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1.
Life Sci ; 255: 117814, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32439300

RESUMO

AIMS: Amiodarone (AMIO) is currently used in medical practice to reverse ventricular tachycardia. Here we determine the effects of AMIO in the electromechanical properties of isolated left ventricle myocyte (LVM) from mice and guinea pig and in a cellular model of Long QT Syndrome Type 3 (LQTS-3) using anemone neurotoxin 2 (ATX II), which induces increase of late sodium current in LVM. MAIN METHODS AND KEY FINDINGS: Using patch-clamp technique, fluorescence imaging to detect cellular Ca2+ transient and sarcomere detection systems we evaluate the effect of AMIO in healthy LVM. AMIO produced a significant reduction in the percentage of sarcomere shortening (0.1, 1 and 10 µM) in a range of pacing frequencies, however, without significant attenuation of Ca2+ transient. Also, 10 µM of AMIO caused the opposite effect on action potential repolarization of mouse and guinea pig LVM. When LVM from mouse and guinea pig were paced in a range of pacing frequencies and exposed to ATX (10 nM), AMIO (10 µM) was only able to abrogate electromechanical arrhythmias in LVM from guinea pig at lower pacing frequency. SIGNIFICANCE: AMIO has negative inotropic effect with opposite effect on action potential waveform in mouse and guinea pig LVM. Furthermore, the antiarrhythmic action of AMIO in LQTS-3 is species and frequency-dependent, which indicates that AMIO may be beneficial for some types of arrhythmias related to late sodium current.


Assuntos
Amiodarona/farmacologia , Antiarrítmicos/farmacologia , Doença do Sistema de Condução Cardíaco/tratamento farmacológico , Síndrome do QT Longo/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Amiodarona/administração & dosagem , Animais , Antiarrítmicos/administração & dosagem , Doença do Sistema de Condução Cardíaco/fisiopatologia , Relação Dose-Resposta a Droga , Cobaias , Ventrículos do Coração/citologia , Síndrome do QT Longo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Especificidade da Espécie
2.
PLoS Genet ; 16(5): e1008782, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32421721

RESUMO

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)-derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ventrículos do Coração/citologia , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Processamento Alternativo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Contagem de Células , Células Cultivadas , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
3.
Nat Cell Biol ; 22(3): 332-340, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123336

RESUMO

Mapping of the holistic cell behaviours sculpting the four-chambered mammalian heart has been a goal or previous studies, but so far only success in transparent invertebrates and lower vertebrates with two-chambered hearts has been achieved. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a mouse embryo culture module, a heartbeat-gated imaging strategy and a digital image processing framework, we realized volumetric imaging of developing mouse hearts at single-cell resolution and with uninterrupted cell lineages for up to 1.5 d. Four-dimensional landscapes of Nppa+ cardiomyocyte cell behaviours revealed a blueprint for ventricle chamber formation by which biased outward migration of the outermost cardiomyocytes is coupled with cell intercalation and horizontal division. The inner-muscle architecture of trabeculae was developed through dual mechanisms: early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction of uninterrupted cell lineages affords a transformative means for deciphering mammalian organogenesis.


Assuntos
Ventrículos do Coração/citologia , Ventrículos do Coração/embriologia , Processamento de Imagem Assistida por Computador/métodos , Miócitos Cardíacos/citologia , Animais , Divisão Celular , Linhagem da Célula , Movimento Celular , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Coração/embriologia , Camundongos , Microscopia , Morfogênese , Miocárdio/citologia , Análise de Célula Única , Técnicas de Cultura de Tecidos
4.
J Vis Exp ; (155)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-32009641

RESUMO

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.


Assuntos
Separação Celular/métodos , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Imagem Óptica , 4-Aminopiridina/farmacologia , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Eletricidade , Corantes Fluorescentes/química , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Pressão , Ratos Sprague-Dawley , Sarcômeros/metabolismo
5.
J Vis Exp ; (155)2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-32009647

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to ß-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.


