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1.
Hum Mol Genet ; 29(8): 1330-1339, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32242214

RESUMEN

Mutations in the RYR1 gene are the most common cause of human congenital myopathies, and patients with recessive mutations are severely affected and often display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle (EOM) involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for Ryr1 mutations. Ex vivo force production in EOMs from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared with that observed in wild-type, single heterozygous mutant carriers or homozygous RyR1p.A4329D mice. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle-specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the EOM phenotype caused by the RyR1p.Q1970fsX16+p.A4329D compound heterozygous Ryr1 mutations is complex and due to a combination of modifications including a direct effect on the macromolecular complex involved in calcium release and indirect effects on the expression of myosin heavy chain isoforms.


Asunto(s)
Debilidad Muscular/genética , Cadenas Pesadas de Miosina/genética , Miotonía Congénita/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Animales , Modelos Animales de Enfermedad , Heterocigoto , Humanos , Ratones , Debilidad Muscular/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación/genética , Miotonía Congénita/patología , Músculos Oculomotores/metabolismo , Músculos Oculomotores/patología , Fenotipo
2.
J Biol Chem ; 295(30): 10331-10339, 2020 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-32499372

RESUMEN

Mutations in the ryanodine receptor 1 (RYR1) gene are associated with several human congenital myopathies, including the dominantly inherited central core disease and exercise-induced rhabdomyolysis, and the more severe recessive phenotypes, including multiminicore disease, centronuclear myopathy, and congenital fiber type disproportion. Within the latter group, those carrying a hypomorphic mutation in one allele and a missense mutation in the other are the most severely affected. Because of nonsense-mediated decay, most hypomorphic alleles are not expressed, resulting in homozygous expression of the missense mutation allele. This should result in 50% reduced expression of the ryanodine receptor in skeletal muscle, but its observed content is even lower. To study in more detail the biochemistry and pathophysiology of recessive RYR1 myopathies, here we investigated a mouse model we recently generated by analyzing the effect of bi-allelic versus mono-allelic expression of the RyR1 p.A4329D mutation. Our results revealed that the expression of two alleles carrying the same mutation or of one allele with the mutation in combination with a hypomorphic allele does not result in functionally equal outcomes and impacts skeletal muscles differently. In particular, the bi-allelic RyR1 p.A4329D mutation caused a milder phenotype than its mono-allelic expression, leading to changes in the biochemical properties and physiological function only of slow-twitch muscles and largely sparing fast-twitch muscles. In summary, bi-allelic expression of the RyR1 p.A4329D mutation phenotypically differs from mono-allelic expression of this mutation in a compound heterozygous carrier.


Asunto(s)
Regulación de la Expresión Génica , Fibras Musculares de Contracción Lenta/metabolismo , Fuerza Muscular , Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , Sustitución de Aminoácidos , Animales , Masculino , Ratones , Ratones Mutantes , Canal Liberador de Calcio Receptor de Rianodina/genética
3.
Hum Mol Genet ; 28(11): 1872-1884, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30689883

RESUMEN

Here we characterized a mouse model knocked-in for a frameshift mutation in RYR1 exon 36 (p.Gln1970fsX16) that is isogenic to that identified in one parent of a severely affected patient with recessively inherited multiminicore disease. This individual carrying the RYR1 frameshifting mutation complained of mild muscle weakness and fatigability. Analysis of the RyR1 protein content in a muscle biopsy from this individual showed a content of only 20% of that present in a control individual. The biochemical and physiological characteristics of skeletal muscles from RyR1Q1970fsX16 heterozygous mice recapitulates that of the heterozygous parent. RyR1 protein content in the muscles of mutant mice reached 38% and 58% of that present in total muscle homogenates of fast and slow muscles from wild-type (WT) littermates. The decrease of RyR1 protein content in total homogenates is not accompanied by a decrease of Cav1.1 content, whereby the Cav1.1/RyR1 stoichiometry ratio in skeletal muscles from RyR1Q1970fsX16 heterozygous mice is lower compared to that from WT mice. Electron microscopy (EM) revealed a 36% reduction in the number/area of calcium release units accompanied by a 2.5-fold increase of dyads (triads that have lost one junctional sarcoplasmic reticulum element); both results suggest a reduction of the RyR1 arrays. Compared to WT, muscle strength and depolarization-induced calcium transients in RyR1Q1970fsX16 heterozygous mice muscles were decreased by 20% and 15%, respectively. The RyR1Q1970fsX16 mouse model provides mechanistic insight concerning the phenotype of the parent carrying the RYR1 ex36 mutation and suggests that in skeletal muscle fibres there is a functional reserve of RyR1.


