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1.
Nat Commun ; 10(1): 4755, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628324

RESUMO

Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that co-localize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations.


Assuntos
Fibrilação Atrial/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linhagem Celular , Cromatina/genética , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Variação Genética , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo
2.
Nat Commun ; 10(1): 4113, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511517

RESUMO

Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.


Assuntos
Endocárdio/crescimento & desenvolvimento , Miocárdio/metabolismo , Transdução de Sinais , Peixe-Zebra/embriologia , Animais , Antígenos CD/metabolismo , Fenômenos Biomecânicos , Caderinas/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Tamanho Celular , Proteínas do Citoesqueleto/metabolismo , Endocárdio/citologia , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Proteína Homeobox Nkx-2.5/metabolismo , Junções Intercelulares/metabolismo , Modelos Biológicos , Mutação/genética , Transativadores/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo
3.
PLoS One ; 14(4): e0213769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30943206

RESUMO

AIMS: Stress granules (SGs) are transient cytoplasmic mRNA and protein complexes that form in eukaryotic cells under stress. SGs are related to multiple diseases, but there are no reports of the existence of SGs in atrial fibrillation (AF). METHODS AND RESULTS: Cell models of AF were established by field stimulation at 600 times per minute. HL-1 cells, cardiomyocytes and cardiac fibroblasts were transfected with G3BP1-cDNA plasmid by Lipofectamine 2000. The presence of SGs was detected by immunofluorescence analysis against GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and poly(A)-binding protein 1 (PABP-1) and electron microscopy. Stable HL-1 cell lines transfected with lentivirus overexpressing G3BP1were constructed to further induce the formation of SGs in AF. Reactive oxygen species (ROS) and calcium overload in tachypaced HL-1 cells were studied by flow cytometry. The effects of G3BP1 overexpression on cardiac fibroblast proliferation and the protein expression level of collagen I/III and fibronectin-1 were also studied. Additionally, we detected protein synthesis in general and in single cells by puromycin incorporation in paced HL-1 cells. Here, we first showed that SGs are present in both tachypaced mouse cardiomyocytes and HL-1 atrial cells, although the presence is partial and at a low level. G3BP1 overexpression promoted SG formation, inhibited the rapid pacing-induced increase in ROS level, and attenuated calcium overload in HL-1 cells (all P<0.05). Furthermore, G3BP1 overexpression inhibited cardiac fibroblast proliferation (P<0.05) and decreased the protein expression level of collagen I and fibronectin-1 in cardiac fibroblasts stimulated by angiotensin II (all P<0.05). The bulk puromycin incorporation analyzed by Western blot did not show a global reduction in protein synthesis. However, puromycin incorporation in single cells detected by immunofluorescence analysis showed that protein synthesis in SG-containing cells significantly reduced (P<0.01). CONCLUSIONS: SGs are rapidly induced and present partially in AF, and G3BP1 overexpression promotes SG formation and confers cytoprotection against oxidative stress, calcium overload and atrial fibrosis in AF.


Assuntos
Fibrilação Atrial/patologia , Grânulos Citoplasmáticos/fisiologia , Fibroblastos/fisiologia , Átrios do Coração/patologia , Miócitos Cardíacos/fisiologia , Animais , Linhagem Celular , Proliferação de Células , DNA Helicases/metabolismo , Modelos Animais de Doenças , Fibroblastos/citologia , Fibrose , Átrios do Coração/citologia , Humanos , Camundongos , Miócitos Cardíacos/citologia , Estresse Oxidativo/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Cultura Primária de Células , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ratos
4.
Nat Commun ; 10(1): 1281, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894542

RESUMO

The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive Gs-protein coupled receptor JellyOp enables optogenetic stimulation of Gs-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological ß-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized Gs-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca2+ currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential Gs-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted Gs stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile Gs activation.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Transdução de Sinal Luminoso , Miócitos Cardíacos/metabolismo , Optogenética/métodos , Animais , Cálcio/metabolismo , Cátions Bivalentes , Diferenciação Celular , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Átrios do Coração/citologia , Átrios do Coração/efeitos da radiação , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos da radiação , Luz , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos da radiação
5.
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
6.
Am J Physiol Cell Physiol ; 316(2): C154-C161, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30427720

RESUMO

Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1-/-) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K+ currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K+ currents in wild-type (WT) atrial cells but not Kcna1-/- cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1-/- atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1-/- atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K+ channels in the heart.


