Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.366
Filtrar
1.
Toxicology ; 447: 152629, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33189796

RESUMO

The authors propose to consider as hormesis phenomenon not only a realization of the Arndt-Schulze rule but any non-monotonic dose-response relationship for a certain outcome that is characterized by changing direction of a response between adjacent ranges of doses of an initiator of this response, the number of such ranges being two or more. This approach is illustrated with results of several in vitro experiments on different established cell lines exposed to CdS or PbS nanoparticles.


Assuntos
Compostos de Cádmio/toxicidade , Hormese/fisiologia , Chumbo/toxicidade , Modelos Teóricos , Miócitos Cardíacos/fisiologia , Nanopartículas/toxicidade , Sulfetos/toxicidade , Animais , Compostos de Cádmio/administração & dosagem , Relação Dose-Resposta a Droga , Hormese/efeitos dos fármacos , Humanos , Chumbo/administração & dosagem , Miócitos Cardíacos/efeitos dos fármacos , Nanopartículas/administração & dosagem , Sulfetos/administração & dosagem
2.
J Nippon Med Sch ; 87(5): 268-276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33311008

RESUMO

BACKGROUND: Some cultured neonatal rat cardiomyocytes continue spontaneous beating even in serum-free medium. The present study explored the cause and genes responsible for this phenomenon. METHODS: Ingenuity Pathway Analysis (IPA) software was used to analyze fold changes in gene expression in beating neonatal rat cardiomyocytes, as compared with non-beating cardiomyocytes, which were obtained from DNA microarray data of total RNA extracts of cardiomyocytes. To confirm the involvement of the 8 genes selected by IPA prediction, cellular protein abundances were determined by Western blot. The gene expression of connective tissue growth factor (CTGF) was substantially higher in beating cardiomyocytes than in non-beating cardiomyocytes; thus, CTGF protein content released from cardiomyocytes into the culture medium was examined. RESULTS: IPA showed that the "Apelin Cardiac Fibroblast Signaling Pathway" was significantly inhibited and that microtubule dynamics and cytoskeleton organization were significantly activated. Each fluctuation in the cellular abundances of the 8 proteins in beating cardiomyocytes, as compared with non-beating cardiomyocytes, was primarily in the same direction as that of gene expression. However, the cellular CTGF protein abundance as well as CTGF content released into the medium did not substantially differ between beating and non-beating cardiomyocytes. CONCLUSIONS: The present results suggest that the large increase in CTGF gene expression in beating cardiomyocytes is not a cause but a result of beating, which may provide a putative pathway for controlling beating. Beating is sustained by developed cardiomyofibrils and directly upregulates CTGF gene expression, which is not followed by CTGF protein synthesis.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação da Expressão Gênica , Expressão Gênica , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Regulação para Cima , Animais , Animais Recém-Nascidos , Células Cultivadas , Ratos
4.
PLoS One ; 15(11): e0241996, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33180823

RESUMO

PURPOSE: Cardiomyocyte organization and performance underlie cardiac function, but the in vivo mobility of these cells during contraction and filling remains difficult to probe. Herein, a novel trigger delay (TD) scout sequence was used to acquire high in-plane resolution (1.6 mm) Spin-Echo (SE) cardiac diffusion tensor imaging (cDTI) at three distinct cardiac phases. The objective was to characterize cardiomyocyte organization and mobility throughout the cardiac cycle in healthy volunteers. MATERIALS AND METHODS: Nine healthy volunteers were imaged with cDTI at three distinct cardiac phases (early systole, late systole, and diastasis). The sequence used a free-breathing Spin-Echo (SE) cDTI protocol (b-values = 350s/mm2, twelve diffusion encoding directions, eight repetitions) to acquire high-resolution images (1.6x1.6x8mm3) at 3T in ~7 minutes/cardiac phase. Helix Angle (HA), Helix Angle Range (HAR), E2 angle (E2A), Transverse Angle (TA), Mean Diffusivity (MD), diffusion tensor eigenvalues (λ1-2-3), and Fractional Anisotropy (FA) in the left ventricle (LV) were characterized. RESULTS: Images from the patient-specific TD scout sequence demonstrated that SE cDTI acquisition was possible at early systole, late systole, and diastasis in 78%, 100% and 67% of the cases, respectively. At the mid-ventricular level, mobility (reported as median [IQR]) was observed in HAR between early systole and late systole (76.9 [72.6, 80.5]° vs 96.6 [85.9, 100.3]°, p<0.001). E2A also changed significantly between early systole, late systole, and diastasis (27.7 [20.8, 35.1]° vs 45.2 [42.1, 49]° vs 20.7 [16.6, 26.4]°, p<0.001). CONCLUSION: We demonstrate that it is possible to probe cardiomyocyte mobility using multi-phase and high resolution cDTI. In healthy volunteers, aggregate cardiomyocytes re-orient themselves more longitudinally during contraction, while cardiomyocyte sheetlets tilt radially during wall thickening. These observations provide new insights into the three-dimensional mobility of myocardial microstructure during systolic contraction.


