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1.
J Pharmacol Toxicol Methods ; 81: 263-73, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27095424

RESUMO

INTRODUCTION: A priority in the development and approval of new drugs is assessment of cardiovascular risk. Current methodologies for screening compounds (e.g. HERG testing) for proarrhythmic risk lead to many false positive and false negative results, resulting in the attrition of potentially therapeutic compounds in early development, and the advancement of other candidates that cause adverse effects. With improvements in the technologies of high content imaging and human stem cell differentiation, it is now possible to directly screen compounds for arrhythmogenic tendencies in human stem cell derived cardiomyocytes (hSC-CMs). METHODS: A training panel of 90 compounds consisting of roughly equal numbers of QT-prolonging and negative control (non-QT-prolonging) compounds, and a follow-up blinded study of 35 compounds including 16 from the 90 compound panel and 2 duplicates, were evaluated for prolongation of the calcium transient in hSC-CMs using kinetic image cytometry (KIC), a specialized form of high content analysis. RESULTS: The KIC-hSC-CM assay identified training compounds that prolong the calcium transient with 98% specificity, 97% precision, 80% sensitivity, and 89% accuracy in predicting clinical QT prolongation by these compounds. The follow-up study of 35 blinded compounds confirmed the reproducibility and strong diagnostic accuracy of the assay. DISCUSSION: The correlation of the KIC-hSC-CM results to clinical observations met or surpassed traditional preclinical assessment of cardiac risk utilizing animal models. Thus, the KIC-hSC-CM assay, which can be accomplished in high throughput and at relatively low cost, is an effective new model system for testing chemicals for cardiovascular risk.


Assuntos
Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Síndrome do QT Longo/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Humanos , Citometria por Imagem , Síndrome do QT Longo/fisiopatologia , Valor Preditivo dos Testes , Controle de Qualidade , Reprodutibilidade dos Testes
2.
Cardiovasc Res ; 111(3): 274-86, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27097650

RESUMO

AIMS: Current mechanisms driving cardiac pacemaker function have focused on ion channel and gap junction channel function, which are essential for action potential generation and propagation between pacemaker cells. However, pacemaker cells also harbour desmosomes that structurally anchor pacemaker cells to each other in tissue, but their role in pacemaker function remains unknown. METHODS AND RESULTS: To determine the role of desmosomes in pacemaker function, we generated a novel mouse model harbouring cardiac conduction-specific ablation (csKO) of the central desmosomal protein, desmoplakin (DSP) using the Hcn4-Cre-ERT2 mouse line. Hcn4-Cre targets cells of the adult mouse sinoatrial node (SAN) and can ablate DSP expression in the adult DSP csKO SAN resulting in specific loss of desmosomal proteins and structures. Dysregulation of DSP via loss-of-function (adult DSP csKO mice) and mutation (clinical case of a patient harbouring a pathogenic DSP variant) in mice and man, respectively, revealed that desmosomal dysregulation is associated with a primary phenotype of increased sinus pauses/dysfunction in the absence of cardiomyopathy. Underlying defects in beat-to-beat regulation were also observed in DSP csKO mice in vivo and intact atria ex vivo. DSP csKO SAN exhibited migrating lead pacemaker sites associated with connexin 45 loss. In vitro studies exploiting ventricular cardiomyocytes that harbour DSP loss and concurrent early connexin loss phenocopied the loss of beat-to-beat regulation observed in DSP csKO mice and atria, extending the importance of DSP-associated mechanisms in driving beat-to-beat regulation of working cardiomyocytes. CONCLUSION: We provide evidence of a mechanism that implicates an essential role for desmosomes in cardiac pacemaker function, which has broad implications in better understanding mechanisms underlying beat-to-beat regulation as well as sinus node disease and dysfunction.


