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1.
Sci Rep ; 10(1): 12060, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694738

RESUMO

Given the poor track record to date of animal models for creating cardioprotective drugs, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been proposed as a therapeutically relevant human platform to guide target validation and cardiac drug development. Mitogen-Activated Protein Kinase Kinase Kinase Kinase-4 (MAP4K4) is an "upstream" member of the MAPK superfamily that is implicated in human cardiac muscle cell death from oxidative stress, based on gene silencing and pharmacological inhibition in hPSC-CMs. A further role for MAP4K4 was proposed in heart muscle cell death triggered by cardiotoxic anti-cancer drugs, given its reported activation in failing human hearts with doxorubicin (DOX) cardiomyopathy, and its activation acutely by DOX in cultured cardiomyocytes. Here, we report successful protection from DOX in two independent hPSC-CM lines, using two potent, highly selective MAP4K4 inhibitors. The MAP4K4 inhibitors enhanced viability and reduced apoptosis at otherwise lethal concentrations of DOX, and preserved cardiomyocyte function, as measured by spontaneous calcium transients, at sub-maximal ones. Notably, in contrast, no intereference was seen in tumor cell killing, caspase activation, or mitochondrial membrane dissipation by DOX, in human cancer cell lines. Thus, MAP4K4 is a plausible, tractable, selective therapeutic target in DOX-induced human heart muscle cell death.

3.
Cell Stem Cell ; 24(4): 579-591.e12, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30853557

RESUMO

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.


Assuntos
Doxorrubicina/farmacologia , Infarto/tratamento farmacológico , Infarto/patologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Peróxido de Hidrogênio/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Infarto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Relação Estrutura-Atividade
4.
Nat Commun ; 9(1): 3123, 2018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30072790

RESUMO

The original version of this article contained an error in the spelling of Juan Pedro Martinez-Barbera, which was incorrectly given as Juan Pedro Martinez Barbera. This error has now been corrected in both the PDF and HTML versions of the Article.

5.
Opt Express ; 26(11): 13819-13840, 2018 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-29877429

RESUMO

We describe an approach that enables the design of optical systems for optimal performance when built, i.e., when user-selected tolerances and compensators are taken into account. The approach does not require significant raytracing or computing time beyond what is used to optimize the nominal design. The approach uses nodal aberration theory to describe the effects of decentered optics; double Zernike polynomials to describe and quantify system performance; and an analytic approach to determining the necessary compensation and residual wavefront error due to a tolerance. We design a triplet using this approach and compare its Monte-Carlo-modeled as-built performance to that of a conventionally-optimized design which optimizes only nominal performance. We also describe several extensions to the theory.

6.
Sci Rep ; 8(1): 8475, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29855498

RESUMO

Bone morphogenetic proteins (BMPs) activate the canonical Smad1/5/8 and non-canonical Tak1-MAPK pathways via BMP receptors I and II to regulate skeletal development and bone remodeling. Specific ablation of Bmpr1a in immature osteoblasts, osteoblasts, or osteocytes results in an increase in cancellous bone mass, yet opposite results have been reported regarding the underlying mechanisms. Moreover, the role for BMPRIA-mediated signaling in bone marrow mesenchymal stromal cells (BM-MSCs) has not been explored. Here, we specifically ablated Bmpr1a in BM-MSCs in adult mice to study the function of BMPR1A in bone remodeling and found that the mutant mice showed an increase in cancellous and cortical bone mass, which was accompanied by a decrease in bone formation rate and a greater decrease in bone resorption. Decreased bone formation was associated with a defect in BM-MSC osteogenic differentiation whereas decreased bone resorption was associated with a decrease in RANKL production and osteoclastogenesis. However, ablation of Tak1, a critical non-canonical signaling molecule downstream of BMP receptors, in BM-MSCs at adult stage did not affect bone remodeling. These results suggest that BMP signaling through BMPRIA controls BM-MSC osteogenic differentiation/bone formation and RANKL expression/osteoclastogenesis in adult mice independent of Tak1 signaling.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Diferenciação Celular , Ligante RANK/metabolismo , Animais , Células da Medula Óssea/citologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem da Célula , Proteínas de Homeodomínio/metabolismo , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligante RANK/genética , Transdução de Sinais
7.
Cell Rep ; 21(6): 1562-1573, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-29117561

RESUMO

Ronin (THAP11), a DNA-binding protein that evolved from a primordial DNA transposon by molecular domestication, recognizes a hyperconserved promoter sequence to control developmentally and metabolically essential genes in pluripotent stem cells. However, it remains unclear whether Ronin or related THAP proteins perform similar functions in development. Here, we present evidence that Ronin functions within the nascent heart as it arises from the mesoderm and forms a four-chambered organ. We show that Ronin is vital for cardiogenesis during midgestation by controlling a set of critical genes. The activity of Ronin coincided with the recruitment of its cofactor, Hcf-1, and the elevation of H3K4me3 levels at specific target genes, suggesting the involvement of an epigenetic mechanism. On the strength of these findings, we propose that Ronin activity during cardiogenesis offers a template to understand how important gene programs are sustained across different cell types within a developing organ such as the heart.