Assuntos
Imagem de Difusão por Ressonância Magnética/métodos , Miócitos Cardíacos/fisiologia , Adulto , Movimento Celular , Imagem de Tensor de Difusão , Feminino , Voluntários Saudáveis , Humanos , Imagem Cinética por Ressonância Magnética , Masculino , Miócitos Cardíacos/citologia , Função Ventricular Esquerda
5.
PLoS Comput Biol ; 16(10): e1008294, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33027247

RESUMO

We propose four novel mathematical models, describing the microscopic mechanisms of force generation in the cardiac muscle tissue, which are suitable for multiscale numerical simulations of cardiac electromechanics. Such models are based on a biophysically accurate representation of the regulatory and contractile proteins in the sarcomeres. Our models, unlike most of the sarcomere dynamics models that are available in the literature and that feature a comparable richness of detail, do not require the time-consuming Monte Carlo method for their numerical approximation. Conversely, the models that we propose only require the solution of a system of PDEs and/or ODEs (the most reduced of the four only involving 20 ODEs), thus entailing a significant computational efficiency. By focusing on the two models that feature the best trade-off between detail of description and identifiability of parameters, we propose a pipeline to calibrate such parameters starting from experimental measurements available in literature. Thanks to this pipeline, we calibrate these models for room-temperature rat and for body-temperature human cells. We show, by means of numerical simulations, that the proposed models correctly predict the main features of force generation, including the steady-state force-calcium and force-length relationships, the length-dependent prolongation of twitches and increase of peak force, the force-velocity relationship. Moreover, they correctly reproduce the Frank-Starling effect, when employed in multiscale 3D numerical simulation of cardiac electromechanics.


Assuntos
Coração/fisiologia , Modelos Cardiovasculares , Miocárdio , Miócitos Cardíacos , Adulto , Animais , Fenômenos Biofísicos/fisiologia , Biologia Computacional , Humanos , Masculino , Miocárdio/química , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Sarcômeros/química , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Adulto Jovem
6.
Sci Rep ; 10(1): 15027, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929098

RESUMO

Ventricular arrhythmias are a major source of early mortality in acute myocardial infarction (MI) and remain a major therapeutic challenge. Thus we investigated effects of ivabradine, a presumably specific bradycardic agent versus metoprolol, a ß-blocker, at doses offering the same heart rate (HR) reduction, on ventricular arrhythmias in the acute non-reperfused MI in the rat. Immediately after MI induction a single dose of ivabradine/ metoprolol was given. ECG was continuously recorded and ventricular arrhythmias were analyzed. After 6 h epicardial monophasic action potentials (MAPs) were recorded and cardiomyocyte Ca2+ handling was assessed. Both ivabradine and metoprolol reduced HR by 17% and arrhythmic mortality (14% and 19%, respectively, versus 33% in MI, p < 0.05) and ventricular arrhythmias in post-MI rats. Both drugs reduced QTc prolongation and decreased sensitivity of ryanodine receptors in isolated cardiomyocytes, but otherwise had no effect on Ca2+ handling, velocity of conduction or repolarization. We did not find any effects of potential IKr inhibition by ivabradine in this setting. Thus Ivabradine is an equally effective antiarrhythmic agent as metoprolol in early MI in the rat. It could be potentially tested as an alternative antiarrhythmic agent in acute MI when ß-blockers are contraindicated.


