RESUMEN
Core myopathies are clinically, pathologically, and genetically heterogeneous muscle diseases. Their onset and clinical severity are variable. Core myopathies are diagnosed by muscle biopsy showing focally reduced oxidative enzyme activity and can be pathologically divided into central core disease, multiminicore disease, dusty core disease, and core-rod myopathy. Although RYR1-related myopathy is the most common core myopathy, an increasing number of other causative genes have been reported, including SELENON, MYH2, MYH7, TTN, CCDC78, UNC45B, ACTN2, MEGF10, CFL2, KBTBD13, and TRIP4. Furthermore, the genes originally reported to cause nemaline myopathy, namely ACTA1, NEB, and TNNT1, have been recently associated with core-rod myopathy. Genetic analysis allows us to diagnose each core myopathy more accurately. In this review, we aim to provide up-to-date information about core myopathies.
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Miopatía del Núcleo Central/genética , Biopsia , Humanos , Proteínas Musculares/genética , Músculo Esquelético/patología , Mutación , Miopatías Nemalínicas/genética , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
The non-encapsulated Streptococcus pneumoniae (NESp) has emerged and increased in the clinical setting. The majority of NESp strains have been isolated from the nasopharynxes of healthy carriers and from respiratory specimens of patients with otitis media. NESp strains were shown to be more effective than encapsulated counterparts at forming biofilms. Therefore, NESp should become one of the leading causes of emerging refractory respiratory disease after the introduction of pneumococcal conjugate vaccines. We report the first case of multidrug-resistant - including fluoroquinolone-resistant - NESp isolated from the intrabronchial aspirate of a patient with pneumonia. Drug-resistant NESp infections can possibly emerge as a clinical problem and thus the continuous monitoring of NESp infections is of utmost importance.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Neumonía Neumocócica/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Adolescente , Ampicilina/farmacología , Ampicilina/uso terapéutico , Antibacterianos/uso terapéutico , Cefalosporinas/uso terapéutico , Quimioterapia Combinada , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , Miopatías Estructurales Congénitas , Oftalmoplejía , Neumonía Neumocócica/diagnóstico , Neumonía Neumocócica/microbiología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Esputo/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Sulbactam/farmacología , Sulbactam/uso terapéutico , CefozopránRESUMEN
Peripheral blood mononuclear cells for reprogramming in this work were donated by a girl with clinically and genetically diagnosed multiminicore disease harboring compound heterozygote mutations of RYR1 gene. Induced pluripotent stem cells (iPSCs) were obtained by non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and c-MYC. The iPSC line (SDQLCHi025-A) presented pluripotent cell morphology, high mRNA levels of pluripotency markers, differentiation potential in vitro, a normal karyotype, and carrying RYR1 gene mutations.
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Células Madre Pluripotentes Inducidas , Diferenciación Celular , Reprogramación Celular , Femenino , Heterocigoto , Humanos , Factor 4 Similar a Kruppel , Leucocitos Mononucleares , Mutación , Miopatías Estructurales Congénitas , Oftalmoplejía , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Congenital myopathies represent a clinically and genetically heterogeneous group of early-onset neuromuscular diseases with characteristic, but not always specific, histopathological features, often presenting with stable and/or slowly progressive truncal and proximal weakness. It is often not possible to have a diagnosis on clinical ground alone. Additional extraocular, respiratory, distal involvement, scoliosis, and distal laxity may provide clues. The "core myopathies" collectively represent the most common form of congenital myopathies, and the name pathologically corresponds to histochemical appearance of focally reduced oxidative enzyme activity and myofibrillar changes on ultrastructural studies. Because of the clinical, pathological, and molecular overlaps, central core disease and multiminicore disease will be discussed together.