Assuntos
Potenciais de Ação/fisiologia , Átrios do Coração/metabolismo , Canal de Potássio Kv1.1/antagonistas & inibidores , Canal de Potássio Kv1.1/genética , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Feminino , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Canal de Potássio Kv1.1/deficiência , Masculino , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética
7.
Stem Cell Reports ; 11(6): 1378-1390, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30416051

RESUMO

Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs' behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening.


Assuntos
Átrios do Coração/anatomia & histologia , Modelos Cardiovasculares , Engenharia Tecidual/métodos , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Átrios do Coração/citologia , Humanos , Cinética , Contração Miocárdica/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismo , Tretinoína/farmacologia
8.
Bull Exp Biol Med ; 165(5): 610-612, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30225709

RESUMO

We studied the effect of neuropeptide Y in concentrations of 10-8-10-6 M on electrical activity of adult rat right atrial cardiomyocytes with preserved spontaneous activity. Neuropeptide Y was found to modulate the amplitude-time parameters of action potential: in concentrations of 10-7 and 10-6 M it reduced the membrane potential, increased the amplitude of action potential and duration of the repolarization phase, and reduced the frequency of action potential generation. In concentration of 10-6 M, neuropeptide Y produced stronger effect on the analyzed parameters, while in concentration of 10-8 M it produced no significant changes.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Átrios do Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Neuropeptídeo Y/farmacologia , Potenciais de Ação/fisiologia , Animais , Animais não Endogâmicos , Relação Dose-Resposta a Droga , Átrios do Coração/citologia , Preparação de Coração Isolado , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Técnicas de Cultura de Órgãos , Cultura Primária de Células , Ratos
9.
Biophys J ; 115(6): 1130-1141, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30195941

RESUMO

When an atrial cell is paced rapidly, calcium (Ca) waves can form on the cell boundary and propagate to the cell interior. These waves are referred to as "triggered waves" because they are initiated by Ca influx from the L-type Ca channel and occur during the action potential. However, the consequences of triggered waves in atrial tissue are not known. Here, we develop a phenomenological model of Ca cycling in atrial myocytes that accounts for the formation of triggered waves. Using this model, we show that a fundamental requirement for triggered waves to induce abnormal electrical activity in tissue is that these waves must be synchronized over large populations of cells. This is partly because triggered waves induce a long action potential duration (APD) followed by a short APD. Thus, if these events are not synchronized between cells, then they will on average cancel and have minimal effects on the APD in tissue. Using our computational model, we identify two distinct mechanisms for triggered wave synchronization. The first relies on cycle length (CL) variability, which can prolong the CL at a given beat. In cardiac tissue, we show that CL prolongation leads to a substantial amplification of APD because of the synchronization of triggered waves. A second synchronization mechanism applies in a parameter regime in which the cell exhibits stochastic alternans in which a triggered wave fires, on average, only every other beat. In this scenario, we identify a slow synchronization mechanism that relies on the bidirectional feedback between the APD in tissue and triggered wave initiation. On large cables, this synchronization mechanism leads to spatially discordant APD alternans with spatial variations on a scale of hundreds of cells. We argue that these spatial patterns can potentially serve as an arrhythmogenic substrate for the initiation of atrial fibrillation.


Assuntos
Sinalização do Cálcio , Átrios do Coração/citologia , Modelos Cardiovasculares , Função Atrial , Fenômenos Eletrofisiológicos , Retroalimentação Fisiológica
10.
Med Eng Phys ; 61: 69-80, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30201284

RESUMO

Cardiac rotors are believed to be a major driver source of persistent atrial fibrillation (AF), and their spatiotemporal characterization is essential for successful ablation procedures. However, electrograms guided ablation have not been proven to have benefit over empirical ablation thus far, and there is a strong need of improving the localization of cardiac arrhythmogenic targets for ablation. A new approach for characterize rotors is proposed that is based on induced spatial temperature gradients (STGs), and investigated by theoretical study using numerical simulations. We hypothesize that such gradients will cause rotor drifting due to induced spatial heterogeneity in excitability, so that rotors could be driven towards the ablating probe. Numerical simulations were conducted in single cell and 2D atrial models using AF remodeled kinetics. STGs were applied either linearly on the entire tissue or as a small local perturbation, and the major ion channel rate constants were adjusted following Arrhenius equation. In the AF-remodeled single cell, recovery time increased exponentially with decreasing temperatures, despite the marginal effect of temperature on the action potential duration. In 2D models, spiral waves drifted with drifting velocity components affected by both temperature gradient direction and the spiral wave rotation direction. Overall, spiral waves drifted towards the colder tissue region associated with global minimum of excitability. A local perturbation with a temperature of T = 28 °C was found optimal for spiral wave attraction for the studied conditions. This work provides a preliminary proof-of-concept for a potential prospective technique for rotor attraction. We envision that the insights from this study will be utilize in the future in the design of a new methodology for AF characterization and termination during ablation procedures.