Assuntos
Coração/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Animais , Bradicardia/metabolismo , Bradicardia/fisiopatologia , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Imunoprecipitação da Cromatina , Ecocardiografia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Coração/diagnóstico por imagem , Histonas/genética , Histonas/metabolismo , Proteína Homeobox Nkx-2.5/deficiência , Proteína Homeobox Nkx-2.5/genética , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/metabolismo , Metilação , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Sítio de Iniciação de Transcrição
8.
Cell Stem Cell ; 21(2): 151-152, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28777937

RESUMO

Cardiomyocyte creation by human pluripotent stem cells (hPSCs) has generated opportunities for heart repair, disease modeling, and drug development. In this issue of Cell Stem Cell,Lee et al. (2017) report prospective markers of atrial versus ventricular myocyte formation from hPSCs and their use in directed differentiation of cardiac sub-lineages.


Assuntos
Miócitos Cardíacos , Células-Tronco Pluripotentes , Diferenciação Celular , Ventrículos do Coração , Humanos , Mesoderma , Estudos Prospectivos
9.
ACS Cent Sci ; 3(4): 338-348, 2017 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-28470052

RESUMO

Retention and survival of transplanted cells are major limitations to the efficacy of regenerative medicine, with short-term paracrine signals being the principal mechanism underlying current cell therapies for heart repair. Consequently, even improvements in short-term durability may have a potential impact on cardiac cell grafting. We have developed a multimodal hydrogel-based platform comprised of a poly(ethylene glycol) network cross-linked with bioactive peptides functionalized with Gd(III) in order to monitor the localization and retention of the hydrogel in vivo by magnetic resonance imaging. In this study, we have tailored the material for cardiac applications through the inclusion of a heparin-binding peptide (HBP) sequence in the cross-linker design and formulated the gel to display mechanical properties resembling those of cardiac tissue. Luciferase-expressing cardiac stem cells (CSC-Luc2) encapsulated within these gels maintained their metabolic activity for up to 14 days in vitro. Encapsulation in the HBP hydrogels improved CSC-Luc2 retention in the mouse myocardium and hind limbs at 3 days by 6.5- and 12- fold, respectively. Thus, this novel heparin-binding based, Gd(III)-tagged hydrogel and CSC-Luc2 platform system demonstrates a tailored, in vivo detectable theranostic cell delivery system that can be implemented to monitor and assess the transplanted material and cell retention.

10.
Dis Model Mech ; 10(3): 259-270, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28250051

RESUMO

Systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) show significant heart involvement and cardiovascular morbidity, which can be due to systemically increased levels of inflammation or direct autoreactivity targeting cardiac tissue. Despite high clinical relevance, cardiac damage secondary to systemic autoimmunity lacks inducible rodent models. Here, we characterise immune-mediated cardiac tissue damage in a new model of SLE induced by topical application of the Toll-like receptor 7/8 (TLR7/8) agonist Resiquimod. We observe a cardiac phenotype reminiscent of autoimmune-mediated dilated cardiomyopathy, and identify auto-antibodies as major contributors to cardiac tissue damage. Resiquimod-induced heart disease is a highly relevant mouse model for mechanistic and therapeutic studies aiming to protect the heart during autoimmunity.


Assuntos
Autoimunidade/efeitos dos fármacos , Cardiomiopatia Dilatada/induzido quimicamente , Imidazóis/efeitos adversos , Miocardite/induzido quimicamente , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Imunidade Adaptativa/efeitos dos fármacos , Transferência Adotiva , Animais , Autoanticorpos/sangue , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/imunologia , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Feminino , Variação Genética , Testes de Função Cardíaca , Imunidade Celular/efeitos dos fármacos , Inflamação/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Masculino , Mutação/genética , Miocardite/complicações , Miocardite/imunologia , Miocardite/fisiopatologia , Miocárdio/patologia , Baço/patologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo
12.
Development ; 143(9): 1435-41, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27143752

RESUMO

In February 2016, The Company of Biologists hosted an intimate gathering of leading international researchers at the forefront of experimental cardiovascular regeneration, with its emphasis on 'Transdifferentiation and Tissue Plasticity in Cardiovascular Rejuvenation'. As I review here, participants at the workshop revealed how understanding cardiac growth and lineage decisions at their most fundamental level has transformed the strategies in hand that presently energize the prospects for human heart repair.


