RESUMEN
Filamins are mechanosensitive actin crosslinking proteins that organize the actin cytoskeleton in a variety of shapes and tissues. In muscles, filamin crosslinks actin filaments from opposing sarcomeres, the smallest contractile units of muscles. This happens at the Z-disc, the actin-organizing center of sarcomeres. In flies and vertebrates, filamin mutations lead to fragile muscles that appear ruptured, suggesting filamin helps counteract muscle rupturing during muscle contractions by providing elastic support and/or through signaling. An elastic region at the C-terminus of filamin is called the mechanosensitive region and has been proposed to sense and counteract contractile damage. Here we use molecularly defined mutants and microscopy analysis of the Drosophila indirect flight muscles to investigate the molecular details by which filamin provides cohesion to the Z-disc. We made novel filamin mutations affecting the C-terminal region to interrogate the mechanosensitive region and detected three Z-disc phenotypes: dissociation of actin filaments, Z-disc rupture, and Z-disc enlargement. We tested a constitutively closed filamin mutant, which prevents the elastic changes in the mechanosensitive region and results in ruptured Z-discs, and a constitutively open mutant which has the opposite elastic effect on the mechanosensitive region and gives rise to enlarged Z-discs. Finally, we show that muscle contraction is required for Z-disc rupture. We propose that filamin senses myofibril damage by elastic changes in its mechanosensory region, stabilizes the Z-disc, and counteracts contractile damage at the Z-disc.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Filaminas , Contracción Muscular , Mutación , Miofibrillas , Animales , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Filaminas/metabolismo , Filaminas/genética , Mecanotransducción Celular/genética , Contracción Muscular/genética , Contracción Muscular/fisiología , Miofibrillas/metabolismo , Miofibrillas/genética , Fenotipo , Sarcómeros/metabolismo , Sarcómeros/genéticaRESUMEN
In striated muscle, the sarcomeric protein myosin-binding protein-C (MyBP-C) is bound to the myosin thick filament and is predicted to stabilize myosin heads in a docked position against the thick filament, which limits crossbridge formation. Here, we use the homozygous Mybpc2 knockout (C2-/-) mouse line to remove the fast-isoform MyBP-C from fast skeletal muscle and then conduct mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers present deficits in force production and calcium sensitivity. Structurally, passive C2-/- fibers present altered sarcomere length-independent and -dependent regulation of myosin head conformations, with a shift of myosin heads towards actin. At shorter sarcomere lengths, the thin filament is axially extended in C2-/-, which we hypothesize is due to increased numbers of low-level crossbridges. These findings provide testable mechanisms to explain the etiology of debilitating diseases associated with MyBP-C.
Asunto(s)
Proteínas Portadoras , Ratones Noqueados , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Ratones , Sarcómeros/metabolismo , Miofibrillas/metabolismo , Miofibrillas/genética , Músculo Esquelético/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/genética , Masculino , Miosinas/metabolismo , Miosinas/genéticaRESUMEN
Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies, where mutations in genes involved in RNA metabolism or characterized by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle MRI, with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterized by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although inter-individual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganization. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients' muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.
