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1.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
2.
Circ Heart Fail ; 12(3): e005529, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30798619

RESUMO

BACKGROUND: Proper dynamics of RNA polymerase II, such as promoter recruitment and elongation, are essential for transcription. PGC-1α (peroxisome proliferator-activated receptor [PPAR]-γ coactivator-1α), also termed PPARGC1a, is a transcriptional coactivator that stimulates energy metabolism, and PGC-1α target genes are downregulated in the failing heart. However, whether the dysregulation of polymerase II dynamics occurs in PGC-1α target genes in heart failure has not been defined. METHODS AND RESULTS: Chromatin immunoprecipitation-sequencing revealed that reduced promoter occupancy was a major form of polymerase II dysregulation on PGC-1α target metabolic gene promoters in the pressure-overload-induced heart failure model. PGC-1α-cKO (cardiac-specific PGC-1α knockout) mice showed phenotypic similarity to the pressure-overload-induced heart failure model in wild-type mice, such as contractile dysfunction and downregulation of PGC-1α target genes, even under basal conditions. However, the protein levels of PGC-1α were neither changed in the pressure-overload model nor in human failing hearts. Chromatin immunoprecipitation assays revealed that the promoter occupancy of polymerase II and PGC-1α was consistently reduced both in the pressure-overload model and PGC-1α-cKO mice. In vitro DNA binding assays using an endogenous PGC-1α target gene promoter sequence confirmed that PGC-1α recruits polymerase II to the promoter. CONCLUSIONS: These results suggest that PGC-1α promotes the recruitment of polymerase II to the PGC-1α target gene promoters. Downregulation of PGC-1α target genes in the failing heart is attributed, in part, to a reduction of the PGC-1α occupancy and the polymerase II recruitment to the promoters, which might be a novel mechanism of metabolic perturbations in the failing heart.


Assuntos
Insuficiência Cardíaca/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/genética , Animais , Modelos Animais de Doenças , Regulação para Baixo , Camundongos , Camundongos Knockout , RNA Polimerase II/metabolismo
3.
J Biol Chem ; 288(6): 3977-88, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23275380

RESUMO

Yap1 is an important regulator of cardiomyocyte proliferation and embryonic heart development, yet the function of endogenous Yap1 in the adult heart remains unknown. We studied the role of Yap1 in maintaining basal cardiac function and in modulating injury after chronic myocardial infarction (MI). Cardiomyocyte-specific homozygous inactivation of Yap1 in the postnatal heart (Yap(F/F)Cre) elicited increased myocyte apoptosis and fibrosis, dilated cardiomyopathy, and premature death. Heterozygous deletion (Yap(+/F)Cre) did not cause an overt cardiac phenotype compared with Yap(F/F) control mice at base line. In response to stress (MI), nuclear Yap1 was found selectively in the border zone and not in the remote area of the heart. After chronic MI (28 days), Yap(+/F)Cre mice had significantly increased myocyte apoptosis and fibrosis, with attenuated compensatory cardiomyocyte hypertrophy, and further impaired function versus Yap(+/F) control mice. Studies in isolated cardiomyocytes demonstrated that Yap1 expression is sufficient to promote increased cell size and hypertrophic gene expression and protected cardiomyocytes against H(2)O(2)-induced cell death, whereas Yap1 depletion attenuated phenylephrine-induced hypertrophy and augmented apoptosis. Finally, we observed a significant decrease in cardiomyocyte proliferation in Yap(+/F)Cre hearts compared with Yap(+/F) controls after MI and demonstrated that Yap1 is sufficient to promote cardiomyocyte proliferation in isolated cardiomyocytes. Our findings suggest that Yap1 is critical for basal heart homeostasis and that Yap1 deficiency exacerbates injury in response to chronic MI.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Cardiomegalia/metabolismo , Proteínas Musculares/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Proteínas de Ciclo Celular , Sobrevivência Celular/genética , Células Cultivadas , Doença Crônica , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Heterozigoto , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Transgênicos , Proteínas Musculares/genética , Isquemia Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Oxidantes/farmacologia , Fosfoproteínas/genética , Isoformas de Proteínas/metabolismo
4.
Cell Metab ; 14(5): 598-611, 2011 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22055503