Assuntos
Função Atrial , Modelos Cardiovasculares , Temperatura Ambiente , Técnicas de Ablação , Difusão , Átrios do Coração/citologia , Cinética , Análise de Célula Única
11.
J Mol Cell Cardiol ; 123: 128-138, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30213724

RESUMO

BACKGROUND: Rivaroxaban, a widely used factor Xa inhibitor in reducing stroke in atrial fibrillation (AF) patients has multiple biological effects with activation of protease-activated receptor (PAR) signaling. Atrial fibrosis plays a critical role in the pathophysiology of AF. In this study, we evaluated whether rivaroxaban regulates atrial fibroblast activity and its underlying mechanisms. METHODS AND RESULTS: Migration, proliferation analyses, nitric oxide (NO) production assay, calcium fluorescence imaging, and western blots were conducted in human atrial fibroblasts with or without rivaroxaban (100 nmol/L or 300 nmol/L) and co-administration of L-NAME (L-NG-nitro arginine methyl ester, 100 µmol/L), EGTA (Ethylene glycol tetra-acetic acid, 1 mmol/L), thrombin (0.5 U/mL), PAR1 agonist peptide (TFLLR-NH2, 100 µmol/L), PAR1 inhibitor (SCH79797, 0.5 µmol/L) and PAR2 inhibitor (GB83, 10 µmol/L). Atrial fibrosis was examined in isoproterenol (100 mg/kg, subcutaneous injection)-treated rats with or without rivaroxaban (10 mg/kg/day orally for 14 consecutive days). Rivaroxaban reduced the migration, pro-collagen type I production, and proliferation of atrial fibroblasts. Rivaroxaban decreased phosphorylated endothelial NO synthase (eNOS) (Thr 495, an inhibitory phosphorylated site of eNOS), and calcium (Ca2+) entry, and increased NO production. Moreover, L-NAME blocked the effects of rivaroxaban on fibroblast collagen and NO production. In the presence of EGTA, the migratory capability was similarly decreased in atrial fibroblasts with and without treatment with rivaroxaban (100 nmol/L), which suggests that rivaroxaban decreases migratory capability of atrial fibroblasts by inhibiting Ca2+ entry. Additionally, rivaroxaban significantly attenuated the effects of thrombin, and TFLLR-NH2 on migratory, proliferative, and pro-collagen type I production capability in atrial fibroblasts. SCH79797 or GB83 decreased pro-collagen type I production, migration, and proliferation capability in fibroblasts, but combined SCH79797 or GB83 with and without rivaroxaban had similar fibroblast activity. Moreover, rivaroxaban significantly decreased atrial fibrosis in isoproterenol-treated rats. CONCLUSIONS: Rivaroxaban (100-300 nmol/L) regulates atrial fibroblast activity and atrial fibrosis by increasing NO production and decreasing Ca2+ entry through inhibition of PAR signaling.


Assuntos
Cálcio/metabolismo , Inibidores do Fator Xa/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Óxido Nítrico/biossíntese , Rivaroxabana/farmacologia , Biomarcadores , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibrose , Átrios do Coração/patologia , Homeostase/efeitos dos fármacos , Humanos , Modelos Biológicos , Imagem Molecular , Transdução de Sinais/efeitos dos fármacos
12.
Basic Res Cardiol ; 113(5): 41, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30178427

RESUMO

Engineered heart tissue (EHT) from rat cells is a useful tool to study ventricular biology and cardiac drug safety. Since atrial and ventricular cells differ significantly, EHT and other 3D cell culture formats generated from ventricular cells have been of limited value to study atrial biology. To date, reliable in vitro models that reflect atrial physiology are lacking. Therefore, we established a novel EHT model using rat atrial cells (atrial EHT, aEHT) to assess atrial physiology, contractility and drug response. The tissue constructs were characterized with regard to gene expression, histology, electrophysiology, and the response to atrial-specific drugs. We observed typical functional properties of atrial tissue in our model such as more regular spontaneous beating with lower force, shorter action potential duration, and faster contraction and relaxation compared to ventricular EHT (vEHT). The expression of atrial-specific genes and proteins was high, whereas ventricle-specific transcripts were virtually absent. The atrial-selective drug carbachol had a strong negative inotropic and chronotropic effect on aEHT only. Taken together, the results demonstrate the feasibility of aEHT as a novel atrial 3D model and as a benchmark for tissue engineering with human induced pluripotent stem cell-derived atrial-like cardiomyocytes. Atrial EHT faithfully recapitulates atrial physiology and shall be useful to study atrial molecular physiology in health and disease as well as drug response.


