Human Muscle Biopsies Research Articles

Development of a primary skeletal muscle cell culture model from human muscle biopsies

Purpose: This work aimed to develop and validate a culture model of primary human skeletal muscle cells. Methods: Muscle biopsies were obtained post-mortem from the vastus lateralis. The biopsies were enzymatically digested with collagenase 1A (1mg/mL) and dispase II (4,8mg/mL) for 60 minutes at 37°C in order to isolate fibroblasts and satellite cells. Then, the satellite cells (CD56+) were separated from fibroblasts (CD56-) by two methods: pre-plating or immunomagnetic cell sorting using an anti-CD56 antibody, a transmembrane glycoprotein expressed on the surface of satellite cells. Indeed, some human muscle biopsies contain a high proportion of fibroblasts that can contaminate the myoblasts cell culture if the separation method is not efficient. The performance of these cell separation methods was evaluated by FACS. We also compared the effects of two culture media on myoblasts proliferation (10%FBS vs. 10%FBS + 2%Ultroser G (UG)) using cell doubling time as parameter. Two differentiation media (2%FBS vs. 0.5%UG) were tested on the myogenic index, which corresponds to the proportion of cells that have fused to form myotubes. This index was determined through the detection of the myosin heavy chain (MYH) by immunofluorescence. Finally, we studied the expression of several muscle cell markers during myogenesis by qRT-PCR and immunofluorescence. Results: The cells obtained from the enzymatic digestion of the biopsy contained a varying proportion of CD56+ satellite cells (86,15 ± 12,09%; 95%CI:73,45 to 98,84). The cell population obtained after immunomagnetic cell sorting method contained more than 92% of CD56+ cells (96,11 ± 3,27%; 95%CI:92,68 to 99,54). In comparison, the ratio of CD56+ obtained by the preplating method was lower (84,19 ± 9,94%; 95%CI:73,76 to 94,62), but no significant difference was observed between both methods. The cell doubling time was significantly shorter with culture media containing 10%FBS and 2%UG than with the media containing only 10%FBS (25.70 ± 3,96h vs. 70.64 ± 21,68h, p = 0.0313). The myogenic index was slightly higher with the 0.5%UG than 2%FBS differentiating medium (62,75% ± 8,57% vs. 56,39 ± 16,45%). Finally, the increase of MyoD1, myogenin and MYH, three markers of differentiation, indicated the phenotypic transition of myoblasts to a myogenic phenotype. Conclusions: Immunomagnetic cell sorting allows better satellite cells and fibroblasts separation than the conventionally used preplating technique. Ultoser G serum substitute accelerates cell proliferation and improves the differentiation of myoblasts into myotubes. The markers MyoD1, myogenin and MYH are good markers of the myogenic differentiation of myoblasts. This in vitro model makes it possible to purify efficiently muscle satellite cells from human muscle biopsies. These cells can be used in in vitro studies to study the pathophysiological mechanisms of muscle disease and the mechanisms of action of treatments.

Importance of immunohistochemical evaluation of developmentally regulated myosin heavy chains in human muscle biopsies

Our retrospective immunohistochemical study of normal quadriceps muscle biopsies shows that embryonic myosin heavy chains are down-regulated by, or soon after, birth. Fetal myosin heavy chains are down-regulated by 4–6 months. Thus the presence of an appreciable number of fibres with embryonic myosin heavy chains at birth or of fetal myosin heavy chains after 6 months of age suggests a delay in maturation or an underlying abnormality. Regenerating fibres in dystrophic muscle often co-express both embryonic and fetal myosin heavy chains but more fibres with fetal than embryonic myosin heavy chains can occur. Embryonic myosin heavy chains are a useful marker of regeneration but effects of denervation, stress, disuse, and fibre maintenance also have to be taken into account. In neurogenic disorders fibres with embryonic myosin heavy chains are rare but fetal myosin heavy chain expression is common, particularly in 5q spinal muscle atrophy. Nuclear clumps in denervated muscle show fetal and sometimes embryonic myosin heavy chains. Developmentally regulated myosins are useful for highlighting the perifascicular atrophy in juvenile dermatomyositis. Our studies highlight the importance of baseline data for embryonic and fetal myosin heavy chains in human muscle biopsies and the importance of assessing them in a spectrum of neuromuscular disorders.