Assuntos
Antiarrítmicos/uso terapêutico , Ivabradina/uso terapêutico , Metoprolol/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Taquicardia Ventricular/tratamento farmacológico , Fibrilação Ventricular/tratamento farmacológico , Potenciais de Ação , Animais , Sinalização do Cálcio , Células Cultivadas , Frequência Cardíaca , Masculino , Infarto do Miocárdio/complicações , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/prevenção & controle , Fibrilação Ventricular/etiologia , Fibrilação Ventricular/prevenção & controle
7.
Am J Physiol Heart Circ Physiol ; 319(5): H1021-H1035, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32946258

RESUMO

Transient receptor potential proteins (TRPs) act as nonselective cation channels. Of the TRP channels, PC2 (also known as polycystin 2) is localized to the sarcoplasmic reticulum (SR); however, its contribution to calcium-induced calcium release and overall cardiac function in the heart is poorly understood. The goal of this study was to characterize the effect of cardiac-specific PC2 deletion in adult cardiomyocytes and in response to chronic ß-adrenergic challenge. We used a temporally inducible model to specifically delete PC2 from cardiomyocytes (Pkd2 KO) and characterized calcium and contractile dynamics in single cells. We found enhanced intracellular calcium release after Pkd2 KO, and near super-resolution microscopy analysis suggested this was due to close localization of PC2 to the ryanodine receptor. At the organ level, speckle-tracking echocardiographical analysis showed increased dyssynchrony in the Pkd2 KO mice. In response to chronic adrenergic stimulus, cardiomyocytes from the Pkd2 KO had no reserve ß-adrenergic calcium responses and significantly attenuated wall motion in the whole heart. Biochemically, without adrenergic stimulus, there was an overall increase in PKA phosphorylated targets in the Pkd2 KO mouse, which decreased following chronic adrenergic stimulus. Taken together, our results suggest that cardiac-specific PC2 limits SR calcium release by affecting the PKA phosphorylation status of the ryanodine receptor, and the effects of PC2 loss are exacerbated upon adrenergic challenge.NEW & NOTEWORTHY Our goal was to characterize the role of the transient receptor potential channel polycystin 2 (PC2) in cardiomyocytes following adult-onset deletion. Loss of PC2 resulted in decreased cardiac shortening and cardiac dyssynchrony and diminished adrenergic reserve. These results suggest that cardiac-specific PC2 modulates intracellular calcium signaling and contributes to the maintenance of adrenergic pathways.


Assuntos
Adrenérgicos/farmacologia , Sinalização do Cálcio , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPP/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Retículo Sarcoplasmático/metabolismo , Canais de Cátion TRPP/genética
8.
PLoS Comput Biol ; 16(9): e1008203, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976482

RESUMO

Novel studies conducting cardiac safety assessment using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising but might be limited by their specificity and predictivity. It is often challenging to correctly classify ion channel blockers or to sufficiently predict the risk for Torsade de Pointes (TdP). In this study, we developed a method combining in vitro and in silico experiments to improve machine learning approaches in delivering fast and reliable prediction of drug-induced ion-channel blockade and proarrhythmic behaviour. The algorithm is based on the construction of a dictionary and a greedy optimization, leading to the definition of optimal classifiers. Finally, we present a numerical tool that can accurately predict compound-induced pro-arrhythmic risk and involvement of sodium, calcium and potassium channels, based on hiPSC-CM field potential data.


Assuntos
Algoritmos , Arritmias Cardíacas , Canais Iônicos , Modelos Cardiovasculares , Miócitos Cardíacos , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologia , Fármacos Cardiovasculares/farmacologia , Biologia Computacional , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Torsades de Pointes/fisiopatologia
9.
Am J Physiol Heart Circ Physiol ; 319(5): H1112-H1122, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32986966

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current (Ik1). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking Ik1-like currents with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1's role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.