Assuntos
Função Atrial , Átrios do Coração/citologia , Contração Miocárdica , Miócitos Cardíacos , Engenharia Tecidual/métodos , Potenciais de Ação , Animais , Animais Recém-Nascidos , Função Atrial/efeitos dos fármacos , Carbacol/farmacologia , Separação Celular/métodos , Células Cultivadas , Estudos de Viabilidade , Regulação da Expressão Gênica , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Frequência Cardíaca , Agonistas Muscarínicos/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos Wistar
13.
Prog Biophys Mol Biol ; 139: 43-58, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30145156

RESUMO

Cardiac cell models are potentially valuable tools for applications such as quantitative safety pharmacology, but have many parameters. Action potentials in real cardiac cells also vary from beat to beat, and from one cell to another. Calibrating cardiac cell models to experimental observations is difficult, because the parameter space is large and high-dimensional. In this study we have demonstrated the use of history matching to calibrate the maximum conductance of ion channels and exchangers in two detailed models of the human atrial action potential against measurements of action potential biomarkers. History matching is an approach developed in other modelling communities, based on constructing fast-running Gaussian process emulators of the model. Emulators were constructed from a small number of model runs (around 102), and then run many times (>106) at low computational cost, each time with a different set of model parameters. Emulator outputs were compared with experimental biomarkers using an implausibility measure, which took into account experimental variance as well as emulator variance. By repeating this process, the region of non-implausible parameter space was iteratively reduced. Both cardiac cell models were successfully calibrated to experimental datasets, resulting in sets of parameters that could be sampled to produce variable action potentials. However, model parameters did not occupy a small range of values. Instead, the history matching process exposed inputs that can co-vary across a wide range and still be consistent with a particular biomarker. We also found correlations between some biomarkers, indicating a need for better descriptors of action potential shape.


Assuntos
Átrios do Coração/citologia , Modelos Cardiovasculares , Potenciais de Ação , Teorema de Bayes , Humanos
14.
Pflugers Arch ; 470(12): 1765-1776, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30155776

RESUMO

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.


Assuntos
Potenciais de Ação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Miócitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animais , Gatos , Células Cultivadas , Feminino , Átrios do Coração/citologia , Magnésio/metabolismo , Masculino , Miócitos Cardíacos/fisiologia , Poliaminas/metabolismo
15.
Methods Mol Biol ; 1816: 39-54, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29987809

RESUMO

High quality cardiomyocyte isolation is of critical importance for successful studies of myocardial function at the cellular and molecular level. Although previous work has established isolation procedures for various species, it still remains challenging to produce consistently a high yield of viable and healthy cardiomyocytes. The basis for the most successful and reproducible isolation of cardiomyocytes from intact hearts is the Langendorff retrograde perfusion technique. Here, we will illustrate in detail all practical aspects of the enzyme-based Langendorff isolation of rat atrial and ventricular cardiomyocytes. This includes a series of obligatory steps starting from quick aortic cannulation to rinse the heart from blood, short perfusion of the heart with Ca2+-free solution to dissociate cells at the level of intercalated discs, followed by longer perfusion with low Ca2+-containing enzyme solution in order to disrupt the extracellular matrix network, extraction of the released cardiomyocytes and gentle Ca2+ reintroduction to allow cells to return gradually to normal cytosolic Ca2+ levels. The average yield of intact viable ventricular myocytes that can be achieved with our protocol is ≈70% (range ≈50-90%). For atrial myocytes, in general, it is slightly (≈10%) lower than for ventricular myocytes. The yield depends on the age of the rat and the degree of cardiac remodeling such that digestion of older and more remodeled hearts (more fibrosis) usually results in lower yields. Isolated atrial and ventricular cardiomyocytes may be employed for studies of cardiomyocyte function (e.g., shortening/contraction, intracellular [Ca2+] transients) as well as for biochemical and molecular biological studies (e.g., immunoblotting, PCR).


