RESUMO
Antiparasitic ivermectin has been reported to induce cardiovascular adverse events, including orthostatic hypotension, tachycardia and cardiopulmonary arrest, of which the underlying pathophysiology remains unknown. Since its drug repurposing as an antiviral agent is underway at higher doses than those for antiparasitic, we evaluated the cardiovascular safety pharmacology of ivermectin using isoflurane-anesthetized beagle dogs (n=4). Ivermectin in doses of 0.1 followed by 1 mg/kg was intravenously infused over 10 min with an interval of 20 min, attaining peak plasma concentrations of 0.94 ± 0.04 and 8.82 ± 1.25 µg/mL, which were 29-31 and 276-288 times higher than those observed after its antiparasitic oral dose of 12 mg/body, respectively. The latter peak concentration was > 2 times greater than those inhibiting proliferation of dengue virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hepatitis B virus in vitro. Ivermectin decreased heart rate without altering mean blood pressure, suggesting that ivermectin does not cause hypotension or tachycardia directly. Ivermectin hardly altered atrioventricular nodal or intraventricular conduction, indicating a lack of inhibitory action on Ca2+ or Na+ channel in vivo. Ivermectin prolonged QT interval/QTcV in a dose-related manner and tended to slow the repolarization speed in a reverse frequency-dependent manner, supporting previously described its IKr inhibition, which would explain Tpeak-Tend prolongation and heart-rate reduction in this study. Meanwhile, ivermectin did not significantly prolong J-Tpeakc or terminal repolarization period, indicating torsadogenic potential of ivermectin leading to the onset of cardiopulmonary arrest would be small. Thus, ivermectin has a broad range of cardiovascular safety profiles, which will help facilitate its drug repurposing.
Assuntos
Parada Cardíaca , Isoflurano , Animais , Cães , Isoflurano/toxicidade , Ivermectina/toxicidade , Seguimentos , Taquicardia/induzido quimicamente , Antiparasitários/toxicidade , Frequência CardíacaRESUMO
Cancer cells acquire drug resistance through the following stages: nonresistant, pre-resistant, and resistant. Although the molecular mechanism of drug resistance is well investigated, the process of drug resistance acquisition remains largely unknown. Here we elucidate the molecular mechanisms underlying the process of drug resistance acquisition by sequential analysis of gene expression patterns in tamoxifen-treated breast cancer cells. Single-cell RNA-sequencing indicates that tamoxifen-resistant cells can be subgrouped into two, one showing altered gene expression related to metabolic regulation and another showing high expression levels of adhesion-related molecules and histone-modifying enzymes. Pseudotime analysis showed a cell transition trajectory to the two resistant subgroups that stem from a shared pre-resistant state. An ordinary differential equation model based on the trajectory fitted well with the experimental results of cell growth. Based on the established model, it was predicted and experimentally validated that inhibition of transition to both resistant subtypes would prevent the appearance of tamoxifen resistance.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Modelos Teóricos , Tamoxifeno/uso terapêutico , Antineoplásicos Hormonais/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Tamoxifeno/administração & dosagemRESUMO
Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48-54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction were disorganized in DMD-iPSC-CMs. Lysophosphatidic acid (LPA), a known lipid mediator on induction of actin polymerization, significantly increased YAP activity and actin dynamics in DMD-iPSC-CMs using live cell imaging. These results suggested that altered YAP activity due to impaired actin dynamics reduced proliferative ability in DMD-iPSC-CMs. Hence, decreased YAP activity in dystrophic heart may contribute to DMD-cardiomyopathy pathogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Cardiomiopatia Dilatada/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Muscular de Duchenne/complicações , Miócitos Cardíacos/patologia , Fatores de Transcrição/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Sistemas CRISPR-Cas/genética , Cardiomiopatia Dilatada/genética , Proliferação de Células , Células Cultivadas , Edição de Genes , Humanos , Masculino , Mecanotransdução Celular , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Cultura Primária de Células , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal relationship between electrical excitation and contractile motion, we simultaneously observed the field potential and motion vector of hiPSC-CMs sheets. Under spontaneous contraction, although an electrical excitation originating from a region propagated unidirectionally over the cell sheet, contraction wave started from multiple sites, and relaxation wave was initiated from a geometric center of hiPSC-CMs sheet. During electrical pacing, contraction and relaxation waves were propagated from the stimulated site. Interestingly, the maximum contraction speed was more increased when the hiPSC-CMs sheet was stimulated at an area relaxation initiated under spontaneous condition. Furthermore, motion vector analysis demonstrated that "positive contraction velocity-frequency relationship" in contraction and "frequency-dependent enhancement of relaxation" were produced in the cell sheet by optimizing the direction and order of the contractile motion with pacing at the relaxation-initiating area. A close analysis of motion vectors along with field potential recording demonstrated that relaxation process consists of fast and slow phases, and suggest that intracellular Ca2+ dynamics may be closely related to functions of Ca2+-ATPase pump and Na+-Ca2+ exchangers. Namely, the slow relaxation phase occurred after the second peak of field potential, suggesting that the slow phase may be associated with extrusion of Ca2+ by Na+-Ca2+ exchangers during repolarization. Increase of oxygen concentration from 20 to 95% as well as ß-adrenergic stimulation with isoproterenol accelerated the fast relaxation, suggesting that it could depend on Ca2+ uptake via Ca2+-ATPase during the depolarization phase. The ratio of maximum contraction speed to field potential duration was increased by the ß-adrenergic stimulation, indicating the elevated contraction efficiency per Ca2+-influx. Thus, these findings revealed potential ability of conventional monolayers of hiPSC-CMs, which will help apply them to translational study filling the gap between physiological as well as pharmacological studies and clinical practice.
RESUMO
Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.
Assuntos
Arritmias Cardíacas/etiologia , Insuficiência Cardíaca , Miócitos Cardíacos/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Arritmias Cardíacas/prevenção & controle , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Camundongos Transgênicos , Ratos , Receptores de Dopamina D1/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Taquicardia Ventricular/etiologia , Taquicardia Ventricular/prevenção & controleRESUMO
Torsade de pointes (TdP) occurred in a long QT syndrome type 3 (LQT3) patient after switching perospirone to blonanserin. We studied how their electropharmacological effects had induced TdP in the LQT3 patient. Perospirone hydrochloride (n = 4) or blonanserin (n = 4) of 0.01, 0.1, and 1 mg/kg, i.v. was cumulatively administered to the halothane-anesthetized dogs over 10 min. The low dose of perospirone decreased total peripheral vascular resistance, but increased heart rate and cardiac output, facilitated atrioventricular conduction, and prolonged J-Tpeakc. The middle dose decreased mean blood pressure and prolonged repolarization period, in addition to those observed after the low dose. The high dose further decreased mean blood pressure with the reduction of total peripheral vascular resistance; however, it did not increase heart rate or cardiac output. It tended to delay atrioventricular conduction and further delayed repolarization with the prolongation of Tpeak-Tend, whereas J-Tpeakc returned to its baseline level. Meanwhile, each dose of blonanserin decreased total peripheral vascular resistance, but increased heart rate, cardiac output and cardiac contractility in a dose-related manner. J-Tpeakc was prolonged by each dose, but Tpeak-Tend was shortened by the middle and high doses. These results indicate that perospirone and blonanserin may cause the hypotension-induced, reflex-mediated increase of sympathetic tone, leading to the increase of inward Ca2+ current in the heart except that the high dose of perospirone reversed them. Thus, blonanserin may have more potential to produce intracellular Ca2+ overload triggering early afterdepolarization than perospirone, which might explain the onset of TdP in the LQT3 patient.
