RESUMO
Obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific overexpression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.
Assuntos
Metabolismo Energético , Resistência à Insulina , MicroRNAs/metabolismo , Miocárdio/metabolismo , Obesidade/genética , Animais , Diabetes Mellitus Tipo 2 , Feminino , Glucose/metabolismo , Coração/fisiologia , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Obesidade/prevenção & controleRESUMO
Regeneration of injured adult skeletal muscle involves fusion of activated satellite cells to form new myofibers. Myomaker is a muscle-specific membrane protein required for fusion of embryonic myoblasts, but its potential involvement in adult muscle regeneration has not been explored. We show that myogenic basic helix-loop-helix (bHLH) transcription factors induce myomaker expression in satellite cells during acute and chronic muscle regeneration. Moreover, genetic deletion of myomaker in adult satellite cells completely abolishes muscle regeneration, resulting in severe muscle destruction after injury. Myomaker is the only muscle-specific protein known to be absolutely essential for fusion of embryonic and adult myoblasts.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Regeneração/genética , Animais , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/lesões , Proteína MyoD/metabolismo , Miogenina/metabolismo , Regiões Promotoras Genéticas/genética , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibres. However, the identity of muscle-specific proteins that directly govern this fusion process in mammals has remained elusive. Here we identify a muscle-specific membrane protein, named myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is downregulated thereafter. Overexpression of myomaker in myoblasts markedly enhances fusion, and genetic disruption of myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibres. Remarkably, forced expression of myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacological perturbation of the actin cytoskeleton abolishes the activity of myomaker, consistent with previous studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein that controls mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.
Assuntos
Proteínas de Membrana/fisiologia , Desenvolvimento Muscular , Proteínas Musculares/fisiologia , Músculo Esquelético/embriologia , Mioblastos/citologia , Animais , Fusão Celular , Linhagem Celular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismoRESUMO
Vascular injury triggers dedifferentiation and cytoskeletal remodeling of smooth muscle cells (SMCs), culminating in vessel occlusion. Serum response factor (SRF) and its coactivator, myocardin, play a central role in the control of smooth muscle phenotypes by regulating the expression of cytoskeletal genes. We show that SRF and myocardin regulate a cardiovascular-specific microRNA (miRNA) cluster encoding miR-143 and miR-145. To assess the functions of these miRNAs in vivo, we systematically deleted them singly and in combination in mice. Mice lacking both miR-143 and miR-145 are viable and do not display overt abnormalities in smooth muscle differentiation, although they show a significant reduction in blood pressure due to reduced vascular tone. Remarkably, however, neointima formation in response to vascular injury is profoundly impeded in mice lacking these miRNAs, due to disarray of actin stress fibers and diminished migratory activity of SMCs. These abnormalities reflect the regulation of a cadre of modulators of SRF activity and actin dynamics by miR-143 and miR-145. Thus, miR-143 and miR-145 act as integral components of the regulatory network whereby SRF controls cytoskeletal remodeling and phenotypic switching of SMCs during vascular disease.
Assuntos
Citoesqueleto/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Lesões das Artérias Carótidas/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Mutação , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Nucleares/metabolismo , Ratos , Alinhamento de Sequência , Transativadores/metabolismoRESUMO
Myocardin-related transcription factors (MRTFs) regulate cellular contractility and motility by associating with serum response factor (SRF) and activating genes involved in cytoskeletal dynamics. We reported previously that MRTF-A contributes to pathological cardiac remodeling by promoting differentiation of fibroblasts to myofibroblasts following myocardial infarction. Here, we show that forced expression of MRTF-A in dermal fibroblasts stimulates contraction of a collagen matrix, whereas contractility of MRTF-A null fibroblasts is impaired under basal conditions and in response to TGF-ß1 stimulation. We also identify an isoxazole ring-containing small molecule, previously shown to induce smooth muscle α-actin gene expression in cardiac progenitor cells, as an agonist of myofibroblast differentiation. Isoxazole stimulates myofibroblast differentiation via induction of MRTF-A-dependent gene expression. The MRTF-SRF signaling axis is activated in response to skin injury, and treatment of dermal wounds with isoxazole accelerates wound closure and suppresses the inflammatory response. These results reveal an important role for MRTF-SRF signaling in dermal myofibroblast differentiation and wound healing and suggest that targeting MRTFs pharmacologically may prove useful in treating diseases associated with inappropriate myofibroblast activity.