Assuntos
Transdiferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Coração/crescimento & desenvolvimento , Regeneração/fisiologia , Transplante de Células-Tronco/métodos , Animais , Coração/fisiologia , Humanos , Linfangiogênese/fisiologia , Miócitos Cardíacos/citologia , Medicina Regenerativa , Células-Tronco/citologia
13.
NPJ Regen Med ; 1: 16001, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-29302333

RESUMO

The insulin-like growth factor Ea propeptide (IGF-1Ea) is a powerful enhancer of cardiac muscle growth and regeneration, also blocking age-related atrophy and beneficial in multiple skeletal muscle diseases. The therapeutic potential of IGF-1Ea compared with mature IGF-1 derives from its local action in the area of synthesis. We have developed an adeno-associated virus (AAV) vector for IGF-1Ea delivery to the heart to treat mice after myocardial infarction and examine the reparative effects of local IGF-1Ea production on left ventricular remodelling. A cardiotropic AAV9 vector carrying a cardiomyocyte-specific IGF-1Ea-luciferase bi-cistronic gene expression cassette (AAV9.IGF-1Ea) was administered intravenously to infarcted mice, 5 h after ischemia followed by reperfusion (I/R), as a model of myocardial infarction. Virally encoded IGF-1Ea in the heart improved global left ventricular function and remodelling, as measured by wall motion and thickness, 28 days after delivery, with higher viral titers yielding better improvement. The present study demonstrates that single intravenous AAV9-mediated IGF-1Ea Gene Therapy represents a tissue-targeted therapeutic approach to prevent the adverse remodelling after myocardial infarct.

14.
Mediators Inflamm ; 2015: 484357, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26491228

RESUMO

Strategies to limit damage and improve repair after myocardial infarct remain a major therapeutic goal in cardiology. Our previous studies have shown that constitutive expression of a locally acting insulin-like growth factor-1 Ea (IGF-1Ea) propeptide promotes functional restoration after cardiac injury associated with decreased scar formation. In the current study, we investigated the underlying molecular and cellular mechanisms behind the enhanced functional recovery. We observed improved cardiac function in mice overexpressing cardiac-specific IGF-1Ea as early as day 7 after myocardial infarction. Analysis of gene transcription revealed that supplemental IGF-1Ea regulated expression of key metalloproteinases (MMP-2 and MMP-9), their inhibitors (TIMP-1 and TIMP-2), and collagen types (Col 1α1 and Col 1α3) in the first week after injury. Infiltration of inflammatory cells, which direct the remodelling process, was also altered; in particular there was a notable reduction in inflammatory Ly6C+ monocytes at day 3 and an increase in anti-inflammatory CD206+ macrophages at day 7. Taken together, these results indicate that the IGF-1Ea transgene shifts the balance of innate immune cell populations early after infarction, favouring a reduction in inflammatory myeloid cells. This correlates with reduced extracellular matrix remodelling and changes in collagen composition that may confer enhanced scar elasticity and improved cardiac function.


Assuntos
Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Inflamação/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Células Mieloides/metabolismo , Infarto do Miocárdio/metabolismo , Animais , Antígenos Ly/metabolismo , Quimiocinas/metabolismo , Colágeno/metabolismo , Citocinas/metabolismo , Ecocardiografia , Citometria de Fluxo , Fator de Crescimento Insulin-Like I/imunologia , Lectinas Tipo C/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Transgênicos , Monócitos/citologia , Monócitos/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Superfície Celular/metabolismo , Fatores de Tempo
15.
Nature ; 525(7570): 479-85, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26375005

RESUMO

The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.


