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1.
Artigo em Inglês | MEDLINE | ID: mdl-32710379

RESUMO

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

3.
FASEB J ; 34(1): 555-570, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914652

RESUMO

Muscle wasting, or muscle atrophy, can occur with age, injury, and disease; it affects the quality of life and complicates treatment. Insulin-like growth factor 1 (IGF1) is a key positive regulator of muscle mass. The IGF1/Igf1 gene encodes multiple protein isoforms that differ in tissue expression, potency, and function, particularly in cellular proliferation and differentiation, as well as in systemic versus localized signaling. Genome engineering is a novel strategy for increasing gene expression and has the potential to recapitulate the diverse biology seen in IGF1 signaling through the overexpression of multiple IGF1 isoforms. Using a CRISPR-Cas9 gene activation approach, we showed that the expression of multiple IGF1 or Igf1 mRNA variants can be increased in human and mouse skeletal muscle myoblast cell lines using a single-guide RNA (sgRNA). We found increased IGF1 protein levels in the cell culture media and increased cellular phosphorylation of AKT1, the main effector of IGF1 signaling. We also showed that the expression of Class 1 or Class 2 mRNA variants can be selectively increased by changing the sgRNA target location. The expression of multiple IGF1 or Igf1 mRNA transcript variants in human and mouse skeletal muscle myoblasts promoted myotube differentiation and prevented dexamethasone-induced atrophy in myotubes in vitro. Our findings suggest that this novel approach for enhancing IGF1 signaling has potential therapeutic applications for treating skeletal muscle atrophy.


Assuntos
Sistemas CRISPR-Cas , Diferenciação Celular , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/citologia , Atrofia Muscular/patologia , Mioblastos/citologia , Ativação Transcricional , Animais , Anti-Inflamatórios/farmacologia , Sequência de Bases , Proliferação de Células , Células Cultivadas , Dexametasona/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Fosforilação , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência , Transdução de Sinais
4.
Cardiovasc Eng Technol ; 11(2): 205-218, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31916039

RESUMO

PURPOSE: The objective of this study was to bioengineer 3D patches from cardiac myocytes that have been reprogrammed from human adipogenic mesenchymal stem cells (hADMSCs). METHODS: Human adipogenic mesenchymal stem cells (hADMSCs) were reprogrammed to form cardiac myocytes using transcription factors ETS2 and MESP1. Reprogrammed cardiac myocytes were cultured in a fibrin gel to bioengineer 3D patch patches. The effect of initial plating density (1-25 million cells per patch), time (28-day culture period) and treatment with 1 µM isoproterenol and 1 µM epinephrine were evaluated. RESULTS: 3D patches were fabricated using cardiac myocytes that have been reprogrammed from hADMSCs. Based on optimization studies, it was determined that 10 million cells were needed to bioengineer a single patch, that measured 2 × 2 cm2. Furthermore, 3D patches fabricated 10 million cells were stable in culture for up to 28 days. Treatment of 3D patches with 1 µM isoproterenol and 1 µM epinephrine resulted in an increase in the electrical properties, as measured by electrical impulse amplitude and frequency. An increase in the expression of mTOR, KCNV1, GJA5, KCNJ16, CTNNT2, KCNV2, MYO3, FOXO1 and KCND2 was noted in response to treatment of 3D patches with isoproterenol and epinephrine. CONCLUSION: Based on the results of this study, there is evidence to support the successful fabrication of a highly functional 3D patches with measurable electrical activity using cardiac myocytes reprogrammed from hADMSCs. 3D patches fabricated using optimal conductions described in this study can be used to improve the functional properties of failing hearts. Predominantly, in case of the infarcted hearts with partial loss of electrical activity, the electrical properties of the 3D patches may restore the electrical activity of the heart.

5.
J Mol Cell Cardiol ; 138: 12-22, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31678351

RESUMO

We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

6.
J Tissue Eng Regen Med ; 14(2): 306-318, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31821703

RESUMO

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, ß-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, ß2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/ß, PCG 1α/ß, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.

