RESUMEN
Mutations in genes encoding nuclear envelope proteins lead to diseases known as nuclear envelopathies, characterized by skeletal muscle and heart abnormalities, such as Emery-Dreifuss muscular dystrophy (EDMD). The tissue-specific role of the nuclear envelope in the etiology of these diseases has not been extensively explored. We previously showed that global deletion of the muscle-specific nuclear envelope protein NET39 in mice leads to neonatal lethality due to skeletal muscle dysfunction. To study the potential role of the Net39 gene in adulthood, we generated a muscle-specific conditional knockout (cKO) of Net39 in mice. cKO mice recapitulated key skeletal muscle features of EDMD, including muscle wasting, impaired muscle contractility, abnormal myonuclear morphology, and DNA damage. The loss of Net39 rendered myoblasts hypersensitive to mechanical stretch, resulting in stretch-induced DNA damage. Net39 was downregulated in a mouse model of congenital myopathy, and restoration of Net39 expression through AAV gene delivery extended life span and ameliorated muscle abnormalities. These findings establish NET39 as a direct contributor to the pathogenesis of EDMD that acts by protecting against mechanical stress and DNA damage.
Asunto(s)
Distrofia Muscular de Emery-Dreifuss , Animales , Ratones , Estrés Mecánico , Distrofia Muscular de Emery-Dreifuss/metabolismo , Núcleo Celular/metabolismo , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Lamina Tipo A/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, that primarily affect the heart and skeletal muscle. A mutation in the NEP LEM domain-containing protein 2 (LEMD2) causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. We generated knockin (KI) mice carrying the human c.T38>G Lemd2 mutation, which causes a missense amino acid exchange (p.L13>R) in the LEM domain of the protein. These mice represent a preclinical model that phenocopies the human disease, as they developed severe dilated cardiomyopathy and cardiac fibrosis leading to premature death. At the cellular level, KI/KI cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of Lemd2 also died shortly after birth due to heart abnormalities. Cardiomyocytes lacking Lemd2 displayed nuclear envelope deformations and extensive DNA damage and apoptosis linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus rescued cardiac function in KI/KI mice. Together, our results reveal the essentiality of LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.
Asunto(s)
Cardiomiopatías , Membrana Nuclear , Humanos , Ratones , Animales , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Daño del ADN , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Skeletal muscle fibers contain hundreds of nuclei, which increase the overall transcriptional activity of the tissue and perform specialized functions. Multinucleation occurs through myoblast fusion, mediated by the muscle fusogens Myomaker (MYMK) and Myomixer (MYMX). We describe a human pedigree harboring a recessive truncating variant of the MYMX gene that eliminates an evolutionarily conserved extracellular hydrophobic domain of MYMX, thereby impairing fusogenic activity. Homozygosity of this human variant resulted in a spectrum of abnormalities that mimicked the clinical presentation of Carey-Fineman-Ziter syndrome (CFZS), caused by hypomorphic MYMK variants. Myoblasts generated from patient-derived induced pluripotent stem cells displayed defective fusion, and mice bearing the human MYMX variant died perinatally due to muscle abnormalities. In vitro assays showed that the human MYMX variant conferred minimal cell-cell fusogenicity, which could be restored with CRISPR/Cas9-mediated base editing, thus providing therapeutic potential for this disorder. Our findings identify MYMX as a recessive, monogenic human disease gene involved in CFZS, and provide new insights into the contribution of myoblast fusion to neuromuscular diseases.
Asunto(s)
Síndrome de Mobius , Enfermedades Musculares , Animales , Humanos , Proteínas de la Membrana/genética , Ratones , Proteínas Musculares/genética , Enfermedades Musculares/genética , Síndrome de Pierre RobinRESUMEN
Lamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery-Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD.
