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1.
Methods Mol Biol ; 2320: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302653

RESUMO

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) have been shown to have great potential to play a key role in investigating cardiac diseases in vitro. Multielectrode array (MEA) system is sometimes preferable to patch-clamp in electrophysiological experiments in terms of several advantages. Here we show our protocol of electrophysiological examinations using MEA.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Análise Serial de Tecidos/métodos , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Técnicas de Patch-Clamp/métodos
2.
Methods Mol Biol ; 2320: 121-133, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302654

RESUMO

Electrophysiological analysis of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a patch-clamp technique enables the most precise evaluation of electrophysiological properties in single cells. Compared to multielectrode array (MEA) and membrane voltage imaging, patch-clamp recordings offer quantitative measurements of action potentials, and the relevant ionic currents which are essential for the research of disease modeling of inherited arrhythmias, safety pharmacology, and drug discovery using hiPSC-CMs. In this chapter, we describe the detail flow of patch-clamp recordings in hiPSC-CMs.


Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp/métodos , Análise Serial de Tecidos/métodos , Potenciais de Ação/fisiologia , Arritmias Cardíacas/terapia , Células Cultivadas , Humanos
4.
Stem Cell Reports ; 13(2): 394-404, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31378668

RESUMO

For long QT syndrome (LQTS), recent progress in genome-sequencing technologies enabled the identification of rare genomic variants with diagnostic, prognostic, and therapeutic implications. However, pathogenic stratification of the identified variants remains challenging, especially in variants of uncertain significance. This study aimed to propose a phenotypic cell-based diagnostic assay for identifying LQTS to recognize pathogenic variants in a high-throughput manner suitable for screening. We investigated the response of LQT2-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) following IKr blockade using a multi-electrode array, finding that the response to IKr blockade was significantly smaller than in Control-iPSC-CMs. Furthermore, we found that LQT1-iPSC-CMs and LQT3-iPSC-CMs could be distinguished from Control-iPSC-CMs by IKs blockade and INa blockade, respectively. This strategy might be helpful in compensating for the shortcomings of genetic testing of LQTS patients.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Síndrome do QT Longo/diagnóstico , Miócitos Cardíacos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Adolescente , Adulto , Estudos de Casos e Controles , Diferenciação Celular , Criança , Feminino , Ensaios de Triagem em Larga Escala/métodos , Humanos , Síndrome do QT Longo/classificação , Masculino , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Adulto Jovem
5.
PLoS One ; 14(3): e0213768, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30875388

RESUMO

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy. The major symptoms of this condition are walking difficulties, dyspnea caused by progressive skeletal muscle weakness, and cardiomyopathy. Recent advances in ventilator support devices have dramatically decreased mortality caused by respiratory distress. Consequently, cardiomyopathy resulting in heart failure is currently the major cause of death among DMD patients. One mechanism by which skeletal muscle is damaged in DMD patients involves elevation of the intracellular Ca2+ concentration. By contrast, the mechanisms underlying the development of cardiomyopathy are unclear. To investigate this, we examined the intracellular Ca2+ concentration and calcium transients in cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). hiPSCs were derived from a DMD patient (DMD-hiPSCs), in whom exon 44 of the gene encoding dystrophin was deleted, and from his parents (control-hiPSCs), who did not carry this mutation. The intracellular Ca2+ concentration was measured using the fluorescent indicator indo-1. The fluorescence ratio (410/490 nm) of indo-1 at rest (R0), the peak of this ratio (Rmax), and the amplitude (Rmax-R0) were significantly higher in cardiomyocytes differentiated from DMD-hiPSCs than in those differentiated from control-hiPSCs. Moreover, mechanical stretching significantly increased the intracellular Ca2+ concentration in cardiomyocytes differentiated from DMD-hiPSCs, but not in those differentiated from control-hiPSCs. These findings indicate that elevation of the intracellular Ca2+ concentration can cause cardiac damage leading to cardiomyopathy in DMD patients.


Assuntos
Cálcio/metabolismo , Cardiomiopatias/patologia , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/patologia , Distrofia Muscular de Duchenne/patologia , Miócitos Cardíacos/patologia , Animais , Cardiomiopatias/metabolismo , Células Cultivadas , Pré-Escolar , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo
6.
Gen Thorac Cardiovasc Surg ; 67(7): 640-643, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30191531

RESUMO

Pediatric pulmonary hypertension after surgery for congenital heart disease is a significant complication. We present a case of living-donor lung transplantation for a 12-year-old girl with pulmonary hypertension after surgical repair of transposition of great arteries. Despite repairing the transposition of great arteries, her growth was severely restricted because of progressive pulmonary hypertension; thus, lung transplantation was discussed. Standard bilateral lobar transplantation seemed unfeasible due to oversized grafts, so we performed a single lobar transplantation. Unexpectedly, she developed complications and died 3 months postoperatively despite another emergent lobar transplantation. We discussed the challenges and potential solutions regarding lobar size mismatching.


Assuntos
Hipertensão Pulmonar/cirurgia , Transplante de Pulmão , Transposição dos Grandes Vasos/cirurgia , Criança , Evolução Fatal , Feminino , Transtornos do Crescimento/etiologia , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Doadores Vivos , Transplante de Pulmão/efeitos adversos , Complicações Pós-Operatórias/etiologia , Transposição dos Grandes Vasos/complicações
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