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1.
Cell ; 160(4): 595-606, 2015 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-25640239

RESUMEN

Functional micropeptides can be concealed within RNAs that appear to be noncoding. We discovered a conserved micropeptide, which we named myoregulin (MLN), encoded by a skeletal muscle-specific RNA annotated as a putative long noncoding RNA. MLN shares structural and functional similarity with phospholamban (PLN) and sarcolipin (SLN), which inhibit SERCA, the membrane pump that controls muscle relaxation by regulating Ca(2+) uptake into the sarcoplasmic reticulum (SR). MLN interacts directly with SERCA and impedes Ca(2+) uptake into the SR. In contrast to PLN and SLN, which are expressed in cardiac and slow skeletal muscle in mice, MLN is robustly expressed in all skeletal muscle. Genetic deletion of MLN in mice enhances Ca(2+) handling in skeletal muscle and improves exercise performance. These findings identify MLN as an important regulator of skeletal muscle physiology and highlight the possibility that additional micropeptides are encoded in the many RNAs currently annotated as noncoding.


Asunto(s)
Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , ARN Largo no Codificante/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Humanos , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Musculares/química , Músculo Esquelético/citología , Miocardio/metabolismo , Estructura Secundaria de Proteína , Proteolípidos/metabolismo , ARN Largo no Codificante/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Alineación de Secuencia
2.
Genes Dev ; 35(11-12): 835-840, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33985971

RESUMEN

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.


Asunto(s)
Regulación de la Expresión Génica/genética , Corazón/fisiología , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/metabolismo , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismo , Factores de Edad , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Factores de Transcripción MEF2/metabolismo , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/genética , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Proteínas Nucleares/genética , ARN Largo no Codificante/genética , Factor de Respuesta Sérica/genética , Transactivadores/genética , Activación Transcripcional
3.
Cell ; 149(3): 671-83, 2012 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-22541436

RESUMEN

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.


Asunto(s)
Metabolismo Energético , Resistencia a la Insulina , MicroARNs/metabolismo , Miocardio/metabolismo , Obesidad/genética , Animales , Diabetes Mellitus Tipo 2 , Femenino , Glucosa/metabolismo , Corazón/fisiología , Homeostasis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Obesidad/prevención & control
4.
Proc Natl Acad Sci U S A ; 121(4): e2315925121, 2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38227654

RESUMEN

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.


Asunto(s)
Rabdomiosarcoma , Adolescente , Niño , Humanos , Diferenciación Celular/genética , Línea Celular Tumoral , Receptores Purinérgicos P1 , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Transducción de Señal
5.
Genes Dev ; 33(11-12): 626-640, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30975722

RESUMEN

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of TWIST2 in fusion-negative RMS. Knockdown of TWIST2 in RMS cells results in up-regulation of MYOGENIN and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.


Asunto(s)
Carcinogénesis , Desarrollo de Músculos , Proteína MioD/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Células Cultivadas , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Transición Epitelial-Mesenquimal , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Secuencias Hélice-Asa-Hélice , Humanos , Proteína MioD/química , Mioblastos/metabolismo , Proteínas Nucleares/genética , Proteínas Represoras/química , Proteína 1 Relacionada con Twist/química
6.
Physiol Rev ; 98(3): 1205-1240, 2018 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-29717930

RESUMEN

Muscular dystrophies represent a large group of genetic disorders that significantly impair quality of life and often progress to premature death. There is no effective treatment for these debilitating diseases. Most therapies, developed to date, focus on alleviating the symptoms or targeting the secondary effects, while the underlying gene mutation is still present in the human genome. The discovery and application of programmable nucleases for site-specific DNA double-stranded breaks provides a powerful tool for precise genome engineering. In particular, the CRISPR/Cas system has revolutionized the genome editing field and is providing a new path for disease treatment by targeting the disease-causing genetic mutations. In this review, we provide a historical overview of genome-editing technologies, summarize the most recent advances, and discuss potential strategies and challenges for permanently correcting genetic mutations that cause muscular dystrophies.