Asunto(s)
Proteínas de la Membrana/deficiencia , Músculo Esquelético/patología , Distrofia Muscular de Emery-Dreifuss/genética , Membrana Nuclear/patología , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatasa/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Lamina Tipo A/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Músculo Esquelético/citología , Distrofia Muscular de Emery-Dreifuss/patología , Proteínas Nucleares/genética , Fosfatidato Fosfatasa/genética , RNA-Seq , Estudios RetrospectivosRESUMEN
The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the muscle-specific membrane protein myomaker. Here, using in silico (BLAST) genome analyses, we show that the myomaker gene from trout includes 14 minisatellites, indicating that it has an unusual structure compared with those of other animal species. We found that the trout myomaker gene encodes a 434-amino acid (aa) protein, in accordance with its apparent molecular mass (â¼40 kDa) observed by immunoblotting. The first half of the trout myomaker protein (1-220 aa) is similar to the 221-aa mouse myomaker protein, whereas the second half (222-234 aa) does not correspond to any known motifs and arises from two protein extensions. The first extension (â¼70 aa) apparently appeared with the radiation of the bony fish clade Euteleostei, whereas the second extension (up to 236 aa) is restricted to the superorder Protacanthopterygii (containing salmonids and pike) and corresponds to the insertion of minisatellites having a length of 30 nucleotides. According to gene expression analyses, trout myomaker expression is consistently associated with the formation of new myofibers during embryonic development, postlarval growth, and muscle regeneration. Using cell-mixing experiments, we observed that trout myomaker has retained the ability to drive the fusion of mouse fibroblasts with C2C12 myoblasts. Our work reveals that trout myomaker has fusogenic function despite containing two protein extensions.
Asunto(s)
Proteínas de Peces , Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana , Repeticiones de Minisatélite , Proteínas Musculares , Oncorhynchus mykiss , Animales , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibrillas/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismoRESUMEN
Maintenance of muscle function requires assembly of contractile proteins into highly organized sarcomeres. Mutations in Kelch-like protein 41 (KLHL41) cause nemaline myopathy, a fatal muscle disorder associated with sarcomere disarray. We generated KLHL41 mutant mice, which display lethal disruption of sarcomeres and aberrant expression of muscle structural and contractile proteins, mimicking the hallmarks of the human disease. We show that KLHL41 is poly-ubiquitinated and acts, at least in part, by preventing aggregation and degradation of Nebulin, an essential component of the sarcomere. Furthermore, inhibition of KLHL41 poly-ubiquitination prevents its stabilization of nebulin, suggesting a unique role for ubiquitination in protein stabilization. These findings provide new insights into the molecular etiology of nemaline myopathy and reveal a mechanism whereby KLHL41 stabilizes sarcomeres and maintains muscle function by acting as a molecular chaperone. Similar mechanisms for protein stabilization likely contribute to the actions of other Kelch proteins.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miopatías Nemalínicas/patología , Sarcómeros/fisiología , Ubiquitina/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular , Proteínas Musculares/genética , Músculo Esquelético/citología , Mutación , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , UbiquitinaciónRESUMEN
Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.
Asunto(s)
Proteínas de la Membrana/genética , Síndrome de Mobius/genética , Morfogénesis/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Mutación , Mioblastos/metabolismo , Síndrome de Pierre Robin/genética , Proteínas de Pez Cebra/genética , Adulto , Secuencia de Aminoácidos , Animales , Fusión Celular , Niño , Modelos Animales de Enfermedad , Embrión no Mamífero , Femenino , Expresión Génica , Genes Recesivos , Prueba de Complementación Genética , Humanos , Lactante , Masculino , Proteínas de la Membrana/deficiencia , Síndrome de Mobius/metabolismo , Síndrome de Mobius/patología , Proteínas Musculares/deficiencia , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/patología , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Mioblastos/patología , Linaje , Síndrome de Pierre Robin/metabolismo , Síndrome de Pierre Robin/patología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Pez Cebra , Proteínas de Pez Cebra/deficienciaRESUMEN
Skeletal muscle formation occurs through fusion of myoblasts to form multinucleated myofibers. From a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) loss-of-function screen for genes required for myoblast fusion and myogenesis, we discovered an 84-amino acid muscle-specific peptide that we call Myomixer. Myomixer expression coincides with myoblast differentiation and is essential for fusion and skeletal muscle formation during embryogenesis. Myomixer localizes to the plasma membrane, where it promotes myoblast fusion and associates with Myomaker, a fusogenic membrane protein. Myomixer together with Myomaker can also induce fibroblast-fibroblast fusion and fibroblast-myoblast fusion. We conclude that the Myomixer-Myomaker pair controls the critical step in myofiber formation during muscle development.
Asunto(s)
Fusión Celular , Proteínas de la Membrana/metabolismo , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/fisiología , Proteínas Musculares/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Mioblastos/fisiología , Animales , Diferenciación Celular , Línea Celular , Membrana Celular/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Fibroblastos/metabolismo , Fibroblastos/fisiología , Masculino , Ratones Noqueados , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Péptidos/genética , Péptidos/metabolismoRESUMEN
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.