Asunto(s)
Canales de Calcio Tipo L/genética , Debilidad Muscular/genética , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Adulto , Alelos , Animales , Modelos Animales de Enfermedad , Mutación del Sistema de Lectura/genética , Heterocigoto , Humanos , Ratones , Microscopía Electrónica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Debilidad Muscular/patología , Miopatías Estructurales Congénitas/fisiopatología , Oftalmoplejía/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura
4.
Hum Mol Genet ; 28(18): 2987-2999, 2019 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-31044239

RESUMEN

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.


Asunto(s)
Calcio/metabolismo , Fuerza Muscular/genética , Músculo Esquelético/metabolismo , Mutación , Miopatía del Núcleo Central/etiología , Miopatía del Núcleo Central/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Alelos , Animales , Señalización del Calcio , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Heterocigoto , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Músculo Esquelético/fisiopatología , Músculo Esquelético/ultraestructura , Miopatía del Núcleo Central/fisiopatología , Fenotipo
5.
Hum Mutat ; 40(7): 962-974, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30932294

RESUMEN

Congenital myopathies are early onset, slowly progressive neuromuscular disorders of variable severity. They are genetically and phenotypically heterogeneous and caused by pathogenic variants in several genes. Multi-minicore Disease, one of the more common congenital myopathies, is frequently caused by recessive variants in either SELENON, encoding the endoplasmic reticulum glycoprotein selenoprotein N or RYR1, encoding a protein involved in calcium homeostasis and excitation-contraction coupling. The mechanism by which recessive SELENON variants cause Multiminicore disease (MmD) is unclear. Here, we extensively investigated muscle physiological, biochemical and epigenetic modifications, including DNA methylation, histone modification, and noncoding RNA expression, to understand the pathomechanism of MmD. We identified biochemical changes that are common in patients harboring recessive RYR1 and SELENON variants, including depletion of transcripts encoding proteins involved in skeletal muscle calcium homeostasis, increased levels of Class II histone deacetylases (HDACs) and DNA methyltransferases. CpG methylation analysis of genomic DNA of patients with RYR1 and SELENON variants identified >3,500 common aberrantly methylated genes, many of which are involved in calcium signaling. These results provide the proof of concept for the potential use of drugs targeting HDACs and DNA methyltransferases to treat patients with specific forms of congenital myopathies.


Asunto(s)
Metilación de ADN , Proteínas Musculares/genética , Enfermedades Musculares/congénito , Enfermedades Musculares/genética , Selenoproteínas/genética , Adolescente , Células Cultivadas , Niño , Preescolar , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , Epigénesis Genética , Código de Histonas , Histona Desacetilasas/genética , Humanos , Canal Liberador de Calcio Receptor de Rianodina/genética , Secuenciación Completa del Genoma
6.
Semin Cell Dev Biol ; 64: 201-212, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-27427513

RESUMEN

The physiological process by which Ca2+ is released from the sarcoplasmic reticulum is called excitation-contraction coupling; it is initiated by an action potential which travels deep into the muscle fiber where it is sensed by the dihydropyridine receptor, a voltage sensing L-type Ca2+channel localized on the transverse tubules. Voltage-induced conformational changes in the dihydropyridine receptor activate the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum. The released Ca2+ binds to troponin C, enabling contractile thick-thin filament interactions. The Ca2+ is subsequently transported back into the sarcoplasmic reticulum by specialized Ca2+ pumps (SERCA), preparing the muscle for a new cycle of contraction. Although other proteins are involved in excitation-contraction coupling, the mechanism described above emphasizes the unique role played by the two Ca2+ channels (the dihydropyridine receptor and the ryanodine receptor), the SERCA Ca2+ pumps and the exquisite spatial organization of the membrane compartments endowed with the proteins responsible for this mechanism to function rapidly and efficiently. Research over the past two decades has uncovered the fine details of excitation-contraction coupling under normal conditions while advances in genomics have helped to identify mutations in novel genes in patients with neuromuscular disorders. While it is now clear that many patients with congenital muscle diseases carry mutations in genes encoding proteins directly involved in Ca2+ homeostasis, it has become apparent that mutations are also present in genes encoding for proteins not thought to be directly involved in Ca2+ regulation. Ongoing research in the field now focuses on understanding the functional effect of individual mutations, as well as understanding the role of proteins not specifically located in the sarcoplasmic reticulum which nevertheless are involved in Ca2+ regulation or excitation-contraction coupling. The principal challenge for the future is the identification of drug targets that can be pharmacologically manipulated by small molecules, with the ultimate aim to improve muscle function and quality of life of patients with congenital muscle disorders. The aim of this review is to give an overview of the most recent findings concerning Ca2+ dysregulation and its impact on muscle function in patients with congenital muscle disorders due to mutations in proteins involved in excitation-contraction coupling and more broadly on Ca2+ homeostasis.