Muscle metabolic remodelling patterns in Duchenne muscular dystrophy revealed by ultra-high-resolution mass spectrometry imaging

Duchenne muscular dystrophy (DMD) is a common and severe X-linked myopathy, characterized by muscle degeneration due to altered or absent dystrophin. DMD has no effective cure, and the underlying molecular mechanisms remain incompletely understood. The aim of this study is to investigate the metabolic changes in DMD using mass spectrometry-based imaging. Nine human muscle biopsies from DMD patients and nine muscle biopsies from control individuals were subjected to untargeted MSI using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry. Both univariate and pattern recognition techniques have been used for data analysis. This study revealed significant changes in 34 keys metabolites. Seven metabolites were decreased in the Duchenne biopsies compared to control biopsies including adenosine triphosphate, and glycerophosphocholine. The other 27 metabolites were increased in the Duchenne biopsies, including sphingomyelin, phosphatidylcholines, phosphatidic acids and phosphatidylserines. Most of these dysregulated metabolites are tightly related to energy and phospholipid metabolism. This study revealed a deep metabolic remodelling in phospholipids and energy metabolism in DMD. This systems-based approach enabled exploring the metabolism in DMD in an unprecedented holistic and unbiased manner with hypothesis-free strategies.

Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles

BackgroundCongenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1−/− mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue.MethodsProteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations.ResultsComprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect.ConclusionsCollectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergström technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

Histopathological features and satellite cell population characteristics in human inferior oblique muscle biopsies: clinicopathological correlation

To investigate the correlation between clinical characteristics and histopathological and immunohistochemical features of inferior oblique muscles in patients with primary and secondary inferior oblique overaction. Inferior oblique muscle specimens of patients who underwent inferior oblique-weakening procedures for primary or secondary inferior oblique overaction were recruited. Subjects were mainly divided into two groups, each of which was further divided into two subgroups: group 1 included patients with primary inferior oblique overaction (subgroups, infantile esotropia vs acquired V-pattern esotropia), and group 2 included patients with secondary inferior oblique overaction (subgroups, congenital vs acquired trochlear nerve palsy). Inferior oblique overaction was graded between 0-4. Histopathologic changes, such as angular fibers, endo- and perimysial fibrosis, and vacuolization were categorized from mild to severe. Immunohistochemical markers Pax7, NCAM, and MyoD1 were used to detect satellite cells, a unique stem cell population in muscles presumably responsible from myofiber regeneration and maintenance, and their activity. Results were reported as stained cells per cross-section ratio. A total of 51 patients were included: 36 in group 1 and 15 in group 2. Satellite cell distribution and activity was significantly higher in group 1 (P<0.001). The angular fiber count and the degree of perimysial fibrosis was higher in the secondary group (P<0.001 and P=0.01, resp.). There was no correlation between clinical amount of inferior oblique muscle overaction and immunohistochemical markers. The differences in immunohistochemical parameters supported with histopathological changes between different strabismus etiologies imply that satellite cell population behavior varies among strabismus types.

Regulation of Lin28a-miRNA let-7b-5p pathway in skeletal muscle cells by peroxisome proliferator-activated receptor delta.

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a-let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a-let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.

CrossTalk proposal: Intramuscular lipid accumulation causes insulin resistance.

CrossTalk proposal: Intramuscular lipid accumulation causes insulin resistance.