Assuntos
Relógios Biológicos , Desmossomos , Frequência Cardíaca , Síndrome do Nó Sinusal/fisiopatologia , Nó Sinoatrial/fisiopatologia , Potenciais de Ação , Fatores Etários , Animais , Função Atrial , Células Cultivadas , Conexinas/metabolismo , Desmoplaquinas/deficiência , Desmoplaquinas/genética , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Predisposição Genética para Doença , Humanos , Camundongos Knockout , Mutação , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Fenótipo , Síndrome do Nó Sinusal/genética , Síndrome do Nó Sinusal/metabolismo , Síndrome do Nó Sinusal/patologia , Nó Sinoatrial/metabolismo , Nó Sinoatrial/ultraestrutura , Fatores de Tempo
3.
Toxicol Sci ; 148(2): 503-16, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26358003

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful in vitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cardiopatias/induzido quimicamente , Ensaios de Triagem em Larga Escala , Citometria por Imagem , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Testes de Toxicidade/métodos , Potenciais de Ação , Alternativas aos Testes com Animais , Cardiotoxicidade , Linhagem Celular , Relação Dose-Resposta a Droga , Cardiopatias/metabolismo , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Cinética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Reprodutibilidade dos Testes , Medição de Risco
4.
Cell Mol Bioeng ; 8(2): 237-246, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-27087858

RESUMO

Volume loading of the cardiac ventricles is known to slow electrical conduction in the rabbit heart, but the mechanisms remain unclear. Previous experimental and modeling studies have investigated some of these mechanisms, including stretch-activated membrane currents, reduced gap junctional conductance, and altered cell membrane capacitance. In order to quantify the relative contributions of these mechanisms, we combined a monomain model of rabbit ventricular electrophysiology with a hyperelastic model of passive ventricular mechanics. First, a simplified geometric model with prescribed homogeneous deformation was used to fit model parameters and characterize individual MEF mechanisms, and showed good qualitative agreement with experimentally measured strain-CV relations. A 3D model of the rabbit left and right ventricles was then compared with experimental measurements from optical electrical mapping studies in the isolated rabbit heart. The model was inflated to an end-diastolic pressure of 30 mmHg, resulting in epicardial strains comparable to those measured in the anterior left ventricular free wall. While the effects of stretch activated channels did alter epicardial conduction velocity, an increase in cellular capacitance was required to explain previously reported experimental results. The new results suggest that for large strains, various mechanisms can combine and produce a biphasic relationship between strain and conduction velocity. However, at the moderate strains generated by high end-diastolic pressure, a stretch-induced increase in myocyte membrane capacitance is the dominant driver of conduction slowing during ventricular volume loading.

5.
PLoS Genet ; 10(2): e1004114, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24586179

RESUMO

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.


Assuntos
Cardiomiopatias/genética , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Animais , Fenômenos Biomecânicos , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto , Regulação da Expressão Gênica , Humanos , Camundongos , Mutação , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo
6.
Hum Mol Genet ; 23(5): 1134-50, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24108106