Assuntos
Doença do Sistema de Condução Cardíaco/fisiopatologia , Antagonistas de Dopamina/toxicidade , Sistema de Condução Cardíaco/efeitos dos fármacos , Síndrome do QT Longo/fisiopatologia , Antagonistas da Serotonina/toxicidade , Torsades de Pointes/induzido quimicamente , Potenciais de Ação/efeitos dos fármacos , Anestésicos Inalatórios , Animais , Agonistas dos Canais de Cálcio/toxicidade , Delírio/tratamento farmacológico , Cães , Relação Dose-Resposta a Droga , Eletrocardiografia , Feminino , Halotano , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Isoindóis , Pessoa de Meia-Idade , Modelos Animais , Piperazinas , Piperidinas , Bloqueadores dos Canais de Potássio/toxicidade , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Tiazóis , Torsades de Pointes/metabolismo , Torsades de Pointes/fisiopatologiaRESUMO
Tyrosine kinase inhibitors are known to clinically induce various types of cardiovascular adverse events; however, it is still difficult to predict them at preclinical stage. In order to explore how to better predict such drug-induced cardiovascular adverse events, we tried to develop a new protocol by assessing acute electrophysiological, cardiohemodynamic, and cytotoxic effects of dasatinib in vivo and in vitro. Dasatinib at 0.03 and 0.3 mg/kg was intravenously administered to the halothane-anesthetized dogs for 10 min with an interval of 20 min between the dosing (n = 4). Meanwhile, that at 0.1, 0.3, and 1 µM was cumulatively applied to the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (n = 7). In the dogs, the low and high doses provided peak plasma concentrations of 40 ± 5 (0.08) and 615 ± 38 ng/mL (1.26 µM), respectively. The low dose decreased the heart rate, impaired the left ventricular mechanical function, and prolonged the ventricular effective refractory period. The high dose prolonged the repolarization period, induced hemorrhagic tendency, and increased plasma cardiac troponin I level in addition to enhancement of the changes observed after the low dose, whereas it neither affected the cardiac conduction nor induced ventricular arrhythmias. In the hiPSC-CMs, dasatinib prolonged the repolarization and refractory periods like in dogs, while it did not induce apoptotic or necrotic process, but that it increased the conduction speed. Clinically observed major cardiovascular adverse events of dasatinib were observed qualitatively by currently proposed assay protocol, which may become a useful guide for predicting the cardiotoxicity of new tyrosine kinase inhibitors.
Assuntos
Antineoplásicos/toxicidade , Arritmias Cardíacas/induzido quimicamente , Dasatinibe/toxicidade , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Inibidores de Proteínas Quinases/toxicidade , Disfunção Ventricular Esquerda/induzido quimicamente , Função Ventricular Esquerda/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Cardiotoxicidade , Células Cultivadas , Cães , Relação Dose-Resposta a Droga , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Terapia de Alvo Molecular/efeitos adversos , Miócitos Cardíacos/metabolismo , Período Refratário Eletrofisiológico , Medição de Risco , Fatores de Tempo , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Correction formulae of QT interval were developed for the halothane-anesthetized microminipigs by adopting atrial pacing (n=5), which were compared with Bazett's and Fridericia's formulae for humans, and Van de Water's one for dogs. The correction formulae: QTc=QT-0.2072 (RR-750) as linear and QTc=QT/(RR/750)0.4007 as non-linear equations, were developed for microminipigs. These formulae can better correct the QT interval of the microminipigs compared with each of the conventional ones for humans and dogs. Moreover, analysis of the slope constant α values indicates that the rate-dependent change in the ventricular repolarization period of microminipig may better mimic that of humans than that of dogs.
Assuntos
Estimulação Cardíaca Artificial/veterinária , Frequência Cardíaca/fisiologia , Porco Miniatura/fisiologia , Anestésicos Inalatórios , Animais , Estimulação Cardíaca Artificial/métodos , Cães/fisiologia , Feminino , Halotano , Humanos , Modelos Lineares , Masculino , Dinâmica não Linear , SuínosRESUMO
We investigated the electro-mechanical relationship of human iPS cell-derived cardiomyocyte sheets under arrhythmic condition, which was induced by digitalis intoxication along with the electrical train stimulation (n = 4). We adopted motion vector analysis by high-speed video microscopy and extracellular field potential recording by 64-microelectrode array system. The motion vector analysis uncovered local contractile events at resting phase, at which the field potential analysis showed no deflection in any cell sheet. Thus, motion vector analysis may provide supplemental information over field potential recording in detecting Ca2+-triggered arrhythmias, which may become a new strategy for assessing arrhythmic liability of test articles.
Assuntos
Arritmias Cardíacas/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , MicroeletrodosRESUMO
Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.
Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
dl-Sotalol which can block both K+ channel and ß-adrenoceptor has been shown to prolong the J-Tpeakc of electrocardiogram in beagle dogs but tended to shorten it in microminipigs, although the drug prolonged the QT interval in both animals under physiologically maintained experimental condition. In order to estimate how the changes in the J-Tpeakc in the normal hearts would be reflected in the pathologic hearts, we compared proarrhythmic effects of dl-sotalol by using proarrhythmia models of beagle dogs and microminipigs, of which atrioventricular node had been ablated > 2 months and 8-9 weeks before, respectively (n = 4 for each species). dl-Sotalol in an oral dose of 10 mg/kg induced torsade de pointes in three out of four beagle dogs, which degenerated into ventricular fibrillation. In microminipigs, the same dose did not trigger torsade de pointes at all, whereas intermittent ventricular pauses were observed in each animal after the drug treatment. These results indicate that assessment of the J-Tpeakc along with the QT-interval prolongation in healthy subjects may provide reliable information of risk prediction for patients susceptible to the drug-induced torsade de pointes.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Antagonistas Adrenérgicos beta/toxicidade , Sistema de Condução Cardíaco/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/toxicidade , Sotalol/toxicidade , Torsades de Pointes/induzido quimicamente , Testes de Toxicidade , Animais , Cardiotoxicidade , Cães , Relação Dose-Resposta a Droga , Feminino , Sistema de Condução Cardíaco/fisiopatologia , Masculino , Medição de Risco , Especificidade da Espécie , Suínos , Porco Miniatura , Torsades de Pointes/fisiopatologiaRESUMO
We analyzed how the enhancement of net sarcoplasmic reticulum (SR) Ca2+ uptake may affect cardiac electrophysiological properties in vivo by using caldaret which can decrease SR diastolic Ca2+ leak, enhance SR Ca2+ reuptake and inhibit reverse-mode Na+/Ca2+ exchanger. Caldaret in doses of 0.5, 5 and 50 µg/kg was intravenously administered over 10 min to the halothane-anesthetized beagle dogs (n = 5), attaining pharmacologically active plasma concentration. The low and middle doses of caldaret increased the ventricular contraction, which could be explained by its on-target pharmacological activities. The high dose enhanced the sinus automaticity followed by its suppression in addition to the increase of the total peripheral resistance, which may be unfavorable for treating diastolic heart failure. The low and middle doses enhanced the atrioventricular conduction, which may have some potential for predisposing the atria to the onset of atrial fibrillation via an induction of mitral and/or tricuspid regurgitation. The middle and high doses of caldaret prolonged the ventricular effective refractory period without altering the intraventricular conduction or repolarization period, which may prevent the onset of ventricular arrhythmias. Thus, modulation of intracellular Ca2+ handling by caldaret can induce not only inotropic effect, but also various electrophysiological actions on the in situ heart.
Assuntos
Benzenossulfonatos/farmacologia , Cálcio/administração & dosagem , Cardiotônicos/farmacologia , Piperazinas/farmacologia , Retículo Sarcoplasmático/efeitos dos fármacos , Animais , Arritmias Cardíacas/prevenção & controle , Benzenossulfonatos/administração & dosagem , Cálcio/metabolismo , Cardiotônicos/administração & dosagem , Cães , Relação Dose-Resposta a Droga , Técnicas Eletrofisiológicas Cardíacas , Feminino , Halotano/administração & dosagem , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Piperazinas/administração & dosagem , Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
Fluvoxamine is a selective serotonin-reuptake inhibitor, of which IC50 values for serotonin- and noradrenaline-uptake process were reported to be 3.8 and 620 nmol/L, respectively, also known to directly inhibit cardiac Na+, Ca2+, and K+ channels. We characterized microminipig as a laboratory animal by analyzing fluvoxamine-induced cardiovascular and dermatological responses under halothane anesthesia. Fluvoxamine maleate was infused in doses of 0.1, 1, and 10 mg/kg over 10 min with a pause of 20 min (n = 4). The peak plasma concentrations were 35, 320, and 1906 ng/mL, of which free plasma concentrations were estimated as 20, 187, and 1108 nmol/L, respectively. The low and middle doses did not alter any cardiovascular variable. The high dose increased heart rate and mean blood pressure, prolonged QRS width, but shortened QT interval, whereas no significant change was detected in PR interval or QTcF. Moreover, it induced systemic erythema on the skin. Pretreatment of H1/5-HT2A antagonist cyproheptadine hydrochloride sesquihydrate in a dose of 0.3 mg/kg significantly attenuated the fluvoxamine-induced pressor response; but tended to further enhance sinus automaticity, atrioventricular nodal conduction; and ventricular repolarization in addition to intraventricular conduction delay; whereas it markedly suppressed onset of systemic erythema (n = 4). In microminipigs, cardiovascular adverse effects of the high dose may be manifested as a sum of its inhibitory action on the cardiac ionic channels and its stimulatory effects on serotonergic and adrenergic systems, whereas dermatologic reaction can be induced primarily through H1/5-HT2A receptor-dependent mechanism. Thus, microminipigs may be used for analyzing such multifarious adverse events of clinical serotonergic pharmacotherapy.