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Miopatías Estructurales Congénitas/diagnóstico , Miopatías Estructurales Congénitas/genética , Miopatía del Núcleo Central/diagnóstico , Miopatía del Núcleo Central/genética , Oftalmoplejía/diagnóstico , Oftalmoplejía/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Humanos , Miopatías Estructurales Congénitas/terapia , Miopatía del Núcleo Central/terapia , Oftalmoplejía/terapia , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
Aims: It is known that mitochondrial reactive oxygen species generation ([ROS]m) causes the release of Ca2+via ryanodine receptor-2 (RyR2) on the sarcoplasmic reticulum (SR) in pulmonary artery smooth muscle cells (PASMCs), playing an essential role in hypoxic pulmonary vasoconstriction (HPV). In this study, we sought to determine whether hypoxia-induced RyR2-mediated Ca2+ release may in turn promote [ROS]m in PASMCs and the underlying signaling mechanism. Results: Our data reveal that application of caffeine or norepinephrine to induce Ca2+ release increased [ROS]m in PASMCs. Likewise, exogenous Ca2+ augmented ROS generation in isolated mitochondria and at complex III from PASMCs. Inhibition of mitochondrial Ca2+ uniporter (MCU) with Ru360 attenuated agonist-induced [ROS]m. Ru360 produced a similar inhibitory effect on hypoxia-induced [ROS]m. Rieske iron-sulfur protein (RISP) gene knockdown inhibited Ca2+- and caffeine-induced [ROS]m. Inhibition of RyR2 by tetracaine or RyR2 gene knockout suppressed hypoxia-induced [ROS]m as well. Innovation: In this article, we present convincing evidence that Ca2+ release following hypoxia or RyR simulation causes a significant increase in MCU, and the increased MCU subsequently RISP-dependent [ROS]m, which provides a positive feedback mechanism to enhance hypoxia-initiated [ROS]m in PASMCs. Conclusion: Our findings demonstrate that hypoxia-induced mitochondrial ROS-dependent SR RyR2-mediated Ca2+ release increases MCU and then RISP-dependent [ROS]m in PASMCs, which may make significant contributions to HPV and associated pulmonary hypertension.
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Calcio/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Hipoxia/metabolismo , Miocitos del Músculo Liso/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Arteria Pulmonar/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Retículo Sarcoplasmático/metabolismoRESUMEN
The core myopathies are a group of congenital myopathies with variable clinical expression - ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity - that are identified by characteristic 'core-like' lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 (RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.
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Conectina/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiopatología , Miopatías Estructurales Congénitas/fisiopatología , Miopatía del Núcleo Central/fisiopatología , Oftalmoplejía/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Selenoproteínas/metabolismo , Alcaloides/farmacología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Estudios de Asociación Genética , Variación Genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Debilidad Muscular , Proteínas Quinasas/metabolismo , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Titin, encoded by the gene TTN, is one of the main sarcomere components. It is involved in not only maintaining the structure of cardiac and skeletal muscles, but also in their development, extensibility, elasticity, and signaling events. Congenital titinopathy increasingly appears an important and common form of axial predominant congenital myopathy. The pathophysiological role of TTN in congenital titinopathy and pediatric heart diseases is yet to be explored. Here, we delineate the phenotype of two female siblings who developed severe congenital multi-minicore disease without cardiac involvement. Genetic investigation by whole exome sequencing demonstrated compound heterozygous TTN mutations (c.15496+1G>A, p.5166_5258del; c.18597_18598insC, p.Thr6200Hisfs*15), corresponding to the Ig domain of the proximal I-band. Aberrant splicing causing exon skipping was verified by in vitro minigene analysis. Our results suggest that TTN mutations affecting the Ig domain of the proximal I-band may be a cause of severe congenital defect in skeletal muscles without severe cardiac involvement, thereby providing evidence for the hypothesis that congenital titinopathy patients carrying biallelic N2BA only mutations are at lower cardiac risk than those with other combinations of mutations. Meanwhile, this study confirm the hypothesis on recessive truncating variants of TTN experimentally and thus support earlier reported genotype-phenotype correlations.