Assuntos
Separação Celular/métodos , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Animais , Cateterismo/métodos , Separação Celular/instrumentação , Células Cultivadas , Colagenases/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Perfusão/métodos , Ratos
16.
J Physiol ; 596(17): 3841-3858, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29989169

RESUMO

KEY POINTS: Although optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all-optical platform to monitor and control electrical activity in real-time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed-loop approach was also applied to simulate a re-entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof-of-concept that a real-time all-optical stimulation can control cardiac rhythm in normal and abnormal conditions. ABSTRACT: Optogenetics has provided new insights in cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide-field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free-run mode with submillisecond temporal resolution or in a closed-loop fashion: a tailored hardware and software platform allowed real-time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real-time intraventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. Furthermore, the closed-loop approach was applied to simulate a re-entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof-of-concept that a real-time optically based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart.


Assuntos
Arritmias Cardíacas/terapia , Bloqueio Atrioventricular/terapia , Terapia por Estimulação Elétrica/métodos , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Optogenética/instrumentação , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Átrios do Coração/fisiopatologia , Átrios do Coração/efeitos da radiação , Ventrículos do Coração/fisiopatologia , Ventrículos do Coração/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Imagem Óptica
17.
PLoS One ; 13(7): e0199896, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29979710

RESUMO

Scaffolds populated with human cardiac progenitor cells (CPCs) represent a therapeutic opportunity for heart regeneration after myocardial infarction. In this work, square-grid scaffolds are prepared by melt-extrusion additive manufacturing from a polyurethane (PU), further subjected to plasma treatment for acrylic acid surface grafting/polymerization and finally grafted with laminin-1 (PU-LN1) or gelatin (PU-G) by carbodiimide chemistry. LN1 is a cardiac niche extracellular matrix component and plays a key role in heart formation during embryogenesis, while G is a low-cost cell-adhesion protein, here used as a control functionalizing molecule. X-ray photoelectron spectroscopy analysis shows nitrogen percentage increase after functionalization. O1s and C1s core-level spectra and static contact angle measurements show changes associated with successful functionalization. ELISA assay confirms LN1 surface grafting. PU-G and PU-LN1 scaffolds both improve CPC adhesion, but LN1 functionalization is superior in promoting proliferation, protection from apoptosis and expression of differentiation markers for cardiomyocytes, endothelial and smooth muscle cells. PU-LN1 and PU scaffolds are biodegraded into non-cytotoxic residues. Scaffolds subcutaneously implanted in mice evoke weak inflammation and integrate with the host tissue, evidencing a significant blood vessel density around the scaffolds. PU-LN1 scaffolds show their superiority in driving CPC behavior, evidencing their promising role in myocardial regenerative medicine.


Assuntos
Regeneração Tecidual Guiada/métodos , Átrios do Coração/citologia , Poliuretanos/química , Transplante de Células-Tronco , Células-Tronco/citologia , Engenharia Tecidual , Tecidos Suporte , Animais , Biomimética , Células Cultivadas , Feminino , Humanos , Masculino , Teste de Materiais , Camundongos , Pessoa de Meia-Idade , Miocárdio , Células-Tronco/fisiologia
18.
Br J Pharmacol ; 175(16): 3422-3432, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29900525

RESUMO

BACKGROUND AND PURPOSE: Adrenergic regulation of cell volume-regulated chloride current (ICl.vol ) is species-dependent. The present study investigates the mechanism underlying adrenergic regulation of ICl.vol in human atrial myocytes. EXPERIMENTAL APPROACH: Conventional whole-cell patch voltage-clamp techniques were used to record membrane current in human atrial myocytes. ICl.vol was evoked by hyposmotic bath solution (0.6 times isosmotic, 0.6 T). KEY RESULTS: ICl.vol was augmented by noradrenaline (1 µM) during cell swelling in 0.6 T but not under isosmotic (1 T) conditions. Up-regulation of ICl.vol in 0.6 T was blocked by the ß-adrenoceptor antagonist propranolol (2 µM), but not by the α1 -adrenoceptor antagonist prazosin (2 µM). This ß-adrenergic response involved cAMP but was independent of PKA; the protein kinase inhibitor H-89 (2 µM) or PKI (10 µM in pipette solution) failed to prevent ICl.vol up-regulation by noradrenaline. Moreover, the PI3K/PKB inhibitor LY294002 (50 µM) and the PKG inhibitor KT5823 (10 µM) did not affect noradrenaline-induced increases in ICl.vol . Interestingly, the exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2'-O-Me-cAMP (50 µM) also up-regulated ICl.vol , and the noradrenaline-induced increase of ICl.vol in 0.6 T was reversed or prevented by the Epac inhibitor ESI-09 (10 µM). CONCLUSION AND IMPLICATIONS: These data show that ICl.vol evoked by cell swelling of human atrial myocytes is up-regulated by noradrenaline via a PKA-independent cAMP/Epac pathway in human atrial myocytes. cAMP/Epac-induced ICl.vol is expected to shorten action potential duration during human atrial myocytes swelling and may be involved in abnormal cardiac electrical activity during cardiac pathologies that evoke ß-adrenoceptor signalling.