Assuntos
Proteínas Relacionadas à Folistatina/metabolismo , Miocárdio/metabolismo , Pericárdio/crescimento & desenvolvimento , Pericárdio/metabolismo , Regeneração , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Proteínas Relacionadas à Folistatina/genética , Humanos , Masculino , Camundongos , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/efeitos dos fármacos , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Pericárdio/citologia , Pericárdio/efeitos dos fármacos , Ratos , Regeneração/efeitos dos fármacos , Transdução de Sinais , Suínos , Transgenes/genética
17.
Biomaterials ; 70: 94-104, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26302234

RESUMO

Generation of de novo cardiomyocytes through viral over-expression of key transcription factors represents a highly promising strategy for cardiac muscle tissue regeneration. Although the feasibility of cell reprogramming has been proven possible both in vitro and in vivo, the efficiency of the process remains extremely low. Here, we report a chemical-free technique in which topographical cues, more specifically parallel microgrooves, enhance the directed differentiation of cardiac progenitors into cardiomyocyte-like cells. Using a lentivirus-mediated direct reprogramming strategy for expression of Myocardin, Tbx5, and Mef2c, we showed that the microgrooved substrate provokes an increase in histone H3 acetylation (AcH3), known to be a permissive environment for reprogramming by "stemness" factors, as well as stimulation of myocardin sumoylation, a post-translational modification essential to the transcriptional function of this key co-activator. These biochemical effects mimicked those of a pharmacological histone deacetylase inhibitor, valproic acid (VPA), and like VPA markedly augmented the expression of cardiomyocyte-specific proteins by the genetically engineered cells. No instructive effect was seen in cells unresponsive to VPA. In addition, the anisotropy resulting from parallel microgrooves induced cellular alignment, mimicking the native ventricular myocardium and augmenting sarcomere organization.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Dimetilpolisiloxanos/química , Dimetilpolisiloxanos/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Clonais , Histonas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Células da Side Population/citologia , Células da Side Population/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Transativadores/metabolismo , Ácido Valproico/farmacologia
18.
Circ Cardiovasc Genet ; 8(5): 643-52, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26175529

RESUMO

BACKGROUND: Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects. METHODS AND RESULTS: We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner. CONCLUSIONS: We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene.


Assuntos
Cardiomiopatias/genética , Insuficiência Cardíaca/genética , Proteínas Nucleares/genética , Animais , Proteínas de Ligação a DNA , Coração/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/fisiologia , Ratos , Estresse Fisiológico , Técnicas de Cultura de Tecidos , Ubiquitina-Proteína Ligases
19.
PLoS One ; 10(6): e0125384, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26047103

RESUMO

UNLABELLED: Adult cardiac stem cells (CSCs) express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd)-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT), and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains). MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized. IN SUMMARY: (1) GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2) Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3) Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.


Assuntos
Reprogramação Celular , Miócitos Cardíacos/citologia , Proteínas Nucleares/metabolismo , Células-Tronco/metabolismo , Proteínas com Domínio T/metabolismo , Transativadores/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Doxiciclina/toxicidade , Embrião de Mamíferos/metabolismo , Fator de Transcrição GATA4/metabolismo , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Lentivirus/genética , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Proteínas Nucleares/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Proteínas com Domínio T/genética , Transativadores/genética , Fatores de Transcrição/metabolismo
20.
Circ J ; 79(7): 1422-30, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26073608

RESUMO

Over the past 2 decades, cardiac regeneration has evolved from an exotic fringe of cardiovascular biology to the forefront of molecular, genetic, epigenetic, translational, and clinical investigations. The unmet patient need is the paucity of self-repair following infarction. Robust regeneration seen in models such as zebrafish and newborn mice has inspired the field, along with encouragement from modern methods that make even low levels of restorative growth discernible, changing the scientific and technical landscape for effective counter-measures. Approaches under study to augment cardiac repair complement each other, and encompass grafting cells of diverse kinds, restarting the cell cycle in post-mitotic ventricular myocytes, reprogramming non-myocytes, and exploiting the dormant progenitor/stem cells that lurk within the adult heart. The latter are the emphasis of the present review. Cardiac-resident stem cells (CSC) can be harvested from heart tissue, expanded, and delivered to the myocardium as a therapeutic product, whose benefits may be hoped to surpass those achieved in human trials of bone marrow. However, important questions are prompted by such cells' discovery. How do they benefit recipient hearts? Do they contribute, measurably, as an endogenous population, to self-repair? Even if "no," might CSCs be targets for activation in situ by growth factors and other developmental catalysts? And, what combination of distinguishing markers best demarcates the cells with robust clonal growth and cardiogenic potential?


Assuntos
Células-Tronco Adultas/citologia , Mioblastos Cardíacos/citologia , Adulto , Células-Tronco Adultas/fisiologia , Células-Tronco Adultas/transplante , Animais , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos , Perfilação da Expressão Gênica , Coração/fisiologia , Humanos , Camundongos , Modelos Animais , Modelos Cardiovasculares , Mioblastos Cardíacos/fisiologia , Mioblastos Cardíacos/transplante , Regeneração/fisiologia , Peixe-Zebra
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