scholarly journals Dysregulation of NRAP degradation by KLHL41 contributes to pathophysiology in Nemaline Myopathy

2018 ◽  
Author(s):  
Caroline Jirka ◽  
Jasmine H Pak ◽  
Claire A Grosgogeat ◽  
Michael M Marchetti ◽  
Vandana A Gupta
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Muscle Development ◽  
Nemaline Myopathy ◽  
Therapeutic Interventions ◽  
Transgenic Zebrafish ◽  
Sarcomeric Protein ◽  
And Function

Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific-adapters for CUL3 E3 ubiquitin ligase to regulate protein turn-over through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM, however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive in vivo. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, NRAP. KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric protein contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

scholarly journals Dysregulation of NRAP degradation by KLHL41 contributes to pathophysiology in nemaline myopathy

2019 ◽  
Vol 28 (15) ◽  
pp. 2549-2560 ◽  
Author(s):  
Caroline Jirka ◽  
Jasmine H Pak ◽  
Claire A Grosgogeat ◽  
Michael Mario Marchetii ◽  
Vandana A Gupta
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Muscle Development ◽  
Nemaline Myopathy ◽  
Therapeutic Interventions ◽  
Transgenic Zebrafish ◽  
Anchoring Protein ◽  
And Function

Abstract Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific adaptors for Cullin 3 (CUL3) E3 ubiquitin ligase to regulate protein turnover through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM; however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin-related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric proteins contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease-causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

scholarly journals Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle

2018 ◽  
Vol 115 (41) ◽  
pp. 10369-10374 ◽  
Author(s):  
Balázs Kiss ◽  
Eun-Jeong Lee ◽  
Weikang Ma ◽  
Frank W. Li ◽  
Paola Tonino ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Thick Filament ◽  
Nemaline Myopathy ◽  
Sarcomeric Protein ◽  
Cross Bridge ◽  
Intact Muscle ◽  
Filament Activation ◽  
Muscle Thin Filament ◽  
And Function

Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in nebulin cause muscle weakness in nemaline myopathy patients, suggesting that nebulin plays important roles in force generation, yet little is known about nebulin’s influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in nebulin (using a conditional nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 Å) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 Å) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.

scholarly journals New Insights into the Structural Roles of Nebulin in Skeletal Muscle

2010 ◽  
Vol 2010 ◽  
pp. 1-6 ◽  
Author(s):  
Coen A. C. Ottenheijm ◽  
Henk Granzier
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Force Production ◽  
Nemaline Myopathy ◽  
Structure And Function ◽  
Filament Length ◽  
Thin Filaments ◽  
Muscle Structure ◽  
And Function

One important feature of muscle structure and function that has remained relatively obscure is the mechanism that regulates thin filament length. Filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments. Recent advances, due in part to the generation of nebulin KO models, reveal that nebulin plays an important role in the regulation of thin filament length. Another structural feature of skeletal muscle that is not well understood is the mechanism involved in maintaining the regular lateral alignment of adjacent sarcomeres, that is, myofibrillar connectivity. Recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils. Thus, novel structural roles of nebulin in skeletal muscle involve the regulation of thin filament length and maintaining myofibrillar connectivity. When these functions of nebulin are absent, muscle weakness ensues, as is the case in patients with nemaline myopathy with mutations in nebulin. Here we review these new insights in the role of nebulin in skeletal muscle structure.

scholarly journals Cofilin loss in Drosophila contributes to myopathy through defective sarcomerogenesis and aggregate formation during muscle growth

2019 ◽  
Author(s):  
Mridula Balakrishnan ◽  
Shannon F. Yu ◽  
Samantha M. Chin ◽  
David B. Soffar ◽  
Stefanie E. Windner ◽  
...  
Keyword(s):  
Point Mutation ◽  
Muscle Function ◽  
Muscle Growth ◽  
Nemaline Myopathy ◽  
Aggregate Formation ◽  
Thin Filaments ◽  
Sarcomeric Protein ◽  
Thick Filaments

SUMMARYSarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila Cofilin (DmCFL), an actin depolymerizing factor, leads to the progressive disruption of sarcomere structure and muscle function in vivo. Loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. Strikingly, activation of the proteasome delayed muscle deterioration in our model. Further, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans, affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation as well as mechanistic implications for disease progression in NM patients.

scholarly journals The Generation and Regulation of Tissue-Resident Tregs and Their Role in Autoimmune Diseases

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Shuang Wang ◽  
Xueyang Zou ◽  
Yi Zhang ◽  
Xiaoya Wang ◽  
Wei Yang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
T Cells ◽  
Immune Responses ◽  
Autoimmune Diseases ◽  
Hot Spot ◽  
Exploratory Stage ◽  
And Function