RESUMO

Arrhythmogenic right ventricular cardiomyopathy (ARVC) termed a 'disease of the desmosome' is an inherited cardiomyopathy that recently underwent reclassification owing to the identification of left-dominant and biventricular disease forms. Homozygous loss-of-function mutations in the desmosomal component, desmoplakin, are found in patients exhibiting a biventricular form of ARVC; however, no models recapitulate the postnatal hallmarks of the disease as seen in these patients. To gain insights into the homozygous loss-of-function effects of desmoplakin in the heart, we generated cardiomyocyte-specific desmoplakin-deficient mice (DSP-cKO) using ventricular myosin light chain-2-Cre mice. Homozygous DSP-cKO mice are viable but display early ultrastructural defects in desmosomal integrity leading to a cardiomyopathy reminiscent of a biventricular form of ARVC, which includes cell death and fibro-fatty replacement within the ventricle leading to biventricular dysfunction, failure and premature death. DSP-cKO mice also exhibited ventricular arrhythmias that are exacerbated with exercise and catecholamine stimulation. Furthermore, DSP-cKO hearts exhibited right ventricular conduction defects associated with loss of connexin 40 expression and electrical wavefront propagation defects associated with loss of connexin 43 expression. Dose-dependent assessment of the effects of loss of desmoplakin in neonatal ventricular cardiomyocytes revealed primary loss of connexin 43 levels, phosphorylation and function independent of the molecular dissociation of the mechanical junction complex and fibro-fatty manifestation associated with ARVC, suggesting a role for desmoplakin as a primary stabilizer of connexin integrity. In summary, we provide evidence for a novel mouse model, which is reminiscent of the postnatal onset of ARVC while highlighting mechanisms underlying a biventricular form of human ARVC.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Conexinas/deficiência , Animais , Animais Recém-Nascidos , Arritmias Cardíacas/genética , Displasia Arritmogênica Ventricular Direita/diagnóstico , Displasia Arritmogênica Ventricular Direita/mortalidade , Síndrome de Brugada , Doença do Sistema de Condução Cardíaco , Catecolaminas/farmacologia , Conexina 43/deficiência , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Desmoplaquinas/deficiência , Modelos Animais de Doenças , Eletrocardiografia , Expressão Gênica , Coração/efeitos dos fármacos , Sistema de Condução Cardíaco/anormalidades , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Fosforilação , Condicionamento Físico Animal/efeitos adversos , Proteína alfa-5 de Junções Comunicantes
7.
J Biomech Eng ; 136(2): 021007, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24337452

RESUMO

Cardiac mechanical contraction is triggered by electrical activation via an intracellular calcium-dependent process known as excitation-contraction coupling. Dysregulation of cardiac myocyte intracellular calcium handling is a common feature of heart failure. At the organ scale, electrical dyssynchrony leads to mechanical alterations and exacerbates pump dysfunction in heart failure. A reverse coupling between cardiac mechanics and electrophysiology is also well established. It is commonly referred as cardiac mechanoelectric feedback and thought to be an important contributor to the increased risk of arrhythmia during pathological conditions that alter regional cardiac wall mechanics, including heart failure. At the cellular scale, most investigations of myocyte mechanoelectric feedback have focused on the roles of stretch-activated ion channels, though mechanisms that are independent of ionic currents have also been described. Here we review excitation-contraction coupling and mechanoelectric feedback at the cellular and organ scales, and we identify the need for new multicellular tissue-scale model systems and experiments that can help us to obtain a better understanding of how interactions between electrophysiological and mechanical processes at the cell scale affect ventricular electromechanical interactions at the organ scale in the normal and diseased heart.


Assuntos
Acoplamento Excitação-Contração/fisiologia , Retroalimentação Fisiológica/fisiologia , Sistema de Condução Cardíaco/fisiologia , Modelos Cardiovasculares , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Função Ventricular/fisiologia , Animais , Humanos
8.
Mol Biol Cell ; 22(22): 4324-34, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21937718

RESUMO

Changes in blood flow regulate gene expression and protein synthesis in vascular endothelial cells, and this regulation is involved in the development of atherosclerosis. How mechanical stimuli are transmitted from the endothelial luminal surface to the nucleus is incompletely understood. The linker of nucleus and cytoskeleton (LINC) complexes have been proposed as part of a continuous physical link between the plasma membrane and subnuclear structures. LINC proteins nesprin-1, -2, and -4 have been shown to mediate nuclear positioning via microtubule motors and actin. Although nesprin-3 connects intermediate filaments to the nucleus, no functional consequences of nesprin-3 mutations on cellular processes have been described. Here we show that nesprin-3 is robustly expressed in human aortic endothelial cells (HAECs) and localizes to the nuclear envelope. Nesprin-3 regulates HAEC morpho-logy, with nesprin-3 knockdown inducing prominent cellular elongation. Nesprin-3 also organizes perinuclear cytoskeletal organization and is required to attach the centrosome to the nuclear envelope. Finally, nesprin-3 is required for flow-induced polarization of the centrosome and flow-induced migration in HAECs. These results represent the most complete description to date of nesprin-3 function and suggest that nesprin-3 regulates vascular endothelial cell shape, perinuclear cytoskeletal architecture, and important aspects of flow-mediated mechanotransduction.