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Conectina/genética , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Gemelos , Femenino , Células HEK293 , Humanos , Técnicas In Vitro , Lactante , Imagen por Resonancia Magnética , Microscopía Electrónica , Hipotonía Muscular/fisiopatología , Músculo Esquelético/diagnóstico por imagen , Miopatías Estructurales Congénitas/diagnóstico por imagen , Miopatías Estructurales Congénitas/patología , Miopatías Estructurales Congénitas/fisiopatología , Oftalmoplejía/diagnóstico por imagen , Oftalmoplejía/patología , Oftalmoplejía/fisiopatología , Sitios de Empalme de ARN/genética , ARN Mensajero/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Escoliosis/diagnóstico por imagen , Escoliosis/fisiopatologíaRESUMEN
INTRODUCTION: The objective of this study was to obtain a 6-month natural history of motor function performance in individuals with RYR1- related myopathy (RYR1-RM) by using the Motor Function Measure-32 (MFM-32) and graded functional tests (GFT) while facilitating preparation for interventional trials. METHODS: In total, 34 participants completed the MFM-32 and GFTs at baseline and 6-month visits. RESULTS: Motor deficits according to MFM-32 were primarily observed in the standing and transfers domain (D1; mean 71%). Among the GFTs, participants required the most time to ascend/descend stairs (>7.5 s). Functional movement, determined by GFT grades, was strongly correlated with MFM-32 (D1; r ≥ 0.770, P < 0.001). Motor Function Measure-32 and GFT scores did not reflect any change in performance between baseline and 6-month visits. DISCUSSION: The MFM-32 and GFTs detected motor impairment in RYR1-RM, which remained stable over 6 months. Thus, these measures may be suitable for assessing change in motor function in response to therapeutic intervention. Muscle Nerve 60: 80-87, 2019.
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Movimiento/fisiología , Miopatías Estructurales Congénitas/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/genética , Adolescente , Adulto , Niño , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Miopatías Estructurales Congénitas/genética , Miopatía del Núcleo Central/genética , Miopatía del Núcleo Central/fisiopatología , Oftalmoplejía/genética , Oftalmoplejía/fisiopatología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Adulto JovenRESUMEN
FXR1 is an alternatively spliced gene that encodes RNA binding proteins (FXR1P) involved in muscle development. In contrast to other tissues, cardiac and skeletal muscle express two FXR1P isoforms that incorporate an additional exon-15. We report that recessive mutations in this particular exon of FXR1 cause congenital multi-minicore myopathy in humans and mice. Additionally, we show that while Myf5-dependent depletion of all FXR1P isoforms is neonatal lethal, mice carrying mutations in exon-15 display non-lethal myopathies which vary in severity depending on the specific effect of each mutation on the protein.
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Genes Recesivos , Predisposición Genética a la Enfermedad/genética , Músculo Esquelético/metabolismo , Mutación , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/genética , Proteínas de Unión al ARN/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Animales , Células Cultivadas , Exones/genética , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones Transgénicos , Miopatías Estructurales Congénitas/congénito , Miopatías Estructurales Congénitas/metabolismo , Oftalmoplejía/congénito , Oftalmoplejía/metabolismo , Proteínas de Unión al ARN/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Multiminicore disease is a myopathy that is pathologically characterized by the presence of multiple areas of small, short, and poorly delineated zones of sarcomeric disorganization lacking mitochondria (minicores) that can be observed in both type 1 and type 2 fibers. Most cases of multiminicore disease typically present with early-onset axial weakness, respiratory insufficiency, scoliosis, and rigid spine. There is no correlation between the frequency of minicores and clinical severity. Multiminicore disease is genetically heterogeneous and can result from recessive or dominant mutations. Genetic testing is needed to establish the precise diagnosis and provide overall prognosis. Here we report a 23-year-old woman with respiratory failure, distal joint hyper-laxity, scoliosis and rigid spine due to multiminicore disease caused by a novel compound heterozygous mutation in the selenoprotein N1-encoding gene (SELN). The preserved ambulation into adulthood and normal creatinine kinase (CK) favor the diagnosis of congenital myopathy over congenital muscular dystrophy (CMD). However, the nonspecific myopathic histopathological changes and extremely rare minicore-like structures can make it challenging to differentiate between SELN-myopathy and congenital muscular dystrophies, such as Ullrich or lamin A/C-CMD.