Asunto(s)
Distrofia Muscular de Cinturas , Humanos , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/patología , Masculino , Femenino , Adulto , Animales , Músculo Esquelético/patología , Músculo Esquelético/metabolismo , Linaje , Drosophila melanogaster , Miofibrillas/patología , Miofibrillas/genética , Miofibrillas/metabolismo , Persona de Mediana Edad , Fenotipo , Adolescente , Adulto Joven , NiñoRESUMEN
BACKGROUND: Congenital myopathies are a clinical, histopathological and genetic heterogeneous group of inherited muscle disorders that are defined on peculiar architectural abnormalities in the muscle fibres. Although there have been at least 33 different genetic causes of the disease, a significant percentage of congenital myopathies remain genetically unresolved. The present study aimed to report a novel TUBA4A variant in two unrelated Chinese patients with sporadic congenital myopathy. METHODS: A comprehensive strategy combining laser capture microdissection, proteomics and whole-exome sequencing was performed to identify the candidate genes. In addition, the available clinical data, myopathological changes, the findings of electrophysiological examinations and thigh muscle MRIs were also reviewed. A cellular model was established to assess the pathogenicity of the TUBA4A variant. RESULTS: We identified a recurrent novel heterozygous de novo c.679C>T (p.L227F) variant in the TUBA4A (NM_006000), encoding tubulin alpha-4A, in two unrelated patients with clinicopathologically diagnosed sporadic congenital myopathy. The prominent myopathological changes in both patients were muscle fibres with focal myofibrillar disorganisation and rimmed vacuoles. Immunofluorescence showed ubiquitin-positive TUBA4A protein aggregates in the muscle fibres with rimmed vacuoles. Overexpression of the L227F mutant TUBA4A resulted in cytoplasmic aggregates which colocalised with ubiquitin in cellular model. CONCLUSION: Our findings expanded the phenotypic and genetic manifestations of TUBA4A as well as tubulinopathies, and added a new type of congenital myopathy to be taken into consideration in the differential diagnosis.
Asunto(s)
Miopatías Estructurales Congénitas , Tubulina (Proteína) , Adulto , Femenino , Humanos , Masculino , Secuenciación del Exoma , Músculo Esquelético/patología , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/metabolismo , Mutación , Miofibrillas/patología , Miofibrillas/genética , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/patología , Miotonía Congénita/genética , Miotonía Congénita/patología , Linaje , Tubulina (Proteína)/genéticaRESUMEN
Myofibrillar myopathies (MFMs) are a group of genetically heterogeneous diseases affecting the skeletal and cardiac muscles. Myofibrillar myopathies are characterized by focal lysis of myogenic fibers and integration of degraded myogenic fiber products into inclusion bodies, which are typically rich in desmin and many other proteins. Herein, we report a case of a 54-year-old woman who experienced bilateral thigh weakness for over three years. She was diagnosed with MFMs based on muscle biopsy findings and the presence of a novel mutation in exon 8 of the LDB3 gene. Myofibrillar myopathies caused by a mutation in the LDB3 gene are extremely uncommon and often lack distinct clinical characteristics and typically exhibit a slow disease progression. When considering a diagnosis of MFMs, particularly in complex instances of autosomal dominant myopathies where muscle biopsies do not clearly indicate MFMs, it becomes crucial for clinicians to utilize genetic test as a diagnostic tool.
Asunto(s)
Miofibrillas , Miopatías Estructurales Congénitas , Femenino , Humanos , Persona de Mediana Edad , Miofibrillas/genética , Miofibrillas/metabolismo , Miofibrillas/patología , Miopatías Estructurales Congénitas/diagnóstico , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Mutación , Exones , Miocardio , Músculo Esquelético/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismoRESUMEN
Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.
Asunto(s)
Miofibrillas , Troponina T , Humanos , Miofibrillas/genética , Miofibrillas/patología , Troponina T/genética , Troponina T/química , Actinas/genética , Mutación , Citoesqueleto de Actina/genética , Miosinas , CalcioRESUMEN
Myofibrils are the intracellular structures formed by actin and myosin filaments. They are paracrystalline contractile cables with unusually well-defined dimensions. The sliding of actin past myosin filaments powers contractions, and the entire system is held in place by a structure called the Z-disc, which anchors the actin filaments. Myosin filaments, in turn, are anchored to another structure called the M-line. Most of the complex architecture of myofibrils can be reduced to studying the Z-disc, and recently, important advances regarding the arrangement and function of Z-discs in insects have been published. On a very small scale, we have detailed protein structure information. At the medium scale, we have cryo-electron microscopy maps, super-resolution microscopy and protein-protein interaction networks, while at the functional scale, phenotypic data are available from precise genetic manipulations. All these data aim to answer how the Z-disc works and how it is assembled. Here, we summarize recent data from insects and explore how it fits into our view of the Z-disc, myofibrils and, ultimately, muscles.
Asunto(s)
Actinas , Sarcómeros , Actinas/metabolismo , Animales , Biología , Microscopía por Crioelectrón , Insectos/metabolismo , Miofibrillas/química , Miofibrillas/genética , Miofibrillas/metabolismo , Miosinas/metabolismoRESUMEN
Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.