7.
EBioMedicine ; 50: 55-66, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31761621

RESUMO

BACKGROUND: Mesoderm Posterior 1 (MESP1) belongs to the family of basic helix-loop-helix transcription factors. It is a master regulator of mesendoderm development, leading to formation of organs such as heart and lung. However, its role in adult pathophysiology remains unknown. Here, we report for the first time a previously-unknown association of MESP1 with non-small cell lung cancer (NSCLC). METHODS: MESP1 mRNA and protein levels were measured in NSCLC-derived cells by qPCR and immunoblotting respectively. Colony formation assay, colorimetric cell proliferation assay and soft agar colony formation assays were used to assess the effects of MESP1 knockdown and overexpression in vitro. RNA-sequencing and chromatin immunoprecipitation (ChIP)-qPCR were used to determine direct target genes of MESP1. Subcutaneous injection of MESP1-depleted NSCLC cells in immuno-compromised mice was done to study the effects of MESP1 mediated tumor formation in vivo. FINDINGS: We found that MESP1 expression correlates with poor prognosis in NSCLC patients, and is critical for proliferation and survival of NSCLC-derived cells, thus implicating MESP1 as a lung cancer oncogene. Ectopic MESP1 expression cooperates with loss of tumor suppressor ARF to transform murine fibroblasts. Xenografts from MESP1-depleted cells showed decreased tumor growth in vivo. Global transcriptome analysis revealed a MESP1 DNA-binding-dependent gene signature associated with various hallmarks of cancer, suggesting that transcription activity of MESP1 is most likely responsible for its oncogenic abilities. INTERPRETATION: Our study demonstrates MESP1 as a previously-unknown lineage-survival oncogene in NSCLC which may serve as a potential prognostic marker and therapeutic target for lung cancer in the future.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Transformação Celular Neoplásica/genética , Regulação da Expressão Gênica , Neoplasias Pulmonares/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Biologia Computacional/métodos , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos
9.
Fundam Clin Pharmacol ; 33(1): 25-30, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29959870

RESUMO

Inbred mouse strains are the most widely used mammalian model organism in biomedical research owing to ease of genetic manipulation and short lifespan; however, each inbred strain possesses a unique repertoire of deleterious homozygous alleles that can make a specific strain more susceptible to a particular disease. In the current study, we report dystrophic cardiac calcinosis (DCC) in C.B-17 SCID male mice at 10 weeks of age with no significant change in cardiac function. Acquisition of DCC was characterized by myocardial injury, fibrosis, calcification, and necrosis of the tissue. At 10 weeks of age, 38% of the C.B-17 SCID mice from two different commercial colonies exhibited significant calcinosis on the ventricular epicardium, predominantly on the right ventricle. The frequency of calcinosis was more than 50% for mice obtained from Taconic's Cambridge City colony and 25% for mice obtained from Taconic's German Town colony. Interestingly, the DCC phenotype did not affect cardiac function at 10 weeks of age. No differences in echocardiography or electrocardiography were observed between the calcinotic and non-calcinotic mice from either colony. Our findings suggest that C.B-17 SCID mice exhibit DCC as early as 10 weeks of age with no significant impact on cardiac function. This strain of mice should be cautiously considered for the study of cardiac physiology.


Assuntos
Calcinose/patologia , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Pericárdio/patologia , Animais , Ecocardiografia/métodos , Eletrocardiografia/métodos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Fenótipo
10.
Dis Model Mech ; 11(9)2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30061196

RESUMO

Deletions of chromosome 1p36 are associated with a high incidence of congenital heart defects (CHDs). The arginine-glutamic acid dipeptide repeats gene (RERE) is located in a critical region for CHD on chromosome 1p36 and encodes a cardiac-expressed nuclear receptor co-regulator. Mutations affecting RERE cause atrial and ventricular septal defects (VSDs) in humans, and RERE-deficient mice also develop VSDs. During cardiac development, mesenchymal cells destined to form part of the atrioventricular (AV) septum are generated when endocardial cells in the AV canal undergo epithelial-to-mesenchymal transition (EMT) and migrate into the space between the endocardium and the myocardium. These newly generated mesenchymal cells then proliferate to fill the developing AV endocardial cushions. Here, we demonstrate that RERE-deficient mouse embryos have reduced numbers of mesenchymal cells in their AV endocardial cushions owing to decreased levels of EMT and mesenchymal cell proliferation. In the endocardium, RERE colocalizes with GATA4, a transcription factor required for normal levels of EMT and mesenchymal cell proliferation. Using a combination of in vivo and in vitro studies, we show that Rere and Gata4 interact genetically in the development of CHDs, RERE positively regulates transcription from the Gata4 promoter and GATA4 levels are reduced in the AV canals of RERE-deficient embryos. Tissue-specific ablation of Rere in the endocardium leads to hypocellularity of the AV endocardial cushions, defective EMT and VSDs, but does not result in decreased GATA4 expression. We conclude that RERE functions in the AV canal to positively regulate the expression of GATA4, and that deficiency of RERE leads to the development of VSDs through its effects on EMT and mesenchymal cell proliferation. However, the cell-autonomous role of RERE in promoting EMT in the endocardium must be mediated by its effects on the expression of proteins other than GATA4.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Transporte/metabolismo , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica no Desenvolvimento , Comunicação Interventricular/embriologia , Comunicação Interventricular/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas Repressoras/deficiência , Alelos , Animais , Proliferação de Células , Embrião de Mamíferos/metabolismo , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Coxins Endocárdicos/patologia , Endocárdio/embriologia , Endocárdio/metabolismo , Endocárdio/patologia , Transição Epitelial-Mesenquimal/genética , Fator de Transcrição GATA4/metabolismo , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/genética
11.
J Biol Chem ; 293(30): 11659-11673, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29891551

