Murine Skeletal Muscle Research Articles

Systemic glucose-insulin-potassium reduces skeletal muscle injury, kidney injury, and pain in a murine ischaemia-reperfusion model.

Glucose-insulin-potassium (GIK) is protective following cardiac myocyte ischaemia-reperfusion (IR) injury, however the role of GIK in protecting skeletal muscle from IR injury has not been evaluated. Given the similar mechanisms by which cardiac and skeletal muscle sustain an IR injury, we hypothesized that GIK would similarly protect skeletal muscle viability. A total of 20 C57BL/6 male mice (10 control, 10 GIK) sustained a hindlimb IR injury using a 2.5-hour rubber band tourniquet. Immediately prior to tourniquet placement, a subcutaneous osmotic pump was placed which infused control mice with saline (0.9% sodium chloride) and treated mice with GIK (40% glucose, 50 U/l insulin, 80 mEq/L KCl, pH 4.5) at a rate of 16 µl/hr for 26.5 hours. At 24 hours following tourniquet removal, bilateral (tourniqueted and non-tourniqueted) gastrocnemius muscles were triphenyltetrazolium chloride (TTC)-stained to quantify percentage muscle viability. Bilateral peroneal muscles were used for gene expression analysis, serum creatinine and creatine kinase activity were measured, and a validated murine ethogram was used to quantify pain before euthanasia. GIK treatment resulted in a significant protection of skeletal muscle with increased viability (GIK 22.07% (SD 15.48%)) compared to saline control (control 3.14% (SD 3.29%)) (p = 0.005). Additionally, GIK led to a statistically significant reduction in gene expression markers of cell death (CASP3, p < 0.001) and inflammation (NOS2, p < 0.001; IGF1, p = 0.007; IL-1β, p = 0.002; TNFα, p = 0.012), and a significant reduction in serum creatine kinase (p = 0.004) and creatinine (p < 0.001). GIK led to a significant reduction in IR-related pain (p = 0.030). Systemic GIK infusion during and after limb ischaemia protects murine skeletal muscle from cell death, kidneys from reperfusion metabolites, and reduces pain by reducing post-ischaemic inflammation.

SENP2 knockdown in human adipocytes reduces glucose metabolism and lipid accumulation, while increases lipid oxidation

Adipose tissue is one of the main regulative sites for energy metabolism. Excess lipid storage and expansion of white adipose tissue (WAT) is the primary contributor to obesity, a strong predisposing factor for development of insulin resistance. Sentrin-specific protease (SENP) 2 has been shown to play a role in metabolism in murine fat and skeletal muscle cells, and we have previously demonstrated its role in energy metabolism of human skeletal muscle cells. In the present work, we have investigated the impact of SENP2 on fatty acid and glucose metabolism in primary human fat cells by using cultured primary human adipocytes to knock down the SENP2 gene. Glucose uptake and oxidation, as well as accumulation and distribution of oleic acid into complex lipids were decreased, while oleic acid oxidation was increased in SENP2-knockdown cells compared to control adipocytes. Furthermore, lipogenesis was reduced by SENP2-knockdown in adipocytes. Although TAG accumulation relative to total uptake was unchanged, there was increased mRNA expression of metabolically relevant genes such as UCP1 and PPARGC1A and mRNA and proteomic data revealed increased levels of mRNA and proteins related to mitochondrial function by SENP2-knockdown. In conclusion, SENP2 is an important regulator of energy metabolism in primary human adipocytes and its knockdown reduce glucose metabolism and lipid accumulation, while increasing lipid oxidation in human adipocytes.

CELLULAR SENESCENCE IMPLICATED IN SEPSIS-INDUCED MUSCLE WEAKNESS AND AMELIORATED WITH METFORMIN.

