Skeletal Muscle最新文献

Limb-girdle muscular dystrophy (MD) type 2B (LGMD2B) and Duchenne MD (DMD) are caused by mutations to the Dysferlin and Dystrophin genes, respectively. We have recently demonstrated in typically mild dysferlin- and dystrophin-deficient mouse models that increased plasma cholesterol levels severely exacerbate muscle wasting, and that DMD patients display primary dyslipidemia characterized by elevated plasma cholesterol and triglycerides. Herein, we investigate lipoprotein abnormalities in LGMD2B and if statin therapy protects dysferlin-deficient mice (Dysf) from muscle damage. Herein, lipoproteins and liver enzymes from LGMD2B patients and dysferlin-null (Dysf) mice were analyzed. Simvastatin, which exhibits anti-muscle wasting effects in mouse models of DMD and corrects aberrant expression of key markers of lipid metabolism and endogenous cholesterol synthesis, was tested in Dysf mice. Muscle damage and fibrosis were assessed by immunohistochemistry and cholesterol signalling pathways via Western blot. LGMD2B patients show reduced serum high-density lipoprotein cholesterol (HDL-C) levels compared to healthy controls and exhibit a greater prevalence of abnormal total cholesterol (CHOL)/HDL-C ratios despite an absence of liver dysfunction. While Dysf mice presented with reduced CHOL and associated HDL-C and LDL-C-associated fractions, simvastatin treatment did not prevent muscle wasting in quadriceps and triceps muscle groups or correct aberrant low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) protein expression. LGMD2B patients present with reduced serum concentrations of HDL-C, a major metabolic comorbidity, and as a result, statin therapy is unlikely to prevent muscle wasting in this population. We propose that like DMD, LGMD2B should be considered as a new type of genetic dyslipidemia.

Background: The AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells (i.e., transiently expressed) remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation.

Methods: We established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivo. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions.

Results: Persistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes.

Conclusions: Our results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process.

Background: Radiotherapy is commonly used to treat childhood cancers and can have adverse effects on muscle function, but the underlying mechanisms have yet to be fully elucidated. We hypothesized that endurance exercise following radiation treatment would improve skeletal muscle function.

Methods: We utilized the Small Animal Radiation Research Platform (SARRP) to irradiate juvenile male mice with a clinically relevant fractionated dose of 3× (every other day over 5 days) 8.2 Gy X-ray irradiation locally from the knee to footpad region of the right hindlimb. Mice were then singly housed for 1 month in cages equipped with either locked or free-spinning voluntary running wheels. Ex vivo muscle contractile function, RT-qPCR analyses, resting cytosolic and sarcoplasmic reticulum (SR) store Ca²⁺ levels, mitochondrial reactive oxygen species levels (MitoSOX), and immunohistochemical and biochemical analyses of muscle samples were conducted to assess the muscle pathology and the relative therapeutic impact of voluntary wheel running (VWR).

Results: Irradiation reduced fast-twitch extensor digitorum longus (EDL) muscle-specific force by 27% compared to that of non-irradiated mice, while VWR post-irradiation improved muscle-specific force by 37%. Radiation treatment similarly reduced slow-twitch soleus muscle-specific force by 14% compared to that of non-irradiated mice, while VWR post-irradiation improved specific force by 18%. We assessed intracellular Ca²⁺ regulation, oxidative stress, and mitochondrial homeostasis as potential mechanisms of radiation-induced pathology and exercise-mediated rescue. We found a significant reduction in resting cytosolic Ca²⁺ concentration following irradiation in sedentary mice. Intriguingly, however, SR Ca²⁺ store content was increased in myofibers from irradiated mice post-VWR compared to mice that remained sedentary. We observed a 73% elevation in the overall protein oxidization in muscle post-irradiation, while VWR reduced protein nitrosylation by 35% and mitochondrial reactive oxygen species (ROS) production by 50%. Finally, we found that VWR significantly increased the expression of PGC1α at both the transcript and protein levels, consistent with an exercise-dependent increase in mitochondrial biogenesis.

Conclusions: Juvenile irradiation stunted muscle development, disrupted proper Ca²⁺ handling, damaged mitochondria, and increased oxidative and nitrosative stress, paralleling significant deficits in muscle force production. Exercise mitigated aberrant Ca²⁺ handling, mitochondrial homeostasis, and increased oxidative and nitrosative stress in a manner that correlated with improved skeletal muscle function after radiation.

Background: Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation. Here, we developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. To test ViaFuse, we utilized immunofluorescence images of differentiated myotubes where the capping actin protein of muscle z-line subunit beta (CAPZB) was depleted in comparison with control cells.

Results: We compared the values achieved using the ViaFuse macros first with manual quantification performed by researchers and second with those obtained utilizing the MATLAB muscle-centric software MyoCount. We observed a high degree of correlation between all methods of quantification.

Conclusions: ViaFuse can detect the borders of myotubes and identify nuclear clumps which have been limitations of previous muscle-centric imaging software. The ViaFuse macros require little computer power or space to run and user inputs to the ViaFuse macros are minimal, thereby automating the analysis process in a quick, easy, and accurate fashion. Additionally, the ViaFuse macros work with Fiji, an existing imaging software widely used by skeletal muscle researchers. Furthermore, ViaFuse is compatible with many computer systems, has a very intuitive interface, and does not require prior complex mathematical knowledge. Therefore, we propose ViaFuse as a robust and meticulous method to quantify skeletal muscle cell viability and differentiation.