RESUMO

Epigenetic regulation is critical in normal cardiac development. We have demonstrated that the deletion of Jarid2 (Jumonji (Jmj) A/T-rich interaction domain 2) in mice results in cardiac malformations recapitulating human congenital cardiac disease and dysregulation of gene expression. However, the precise developmental and epigenetic functions of Jarid2 within the developing heart remain to be elucidated. Here, we determined the cardiac-specific functions of Jarid2 and the genetic networks regulated by Jarid2. Jarid2 was deleted using different cardiac-specific Cre mice. The deletion of Jarid2 by Nkx2.5-Cre mice (Jarid2Nkx) caused cardiac malformations including ventricular septal defects, thin myocardium, hypertrabeculation, and neonatal lethality. Jarid2Nkx mice exhibited elevated expression of neural genes, cardiac jelly, and other key factors including Isl1 and Bmp10 in the developing heart. By employing combinatorial genome-wide approaches and molecular analyses, we showed that Jarid2 in the myocardium regulates a subset of Jarid2 target gene expression and H3K27me3 enrichment during heart development. Specifically, Jarid2 was required for PRC2 occupancy and H3K27me3 at the Isl1 promoter locus, leading to the proper repression of Isl1 expression. In contrast, Jarid2 deletion in differentiated cardiomyocytes by cTnt-Cre mice caused no gross morphological defects or neonatal lethality. Thus, the early deletion of Jarid2 in cardiac progenitors, prior to the differentiation of cardiac progenitors into cardiomyocytes, results in morphogenetic defects manifested later in development. Our studies reveal that there is a critical window during early cardiac progenitor differentiation when Jarid2 is crucial to establish the epigenetic landscape at later stages of development.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Coração/embriologia , Complexo Repressor Polycomb 2/genética , Animais , Desenvolvimento Embrionário , Feminino , Deleção de Genes , Redes Reguladoras de Genes , Cardiopatias Congênitas/patologia , Código das Histonas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia
13.
Stem Cells ; 35(8): 1913-1923, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28580685

RESUMO

During cardiac development, DNA binding transcription factors and epigenetic modifiers regulate gene expression in cardiac progenitor cells (CPCs). We have previously shown that Yin Yang 1 (YY1) is essential for the commitment of mesodermal precursors into CPCs. However, the role of YY1 in the maintenance of CPC phenotype and their differentiation into cardiomyocytes is unknown. In this study, we found, by genome-wide transcriptional profiling and phenotypic assays, that YY1 overexpression prevents cardiomyogenic differentiation and maintains the proliferative capacity of CPCs. We show further that the ability of YY1 to regulate CPC phenotype is associated with its ability to modulate histone modifications specifically at a developmentally critical enhancer of Nkx2-5 and other key cardiac transcription factor such as Tbx5. Specifically, YY1 overexpression helps to maintain markers of gene activation such as the acetylation of histone H3 at lysine 9 (H3K9Ac) and lysine 27 (H3K27Ac) as well as trimethylation at lysine 4 (H3K4Me3) at the Nkx2-5 cardiac enhancer. Furthermore, transcription factors associated proteins such as PoIII, p300, and Brg1 are also enriched at the Nkx2-5 enhancer with YY1 overexpression. The biological activities of YY1 in CPCs appear to be cell autonomous, based coculture assays in differentiating embryonic stem cells. Altogether, these results demonstrate that YY1 overexpression is sufficient to maintain a CPC phenotype through its ability to sustain the presence of activating epigenetic/chromatin marks at key cardiac enhancers. Stem Cells 2017;35:1913-1923.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Miocárdio/citologia , Fator de Transcrição YY1/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Mutação com Ganho de Função , Regulação da Expressão Gênica , Proteína Homeobox Nkx-2.5/genética , Camundongos
14.
J Cell Sci ; 130(15): 2551-2563, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600325

