Human Skeletal Muscle Research Articles

Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.

A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Characterization of three-dimensional engineered human skeletal muscle at different scales

Characterization of three-dimensional engineered human skeletal muscle at different scales

Assessing thin filament lengths in young healthy human skeletal muscle—a role for fiber type?

Assessing thin filament lengths in young healthy human skeletal muscle—a role for fiber type?

Human myofiber-enriched aging-induced lncRNA FRAIL1 promotes loss of skeletal muscle function.

The loss of skeletal muscle mass during aging is a significant health concern linked to adverse outcomes in older individuals. Understanding the molecular basis of age-related muscle loss is crucial for developing strategies to combat this debilitating condition. Long noncoding RNAs (lncRNAs) are a largely uncharacterized class of biomolecules that have been implicated in cellular homeostasis and dysfunction across a many tissues and cell types. To identify lncRNAs that might contribute to skeletal muscle aging, we screened for lncRNAs whose expression was altered in vastus lateralis muscle from older compared to young adults. We identified FRAIL1 as an aging-induced lncRNA with high abundance in human skeletal muscle. In healthy young and older adults, skeletal muscle FRAIL1 was increased with age in conjunction with lower muscle function. Forced expression of FRAIL1 in mouse tibialis anterior muscle elicits a dose-dependent reduction in skeletal muscle fiber size that is independent of changes in muscle fiber type. Furthermore, this reduction in muscle size is dependent on an intact region of FRAIL1 that is highly conserved across non-human primates. Unbiased transcriptional and proteomic profiling of the effects of FRAIL1 expression in mouse skeletal muscle revealed widespread changes in mRNA and protein abundance that recapitulate age-related changes in pathways and processes that are known to be altered in aging skeletal muscle. Taken together, these findings shed light on the intricate molecular mechanisms underlying skeletal muscle aging and implicate FRAIL1 in age-related skeletal muscle phenotypes.

Pro-Brain-Derived Neurotrophic Factor (BDNF), but Not Mature BDNF, Is Expressed in Human Skeletal Muscle: Implications for Exercise-Induced Neuroplasticity.

Exercise promotes brain plasticity partly by stimulating increases in mature brain-derived neurotrophic factor (mBDNF), but the role of the pro-BDNF isoform in the regulation of BDNF metabolism in humans is unknown. We quantified the expression of pro-BDNF and mBDNF in human skeletal muscle and plasma at rest, after acute exercise (+/- lactate infusion), and after fasting. Pro-BDNF and mBDNF were analyzed with immunoblotting, enzyme-linked immunosorbent assay, immunohistochemistry, and quantitative polymerase chain reaction. Pro-BDNF was consistently and clearly detected in skeletal muscle (40-250 pg mg-1 dry muscle), whereas mBDNF was not. All methods showed a 4-fold greater pro-BDNF expression in type I muscle fibers compared to type II fibers. Exercise resulted in elevated plasma levels of mBDNF (55%) and pro-BDNF (20%), as well as muscle levels of pro-BDNF (∼10%, all P<0.05). Lactate infusion during exercise induced a significantly greater increase in plasma mBDNF (115%, P<0.05) compared to control (saline infusion), with no effect on pro-BDNF levels in plasma or muscle. A 3-day fast resulted in a small increase in plasma pro-BDNF (∼10%, P<0.05), with no effect on mBDNF. Pro-BDNF is highly expressed in human skeletal muscle, particularly in type I fibers, and is increased after exercise. While exercising with higher lactate augmented levels of plasma mBDNF, exercise-mediated increases in circulating mBDNF likely derive partly from release and cleavage of pro-BDNF from skeletal muscle, and partly from neural and other tissues. These findings have implications for preclinical and clinical work related to a wide range of neurological disorders such as Alzheimer's, clinical depression, and amyotrophic lateral sclerosis.

Antibodies against SARS-CoV-2 non-structural protein 3 cross-react with human muscle cells and neuroglial cells

Coronavirus Disease 2019 (COVID-19) vaccines protect the public and limit viral spread. However, inactivated viral vaccines use the whole virus particle, which contains many non-capsid proteins that may cause adverse immune responses. A report has found that the ADP-ribose-binding domains of SARS-CoV-2 non-structural protein 3 (NSP3) and human poly(ADP-ribose) polymerase family member 14 (PARP14) share a significant degree of homology. Here, we further show that antibodies against 2019 novel SARS-like coronavirus (SARS-CoV-2) NSP3 can bind human PARP14 protein. However, when G159R + G162R mutations were introduced into NSP3, the antibody titer against human PARP14 decreased 14-fold. Antibodies against SARS-CoV-2 NSP3 can cross-react with human skeletal muscle cells and astrocytes, but not human embryonic kidney 293T cells. However, when G159R + G162R mutations were introduced into NSP3, the cross-reaction was largely inhibited. The results imply that COVID-19 patients with high antibody titers against NSP3 may have high risks of muscular and/or neurological complications. And the possible strategies to improve the safety of inactivated viral vaccines are also discussed.