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Toxidermias/etiologia , Eritema/induzido quimicamente , Fluvoxamina/toxicidade , Hipertensão/induzido quimicamente , Inibidores Seletivos de Recaptação de Serotonina/toxicidade , Pele/efeitos dos fármacos , Taquicardia/induzido quimicamente , Testes de Toxicidade/métodos , Animais , Pressão Sanguínea/efeitos dos fármacos , Cardiotoxicidade , Sistema Cardiovascular/fisiopatologia , Relação Dose-Resposta a Droga , Toxidermias/patologia , Eritema/patologia , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/fisiopatologia , Masculino , Modelos Animais , Medição de Risco , Pele/patologia , Especificidade da Espécie , Suínos , Porco Miniatura , Taquicardia/fisiopatologia , Fatores de TempoRESUMO
BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure. METHODS AND RESULTS: We applied three novel single-cell analysis methods, namely, single-cell quantitative PCR (sc-qPCR), single-cell RNA sequencing (scRNA-seq), and single-molecule fluorescence in situ hybridization (smFISH). Isolated cardiomyocytes and cross sections from pressure overloaded murine hearts after transverse aortic constriction (TAC) were analyzed at an early hypertrophy stage (2â¯weeks, TAC2W) and at a late heart failure stage (8â¯weeks, TAC8W). Expression of myosin heavy chain ß (Myh7), a representative fetal gene, was induced in some cardiomyocytes in TAC2W hearts and in more cardiomyocytes in TAC8W hearts. Expression levels of Myh7 varied considerably among cardiomyocytes. Myh7-expressing cardiomyocytes were significantly more abundant in the middle layer, compared with the inner or outer layers of TAC2W hearts, while such spatial differences were not observed in TAC8W hearts. Expression levels of Myh7 were inversely correlated with cardiomyocyte size and expression levels of mitochondria-related genes. CONCLUSIONS: We developed a new image-analysis pipeline to allow automated and unbiased quantification of gene expression at the single-cell level and determined the spatial and temporal regulation of heterogenous Myh7 expression in cardiomyocytes after pressure overload.
Assuntos
Aorta/diagnóstico por imagem , Cardiomegalia/genética , Insuficiência Cardíaca/diagnóstico por imagem , Imagem Molecular/métodos , Cadeias Pesadas de Miosina/genética , Animais , Aorta/metabolismo , Aorta/patologia , Cardiomegalia/diagnóstico , Cardiomegalia/diagnóstico por imagem , Regulação da Expressão Gênica/genética , Coração/diagnóstico por imagem , Coração/fisiopatologia , Insuficiência Cardíaca/patologia , Hemodinâmica , Hibridização in Situ Fluorescente , Camundongos , Mitocôndrias/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Cadeias Pesadas de Miosina/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , Análise de Sequência de RNA , Imagem Individual de Molécula , Análise de Célula ÚnicaRESUMO
BACKGROUND: Since regenerative capacity of adult mammalian myocardium is limited, activation of the endogenous proliferative capacity of existing cardiomyocytes is a potential therapeutic strategy for treating heart diseases accompanied by cardiomyocyte loss. Recently, we performed a compound screening and developed a new drug named TT-10 (C11H10FN3OS2) which promotes the proliferation of murine cardiomyocytes via enhancement of YES-associated protein (YAP)-transcriptional enhancer factor domain (TEAD) activity and improves cardiac function after myocardial infarction in adult mice. METHODS AND RESULTS: To test whether TT-10 can also promote the proliferative capacity of human cardiomyocytes, we investigated the efficacy of TT-10 on human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSCMs). The hiPSCs were established from monocytes obtained from healthy donors and cardiac differentiation was performed using a chemically defined protocol. As was observed in murine cardiomyocytes, TT-10 markedly promoted cell cycle activation and increased cell division of hiPSCMs. We then evaluated other effects of TT-10 on the functional properties of hiPSCMs by gene expression and cell motion analyses. We observed that TT-10 had no unfavorable effects on the expression of functional and structural genes or the contractile properties of hiPSCMs. CONCLUSIONS: Our results suggest that the novel drug TT-10 effectively activated the cell cycle of hiPSCMs without apparent functional impairment of myocardium, suggesting the potential of clinical usefulness of this drug.