Assuntos
Diferenciação Celular , Derme/lesões , Derme/metabolismo , Regulação da Expressão Gênica , Miofibroblastos/metabolismo , Transativadores/metabolismo , Cicatrização , Androstenóis/farmacologia , Animais , Derme/patologia , Camundongos , Miofibroblastos/patologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Fosfoproteínas/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Proteínas de Ciclo Celular , Primers do DNA/genética , Ecocardiografia , Via de Sinalização Hippo , Técnicas Histológicas , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sais de Tetrazólio , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.
Assuntos
MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Northern Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Transdução de Sinais , Transativadores/metabolismoRESUMO
RATIONALE: Establishment of a functional vasculature requires the interconnection and remodeling of nascent blood vessels. Precise regulation of factors that influence endothelial cell migration and function is essential for these stereotypical vascular patterning events. The secreted Slit ligands and their Robo receptors constitute a critical signaling pathway controlling the directed migration of both neurons and vascular endothelial cells during embryonic development, but the mechanisms of their regulation are incompletely understood. OBJECTIVE: To identify microRNAs regulating aspects of the Slit-Robo pathway and vascular patterning. METHODS AND RESULTS: Here, we provide evidence that microRNA (miR)-218, which is encoded by an intron of the Slit genes, inhibits the expression of Robo1 and Robo2 and multiple components of the heparan sulfate biosynthetic pathway. Using in vitro and in vivo approaches, we demonstrate that miR-218 directly represses the expression of Robo1, Robo2, and glucuronyl C5-epimerase (GLCE), and that an intact miR-218-Slit-Robo regulatory network is essential for normal vascularization of the retina. Knockdown of miR-218 results in aberrant regulation of this signaling axis, abnormal endothelial cell migration, and reduced complexity of the retinal vasculature. CONCLUSIONS: Our findings link Slit gene expression to the posttranscriptional regulation of Robo receptors and heparan sulfate biosynthetic enzymes, allowing for precise control over vascular guidance cues influencing the organization of blood vessels during development.
Assuntos
Glicoproteínas/antagonistas & inibidores , MicroRNAs/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Vasos Retinianos/embriologia , Transdução de Sinais/genética , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicoproteínas/fisiologia , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/biossíntese , Heparitina Sulfato/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Dados de Sequência Molecular , Neovascularização Fisiológica/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Receptores Imunológicos/genética , Vasos Retinianos/fisiologia , Transcrição Gênica , Proteínas RoundaboutRESUMO
RATIONALE: Myocardial infarction (MI) results in loss of cardiac myocytes in the ischemic zone of the heart, followed by fibrosis and scar formation, which diminish cardiac contractility and impede angiogenesis and repair. Myofibroblasts, a specialized cell type that switches from a fibroblast-like state to a contractile, smooth muscle-like state, are believed to be primarily responsible for fibrosis of the injured heart and other tissues, although the transcriptional mediators of fibrosis and myofibroblast activation remain poorly defined. Myocardin-related transcription factors (MRTFs) are serum response factor (SRF) cofactors that promote a smooth muscle phenotype and are emerging as components of stress-responsive signaling. OBJECTIVE: We aimed to examine the effect of MRTF-A on cardiac remodeling and fibrosis. METHODS AND RESULTS: Here, we show that MRTF-A controls the expression of a fibrotic gene program that includes genes involved in extracellular matrix production and smooth muscle cell differentiation in the heart. In MRTF-A-null mice, fibrosis and scar formation following MI or angiotensin II treatment are dramatically diminished compared with wild-type littermates. This protective effect of MRTF-A deletion is associated with a reduction in expression of fibrosis-associated genes, including collagen 1a2, a direct transcriptional target of SRF/MRTF-A. CONCLUSIONS: We conclude that MRTF-A regulates myofibroblast activation and fibrosis in response to the renin-angiotensin system and post-MI remodeling.