Assuntos
Cálcio/metabolismo , Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Animais , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ratos Sprague-Dawley , Fatores de Tempo
6.
PLoS One ; 15(2): e0229214, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084220

RESUMO

INTRODUCTION: The application of brief high voltage electrical pulses to tissue can lead to an irreversible or reversible electroporation effect in a cell-specific manner. In the management of ventricular arrhythmias, the ability to target different tissue types, specifically cardiac conduction tissue (His-Purkinje System) vs. cardiac myocardium would be advantageous. We hypothesize that pulsed electric fields (PEFs) can be applied safely to the beating heart through a catheter-based approach, and we tested whether the superficial Purkinje cells can be targeted with PEFs without injury to underlying myocardial tissue. METHODS: In an acute (n = 5) and chronic canine model (n = 6), detailed electroanatomical mapping of the left ventricle identified electrical signals from myocardial and overlying Purkinje tissue. Electroporation was effected via percutaneous catheter-based Intracardiac bipolar current delivery in the anesthetized animal. Repeat Intracardiac electrical mapping of the heart was performed at acute and chronic time points; followed by histological analysis to assess effects. RESULTS: PEF demonstrated an acute dose-dependent functional effect on Purkinje, with titration of pulse duration and/or voltage associated with successful acute Purkinje damage. Electrical conduction in the insulated bundle of His (n = 2) and anterior fascicle bundle (n = 2), was not affected. At 30 days repeat cardiac mapping demonstrated resilient, normal electrical conduction throughout the targeted area with no significant change in myocardial amplitude (pre 5.9 ± 1.8 mV, 30 days 5.4 ± 1.2 mV, p = 0.92). Histopathological analysis confirmed acute Purkinje fiber targeting, with chronic studies showing normal Purkinje fibers, with minimal subendocardial myocardial fibrosis. CONCLUSION: PEF provides a novel, safe method for non-thermal acute modulation of the Purkinje fibers without significant injury to the underlying myocardium. Future optimization of this energy delivery is required to optimize conditions so that selective electroporation can be utilized in humans the treatment of cardiac disease.


Assuntos
Eletroporação , Ventrículos do Coração/citologia , Animais , Cães , Estudos de Viabilidade , Masculino , Células de Purkinje/citologia , Segurança , Sobrevivência de Tecidos , Função Ventricular
7.
Nat Commun ; 11(1): 75, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911598

RESUMO

The functions of the heart are achieved through coordination of different cardiac cell subtypes (e.g., ventricular, atrial, conduction-tissue cardiomyocytes). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer unique opportunities for cardiac research. Traditional studies using these cells focused on single-cells and utilized mixed cell populations. Our goal was to develop clinically-relevant engineered heart tissues (EHTs) comprised of chamber-specific hPSC-CMs. Here we show that such EHTs can be generated by directing hPSCs to differentiate into ventricular or atrial cardiomyocytes, and then embedding these cardiomyocytes in a collagen-hydrogel to create chamber-specific, ring-shaped, EHTs. The chamber-specific EHTs display distinct atrial versus ventricular phenotypes as revealed by immunostaining, gene-expression, optical assessment of action-potentials and conduction velocity, pharmacology, and mechanical force measurements. We also establish an atrial EHT-based arrhythmia model and confirm its usefulness by applying relevant pharmacological interventions. Thus, our chamber-specific EHT models can be used for cardiac disease modeling, pathophysiological studies and drug testing.


Assuntos
Átrios do Coração/citologia , Ventrículos do Coração/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Potenciais de Ação , Diferenciação Celular , Átrios do Coração/crescimento & desenvolvimento , Ventrículos do Coração/crescimento & desenvolvimento , Humanos , Engenharia Tecidual
8.
Am J Physiol Heart Circ Physiol ; 318(2): H264-H282, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31834834

RESUMO

The ß1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the ß1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of ß1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the ß1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of ß1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the ß1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases.NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the ß1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle.