RESUMO

Nucleosome assembly proceeds through DNA replication-coupled or replication-independent mechanisms. For skeletal myocytes, whose nuclei have permanently exited the cell cycle, replication-independent assembly is the only mode available for chromatin remodeling. For this reason, any nucleosome composition alterations accompanying transcriptional responses to physiological signals must occur through a DNA replication-independent pathway. HIRA is the histone chaperone primarily responsible for replication-independent incorporation of histone variant H3.3 across gene bodies and regulatory regions. Thus, HIRA would be expected to play an important role in epigenetically regulating myocyte gene expression. The objective of this study was to determine the consequence of eliminating HIRA from mouse skeletal myocytes. At 6 weeks of age, myofibers lacking HIRA showed no pathological abnormalities; however, genes involved in transcriptional regulation were downregulated. By 6 months of age, myofibers lacking HIRA exhibited hypertrophy, sarcolemmal perforation and oxidative damage. Genes involved in muscle growth and development were upregulated, but those associated with responses to cellular stresses were downregulated. These data suggest that elimination of HIRA produces a hypertrophic response in skeletal muscle and leaves myofibers susceptible to stress-induced degeneration.


Assuntos
Proteínas de Ciclo Celular/deficiência , Chaperonas de Histonas/deficiência , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Estresse Oxidativo , Fatores de Transcrição/deficiência , Animais , Hipertrofia , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Doenças Musculares/genética , Doenças Musculares/patologia
15.
ASAIO J ; 63(3): 333-341, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28459744

RESUMO

Development of tissue-engineered hearts for treatment of myocardial infarction or biologic pacemakers has been hindered by the production of mostly arrhythmic or in-synergistic constructs. Electrical stimulation (ES) of these constructs has been shown to produce tissues with greater twitch force and better adrenergic response. To further our understanding of the mechanisms underlying the effect of ES, we fabricated a bioreactor capable of delivering continuous or intermittent waveforms of various types to multiple constructs simultaneously. In this study, we examined the effect of an intermittent biphasic square wave on our artificial heart muscle (AHM) composed of neonatal rat cardiac cells and fibrin gel. Twitch forces, spontaneous contraction rates, biopotentials, gene expression profiles, and histologic observations were examined for the ES protocol over a 12 day culture period. We demonstrate improved consistency between samples for twitch force and contraction rate, and higher normalized twitch force amplitudes for electrically stimulated AHMs. Improvements in electrophysiology within the AHM were noted by higher conduction velocities and lower latency in electrical response for electrically stimulated AHMs. Genes expressing key electrophysiologic and structural markers peaked at days 6 and 8 of culture, only a few days after the initiation of ES. These results may be used for optimization strategies to establish protocols for producing AHMs capable of replacing damaged heart tissue in either a contractile or electrophysiologic capacity. Optimized AHMs can lead to alternative treatments to heart failure and alleviate the limited donor supply crisis.


Assuntos
Coração Artificial , Miócitos Cardíacos/citologia , Animais , Reatores Biológicos , Estimulação Elétrica , Expressão Gênica , Insuficiência Cardíaca/terapia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Ratos , Ratos Sprague-Dawley
16.
Nucleic Acids Res ; 45(6): 3046-3058, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-27940555

RESUMO

The expression of nearly all smooth muscle genes are controlled by serum response factor binding sites in their promoter regions. However, SRF alone is not sufficient for regulating smooth muscle cell development. It associates with other cardiovascular specific cofactors to regulate smooth muscle gene expression. Previously, we showed that the transcription co-factor CRP2 was a regulator of smooth muscle gene expression. Here, we report that CSRP2BP, a coactivator for CRP2, is a histone acetyltransferase and a driver of smooth muscle gene expression. CSRP2BP directly interacted with SRF, CRP2 and myocardin. CSRP2BP synergistically activated smooth muscle gene promoters in an SRF-dependent manner. A combination of SRF, GATA6 and CRP2 required CSRP2BP for robust smooth muscle gene promoter activity. Knock-down of Csrp2bp in smooth muscle cells resulted in reduced smooth muscle gene expression. We conclude that the CSRP2BP histone acetyltransferase is a coactivator for CRP2 that works synergistically with SRF and myocardin to regulate smooth muscle gene expression.