Asunto(s)
Señalización del Calcio , Enfermedades Musculares/metabolismo , Edad de Inicio , Animales , Calcio/metabolismo , Humanos , Modelos Biológicos , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutación/genética , Retículo Sarcoplasmático/metabolismo
7.
Hum Mol Genet ; 26(2): 320-332, 2017 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-28007904

RESUMEN

Centronuclear myopathies are early-onset muscle diseases caused by mutations in several genes including MTM1, DNM2, BIN1, RYR1 and TTN. The most severe and often fatal X-linked form of myotubular myopathy (XLMTM) is caused by mutations in the gene encoding the ubiquitous lipid phosphatase myotubularin, an enzyme specifically dephosphorylating phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate. Because XLMTM patients have a predominantly muscle-specific phenotype a number of pathogenic mechanisms have been proposed, including a direct effect of the accumulated lipid on the skeletal muscle calcium channel ryanodine receptor 1, a negative effect on the structure of intracellular organelles and defective autophagy. Animal models knocked out for MTM1 show severe reduction of ryanodine receptor 1 mediated calcium release but, since knocking out genes in animal models does not necessarily replicate the human phenotype, we considered it important to study directly the effect of MTM1 mutations on patient muscle cells. The results of the present study show that at the level of myotubes MTM1 mutations do not dramatically affect calcium homeostasis and calcium release mediated through the ryanodine receptor 1, though they do affect myotube size and nuclear content. On the other hand, mature muscles such as those obtained from patient muscle biopsies exhibit a significant decrease in expression of the ryanodine receptor 1, a decrease in muscle-specific microRNAs and a considerable up-regulation of histone deacetylase-4. We hypothesize that the latter events consequent to the primary genetic mutation, are the cause of the severe decrease in muscle strength that characterizes these patients.


Asunto(s)
Histona Desacetilasas/genética , Músculo Esquelético/metabolismo , Miopatías Estructurales Congénitas/genética , Proteínas Tirosina Fosfatasas no Receptoras/genética , Proteínas Represoras/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Animales , Biopsia , Calcio/metabolismo , Niño , Preescolar , Femenino , Regulación de la Expresión Génica , Histona Desacetilasas/biosíntesis , Humanos , Lactante , Recién Nacido , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Músculo Esquelético/patología , Mutación , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Proteínas Represoras/biosíntesis , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , Pez Cebra
8.
J Biol Chem ; 291(28): 14555-65, 2016 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-27189940

RESUMEN

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La(3+)- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca(2+) concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca(2+) concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Calsecuestrina/metabolismo , Proteínas de la Membrana/metabolismo , Fibras Musculares de Contracción Lenta/fisiología , Animales , Proteínas de Unión al Calcio/genética , Calsecuestrina/genética , Técnicas de Inactivación de Genes , Proteínas de la Membrana/genética , Ratones , Contracción Muscular , Músculo Esquelético/fisiología , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo
9.
Hum Mol Genet ; 24(16): 4636-47, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26019235

RESUMEN

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.


Asunto(s)
Epigénesis Genética , Histona Desacetilasas/biosíntesis , Debilidad Muscular/metabolismo , Miotonía Congénita/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , Animales , Histona Desacetilasas/genética , Ratones , Debilidad Muscular/genética , Debilidad Muscular/patología , Mutación , Miotonía Congénita/genética , Miotonía Congénita/patología , Canal Liberador de Calcio Receptor de Rianodina/genética
10.
J Cell Sci ; 128(5): 951-63, 2015 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-25609705

RESUMEN

Junctin, a non-catalytic splice variant encoded by the aspartate-ß-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.