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Miopatías Estructurales Congénitas/diagnóstico , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/diagnóstico , Oftalmoplejía/genética , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Selenoproteínas/genética , Femenino , Humanos , Distrofia Muscular de Cinturas/diagnóstico , Distrofia Muscular de Cinturas/patología , Mutación , Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/genética , Adulto JovenRESUMEN
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.
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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/ultraestructuraRESUMEN
INTRODUCTION: Ca2+ release from sarcoplasmic reticulum (SR) is known to contribute to automaticity via the cytoplasmic Na+-Ca2+ exchanger (NCX). Mitochondria participate in Ca2+ cycling. We studied the role of mitochondrial Ca2+ flux in ventricular spontaneous electrical activity. METHODS: Spontaneously contracting mouse embryonic stem cells (ESC)-derived ventricular cardiomyocytes (CMs) were differentiated from wild type and ryanodine receptor type 2 (RYR2) knockout mouse ESCs and differentiated for 19-21 days. Automaticity was also observed in human induced pluripotent stem cell (hiPSC)-derived ventricular CMs differentiated for 30 days, and acute isolated adult mouse ventricular cells in ischemic simulated buffer. Action potentials (APs) were recorded by perforated whole cell current-clamp. Cytoplasmic and mitochondrial Ca2+ transients were determined by fluorescent imaging. RESULTS: In mouse ESC-derived ventricular CMs, spontaneous beating was dependent on the L-type Ca2+ channel, cytoplasmic NCX and mitochondrial NCX. Spontaneous beating was modulated by SR Ca2+ release from RYR2 or inositol trisphosphate receptors (IP3R), the pacemaker current (If) and mitochondrial Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU). In RYR2 knockout mouse ESC-derived ventricular CMs, mitochondrial Ca2+ flux influenced spontaneous beating independently of the SR Ca2+ release from RYR2, and the mitochondrial effect was dependent on IP3R SR Ca2+ release. Depolarization of mitochondria and preservation of ATP could terminate spontaneous beating. A contribution of mitochondrial Ca2+ flux to automaticity was confirmed in hiPSC-derived ventricular CMs and ischemic adult mouse ventricular CMs, confirming the findings across species and cell maturity levels. CONCLUSIONS: Mitochondrial and sarcolemma NCX fluxes are required for ventricular automaticity. Mitochondrial Ca2+ uptake plays a modulatory role. Mitochondrial Ca2+ uptake through MCU is influenced by IP3R-dependent SR Ca2+ release.
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Potenciales de Acción/fisiología , Calcio/metabolismo , Ventrículos Cardíacos/metabolismo , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Cationes Bivalentes/metabolismo , Línea Celular , Citoplasma/metabolismo , Células Madre Embrionarias/metabolismo , Ventrículos Cardíacos/citología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Miocitos Cardíacos/citología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genética , Sarcolema/metabolismoRESUMEN
Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (CaV1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth.