Assuntos
Polaridade Celular , Citoesqueleto/metabolismo , Células Endoteliais/citologia , Proteínas dos Microfilamentos/metabolismo , Membrana Nuclear/metabolismo , Aorta/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Forma Celular , Células Cultivadas , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Mecanotransdução Celular , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno
9.
Biophys J ; 97(4): 1125-9, 2009 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-19686660

RESUMO

Calcium is essential for many biological processes involved in cellular motility. However, the pathway by which calcium influences motility, in processes such as muscle contraction and neuronal growth, is often indirect and complex. We establish a simple and direct mechanochemical link that shows how calcium quantitatively regulates the dynamics of a primitive motile system, the actin-based acrosomal bundle of horseshoe crab sperm. The extension of this bundle requires the continuous presence of external calcium. Furthermore, the extension rate increases with calcium concentration, but at a given concentration, we find that the volumetric rate of extension is constant. Our experiments and theory suggest that calcium sequentially binds to calmodulin molecules decorating the actin filaments. This binding leads to a collective wave of untwisting of the actin filaments that drives bundle extension.


Assuntos
Actinas/fisiologia , Cálcio/fisiologia , Modelos Biológicos , Modelos Químicos , Proteínas Motores Moleculares/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Actinas/química , Animais , Cálcio/química , Células Cultivadas , Módulo de Elasticidade , Caranguejos Ferradura , Masculino , Proteínas Motores Moleculares/química , Espermatozoides/química , Estresse Mecânico
10.
Antimicrob Agents Chemother ; 51(9): 3410-2, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17606687

RESUMO

We used an assay to measure quinolone sensitivity as a shift in the position of the cleavage-religation equilibrium. This assay was found to be useful in identifying the primary target of a quinolone drug and assessing the effect of quinolone resistance-conferring mutations.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Mutação/genética , Quinolonas/farmacologia , DNA Girase/genética , DNA Topoisomerase IV/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
11.
Antimicrob Agents Chemother ; 48(2): 608-11, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14742217

RESUMO

Replacement of the alpha4 helix of ParC with that of GyrA increased the stability of topoisomerase IV-quinolone-DNA ternary complexes. This mutant topoisomerase IV-mediated cell killing was more efficient than topoisomerase IV-mediated cell killing in Escherichia coli. Thus, the alpha4 helix plays critical roles in determining the stability and the cytotoxicity of ternary complexes.


Assuntos
DNA Girase/genética , DNA Topoisomerase IV/química , DNA Topoisomerase IV/genética , DNA Bacteriano/genética , Escherichia coli/genética , Quinolonas/química , Anti-Infecciosos/farmacologia , Clonagem Molecular , DNA Girase/química , DNA Bacteriano/química , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Mutação/genética , Norfloxacino/farmacologia
12.
J Biol Chem ; 278(48): 48154-61, 2003 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-12972433

RESUMO

Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ETA receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-beta 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-epsilon-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte "surface" mimicking the negative inotropic effects of bath-applied PKC activators and "deep" release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such "detubulated" cardiac myocytes showed no positive inotropic response to 20 nM ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-beta 1-PKC-epsilon signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.


Assuntos
Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Receptores de Endotelina/metabolismo , Animais , Western Blotting , Cães , Relação Dose-Resposta a Droga , Endotelina-1/metabolismo , Fluoresceína/farmacologia , Proteínas de Fluorescência Verde , Ventrículos do Coração/metabolismo , Isoenzimas/metabolismo , Isoproterenol/farmacologia , Cinética , Proteínas Luminescentes/metabolismo , Masculino , Microscopia de Fluorescência , Modelos Biológicos , Modelos Químicos , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Fosfolipase C beta , Fótons , Ligação Proteica , Proteína Quinase C/metabolismo , Proteína Quinase C-épsilon , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
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