RESUMO

High energy production in mitochondria is essential for maintaining cardiac contraction in the heart. Genes regulating mitochondrial function are commonly downregulated during heart failure. Here we show that both PPARα and Sirt1 are upregulated by pressure overload in the heart. Haploinsufficiency of either PPARα or Sirt1 attenuated pressure overload-induced cardiac hypertrophy and failure, whereas simultaneous upregulation of PPARα and Sirt1 exacerbated the cardiac dysfunction. PPARα and Sirt1 coordinately suppressed genes involved in mitochondrial function that are regulated by estrogen-related receptors (ERRs). PPARα bound and recruited Sirt1 to the ERR response element (ERRE), thereby suppressing ERR target genes in an RXR-independent manner. Downregulation of ERR target genes was also observed during fasting, and this appeared to be an adaptive response of the heart. These results suggest that suppression of the ERR transcriptional pathway by PPARα/Sirt1, a physiological fasting response, is involved in the progression of heart failure by promoting mitochondrial dysfunction.


Assuntos
Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , PPAR alfa/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais/genética , Sirtuína 1/metabolismo , Transcrição Genética , Adaptação Fisiológica , Animais , Aorta/fisiopatologia , Aorta/cirurgia , Sítios de Ligação , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Jejum/metabolismo , Regulação da Expressão Gênica , Haploinsuficiência , Haplótipos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas , Miocárdio/patologia , Técnicas de Cultura de Órgãos , PPAR alfa/genética , Ligação Proteica , Receptores de Estrogênio/genética , Sirtuína 1/genética , Regulação para Cima
5.
Circ Res ; 109(2): 161-71, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21617130

RESUMO

RATIONALE: Overexpression of muscle atrophy F-box (MAFbx/atrogin-1), an E3 ubiquitin ligase, induces proteasomal degradation in cardiomyocytes. The role of endogenous MAFbx in regulating cardiac hypertrophy and failure remains unclear. OBJECTIVE: We investigated the role of MAFbx in regulating cardiac hypertrophy and function in response to pressure overload. Transverse aortic constriction (TAC) was applied to MAFbx knockout (KO) and wild-type (WT) mice. METHODS AND RESULTS: Expression of MAFbx in WT mice was significantly increased by TAC. TAC-induced increases in cardiac hypertrophy were significantly smaller in MAFbx KO than in WT mice. There was significantly less lung congestion and interstitial fibrosis in MAFbx KO than in WT mice. MAFbx KO also inhibited ß-adrenergic cardiac hypertrophy. DNA microarray analysis revealed that activation of genes associated with the transcription factor binding site for the nuclear factor-κB family were inhibited in MAFbx KO mice compared with WT mice after TAC. Although the levels of IκB-α were significantly decreased after TAC in WT mice, they were increased in MAFbx KO mice. MAFbx regulates ubiquitination and proteasomal degradation of IκB-α in cardiomyocytes. In primary cultured rat cardiomyocytes, phenylephrine-induced activation of nuclear factor-κB and hypertrophy were significantly suppressed by MAFbx knockdown but were partially rescued by overexpression of nuclear factor-κB p65. CONCLUSIONS: MAFbx plays an essential role in mediating cardiac hypertrophy in response to pressure overload. Downregulation of MAFbx inhibits cardiac hypertrophy in part through stabilization of IκB-α and inactivation of nuclear factor-κB. Taken together, inhibition of MAFbx attenuates pathological hypertrophy, thereby protecting the heart from progression into heart failure.