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Calcio/metabolismo , Unión Neuromuscular/fisiología , Proyección Neuronal/fisiología , Receptores Colinérgicos/metabolismo , Animales , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Diafragma/metabolismo , Embrión de Mamíferos/metabolismo , Desarrollo Fetal , Ratones , Ratones Noqueados , Neuronas Motoras/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
In skeletal muscle the coordinated actions of two mechanically coupled Ca2+ channels-the 1,4-dihydropyridine receptor (Cav1.1) and the type 1 ryanodine receptor (RYR1)-underlie the molecular mechanism of rapid cytosolic [Ca2+] increase leading to contraction. While both [Ca2+]i and contractile activity have been implicated in the regulation of myogenesis, less is known about potential specific roles of Cav1.1 and RYR1 in skeletal muscle development. In this study, we analyzed the histology and the transcriptomic changes occurring at E14.5 -the end of primary myogenesis and around the onset of intrauterine limb movement, and at E18.5 -the end of secondary myogenesis, in WT, RYR1-/-, and Cav1.1-/- murine limb skeletal muscle. At E14.5 the muscle histology of both mutants exhibited initial alterations, which became much more severe at E18.5. Immunohistological analysis also revealed higher levels of activated caspase-3 in the Cav1.1-/- muscles at E14.5, indicating an increase in apoptosis. With WT littermates as controls, microarray analyses identified 61 and 97 differentially regulated genes (DEGs) at E14.5, and 493 and 1047 DEGs at E18.5, in RYR1-/- and Cav1.1-/- samples, respectively. Gene enrichment analysis detected no overlap in the affected biological processes and pathways in the two mutants at E14.5, whereas at E18.5 there was a significant overlap of DEGs in both mutants, affecting predominantly processes linked to muscle contraction. Moreover, the E18.5 vs. E14.5 comparison revealed multiple genotype-specific DEGs involved in contraction, cell cycle and miRNA-mediated signaling in WT, neuronal and bone development in RYR1-/-, and lipid metabolism in Cav1.1-/- samples. Taken together, our study reveals discrete changes in the global transcriptome occurring in limb skeletal muscle from E14.5 to E18.5 in WT, RYR1-/- and Cav1.1-/- mice. Our results suggest distinct functional roles for RYR1 and Cav1.1 in skeletal primary and secondary myogenesis.
Asunto(s)
Canales de Calcio Tipo L/genética , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Transcriptoma , Animales , Canales de Calcio Tipo L/deficiencia , Ontología de Genes , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Desarrollo de Músculos/genética , Músculo Esquelético/embriología , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Factores de TiempoRESUMEN
Reduced protein expression of the cardiac ryanodine receptor type 2 (RyR2) is thought to affect the susceptibility to stress-induced ventricular tachyarrhythmia (VT) and cardiac alternans, but direct evidence for the role of RyR2 protein expression in VT and cardiac alternans is lacking. Here, we used a mouse model (crrm1) that expresses a reduced level of the RyR2 protein to determine the impact of reduced RyR2 protein expression on the susceptibility to VT, cardiac alternans, cardiac hypertrophy, and sudden death. Electrocardiographic analysis revealed that after the injection of relatively high doses of caffeine and epinephrine (agents commonly used for stress test), wild-type (WT) mice displayed long-lasting VTs, whereas the crrm1 mutant mice exhibited no VTs at all, indicating that the crrm1 mutant mice are resistant to stress-induced VTs. Intact heart Ca2+ imaging and action potential (AP) recordings showed that the crrm1 mutant mice are more susceptible to fast-pacing induced Ca2+ alternans and AP duration alternans compared with WT mice. The crrm1 mutant mice also showed an increased heart-to-body-weight ratio and incidence of sudden death at young ages. Furthermore, the crrm1 mutant hearts displayed altered Ca2+ transients with increased time-to-peak and decay time (T50), increased ventricular wall thickness and ventricular cell area compared with WT hearts. These results indicate that reduced RyR2 protein expression suppresses stress-induced VTs, but enhances the susceptibility to cardiac alternans, hypertrophy, and sudden death.