Assuntos
Transdiferenciação Celular , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Transativadores/metabolismo , Remodelação Ventricular , Amidas/farmacologia , Angiotensina II/administração & dosagem , Animais , Sequência de Bases , Células COS , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Chlorocebus aethiops , Colágeno/genética , Colágeno Tipo I , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fenótipo , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Fatores de Tempo , Transativadores/deficiência , Transativadores/genética , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta1/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/genética , Quinases Associadas a rho/metabolismoRESUMO
RATIONALE: Gender differences in cardiovascular disease have long been recognized and attributed to beneficial cardiovascular actions of estrogen. Class II histone deacetylases (HDACs) act as key modulators of heart disease by repressing the activity of the myocyte enhancer factor (MEF)2 transcription factor, which promotes pathological cardiac remodeling in response to stress. Although it is proposed that HDACs additionally influence nuclear receptor signaling, the effect of class II HDACs on gender differences in cardiovascular disease remains unstudied. OBJECTIVE: We aimed to examine the effect of class II HDACs on post-myocardial infarction remodeling in male and female mice. METHODS AND RESULTS: Here we show that the absence of HDAC5 or -9 in female mice protects against maladaptive remodeling following myocardial infarction, during which there is an upregulation of estrogen-responsive genes in the heart. This genetic reprogramming coincides with a pronounced increase in expression of the estrogen receptor (ER)alpha gene, which we show to be a direct MEF2 target gene. ERalpha also directly interacts with class II HDACs. Cardioprotection resulting from the absence of HDAC5 or -9 in female mice can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of the ER target gene vascular endothelial growth factor-a. CONCLUSIONS: Our results reveal a novel gender-specific pathway of cardioprotection mediated by ERalpha and its regulation by MEF2 and class II HDACs.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Histona Desacetilases/metabolismo , Infarto do Miocárdio/metabolismo , Fatores de Regulação Miogênica/metabolismo , Proteínas Repressoras/metabolismo , Caracteres Sexuais , Animais , Receptor alfa de Estrogênio/genética , Feminino , Histona Desacetilases/genética , Fatores de Transcrição MEF2 , Masculino , Camundongos , Camundongos Knockout , Infarto do Miocárdio/genética , Fatores de Regulação Miogênica/genética , Neovascularização Fisiológica/genética , Proteínas Repressoras/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Acute myocardial infarction (MI) due to coronary artery occlusion is accompanied by a pathological remodeling response that includes hypertrophic cardiac growth and fibrosis, which impair cardiac contractility. Previously, we showed that cardiac hypertrophy and heart failure are accompanied by characteristic changes in the expression of a collection of specific microRNAs (miRNAs), which act as negative regulators of gene expression. Here, we show that MI in mice and humans also results in the dysregulation of specific miRNAs, which are similar to but distinct from those involved in hypertrophy and heart failure. Among the MI-regulated miRNAs are members of the miR-29 family, which are down-regulated in the region of the heart adjacent to the infarct. The miR-29 family targets a cadre of mRNAs that encode proteins involved in fibrosis, including multiple collagens, fibrillins, and elastin. Thus, down-regulation of miR-29 would be predicted to derepress the expression of these mRNAs and enhance the fibrotic response. Indeed, down-regulation of miR-29 with anti-miRs in vitro and in vivo induces the expression of collagens, whereas over-expression of miR-29 in fibroblasts reduces collagen expression. We conclude that miR-29 acts as a regulator of cardiac fibrosis and represents a potential therapeutic target for tissue fibrosis in general.