Asunto(s)
Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Línea Celular , Perros , Unión Proteica , Estructura Terciaria de Proteína , Canal Liberador de Calcio Receptor de Rianodina/genética
11.
Acta Neuropathol ; 133(4): 517-533, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28012042

RESUMEN

Muscle contraction upon nerve stimulation relies on excitation-contraction coupling (ECC) to promote the rapid and generalized release of calcium within myofibers. In skeletal muscle, ECC is performed by the direct coupling of a voltage-gated L-type Ca2+ channel (dihydropyridine receptor; DHPR) located on the T-tubule with a Ca2+ release channel (ryanodine receptor; RYR1) on the sarcoplasmic reticulum (SR) component of the triad. Here, we characterize a novel class of congenital myopathy at the morphological, molecular, and functional levels. We describe a cohort of 11 patients from 7 families presenting with perinatal hypotonia, severe axial and generalized weakness. Ophthalmoplegia is present in four patients. The analysis of muscle biopsies demonstrated a characteristic intermyofibrillar network due to SR dilatation, internal nuclei, and areas of myofibrillar disorganization in some samples. Exome sequencing revealed ten recessive or dominant mutations in CACNA1S (Cav1.1), the pore-forming subunit of DHPR in skeletal muscle. Both recessive and dominant mutations correlated with a consistent phenotype, a decrease in protein level, and with a major impairment of Ca2+ release induced by depolarization in cultured myotubes. While dominant CACNA1S mutations were previously linked to malignant hyperthermia susceptibility or hypokalemic periodic paralysis, our findings strengthen the importance of DHPR for perinatal muscle function in human. These data also highlight CACNA1S and ECC as therapeutic targets for the development of treatments that may be facilitated by the previous knowledge accumulated on DHPR.


Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Miotonía Congénita/genética , Miotonía Congénita/metabolismo , Adolescente , Adulto , Calcio/metabolismo , Canales de Calcio Tipo L , Células Cultivadas , Niño , Estudios de Cohortes , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Musculares/metabolismo , Células Musculares/patología , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación , Miotonía Congénita/diagnóstico por imagen , Miotonía Congénita/patología , Fenotipo , Homología de Secuencia de Aminoácido , Adulto Joven
12.
Biochem J ; 466(1): 29-36, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25387602

RESUMEN

Excitation-contraction coupling (ECC) is the physiological mechanism whereby an electrical signal detected by the dihydropyridine receptor, is converted into an increase in [Ca2+], via activation of ryanodine receptors (RyRs). Mutations in RYR1, the gene encoding RyR1, are the underlying cause of various congenital myopathies including central core disease, multiminicore disease (MmD), some forms of centronuclear myopathy (CNM) and congenital fibre-type disproportion. Interestingly, patients with recessive, but not dominant, RYR1 mutations show a significant reduction in RyR protein in muscle biopsies as well as ophthalmoplegia. This specific involvement of the extraocular muscles (EOMs) indicates that this group of muscles may express different amounts of proteins involved in ECC compared with limb muscles. In the present paper, we report that this is indeed the case; in particular the transcripts encoding RyR3, cardiac calsequestrin (CSQ2) and the α1 subunit of the cardiac dihydropyridine receptor are up-regulated by at least 100-fold, whereas excitation-coupled Ca2+ entry is 3-fold higher. These findings support the hypothesis that EOMs have a unique mode of calcium handling.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Regulación de la Expresión Génica , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Calsecuestrina/genética , Calsecuestrina/metabolismo , Diferenciación Celular , Perfilación de la Expresión Génica , Humanos , Células Musculares/citología , Músculo Esquelético/citología , Mutación , Órbita , Cultivo Primario de Células , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transcripción Genética
13.
Biochem J ; 466(1): 123-35, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25431931

RESUMEN

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Canales de Calcio Tipo L/genética , Complejos Multiproteicos/genética , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Serina-Treonina Quinasas TOR/genética , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Potenciales Evocados/fisiología , Acoplamiento Excitación-Contracción/fisiología , Regulación de la Expresión Génica , Glucógeno Fosforilasa/genética , Glucógeno Fosforilasa/metabolismo , Contracción Isométrica , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Proteína Reguladora Asociada a mTOR , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo
14.
Proc Natl Acad Sci U S A ; 110(50): 20314-9, 2013 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-24277823