Background: Sepsis is a life-threatening medical emergency, frequently complicated with intensive care unit-acquired weakness syndrome (ICU-AW). ICU-AW patients display flaccid weakness of the limbs, especially in the proximal limb muscles. However, little is known regarding its pathogenesis. Here, we aimed to identify the potential signaling pathway involved in ICU-AW regulation and identify a potential therapeutic drug for intervention. Methods: Both in vivo and in vitro septic mice were used. For the in vivo septic mice, either cecum ligation and puncture or intraperitoneal injection of LPS was conducted in mice. The body weight and muscle mass were then measured and recorded. Muscle strength was evaluated by limb grip strength test. The expression of proteins extracted from cells and muscles was checked through Western blot analysis. Quantitative reverse transcription-polymerase chain reaction was carried out to test the transcriptional level of genes. Senescence-associated β-galactosidase (SA-β-gal) staining and Sirius red for collagen staining were conducted. Metformin, as an antiaging agent, was then tested for any attenuation of sepsis-related symptoms. For in vitro sepsis modeling, myoblasts were treated with LPS, analyzed for senescence-related protein expression, and subsequently retested upon metformin treatment. Results: We found that both the weight and strength of muscle were dramatically reduced in cecum ligation and puncture- or LPS-induced septic mice. RNA-seq analysis revealed that various cellular senescent genes were involved in sepsis. In line with this, expression of senescence-related genes, p53 and p21 were both upregulated. Both SA-β-gal and Sirius red for collagen staining were enhanced in tibialis anterior muscles. Notably, inhibition of p53 expression by siRNA prominently reduced the number of SA-β-gal-positive myoblasts upon LPS treatment. This indicated sepsis-induced cellular senescence to be dependent on p53. Consistent with the function of metformin in antiaging, metformin attenuated cellular senescence in both murine myoblasts and skeletal muscles during sepsis. Muscle strength of septic mice was improved upon metformin treatment. Metformin intervention is therefore proposed as a potential therapeutic strategy for ICU-AW. Conclusion: Taken together, we revealed a previously unappreciated linkage between cellular senescence and sepsis-induced muscle weakness and propose metformin as a potential therapeutic drug for the treatment of ICU-AW.

Role of TFEB and TFE3 in mediating lysosomal and mitochondrial adaptations to contractile activity in skeletal muscle myotubes

Exercise is potent stimulus for mitochondrial adaptations, serving to activate mitochondrial biogenesis as well as mitochondrial turnover. Through the process of mitophagy, dysfunctional mitochondria are selectively targeted and recycled via the lysosomes, which is activated following a single bout of exercise. The microphthalamia (MiT) family of transcription factors, including TFEB and TFE3, are widely recognized as the master regulators of lysosomal biogenesis, as they homo- and hetero-dimerize to transcriptionally regulate lysosomal and macroautophagy-related genes. It is currently unknown to what extent TFEB and TFE3 regulate mitophagy, and whether these transcription factors mediate mitochondrial adaptations to contractile activity (CA). Here we show that following an acute bout of contractile activity in cultured C2C12 murine skeletal muscle myotubes, LC3-II mitophagy flux is induced and the absence of TFEB or TFE3 impairs this acute mitophagic response. However, the loss of either transcription factor alone does not mitigate the improvements in oxygen consumption seen following chronic contractile activity (CCA). Chronic contractile activity also elicited functional improvements in lysosomes including a reduction in size and increased proteolytic activity, evidenced by increased digestion and unquenching of DQ-BSA fluorophore, thereby illustrating a level of redundancy between the two transcription factors in mediating chronic contractile activity-induced adaptations. However, in the absence of both TFEB and TFE3, lysosomal adaptations were not observed following chronic contractile activity and subsequent mitochondrial adaptations were attenuated. These findings underscore the importance of the lysosomes, and of TFEB and TFE3, in mediating mitochondrial adaptations to chronic contractile activity.

An &lt;em&gt;ex vivo&lt;/em&gt; experimental system to track fluorescent nanoparticles inside skeletal muscle

The development of novel nanoconstructs for biomedical applications requires the assessment of their biodistribution, metabolism and clearance in single cells, organs and entire organisms in a living environment. To reduce the number of in vivo experiments performed and to refine the methods used, in accordance with the 3Rs principle, this work proposes an ex vivo experimental system to monitor, using fluorescence microscopy, the distribution of nanoparticles in explanted murine skeletal muscle maintained in a bioreactor that can preserve the structural and functional features of the organ for long periods of time. Fluorescently-labelled liposomes and poly(lactide-co-glycolide) (PLGA)-based nanoparticles were injected into the intact soleus muscle (in the distal region close to the tendon) immediately after explants, and their distribution was analysed at increasing incubation times in cross cryosections from the proximal region of the belly. Both nanocarriers were clearly recognized in the muscle and were found to enter and migrate inside the myofibres, whereas their migration in the connective tissue seemed to be limited. In addition, some fluorescent signals were observed inside the macrophages, demonstrating the physiological clearance of the nanocarriers that did not enter the myofibres. Our ex vivo system therefore provides more information than previous in vitro experiments on cultured muscle cells, highlighting the need for the appropriate functionalization of nanocarriers if myofibre targeting is to be improved.