Assuntos
Fibrose Endomiocárdica/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Infarto do Miocárdio/genética , Animais , Células COS , Chlorocebus aethiops , Colágeno/genética , Colágeno/metabolismo , Regulação para Baixo , Fibrose Endomiocárdica/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Infarto do Miocárdio/patologia , RNA MensageiroRESUMO
The muscle-specific microRNAs, miR-1 and miR-133, play important roles in muscle growth and differentiation. Here, we show that the MEF2 transcription factor, an essential regulator of muscle development, directly activates transcription of a bicistronic primary transcript encoding miR-1-2 and 133a-1 via an intragenic muscle-specific enhancer located between the miR-1-2 and 133a-1 coding regions. This MEF2-dependent enhancer is activated in the linear heart tube during mouse embryogenesis and thereafter controls transcription throughout the atrial and ventricular chambers of the heart. MEF2 together with MyoD also regulates the miR-1-2/-133a-1 intragenic enhancer in the somite myotomes and in all skeletal muscle fibers during embryogenesis and adulthood. A similar muscle-specific intragenic enhancer controls transcription of the miR-1-1/-133a-2 locus. These findings reveal a common architecture of regulatory elements associated with the miR-1/-133 genes and underscore the central role of MEF2 as a regulator of the transcriptional and posttranscriptional pathways that control cardiac and skeletal muscle development.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/fisiologia , Transcrição Gênica , Animais , Deleção de Genes , Coração/embriologia , Fatores de Transcrição MEF2 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Miocárdio/metabolismoRESUMO
Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an amino-terminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.
Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Miócitos de Músculo Liso/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Artérias/metabolismo , Biomarcadores , Células Cultivadas , Embrião de Mamíferos/anatomia & histologia , Expressão Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Fenótipo , Ligação Proteica , Proteínas Metiltransferases , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Veias/metabolismoRESUMO
The Hippo signaling pathway regulates growth of the heart and other tissues. Hippo pathway kinases influence the activity of various targets, including the transcriptional coactivator Yap, but the specific role of Yap in heart growth has not been investigated. We show that Yap is necessary and sufficient for embryonic cardiac growth in mice. Deletion of Yap in the embryonic mouse heart impeded cardiomyocyte proliferation, causing myocardial hypoplasia and lethality at embryonic stage 10.5. Conversely, forced expression of a constitutively active form of Yap in the embryonic heart increased cardiomyocyte number and heart size. Yap activated the insulin-like growth factor (IGF) signaling pathway in cardiomyocytes, resulting in inactivation of glycogen synthase kinase 3ß, which led to increased abundance of ß-catenin, a positive regulator of cardiac growth. Our results point to Yap as a critical downstream effector of the Hippo pathway in the control of cardiomyocyte proliferation and a nexus for coupling the IGF, Wnt, and Hippo signaling pathways with the developmental program for heart growth.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Somatomedinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Western Blotting , Proteínas de Ciclo Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Coração Fetal/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Fosfoproteínas/genética , Ratos , Ratos Sprague-Dawley , Somatomedinas/genética , Proteínas de Sinalização YAP , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Animais , Sequência de Bases , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Linhagem Celular , Chlorocebus aethiops , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismoRESUMO
The heart responds to diverse forms of stress by hypertrophic growth accompanied by fibrosis and eventual diminution of contractility, which results from down-regulation of alpha-myosin heavy chain (alphaMHC) and up-regulation of betaMHC, the primary contractile proteins of the heart. We found that a cardiac-specific microRNA (miR-208) encoded by an intron of the alphaMHC gene is required for cardiomyocyte hypertrophy, fibrosis, and expression of betaMHC in response to stress and hypothyroidism. Thus, the alphaMHC gene, in addition to encoding a major cardiac contractile protein, regulates cardiac growth and gene expression in response to stress and hormonal signaling through miR-208.