Asunto(s)
Proteína CapZ/metabolismo , Enfermedades Musculares , Miofibrillas , Actinas/genética , Actinas/metabolismo , Animales , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Miofibrillas/genética , Miofibrillas/metabolismo , Tropomodulina/genética , Tropomodulina/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.
Asunto(s)
Proteínas de Drosophila , Miofibrillas , Proteínas de Unión al ARN , Animales , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Masculino , Miofibrillas/genética , Miofibrillas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. METHODS: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. RESULTS: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. CONCLUSIONS: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs.
Asunto(s)
Cardiomiopatía Dilatada/genética , Conectina/genética , Edición Génica , Sistemas de Lectura/genética , Edición Génica/métodos , Variación Genética/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Miofibrillas/genética , Miofibrillas/metabolismoRESUMEN
Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1-related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses (MYH1E321G-/-) and Quarter Horses homozygous (MYH1E321G+/+) or heterozygous (MYH1E321G+/-) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca2+ sensitivity for MYH1E321G+/+ in comparison with MYH1E321G+/- and MYH1E321G-/- horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.
Asunto(s)
Caballos/genética , Contracción Muscular/genética , Enfermedades Musculares/genética , Cadenas Pesadas de Miosina/genética , Sustitución de Aminoácidos/genética , Animales , Regulación de la Expresión Génica/genética , Heterocigoto , Homocigoto , Contracción Muscular/fisiología , Enfermedades Musculares/patología , Enfermedades Musculares/veterinaria , Mutación/genética , Miofibrillas/genética , Miofibrillas/metabolismoRESUMEN
Myofiber atrophy occurs with aging and in many diseases but the underlying mechanisms are incompletely understood. Here, we have used >1,100 muscle-targeted RNAi interventions to comprehensively assess the function of 447 transcription factors in the developmental growth of body wall skeletal muscles in Drosophila. This screen identifies new regulators of myofiber atrophy and hypertrophy, including the transcription factor Deaf1. Deaf1 RNAi increases myofiber size whereas Deaf1 overexpression induces atrophy. Consistent with its annotation as a Gsk3 phosphorylation substrate, Deaf1 and Gsk3 induce largely overlapping transcriptional changes that are opposed by Deaf1 RNAi. The top category of Deaf1-regulated genes consists of glycolytic enzymes, which are suppressed by Deaf1 and Gsk3 but are upregulated by Deaf1 RNAi. Similar to Deaf1 and Gsk3 overexpression, RNAi for glycolytic enzymes reduces myofiber growth. Altogether, this study defines the repertoire of transcription factors that regulate developmental myofiber growth and the role of Gsk3/Deaf1/glycolysis in this process.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Glucógeno Sintasa Quinasa 3/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Animales , Animales Modificados Genéticamente/genética , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Desarrollo Embrionario/genética , Glucólisis/genética , Humanos , Músculo Esquelético/crecimiento & desarrollo , Atrofia Muscular/patología , Miofibrillas/genética , Miofibrillas/metabolismo , Interferencia de ARN , Factores de Transcripción/genéticaRESUMEN
This study aimed to analyze the effects of fibrin constructs enhanced with laminin-nidogen, implanted in the wounded rat soft palate. Fibrin constructs with and without laminin-nidogen were implanted in 1 mm excisional wounds in the soft palate of 9-week-old rats and compared with the wounded soft palate without implantation. Collagen deposition and myofiber formation were analyzed at days 3, 7, 28 and 56 after wounding by histochemistry. In addition, immune staining was performed for a-smooth muscle actin (a-SMA), myosin heavy chain (MyHC) and paired homeobox protein 7 (Pax7). At day 56, collagen areas were smaller in both implant groups (31.25 ± 7.73% fibrin only and 21.11 ± 6.06% fibrin with laminin-nidogen)) compared to the empty wounds (38.25 ± 8.89%, p < 0.05). Moreover, the collagen area in the fibrin with laminin-nidogen group was smaller than in the fibrin only group (p Ë 0.05). The areas of myofiber formation in the fibrin only group (31.77 ± 10.81%) and fibrin with laminin-nidogen group (43.13 ± 10.39%) were larger than in the empty wounds (28.10 ± 11.68%, p Ë 0.05). Fibrin-based constructs with laminin-nidogen reduce fibrosis and improve muscle regeneration in the wounded soft palate. This is a promising strategy to enhance cleft soft palate repair and other severe muscle injuries.