Regulatory T cells (Tregs), as an important subset of T cells, play an important role in maintaining body homeostasis by regulating immune responses and preventing autoimmune diseases. In-depth research finds that Tregs have strong instability and plasticity, and according to their developmental origin, Tregs can be classified into thymic-derived Tregs (tTregs), endogenous-induced Tregs (pTregs), which are produced by antigen-stimulated T cells in the periphery in vivo, and induced Tregs (iTregs), which differentiate from naïve T cells in vitro. In recent years, studies have found that Tregs are divided into lymphatic and tissue-resident Tregs according to their location. Research on the generation and function of lymphoid Tregs has been more comprehensive and thorough, but the role of tissue Tregs is still in the exploratory stage, and it has become a research hot spot. In this review, we discuss the instability and plasticity of Tregs and the latest developments of tissue-resident Tregs in the field of biology, including adipose tissue, colon, skeletal muscle, and other Tregs that have been recently discovered as well as their production, regulation, and function in specific tissues and their role in the pathogenesis of autoimmune diseases.

scholarly journals The role of TORC1 in muscle development in Drosophila.

2014 ◽  
Author(s):  
Isabelle Hatfield ◽  
Innocence Harvey ◽  
Erika R. Yates ◽  
JeAnna R. Redd ◽  
Lawrence T. Reiter ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Muscle Development ◽  
Myosin Heavy Chain Isoforms ◽  
C2c12 Cells ◽  
Important Process ◽  
Mtorc1 Pathway ◽  
And Function ◽  
Muscle Precursor Cells

Myogenesis is an important process during both development and muscle repair. Previous studies suggest that mTORC1 plays a role in the formation of mature muscle from immature muscle precursor cells. Here we show that gene expression for several myogenic transcription factors including Myf5, Myog and Mef2c but not MyoD and myosin heavy chain isoforms decrease when C2C12 cells are treated with rapamycin, supporting a role for mTORC1 pathway during muscle development. To investigate the possibility that mTORC1 can regulate muscle in vivo we ablated the essential dTORC1 subunit Raptor in Drosophila melanogaster and found that muscle-specific knockdown of Raptor causes flies to be too weak to emerge from their pupal cases during eclosion. Using a series of GAL4 drivers we also show that muscle-specific Raptor knockdown also causes shortened lifespan, even when eclosure is unaffected. Together these results highlight an important role for TORC1 in muscle development, integrity and function in both Drosophila and mammalian cells.

scholarly journals Differential physiological role of BIN1 isoforms in skeletal muscle development, function and regeneration

2018 ◽  
Author(s):  
Ivana Prokic ◽  
Belinda Cowling ◽  
Candice Kutchukian ◽  
Christine Kretz ◽  
Hichem Tasfaout ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Regeneration ◽  
Muscle Development ◽  
Physiological Role ◽  
Membrane Remodeling ◽  
Skeletal Muscle Development ◽  
Developmental Defects ◽  
Intracellular Organization ◽  
And Function

AbstractSkeletal muscle development and regeneration are tightly regulated processes. How the intracellular organization of muscle fibers is achieved during these steps is unclear. Here we focus on the cellular and physiological roles of amphiphysin 2 (BIN1), a membrane remodeling protein mutated in both congenital and adult centronuclear myopathies, that is ubiquitously expressed and has skeletal muscle-specific isoforms. We created and characterized constitutive, muscle-specific and inducible Bin1 homozygous and heterozygous knockout mice targeting either ubiquitous or muscle-specific isoforms. Constitutive Bin1-deficient mice died at birth from lack of feeding due to a skeletal muscle defect. T-tubules and other organelles were misplaced and altered, supporting a general early role of BIN1 on intracellular organization in addition to membrane remodeling. Whereas restricted deletion of Bin1 in unchallenged adult muscles had no impact, the forced switch from the muscle-specific isoforms to the ubiquitous isoforms through deletion of the in-frame muscle–specific exon delayed muscle regeneration. Thus, BIN1 ubiquitous function is necessary for muscle development and function while its muscle-specific isoforms fine-tune muscle regeneration in adulthood, supporting that BIN1 centronuclear myopathy with congenital onset are due to developmental defects while later onset may be due to regeneration defects.