Assuntos
Células Gigantes/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Potenciais de Ação , Animais , Células Cultivadas , Reprogramação Celular , Técnicas de Reprogramação Celular/métodos , Técnicas de Reprogramação Celular/normas , Feminino , Células Gigantes/citologia , Células Gigantes/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Cultura Primária de Células/métodos , Cultura Primária de Células/normas , Ratos
10.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 36(3): 228-231, 2020 May.
Artigo em Chinês | MEDLINE | ID: mdl-32981277

RESUMO

Objective: To observe the effects of hypothermia on the repolarization duration and the expression of Kir2.1 protein of ventricular myocytes in isolated rat heart and explore the role of Kir2.1 protein.Methods: Eighteen healthy adult male Sprague-Dawley rats were randomly divided into three groups (n=6 per group): Control group (C group), 35℃ group (H1 group), 32℃ group (H2 group). Langendorff isolated heart models were established. After 15 min 37℃ K-H fluid banlanced perfusion, C group continued to perfuse the K-H solution at 37℃ for 30 minutes, H1 group continued to perfuse the K-H solution at 35℃ for 30 minutes, H2 group continued to perfuse the K-H solution at 32℃ for 30 minutes. At 15 min of balanced perfusion (T1), and 30 min of continuous perfusion (T2), the heart rate,and the MAP in the three layers of the left ventricular anterior wall were recorded, the action potential duration at 50% repolarization (MAPD50), the action potential duration at 90% repolarization (MAPD90) and transmural dispersion of repolarization(TDR) were calculated. At the same time, the occurrence of arrhythmia was recorded. The expression of Kir2.1 protein was measured by Western blot. The average optical density (AOD) and the distribution of Kir2.1 protein were measured by immunohistochemistry in the ventricular tissue measured by electrophysiology. Results: Compared with T0, the heart rate was decreased, MAPD50 and MAPD90 were prolonged significantly (P<0.05), and TDR was increased significantly (P<0.05) in H1 group, H2 group at T1. Compared with C group, the HR was decreased, the MAPD90 was prolonged significantly (P<0.05), TDR was increased significantly (P<0.05),the expression and the AOD of Kir2.1 protein were decreased significantly (P<0.05) in H1group, H2group at T1. Compared with H1 group, the heart rate of H2 group was decreased significantly (P<0.05), MAPD50 and MAPD90 were prolonged significantly (P<0.05), and TDR was increased significantly (P<0.05) at T1. The distribution of Kir2.1 protein in group C was normal, while the distribution of Kir2.1 in H1 group and H2 group was disordered. Conclusion: Hypothermia prolonged the ventricular duration of repolarization and increased the dispersion of repolarization. The mechanism is related to the down-regulation the expression of Kir2.1 protein and the disorder of the distribution of Kir2.1 protein.


Assuntos
Hipotermia , Miócitos Cardíacos , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas , Temperatura Baixa , Ventrículos do Coração/citologia , Ventrículos do Coração/fisiopatologia , Masculino , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley
11.
Am J Physiol Heart Circ Physiol ; 319(5): H927-H937, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32822546

RESUMO

Sympathetic neurons (SNs) capable of modulating the heart rate of murine cardiomyocytes (CMs) can be differentiated from human stem cells. The electrophysiological properties of human stem cell-derived SNs remain largely uncharacterized, and human neurocardiac cocultures remain to be established. Here, we have adapted previously published differentiation and coculture protocols to develop feeder-free SNs using human-induced pluripotent stem cells (hiPSCs). hiPSC-SNs were characterized in monoculture and coculture with hiPSC-CMs, using antibody labeling, enzyme-linked immunosorbent assay, and whole cell patch-clamp electrophysiology techniques. hiPSC-SNs stained positive for peripherin, tyrosine hydroxylase, and nicotinic acetylcholine receptors, the latter two colocalizing in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average = -61 ± 0.7 mV), secreted norepinephrine upon activation, and generated synaptic and action currents and inward and outward voltage-dependent currents. All hiPSC-SNs fired action potentials in response to current injection, local application of potassium, or spontaneously, followed by short-medium afterhyperpolarizations. hiPSC-SNs could successfully be maintained in coculture with hiPSC-CMs, and this induced further development of hiPSC-SN action potential kinetics. To test functional coupling between the neurons and cardiomyocytes, the hiPSC-CM beating response to nicotine-induced norepinephrine release was assessed. In neurocardiac cocultures, nicotine exposure significantly increased the hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monocultures, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterization of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multicellular model to study neurocardiac modulation under physiological and pathological conditions.NEW & NOTEWORTHY We present data on a functional coculture between human-induced pluripotent stem cell-derived sympathetic neurons and cardiomyocytes. Moreover, this study adds significantly to the available data on the electrophysiological function of human-induced pluripotent stem cell-derived sympathetic neurons.