Asunto(s)
Edición Génica/métodos , Terapia Genética , Distrofias Musculares/prevención & control , Animales , Modelos Animales de Enfermedad , Humanos , Distrofia Muscular Animal
7.
Circ Res ; 133(12): 1006-1021, 2023 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-37955153

RESUMEN

BACKGROUND: The p.Arg14del variant of the PLN (phospholamban) gene causes cardiomyopathy, leading to severe heart failure. Calcium handling defects and perinuclear PLN aggregation have both been suggested as pathological drivers of this disease. Dwarf open reading frame (DWORF) has been shown to counteract PLN regulatory calcium handling function in the sarco/endoplasmic reticulum (S/ER). Here, we investigated the potential disease-modulating action of DWORF in this cardiomyopathy and its effects on calcium handling and PLN aggregation. METHODS: We studied a PLN-R14del mouse model, which develops cardiomyopathy with similar characteristics as human patients, and explored whether cardiac DWORF overexpression could delay cardiac deterioration. To this end, R14Δ/Δ (homozygous PLN-R14del) mice carrying the DWORF transgene (R14Δ/ΔDWORFTg [R14Δ/Δ mice carrying the DWORF transgene]) were used. RESULTS: DWORF expression was suppressed in hearts of R14Δ/Δ mice with severe heart failure. Restoration of DWORF expression in R14Δ/Δ mice delayed cardiac fibrosis and heart failure and increased life span >2-fold (from 8 to 18 weeks). DWORF accelerated sarcoplasmic reticulum calcium reuptake and relaxation in isolated cardiomyocytes with wild-type PLN, but in R14Δ/Δ cardiomyocytes, sarcoplasmic reticulum calcium reuptake and relaxation were already enhanced, and no differences were detected between R14Δ/Δ and R14Δ/ΔDWORFTg. Rather, DWORF overexpression delayed the appearance and formation of large pathogenic perinuclear PLN clusters. Careful examination revealed colocalization of sarcoplasmic reticulum markers with these PLN clusters in both R14Δ/Δ mice and human p.Arg14del PLN heart tissue, and hence these previously termed aggregates are comprised of abnormal organized S/ER. This abnormal S/ER organization in PLN-R14del cardiomyopathy contributes to cardiomyocyte cell loss and replacement fibrosis, consequently resulting in cardiac dysfunction. CONCLUSIONS: Disorganized S/ER is a major characteristic of PLN-R14del cardiomyopathy in humans and mice and results in cardiomyocyte death. DWORF overexpression delayed PLN-R14del cardiomyopathy progression and extended life span in R14Δ/Δ mice, by reducing abnormal S/ER clusters.


Asunto(s)
Cardiomiopatías , Insuficiencia Cardíaca , Humanos , Ratones , Animales , Retículo Sarcoplasmático/metabolismo , Calcio/metabolismo , Longevidad , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo
8.
Nat Rev Mol Cell Biol ; 14(8): 529-41, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23839576

RESUMEN

As the adult mammalian heart has limited potential for regeneration and repair, the loss of cardiomyocytes during injury and disease can result in heart failure and death. The cellular processes and regulatory mechanisms involved in heart growth and development can be exploited to repair the injured adult heart through 'reawakening' pathways that are active during embryogenesis. Heart function has been restored in rodents by reprogramming non-myocytes into cardiomyocytes, by expressing transcription factors (GATA4, HAND2, myocyte-specific enhancer factor 2C (MEF2C) and T-box 5 (TBX5)) and microRNAs (miR-1, miR-133, miR-208 and miR-499) that control cardiomyocyte identity. Stimulating cardiomyocyte dedifferentiation and proliferation by activating mitotic signalling pathways involved in embryonic heart growth represents a complementary approach for heart regeneration and repair. Recent advances in understanding the mechanistic basis of heart development offer exciting opportunities for effective therapies for heart failure.


Asunto(s)
Desarrollo Embrionario/fisiología , Corazón/embriología , Corazón/fisiología , Regeneración/fisiología , Medicina Regenerativa/métodos , Adulto , Animales , Desarrollo Embrionario/genética , Humanos , Modelos Biológicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Regeneración/genética , Medicina Regenerativa/tendencias , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología
9.
Cell ; 143(1): 35-45, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20887891

RESUMEN

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.