Assuntos
Regulação da Expressão Gênica , Cardiopatias/genética , MicroRNAs/fisiologia , Miocárdio/metabolismo , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Miosinas Ventriculares/genética , Animais , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Fibrose , Deleção de Genes , Coração/fisiopatologia , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Hipotireoidismo/fisiopatologia , Íntrons , Complexo Mediador , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Contração Miocárdica , Miócitos Cardíacos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Transdução de Sinais , Estresse Fisiológico/fisiopatologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tri-Iodotironina/metabolismo , Regulação para Cima , Miosinas Ventriculares/metabolismoRESUMO
Myocardin is a transcriptional co-activator of serum response factor (Srf), which is a key regulator of the expression of smooth and cardiac muscle genes. Consistent with its role in regulating cardiovascular development, myocardin is the earliest known marker specific to both the cardiac and smooth muscle lineages during embryogenesis. To understand how the expression of this early transcriptional regulator is initiated and maintained, we scanned 90 kb of genomic DNA encompassing the myocardin gene for cis-regulatory elements capable of directing myocardin transcription in cardiac and smooth muscle lineages in vivo. Here, we describe an enhancer that controls cardiovascular expression of the mouse myocardin gene during mouse embryogenesis and adulthood. Activity of this enhancer in the heart and vascular system requires the combined actions of the Mef2 and Foxo transcription factors. In addition, the Tead transcription factor is required specifically for enhancer activation in neural-crest-derived smooth muscle cells and dorsal aorta. Notably, myocardin also regulates its own enhancer, but in contrast to the majority of myocardin target genes, which are dependent on Srf, myocardin acts through Mef2 to control its enhancer. These findings reveal an Srf-independent mechanism for smooth and cardiac muscle-restricted transcription and provide insight into the regulatory mechanisms responsible for establishing the smooth and cardiac muscle phenotypes during development.
Assuntos
Sistema Cardiovascular/embriologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Regulação Miogênica/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sistema Cardiovascular/anatomia & histologia , Sistema Cardiovascular/crescimento & desenvolvimento , Linhagem da Célula , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Humanos , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Músculo Liso/embriologia , Músculo Liso/crescimento & desenvolvimento , Músculo Liso/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Fatores de Regulação Miogênica/genética , Proteínas Nucleares/genética , Alinhamento de Sequência , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição de Domínio TEA , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Myocardin is a cardiac- and smooth muscle-specific SAP domain transcription factor that functions as a coactivator for serum response factor (SRF), which controls genes involved in muscle differentiation and cell proliferation. The DNA binding domain of SRF, which interacts with myocardin, shares homology with the MEF2 transcription factor, which also controls muscle and growth-associated genes. Here we show that alternative splicing produces a cardiac-enriched isoform of myocardin containing a unique peptide sequence that confers the ability to interact with and stimulate the transcriptional activity of MEF2. This MEF2 binding motif is also contained in a previously unknown SAP domain transcription factor, referred to as MASTR, which functions as a MEF2 coactivator. This unique protein-protein interaction motif expands the regulatory potential of myocardin, and its presence in MASTR reveals a new mechanism for the control of MEF2 activity.
Assuntos
Proteínas de Domínio MADS/metabolismo , Fatores de Regulação Miogênica/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores Ativadores da Transcrição/fisiologia , Processamento Alternativo , Animais , Sequência de Bases , Humanos , Fatores de Transcrição MEF2 , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Diverse forms of injury and stress evoke a hypertrophic growth response in adult cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, assembly of sarcomeres, and reactivation of fetal genes, often culminating in heart failure and sudden death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs that were regulated during cardiac hypertrophy and heart failure. We describe >12 miRNAs that are up- or down-regulated in cardiac tissue from mice in response to transverse aortic constriction or expression of activated calcineurin, stimuli that induce pathological cardiac remodeling. Many of these miRNAs were similarly regulated in failing human hearts. Forced overexpression of stress-inducible miRNAs was sufficient to induce hypertrophy in cultured cardiomyocytes. Similarly, cardiac overexpression of miR-195, which was up-regulated during cardiac hypertrophy, resulted in pathological cardiac growth and heart failure in transgenic mice. These findings reveal an important role for specific miRNAs in the control of hypertrophic growth and chamber remodeling of the heart in response to pathological signaling and point to miRNAs as potential therapeutic targets in heart disease.
Assuntos
Cardiopatias/genética , Cardiopatias/fisiopatologia , MicroRNAs/genética , Estresse Fisiológico/genética , Animais , Regulação da Expressão Gênica , Cardiopatias/patologia , Humanos , Hipertrofia/genética , RatosRESUMO
Myocardin is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, we show that myocardin is sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. We also demonstrate that, for the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. We conclude that myocardin is an essential component of the regulatory pathway for myocardial differentiation.