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Calcio/metabolismo , Cardiomegalia/genética , Ventrículos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Potenciales de Acción/efectos de los fármacos , Animales , Cafeína/farmacología , Señalización del Calcio , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatología , Muerte Súbita Cardíaca/patología , Modelos Animales de Enfermedad , Epinefrina/farmacología , Expresión Génica , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ratones , Ratones Transgénicos , Contracción Muscular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Técnicas de Cultivo de Órganos , Periodicidad , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Estrés Fisiológico/efectos de los fármacos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologíaRESUMEN
BACKGROUND: Defects of the slow myosin heavy chain isoform coding MYH7 gene primarily cause skeletal myopathies including Laing Distal Myopathy, Myosin Storage Myopathy and are also responsible for cardiomyopathies. Scapuloperoneal and limb-girdle muscle weakness, congenital fiber type disproportion, multi-minicore disease were also reported in connection of MYH7. Pathogeneses of the defects in the head and proximal rod region of the protein are well described. However, the C-terminal mutations of the MYH7 gene are less known. Moreover, only two articles describe the phenotypic impact of the elongated mature protein product caused by termination signal loss. CASE PRESENTATION: Here we present a male patient with an unusual phenotypic variant of early-onset and predominant involvement of neck muscles with muscle biopsy indicating myopathy and sarcoplasmic storage material. Cardiomyopathic involvements could not be observed. Sequencing of MYH7 gene revealed a stop-loss mutation on the 3-prime end of the rod region, which causes the elongation of the mature protein. CONCLUSIONS: The elongated protein likely disrupts the functions of the sarcomere by multiple functional abnormalities. This elongation could also affect the thick filament degradation leading to protein deposition and accumulation in the sarcomere, resulting in the severe myopathy of certain axial muscles. The phenotypic expression of the detected novel MYH7 genotype could strengthen and further expand our knowledge about mutations affecting the structure of MyHCI by termination signal loss in the MYH7 gene.
Asunto(s)
Miosinas Cardíacas/genética , Variación Genética , Enfermedades Musculares/congénito , Cadenas Pesadas de Miosina/genética , Miopatías Distales/diagnóstico por imagen , Miopatías Distales/genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Enfermedades Musculares/diagnóstico por imagen , Enfermedades Musculares/genética , Mutación , Miopatías Estructurales Congénitas/diagnóstico por imagen , Miopatías Estructurales Congénitas/genética , Oftalmoplejía/diagnóstico por imagen , Oftalmoplejía/genética , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
RATIONALE: Mutations in the cardiac Ryanodine Receptor gene (RYR2) cause dominant catecholaminergic polymorphic ventricular tachycardia (CPVT), a leading cause of sudden death in apparently healthy individuals exposed to emotions or physical exercise. OBJECTIVE: We investigated the efficacy of allele-specific silencing by RNA interference to prevent CPVT phenotypic manifestations in our dominant CPVT mice model carriers of the heterozygous mutation R4496C in RYR2. METHODS AND RESULTS: We developed an in vitro mRNA and protein-based assays to screen multiple siRNAs for their ability to selectively silence mutant RYR2-R4496C mRNA over the corresponding wild-type allele. For the most performant of these siRNAs (siRYR2-U10), we evaluated the efficacy of an adeno-associated serotype 9 viral vector (AAV9) expressing miRYR2-U10 in correcting RyR2 (Ryanodine Receptor type 2 protein) function after in vivo delivery by intraperitoneal injection in neonatal and adult RyR2R4496C/+ (mice heterozygous for the R4496C mutation in the RyR2) heterozygous CPVT mice. Transcriptional analysis showed that after treatment with miRYR2-U10, the ratio between wild-type and mutant RYR2 mRNA was doubled (from 1:1 to 2:1) confirming the ability of miRYR2-U10 to selectively inhibit RYR2-R4496C mRNA, whereas protein quantification showed that total RyR2 was reduced by 15% in the heart of treated mice. Furthermore, AAV9-miRYR2-U10 effectively (1) reduced isoproterenol-induced delayed afterdepolarizations and triggered activity in infected cells, (2) reduced adrenergically mediated ventricular tachycardia in treated mice, (3) reverted ultrastructural abnormalities of junctional sarcoplasmic reticulum and transverse tubules, and (4) attenuated mitochondrial abnormalities. CONCLUSIONS: The study demonstrates that allele-specific silencing with miRYR2-U10 prevents life-threatening arrhythmias in CPVT mice, suggesting that the reduction of mutant RyR2 may be a novel therapeutic approach for CPVT.
