Skeletal Muscle最新文献

Biallelic pathogenic variants in the nebulin (NEB) gene lead to the congenital muscle disease nemaline myopathy. In-frame deletion of exon 55 (ΔExon55) is the most common disease-causing variant in NEB. Previously, a mouse model of Neb^ΔExon55 was developed; however, it presented an uncharacteristically severe phenotype with a near complete reduction in Neb transcript expression that is not observed in NEB exon 55 patients. We identified by RNA sequencing that the cause of this unexpectedly severe presentation in mice is the generation of a pseudoexon containing two premature termination codons (and promoting nonsense mediated decay) at the Neb exon 55 deletion site. To prove that this is the cause of the loss of Neb transcript, and to generate a more faithful model of the human disease, we used CRISPR gene editing to remove the pseudoexon sequence and replace it with human intron 54 sequence containing a validated cas9 gRNA protospacer. The resulting "hmz" mice have a significant reduction in pseudoexon formation (93.6% reduction), and a re-introduction of stable Neb transcript expression. This new model has the characteristic features of nemaline myopathy at the physiological, histological, and molecular levels. Importantly, unlike the existing exon 55 deletion mice (which die by age 7 days), it survives beyond the first months and exhibits obvious signs of neuromuscular dysfunction. It thus provides a new, robust model for studying pathomechanisms and developing therapies for NEB related nemaline myopathy.

Background: SELENON-Congenital Myopathy (SELENON-CM) is a rare congenital myopathy caused by mutations of the SELENON gene characterized by axial muscle weakness and progressive respiratory insufficiency. Muscle histopathology may be non-specific, but commonly includes multiminicores or a dystrophic pattern. The SELENON gene encodes selenoprotein N (SelN), a selenocysteine-containing redox enzyme located in the endo/sarcoplasmic reticulum membrane where it colocalizes with mitochondria-associated membranes. However, the molecular mechanism(s) by which SelN deficiency cause SELENON-CM remain poorly understood. A hurdle is the lack of cellular and animal models that show easily assayable phenotypes.

Methods: Using CRISPR-Cas9 we generated three zebrafish models of SELENON-CM, which were then studied by spontaneous coiling, hatching, and activity assays. We also performed selenon coexpression analysis using a single cell RNAseq zebrafish embryo-atlas. SelN-deficient myoblasts were generated and assayed for glutathione, reactive oxygen species, carbonylation, and nytrosylation levels. Finally, we tested Selenon-deficient myoblasts' metabolism using a Seahorse cell respirometer.

Results: We report deep-phenotyping of SelN-deficient zebrafish and muscle cells. SelN-deficient zebrafish exhibit changes in embryonic muscle function and swimming activity in larvae. Analysis of single cell RNAseq data in a zebrafish embryo-atlas revealed coexpression of selenon and genes involved in the glutathione redox pathway. SelN-deficient zebrafish and mouse myoblasts exhibit altered glutathione and redox homeostasis, as well as abnormal patterns of energy metabolism, suggesting roles for SelN in these functions.

Conclusions: These data demonstrate a role for SelN in zebrafish early development and myoblast metabolism and provide a basis for cellular and animal model assays for SELENON-CM.

Background: Sarcoglycanopathies are muscle dystrophies caused by mutations in the genes encoding sarcoglycans (α, β, γ, and δ) that can destabilize the dystrophin-associated glycoprotein complex at the sarcolemma, leaving muscle fibers vulnerable to damage after contraction, followed by inflammatory and fibrotic responses and resulting in muscle weakness and atrophy. Two signaling pathways have been implicated in fibrosis and inflammation in various tissues: autotaxin/lysophosphatidic acid (ATX-LPA) and yes-associated protein 1/transcriptional co-activator with PDZ-binding motif (YAP/TAZ). LPA, synthesized by ATX, can act as a pleiotropic molecule due to its multiple receptors. Two Hippo pathway effectors, YAP/TAZ, can be dephosphorylated by LPA and translocated to the nucleus. They induce several target genes, such as CCN2/CTGF, involved in fibrosis and inflammation. However, no detailed characterization of these processes or whether these pathways change early in the development of sarcoglycanopathy has been evaluated in skeletal muscle.

Methods: Using the δ-sarcoglycan knockout mouse model (Sgcd^-/-), we investigated components of these pathways, inflammatory and fibrotic markers, and contractile properties of different skeletal muscles (triceps-TR, gastrocnemius-GST, diaphragm-DFG, tibialis anterior-TA, and extensor digitorum longus-EDL) at one and two months of age.