Assuntos
Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Miócitos de Músculo Liso/metabolismo , Acetilação , Animais , Linhagem Celular , Núcleo Celular/enzimologia , Células Cultivadas , Cromatina/enzimologia , Expressão Gênica , Histonas/metabolismo , Humanos , Camundongos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ratos , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
18.
Sci Rep ; 6: 31457, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27538477

RESUMO

Mesp1 directs multipotential cardiovascular cell fates, even though it's transiently induced prior to the appearance of the cardiac progenitor program. Tracing Mesp1-expressing cells and their progeny allows isolation and characterization of the earliest cardiovascular progenitor cells. Studying the biology of Mesp1-CPCs in cell culture and ischemic disease models is an important initial step toward using them for heart disease treatment. Because of Mesp1's transitory nature, Mesp1-CPC lineages were traced by following EYFP expression in murine Mesp1(Cre/+); Rosa26(EYFP/+) ES cells. We captured EYFP+ cells that strongly expressed cardiac mesoderm markers and cardiac transcription factors, but not pluripotent or nascent mesoderm markers. BMP2/4 treatment led to the expansion of EYFP+ cells, while Wnt3a and Activin were marginally effective. BMP2/4 exposure readily led EYFP+ cells to endothelial and smooth muscle cells, but inhibition of the canonical Wnt signaling was required to enter the cardiomyocyte fate. Injected mouse pre-contractile Mesp1-EYFP+ CPCs improved the survivability of injured mice and restored the functional performance of infarcted hearts for at least 3 months. Mesp1-EYFP+ cells are bona fide CPCs and they integrated well in infarcted hearts and emerged de novo into terminally differentiated cardiac myocytes, smooth muscle and vascular endothelial cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Coração/diagnóstico por imagem , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos SCID , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Transcriptoma , Proteína Wnt3A/metabolismo
19.
Proc Natl Acad Sci U S A ; 113(34): 9551-6, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27512039

RESUMO

Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Embrião de Mamíferos , Perfilação da Expressão Gênica , Genes Reporter , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , MicroRNAs/metabolismo , Morfogênese/genética , Células-Tronco Embrionárias Murinas/citologia , Miócitos Cardíacos/citologia , Cultura Primária de Células , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transdução de Sinais
20.
Elife ; 52016 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-27331609

RESUMO

Transcription factors organize gene expression profiles by regulating promoter activity. However, the role of transcription factors after transcription initiation is poorly understood. Here, we show that the homeoprotein Nkx2-5 and the 5'-3' exonuclease Xrn2 are involved in the regulation of alternative polyadenylation (APA) during mouse heart development. Nkx2-5 occupied not only the transcription start sites (TSSs) but also the downstream regions of genes, serving to connect these regions in primary embryonic cardiomyocytes (eCMs). Nkx2-5 deficiency affected Xrn2 binding to target loci and resulted in increases in RNA polymerase II (RNAPII) occupancy and in the expression of mRNAs with long 3'untranslated regions (3' UTRs) from genes related to heart development. siRNA-mediated suppression of Nkx2-5 and Xrn2 led to heart looping anomaly. Moreover, Nkx2-5 genetically interacts with Xrn2 because Nkx2-5(+/-)Xrn2(+/-), but neither Nkx2-5(+/-)nor Xrn2(+/-), newborns exhibited a defect in ventricular septum formation, suggesting that the association between Nkx2-5 and Xrn2 is essential for heart development. Our results indicate that Nkx2-5 regulates not only the initiation but also the usage of poly(A) sites during heart development. Our findings suggest that tissue-specific transcription factors is involved in the regulation of APA.


Assuntos
Exorribonucleases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteína Homeobox Nkx-2.5/metabolismo , Poliadenilação , Animais , Exorribonucleases/genética , Proteína Homeobox Nkx-2.5/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout
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