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1.
Biochim Biophys Acta Mol Basis Dis ; 1863(11): 2964-2972, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28754452

RESUMEN

Dilated cardiomyopathy (DCM) is cardiac disease characterized by increased left ventricular chamber volume and decreased systolic function. DCM patient-specific human induced-pluripotent stem cells-derived cardiomyocytes (DCM-hiPSC-CMs) were generated. We found that uniaxial stretch elicited a cytosolic [Ca2+]i rise in hiPSC-CMs. Compared to control-hiPSC-CMs, DCM-hiPSC-CMs displayed a greater magnitude of [Ca2+]i responses to the cell stretch of 10-15% elongation in length. This stretch-induced [Ca2+]i rise was abolished by removal of extracellular Ca2+ and markedly attenuated by TRPV4 inhibitors HC-067047 and RN-1734. Application of nifedipine and tranilast also reduced the [Ca2+]i response but to a lesser degree. Moreover, the augmented [Ca2+]i was decreased by cytochalasin D treatment. Taken together, our study for the first time demonstrated an abnormal TRPV4-related mechanosensitive Ca2+ signaling in DCM-hiPSC-CMs.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Citosol/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mecanotransducción Celular , Miocitos Cardíacos/metabolismo , Canales Catiónicos TRPV/metabolismo , Cardiomiopatía Dilatada/patología , Citosol/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Miocitos Cardíacos/patología
2.
Stem Cells ; 34(11): 2693-2706, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27333945

RESUMEN

Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3ß inhibitor-(2'Z,3'E)- 6-Bromoindirubin-3'-oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 × 106 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3ß inhibitor-6-Bromoindirubin-3'-oxime and Oncostatin M may represent a novel source for cell therapy in MI. Stem Cells 2016;34:2693-2706.


Asunto(s)
Reprogramación Celular , Fibroblastos/metabolismo , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Regeneración/fisiología , Esferoides Celulares/trasplante , Potenciales de Acción , Animales , Calcio/metabolismo , Diferenciación Celular , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Indoles/farmacología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oncostatina M/farmacología , Oximas/farmacología , Cultivo Primario de Células , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismo , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Transducción Genética , Función Ventricular Izquierda/fisiología
3.
Cardiovasc Res ; 117(3): 767-779, 2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-32173736

RESUMEN

AIMS: In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action. METHODS AND RESULTS: We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects. CONCLUSION: Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway.


Asunto(s)
Genes Modificadores , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/genética , Mutación , Polimorfismo de Nucleótido Simple , Proteínas Tirosina Fosfatasas no Receptoras/genética , Células Cultivadas , Predisposición Genética a la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Canal de Potasio KCNQ1/metabolismo , Síndrome de QT Prolongado/metabolismo , Miocitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Fenotipo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteolisis
4.
Stem Cell Res ; 49: 102090, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33232900

RESUMEN

GADD45A is a DNA damage and stressful growth arrest inducible protein, also it is shown to a be tumor suppressor gene and a chromatin relaxer associated with opening chromatin during the somatic reprogramming. However, its role in human embryonic stem cells and human embryonic stem cell modeled development has been merely documented. To illustrate the function of GADD45A in the human embryonic stem cell biology, we reported a GADD45A knockout human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting. This cell line displayed normal karyotype, pluripotent stem cell marker expression and differentiation potential both in vivo and vitro.


Asunto(s)
Línea Celular , Células Madre Embrionarias Humanas , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular , Técnicas de Inactivación de Genes , Humanos
5.
Stem Cell Res ; 50: 102120, 2020 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-33352530

RESUMEN

The human transcription factor NKX2-5 plays an important role in cardiac formation and development, and thus it can be used for isolation of cardiomyocytes (CMs) differentiated from human pluripotent stem cells (hPSCs). Here, we knocked-in enhanced GFP (eGFP) and Pac (a puromycin resistant gene; PuroR) into the exon 1 coding region of NKX2-5 from a human iPSC line iPSN0003 using TALENs. The generated GIBHi002-A-2 enables us to monitor and optimize cardiac differentiation procedures via the cardiac progenitor cells (CPCs), as well as to isolate iPSC-derived CMs for drug screening.

6.
Sci Rep ; 8(1): 10846, 2018 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-30022097

RESUMEN

Targeted next generation sequencing of gene panels has become a popular tool for the genetic diagnosis of hypertrophic (HCM) and dilated cardiomyopathy (DCM). However, it is uncertain whether the use of Whole Exome Sequencing (WES) represents a more effective approach for diagnosis of cases with HCM and DCM. In this study, we performed indirect comparisons of the coverage and diagnostic yield of WES on genes and variants related to HCM and DCM versus 4 different commercial gene panels using 40 HCM and DCM patients, assuming perfect coverage in those panels. We identified 6 pathogenic or likely pathogenic among 14 HCM patients (diagnostic yield 43%). 3 pathogenic or likely pathogenic were found among the 26 DCM patients (diagnostic yield 12%). The coverage was similar to that of four existing commercial gene panels due to the clustering of mutation within MYH7, MYBPC3, TPM1, TNT2, and TTN. Moreover, the coverage of WES appeared inadequate for TNNI3 and PLN. We conclude that most of the pathogenic variants for HCM and DCM can be found within a small number of genes which were covered by all the commercial gene panels, and the application of WES did not increase diagnostic yield.