Assuntos
Técnicas de Reprogramação Celular/métodos , Técnicas de Cocultura/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Neurônios/citologia , Potenciais de Ação , Adulto , Células Cultivadas , Reprogramação Celular , Humanos , Masculino , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Norepinefrina/metabolismo , Periferinas/genética , Periferinas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
12.
PLoS One ; 15(8): e0236949, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750067

RESUMO

Heart arrhythmia is a pathological condition where the sequence of electrical impulses in the heart deviates from the normal rhythm. It is often associated with specific channelopathies in cardiac tissue, yet how precisely the changes in ionic channels affect the electrical activity of cardiac cells is still an open question. Even though sodium channel mutations that underlie cardiac syndromes like the Long-Q-T and the Brugada-syndrome are known to affect a number of channel parameters simultaneously, previous studies have predominantly focused on the persistent late component of the sodium current as the causal explanation for an increased risk of heart arrhythmias in these cardiac syndromes. A systematic analysis of the impact of other important sodium channel parameters is currently lacking. Here, we investigate the reduced ten-Tusscher-model for single human epicardium ventricle cells and use mathematical bifurcation analysis to predict the dependence of the cardiac action potential on sodium channel activation and inactivation time-constants and voltage dependence. We show that, specifically, shifts of the voltage dependence of activation and inactivation curve can lead to drastic changes in the action potential dynamics, inducing oscillations of the membrane potential as well as bistability. Our results not only demonstrate a new way to induce multiple co-existing states of excitability (biexcitability) but also emphasize the critical role of the voltage dependence of sodium channel activation and inactivation curves for the induction of heart-arrhythmias.


Assuntos
Potenciais de Ação , Arritmias Cardíacas/fisiopatologia , Modelos Biológicos , Miócitos Cardíacos/fisiologia , Canais de Sódio Disparados por Voltagem/fisiologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Síndrome de Brugada , Ventrículos do Coração/metabolismo , Humanos , Síndrome do QT Longo , Mutação , Miócitos Cardíacos/metabolismo , Função Ventricular , Canais de Sódio Disparados por Voltagem/genética
13.
PLoS One ; 15(8): e0231806, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817622

RESUMO

The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Diabetes Mellitus/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Insulina/metabolismo , Insulina/farmacologia , Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos
14.
Nat Commun ; 11(1): 3881, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753572

RESUMO

Cells typically respond to chemical or physical perturbations via complex signaling cascades which can simultaneously affect multiple physiological parameters, such as membrane voltage, calcium, pH, and redox potential. Protein-based fluorescent sensors can report many of these parameters, but spectral overlap prevents more than ~4 modalities from being recorded in parallel. Here we introduce the technique, MOSAIC, Multiplexed Optical Sensors in Arrayed Islands of Cells, where patterning of fluorescent sensor-encoding lentiviral vectors with a microarray printer enables parallel recording of multiple modalities. We demonstrate simultaneous recordings from 20 sensors in parallel in human embryonic kidney (HEK293) cells and in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and we describe responses to metabolic and pharmacological perturbations. Together, these results show that MOSAIC can provide rich multi-modal data on complex physiological responses in multiple cell types.


Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Imagem Óptica/métodos , Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/farmacologia , Técnicas Biossensoriais/instrumentação , Cálcio/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Imagem Óptica/instrumentação , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Propanolaminas/farmacologia
15.
PLoS Comput Biol ; 16(8): e1008074, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804929

RESUMO

Congestive heart failure is characterized by suppressed cardiac output and arterial filling pressure, leading to renal retention of salt and water, contributing to further volume overload. Mathematical modeling provides a means to investigate the integrated function and dysfunction of heart and kidney in heart failure. This study updates our previously reported integrated model of cardiac and renal functions to account for the fluid exchange between the blood and interstitium across the capillary membrane, allowing the simulation of edema. A state of heart failure with reduced ejection fraction (HF-rEF) was then produced by altering cardiac parameters reflecting cardiac injury and cardiovascular disease, including heart contractility, myocyte hypertrophy, arterial stiffness, and systemic resistance. After matching baseline characteristics of the SOLVD clinical study, parameters governing rates of cardiac remodeling were calibrated to describe the progression of cardiac hemodynamic variables observed over one year in the placebo arm of the SOLVD clinical study. The model was then validated by reproducing improvements in cardiac function in the enalapril arm of SOLVD. The model was then applied to prospectively predict the response to the sodium-glucose co-transporter 2 (SGLT2) inhibitor dapagliflozin, which has been shown to reduce heart failure events in HF-rEF patients in the recent DAPAHF clinical trial by incompletely understood mechanisms. The simulations predict that dapagliflozin slows cardiac remodeling by reducing preload on the heart, and relieves congestion by clearing interstitial fluid without excessively reducing blood volume. This provides a quantitative mechanistic explanation for the observed benefits of SGLT2i in HF-rEF. The model also provides a tool for further investigation of heart failure drug therapies.