The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation.

Adult skeletal muscle harbours a population of muscle stem cells (MuSCs) that are required for repair after tissue injury. In youth, MuSCs return to a reversible state of cell-cycle arrest termed 'quiescence' after injury resolution. Conversely, some MuSCs in aged muscle remain semi-activated, causing a premature response to injuries that results in incomplete repair and eventual stem cell depletion. Regulating this balance between MuSC quiescence and activation may hold the key to restoring tissue homeostasis with age, but is incompletely understood. To fill this gap, we developed a simple and tractable in vitro method, to rapidly inactivate MuSCs freshly isolated from young murine skeletal muscle, and return them to a quiescent-like state for at least 1-week, which we name mini-IDLE (Inactivation and Dormancy LEveraged in vitro). This was achieved by introducing MuSCs into a 3D bioartificial niche comprised of a thin sheet of mouse myotubes, which we demonstrate provides the minimal cues necessary to induce quiescence. With different starting numbers of MuSCs, the assay revealed cellular heterogeneity and population-level adaptations that converged on a common niche repopulation density; behaviours previously observed only in vivo. Quiescence-associated hallmarks included a Pax7+CalcR+DDX6+MyoD-c-FOS- signature, quiescent-like morphologies, and polarized niche markers. Leveraging high-content bioimaging pipelines, we demonstrate a relationship between morphology and cell fate signatures for possible real-time morphology-based screening. When using MuSCs from aged muscle, they displayed aberrant proliferative activities and delayed inactivation kinetics, among other quiescence-associated defects that we show are partially rescued by wortmannin treatment. Thus, the assay offers an unprecedented opportunity to systematically investigate long-standing queries in areas such as regulation of pool size and functional heterogeneity within the MuSC population, and to uncover quiescence regulators in youth and age.

Genetic Manipulation of CB1 Cannabinoid Receptors Reveals a Role in Maintaining Proper Skeletal Muscle Morphology and Function in Mice.

The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca2+ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre+/-) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre+/- mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre+/- mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described.

Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation

IntroductionFuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates.ObjectivesThe goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism.MethodsIsolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles.ResultsPyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation.ConclusionIsotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

Covalent Stem Cell-Combining Injectable Materials with Enhanced Stemness and Controlled Differentiation In Vivo.

Biohybrid materials, which are defined as engineered functional materials combining living components with nonliving synthetic materials, are considered promising bioactive materials for applications in in vivo tissue engineering. However, the rational design of biohybrid materials applicable to in vivo tissue engineering faces major challenges associated with techniques for combining living cells with nonliving synthetic materials and cell sources. Here, we report injectable covalent stem cell-combing biohybrid materials prepared via a bio-orthogonal click cross-linking reaction of azide-modified adipose-derived stem cells (N3[+]ADSCs), one of the most promising cell sources utilized clinically, with alkyne-modified biocompatible alginate polymers. The mechanical properties of the covalent stem cell-combining biohybrid materials can be adapted to the mechanical properties of the surrounding environment in which they are transplanted by alternating the number of N3[+]ADSCs, the concentration of alkyne-modified alginate, and the number of alkyne groups. Importantly, ADSCs in the covalent biohybrid materials expressed a high level of CD-105, a marker for undifferentiated mesenchymal stem cells, in the body in the absence of differentiation signals, whereas very little CD-105 was expressed in the control physical cell-loading materials, demonstrating that this covalent stem cell-combining approach results in enhanced retention of the material's "stemness" and controlled differentiation in the body. We assessed the potential utility of the covalent stem cell-combining biohybrid materials for in vivo tissue engineering using a murine severe skeletal muscle defect-healing model. Importantly, all of the tissues regenerated by the covalent biohybrid material treatment expressed MYH3, a myogenic marker protein, whereas no expression of MYH3 was detected in the tissues reconstructed by treatment with control physical stem cell-loading materials and Matrigel, indicating that this covalent stem cell-combining approach results in controlled differentiation in the body. Our data demonstrate the potential utility of covalent stem cell-combining biohybrid materials with host tissue-integrative and controlled differentiation capabilities available for in vivo tissue engineering.