Assuntos
Ciclo Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Regeneração/efeitos dos fármacos , Regeneração/genéticaRESUMO
CD36 is one of the important transporters of long-chain fatty acids (LCFAs) in the myocardium. We previously reported that CD36-deficient patients demonstrate a marked reduction of myocardial uptake of LCFA, while myocardial glucose uptake shows a compensatory increase, and are often accompanied by cardiomyopathy. However, the molecular mechanisms and functional role of CD36 in the myocardium remain unknown.The current study aimed to explore the pathophysiological role of CD36 in the heart. Methods: Using wild type (WT) and knockout (KO) mice, we generated pressure overload by transverse aortic constriction (TAC) and analyzed cardiac functions by echocardiography. To assess cardiac hypertrophy and fibrosis, histological and molecular analyses and measurement of ATP concentration in mouse hearts were performed.By applying TAC, the survival rate was significantly lower in KO than that in WT mice. After TAC, KO mice showed significantly higher heart weight-to-tibial length ratio and larger cross-sectional area of cardiomyocytes than those of WT. Although left ventricular (LV) wall thickness in the KO mice was similar to that in the WT mice, the KO mice showed a significant enlargement of LV cavity and reduced LV fractional shortening compared to the WT mice with TAC. A tendency for decreased myocardial ATP concentration was observed in the KO mice compared to the WT mice after TAC operation.These data suggest that the LCFA transporter CD36 is required for the maintenance of energy provision, systolic function, and myocardial structure.
Assuntos
Antígenos CD36/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD36/fisiologia , Metabolismo Energético/fisiologia , Fibrose , Glucose/metabolismo , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Miócitos Cardíacos/patologia , Pressão/efeitos adversos , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/fisiopatologia , Remodelação VentricularRESUMO
Although azithromycin can suppress cardiac INa, IKr, IKs, ICa,L and IK1, its onset mechanisms for cardiovascular death have not been fully investigated. We examined electropharmacological effects of azithromycin in intravenous doses of 0.3, 3 and 30 mg/kg using microminipigs under the halothane anesthesia (n = 4), which provided plasma concentrations of 3.1, 11.2 and 120.4 µg/mL, respectively. The low dose did not alter any of the cardiohemodynamic or electrocardiographic variables. The middle dose significantly shortened QT interval for 10-20 min and QTc for 10-30 min. The high dose significantly decreased mean blood pressure for 5-60 min, prolonged QRS width at 20 min, but shortened QT interval for 15-20 min and QTc for 15-30 min (n = 3). Cardiohemodynamic collapse occurred in 1 animal after the start of the high dose infusion, which might be associated with the cardiovascular death in patients with vasomotor dysfunction. Prolongation of QRS width indicates that azithromycin may suppress ventricular INa in vivo, which may unmask latent type of Brugada electrocardiographic genotype. Meanwhile, abbreviation of the QTc might cause potentially lethal, short QT-related, cardiac arrhythmia syndrome. These findings with microminipigs suggest the possible entry point for analyzing the mechanisms of cardiovascular death clinically seen with this antibiotic.
Assuntos
Azitromicina/toxicidade , Pressão Sanguínea/efeitos dos fármacos , Doenças Cardiovasculares/induzido quimicamente , Eletrocardiografia/efeitos dos fármacos , Animais , Azitromicina/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Suínos , Porco MiniaturaRESUMO
Accumulating data suggest that new cardiomyocytes in adults are generated from existing cardiomyocytes throughout life. To enhance the endogenous cardiac regeneration, we performed chemical screenings to identify compounds that activate pro-proliferative YES-associated protein and transcriptional enhancer factor domain activities in cardiomyocytes. We synthesized a novel fluorine-containing TT-10 (C11H10FN3OS2) from the biologically hit compound. TT-10 promoted cardiomyocyte proliferation and simultaneously exerted antioxidant and antiapoptotic effects in vitro. TT-10 treatment in mice ameliorated myocardial infarction-induced cardiac dysfunction at least in part via enhancing clonal expansion of existing cardiomyocytes with nuclear YES-associated protein expression. Stimulating cardiomyocyte proliferation and/or protection with TT-10 might complement current therapies for myocardial infarction.
RESUMO
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