Results: We found that Sgcd^-/- mice show early dystrophic features (fiber damage/necrosis, centrally nucleated fibers, inflammatory infiltrate, and regenerated fibers) followed by later fiber size reduction in TR, GST, and DFG. These changes are concomitant with an early inflammatory and fibrotic response in these muscles. Sgcd^-/- mice also have early impaired force generation in the TA and EDL, and resistance to mechanical damage in the EDL. In addition, an early dysregulation of the ATX-LPA axis and the YAP/TAZ signaling pathway in the TR, GST, and DFG was observed in these mice.

Conclusions: The ATX-LPA axis and the YAP/TAZ signaling pathway, which are involved in inflammation and fibrosis, are dysregulated in skeletal muscle from an early age in Sgcd^-/- mice. These changes are concomitant with a fibrotic and inflammatory response in these mice. Unraveling the role of the LPA axis and YAP/TAZ in sarcoglycanopathy holds great promise for improving our understanding of disease pathogenesis and identifying novel therapeutic targets for this currently incurable group of muscle disorders.

Background: Highly expressed in skeletal muscles, the gene Obscurin (i.e. OBSCN) has 121 non-overlapping exons and codes for some of the largest known mRNAs in the human genome. Furthermore, it plays an essential role in muscle development and function. Mutations in OBSCN are associated with several hypertrophic cardiomyopathies and muscular disorders. OBSCN undergoes extensive and complex alternative splicing, which is the main reason that its splicing regulation associated with skeletal and cardiac muscle development has not previously been thoroughly studied.

Methods: We analyzed RNA-Seq data from skeletal and cardiac muscles extracted from 44 postnatal individuals and six fetuses. We applied the intron/exon level splicing analysis software IntEREst to study the splicing of OBSCN in the studied samples. The differential splicing analysis was adjusted for batch effects. Our comparisons revealed the splicing variations in OBSCN between the human skeletal and cardiac muscle, as well as between post-natal muscle (skeletal and cardiac) and the pre-natal equivalent muscle.

Results: We detected several splicing regulations located in the 5'end, 3' end, and the middle of OBSCN that are associated with human cardiac or skeletal muscle development. Many of these alternative splicing events have not previously been reported. Our results also suggest that many of these muscle-development associated splicing events may be regulated by BUB3.

Conclusions: We conclude that the splicing of OBSCN is extensively regulated during the human skeletal/cardiac muscle development. We developed an interactive visualization tool that can be used by clinicians and researchers to study the inclusion of specific OBSCN exons in pre- and postnatal cardiac and skeletal muscles and access the statistics for the differential inclusion of the exons across the studied sample groups. The OBSCN exon inclusion map related to the human cardiac and skeletal muscle development is available at http://psivis.it.helsinki.fi:3838/OBSCN_PSIVIS/ . These findings are essential for an accurate pre- and postnatal clinical interpretation of the OBSCN exonic variants.

Background: Prior studies suggested that canonical Activin Receptor II (ActRII) and BMP receptor (BMPR) ligands can have opposing, distinct effects on skeletal muscle depending in part on differential downstream SMAD activation. It was therefore of interest to test ActRII ligands versus BMP ligands in settings of muscle differentiation and in vivo.

Methods and results: In human skeletal muscle cells, both ActRII ligands and BMP ligands inhibited myogenic differentiation: ActRII ligands in a SMAD2/3-dependent manner, and BMP ligands via SMAD1/5. Surprisingly, a neutralizing ActRIIA/B antibody mitigated the negative effects of both classes of ligands, indicating that some BMPs act at least partially through the ActRII receptors in skeletal muscle. Gene expression analysis showed that both ActRII and BMP ligands repress muscle differentiation genes in human myoblasts and myotubes. In mice, hepatic BMP9 over-expression induced liver toxicity, caused multi-organ wasting, and promoted a pro-atrophy gene signature despite elevated SMAD1/5 signaling in skeletal muscle. Local overexpression of BMP7 or BMP9, achieved by intramuscular AAV delivery, induced heterotopic ossification. Elevated SMAD1/5 signaling with increased expression of BMP target genes was also observed in sarcopenic muscles of old rats.

Conclusions: The canonical ActRII ligand-SMAD2/3 and BMP ligand-SMAD1/5 axes can both block human myoblast differentiation. Our observations further demonstrate the osteoinductive function of BMP ligands while pointing to a potential relevancy of blocking the BMP-SMAD1/5 axis in the setting of therapeutic anti-ActRIIA/B inhibition.

Background: Skeletal muscle is a plastic tissue that adapts to increased mechanical loading/contractile activity through fusion of muscle stem cells (MuSCs) with myofibers, a physiological process referred to as myonuclear accretion. However, it is still unclear whether myonuclear accretion is driven by increased mechanical loading per se, or occurs, at least in part, in response to muscle injury/regeneration. Here, we developed a non-damaging protocol to evaluate contractile activity-induced myonuclear accretion/hypertrophy in physiological conditions.

Methods: Contractile activity was generated by applying repeated electrical stimuli over the mouse plantar flexor muscles. This method is commonly referred to as NeuroMuscular Electrical Simulation (NMES) in Human. Each NMES training session consisted of 80 isometric contractions delivered at ∼15% of maximal tetanic force to avoid muscle damage. C57BL/6J male mice were submitted to either a short (i.e., 6 sessions) or long (i.e., 12 sessions) individualized NMES training program while unstimulated mice were used as controls. Histological investigations were performed to assess the impact of NMES on MuSC number and status, myonuclei content and muscle tissue integrity, typology and size.

Results: NMES led to a robust proliferation of MuSCs and myonuclear accretion in the absence of overt signs of muscle damage/regeneration. NMES-induced myonuclear accretion was specific to type IIB myofibers and was an early event preceding muscle hypertrophy inasmuch as a mild increase in myofiber cross-sectional area was only observed in response to the long-term NMES training protocol.

Conclusion: We conclude that NMES-induced myonuclear accretion and muscle hypertrophy are driven by a mild increase in mechanical loading in the absence of overt signs of muscle injury.

Background: Duchenne muscular dystrophy (DMD) is a prevalent, fatal degenerative muscle disease with no effective treatments. Mdx mouse model of DMD exhibits impaired muscle performance, oxidative stress, and dysfunctional autophagy. Although antioxidant treatments may improve the mdx phenotype, the precise molecular mechanisms remain unclear. This study investigates the effects of aminoguanidine hemisulfate (AGH), an inhibitor of reactive oxygen species (ROS), on mitochondrial autophagy, oxidative stress, and muscle force in mdx mice.

Methods: Male wild-type (WT) and mdx mice were divided into three groups: WT, mdx, and AGH-treated mdx mice (40 mg/kg intraperitoneally for two weeks) at 6 weeks of age. Gene expression, western blotting, H&E staining, immunofluorescence, ROS assays, TUNEL apoptosis, glutathione activity, and muscle force measurements were performed. Statistical comparisons used one-way ANOVA.

Results: AGH treatment significantly reduced the protein levels of LC3, and p62 in mdx mice, indicating improved autophagy activity and the ability to clear damaged mitochondria. AGH restored the expression of mitophagy-related genes Pink1 and Parkin and increased Mfn1, rebalancing mitochondrial dynamics. It also increased Pgc1α and mtTFA levels, promoting mitochondrial biogenesis. ROS levels were reduced, with higher Prdx3 and MnSOD expression, improving mitochondrial antioxidant defenses. AGH normalized the GSSG/GSH ratio and decreased glutathione reductase and peroxidase activities, further improving redox homeostasis. Additionally, AGH reduced apoptosis, shown by fewer TUNEL-positive cells and lower caspase-3 expression. Histological analysis revealed decreased muscle damage and fewer embryonic and neonatal myosin-expressing fibers. AGH altered fiber composition, decreasing MyH7 while increasing MyH4 and MyH2. Muscle force improved significantly, with greater twitch and tetanic forces. Mechanistically, AGH modulated the AKT/FOXO1 pathway, decreasing myogenin and Foxo1 while increasing MyoD.

Conclusions: AGH treatment restored mitochondrial autophagy, reduced oxidative stress, apoptosis, and altered muscle fiber composition via the AKT/FOXO1 pathway, collectively improving muscle force in mdx mice. We propose AGH as a potential therapeutic strategy for DMD and related muscle disorders.

Background: Maintaining the connection between skeletal muscle fibers and the surrounding basement membrane is essential for muscle function. Dystroglycan (DG) serves as a basement membrane extracellular matrix (ECM) receptor in many cells, and is also expressed in the outward-facing membrane, or sarcolemma, of skeletal muscle fibers. DG is a transmembrane protein comprised of two subunits: alpha-DG (α-DG), which resides in the peripheral membrane, and beta-DG (β-DG), which spans the membrane to intracellular regions. Extensive post-translational processing and O-mannosylation are required for α-DG to bind ECM proteins, which is mediated by a glycan structure known as matriglycan. O-mannose glycan biosynthesis is initiated by the protein O-mannosyltransferase 1 (POMT1) and POMT2 enzyme complex and leads to three subtypes of glycans called core M1, M2, and M3. The lengthy core M3 is capped with matriglycan. Genetic defects in post-translational O-mannosylation of DG interfere with its receptor function and result in muscular dystrophy with central nervous system and skeletal muscle pathophysiology.

Methods: To evaluate how the loss of O-mannosylated DG in skeletal muscle affects the development and progression of myopathology, we generated and characterized mice in which the Pomt1 gene was specifically deleted in skeletal muscle (Pomt1^skm) to interfere with POMT1/2 enzyme activity. To investigate whether matriglycan is the primary core M glycan structure that provides the stabilizing link between the sarcolemma and ECM, we generated mice that retained cores M1, M2, and M3, but lacked matriglycan (conditional deletion of like-acetylglucosaminyltransferase 1; Large1^skm). Next, we restored Pomt1 using gene transfer via AAV2/9-MCK-mPOMT1 and determined the effect on Pomt1^skm pathophysiology.

Results: Our data showed that in Pomt1^skm mice O-mannosylated DG is required for sarcolemma resilience, remodeling of muscle fibers and muscle tissue, and neuromuscular function. Notably, we observed similar body size limitations, sarcolemma weakness, and neuromuscular weakness in Large1^skm mice that only lacked matriglycan. Furthermore, our data indicate that genetic rescue of Pomt1 in Pomt1^skm mice limits contraction-induced sarcolemma damage and skeletal muscle pathology.

Conclusions: Collectively, our data indicate that DG modification by Pomt1/2 results in core M3 capped with matriglycan, and that this is required to reinforce the sarcolemma and enable skeletal muscle health and neuromuscular strength.

Background: INTER- and INTRAmuscular fat (IMF) is elevated in high metabolic states and can promote inflammation. While magnetic resonance imaging (MRI) excels in depicting IMF, the lack of reproducible tools prevents the ability to measure change and track intervention success.

Methods: We detail an open-source fully-automated iterative threshold-seeking algorithm (ITSA) for segmenting IMF from T1-weighted MRI of the calf and thigh within three cohorts (CaMos Hamilton (N = 54), AMBERS (N = 280), OAI (N = 105)) selecting adults 45-85 years of age. Within the CaMos Hamilton cohort, same-day and 1-year repeated images (N = 38) were used to evaluate short- and long-term precision error with root mean square coefficients of variation; and to validate against semi-automated segmentation methods using linear regression. The effect of algorithmic improvements to fat ascertainment using 3D connectivity and partial volume correction rules on analytical precision was investigated. Robustness and versatility of the algorithm was demonstrated by application to different MR sequences/magnetic strength and to calf versus thigh scans.

Results: Among 439 adults (319 female(89%), age: 71.6 ± 7.6 yrs, BMI: 28.06 ± 4.87 kg/m², IMF%: 10.91 ± 4.57%), fully-automated ITSA performed well across MR sequences and anatomies from three cohorts. Applying both 3D connectivity and partial volume fat correction improved precision from 4.99% to 2.21% test-retest error. Validation against semi-automated methods showed R² from 0.92 to 0.98 with fully-automated ITSA routinely yielding more conservative computations of IMF volumes. Quality control shows 7% of cases requiring manual correction, primarily due to IMF merging with subcutaneous fat. A full workflow described methods to export tags for manual correction.

Conclusions: The greatest challenge in segmenting IMF from MRI is in selecting a dynamic threshold that consistently performs across repeated imaging. Fully-automated ITSA achieved this, demonstrated low short- and long-term precision error, conducive of use within RCTs.

Background: Muscle stem cells (MuSCs) undergo numerous state transitions throughout life, which are critical for supporting normal muscle growth and regeneration. Epigenetic modifications in skeletal muscle play a significant role in influencing the niche and cellular states of MuSCs. Mixed-lineage leukemia 4 (Mll4) is a histone methyltransferase critical for activating the transcription of various target genes and is highly expressed in skeletal muscle. This raises the question of whether Mll4 has a regulatory function in modulating the state transitions of MuSCs, warranting further investigation.

Methods: To assess if myofiber-expressed Mll4, a histone methyltransferase, contributes to the maintenance of MuSCs, we crossed MCK^Cre/+ or HSA^MerCreMer/+ mice to Mll4^f/f mice to generate myofiber-specific Mll4-deleted mice. Investigations were conducted using 8-week-old and 4-week-old MCK^Cre/+;Mll4^f/f mice, and adult HSA^MerCreMer/+;Mll4^f/f mice between the ages of 3 months and 6 months.

Results: During postnatal myogenesis, Mll4 deleted muscles were observed with increased number of cycling MuSCs that proceeded to a differentiation state, leading to MuSC deprivation. This phenomenon occurred independently of gender. When Mll4 was ablated in adult muscles using the inducible method, adult MuSCs lost their quiescence and differentiated into myoblasts, also causing the depletion of MuSCs. Such roles of Mll4 in myofibers coincided with decreased expression levels of distinct Notch ligands: Jag1 and Dll1 in pubertal and Jag2 and Dll4 in adult muscles.

Conclusions: Our study suggests that Mll4 is crucial for maintaining MuSCs in both pubertal and adult muscles, which may be accomplished through the modulation of distinct Notch ligand expressions in myofibers. These findings offer new insights into the role of myofiber-expressed Mll4 as a master regulator of MuSCs, highlighting its significance not only in developmental myogenesis but also in adult muscle, irrespective of sex.