RESUMEN

Skeletal muscle mass loss and dysfunction have been linked to many diseases. Conversely, resistance exercise, mainly by activating mammalian target of rapamycin complex 1 (mTORC1), promotes skeletal muscle hypertrophy and exerts several therapeutic effects. Moreover, mTORC1, along with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), regulates skeletal muscle metabolism. However, it is unclear whether PGC-1α is required for skeletal muscle adaptations after overload. Here we show that although chronic overload of skeletal muscle via synergist ablation (SA) strongly induces hypertrophy and a switch toward a slow-contractile phenotype, these effects were independent of PGC-1α. In fact, SA down-regulated PGC-1α expression and led to a repression of energy metabolism. Interestingly, however, PGC-1α deletion preserved peak force after SA. Taken together, our data suggest that PGC-1α is not involved in skeletal muscle remodeling induced by SA.


Asunto(s)
Metabolismo Energético/fisiología , Complejos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Cartilla de ADN/genética , Hipertrofia/metabolismo , Inmunohistoquímica , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Transgénicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosfofructoquinasas/metabolismo , Reacción en Cadena de la Polimerasa , Succinato Deshidrogenasa/metabolismo
15.
J Cell Sci ; 126(Pt 15): 3485-92, 2013 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-23704352

RESUMEN

Mutations in RYR1, the gene encoding ryanodine receptor 1, are linked to a variety of neuromuscular disorders including malignant hyperthermia (MH), a pharmacogenetic hypermetabolic disease caused by dysregulation of Ca(2+) in skeletal muscle. RYR1 encodes a Ca(2+) channel that is predominantly expressed in skeletal muscle sarcoplasmic reticulum, where it is involved in releasing the Ca(2+) necessary for muscle contraction. Other tissues, however, including cells of the immune system, have been shown to express ryanodine receptor 1; in dendritic cells its activation leads to increased surface expression of major histocompatibility complex II molecules and provides synergistic signals leading to cell maturation. In the present study, we investigated the impact of an MH mutation on the immune system by studying the RYR1Y522S knock-in mouse. Our results show that there are subtle but significant differences both in resting 'non-challenged' mice as well as in mice treated with antigenic stimuli, in particular the knock-in mice: (i) have dendritic cells that are more efficient at stimulating T cell proliferation, (ii) have higher levels of natural IgG1 and IgE antibodies, and (iii) are faster and more efficient at mounting a specific immune response in the early phases of immunization. We suggest that some gain-of-function MH-linked RYR1 mutations might offer selective immune advantages to their carriers. Furthermore, our results raise the intriguing possibility that pharmacological activation of RyR1 might be exploited for the development of new classes of vaccines and adjuvants.


Asunto(s)
Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/inmunología , Animales , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Inmunoglobulinas/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nematospiroides dubius/inmunología , Infecciones por Strongylida/sangre , Infecciones por Strongylida/inmunología
16.
Biochem J ; 455(2): 169-77, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-23905709

RESUMEN

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.


Asunto(s)
Calcio/metabolismo , Músculo Esquelético/metabolismo , Canales de Calcio Tipo L/metabolismo , Línea Celular , Fenómenos Electrofisiológicos , Femenino , Humanos , Contracción Muscular , Proteínas Musculares/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Adulto Joven
17.
Proc Natl Acad Sci U S A ; 108(51): 20808-13, 2011 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-22143799

RESUMEN

Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy.


Asunto(s)
Bezafibrato/farmacología , Regulación de la Expresión Génica , Enfermedades Musculares/metabolismo , Proteínas/metabolismo , Animales , Glucógeno/química , Glucógeno/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias Musculares/metabolismo , Modelos Genéticos , Complejos Multiproteicos , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Serina-Treonina Quinasas TOR , Transactivadores/metabolismo , Factores de Transcripción
18.
J Gen Physiol ; 156(4)2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38445312

