenzyme displayed aberrant action potentials after adrenergic stimulation. Unbiased computational modeling of B56α KO (knockout) mouse myocyte action potentials revealed an unexpected role of PP2A in INa,L regulation that was confirmed by direct INa,L recordings from B56α KO myocytes. Further, B56α KO myocytes display decreased sensitivity to isoproterenol-induced induction of arrhythmogenic INa,L, and reduced CaMKII-dependent phosphorylation of Nav1.5. At the molecular level, PP2A/B56α complex was found to localize and coimmunoprecipitate with the primary cardiac Nav channel, Nav1.5. CONCLUSIONS: PP2A regulates Nav1.5 activity in mouse cardiomyocytes. This regulation is critical for pathogenic Nav1.5 late current and requires PP2A-B56α. Our study supports B56α as a novel target for the treatment of arrhythmia.


Assuntos
Arritmias Cardíacas/enzimologia , Frequência Cardíaca , Ativação do Canal Iônico , Miócitos Cardíacos/enzimologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteína Fosfatase 2/metabolismo , Potenciais de Ação , Agonistas Adrenérgicos beta/farmacologia , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Fosforilação , Proteína Fosfatase 2/deficiência , Proteína Fosfatase 2/genética , Fatores de Tempo
13.
Int J Cardiol ; 284: 68-73, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30638748

RESUMO

BACKGROUND: Cardiac accessory ß-subunits are part of macromolecular Nav1.5 channel complexes modulating biophysical properties and contributing to arrhythmias. Recent studies demonstrated the structural interaction between ß-subunits of Na+ (Nav1.5) and K+ (Kv4.3) channels. Here, we identified the dipeptidyl peptidase-like protein-10 (DPP10), which is known to modulate Kv4.3-current kinetics, as a new regulator of Nav1.5 channels. METHODS: We assessed DPP10 expression in the healthy and diseased human heart and we studied the functional effects of DPP10 on the Na+ current in isolated rat cardiomyocytes expressing DPP10 after adenoviral gene-transfer (DPP10ad). RESULTS: DPP10 mRNA and proteins were detected in human ventricle, with higher levels in patients with heart failure. In rat cardiomyocytes, DPP10ad significantly reduced upstroke velocity of action potentials indicating reduction in Na+-current density. DPP10 significantly shifted the voltage-dependent Na+ channel activation and inactivation curve to more positive potentials, resulting in greater availability of Na+ channels for activation, along with increasing window Na+ current. In addition, time-to-peak Na+ current was reduced, whereas time course of recovery from inactivation was significantly accelerated by DPP10ad. DPP10 co-immunoprecipitated with Nav1.5 channels in human ventricles, confirming their physical interaction. CONCLUSION: We provide first evidence that DPP10 interacts with Nav1.5 channels, linking Na+- and K+-channel complexes in the heart. Our data suggest that increased ventricular DPP10 expression in heart failure might promote arrhythmias by decreasing peak Na+ current, while increasing window Na+ current and channel re-openings due to accelerated recovery from inactivation.


Assuntos
Arritmias Cardíacas/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Regulação da Expressão Gênica , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , RNA/genética , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Linhagem Celular , Cricetinae , Dipeptidil Peptidases e Tripeptidil Peptidases/biossíntese , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Miocárdio/patologia , Ratos , Ratos Wistar
14.
Am J Physiol Heart Circ Physiol ; 316(2): H371-H379, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30499712

RESUMO

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.


Assuntos
Fibrilação Atrial/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Potenciais de Ação , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética
15.
Immunobiology ; 224(1): 80-93, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391100

RESUMO

Prior work demonstrated that a splice variant of SCN5A, a voltage-gated sodium channel gene, acts as a cytoplasmic sensor for viral dsRNA in human macrophages. Expression of this channel also polarizes macrophages to an anti-inflammatory phenotype in vitro and in vivo. Here we utilized global expression analysis of splice variants to identify novel channel-dependent signaling mechanisms. Pharmacological activation of voltage-gated sodium channels in human macrophages, but not treatment with cytoplasmic poly I:C, was associated with splicing of a retained intron in transcripts of PPP1R10, a regulator of phosphatase activity and DNA repair. Microarray analysis also demonstrated expression of a novel sodium channel splice variant, human macrophage SCN10A, that contains a similar exon deletion as SCN5A. SCN10A localizes to cytoplasmic and nuclear vesicles in human macrophages. Simultaneous expression of human macrophage SCN5A and SCN10A was required to decrease expression of the retained intron and increase protein expression of PPP1R10. Channel activation also increased protein expression of the splicing factor EFTUD2, and knockdown of EFTUD2 prevented channel dependent splicing of the retained PPP1R10 intron. Knockdown of the SCN5A and SCN10A variants in human macrophages reduced the severity of dsDNA breaks induced by treatment with bleomycin and type 1 interferon. These results suggested that human macrophage SCN5A and SCN10A variants mediate an innate immune signaling pathway that limits DNA damage through increased expression of PPP1R10. The functional significance of this pathway is that it may prevent cytotoxicity during inflammatory responses.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Inflamação/metabolismo , Macrófagos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Células Cultivadas , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Inflamação/genética , Análise em Microsséries , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Fatores de Alongamento de Peptídeos/genética , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Transdução de Sinais , Regulação para Cima
16.
J Cardiovasc Electrophysiol ; 30(1): 118-127, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30203441