Prospective isolation of human fibroadipogenic progenitors with CD73

Skeletal muscle relies on coordination between myogenic and non-myogenic interstitial cells for homeostasis and for regeneration and response to injury. Fibroadipogenic progenitors (FAPs) have recently been recognized as key modulators of signaling to promote myogenesis following injury. FAPs are also responsible for the fibrosis and fatty replacement of muscle tissue seen in many diseased states. While extensive use of surface markers to purify FAPs has been undertaken in the mouse system, in particular PDGFRA, markers for human FAPs are less well understood. Here, we show that CD73 can be used as a single positive marker to purify FAPs from the lineage-negative (CD45-neg, CD31-neg) fraction of skeletal muscle mononuclear cells. Although CD73 was previously found to be expressed in cultured myogenic cells, we find that this marker is only acquired upon culture and that the CD73+ fraction of human skeletal muscle has no myogenic activity. We show that Lin-neg CD73+ cells from human muscle undergo fat differentiation as well as fibrogenesis when exposed to appropriate activating signals in vitro. This simple single positive marker approach effectively enables isolation of human FAPs from fresh human skeletal muscle biopsies.

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle.

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin's concordance correlation coefficients and Bland and Altman's graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

1883-P: Metformin Increases Protein Phosphatase 2A Activity in Primary Skeletal Muscle Cells Derived from Lean Healthy Insulin-Sensitive Human Participants

Skeletal muscle insulin resistance is one of the primary contributors of type 2 diabetes (T2D). Metformin is the first-line drug for the treatment of T2D. Main known effects of metformin include inhibition of glucose production in liver and attenuation of insulin resistance in skeletal muscle. However, molecular mechanism of metformin’s action in skeletal muscle is not well understood. Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, plays a pivotal role in cellular processes (e.g., signal transduction, apoptosis), and acts through dephosphorylating key signaling molecules such as AKT, AMPK, etc. However, whether or not PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells remains to be elucidated. In the present study, hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies from 8 lean insulin sensitive participants (4 Female and 4 male; age: 21.4±0.8 yrs; BMI: 22.1±0.7 kg/m2; 2-hour OGTT: 96.5± 6.3 mg/dl; HbA1c: 5.3±0.1 %; fasting plasma glucose: 87.5±1.4 mg/dl; M-value; 10.9±0.8 mg/kgBW/min). The cells were expanded, differentiated into myotubes, and treated with 50µM metformin (the physiologic concentration) for 24 hours before harvesting. PP2A activity was measured by a phosphatase activity assay (Millipore). The results indicated that metformin increased the activity of PP2A in the myotubes derived from all 8 participants, and the average fold increase is 1.59±0.13 (p&amp;lt;0.05). These results provided the first evidence to suggest that metformin promotes activation of PP2A in human skeletal muscle cells derived from lean insulin sensitive participants, and may help to understand metformin’s action in skeletal muscle in humans. Disclosure A.H. Mestareehi: None. B. Seyoum: None. Z. Msallaty: None. A.P. Mallisho: None. X. Zhang: None. A. Kowluru: None. Z. Yi: None. Funding American Diabetes Association (1-13-TS-27 to Z.Y.); National Institutes of Health (R01DK107666, R01DK081750 to Z.Y.), (T32HL120822 to A.H.M.); DOTS Scholars (to A.H.M., Z.Y.)

Muscle Weakness in Myositis: MicroRNA-Mediated Dystrophin Reduction in a Myositis Mouse Model and Human Muscle Biopsies.