RESUMEN

RYR1 is the most commonly mutated gene associated with congenital myopathies, a group of early-onset neuromuscular conditions of variable severity. The functional effects of a number of dominant RYR1 mutations have been established; however, for recessive mutations, these effects may depend on multiple factors, such as the formation of a hypomorphic allele, or on whether they are homozygous or compound heterozygous. Here, we functionally characterize a new transgenic mouse model knocked-in for mutations identified in a severely affected child born preterm and presenting limited limb movement. The child carried the homozygous c.14928C>G RYR1 mutation, resulting in the p.F4976L substitution. In vivo and ex vivo assays revealed that homozygous mice fatigued sooner and their muscles generated significantly less force compared with their WT or heterozygous littermates. Electron microscopy, biochemical, and physiological analyses showed that muscles from RyR1 p.F4976L homozygous mice have the following properties: (1) contain fewer calcium release units and show areas of myofibrillar degeneration, (2) contain less RyR1 protein, (3) fibers show smaller electrically evoked calcium transients, and (4) their SR has smaller calcium stores. In addition, single-channel recordings indicate that RyR1 p.F4976L exhibits higher Po in the presence of 100 µM [Ca2+]. Our mouse model partly recapitulates the clinical picture of the homozygous human patient and provides significant insight into the functional impact of this mutation. These results will help understand the pathology of patients with similar RYR1 mutations.


Asunto(s)
Calcio , Enfermedades Musculares , Animales , Niño , Humanos , Ratones , Modelos Animales de Enfermedad , Homeostasis , Ratones Transgénicos , Músculos , Canal Liberador de Calcio Receptor de Rianodina/genética
19.
Hum Mutat ; 34(1): 184-90, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22927026

RESUMEN

JP-45 (also JP45; encoded by JSRP1) is an integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane interacting with Ca(v) 1.1 (the α.1 subunit of the voltage-sensing dihydropyridine receptor, DHPR) and the luminal calcium-binding protein calsequestrin. Two JSRP1 variants have been found in the human population: c.323C>T (p.P108L) in exon 5 and c.449G>C (p.G150A) in exon 6, but nothing is known concerning the incidence of these polymorphisms in the general population or in patients with neuromuscular diseases nor the impact of the polymorphisms on excitation-contraction (EC) coupling. In the present report, we investigated the frequencies of these two JSRP1 polymorphisms in the Swiss malignant hyperthermia population and studied the functional impact of the variants on EC coupling. Our results show that the polymorphisms are equally distributed among malignant hyperthermia negative, malignant hyperthermia equivocal, and malignant hyperthermia susceptible individuals. Interestingly, however, the presence of either one of these JP-45 variants decreased the sensitivity of the DHPR to activation. The presence of a JSRP1 variant may explain the variable phenotype seen in patients with malignant hyperthermia carrying the same mutation and, more importantly, may counteract the hypersensitivity of EC coupling caused by mutations in the RYR1 gene.


Asunto(s)
Canales de Calcio Tipo L/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Hipertermia Maligna/fisiopatología , Proteínas de la Membrana/genética , Músculo Esquelético/fisiología , Mutación Missense , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Células Cultivadas , Análisis Mutacional de ADN , Acoplamiento Excitación-Contracción/genética , Frecuencia de los Genes , Humanos , Hipertermia Maligna/genética , Hipertermia Maligna/metabolismo , Potenciales de la Membrana , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/metabolismo , Técnicas de Placa-Clamp , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/metabolismo
20.
Hum Mol Genet ; 20(3): 589-600, 2011 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-21088110

RESUMEN

Prolonged depolarization of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. Skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4-dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. In this study, we directly measured excitation-coupled calcium entry by total internal reflection fluorescence microscopy in human skeletal muscle myotubes harbouring mutations in the RYR1 gene linked to malignant hyperthermia (MH) and central core disease (CCD). We found that excitation-coupled calcium entry is strongly enhanced in cells from patients with CCD compared with individuals with MH and controls. Furthermore, excitation-coupled calcium entry induces generation of reactive nitrogen species and enhances nuclear localization of NFATc1, which in turn may be responsible for the increased IL-6 released by myotubes from patients with CCD.


Asunto(s)
Calcio/metabolismo , Interleucina-6/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miopatía del Núcleo Central/metabolismo , Factores de Transcripción NFATC/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Hipertermia Maligna/genética , Microscopía Fluorescente , Músculo Esquelético/metabolismo , Mutación , Miopatía del Núcleo Central/genética , Reacción en Cadena de la Polimerasa , Especies de Nitrógeno Reactivo/biosíntesis , Especies de Nitrógeno Reactivo/metabolismo
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