Assuntos
AMP Cíclico/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Norepinefrina/farmacologia , Células Cultivadas , Cloretos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico , Átrios do Coração/citologia , Humanos , Miócitos Cardíacos/fisiologia , Regulação para Cima/efeitos dos fármacos
19.
Cardiovasc Res ; 114(14): 1848-1859, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29917042

RESUMO

Aims: The generation of homogeneous cardiomyocyte populations from fresh tissue or stem cells is laborious and costly. A potential solution to this problem would be to establish lines of immortalized cardiomyocytes. However, as proliferation and (terminal) differentiation of cardiomyocytes are mutually exclusive processes, their permanent immortalization causes loss of electrical and mechanical functions. We therefore aimed at developing conditionally immortalized atrial myocyte (iAM) lines allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition. Methods and results: Freshly isolated neonatal rat atrial cardiomyocytes (AMs) were transduced with a lentiviral vector conferring doxycycline (dox)-controlled expression of simian virus 40 large T antigen. Under proliferative conditions (i.e. in the presence of dox), the resulting cells lost most cardiomyocyte traits and doubled every 38 h. Under differentiation conditions (i.e. in the absence of dox), the cells stopped dividing and spontaneously reacquired a phenotype very similar to that of primary AMs (pAMs) in gene expression profile, sarcomeric organization, contractile behaviour, electrical properties, and response to ion channel-modulating compounds (as assessed by patch-clamp and optical voltage mapping). Moreover, differentiated iAMs had much narrower action potentials and propagated them at >10-fold higher speeds than the widely used murine atrial HL-1 cells. High-frequency electrical stimulation of confluent monolayers of differentiated iAMs resulted in re-entrant conduction resembling atrial fibrillation, which could be prevented by tertiapin treatment, just like in monolayers of pAMs. Conclusion: Through controlled expansion and differentiation of AMs, large numbers of functional cardiomyocytes were generated with properties superior to the differentiated progeny of existing cardiomyocyte lines. iAMs provide an attractive new model system for studying cardiomyocyte proliferation, differentiation, metabolism, and (electro)physiology as well as to investigate cardiac diseases and drug responses, without using animals.


Assuntos
Diferenciação Celular , Proliferação de Células , Átrios do Coração/metabolismo , Desenvolvimento Muscular , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Linhagem Celular Transformada , Regulação da Expressão Gênica , Átrios do Coração/citologia , Frequência Cardíaca , Potenciais da Membrana , Fenótipo , Ratos , Transdução de Sinais , Fatores de Tempo
20.
Micron ; 111: 19-27, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29859424

RESUMO

Detection of patterns of subcellular calcium distribution in the cardiovascular system can contribute to understanding its role in cardiac and blood function. The present study localized calcium in heart atrium, ventricle, and bulbus arteriosus as well as in erythrocytes of zebrafish Danio rerio using an oxalate-pyroantimonate technique combined with transmission electron microscopy. Intracellular calcium stores were detected in caveolae, mitochondria, and the nuclei of several zebrafish cardiac cell types. Melanin pigmentation containing calcium stores was detected in the pericardial cavity. Melanin might be an extracellular source of calcium for heart beating and/or a lubricant to prevent friction during beating process. Calcium deposits were also detected in the plasma membrane, cytoplasm and nucleus of erythrocytes as well as in blood plasma. Possible exchange of calcium between erythrocytes and blood plasma was observed. Interactions of such calcium stores and possible contribution of extracellular calcium stores such as melanin pigmentation to supply calcium for vital functions of heart cells should be addressed in future studies.


Assuntos
Cálcio/análise , Eritrócitos/metabolismo , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/fisiologia , Animais , Cavéolas/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Melaninas/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo
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