Assuntos
Ventrículos do Coração , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 1/fisiologia , Algoritmos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Cardiotônicos/farmacologia , Proteínas de Transporte/metabolismo , Simulação por Computador , Frequência Cardíaca/fisiologia , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Camundongos , Modelos Teóricos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação , Bloqueadores dos Canais de Potássio/farmacologia , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Sarcômeros/fisiologia , Troponina I/metabolismo , Troponina I/fisiologia
9.
Phys Rev Lett ; 123(21): 218101, 2019 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-31809131

RESUMO

We develop an iterated map model to describe the bifurcations and complex dynamics caused by the feedback between voltage and intracellular Ca^{2+} and Na^{+} concentrations in paced ventricular myocytes. Voltage and Ca^{2+} can form either a positive or a negative feedback loop, while voltage and Na^{+} form a negative feedback loop. Under certain diseased conditions, when the feedback between voltage and Ca^{2+} is positive, Hopf bifurcations occur, leading to periodic oscillatory behaviors. When this feedback is negative, period-doubling bifurcation routes to alternans and chaos occur.


Assuntos
Cálcio/metabolismo , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Sódio/metabolismo , Relógios Biológicos , Cátions Bivalentes/metabolismo , Cátions Monovalentes/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Retroalimentação Fisiológica , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Potenciais da Membrana , Miócitos Cardíacos/citologia , Trocador de Sódio e Cálcio/metabolismo
10.
Nat Commun ; 10(1): 5754, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848331

RESUMO

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.


Assuntos
Insuficiência Cardíaca/etiologia , Músculo Esquelético/patologia , Distrofias Musculares/complicações , Miócitos Cardíacos/patologia , Transferases/genética , Adulto , Fatores Etários , Animais , Animais Recém-Nascidos , Sistemas CRISPR-Cas/genética , Células Cultivadas , Modelos Animais de Doenças , Distroglicanas/metabolismo , Feminino , Técnicas de Inativação de Genes , Glicosilação , Células HEK293 , Insuficiência Cardíaca/patologia , Ventrículos do Coração/citologia , Ventrículos do Coração/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Contração Miocárdica/genética , Miócitos Cardíacos/citologia , Cultura Primária de Células , Sarcolema/patologia , Transferases/metabolismo
11.
Bull Exp Biol Med ; 168(2): 187-192, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31776956

RESUMO

The whole-cell patch-clamp technique was used to examine the effect of gadolinium Gd3+ (a non-specific blocker of mechanically gated current IMGCh, a component of late current IL) on ionic currents in insolated rat ventricular cardiomyocytes alone and in combination with the blockers of L-type calcium currents (ICaL) nifedipine (10 µM) or verapamil (1 µM). In K+in/K+out or Cs+in/Cs+out media, blockade of ICaL produced no effect on IL at negative potentials, but inhibited IL at positive ones. In K+in/K+out medium, Gd3+ (5 µM) decreased the net persistent current (Inp) at -45 mV from 198.6±6.4 to 96.7±9.5 pA over 15 min. Gd3+ alone or in combination with ICaL blockers shifted the reversal potential of IL to more negative values. At negative potentials, Gd3+ decreased IK1 and inward current including IMGCh. At positive potentials, Gd3+ alone or in combination with ICaL blockers decreased IL. When applied for 15 min in Cs+in/Cs+out medium at -45 mV, Gd3+ produced no effect on net current and inward and outward components of IL. Thus, Gd3+ can be viewed as a specific blocker of IMGCh only in Cs+ medium.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Gadolínio/farmacologia , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Césio/metabolismo , Ventrículos do Coração/citologia , Masculino , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Ratos , Verapamil/farmacologia
12.
Int J Nanomedicine ; 14: 5595-5609, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413565