Asunto(s)
Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Hipertrófica/diagnóstico , Secuenciación del Exoma/métodos , Marcadores Genéticos , Mutación , Análisis de Secuencia de ADN/métodos , Cardiomiopatía Dilatada/genética , Cardiomiopatía Hipertrófica/genética , Femenino , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad
7.
Stem Cell Res Ther ; 7(1): 139, 2016 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-27649756

RESUMEN

Laminopathy is a disease closely related to deficiency of the nuclear matrix protein lamin A/C or failure in prelamin A processing, and leads to accumulation of the misfold protein causing progeria. The resultant disrupted lamin function is highly associated with abnormal nuclear architecture, cell senescence, apoptosis, and unstable genome integrity. To date, the effects of loss in nuclear integrity on the susceptible organ, striated muscle, have been commonly associated with muscular dystrophy, dilated cardiac myopathy (DCM), and conduction defeats, but have not been studied intensively. In this review, we aim to summarize recent breakthroughs in an in vivo laminopathy model and in vitro study using patient-specific human induced pluripotent stem cells (iPSCs) that reproduce the pathophysiological phenotype for further drug screening. We describe several in-vivo transgenic mouse models to elucidate the effects of Lmna H222P, N195K mutations, and LMNA knockout on cardiac function, in terms of hemodynamic and electrical signal propagation; certain strategies targeted on stress-related MAPK are mentioned. We will also discuss human iPSC cardiomyocytes serving as a platform to reveal the underlying mechanisms, such as the altered mechanical sensation in electrical coupling of the heart conduction system and ion channel alternation in relation to altered nuclear architecture, and furthermore to enable screening of drugs that can attenuate this cardiac premature aging phenotype by inhibition of prelamin misfolding and oxidative stress, and also enhancement of autophagy protein clearance and cardiac-protective microRNA.


Asunto(s)
Cardiomiopatía Dilatada/genética , Lamina Tipo A/genética , Modelos Cardiovasculares , Mutación , Progeria/genética , Deficiencias en la Proteostasis/genética , Animales , Cardiomiopatía Dilatada/tratamiento farmacológico , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiotónicos/farmacología , Expresión Génica , Inestabilidad Genómica , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/metabolismo , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Lamina Tipo A/deficiencia , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Progeria/tratamiento farmacológico , Progeria/metabolismo , Progeria/patología , Pliegue de Proteína , Deficiencias en la Proteostasis/tratamiento farmacológico , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología
8.
J Toxicol Sci ; 36(6): 713-24, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22129735

RESUMEN

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs) and similar compounds are toxic to animals and humans. Based on a yeast reporter system, AhR-activating ligands similar in concentration to 2 ng/l of TCDD were detected in two canal waters in Guangzhou, China. In this study, a three-generation experiment was conducted to assess the reproductive and developmental risks associated with these waters in C57BL/6J female mice, including female reproduction, pup indices, reproductive hormone levels, and levels of AhR, ARNT, and CYP1A2 in the uterus. Similar reproductive toxic effects were produced in the offspring of mice that drank the canal water as would occur if they drank 2 ng/l/day TCDD. The major reproductive indices that were affected included mating time and gestation length over all the generations. A striking finding is the TCDD (2 ng/l) and the water samples significantly reduced Day 4 pup survival rates in the F2 and F3. Both TCDD exposure and drinking canal water decreased estradiol-17ß (E2) levels in the multiparous females and decreased follicle-stimulating hormone (FSH), luteinizing hormone (LH) and E2 levels in the virgin females. Immunochemical staining revealed that the AhR and CYP1A2 positive signals were enhanced, and the ARNT positive signal was weakened in the uteri of mice drinking water with TCDD (2 ng/l) and the canal water samples. These results imply that the canal water contains AhR ligands that could induce similar toxic effects as do low levels of TCDD. Exposure to these contaminants can significantly impair the reproductive health of female mice. Considering this canals are open directly to Pearl River, whether these effects could be caused in human reproduction and development warrants further study.


Asunto(s)
Disruptores Endocrinos/toxicidad , Exposición Materna , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Contaminantes Químicos del Agua/toxicidad , Animales , Proteínas Portadoras/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Drenaje de Agua , Estradiol/sangre , Femenino , Proteínas Fetales/metabolismo , Hormona Folículo Estimulante/sangre , Hormona Luteinizante/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos , Dibenzodioxinas Policloradas/análogos & derivados , Embarazo , Reproducción/efectos de los fármacos , Útero/efectos de los fármacos , Útero/metabolismo
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