RESUMO

BACKGROUND: A large number of SCN5A variants have been reported to underlie Brugada syndrome (BrS). However, the evidence supporting individual variants is highly heterogeneous. OBJECTIVE: We systematically re-evaluated all SCN5A variants reported in BrS using the 2015 American college of medical genetics and genomics and the association for molecular pathology (ACMG-AMP) guidelines. METHODS: A PubMed/Embase search was performed to identify all reported SCN5A variants in BrS. Standardized bioinformatic re-analysis (SIFT, PolyPhen, Mutation Taster, Mutation assessor, FATHMM, GERP, PhyloP, and SiPhy) and re-evaluation of frequency in the gnomAD database were performed. Fourteen ACMG-AMP rules were deemed applicable for SCN5A variant analysis. RESULTS: Four hundred and eighty unique SCN5A variants were identified, the majority of which 425 (88%) were coding variants. One hundred and fifty-six of 425 (37%) variants were classified as pathogenic/likely pathogenic. Two hundred and fifty-eight (60%) were classified as variants of uncertain significance, while a further 11 (3%) were classified as benign/likely benign. When considering the subset of variants that were considered "null" variants separately, 95% fulfilled criteria for pathogenicity/likely pathogenicity. In contrast, only 17% of missense variants fulfilled criteria for pathogenicity/likely pathogenicity. Importantly, however, only 25% of missense variants had available functional data, which was a major score driver for pathogenic classification. CONCLUSION: Based on contemporary ACMG-AMP guidelines, only a minority of SCN5A variants implicated in BrS fulfill the criteria for pathogenicity or likely pathogenicity.


Assuntos
Síndrome de Brugada/genética , Variação Genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Potenciais de Ação , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/metabolismo , Síndrome de Brugada/fisiopatologia , Predisposição Genética para Doença , Frequência Cardíaca , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Fenótipo , Fatores de Risco
17.
J Mol Cell Cardiol ; 126: 86-95, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30452906

RESUMO

BACKGROUND: The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. METHODS: Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. RESULTS: First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. CONCLUSIONS: In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.


Assuntos
Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Animais , Animais Recém-Nascidos , Conexina 43/metabolismo , Humanos , Ativação do Canal Iônico , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ratos Wistar
19.
Eur J Pharmacol ; 842: 221-230, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30391349

RESUMO

Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs' actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.


Assuntos
Benzoquinonas/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Pentamidina/farmacologia , Segurança , Células-Tronco/citologia , Animais , Benzoquinonas/efeitos adversos , Células CHO , Canais de Cálcio Tipo L/metabolismo , Cricetulus , Canal de Potássio ERG1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lactamas Macrocíclicas/efeitos adversos , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Pentamidina/efeitos adversos
20.
Circ Res ; 124(4): 539-552, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30566038

RESUMO

RATIONALE: Mutations in the SCN5A gene, encoding the α subunit of the Nav1.5 channel, cause a life-threatening form of cardiac arrhythmia, long QT syndrome type 3 (LQT3). Mexiletine, which is structurally related to the Na+ channel-blocking anesthetic lidocaine, is used to treat LQT3 patients. However, the patient response is variable, depending on the genetic mutation in SCN5A. OBJECTIVE: The goal of this study is to understand the molecular basis of patients' variable responses and build a predictive statistical model that can be used to personalize mexiletine treatment based on patient's genetic variant. METHODS AND RESULTS: We monitored the cardiac Na+ channel voltage-sensing domain (VSD) conformational dynamics simultaneously with other gating properties for the LQT3 variants. To systematically identify the relationship between mexiletine block and channel biophysical properties, we used a system-based statistical modeling approach to connect the multivariate properties to patient phenotype. We found that mexiletine altered the conformation of the Domain III VSD, which is the same VSD that many tested LQT3 mutations affect. Analysis of 15 LQT3 variants showed a strong correlation between the activation of the Domain III-VSD and the strength of the inhibition of the channel by mexiletine. Based on this improved molecular-level understanding, we generated a systems-based model based on a dataset of 32 LQT3 patients, which then successfully predicted the response of 7 out of 8 patients to mexiletine in a blinded, retrospective trial. CONCLUSIONS: Our results imply that the modulated receptor theory of local anesthetic action, which confines local anesthetic binding effects to the channel pore, should be revised to include drug interaction with the Domain III-VSD. Using an algorithm that incorporates this mode of action, we can predict patient-specific responses to mexiletine, improving therapeutic decision making.


Assuntos
Antiarrítmicos/uso terapêutico , Síndrome do QT Longo/genética , Mexiletina/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Variantes Farmacogenômicos , Bloqueadores dos Canais de Sódio/uso terapêutico , Adolescente , Adulto , Animais , Antiarrítmicos/farmacologia , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Síndrome do QT Longo/tratamento farmacológico , Masculino , Mexiletina/farmacologia , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Xenopus
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