RESUMO

Background: Platinum nanoparticles (PtNPs) have been considered a nontoxic nanomaterial and been clinically used in cancer chemotherapy. PtNPs can also be vehicle exhausts and environmental pollutants. These situations increase the possibility of human exposure to PtNPs. However, the potential biotoxicities of PtNPs including that on cardiac electrophysiology have been poorly understood. Methods: Ion channel currents of cardiomyocytes were recorded by patch clamp. Heart rhythm was monitored by electrocardiogram recording. Morphology and characteristics of PtNPs were examined by transmission electron microscopy, dynamic light scattering and electrophoretic light scattering analyses. Results: In cultured neonatal mice ventricular cardiomyocytes, PtNPs with diameters 5 nm (PtNP-5) and 70 nm (PtNP-70) concentration-dependently (10-9 - 10-5 g/mL) depolarized the resting potentials, suppressed the depolarization of action potentials and delayed the repolarization of action potentials. At the ion channel level, PtNPs decreased the current densities of INa, IK1 and Ito channels, but did not affect the channel activity kinetics. In vivo, PtNP-5 and PtNP-70 dose-dependently (3-10 mg/kg, i.v.) decreased the heart rate and induced complete atrioventricular conduction block (AVB) at higher doses. Both PtNP-5 and PtNP-70 (10-9 - 10-5 g/mL) did not significantly increase the generation of ROS and leak of lactate dehydrogenase (LDH) from cardiomyocytes within 5 mins after exposure except that only very high PtNP-5 (10-5 g/mL) slightly increased LDH leak. The internalization of PtNP-5 and PtNP-70 did not occur within 5 mins but occurred 1 hr after exposure. Conclusion: PtNP-5 and PtNP-70 have similar acute toxic effects on cardiac electrophysiology and can induce threatening cardiac conduction block. These acute electrophysiological toxicities of PtNPs are most likely caused by a nanoscale interference of PtNPs on ion channels at the extracellular side, rather than by oxidative damage or other slower biological processes.


Assuntos
Frequência Cardíaca/efeitos dos fármacos , Canais Iônicos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Miócitos Cardíacos/metabolismo , Platina/toxicidade , Testes de Toxicidade Aguda , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletrocardiografia , Endocitose/efeitos dos fármacos , Sistema de Condução Cardíaco/efeitos dos fármacos , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Platina/administração & dosagem
13.
PLoS One ; 14(8): e0220573, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31374110

RESUMO

RATIONALE: In virtually all models of heart failure, prognosis is determined by right ventricular (RV) function; thus, understanding the cellular mechanisms contributing to RV dysfunction is critical. Whole organ remodeling is associated with cell-specific changes, including cardiomyocyte dedifferentiation and activation of cardiac fibroblasts (Cfib) which in turn is linked to disorganization of cytoskeletal proteins and loss of sarcomeric structures. However, how these cellular changes contribute to RV function remains unknown. We've previously shown significant organ-level RV dysfunction in a large animal model of pulmonary hypertension (PH) which was not mirrored by reduced function of isolated cardiomyocytes. We hypothesized that factors produced by the endogenous Cfib contribute to global RV dysfunction by generating a heterogeneous cellular environment populated by dedifferentiated cells. OBJECTIVE: To determine the effect of Cfib conditioned media (CM) from the PH calf (PH-CM) on adult rat ventricular myocytes (ARVM) in culture. METHODS AND RESULTS: Brief exposure (<2 days) to PH-CM results in rapid, marked dedifferentiation of ARVM to a neonatal-like phenotype exhibiting spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI and α-actinin yet exhibit myofibroblast characteristics including expression of α-smooth muscle actin. Using a bioinformatics approach to identify factor(s) that contribute to dedifferentiation, we found activation of the PH Cfib results in a unique transcriptome correlating with factors both in the secretome and with activated pathways in the dedifferentiated myocyte. Further, we identified upregulation of periostin in the Cfib and CM, and demonstrate that periostin is sufficient to drive cardiomyocyte dedifferentiation. CONCLUSIONS: These data suggest that paracrine factor(s) released by Cfib from the PH calf signal a phenotypic transformation in a population of cardiomyocytes that likely contributes to RV dysfunction. Therapies targeting this process, such as inhibition of periostin, have the potential to prevent RV dysfunction.