ObjectiveMuscle inflammation is a feature in myositis and Duchenne muscular dystrophy (DMD). Autoimmune mechanisms are thought to contribute to muscle weakness in patients with myositis. However, a lack of correlation between the extent of inflammatory cell infiltration and muscle weakness indicates that nonimmune pathologic mechanisms may play a role. The present study focused on 2 microRNA (miRNA) sets previously identified as being elevated in the muscle of patients with DMD—an “inflammatory” miRNA set that is dampened with glucocorticoids, and a “dystrophin‐targeting” miRNA set that inhibits dystrophin translation—to test the hypothesis that these miRNAs are similarly dysregulated in the muscle of patients with myositis, and could contribute to muscle weakness and disease severity.MethodsA major histocompatibility complex class I–transgenic mouse model of myositis was utilized to study gene and miRNA expression and histologic features in the muscle tissue, with the findings validated in human muscle biopsy tissue from 6 patients with myositis. Mice were classified as having mild or severe myositis based on transgene expression, body weight, histologic disease severity, and muscle strength/weakness.ResultsIn mice with severe myositis, muscle tissue showed mononuclear cell infiltration along with elevated expression of type I interferon and NF‐κB–regulated genes, including Tlr7 (3.8‐fold increase, P < 0.05). Furthermore, mice with severe myositis showed elevated expression of inflammatory miRNAs (miR‐146a, miR‐142‐3p, miR‐142‐5p, miR‐455‐3p, and miR‐455‐5p; ~3–40‐fold increase, P < 0.05) and dystrophin‐targeting miRNAs (miR‐146a, miR‐146b, miR‐31, and miR‐223; ~3–38‐fold increase, P < 0.05). Bioinformatics analyses of chromatin immunoprecipitation sequencing (ChIP‐seq) data identified at least one NF‐κB consensus element within the promoter/enhancer regions of these miRNAs. Western blotting and immunofluorescence analyses of the muscle tissue from mice with severe myositis demonstrated reduced levels of dystrophin. In addition, elevated levels of NF‐κB–regulated genes, TLR7, and miRNAs along with reduced dystrophin levels were observed in muscle biopsy tissue from patients with histologically severe myositis.ConclusionThese data demonstrate that an acquired dystrophin deficiency may occur through NF‐κB–regulated miRNAs in myositis, thereby suggesting a unifying theme in which muscle injury, inflammation, and weakness are perpetuated both in myositis and in DMD.

MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans.

Using an unbiased high‐throughput microRNA (miRNA)‐silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA‐204‐5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA‐204‐5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA‐204‐5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC‐1α (3.71‐fold; p < .01), predicted as an miR‐204‐5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA‐204‐5p silencing in C2C12 myotubes. Silencing of miRNA‐204‐5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA‐204‐5p silencing. Finally, miRNA‐204‐5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA‐204‐5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA‐204‐5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA‐204‐5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

The acute effects of cigarette smoke exposure on muscle fiber type dynamics in rats.

Reduced exercise capacity is common in people with chronic obstructive pulmonary diseases (COPD) and chronic smokers and is suggested to be related to skeletal muscle dysfunction. Previous studies using human muscle biopsies have shown fiber-type shifting in chronic smokers particularly those with COPD. These results, however, are confounded with aging effects because people with COPD tend to be older. In the present study, we implemented an acute 7-day cigarette smoke-exposed model using Sprague-Dawley rats to evaluate early effects of cigarette smoking on soleus muscles. Rats (n = 5 per group) were randomly assigned to either a sham air (SA) or cigarette smoking (CS) groups of three different concentrations of total particulate matters (TPM) (CSTPM2.5, CSTPM5, CSTPM10). Significantly lower percentages of type I and higher type IIa fiber were detected in the soleus muscle in CS groups when compared with SA group. Of these, only CSTMP10 group exhibited significantly lower citrate synthase activity and higher muscle tumor necrosis factor-α level than that of SA group. Tumor necrosis factor-α level was correlated with the percentage of type I and IIa fibers. However, no significant between-group differences were found in fiber cross-sectional area, physical activities, or lung function assessments. In conclusion, acute smoking may directly trigger the onset of glycolytic fiber type shift in skeletal muscle independent of aging.

Unlike a high-fat diet model, mitochondrial ROS production does not appear to contribute to bed rest-induced insulin resistance.

Unlike a high-fat diet model, mitochondrial ROS production does not appear to contribute to bed rest-induced insulin resistance.