Assuntos
Insuficiência Cardíaca/fisiopatologia , Rim/fisiopatologia , Modelos Cardiovasculares , Volume Sistólico/fisiologia , Compostos Benzidrílicos/uso terapêutico , Cardiomegalia/fisiopatologia , Líquido Extracelular/fisiologia , Glucosídeos/uso terapêutico , Coração/fisiopatologia , Insuficiência Cardíaca/tratamento farmacológico , Hemodinâmica/fisiologia , Humanos , Miócitos Cardíacos/fisiologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico
16.
Cell Physiol Biochem ; 54(4): 665-681, 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32639114

RESUMO

BACKGROUND/AIMS: Aortic stenosis-induced chronic pressure overload leads to cardiac dysfunction and congestive heart failure. The pathophysiological mechanisms of the myocardial impairment are multifactorial and include maladaptive ß-adrenergic signaling. Exercise training (ET) has been used as a non-pharmacological therapy for heart failure management. The present study tested the hypothesis that exercise training attenuates diastolic dysfunction through ß-adrenergic signaling preservation. METHODS: Wistar rats were submitted to ascending aortic stenosis (AS) surgery, and after 18 weeks, a moderate aerobic exercise training protocol was performed for ten weeks. RESULTS: ET attenuated diastolic dysfunction, evaluated by echocardiogram and isolated papillary muscle (IPM) assay. Also, ET reduced features of heart failure, cross-sectional cardiomyocyte area, and exercise intolerance, assessed by treadmill exercise testing. The ß2 adrenergic receptor protein expression was increased in AS rats independently of exercise. Interestingly, ET restored the protein levels of phosphorylated phospholamban at Serine 16 and preserved the ß-adrenergic receptor responsiveness as visualized by the lower myocardial compliance decline and time to 50% tension development and relaxation during ß-adrenergic stimulation in the IPM than untrained rats. Additionally, AS rats presented higher levels of TNFα and iNOS, which were attenuated by ET. CONCLUSION: Moderate ET improves exercise tolerance, reduces heart failure features, and attenuates diastolic dysfunction. In the myocardium, ET decreases the cross-sectional area of the cardiomyocyte and preserves the ß-adrenergic responsiveness, which reveals that the adjustments in ß-adrenergic signaling contribute to the amelioration of cardiac dysfunction by mild exercise training in aortic stenosis rats.


Assuntos
Estenose Aórtica Supravalvular/metabolismo , Insuficiência Cardíaca Diastólica/terapia , Miócitos Cardíacos/metabolismo , Condicionamento Físico Animal/fisiologia , Receptores Adrenérgicos beta/metabolismo , Animais , Estenose Aórtica Supravalvular/terapia , Proteínas de Ligação ao Cálcio/metabolismo , Ecocardiografia , Teste de Esforço , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Músculos Papilares/fisiologia , Fosforilação , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
17.
Cell Physiol Biochem ; 54(4): 696-706, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32706220