Assuntos
Desdiferenciação Celular/fisiologia , Fibroblastos/metabolismo , Ventrículos do Coração/metabolismo , Hipertensão Pulmonar/metabolismo , Miócitos Cardíacos/citologia , Disfunção Ventricular Direita/metabolismo , Animais , Bovinos , Modelos Animais de Doenças , Fibroblastos/citologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/metabolismo , Função Ventricular Direita/fisiologia , Remodelação Ventricular
14.
Basic Res Cardiol ; 114(5): 36, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399804

RESUMO

Individual adult ventricular cardiomyocytes are either mono- or multi-nucleated and undergo morphological changes during cardiac hypertrophy. However, corresponding transcriptional signatures, reflecting potentially different functions or the ability for cell-cycle entry, are not known. The aim of this study was to determine the transcriptional profile of mono- and multi-nucleated adult cardiomyocytes by single-cell RNA-sequencing (scRNA-seq) and to investigate heterogeneity among cardiomyocytes under baseline conditions and in pressure-induced cardiac hypertrophy. We developed an array-based approach for scRNA-seq of rod-shaped multi-nucleated cardiomyocytes from both healthy and hypertrophic hearts. Single-cell transcriptomes of mono- or multi-nucleated cardiomyocytes were highly similar, although a certain degree of variation was noted across both populations. Non-image-based quality control allowing inclusion of damaged cardiomyocytes generated artificial cell clusters demonstrating the need for strict exclusion criteria. In contrast, cardiomyocytes isolated from hypertrophic heart after transverse aortic constriction showed heterogeneous transcriptional signatures, characteristic for hypoxia-induced responses. Immunofluorescence analysis revealed an inverse correlation between HIF1α+ cells and CD31-stained vessels, suggesting that imbalanced vascular growth in the hypertrophied heart induces cellular heterogeneity. Our study demonstrates that individual mono- and multi-nucleated cardiomyocytes express nearly identical sets of genes. Homogeneity among cardiomyocytes was lost after induction of hypertrophy due to differential HIF1α-dependent responses most likely caused by none-homogenous vessel growth.


Assuntos
Cardiomegalia/patologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Transcriptoma , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL
15.
Circ Res ; 125(4): 431-448, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31284834

RESUMO

RATIONALE: Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. OBJECTIVES: This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. METHODS AND RESULTS: By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. CONCLUSIONS: This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.


Assuntos
Miócitos Cardíacos/metabolismo , Ribossomos/metabolismo , Análise de Sequência de RNA/métodos , Disfunção Ventricular/genética , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Hemodinâmica , Masculino , Camundongos , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/química , Estresse Fisiológico , Disfunção Ventricular/metabolismo
16.
Methods Mol Biol ; 2029: 161-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31273741

RESUMO

Cardiac progenitor cells (CPCs) are gaining interest as a therapeutic option for the treatment of the heart. Due to the limited pool of CPCs residing in the heart, it is essential to isolate and expand the CPCs in vitro. Here we describe the protocol for isolation and culture of the heterogeneous population of CPCs from right atrial appendage and left ventricular tissue collected from patients undergoing on-pump coronary artery bypass graft surgery for the treatment of ischemic heart disease. Our protocol is developed to simultaneously isolate, culture, and characterize the CPCs from both atrial and ventricular tissues. We also describe the protocol for flow cytometry and immunohistochemical characterization of the isolated CPCs.