MO588DECREASED IL-15 PRODUCTION INDUCED BY HYPERPHOSPHATEMIA AS A POSSIBLE MECHANISM OF AGED-RELATED SARCOPENIA*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Elena Alcalde-Estévez ◽  
Ana Asenjo-Bueno ◽  
Gemma Olmos ◽  
Diego Rodríguez-Puyol ◽  
Susana López-Ongil ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Muscle Mass ◽  
Quadriceps Muscle ◽  
Serum Phosphate ◽  
In Vivo Studies ◽  
And Function ◽  
Old Mice

Abstract Background and Aims The loss of muscle mass and function, termed sarcopenia, is an aging-related condition associated to some important diseases such as chronic kidney disease (CKD). Hyperphosphatemia has been related to both pathologies. A chronic subclinical inflammation and a dysregulated immune system function are associated to aging affecting to multiple pathways in the skeletal muscle, and this fact has been linked to the development of sarcopenia. Interleukin-15 (IL-15) is a skeletal muscle-derived cytokine which promotes muscle regeneration. The aim of this work was to analyze the role of hyperphosphatemia on the IL-15 production of the skeletal muscle and its implication in aging-related sarcopenia. Method Cultured C2C12 myoblasts were used for in vitro experiments. Cells were treated with 10 mM beta-glycerophosphate (BGP) as a phosphate donor for 2, 4, 6, 8 and 24 hours. IL-15 mRNA levels were assessed by RT-qPCR. Three groups of C57BL6 male mice were used for the in vivo studies: 5-months-old mice (young), 24-month-old mice fed with a standard diet containing 0.6 % of phosphate (old) and 24-month-old mice fed with a hypophosphatemic diet, containing a 0.2% of phosphate (old+lowPi), for the last three months before sacrifice. Muscle force was measured by a grip test. Serum phosphate levels were analyzed with a commercial kit. Quadriceps muscle samples were collected to evaluate in them the IL-15 mRNA expression by RT-qPCR. Results C2C12 cells treated with BGP show a significant decrease in the IL-15 mRNA expression. On the other hand, in vivo studies showed that old mice had an increase in serum phosphate concentration and a reduction in forelimb strength and muscle mass, compared to young mice. Old animals fed with the hypophosphatemic diet displayed lower levels of phosphate serum linked to an improvement in the muscle mass and function. IL-15 expression of quadriceps muscle was reduced in old mice compared to young mice, whereas those values were increased in old mice fed with the low phosphate diet. Furthermore, there was a negative correlation between IL-15 expression levels and serum phosphate concentrations and a positive correlation between IL-15 and forelimb strength and muscle mass, suggesting that a decreased IL-15 expression affects muscle function. Conclusion High extracellular phosphate concentrations decrease IL-15 mRNA expression in myoblasts, and it is correlated with low IL-15 mRNA expression in the quadriceps muscle isolated from old mice. This reduction was associated to a decreased muscular strength and muscle mass, whereas the dietary restriction of phosphate improved these features. These results point to a role of hyperphosphatemia in the impaired immune system function, disrupting the skeletal muscle function, and this could be involved in aging and CKD-related sarcopenia.

Insights into wild-type dynamin 2 and the consequences of DNM2 mutations from transgenic zebrafish

2019 ◽  
Vol 28 (24) ◽  
pp. 4186-4196 ◽  
Author(s):  
Mo Zhao ◽  
Lindsay Smith ◽  
Jonathan Volpatti ◽  
Lacramioara Fabian ◽  
James J Dowling
Keyword(s):  
Muscle Development ◽  
Neuromuscular Disorders ◽  
Transgenic Zebrafish ◽  
Wild Type ◽  
Charcot Marie Tooth Disease ◽  
Membrane Fission ◽  
First Time ◽  
Dynamin 2

Abstract Dynamin 2 (DNM2) encodes a ubiquitously expressed large GTPase with membrane fission capabilities that participates in the endocytosis of clathrin-coated vesicles. Heterozygous mutations in DNM2 are associated with two distinct neuromuscular disorders, Charcot–Marie–Tooth disease (CMT) and autosomal dominant centronuclear myopathy (CNM). Despite extensive investigations in cell culture, the role of dynamin 2 in normal muscle development is poorly understood and the consequences of DNM2 mutations at the molecular level in vivo are not known. To address these gaps in knowledge, we developed transgenic zebrafish expressing either wild-type dynamin 2 or dynamin 2 with either a CNM or CMT mutation. Taking advantage of the live imaging capabilities of the zebrafish embryo, we establish the localization of wild-type and mutant dynamin 2 in vivo, showing for the first time distinctive dynamin 2 subcellular compartments. Additionally, we demonstrate that CNM-related DNM2 mutations are associated with protein mislocalization and aggregation. Lastly, we define core phenotypes associated with our transgenic mutant fish, including impaired motor function and altered muscle ultrastructure, making them the ideal platform for drug screening. Overall, using the power of the zebrafish, we establish novel insights into dynamin 2 localization and dynamics and provide the necessary groundwork for future studies examining dynamin 2 pathomechanisms and therapy development.

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