Asunto(s)
Histona Desacetilasas/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/inervación , Músculo Esquelético/patología , Miogenina/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Atrofia , Ratones , Ratones Noqueados , Proteínas de Motivos Tripartitos
10.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-35101990

RESUMEN

Emerging evidence indicates that a subset of RNA molecules annotated as noncoding contain short open reading frames that code for small functional proteins called microproteins, which have largely been overlooked due to their small size. To search for cardiac-expressed microproteins, we used a comparative genomics approach and identified mitolamban (Mtlbn) as a highly conserved 47-amino acid transmembrane protein that is abundantly expressed in the heart. Mtlbn localizes specifically to the inner mitochondrial membrane where it interacts with subunits of complex III of the electron transport chain and with mitochondrial respiratory supercomplexes. Genetic deletion of Mtlbn in mice altered complex III assembly dynamics and reduced complex III activity. Unbiased metabolomic analysis of heart tissue from Mtlbn knockout mice further revealed an altered metabolite profile consistent with deficiencies in complex III activity. Cardiac-specific Mtlbn overexpression in transgenic (TG) mice induced cardiomyopathy with histological, biochemical, and ultrastructural pathologic features that contributed to premature death. Metabolomic analysis and biochemical studies indicated that hearts from Mtlbn TG mice exhibited increased oxidative stress and mitochondrial dysfunction. These findings reveal Mtlbn as a cardiac-expressed inner mitochondrial membrane microprotein that contributes to mitochondrial electron transport chain activity through direct association with complex III and the regulation of its assembly and function.


Asunto(s)
Cardiomiopatías/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Miocardio/metabolismo , Animales , Cardiomiopatías/genética , Células Cultivadas , Complejo III de Transporte de Electrones/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mitocondrias Cardíacas/genética , Proteínas Mitocondriales/genética , Especificidad de Órganos
11.
Genes Dev ; 31(17): 1770-1783, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28982760

RESUMEN

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.


Asunto(s)
Reprogramación Celular/genética , Regulación de la Expresión Génica/genética , Factores de Transcripción/metabolismo , Antiinflamatorios/farmacología , Reprogramación Celular/efectos de los fármacos , Fibroblastos/fisiología , Factor de Transcripción GATA4/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Proteínas Represoras , Transcriptoma
12.
Semin Cell Dev Biol ; 122: 3-13, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34246567

RESUMEN

Ischemic heart disease is the leading cause of morbidity, mortality, and healthcare expenditure worldwide due to an inability of the heart to regenerate following injury. Thus, novel heart failure therapies aimed at promoting cardiomyocyte regeneration are desperately needed. In recent years, direct reprogramming of resident cardiac fibroblasts to induced cardiac-like myocytes (iCMs) has emerged as a promising therapeutic strategy to repurpose the fibrotic response of the injured heart toward a functional myocardium. Direct cardiac reprogramming was initially achieved through the overexpression of the transcription factors (TFs) Gata4, Mef2c, and Tbx5 (GMT). However, this combination of TFs and other subsequent cocktails demonstrated limited success in reprogramming adult human and mouse fibroblasts, constraining the clinical translation of this therapy. Over the past decade, significant effort has been dedicated to optimizing reprogramming cocktails comprised of cardiac TFs, epigenetic factors, microRNAs, or small molecules to yield efficient cardiac cell fate conversion. Yet, efficient reprogramming of adult human fibroblasts remains a significant challenge. Underlying mechanisms identified to accelerate this process have been centered on epigenetic remodeling at cardiac gene regulatory regions. Further studies to achieve a refined understanding and directed means of overcoming epigenetic barriers are merited to more rapidly translate these promising therapies to the clinic.


Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , Reprogramación Celular/fisiología , Miocitos Cardíacos/metabolismo , Animales , Humanos , Ratones
13.
Circulation ; 148(19): 1490-1504, 2023 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-37712250

RESUMEN

BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.


Asunto(s)
Insuficiencia Cardíaca , Células Madre Pluripotentes Inducidas , Ratones , Humanos , Animales , Edición Génica , Sistemas CRISPR-Cas , Ratones Noqueados , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Fosforilación , Fibrosis , Adenina , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo
14.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34088848

RESUMEN

Homeothermic vertebrates produce heat in cold environments through thermogenesis, in which brown adipose tissue (BAT) increases mitochondrial oxidation along with uncoupling of the electron transport chain and activation of uncoupling protein 1 (UCP1). Although the transcription factors regulating the expression of UCP1 and nutrient oxidation genes have been extensively studied, only a few other proteins essential for BAT function have been identified. We describe the discovery of FAM195A, a BAT-enriched RNA binding protein, which is required for cold-dependent thermogenesis in mice. FAM195A knockout (KO) mice display whitening of BAT and an inability to thermoregulate. In BAT of FAM195A KO mice, enzymes involved in branched-chain amino acid (BCAA) metabolism are down-regulated, impairing their response to cold. Knockdown of FAM195A in brown adipocytes in vitro also impairs expression of leucine oxidation enzymes, revealing FAM195A to be a regulator of BCAA metabolism and a potential target for metabolic disorders.


