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1.
Hum Mol Genet ; 2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34270708

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by severe, progressive muscle wasting. Cardiomyopathy has emerged as a leading cause of death in patients with DMD. The mechanisms contributing to DMD cardiac disease remain under investigation and specific therapies available are lacking. Our prior work has shown that DMD-iPSC derived cardiomyocytes (DMD-iCMs) are vulnerable to oxidative stress injury and chronic exposure to DMD secreted exosomes impaired the cell's ability to protect against stress. In this study, we sought to examine a mechanism by which DMD cardiac exosomes impair cellular response through altering important stress-responsive genes in the recipient cells. Here, we report that DMD-iCMs secrete exosomes containing altered microRNA (miR) profiles in comparison to healthy controls. In particular, miR-339-5p was upregulated in DMD-iCMs, DMD exosomes, and in mdx mouse cardiac tissue. Restoring dystrophin in DMD-iCMs improved the cellular response to stress and was associated with downregulation of miR-339-5p, suggesting that it is disease-specific. Knockdown of miR-339-5p was associated with increased expression of MDM2, GSK3A and MAP2K3, which are genes involved in important stress-responsive signaling pathways. Finally, knockdown of miR-339-5p led to mitochondrial protection and a reduction in cell death in DMD-iCMs, indicating miR-339-5p is involved in direct modulation of stress-responsiveness. Together, these findings identify a potential mechanism by which exosomal miR-339-5p may be modulating cell signaling pathways which are important for robust stress responses. Additionally, these exosomal miRs may provide important disease specific targets for future therapeutic advancements for the management and diagnosis of DMD cardiomyopathy.

2.
Methods Mol Biol ; 2319: 51-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331242

RESUMO

Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Desenvolvimento Muscular , Miócitos Cardíacos/citologia , Via de Sinalização Wnt , Imunofluorescência , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
3.
Dis Model Mech ; 13(11)2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188007

RESUMO

Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.

4.
J Muscle Res Cell Motil ; 41(4): 269-284, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31564031

RESUMO

microRNAs are short, (18-22 nt) non-coding RNAs involved in important cellular processes due to their ability to regulate gene expression at the post-transcriptional level. Exosomes are small (50-200 nm) extracellular vesicles, naturally secreted from a variety of living cells and are believed to mediate cell-cell communication through multiple mechanisms, including uptake in destination cells. Circulating microRNAs and exosome-derived microRNAs can have key roles in regulating muscle cell development and differentiation. Several microRNAs are highly expressed in muscle and their regulation is important for myocyte homeostasis. Changes in muscle associated microRNA expression are associated with muscular diseases including muscular dystrophies, inflammatory myopathies, and congenital myopathies. In this review, we aim to highlight the biology of microRNAs and exosomes as well as their roles in muscle health and diseases. We also discuss the potential crosstalk between skeletal and cardiac muscle through exosomes and their contents.

5.
Sci Rep ; 8(1): 16519, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410044

RESUMO

As mediators of intercellular communication, exosomes containing molecular cargo are secreted by cells and taken up by recipient cells to influence cellular phenotype and function. Here we have investigated the effects of exosomes in dystrophin-deficient (Dys) induced pluripotent stem cell derived cardiomyocytes (iCMs). Our data demonstrate that exosomes secreted from either wild type (WT) or Dys-iCMs protect the Dys-iCM from stress-induced injury by decreasing reactive oxygen species and delaying mitochondrial permeability transition pore opening to maintain the mitochondrial membrane potential and decrease cell death. The protective effects of exosomes were dependent on the presence of exosomal surface proteins and activation of ERK1/2 and p38 MAPK signaling. Based on our findings, the acute effects of exosomes on recipient cells can be initiated from exosome membrane proteins and not necessarily their internal cargo.


Assuntos
Distrofina/deficiência , Exossomos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Sistema de Sinalização das MAP Quinases , Mitocôndrias/metabolismo , Miócitos Cardíacos/citologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Distrofina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Potencial da Membrana Mitocondrial , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/metabolismo , Deleção de Sequência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Stem Cell Res ; 18: 33-36, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28395799

RESUMO

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from non-affected control subjects to use as a comparison group for the iPSC lines containing a Plasminogen Activator Inhibitor-1 (PAI-1 homozygous/heterozygous) mutation. The Sendai Virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the UCs.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Urina/citologia , Sequência de Bases , Técnicas de Cultura de Células/métodos , Linhagem Celular , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Genótipo , Heterozigoto , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Vírus Sendai/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Stem Cell Res ; 18: 41-44, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28395801

RESUMO

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from subjects heterozygous for a novel Plasminogen Activator Inhibitor-1 (PAI-1) mutation. The Sendai Virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the PAI-1 UCs.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Inibidor 1 de Ativador de Plasminogênio/genética , Urina/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Polimorfismo Genético , Vírus Sendai/genética
9.
J Cardiovasc Transl Res ; 10(3): 295-304, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28032312

RESUMO

Heart failure with preserved left ventricular ejection fraction (HFpEF) has emerged as one of the largest unmet needs in cardiovascular medicine. HFpEF is increasing in prevalence and causes significant morbidity, mortality, and health care resource utilization. Patients have multiple co-morbidities which contribute to the disease complexity. To date, no effective treatment for HFpEF has been identified. The paucity of cardiac biopsies from this patient population and the absence of well-accepted animal models limit our understanding of the underlying molecular mechanisms of HFpEF. In this review, we discuss combining state-of-the-art technologies of microRNA profiling and human induced pluripotent cell-derived cardiomyocytes (iPSC-CMs) in order to uncover novel molecular pathways that may contribute to the development of HFpEF. Here, we focus the advantages and limitations of microRNA profiling and iPSC-CMs as a disease model system to discover molecular mechanisms in HFpEF.


Assuntos
Insuficiência Cardíaca/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Volume Sistólico , Animais , Linhagem Celular , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , MicroRNAs/genética , Miócitos Cardíacos/patologia , Fenótipo , Transdução de Sinais
10.
Stem Cell Res ; 17(3): 657-660, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27934602

RESUMO

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from subject with a novel homozygous Plasminogen Activator Inhibitor-1 (PAI-1 null) mutation. The Sendai virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the PAI-1 UCs.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Inibidor 1 de Ativador de Plasminogênio/genética , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Análise Mutacional de DNA , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Feminino , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Mutagênese Insercional , Vírus Sendai/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Urina/citologia
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