Abstract 12013: Nuclear Localization of PDGFRa Tyrosine Kinase Represents a Novel Mechanism Controlling the Fibrotic Response of Mesenchymal Stromal Cells to Myocardial Ischemia

Background: We discovered a time dependent dual role role for mesenchymal stromal cells expressing the platelet-derived growth factor alpha (PDGFRα + MSCs), which play essential functions in tissue revascularization and in fibro-adipogenic repair. Hypothesis: Based on our preliminary data, we hypothesized that specific and distinct stromal cells exist in the vertebrate heart, which retain regenerative capacities relevant to the extension of disease. Methods: All animal studies were performed in compliance with the regulations of the NIH and the local institutional animal care. Myocardial ischemia/reperfusion (MI/R) was induced in mice (n=10, FVB female mice) and pigs (n=10 female Yorkshire pigs) with standard procedures. Control animals were not operated (n=3 mice and n=4 pigs). Bulk RNA sequencing (n=4) was performed using a HiSeq2500 (Illumina). Results: Here, we showed that cardiac PDGFRα + MSCs have the potential to revascularize cardiac tissue and likely control cardiac electromechanical properties only earlier after MI/R. During the subacute phase, we discovered nuclear shuttling of the receptor from the plasma membrane before lineage specification into fibrotic cells (nuclear PDGFRα in non-ischemic 3±1 versus ischemic 7.7±2.5 hearts; p&lt;0.05). Interestingly, a similar response was observed in the heart of large animals (i.e., pigs), suggesting a cross-species mechanism controlling PDGFRα + MSCs commitment via intracellular shuffling of the tyrosine kinase receptor. A roadblock to the clinical translation of these discoveries is that there is no mechanism to manipulate PDGFRα + MSCs dual function. To cover this gap, we compared the transcriptomics of murine cardiac and skeletal muscle PDGFRα + MSCs using bulk RNA sequencing in physiological conditions. We discovered that key pathways, likely a signature of non-regenerating stromal cells, are differentially regulated in cardiac PDGFRα + MSCs, including Notch mediators of mesenchymal lineages and IGF-1 signaling. Conclusions: These data suggest that by modulating PDGFRα intracellular shuttling or altering the expression of specific genes, it may be possible to therapeutically direct PDGFRα + MSCs duality to reduce fibrosis, while harnessing their beneficial properties.

Three-dimensional mechanical characterization of murine skeletal muscle using quantitative micro-elastography.

Skeletal muscle function is governed by both the mechanical and structural properties of its constituent tissues, which are both modified by disease. Characterizing the mechanical properties of skeletal muscle tissue at an intermediate scale, i.e., between that of cells and organs, can provide insight into diseases such as muscular dystrophies. In this study, we use quantitative micro-elastography (QME) to characterize the micro-scale elasticity of ex vivo murine skeletal muscle in three-dimensions in whole muscles. To address the challenge of achieving high QME image quality with samples featuring uneven surfaces and geometry, we encapsulate the muscles in transparent hydrogels with flat surfaces. Using this method, we study aging and disease in quadriceps tissue by comparing normal wild-type (C57BL/6J) mice with dysferlin-deficient BLAJ mice, a model for the muscular dystrophy dysferlinopathy, at 3, 10, and 24 months of age (sample size of three per group). We observe a 77% decrease in elasticity at 24 months in dysferlin-deficient quadriceps compared to wild-type quadriceps.

Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms.

Real-time quantitative PCR (RT-qPCR) is a widely applied technique for relative quantification of gene expression. In this context, the selection of a suitable reference gene (RG) is an essential step for obtaining reliable and biologically relevant RT-qPCR results. The present study aimed to determine the expression stability of commonly used RGs in mouse skeletal muscle tissue. The expression pattern of eight RGs (ACTB, GAPDH, HPRT, YWHAZ, B2M, PPIA, TUBA and 18S) were evaluated by RT-qPCR in different sample groups classified based on genetic background, muscle tissue type, and growth stage, as well as in a C2C12 myoblast cell line model. Five computational programs were included in the study (comparative ΔCq value, NormFinder, BestKeeper, geNorm, RefFinder) to evaluate the expression stability of RGs. Furthermore, the normalization effects of RGs in soleus (SOL) and gastrocnemius (GAS) muscle tissue were evaluated. Collectively, ACTB, HPRT and YWHAZ were shown to be the most stable RGs, while GADPH and 18S were the least stable. Therefore, the combined use of ACTB, HPRT and YWHAZ is recommended for the normalization of gene expression results in experiments with murine skeletal muscle. The results discussed herein provide a foundation for gene expression analysis by RT-qPCR in mammalian skeletal muscle.

Shared and unique phosphoproteomics responses in skeletal muscle from exercise models and in hyperammonemic myotubes

Skeletal muscle generation of ammonia, an endogenous cytotoxin, is increased during exercise. Perturbations in ammonia metabolism consistently occur in chronic diseases, and may blunt beneficial skeletal muscle molecular responses and protein homeostasis with exercise. Phosphorylation of skeletal muscle proteins mediates cellular signaling responses to hyperammonemia and exercise. Comparative bioinformatics and machine learning-based analyses of published and experimentally derived phosphoproteomics data identified differentially expressed phosphoproteins that were unique and shared between hyperammonemic murine myotubes and skeletal muscle from exercise models. Enriched processes identified in both hyperammonemic myotubes and muscle from exercise models with selected experimental validation included protein kinase A (PKA), calcium signaling, mitogen-activated protein kinase (MAPK) signaling, and protein homeostasis. Our approach of feature extraction from comparative untargeted "omics" data allows for selection of preclinical models that recapitulate specific human exercise responses and potentially optimize functional capacity and skeletal muscle protein homeostasis with exercise in chronic diseases.

Increasing plasma L-kynurenine impairs mitochondrial oxidative phosphorylation prior to the development of atrophy in murine skeletal muscle: A pilot study.

Introduction: L-Kynurenine (L-Kyn), a product of tryptophan (Trp) catabolism, has been linked with impairments in walking speed, muscle strength/size, and physical function. The purpose of this pilot study was to develop a dietary model that elevates plasma L-Kyn levels in mice and characterize its impact on muscle health and function. Methods: Four-month-old C57BL6J male mice were randomized to either a L-Kyn supplemented (150 mg/kg) or chow diet for 10 weeks. Plasma L-Kyn and Trp levels were measured via mass spectrometry. Primary outcomes included assessments of muscle weights, myofiber cross-sectional area (CSA), nerve-stimulated contractile performance, and mitochondrial oxidative phosphorylation (OXPHOS) and hydrogen peroxide (H2O2) production. Additional experiments in cultured myotubes explored the impact of enhancing L-Kyn metabolism. Results: Mice randomized to the L-Kyn diet displayed significant increases in plasma L-Kyn levels (p = 0.0028) and the L-Kyn/Trp ratio (p = 0.011) when compared to chow fed mice. Food intake and body weights were not different between groups. There were no detectable differences in muscle weights, myofiber CSA, or contractile performance. L-Kyn fed mice displayed reductions in mitochondrial OXPHOS (p = 0.05) and maximal ADP-stimulated respiration (p = 0.0498). In cultured myotubes, overexpression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha prevented atrophy and proteolysis, as well as deficits in mitochondrial respiration with L-Kyn treatment. Conclusion: Dietary feeding of L-Kyn increases plasma L-Kyn levels and the L-Kyn/Trp ratio in healthy male mice. Mitochondrial impairments in muscle were observed in mice with elevated L-Kyn without changes in muscle size or function. Enhancing L-Kyn metabolism can protect against these effects in culture myotubes.

Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders.

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors—AAVMYO2 and AAVMYO3—prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Counteractions of a Novel Hydroalcoholic Extract from Lens Culinaria against the Dexamethasone-Induced Osteoblast Loss of Native Murine Cells.