Asunto(s)
Adipocitos Marrones , Tejido Adiposo Pardo , Frío , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Termogénesis , Aminoácidos de Cadena Ramificada/genética , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Línea Celular Transformada , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados
15.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-34260377

RESUMEN

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.


Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/patología , Prednisolona/farmacología , Fenómenos Biomecánicos , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Distrofina/deficiencia , Distrofina/metabolismo , Glicoproteínas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/genética , Mutación/genética , Optogenética , Fenotipo
16.
Genes Dev ; 30(4): 434-46, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26883362

RESUMEN

The Mediator complex governs gene expression by linking upstream signaling pathways with the basal transcriptional machinery. However, how individual Mediator subunits may function in different tissues remains to be investigated. Through skeletal muscle-specific deletion of the Mediator subunit MED13 in mice, we discovered a gene regulatory mechanism by which skeletal muscle modulates the response of the liver to a high-fat diet. Skeletal muscle-specific deletion of MED13 in mice conferred resistance to hepatic steatosis by activating a metabolic gene program that enhances muscle glucose uptake and storage as glycogen. The consequent insulin-sensitizing effect within skeletal muscle lowered systemic glucose and insulin levels independently of weight gain and adiposity and prevented hepatic lipid accumulation. MED13 suppressed the expression of genes involved in glucose uptake and metabolism in skeletal muscle by inhibiting the nuclear receptor NURR1 and the MEF2 transcription factor. These findings reveal a fundamental molecular mechanism for the governance of glucose metabolism and the control of hepatic lipid accumulation by skeletal muscle. Intriguingly, MED13 exerts opposing metabolic actions in skeletal muscle and the heart, highlighting the customized, tissue-specific functions of the Mediator complex.


Asunto(s)
Glucosa/metabolismo , Homeostasis/genética , Hígado/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Músculo Esquelético/metabolismo , Animales , Dieta Alta en Grasa , Hígado Graso/genética , Eliminación de Gen , Regulación de la Expresión Génica Arqueal/genética , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BL
18.
Proc Natl Acad Sci U S A ; 117(47): 29691-29701, 2020 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-33148801

RESUMEN

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.


Asunto(s)
Degeneración Macular/genética , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Regeneración/genética , Transducción de Señal/genética , Animales , Modelos Animales de Enfermedad , Exones/genética , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Mutación/genética , Miofibrillas/genética , Análisis de Secuencia de ARN/métodos , Transcripción Genética/genética , Transcriptoma/genética
19.
Circ Res ; 126(1): 6-24, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31730408

RESUMEN

RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.


Asunto(s)
Sistemas CRISPR-Cas , Regulación de la Expresión Génica , Miocardio/metabolismo , Activación Transcripcional , Animales , Línea Celular , Dependovirus/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Genes Sintéticos , Vectores Genéticos/genética , Corazón/crecimiento & desarrollo , Factores de Transcripción de Tipo Kruppel/biosíntesis , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción MEF2/biosíntesis , Factores de Transcripción MEF2/genética , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Regiones Promotoras Genéticas , Dominios Proteicos , ARN Polimerasa III/genética , ARN Guía de Kinetoplastida/genética
20.
Exp Cell Res ; 408(1): 112844, 2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34571006

RESUMEN

Muscular dystrophies are a heterogeneous group of monogenic neuromuscular disorders which lead to progressive muscle loss and degeneration of the musculoskeletal system. The genetic causes of muscular dystrophies are well characterized, but no effective treatments have been developed so far. The discovery and application of the CRISPR/Cas system for genome editing offers a new path for disease treatment with the potential to permanently correct genetic mutations. The post-mitotic and multinucleated features of skeletal muscle provide an ideal target for CRISPR/Cas therapeutic genome editing because correction of a subpopulation of nuclei can provide benefit to the whole myofiber. In this review, we provide an overview of the CRISPR/Cas system and its derivatives in genome editing, proposing potential CRISPR/Cas-based therapies to correct diverse muscular dystrophies, and we discuss challenges for translating CRISPR/Cas genome editing to a viable therapy for permanent correction of muscular dystrophies.


Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica , Terapia Genética , Distrofia Muscular de Duchenne/genética , Animales , Modelos Animales de Enfermedad , Distrofina/metabolismo , Edición Génica/métodos , Humanos , Distrofia Muscular de Duchenne/terapia , Mutación/genética
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