Functional hypoxia reduces mitochondrial calcium uptake

Mitochondrial respiration extends beyond ATP generation, with the organelle participating in many cellular and physiological processes. Parallel changes in components of the mitochondrial electron transfer system with respiration render it an appropriate hub for coordinating cellular adaption to changes in oxygen levels. How changes in respiration under functional hypoxia (i.e., when intracellular O2 levels limit mitochondrial respiration) are relayed by the electron transfer system to impact mitochondrial adaption and remodeling after hypoxic exposure remains poorly defined. This is largely due to challenges integrating findings under controlled and defined O2 levels in studies connecting functions of isolated mitochondria to humans during physical exercise. Here we present experiments under conditions of hypoxia in isolated mitochondria, myotubes and exercising humans. Performing steady-state respirometry with isolated mitochondria we found that oxygen limitation of respiration reduced electron flow and oxidative phosphorylation, lowered the mitochondrial membrane potential difference, and decreased mitochondrial calcium influx. Similarly, in myotubes under functional hypoxia mitochondrial calcium uptake decreased in response to sarcoplasmic reticulum calcium release for contraction. In both myotubes and human skeletal muscle this blunted mitochondrial adaptive responses and remodeling upon contractions. Our results suggest that by regulating calcium uptake the mitochondrial electron transfer system is a hub for coordinating cellular adaption under functional hypoxia.

Skeletal muscle TET3 promotes insulin resistance through destabilisation of PGC-1α

Aim/hypothesisThe peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α) plays a critical role in the maintenance of glucose, lipid and energy homeostasis by orchestrating metabolic programs in multiple tissues in response to environmental cues. In skeletal muscles, PGC-1α dysregulation has been associated with insulin resistance and type 2 diabetes but the underlying mechanisms have remained elusive. This research aims to understand the role of TET3, a member of the ten-eleven translocation (TET) family dioxygenases, in PGC-1α dysregulation in skeletal muscles in obesity and diabetes.MethodsTET expression levels in skeletal muscles were analysed in humans with or without type 2 diabetes, as well as in mouse models of high-fat diet (HFD)-induced or genetically induced (ob/ob) obesity/diabetes. Muscle-specific Tet3 knockout (mKD) mice were generated to study TET3’s role in muscle insulin sensitivity. Genome-wide expression profiling (RNA-seq) of muscle tissues from wild-type (WT) and mKD mice was performed to mine deeper insights into TET3-mediated regulation of muscle insulin sensitivity. The correlation between PGC-1α and TET3 expression levels was investigated using muscle tissues and in vitro-derived myotubes. PGC-1α phosphorylation and degradation were analysed using in vitro assays.ResultsTET3 expression was elevated in skeletal muscles of humans with type 2 diabetes and in HFD-fed and ob/ob mice compared with healthy controls. mKD mice exhibited enhanced glucose tolerance, insulin sensitivity and resilience to HFD-induced insulin resistance. Pathway analysis of RNA-seq identified ‘Mitochondrial Function’ and ‘PPARα Pathway’ to be among the top biological processes regulated by TET3. We observed higher PGC-1α levels (~25%) in muscles of mKD mice vs WT mice, and lower PGC-1α protein levels (~25–60%) in HFD-fed or ob/ob mice compared with their control counterparts. In human and murine myotubes, increased PGC-1α levels following TET3 knockdown contributed to improved mitochondrial respiration and insulin sensitivity. TET3 formed a complex with PGC-1α and interfered with its phosphorylation, leading to its destabilisation.Conclusions/interpretationOur results demonstrate an essential role for TET3 in the regulation of skeletal muscle insulin sensitivity and suggest that TET3 may be used as a potential therapeutic target for the metabolic syndrome.Data availabilitySequences are available from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with accession number of GSE224042.Graphical

Huntington's disease affects mitochondrial network dynamics predisposing to pathogenic mitochondrial DNA mutations.

Huntington's disease (HD) predominantly affects the brain, causing a mixed movement disorder, cognitive decline and behavioural abnormalities. It also causes a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been reported in tissues of HD models, including skeletal muscle, and lymphoblast and fibroblast cultures from patients with HD. Mutant huntingtin protein (mutHTT) expression can impair mitochondrial quality control and accelerate mitochondrial ageing. Here, we obtained fresh human skeletal muscle, a post-mitotic tissue expressing the mutated HTT allele at physiological levels since birth, and primary cell lines from HTT CAG repeat expansion mutation carriers and matched healthy volunteers to examine whether such a mitochondrial phenotype exists in human HD. Using ultra-deep mitochondrial DNA (mtDNA) sequencing, we showed an accumulation of mtDNA mutations affecting oxidative phosphorylation. Tissue proteomics indicated impairments in mtDNA maintenance with increased mitochondrial biogenesis of less efficient oxidative phosphorylation (lower complex I and IV activity). In full-length mutHTT expressing primary human cell lines, fission-inducing mitochondrial stress resulted in normal mitophagy. In contrast, expression of high levels of N-terminal mutHTT fragments promoted mitochondrial fission and resulted in slower, less dynamic mitophagy. Expression of high levels of mutHTT fragments due to somatic nuclear HTT CAG instability can thus affect mitochondrial network dynamics and mitophagy, leading to pathogenic mtDNA mutations. We show that life-long expression of mutant HTT causes a mitochondrial phenotype indicative of mtDNA instability in fresh post-mitotic human skeletal muscle. Thus, genomic instability may not be limited to nuclear DNA, where it results in somatic expansion of the HTT CAG repeat length in particularly vulnerable cells such as striatal neurons. In addition to efforts targeting the causative mutation, promoting mitochondrial health may be a complementary strategy in treating diseases with DNA instability such as HD.

Predicting myosin heavy chain isoform from postdissection fiber length in human skeletal muscle fibers.

Experimental techniques in single human skeletal muscle cells require manual dissection. Unlike other mammalian species, human skeletal muscle is characterized by a heterogeneous mixture of myosin heavy chain (MHC) isoforms, typically used to define "fiber type," which profoundly influences cellular function. Therefore, it is beneficial to predict MHC isoform at the time of dissection, facilitating a more balanced fiber-type distribution from a potentially imbalanced sample. Although researchers performing single fiber dissection report predicting fiber-type based on mechanical properties of fibers upon dissection, a rigorous examination of this approach has not been performed. Therefore, we measured normalized fiber length (expressed as a % of the length of the bundle from which the fiber was dissected) in single fibers immediately following dissection. Six hundred sixty-eight individual fibers were dissected from muscle tissue samples from healthy, young adults to assess whether this characteristic could differentiate fibers containing MHC I ("slow" fiber type) or not ("fast" fiber type). Using receiver operator characteristic (ROC) curves, we found that differences in normalized fiber length (114 ± 13%, MHC I; 124 ± 17%, MHC IIA, P < 0.01) could be used to predict fiber type with excellent reliability (area under the curve = 0.72). We extended these analyses to include older adults (2 females, 1 male) to demonstrate the durability of this approach in fibers with likely different morphology and mechanical characteristics. We report that MHC isoform expression in human skeletal muscle fibers can be predicted at the time of dissection, regardless of origin.NEW & NOTEWORTHY A priori estimation of myosin heavy chain (MHC) isoform in individual muscle fibers may bias the relative abundance of fiber types in subsequent assessment. Until now, no standardized assessment approach has been proposed to characterize fibers at the time of dissection. We demonstrate an approach based on normalized fiber length that may dramatically bias a sample toward slow twitch (MHC I) or fast twitch (not MHC I) fiber populations.

Oldies, but goldies-preserved morphology and stability of antigenic determinants in decades-old cryosections of human m. vastus lateralis.

Fibre typing by immunohistochemistry on cryosections from human skeletal muscle biopsies is an essential tool in the diagnosis and research of muscular diseases, ageing, and responses to exercise training and disuse. Preserving a good quality in these frozen specimens can be challenging especially if they are stored for longer periods before histological processing, which is often the case in studies with a large number of test subjects and/or repeated sampling separated by multiple years. We demonstrate in this article that both, the morphology and reactivity of epitopes to myosin heavy chain isoforms and dystrophin are well preserved in up to 18-year-stored unfixed and unstained cryosections of human m. vastus lateralis (n = 241). Any variation in staining intensity between samples was unrelated to the age of the biopsy donor or the storage period of the unstained cryosections, and in all cases, the obtained images were appropriate for image analysis, such as the determination of the fibre type composition and the fibre cross-sectional area, and quantitative analysis of muscle capillarisation.