RESUMO

BACKGROUND/AIMS: Mutations of desmosomal genes are known to cause arrhythmogenic cardiomyopathy characterized by arrhythmias and sudden cardiac death. Previously, we described a novel genetic variant H1684R in desmoplakin gene (DSP), associated with a progressive cardiac conduction disease (PCCD). In the present study, we aimed to investigate an effect of the DSP-H1684R genetic variant on the activity of ion channels. METHODS: We used cardiomyocytes derived from induced pluripotent stem cells (iPSC cardiomyocytes) from a patient with DSP-H1684R genetic variant and from two healthy donors. Immunofluorescent staining and western blot analyses were used to characterize patient-specific cardiomyocytes. By the whole-cell voltage-clamp technique we estimated the activity of voltage-gated sodium, calcium, and potassium channels that are responsible for action potential generation and its shape. Action potentials' parameters were measured using whole-cell current-clamp technique. RESULTS: In patient-specific cardiomyocytes we observed both lower amplitudes of currents through sodium Nav1.5 channels and L-type calcium channels, but higher amplitude of current through transient-outward potassium channels in comparison to donor cardiomyocytes. Current-clamp measurements revealed shortening of action-potential in DSP-H1684R-carrying iPSC cardiomyocytes. Therefore, observed alterations in the channels activity might have a great impact on the properties of action potential and development of PCCD. CONCLUSION: Our results show that desmoplakin genetic variants, besides conduction slowing caused by structural heart remodeling, could affect multiple ion channel activity aggravating arrhythmia manifestation in PCCD.


Assuntos
Doença do Sistema de Condução Cardíaco/genética , Desmoplaquinas/genética , Bloqueio Cardíaco/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Canais Iônicos/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Potenciais de Ação/fisiologia , Canais de Cálcio/fisiologia , Doença do Sistema de Condução Cardíaco/metabolismo , Desmoplaquinas/metabolismo , Imunofluorescência , Bloqueio Cardíaco/metabolismo , Humanos , Canais Iônicos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Canais de Sódio Disparados por Voltagem/fisiologia
18.
Circ Heart Fail ; 13(7): e006935, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32635769

RESUMO

BACKGROUND: NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed. METHODS: Nexn inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca2+ transient studies were conducted. RESULTS: Inducible cardiomyocyte-specific loss of Nexn in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; P<0.05). In vivo and in vitro analyses of adult mouse heart samples revealed that NEXN was essential for optimal contraction and calcium handling and was required for maintenance of T-tubule network organization (transverse tubular component in Nexn inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, P<0.05). CONCLUSIONS: Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.


Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Proteínas dos Microfilamentos/fisiologia , Microtúbulos/fisiologia , Miócitos Cardíacos/fisiologia , Disfunção Ventricular Esquerda/fisiopatologia , Fatores Etários , Animais , Cálcio/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo
19.
J Vis Exp ; (160)2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32628167

RESUMO

In this article, we describe the steps required to isolate a single permeabilized ("skinned") cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Fenômenos Fisiológicos Cardiovasculares , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Animais , Camundongos , Miócitos Cardíacos/citologia , Miofibrilas
20.
Braz J Med Biol Res ; 53(9): e9693, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32696821

RESUMO

Ischemic heart disease (IHD) is one of the leading causes of death worldwide. C-type lectin domain family 3 member B (CLEC3B) is a C-type lectin superfamily member and is reported to promote tissue remodeling. The serum levels of CLEC3B are downregulated in patients with cardiovascular disease. However, the molecular mechanisms of CLEC3B in IHD is not well-characterized. Therefore, we overexpressed CLEC3B and silenced CLEC3B in H9c2 rat cardiomyocytes for the first time. We then constructed a model of IHD in vitro through culturing H9c2 cardiomyocytes in serum-free medium under oxygen-deficit conditions. Then, Cell Counting Kit-8 (CCK-8), flow cytometry, qRT-PCR, and western blot assays were performed to investigate cell viability, apoptosis, and expression levels of CLEC3B, phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), and cleaved-caspase 3. We observed that the mRNA expression of CLEC3B was decreased in hypoxic H9c2 cardiomyocytes (P<0.05). Overexpression of CLEC3B increased cell viability (P<0.01), inhibited cell apoptosis (P<0.05), upregulated the levels of p-PI3K/PI3K and p-Akt/Akt (P<0.01 or P<0.05), and downregulated expression of cleaved-caspase 3 (P<0.001) in hypoxic H9c2 cardiomyocytes while silencing of CLEC3B caused the opposite results. Inhibition of the PI3K/Akt pathway reversed the protective effect of CLEC3B on hypoxic H9c2 cardiomyocytes. Our study demonstrated that CLEC3B alleviated the injury of hypoxic H9c2 cardiomyocytes via the PI3K/Akt pathway.


Assuntos
Apoptose/fisiologia , Lectinas Tipo C/metabolismo , Animais , Humanos , Hipóxia , Miócitos Cardíacos/fisiologia , Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA