Skeletal Muscle最新文献

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common and progressive muscle wasting disease that is characterized by muscle weakness often first noticed in the face, the shoulder girdle and upper arms before progressing to the lower limb muscles. FSHD is caused by the misexpression of the Double Homeobox 4 (DUX4) transcription factor in skeletal muscle. While epigenetic derepression of D4Z4 macrosatellite repeats underlies DUX4 misexpression, our understanding of the complex transcriptional activation of DUX4 is incomplete.

Methods: To identify potential DUX4-regulatory factors, we used small interfering RNAs (siRNAs) to knockdown SIX family transcription factors (SIX1, 2, 4, 5) in patient-derived FSHD1 and FSHD2 myoblasts that were differentiated to form multinucleated myotubes. Quantitative real-time polymerase chain reaction was used to measure changes in DUX4 mRNA, DUX4 target gene expression and myogenic markers. Staining for SIX1 and SIX2 with specific antibodies was performed in FSHD myoblasts and myotubes. To assess reciprocal effects of DUX4 on SIX1, 2, and 4 expression, we utilized a doxycycline-inducible DUX4 myoblast cell line.

Result: We show that SIX1, 2 and 4 transcription factors, regulators of embryonic development, muscle differentiation, regeneration and homeostasis, are necessary for myogenic differentiation-dependent DUX4 expression in FSHD muscle cells. Using siRNA, we demonstrate SIX1, SIX2, and SIX4 to be critical factors involved in the induction of DUX4 transcription in differentiating FSHD myotubes in vitro. siRNA dual knockdown of SIX1 and SIX2 resulted in a ~ 98% decrease of DUX4 and DUX4 target genes, suggesting that SIX1 and SIX2 are the most critical in promoting DUX4 expression. Importantly, we show that DUX4 downregulates SIX RNA levels, suggesting negative feedback regulation.

Conclusions: In this study, we identified a family of developmental regulators that promote aberrant DUX4 expression in FSHD1 and FSHD2 differentiating muscle cells. Our findings highlight the critical involvement of SIX transcription factors (SIX1, 2, 4) in the pathogenesis of FSHD by serving as necessary factors that function in the promotion of DUX4 expression following epigenetic derepression of the D4Z4 repeats.

Background: Severe acute pancreatitis (SAP) is characterized by high mortality rates and various complications, including skeletal muscle atrophy, which significantly exacerbates its outcomes. Despite its clinical relevance, the mechanistic understanding of the relationship between skeletal muscle and the pancreas in SAP remains limited. Our study aimed to elucidate this "organ crosstalk" and its potential implications.

Methods: We established an SAP mouse model through pancreatic duct ligation (PDL) and evaluated pancreatic necrosis, skeletal muscle atrophy, and myonectin expression levels. Recombinant myonectin protein was administered in vivo and in vitro to assess its effects on acinar cell necrosis. Mechanistic insights were gained through RNA-seq data analysis and experimental validation. Serum samples from AP patients and healthy controls were collected to investigate the correlation between serum myonectin levels and disease severity.

Results: The mouse model exhibited severe pancreatic necrosis, skeletal muscle atrophy, and elevated myonectin levels, with myonectin administration exacerbating model severity. We identified iron accumulation-induced ferroptosis as a key pathway contributing to myonectin-mediated acinar cell necrosis. A total of 22 healthy controls and 52 patients with varying degrees of AP were included in the serum samples and clinical data (36.5% females, age 49.79 ± 16.53). Analysis of serum samples revealed significantly higher myonectin levels in AP patients, correlating with disease severity (R = 0.28, P = 0.041).

Conclusions: Our findings underscore the significant role of myonectin in SAP progression and its potential as a prognostic marker for disease severity in AP patients. This study contributes to a deeper understanding of the pathophysiology of SAP and highlights potential therapeutic targets for intervention.

Background: Antisense oligonucleotides (AON) represent a promising treatment for Duchenne muscular dystrophy (DMD) carrying out-of-frame deletions, but also show limitations. In a completed clinical trial golodirsen, approved by FDA to induce skipping of DMD gene exon 53 in eligible patients, we demonstrated increase in DMD expression and protein production, albeit with inter-patient variability.

Methods: Here, we investigate further the golodirsen mechanism of action using myotubes derived from MyoD transfected fibroblasts isolated from DMD patients at the baseline of the clinical trial SRP-4053.

Results: We confirm golodirsen's selectivity and efficiency in removing only exon 53. For the first time in human cells, we revealed a significant reduction in the so called DMD "transcript imbalance", in golodirsen-treated DMD muscle cultures. The transcript imbalance is a unique DMD phenomenon characterized by non-homogeneous transcript expression along its entire length and responsible for the reduced stability of the transcript. Our in-vivo study also showed that the efficiency of exon skipping did not always correspond to a proportional restoration of the dystrophin protein. Predominant nuclear localization of the DMD transcript, observed in patients and animal models, persists even after exon skipping.

Conclusion: All these findings suggest challenges other than AON delivery for high level of protein restoration in DMD, highlighting the importance of investigating the biological mechanisms upstream of protein production to further enhance the efficiency of any AON treatment in this condition.

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation.

Background: In recent years, the African spiny mouse Acomys cahirinus has been shown to regenerate a remarkable array of severe internal and external injuries in the absence of a fibrotic response, including the ability to regenerate full-thickness skin excisions, ear punches, severe kidney injuries, and complete transection of the spinal cord. While skeletal muscle is highly regenerative in adult mammals, Acomys displays superior muscle regeneration properties compared with standard laboratory mice following several injuries, including serial cardiotoxin injections of skeletal muscle and volumetric muscle loss (VML) of the panniculus carnosus muscle following full-thickness excision injuries. VML is an extreme muscle injury defined as the irrecoverable ablation of muscle mass, most commonly resulting from combat injuries or surgical debridement. Barriers to the treatment of VML injury include early and prolonged inflammatory responses that promote fibrotic repair and the loss of structural and mechanical cues that promote muscle regeneration. While the regeneration of the panniculus carnosus in Acomys is impressive, its direct relevance to the study of VML in patients is less clear as this muscle has largely been lost in humans, and, while striated, is not a true skeletal muscle. We therefore sought to test the ability of Acomys to regenerate a skeletal muscle more commonly used in VML injury models.

Methods: We performed two different VML injuries of the Acomys tibialis anterior muscle and compared the regenerative response to a standard laboratory mouse strain, Mus C57BL6/J.

Results: Neither Acomys nor Mus recovered lost muscle mass or myofiber number within three months following VML injury, and Acomys also failed to recover force production better than Mus. In contrast, Acomys continued to express eMHC within the injured area even three months following injury, whereas Mus ceased expressing eMHC less than one-month post-injury, suggesting that Acomys muscle was primed, but failed, to regenerate.

Conclusions: While the panniculus carnosus muscle in Acomys regenerates following VML injury in the context of full-thickness skin excision, this regenerative ability does not translate to regenerative repair of a skeletal muscle.

Background: Skeletal muscles possess unique abilities known as adaptation or plasticity. When exposed to external stimuli, such as mechanical loading, both myofiber size and myonuclear number increase. Muscle stem cells, also known as muscle satellite cells (MuSCs), play vital roles in these changes. HeyL, a direct target of Notch signaling, is crucial for efficient muscle hypertrophy because it ensures MuSC proliferation in surgically overloaded muscles by inhibiting the premature differentiation. However, it remains unclear whether HeyL is essential for MuSC expansion in physiologically exercised muscles. Additionally, the influence of myofiber type on the requirement for HeyL in MuSCs within exercised muscles remains unclear.

Methods: We used a voluntary wheel running model and HeyL-knockout mice to investigate the impact of HeyL deficiency on MuSC-derived myonuclei, MuSC behavior, muscle weight, myofiber size, and myofiber type in the running mice.

Results: The number of new MuSC-derived myonuclei was significantly lower in both slow-twitch soleus and fast-twitch plantaris muscles from exercised HeyL-knockout mice than in control mice. However, expect for the frequency of Type IIb myofiber in plantaris muscle, exercised HeyL-knockout mice exhibited similar responses to control mice regarding myofiber size and type.

Conclusions: HeyL expression is crucial for MuSC expansion during physiological exercise in both slow and fast muscles. The frequency of Type IIb myofiber in plantaris muscle of HeyL-knockout mice was not significantly reduced compared to that of control mice. However, the absence of HeyL did not affect the increased size and frequency of Type IIa myofiber in plantaris muscles. In this model, no detectable changes in myofiber size or type were observed in the soleus muscles of either control or HeyL-knockout mice. These findings imply that the requirement for MuSCs in the wheel-running model is difficult to observe due to the relatively low degree of hypertrophy compared to surgically overloaded models.

Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14 weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in the Gastrocnemius and soleus muscles were analyzed by LC/MS-MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius muscle were decreased after 14 weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5-2fold in the gastrocnemius muscle after 14 weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7 nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4 nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting.

Background: Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1^G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1^G93A ALS mice could lead to differential and potentially improved benefits.

Methods: We thus investigated the contribution of Fn14 to disease phenotypes in the SOD1^G93A ALS mice. To do so, Fn14 knockout mice (Fn14^-/-) were crossed onto the SOD1^G93A background to generate SOD1^G93A;Fn14^-/- mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14^-/-, SOD1^G93A and SOD1^G93A;Fn14^-/-).

Results: Here, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1^G93A mice. We then show that Fn14-depleted SOD1^G93A mice display increased lifespan, myofiber size, neuromuscular junction endplate area as well as altered expression of known molecular effectors of the TWEAK/Fn14 pathway, without an impact on motor function. Importantly, we also observe a complex interaction between exercise (rotarod and grid test), genotype, disease state and sex that influences the overall effects of Fn14 deletion on survival, expression of known molecular effectors of the TWEAK/Fn14 pathway, expression of myosin heavy chain isoforms and myofiber size.

Conclusions: Our study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease, sex and exercise. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism, sex and exercise will be of importance in future studies.

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Background: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies.

Methods: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status.

Results: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55.

Conclusions: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.