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1.
PLoS Pathog ; 19(8): e1011328, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37549173

RESUMO

The Coronavirus envelope (E) protein is a small structural protein with ion channel activity that plays an important role in virus assembly, budding, immunopathogenesis and disease severity. The viroporin E is also located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we evaluated in vitro antiviral activity, mechanism of action and in vivo efficacy of BIT225 for the treatment of SARS-CoV-2 infection. BIT225 showed broad-spectrum direct-acting antiviral activity against SARS-CoV-2 in Calu3 and Vero cells with similar potency across 6 different virus strains. BIT225 inhibited ion channel activity of E protein but did not inhibit endogenous currents or calcium-induced ion channel activity of TMEM16A in Xenopus oocytes. BIT225 administered by oral gavage for 12 days starting 12 hours before infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (100% survival, n = 12), while all vehicle-dosed animals reached a mortality endpoint by Day 9 across two studies (n = 12). When treatment started at 24 hours after infection, body weight loss, and mortality were also prevented (100% survival, n = 5), while 4 of 5 mice maintained and increased body weight and survived when treatment started 48 hours after infection. Treatment efficacy was dependent on BIT225 dose and was associated with significant reductions in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. These results validate viroporin E as a viable antiviral target and support the clinical study of BIT225 for treatment and prophylaxis of SARS-CoV-2 infection.


Assuntos
COVID-19 , Hepatite C Crônica , Chlorocebus aethiops , Camundongos , Animais , Antivirais/farmacologia , Células Vero , SARS-CoV-2 , Camundongos Transgênicos , Proteínas Viroporinas , Fatores de Transcrição , Gravidade do Paciente , Redução de Peso , Canais Iônicos , Modelos Animais de Doenças
2.
J Cardiovasc Electrophysiol ; 34(1): 126-134, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36482155

RESUMO

BACKGROUND: Obstructive sleep apnea (OSA) creates a complex substrate for atrial fibrillation (AF), which is refractory to many clinically available pharmacological interventions. We investigated atrial antiarrhythmogenic properties and ventricular electrophysiological safety of small-conductance Ca2+ -activated K+ (SK)-channel inhibition in a porcine model for obstructive respiratory events. METHODS: In spontaneously breathing pigs, obstructive respiratory events were simulated by intermittent negative upper airway pressure (INAP) applied via a pressure device connected to the intubation tube. INAP was applied for 75 s, every 10 min, three times before and three times during infusion of the SK-channel inhibitor AP14145. Atrial effective refractory periods (AERP) were acquired before (pre-INAP), during (INAP) and after (post-) INAP. AF-inducibility was determined by a S1S2 atrial pacing protocol. Ventricular arrhythmicity was evaluated by heart rate adjusted QT-interval duration (QT-paced) and electromechanical window (EMW) shortening. RESULTS: During vehicle infusion, INAP transiently shortened AERP (pre-INAP: 135 ± 10 ms vs. post-INAP 101 ± 11 ms; p = .008) and increased AF-inducibility. QT-paced prolonged during INAP (pre-INAP 270 ± 7 ms vs. INAP 275 ± 7 ms; p = .04) and EMW shortened progressively throughout INAP and post-INAP (pre-INAP 80 ± 4 ms; INAP 59 ± 6 ms, post-INAP 46 ± 10 ms). AP14145 prolonged baseline AERP, partially prevented INAP-induced AERP-shortening and reduced AF-susceptibility. AP14145 did not alter QT-paced at baseline (pre-AP14145 270 ± 7 ms vs. AP14145 268 ± 6 ms, p = .83) or QT-paced and EMW-shortening during INAP. CONCLUSION: In a pig model for obstructive respiratory events, the SK-channel-inhibitor AP14145 prevented INAP-associated AERP-shortening and AF-susceptibility without impairing ventricular electrophysiology. Whether SK-channels represent a target for OSA-related AF in humans warrants further study.


Assuntos
Fibrilação Atrial , Apneia Obstrutiva do Sono , Humanos , Suínos , Animais , Fibrilação Atrial/prevenção & controle , Acetamidas
3.
Front Genet ; 13: 806429, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35154276

RESUMO

Background: Atrial Fibrillation (AF) is the most prevalent sustained cardiac arrhythmia, responsible for considerable morbidity and mortality. The heterogenic and complex pathogenesis of AF remains poorly understood, which contributes to the current limitation in effective treatments. We aimed to identify rare genetic variants associated with AF in patients with familial AF. Methods and results: We performed whole exome sequencing in a large family with familial AF and identified a rare variant in the gene CACNA1A c.5053G > A which co-segregated with AF. The gene encodes for the protein variants CaV2.1-V1686M, and is important in neuronal function. Functional characterization of the CACNA1A, using patch-clamp recordings on transiently transfected mammalian cells, revealed a modest loss-of-function of CaV2.1-V1686M. Conclusion: We identified a rare loss-of-function variant associated with AF in a gene previously linked with neuronal function. The results allude to a novel link between dysfunction of an ion channel previously associated with neuronal functions and increased risk of developing AF.

