Project description:This SuperSeries is composed of the following subset Series: GSE36741: In vivo and in vitro investigations of heterozygous nebulin knock-out mice reveal similarities with mild human form of nemaline myopathy [miRNA] GSE36743: In vivo and in vitro investigations of heterozygous nebulin knock-out mice reveal similarities with mild human form of nemaline myopathy [mRNA] Refer to individual Series
Project description:The sarcomere is the muscle contractile unit, whose assembly and disassembly are intimately linked to changes in myofiber size and function. Mutations in sarcomeric proteins are common causes of myopathies, ranging from severe neonatal to adult-onset forms. Here, we identify HECTD1 as an E3 ubiquitin ligase necessary for sarcomere maintenance. We show that silencingHectd1 in myotubesreduces the levels of proteins critical for sarcomere integrity including a-actin, titin, troponins, tropomyosins, and several myosin heavy chain isoforms. We further establish that Hectd1 ubiquitylates and stabilizes the molecular chaperones KLHL40/41, that when dysfunctional destabilize nebulin and other sarcomere thin filament proteins (causing nemaline myopathy). Consequently, skeletal muscle-specific Hectd1 knockout mice (Hectd1 mKO) show progressive muscle weakness, exercise intolerance, and unresolved tissue remodeling. Molecular and histological analysis of Hectd1 mKO muscle revealed subsarcolemmal storage of the scaffold protein desmin, mitochondrial abnormalities, and severe sarcomere disorganization. This work uncovers Hectd1 as an E3 ligase critical for sarcomere assembly, and a novel diagnostic gene for myopathy with features of nemaline and desmin myopathy.
2024-07-26 | GSE272775 | GEO
Project description:RNA seq of Nemaline Myopathy samples
Project description:Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical NEM adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin’s functional roles in adult muscle we performed studies on a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscle but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survives to adulthood with low nebulin levels (<5% of control), contain nemaline rods, and undergo fiber-type switching towards oxidative types. These microarrays investigate the changes in gene expression when nebulin is deficient.
Project description:Maintenance of muscle function requires assembly of contractile proteins into highly organized sarcomeres. Mutations in Kelch-like protein 41 (KLHL41) cause nemaline myopathy, a fatal muscle disorder associated with sarcomere disarray. We generated KLHL41 mutant mice, which display lethal disruption of sarcomeres and aberrant expression of muscle structural and contractile proteins, mimicking the hallmarks of the human disease. We show that KLHL41 is poly-ubiquitinated and acts, at least in part, by preventing aggregation and degradation of nebulin, an essential component of the sarcomere. Furthermore, inhibition of KLHL41 poly-ubiquitination prevents its stabilization of nebulin, suggesting a unique role for ubiquitination in protein stabilization. These findings provide new insights into the molecular etiology of nemaline myopathy and reveal a mechanism whereby KLHL41 stabilizes sarcomeres and maintains muscle function by acting as a molecular chaperone. Similar mechanisms for protein stabilization likely contribute to the actions of other Kelch proteins.
Project description:Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, and their role in the disease remains unresolved. Here, we demonstrate that loss of a muscle-specific protein, Klhl40, results in a nemaline-like myopathy in mice that closely phenocopies the muscle abnormalities observed KLHL40 deficient patients. We show that Klhl40 dynamically localizes to the sarcomere I-band and A-band and binds to Nebulin (Neb), a protein frequently implicated in NM, as well as a putative thin filament protein, Lmod3. Klhl40 belongs to the BTB-BACK-Kelch (BBK) family of proteins, some of which have been previously shown to promote degradation of their substrates. In contrast, we find that Klhl40 promotes stability of Neb and Lmod3 and blocks Lmod3 ubiquitination. Accordingly, loss of Klhl40 reduces Neb and Lmod3 protein in skeletal muscle of mice and KLHL40 deficient patients. Because loss of sarcomere thin filament proteins is a frequent cause of NM, our data establishes a possible molecular basis for NM in KLHL40 deficient patients by establishing a novel pro-stability function of Klhl40 for Neb and Lmod3. Total RNA was harvested from quadriceps muscle of three Klhl40 WT (control) and three Klhl40 KO mice. Each KO mouse was sacrificed with a corresponding WT littermate. Tissues were also taken at 0 days of age to minimize confounding gene changes occurring due to malnourishment as the phenotype worsens.
Project description:Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical NEM adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin’s functional roles in adult muscle we performed studies on a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscle but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survives to adulthood with low nebulin levels (<5% of control), contain nemaline rods, and undergo fiber-type switching towards oxidative types. These microarrays investigate the changes in gene expression when nebulin is deficient. Two skeletal muscle groups were studied: Quadriceps (which is markedly smaller in the Neb cKO mice relative to control) and Soleus (which is not significantly smaller in the Neb cKO relative to control). Six biological replicates for each muscle group were selected; all are age-matched males.
Project description:Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, and their role in the disease remains unresolved. Here, we demonstrate that loss of a muscle-specific protein, Klhl40, results in a nemaline-like myopathy in mice that closely phenocopies the muscle abnormalities observed KLHL40 deficient patients. We show that Klhl40 dynamically localizes to the sarcomere I-band and A-band and binds to Nebulin (Neb), a protein frequently implicated in NM, as well as a putative thin filament protein, Lmod3. Klhl40 belongs to the BTB-BACK-Kelch (BBK) family of proteins, some of which have been previously shown to promote degradation of their substrates. In contrast, we find that Klhl40 promotes stability of Neb and Lmod3 and blocks Lmod3 ubiquitination. Accordingly, loss of Klhl40 reduces Neb and Lmod3 protein in skeletal muscle of mice and KLHL40 deficient patients. Because loss of sarcomere thin filament proteins is a frequent cause of NM, our data establishes a possible molecular basis for NM in KLHL40 deficient patients by establishing a novel pro-stability function of Klhl40 for Neb and Lmod3.
Project description:Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed based on the presence of nemaline rods on skeletal muscle biopsy. While NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted well. The common pathological endpoint of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggests that shared secondary processes contributed to the pathogenesis of NM. We speculated that these processes could be identified through a proteome wide interrogation utilizing a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb cKO, KI.Acta1H40Y, and TgACTA1D286G mouse models of nemaline myopathy as compared to their respective wild-type counterparts (Neb WT, WT.Acta1H40Y, and C57) to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. Downstream analyses utilizing Scaffold, RStudio, and Ingenuity Pathway Analysis identified mitochondrial dysfunction and stress-related signaling as being enriched in NM mouse datasets. Pathway validation revealed that proteins in mitochondrial and stress-related signaling pathways aggregated in NM muscle in a severity dependent manner and an increase in protein content was generally associated with more severe disease. Structural and functional mitochondrial analyses revealed that mitochondrial dysfunction also grades with disease severity. RCI measured by respirometry, ATP/ADP/phosphate content, and mitochondrial transmembrane potential were affected in a severity dependent manner with the Neb cKOs being the most abnormal, KI.Acta1H40Y being mildly affected, and the TgACTA1D286G being minimally affected. These studies identify mitochondrial dysfunction as a secondary process impacting disease severity in NM.