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 SecuenciaRESUMEN
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.
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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 TranscripcionalRESUMEN
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 TripartitosRESUMEN
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/metabolismoRESUMEN
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 NoqueadosRESUMEN
[Figure: see text].
Asunto(s)
Infarto del Miocardio/metabolismo , Miofibroblastos/metabolismo , Pirofosfatasas/metabolismo , Animales , Células Cultivadas , Fibrosis , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Miofibroblastos/patología , Pirofosfatasas/genéticaRESUMEN
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/genéticaRESUMEN
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/genéticaRESUMEN
HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes. Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality. Hand2 expression is tightly regulated by upstream enhancers that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications. Here we show that transcription of a Hand2-associated long non-coding RNA, which we named upperhand (Uph), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome, the Uph-Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cromatina/genética , Corazón/embriología , Organogénesis/genética , ARN Largo no Codificante/genética , Transcripción Genética/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Pérdida del Embrión/genética , Elementos de Facilitación Genéticos/genética , Técnicas de Inactivación de Genes , Cardiopatías Congénitas/genética , Ventrículos Cardíacos/anomalías , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/biosíntesis , Elongación de la Transcripción GenéticaRESUMEN
Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational "hotspot" regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.
Asunto(s)
Exones , Eliminación de Gen , Edición Génica/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Miocitos Cardíacos/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.
Asunto(s)
Proteínas de la Membrana/metabolismo , Músculo Esquelético/fisiopatología , Células Satélite del Músculo Esquelético/metabolismo , Animales , Fusión Celular , Femenino , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Regeneración , Células Satélite del Músculo Esquelético/citologíaRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.
Asunto(s)
Envejecimiento/genética , Sistemas CRISPR-Cas/genética , Eliminación de Gen , Miocardio/metabolismo , Animales , Cardiomegalia/complicaciones , Cardiomegalia/patología , Separación Celular , Dependovirus/metabolismo , Técnicas de Silenciamiento del Gen , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/patología , Ratones Transgénicos , Modelos Animales , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Especificidad de Órganos/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Innervation of skeletal muscle by motor neurons occurs through the neuromuscular junction, a cholinergic synapse essential for normal muscle growth and function. Defects in nerve-muscle signaling cause a variety of neuromuscular disorders with features of ataxia, paralysis, skeletal muscle wasting, and degeneration. Here we show that the nuclear zinc finger protein ZFP106 is highly enriched in skeletal muscle and is required for postnatal maintenance of myofiber innervation by motor neurons. Genetic disruption of Zfp106 in mice results in progressive ataxia and hindlimb paralysis associated with motor neuron degeneration, severe muscle wasting, and premature death by 6 mo of age. We show that ZFP106 is an RNA-binding protein that associates with the core splicing factor RNA binding motif protein 39 (RBM39) and localizes to nuclear speckles adjacent to spliceosomes. Upon inhibition of pre-mRNA synthesis, ZFP106 translocates with other splicing factors to the nucleolus. Muscle and spinal cord of Zfp106 knockout mice displayed a gene expression signature of neuromuscular degeneration. Strikingly, altered splicing of the Nogo (Rtn4) gene locus in skeletal muscle of Zfp106 knockout mice resulted in ectopic expression of NOGO-A, the neurite outgrowth factor that inhibits nerve regeneration and destabilizes neuromuscular junctions. These findings reveal a central role for Zfp106 in the maintenance of nerve-muscle signaling, and highlight the involvement of aberrant RNA processing in neuromuscular disease pathogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Síndrome Debilitante/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Desnervación Muscular , Músculo Esquelético/inervación , Músculo Esquelético/patología , Atrofia Muscular/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Síndrome Debilitante/metabolismoRESUMEN
Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.
Asunto(s)
Diferenciación Celular/fisiología , Inducción Embrionaria/fisiología , Fibroblastos/citología , Corazón/embriología , Miocitos Cardíacos/fisiología , Factores de Transcripción/metabolismo , Potenciales de Acción/fisiología , Análisis de Varianza , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cartilla de ADN/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Inmunohistoquímica , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Miocitos Cardíacos/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genéticaRESUMEN
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.
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Citoesqueleto/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Miocitos del Músculo Liso/metabolismo , Actinas/metabolismo , Animales , Secuencia de Bases , Traumatismos de las Arterias Carótidas/metabolismo , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Datos de Secuencia Molecular , Mutación , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/patología , Proteínas Nucleares/metabolismo , Ratas , Alineación de Secuencia , Transactivadores/metabolismoRESUMEN
Excitation-contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca(2+) transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca(2+) release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca(2+) store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.
Asunto(s)
Calcio/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Potenciales de Acción/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Northern Blotting , Western Blotting , Cartilla de ADN/genética , Electroporación , Genotipo , Hibridación in Situ , Ratones , Ratones Noqueados , Microscopía Electrónica , Músculo Esquelético/fisiología , Músculo Esquelético/ultraestructura , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , beta-GalactosidasaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Corazón/fisiología , Miocitos Cardíacos/fisiología , Fosfoproteínas/metabolismo , Regeneración/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Proteínas de Ciclo Celular , Cartilla de ADN/genética , Ecocardiografía , Vía de Señalización Hippo , Técnicas Histológicas , Ratones , Ratones Transgénicos , Mutación Missense/genética , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Sales de Tetrazolio , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
AIMS: RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. METHODS AND RESULTS: We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. CONCLUSION: This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.
Asunto(s)
Empalme Alternativo , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Conectina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Noqueados , Miocitos Cardíacos/metabolismo , ARN/metabolismo , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.
Asunto(s)
Cardiomiopatías , Enfermedades Cardiovasculares , Ratones , Animales , Humanos , Sistemas CRISPR-Cas , Edición Génica , Corazón , Cardiomiopatías/genética , Enfermedades Cardiovasculares/genéticaRESUMEN
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.