4.
Int J Cardiol Heart Vasc ; 37: 100898, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34746364

RESUMO

BACKGROUND: Atrial dilation is an important risk factor for atrial fibrillation (AF) and animal studies have found that acute atrial dilation shortens the atrial effective refractory period (AERP) and increases the risk of AF. Stretch activated ion channels (SACs) and calcium channels play a role in this. The expression profile and calcium dependent activation makes the small conductance calcium activated K+ channel (KCa2.x) a candidate for coupling stretch induced increases in intracellular calcium through K+-efflux and thereby shortening of atrial refractoriness. OBJECTIVES: We hypothesized that KCa2.x channel inhibitors can prevent the stretch induced shortening of AERP and protect the heart from AF. METHODS: The effect of KCa2 channel inhibitor (N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) 1 µM) was investigated using the isolated perfused rabbit heart preparation. To stretch the left atrium (LA) a balloon was inserted and inflated. AERP and action potential duration (APD) were recorded before and after atrial stretch. AF was induced by burst pacing the LA at different degrees of atrial stretch. RESULTS: Stretching of the LA by increasing the balloon pressure from 0 to 20 mmHg shortened the AERP by 8.6 ± 1 ms. In comparison, the KCa2 inhibitor ICA significantly attenuated the stretch induced shortening of AERP to 2.5 ± 1.1 ms. Total AF duration increased linearly with atrial balloon pressure. This relationship was not found in the presence of ICA. ICA lowered the incidence of AF induction and total AF duration. CONCLUSION: The KCa2 channel inhibitor ICA attenuates the acute stretch induced shortening of AERP and decreases stretch induced vulnerability to AF.

5.
ACS Chem Biol ; 16(9): 1654-1662, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34423964

RESUMO

Marine tunicates produce defensive amino-acid-derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes, and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline intracellular calcium concentration [Ca2+]i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 °C. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.


Assuntos
Aminas/metabolismo , Canais de Cálcio/metabolismo , Células Receptoras Sensoriais/metabolismo , Aminas/administração & dosagem , Animais , Cálcio/metabolismo , Gânglios Espinais/metabolismo , Masculino , Camundongos , Técnicas de Patch-Clamp , Transdução de Sinais , Sensação Térmica/fisiologia , Urocordados , Vertebrados
6.
Sci Rep ; 10(1): 1453, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996765

RESUMO

Atrial Fibrillation (AF) is the most common cardiac arrhythmia. Its pathogenesis is complex and poorly understood. Whole exome sequencing of Danish families with AF revealed a novel four nucleotide deletion c.1041_1044del in CLCN2 shared by affected individuals. We aimed to investigate the role of genetic variation of CLCN2 encoding the inwardly rectifying chloride channel ClC-2 as a risk factor for the development of familiar AF. The effect of the CLCN2 variant was evaluated by electrophysiological recordings on transiently transfected cells. We used quantitative PCR to assess CLCN2 mRNA expression levels in human atrial and ventricular tissue samples. The nucleotide deletion CLCN2 c.1041_1044del results in a frame-shift and premature stop codon. The truncated ClC-2 p.V347fs channel does not conduct current. Co-expression with wild-type ClC-2, imitating the heterozygote state of the patients, resulted in a 50% reduction in macroscopic current, suggesting an inability of truncated ClC-2 protein to form channel complexes with wild type channel subunits. Quantitative PCR experiments using human heart tissue from healthy donors demonstrated that CLCN2 is expressed across all four heart chambers. Our genetic and functional data points to a possible link between loss of ClC-2 function and an increased risk of developing AF.


Assuntos
Fibrilação Atrial/genética , Canais de Cloreto/genética , Genótipo , Coração/fisiologia , Mutação com Perda de Função/genética , Adolescente , Adulto , Canais de Cloro CLC-2 , Dinamarca , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Heterozigoto , Humanos , Transporte de Íons/genética , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo Genético , Risco
7.
Artigo em Inglês | MEDLINE | ID: mdl-26213205

RESUMO

We propose a novel parallel computing framework for a nonlinear finite element method (FEM)-based cell model and apply it to simulate avascular tumor growth. We derive computation formulas to simplify the simulation and design the basic algorithms. With the increment of the proliferation generations of tumor cells, the FEM elements may become larger and more distorted. Then, we describe a remesh and refinement processing of the distorted or over large finite elements and the parallel implementation based on Message Passing Interface to improve the accuracy and efficiency of the simulation. We demonstrate the feasibility and effectiveness of the FEM model and the parallelization methods in simulations of early tumor growth.


Assuntos
Simulação por Computador , Análise de Elementos Finitos , Neoplasias/patologia , Dinâmica não Linear , Algoritmos , Divisão Celular , Proliferação de Células , Humanos , Modelos Teóricos
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