Assuntos
Miócitos Cardíacos/citologia , Células Cultivadas , Ponte de Artéria Coronária/métodos , Vasos Coronários/citologia , Citometria de Fluxo/métodos , Ventrículos do Coração/citologia , Humanos , Miocárdio/citologia
17.
J Cardiovasc Magn Reson ; 21(1): 35, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31256759

RESUMO

BACKGROUND: The three-dimensional rearrangement of the right ventricular (RV) myocardium during cardiac deformation is unknown. Previous in-vivo studies have shown that myocardial left ventricular (LV) deformation is driven by rearrangement of aggregations of cardiomyocytes that can be characterised by changes in the so-called E3-angle. Ex-vivo imaging offers superior spatial resolution compared with in-vivo measurements, and can thus provide novel insight into the deformation of the myocardial microstructure in both ventricles. This study sought to describe the dynamic changes of the orientations of the cardiomyocytes in both ventricles brought upon by cardiac contraction, with particular interest in the thin-walled RV, which has not previously been described in terms of its micro-architecture. METHODS: The hearts of 14 healthy 20 kg swine were excised and preserved in either a relaxed state or a contracted state. Myocardial architecture was assessed and compared between the two contractional states by quantification of the helical, transmural and E3-angles of the cardiomyocytes using high-resolution diffusion tensor imaging. RESULTS: The differences between the two states of contraction were most pronounced in the endocardium where the E3-angle decreased from 78.6° to 24.8° in the LV and from 82.6° to 68.6° in the RV. No significant change in neither the helical nor the transmural angle was found in the cardiomyocytes of the RV. In the endocardium of the LV, however, the helical angle increased from 35.4° to 47.8° and the transmural angle increased from 3.1° to 10.4°. CONCLUSION: The entire myocardium rearranges through the cardiac cycle with the change in the orientation of the aggregations of cardiomyocytes being the predominant mediator of myocardial wall thickening. Interestingly, differences also exist between the RV and LV, which helps in the explanation of the different physiological capabilities of the ventricles.


Assuntos
Imagem de Tensor de Difusão , Ventrículos do Coração/diagnóstico por imagem , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Função Ventricular Esquerda , Função Ventricular Direita , Remodelação Ventricular , Animais , Feminino , Ventrículos do Coração/citologia , Valor Preditivo dos Testes , Sus scrofa , Fatores de Tempo
18.
Biomed Eng Online ; 18(1): 72, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174533

RESUMO

PURPOSE: Although the quantitative analysis of electromechanical alternans is important, previous studies have focused on electrical alternans, and there is a lack quantitative analysis of mechanical alternans at the subcellular level according to various basic cycle lengths (BCLs). Therefore, we used the excitation-contraction (E-C) coupling model of human ventricular cells to quantitatively analyze the mechanical alternans of ventricular cells according to various BCLs. METHODS: To implement E-C coupling, we used calcium transient data, which is the output data of electrical simulation using the electrophysiological model of human ventricular myocytes, as the input data of mechanical simulation using the contractile myofilament dynamics model. Moreover, we applied various loads on ventricular cells for implementation of isotonic and isometric contraction. RESULTS: As the BCL was reduced from 1000 to 200 ms at 30 ms increments, mechanical alternans, as well as electrical alternans, were observed. At this time, the myocardial diastolic tension increased, and the contractile ATP consumption rate remained greater than zero even in the resting state. Furthermore, the time of peak tension, equivalent cell length, and contractile ATP consumption rate were all reduced. There are two tendencies that endocardial, mid-myocardial, and epicardial cells have the maximum amplitude of tension and the peak systolic tension begins to appear at a high rate under the isometric condition at a particular BCL. CONCLUSIONS: We observed mechanical alternans of ventricular myocytes as well as electrical alternans, and identified unstable conditions associated with mechanical alternans. We also determined the amount of BCL given to each ventricular cell to generate stable and high tension state in the case of isometric contraction.