The cytoprotective effects of a novel hydroalcoholic extract (0.01–5 mg/mL) from Lens culinaria (Terre di Altamura Srl) were investigated within murine native skeletal muscle fibers, bone marrow cells, and osteoblasts, and in cell lines treated with the apoptotic agent staurosporine (2.14 × 10−6 M), the alkylating drug cisplatin (10−4 M), the topoisomerase I inhibitor irinotecan (10−4 M), the antimitotic pro-oxidant doxorubicin (10−6 M), and the immunosuppressant dexamethasone (2 × 10−6 M). An amount of 10g of plant material was used to obtain a 70% ethanol/water product, following two-step extraction, evaporation, lyophilization, and storage at −20 °C. For the murine osteoblasts, doxorubicin reduced survival by −65%, dexamethasone by −32% and −60% after 24 and 48 h of incubation time, respectively. The extract was effective in preventing the osteoblast count-reduction induced by dexamethasone; it was also effective at preventing the inhibition of mineralization induced by dexamethasone. Doxorubicin and cisplatin caused a significant reduction in cell growth by −77% for bone marrow cells, −43% for irinotecan, and −60% for dexamethasone, but there was no evidence for the cytoprotective effects of the extract in these cells. Staurosporine and doxorubicin caused a fiber death rate of >−40% after 18 and 24 h of incubation, yet the extract was not effective at preventing these effects. The extract was effective in preventing the staurosporine-induced reduction of HEK293 proliferation and colony formation in the crystal violet DNA staining and the clonogenic assays. It was also effective for the cisplatin-induced reduction in HEK293 cell proliferation. The extract, however, failed to protect the SHSY5Y neurons against cisplatin and irinotecan-induced cytotoxicity. A UV/VIS spectroscopy analysis showed three peaks at the wavelengths of 350, 260, and 190 nm, which correspond to flavonoids, proanthocyanins, salicylates, and AA, constituting the extract. These data suggest the possible development of this extract for use against dexamethasone-induced bone loss and renal chemotherapy-induced damage.

Cellular and Molecular Variations in Male and Female Murine Skeletal Muscle after Long-Term Feeding with a High-Fat Diet.

Current information regarding the effects of a high-fat diet (HFD) on skeletal muscle is contradictory. This study aimed to investigate the effects of a long-term HFD on skeletal muscle in male and female mice at the morphological, cellular, and molecular levels. Adult mice of the C57BL/6 strain were fed standard chow or an HFD for 20 weeks. The tibialis anterior muscles were dissected, weighed, and processed for cellular and molecular analyses. Immunocytochemical and morphometric techniques were applied to quantify fiber size, satellite cells (SCs), and myonuclei. Additionally, PCR array and RT-qPCR tests were performed to determine the expression levels of key muscle genes. Muscles from HFD mice showed decreases in weight, SCs, and myonuclei, consistent with the atrophic phenotype. This atrophy was associated with a decrease in the percentage of oxidative fibers within the muscle. These findings were further confirmed by molecular analyses that showed significant reductions in the expression of Pax7, Myh1, and Myh2 genes and increased Mstn gene expression. Male and female mice showed similar trends in response to HFD-induced obesity. These findings indicate that the long-term effects of obesity on skeletal muscle resemble those of age-related sarcopenia.

Nano Modification of Antrodia Cinnamomea Exhibits Anti-Inflammatory Action and Improves the Migratory Potential of Myogenic Progenitors.

The skeletal muscle progenitors’ proliferation and migration are crucial stages of myogenesis. Identifying drug candidates that contribute to myogenesis can have a positive impact on atrophying muscle. The purpose of the study is to synthesize the Antrodia cinnamomea (AC)-β-cyclodextrin (BCD) inclusion complex (IC) and understand its in vitro pro-regenerative influence in murine skeletal C2C12 myoblasts. The IC was subjected to various nano-characterization studies. Fluorescent IC was synthesized to understand the cellular uptake of IC. Furthermore, 25 µg/mL, 12.5 µg/mL, and 6.25 µg/mL of IC were tested on murine C2C12 skeletal muscle cells for their anti-inflammatory, pro-migratory, and pro-proliferative action. The cellular internalization of IC occurred rapidly via pinocytosis. IC (252.6 ± 3.2 nm size and −37.24 ± 1.55 surface charge) exhibited anti-inflammatory action by suppressing the secretion of interleukin-6 and enhanced cell proliferation with promising cytocompatibility. A 12.5 μg/mL dose of IC promoted cell migration in 24 h, but the same dose of AC significantly reduced cell migration, suggesting modification by BCD. Molecular studies revealed that IC promoted C2C12 myoblasts migration by upregulating long non-coding RNA (lncRNA) NEAT-1, SYISL, and activating the pPKC/β-catenin pathway. Our study is the first report on the pro-proliferative and pro-migratory effects of BCD-modified extracts of AC.

Sphingosine Phosphate Lyase Is Upregulated in Duchenne Muscular Dystrophy, and Its Inhibition Early in Life Attenuates Inflammation and Dystrophy in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.

A novel method for determining murine skeletal muscle fiber type using autofluorescence lifetimes.

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus,τ2was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle,τ2was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). Fromτ2distributions, it follows that an FDB fiber withτ2> 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.