Asunto(s)
Fibrina/genética , Fibrosis/genética , Paladar Blando/lesiones , Cicatrización de Heridas/genética , Actinas/genética , Animales , Colágeno/genética , Fibrina/farmacología , Fibrosis/patología , Fibrosis/terapia , Humanos , Laminina/genética , Laminina/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/crecimiento & desarrollo , Miofibrillas/genética , Cadenas Pesadas de Miosina/genética , Factores de Transcripción Paired Box/genética , Paladar Blando/efectos de los fármacos , Paladar Blando/patología , Ratas , Regeneración/genéticaRESUMEN
Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Microfilamentos/genética , Músculo Esquelético/metabolismo , Estrés Mecánico , Proteínas de Transporte Vesicular/genética , Citoesqueleto de Actina/genética , Actinina/genética , Actinas/genética , Animales , Autofagia/genética , Línea Celular , Citoesqueleto/genética , Humanos , Ratones , Fibras Musculares Esqueléticas/metabolismo , Músculo Estriado/metabolismo , Miofibrillas/genética , Miofibrillas/metabolismo , Dominios PDZ/genética , Isoformas de Proteínas/genética , Sinaptofisina/genéticaRESUMEN
A comprehensive transcriptomic survey of pigs can provide a mechanistic understanding of tissue specialization processes underlying economically valuable traits and accelerate their use as a biomedical model. Here we characterize four transcript types (lncRNAs, TUCPs, miRNAs, and circRNAs) and protein-coding genes in 31 adult pig tissues and two cell lines. We uncover the transcriptomic variability among 47 skeletal muscles, and six adipose depots linked to their different origins, metabolism, cell composition, physical activity, and mitochondrial pathways. We perform comparative analysis of the transcriptomes of seven tissues from pigs and nine other vertebrates to reveal that evolutionary divergence in transcription potentially contributes to lineage-specific biology. Long-range promoter-enhancer interaction analysis in subcutaneous adipose tissues across species suggests evolutionarily stable transcription patterns likely attributable to redundant enhancers buffering gene expression patterns against perturbations, thereby conferring robustness during speciation. This study can facilitate adoption of the pig as a biomedical model for human biology and disease and uncovers the molecular bases of valuable traits.
Asunto(s)
Tejido Adiposo/metabolismo , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , ARN Circular/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Transcriptoma/genética , Empalme Alternativo , Animales , Evolución Biológica , Línea Celular , Linaje de la Célula , Núcleo Celular/genética , Núcleo Celular/metabolismo , Elementos de Facilitación Genéticos , Evolución Molecular , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , Mitocondrias/metabolismo , Conformación Molecular , Miofibrillas/genética , Miofibrillas/metabolismo , Filogenia , Regiones Promotoras Genéticas , ARN Circular/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Análisis Espacial , PorcinosRESUMEN
One of the complexities of understanding the pathology of familial forms of cardiac diseases is the level of mutation incorporation in sarcomeres. Computational models of the sarcomere that are spatially explicit offer an approach to study aspects of mutational incorporation into myofilaments that are more challenging to get at experimentally. We studied two well characterized mutations of cardiac TnC, L48Q and I61Q, that decrease or increase the release rate of Ca2+ from cTnC, k-Ca, resulting in HCM and DCM respectively [1]. Expression of these mutations in transgenic mice was used to provide experimental data for incorporation of 30 and 50% (respectively) into sarcomeres. Here we demonstrate that fixed length twitch contractions of trabeculae from mice containing mutant differ from WT; L48Q trabeculae have slower relaxation while I61Q trabeculae have markedly reduced peak tension. Using our multiscale modelling approach [2] we were able to describe the tension transients of WT mouse myocardium. Tension transients for the mutant cTnCs were simulated with changes in k-Ca, measured experimentally for each cTnC mutant in whole troponin complex, a change in the affinity of cTnC for cTnI, and a reduction in the number of detached crossbridges available for binding. A major advantage of the multiscale explicit 3-D model is that it predicts the effects of variable mutation incorporation, and the effects of variations in mutation distribution within thin filaments in sarcomeres. Such effects are