Assuntos
Cardiomegalia/metabolismo , Proteínas Musculares/fisiologia , NF-kappa B/metabolismo , Proteínas Ligases SKP Culina F-Box/fisiologia , Animais , Cardiomegalia/etiologia , Células Cultivadas , Constrição Patológica , Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Proteínas I-kappa B/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/deficiência , Proteínas Musculares/metabolismo , Inibidor de NF-kappaB alfa , Substâncias Protetoras , Ratos , Proteínas Ligases SKP Culina F-Box/deficiência , Proteínas Ligases SKP Culina F-Box/metabolismo
6.
Circ Res ; 108(4): 478-89, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21233455

RESUMO

RATIONALE: Glycogen synthase kinase (GSK)-3ß upregulates cardiac genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. Ex vivo modification of signaling mechanisms in MSCs may improve the efficiency of cardiac cell-based therapy (CBT). OBJECTIVE: To test the effect of GSK-3ß on the efficiency of CBT with MSCs after myocardial infarction (MI). METHODS AND RESULTS: MSCs overexpressing either GSK-3ß (GSK-3ß-MSCs), LacZ (LacZ-MSCs), or saline was injected into the heart after coronary ligation. A significant improvement in the mortality and left ventricular (LV) function was observed at 12 weeks in GSK-3ß-MSC-injected mice compared with in LacZ-MSC- or saline-injected mice. MI size and LV remodeling were reduced in GSK-3ß-MSC-injected mice compared with in LacZ-MSC- or saline-injected ones. GSK-3ß increased survival and increased cardiomyocyte differentiation of MSCs, as evidenced by activation of an Nkx2.5-LacZ reporter and upregulation of troponin T. Injection of GSK-3ß-MSCs induced Ki67-positive myocytes and c-Kit-positive cells, suggesting that GSK-3ß-MSCs upregulate cardiac progenitor cells. GSK-3ß-MSCs also increased capillary density and upregulated paracrine factors, including vascular endothelial growth factor A (Vegfa). Injection of GSK-3ß-MSCs in which Vegfa had been knocked down abolished the increase in survival and capillary density. However, the decrease in MI size and LV remodeling and the improvement of LV function were still observed in MI mice injected with GSK-3ß-MSCs without Vegfa. CONCLUSIONS: GSK-3ß significantly improves the efficiency of CBT with MSCs in the post-MI heart. GSK-3ß not only increases survival of MSCs but also induces cardiomyocyte differentiation and angiogenesis through Vegfa-dependent and -independent mechanisms.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Quinase 3 da Glicogênio Sintase/metabolismo , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/complicações , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Hipertrofia Ventricular Esquerda/patologia , Injeções , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Transdução de Sinais/fisiologia , Remodelação Ventricular
7.
J Biol Chem ; 284(52): 36647-36658, 2009 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19858210

RESUMO

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and alpha-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3beta and down-regulation of beta-catenin, whereas it stimulated GSK-3alpha expression only modestly. The promoter region of GSK-3beta was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3beta potently induced CM differentiation, that of GSK-3alpha induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3beta abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3alpha facilitated CM differentiation in MSCs. Although GSK-3beta is found in both the cytosol and nucleus in MSCs, GSK-3alpha is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3beta failed to stimulate CM differentiation. Down-regulation of beta-catenin mediates GSK-3beta-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3alpha knockdown. These results suggest that GSK-3alpha and GSK-3beta have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3beta in the cytosol induces CM differentiation of MSCs through down-regulation of beta-catenin. In contrast, GSK-3alpha in the nucleus inhibits CM differentiation through down-regulation of c-Jun.


Assuntos
Células da Medula Óssea/enzimologia , Diferenciação Celular/fisiologia , Quinase 3 da Glicogênio Sintase/biossíntese , Células-Tronco Mesenquimais/enzimologia , Miócitos Cardíacos/enzimologia , Animais , Azacitidina/farmacologia , Células da Medula Óssea/citologia , Núcleo Celular/enzimologia , Núcleo Celular/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/fisiologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , beta Catenina/genética , beta Catenina/metabolismo
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