Asunto(s)
Alelos , Arritmias Cardíacas/genética , Heterocigoto , Mutación/genética , ARN Mensajero/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Animales , Animales Recién Nacidos , Arritmias Cardíacas/patología , Arritmias Cardíacas/prevención & control , Células Cultivadas , Silenciador del Gen/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , ARN Mensajero/ultraestructura , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/ultraestructuraRESUMEN
Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.
Asunto(s)
Señalización del Calcio/fisiología , Mitocondrias Cardíacas/fisiología , Animales , Acoplamiento Excitación-Contracción/fisiología , Ratones , Microscopía Confocal , Microscopía Electrónica , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/ultraestructura , Dinámicas Mitocondriales/fisiología , Mutagénesis Sitio-Dirigida , Mutación Missense , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Taquicardia Ventricular/genéticaRESUMEN
BACKGROUND: Phosphorylation of the cardiac ryanodine receptor (RyR2) phospho-site S2808 has been touted by the Marks group as a hallmark of heart failure (HF) and a critical mediator of the physiological fight-or-flight response of the heart. In support of this hypothesis, mice unable to undergo phosphorylation at RyR2-S2808 (S2808A) were significantly protected against HF and displayed a blunted response to adrenergic stimulation. However, the issue remains highly controversial because several groups have been unable to reproduce these findings. An important variable not considered before is the genetic background of the mice used to obtain these divergent results. METHODS AND RESULTS: We backcrossed a RyR2-S2808A mouse into a congenic C57Bl/6 strain, the same strain used by the Marks group to conduct their experiments. We then performed several key experiments to confirm or discard the genetic background of the mouse as a relevant variable, including induction of HF by myocardial infarction and tests of integrity of adrenergic response. Congenic C57Bl/6 harboring the S2808A mutation showed similar echocardiographic parameters that indicated identical progression towards HF compared to wild type controls, and had a normal response to adrenergic stimulation in whole animal and cellular experiments. CONCLUSIONS: The genetic background of the different mouse models is unlikely to be the source of the divergent results obtained by the Marks group in comparison to several other groups. Cardiac adrenergic response and progression towards HF proceed unaltered in mice harboring the RyR2-S2808A mutation. Preventing RyR2-S2808 phosphorylation does not preclude a normal sympathetic response nor mitigates the pathophysiological consequences of MI.
Asunto(s)
Adrenérgicos/farmacología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Acoplamiento Excitación-Contracción/efectos de los fármacos , Acoplamiento Excitación-Contracción/genética , Insuficiencia Cardíaca/mortalidad , Insuficiencia Cardíaca/patología , Pruebas de Función Cardíaca , Ratones , Ratones Transgénicos , Infarto del Miocardio/complicaciones , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Fosforilación/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/deficiencia , Análisis de Secuencia de ADNRESUMEN
The TTN gene with 363 coding exons encodes titin, a giant muscle protein spanning from the Z-disk to the M-band within the sarcomere. Mutations in the TTN gene have been associated with different genetic disorders, including hypertrophic and dilated cardiomyopathy and several skeletal muscle diseases.Before the introduction of next generation sequencing (NGS) methods, the molecular analysis of TTN has been laborious, expensive and not widely used, resulting in a limited number of mutations identified. Recent studies however, based on the use of NGS strategies, give evidence of an increasing number of rare and unique TTN variants. The interpretation of these rare variants of uncertain significance (VOUS) represents a challenge for clinicians and researchers.The main aim of this review is to describe the wide spectrum of muscle diseases caused by TTN mutations so far determined, summarizing the molecular findings as well as the clinical data, and to highlight the importance of joint efforts to respond to the challenges arising from the use of NGS. An international collaboration through a clinical and research consortium and the development of a single accessible database listing variants in the TTN gene, identified by high throughput approaches, may be the key to a better assessment of titinopathies and to systematic genotype- phenotype correlation studies.