currently impossible to explore experimentally. We explored random and clustered distributions of mutant cTnCs in thin filaments, as well as distributions of individual thin filaments with only WT or mutant cTnCs present. The effects of variable amounts of incorporation and non-random distribution of mutant cTnCs are more marked for I61Q than L48Q cTnC. We conclude that this approach can be effective for study on mutations in multiple proteins of the sarcomere. SUMMARY: A challenge in experimental studies of diseases is accounting for the effect of variable mutation incorporation into myofilaments. Here we use a spatially explicit computational approach, informed by experimental data from transgenic mice expressing one of two mutations in cardiac Troponin C that increase or decrease calcium sensitivity. We demonstrate that the model can accurately describe twitch contractions for the data and go on to explore the effect of variable mutant incorporation and localization on simulated cardiac muscle twitches.
Asunto(s)
Modelos Biológicos , Mutación , Contracción Miocárdica , Miofibrillas/genética , Miofibrillas/metabolismo , Troponina C/genética , Algoritmos , Alelos , Animales , Biomarcadores , Calcio/metabolismo , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Miofibrillas/química , Unión Proteica , Sarcómeros/metabolismo , Relación Estructura-Actividad , Troponina C/química , Troponina I/genética , Troponina I/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.
Asunto(s)
Degeneración Macular/genética , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Regeneración/genética , Transducción de Señal/genética , Animales , Modelos Animales de Enfermedad , Exones/genética , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Mutación/genética , Miofibrillas/genética , Análisis de Secuencia de ARN/métodos , Transcripción Genética/genética , Transcriptoma/genéticaRESUMEN
DNA endoreplication has been implicated as a cell strategy for cell growth and in tissue injury. Here, we demonstrate that barrier-to-autointegration factor (BAF) represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope is eliminated in flies with mutations of the linker of nucleoskeleton and cytoskeleton (LINC) complex in which the LEM-domain protein Otefin is excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope requires the activity of the BAF kinase VRK1/Ball, and, consistently, non-phosphorylatable BAF-GFP is excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication, overlaps BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope results in increased E2F1 levels in the nucleoplasm and subsequent elevated DNA content. We suggest that LINC-dependent and phosphosensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation in the nucleoplasm.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Endorreduplicación/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Citoesqueleto/genética , Replicación del ADN/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica/genética , Mutación/genética , Miofibrillas/genética , Membrana Nuclear/genética , Matriz Nuclear/genética , Protamina Quinasa/genéticaRESUMEN
Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension-generating capacity. We demonstrated that cardiac twitch tension-time integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using multiscale computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L48Q can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L48Q cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.
Asunto(s)
Señalización del Calcio/genética , Cardiomiopatía Dilatada/genética , Corazón/crecimiento & desarrollo , Troponina C/genética , Sustitución de Aminoácidos/genética , Animales , Calcio/metabolismo , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Corazón/fisiopatología , Humanos , Ratones , Ratones Transgénicos , Mutación/genética , Contracción Miocárdica/genética , Miocardio/metabolismo , Miocardio/patología , Miofibrillas/genética , Miofibrillas/patología , Sarcómeros/genética , Sarcómeros/patologíaRESUMEN
Muscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and Ca2+-mediated force were largely unaffected. Mutant single fibers showed tendencies towards faster unloaded shortening over wild type fibers. Effects of aging seen in the wild type appeared earlier in the mutant desmin fibers. Our single-fiber experiments, free of extracellular matrix, suggest that compromised muscle biomechanics is not exclusively attributed to fibrosis but also originates from an impaired intermediate filament network.