Assuntos
Fenômenos Eletrofisiológicos , Ventrículos do Coração/citologia , Fenômenos Mecânicos , Modelos Cardiovasculares , Miócitos Cardíacos/citologia , Função Ventricular , Fenômenos Biomecânicos , Eletrocardiografia , Humanos
19.
Cell Tissue Res ; 378(1): 59-66, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31041505

RESUMO

Several studies have been reported in various domains from induction methods to utilities of somatic cell pluripotent reprogramming. However, one of the major struggles facing the research field of induced pluripotent stem cell (iPSC)-derived target cells is the lack of consistency in observations. This could be due to variety of reasons including varied culture periods post-differentiation. The cardiomyocytes (CMs) derived from iPSCs are commonly studied and proposed to be utilized in the comprehensive in vitro proarrhythmia initiative for drug safety screening. As the influence of varied culture periods on the electrophysiological properties of iPSC-CMs is not clearly known, using whole-cell patch clamp technique, we compared two groups of differentiated ventricular-like iPSC-CMs that are cultured for 10 to 15 days (D10-15) and more than 30 days (≥ D30) both under current and voltage clamps. The prolonged culture imparts increased excitability with high-frequency spontaneous action potentials, robust increase in the magnitude of peak Na+ current density, relatively shallow inactivation kinetics of Na+ channels, faster recovery from inactivation, and augmented Ca2+ current density. Quantitative real-time PCR studies of α-subunit transcripts showed enhanced mRNA expression of SCN1A, SCN5A Na+ channel subtypes, and CACNA1C, CACNA1G, and CACNA1I Ca2+ channel subtypes, in ≥ D30 group. Conclusively, the prolonged culture of differentiated iPSC-CMs affects the excitability, single-cell electrophysiological properties, and ion channel expressions. Therefore, following standard periods of culture across research studies while utilizing ventricular-like iPSC-CMs for in vitro health/disease modeling to study cellular functional mechanisms or test high-throughput drugs' efficacy and toxicity becomes crucial.


Assuntos
Canais de Cálcio/metabolismo , Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Canais de Potássio/metabolismo , Potenciais de Ação , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ativação do Canal Iônico , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo
20.
Arch Biochem Biophys ; 666: 31-39, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30930285

RESUMO

A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨM) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 ″super-resolution" or "high-resolution" imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 s (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 min. The time to first depolarization events was 8.4 min (±1.1 SEM n = 21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 min) and virtually no permanent depolarization events occurred for over 1 h of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon "stress" in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1-5], we show that cyclosporine-A (CsA, 10 µM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80% and 93% respectively. Furthermore, CsA and NAC treatment led to the virtual disappearance of permanent depolarization events. Nevertheless, all transient depolarization events in any condition (control, CsA and NAC) appeared to repolarize with a similar half-time of 30 ±â€¯6 s (n = 478) at 37 °C. Further experiments showed quantitatively similar results in cerebral vascular smooth muscle cells, using a different confocal system, and different photon absorbing reagent (TMRE; tetramethylrhodamine ethyl ester). In these experiments, using modest power (1% laser power) transient depolarization events were seen in only 8 out of 23 cells while with higher power (8%), all cells showed transient events, which align with the level of photon stress being the driver of the effect. Together, our findings suggest that photon-induced ROS is sufficient to cause depolarization events of individual mitochondria in quiescent cells; without electrical or mechanical activity to stimulates mitochondrial metabolism, and without raising the mitochondrial matrix Ca2+. In a broad context, these findings neither support nor deny the relevance or occurrence of ΔΨM depolarization events in specific putatively physiologic mitochondrial behaviors such as MitoFlashes [6,7] or MitoWinks [8]. Instead, our findings raise a caution with regards to the physiological and pathophysiological functions attributed to singular ΔΨM depolarization events when those functions are investigated using photon absorbing substances. Nevertheless, using photon stress as a tool ("Optical Stress-Probe"), we can extract information on the activation, reversibility, permanency and kinetics of mitochondrial depolarization. These data may provide new information on mPTP, help identify the mPTP protein complex, and establish the physiological function of the mPTP protein complex and their links to MitoFlashes and MitoWinks.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Potencial da Membrana Mitocondrial , Ratos , Ratos Sprague-Dawley
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