Foci of Segmentally Contracted Sarcomeres in Trapezius Muscle Biopsy Specimens in Myalgic and Nonmyalgic Human Subjects: Preliminary Results.

The myofascial trigger point hypothesis postulates that there are small foci of contracted sarcomeres in resting skeletal muscle. Only one example, in canine muscle, has been published previously. This study evaluated human muscle biopsies for foci of contracted sarcomeres. The Departments of Rehabilitation Sciences and Physiotherapy at Ghent University, Ghent, Belgium. Biopsies from 28 women with or without trapezius myalgia were evaluated, 14 in each group. Muscle biopsies were obtained from regions of taut bands in the trapezius muscle and processed for light and electron microscopy and for histochemical analysis. Examination of the biopsies was blinded as to group. A small number of foci of segmentally contracted sarcomeres were identified. One fusiform segmental locus involved the entire muscle fiber in tissue from a myalgic subject. Several transition zones from normal to contracted sarcomeres were found in both myalgic and nonmyalgic subjects. The distance between Z-lines in contracted sarcomeres was about 25-45% of the same distance in normal sarcomeres. Z-lines were disrupted and smeared in the contracted sarcomeres. A small number of foci of segmentally contracted sarcomeres were found in relaxed trapezius muscle in human subjects, a confirmation of the only other example of spontaneous segmental contraction of sarcomeres (in a canine muscle specimen), consistent with the hypothesis of trigger point formation and with the presence of trigger point end plate noise.

Transcriptional and cytopathological hallmarks of FSHD in chronic DUX4-expressing mice.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by loss of repression of the DUX4 gene; however, the DUX4 protein is rare and difficult to detect in human muscle biopsies, and pathological mechanisms are obscure. FSHD is also a chronic disease that progresses slowly over decades. We used the sporadic, low-level, muscle-specific expression of DUX4 enabled by the iDUX4pA-HSA mouse to develop a chronic long-term muscle disease model. After 6 months of extremely low sporadic DUX4 expression, dystrophic muscle presented hallmarks of FSHD histopathology, including muscle degeneration, capillary loss, fibrosis, and atrophy. We investigated the transcriptional profile of whole muscle as well as endothelial cells and fibroadiopogenic progenitors (FAPs). Strikingly, differential gene expression profiles of both whole muscle and, to a lesser extent, FAPs, showed significant overlap with transcriptional profiles of MRI-guided human FSHD muscle biopsies. These results demonstrate a pathophysiological similarity between disease in muscles of iDUX4pA-HSA mice and humans with FSHD, solidifying the value of chronic rare DUX4 expression in mice for modeling pathological mechanisms in FSHD and highlighting the importance FAPs in this disease.

In vivo analysis of γH2AX+ cells in skeletal muscle from aged and obese humans.

Over the past 20years, various identifiers of cellular senescence have been used to quantify the abundance of these cells in different tissues. These include classic markers such as p16, senescence-associated β-gal, and γH2AX, in addition to more recent markers (Sudan Black B and HMGB1). In vivo data on the usefulness of these markers in skeletal muscle are very limited and inconsistent. In the present study, we attempted to identify senescent cells in frozen human skeletal muscle biopsies using these markers to determine the effects of age and obesity on senescent cell burden; however, we were only able to assess the abundance of DNA-damaged nuclei using γH2AX immunohistochemistry. The abundance of γH2AX+ cells, including satellite cells, was not higher in muscle from old compared to young individuals; however, γH2AX+ cells were higher with obesity. Additionally, terminally differentiated, postmitotic myofiber nuclei from obese individuals had elevated γH2AX abundance compared to muscle from lean individuals. Analyses of gene expression support the conclusion that the elevated DNA damage and the senescence-associated secretory phenotype are preferentially associated with obesity in skeletal muscle. These data implicate obesity as a larger contributor to DNA damage in skeletal muscle than aging; however, more sensitive senescence markers for human skeletal muscle are needed to determine if these cells are in fact senescent.