Myopathy-causing mutation R91P in the TPM3 gene drastically impairs structural and functional properties of slow skeletal muscle tropomyosin γβ-heterodimer

Tropomyosin (Tpm) is a regulatory actin-binding protein involved in Ca2+ activation of contraction of striated muscle. In human slow skeletal muscles, two distinct Tpm isoforms, γ and β, are present. They interact to form three types of dimeric Tpm molecules: γγ-homodimers, γβ-heterodimers, or ββ-homodimers, and a majority of the molecules are present as γβ-Tpm heterodimers. Point mutation R91P within the TPM3 gene encoding γ-Tpm is linked to the condition known as congenital fiber-type disproportion (CFTD), which is characterized by severe muscle weakness. Here, we investigated the influence of the R91P mutation in the γ-chain on the properties of the γβ-Tpm heterodimer. We found that the R91P mutation impairs the functional properties of γβ-Tpm heterodimer more severely than those of earlier studied γγ-Tpm homodimer carrying this mutation in both γ-chains. Since a significant part of Tpm molecules in slow skeletal muscle is present as γβ-heterodimers, our results explain why this mutation leads to muscle weakness in CFTD.

Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice.

Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice.

The C5aR1 complement receptor: A novel immunomodulator of insulin action in skeletal muscle.

The complement system constitutes an integral component of the innate immune system and plays a critical role in adaptive immunity. Activation of this system engenders the production of complement peptide fragments, including C5a, which engage G-protein coupled receptors predominantly expressed in immune-associated cells, such as neutrophils, initiating pro-inflammatory responses. Intriguingly, our investigation has unveiled the presence of C5a receptor 1 (C5aR1) expression within skeletal muscle, a key metabolic tissue and primary target of insulin. Herein, we demonstrate that C5aR1 activation by C5a in differentiated human skeletal muscle cells elicits acute suppression of insulin signalling. This suppression manifests as impaired insulin-dependent association between IRS1 and the p85 subunit of PI3-kinase, a 50% reduction in Akt phosphorylation, and a 60% decline in insulin-stimulated glucose uptake. This impairment in insulin signalling is associated with a three-fold elevation in intramyocellular diacylglycerol (DAG) levels and a two-fold increase in cytosolic calcium content, which promote PKC-mediated IRS1 inhibition via enhanced phosphorylation at IRS1 Ser1101. Significantly, our findings demonstrate that structurally diverse C5aR1 antagonists, along with genetic deletion or stable silencing of C5aR1 by 80% using short-hairpin RNA, effectively attenuate repression of insulin signalling by C5a in LHCN-M2 human skeletal myotubes. These results underscore the potential of heightened C5aR1 activation, characteristic of obesity and chronic inflammatory conditions, to detrimentally impact insulin function within skeletal muscle cells. Additionally, the study suggests that agents targeting the C5a-C5aR axis, originally devised for mitigating complement-dependent inflammatory conditions, may offer therapeutic avenues to ameliorate immune-driven insulin resistance in key peripheral metabolic tissues, including skeletal muscle.

Mechanism of static loading injury in human skeletal muscle cells.

To establish a cellular-level mechanical injury model for human skeletal muscle cells and investigate changes in the mechanical effect mechanism after such injuries. The FX-5000™ Compression System was used to apply constant static mechanical pressure to human skeletal muscle cells. A factorial design analysis was conducted to discover the optimal injury model by evaluating the correlation between the amount of pressure, the duration of mechanical stimulation, and the number of days of observation. Skeletal muscle cell injury was evaluated by measuring cell metabolism, morphology, and calcium homeostasis. Mechanical injury was modeled as continuous pressure of 1 MPa for 2 hours with observation for 3 days. The results show that mechanical injury increased creatine kinase, intracellular Ca2+ concentration, and malondialdehyde content, decreased superoxide dismutase, and caused cell swelling and severe cytoplasmic vacuolization (all P < 0.05). This model of mechanically-injured human skeletal muscle cells provides an experimental model for the clinically common skeletal muscle injury caused by static loading pressure. It may be used to study the mechanism of action of treatment methods for mechanically injured skeletal muscle.

High-throughput Analysis of Capillary Density in Skeletal Muscle Cross Sections.

Capillary density in skeletal muscles is key to estimate exercise capacity in healthy individuals, athletes, and those with muscle-related pathologies. Here, we present a step-by-step, high-throughput semi-automated method for quantifying capillary density from whole human skeletal muscle cross-sections, in areas of the muscle occupied by myofibers. We provide a detailed protocol for immunofluorescence staining, image acquisition, processing, and quantification. Image processing is performed in ImageJ, and data analysis is conducted in R. The provided protocol allows high-throughput quantification of capillary density. Key features • This protocol builds upon the method and results described in Abbassi-Daloii et al. (2023b). • It includes step-by-step details on image acquisition and image processing of the entire muscle section. • It enables high-throughput and semi-automated image quantification of capillary density. • It provides a robust analysis for determining capillary density over the entire muscle cross section.

The impact of bed rest on human skeletal muscle metabolism

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

Sporadic inclusion body myositis-derived myotube culture revealed muscle cell-autonomous expression profiles.

Sporadic inclusion body myositis (sIBM) is a muscle disease in older people and is characterized by inflammatory cell invasion into intact muscle fibers and rimmed vacuoles. The pathomechanism of sIBM is not fully elucidated yet, and controversy exists as to whether sIBM is a primary autoimmune disease or a degenerative muscle disease with secondary inflammation. Previously, we established a method of collecting CD56-positive myoblasts from human skeletal muscle biopsy samples. We hypothesized that the myoblasts derived from these patients are useful to see the cell-autonomous pathomechanism of sIBM. With these resources, myoblasts were differentiated into myotubes, and the expression profiles of cell-autonomous pathology of sIBM were analyzed. Myoblasts from three sIBM cases and six controls were differentiated into myotubes. In the RNA-sequencing analysis of these "myotube" samples, 104 differentially expressed genes (DEGs) were found to be significantly upregulated by more than twofold in sIBM, and 13 DEGs were downregulated by less than twofold. For muscle biopsy samples, a comparative analysis was conducted to determine the extent to which "biopsy" and "myotube" samples differed. Fifty-three DEGs were extracted of which 32 (60%) had opposite directions of expression change (e.g., increased in biopsy vs decreased in myotube). Apolipoprotein E (apoE) and transmembrane protein 8C (TMEM8C or MYMK) were commonly upregulated in muscle biopsies and myotubes from sIBM. ApoE and myogenin protein levels were upregulated in sIBM. Given that enrichment analysis also captured changes in muscle contraction and development, the triggering of muscle atrophy signaling and abnormal muscle differentiation via MYMK or myogenin may be involved in the pathogenesis of sIBM. The presence of DEGs in sIBM suggests that the myotubes formed from sIBM-derived myoblasts revealed the existence of muscle cell-autonomous degeneration in sIBM. The catalog of DEGs will be an important resource for future studies on the pathogenesis of sIBM focusing on primary muscle degeneration.

Cellular activation patterns of CD10+ fibro-adipogenic progenitors across acquired disease states in human skeletal muscle biopsies.

Background: Fibro-adipogenic progenitors (FAP) are muscle resident mesenchymal stem cells pivotal for regulation of myofiber repair. Experimental results show in addition involvement in a range of other pathological conditions and potential for pharmacological intervention. FAP histopathology in human muscle biopsies is largely unknown, but has potential to inform translational research. Methods: CD10+ FAPs in 32 archival muscle biopsies from 8 groups (normal, dermatomyositis, inclusion body myositis (IBM), anti-synthetase syndrome, immune-mediated necrotizing myopathy (IMNM), denervation, type 2 atrophy, rhabdomyolysis) were visualized by CD10 immunohistochemistry and their histology compared. Groups are compared by semi-quantitative scoring. Results: Histological activation of endomysial CD10+ FAPs includes prominent expansion of a network of cell processes surrounding muscle fibers, as well as endomysial cell clusters evidencing proliferation. Prominence of periarteriolar processes is a notable feature in some pathologies. FAP activation is often associated with fiber degeneration/regeneration, foci of inflammation, and denervation in keeping with experimental results. Type 2 atrophy shows no evidence of FAP activation. Dermatomyositis and anti-synthetase syndrome associated myositis demonstrate diffuse activation. Conclusion: Assessment of CD10+ FAP activation is routinely possible using CD10 immunohistochemistry and demonstrates several patterns in keeping with preclinical results. Prominent expansion of FAP processes surrounding myofibers suggests enhanced interaction between myofiber/basement membranes and FAPs during activation. The presence of diffuse FAP activation in dermatomyositis biopsies unrelated to fiber repair raises the possibility of FAP activation as part of the autoimmune process. Future diagnostic applications, clinical significance and therapeutic potential remain to be elucidated.

Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle.

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.