Myopathy as a cause of fatigue in long-term post-COVID-19 symptoms: Evidence of skeletal muscle histopathology.

BACKGROUND AND PURPOSE: Among post-COVID-19 symptoms, fatigue is reported as one of the most common, even after mild acute infection, and as the cause of fatigue, myopathy diagnosed by electromyography has been proposed in previous reports. This study aimed to explore the histopathological changes in patients with post-COVID-19 fatigue. METHODS: Sixteen patients (mean age = 46 years) with post-COVID-19 complaints of fatigue, myalgia, or weakness persisting for up to 14 months were included. In all patients, quantitative electromyography and muscle biopsies analyzed with light and electron microscopy were taken. RESULTS: Muscle weakness was present in 50% and myopathic electromyography in 75%, and in all patients there were histological changes. Muscle fiber atrophy was found in 38%, and 56% showed indications of fiber regeneration. Mitochondrial changes, comprising loss of cytochrome c oxidase activity, subsarcollemmal accumulation, and/or abnormal cristae, were present in 62%. Inflammation was found in 62%, seen as T lymphocytes and/or muscle fiber human leukocyte antigen ABC expression. In 75%, capillaries were affected, involving basal lamina and cells. In two patients, uncommon amounts of basal lamina were found, not only surrounding muscle fibers but also around nerves and capillaries. CONCLUSIONS: The wide variety of histological changes in this study suggests that skeletal muscles may be a major target of SARS-CoV-2, causing muscular post-COVID-19 symptoms. The mitochondrial changes, inflammation, and capillary injury in muscle biopsies can cause fatigue in part due to reduced energy supply. Because most patients had mild-moderate acute affection, the new variants that might cause less severe acute disease could still have the ability to cause long-term myopathy.

Choline transporter-like 1 deficiency causes a new type of childhood-onset neurodegeneration.

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.

Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

SPARC Interacts with Actin in Skeletal Muscle in Vitro and in Vivo.

The cytoskeleton is an integral part of skeletal muscle structure, and reorganization of the cytoskeleton occurs during various modes of remodeling. We previously found that the extracellular matrix protein secreted protein acidic and rich in cysteine (SPARC) is up-regulated and expressed intracellularly in developing muscle, during regeneration and in myopathies, which together suggests that SPARC might serve a specific role within muscle cells. Using co-immunoprecipitation combined with mass spectrometry and verified by staining for direct protein-protein interaction, we find that SPARC binds to actin. This interaction is present in regenerating myofibers of patients with Duchenne muscular dystrophy, polymyositis, and compartment syndrome. Analysis of the α-, ß-, and γ-actin isoforms in SPARC knockout myoblasts reveals a changed expression pattern with dominance of γ-actin. In SPARC knockout mice, we performed an injury study to investigate whether lack of SPARC would compromise the ability to repair muscle. We report that these mice develop normal skeletal muscle with retained ability to regenerate. However, when we subject muscle from SPARC-deficient mice to an in vitro fatigue stimulation protocol, we find a defective force recovery. Therefore, SPARC appears to be an important modulator of the actin cytoskeleton, implicating maintenance of muscular function. This direct interaction with actin suggests a new role of SPARC during tissue remodeling.

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle.

BACKGROUND: Transplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT. METHOD: Primary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury. RESULTS: Li-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1. CONCLUSION: Treatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro.

Quality of life in patients with myositis is associated with functional capacity, body composition, and disease activity-Baseline data from a randomized controlled trial.

OBJECTIVE: To investigate the potential associations between functional capacity, muscle strength, body composition, and disease-related measures and quality of life in patients with myositis. METHODS: Baseline measures of functional capacity (functional index 3 (FI3), 2-minute walk test (2MWT), timed up and go (TUG) and 30-s sit-to-stand (30-STS)), muscle strength (incl. leg and handgrip strength), maximal leg extensor power, body composition (appendicular lean mass, fat percentage/mass) and disease-related measures (disease activity & damage core sets) were examined for their associations with quality of life (physical- and mental component summary scores, Short Form 36 questionnaire (SF-36)) by means of Spearman's correlation analysis. RESULTS: A total of 32 patients with myositis were included. Positive correlations between SF-36 physical component summary score (PCS) and FI3, 30-STS, TUG, 2MWT, leg extensor power, leg strength, bench press strength, and handgrip strength were observed. In contrast, fat percentage and fat mass correlated negatively with PCS. In disease-related measures, Extramuscular global assessment, health assessment questionnaire, physician global damage, and patient global damage scores were negatively associated with SF-36 PCS. No correlations to the mental component summary score of SF-36 were observed. CONCLUSION: All measures of functional capacity were positively related to the SF-36 physical component summary score, indicating higher functional capacity positively affects quality of life in patients with myositis. Health assessment questionnaire and patient global damage scores demonstrated the strongest correlations with SF-36 physical component summary scores, further supporting these patient-reported outcomes as viable monitoring tools in patients with myositis.

Prognostic value of Musashi-1 in gliomas.

The aim of this study was to investigate the prognostic value of the RNA-binding protein Musashi-1 in adult patients with primary gliomas. Musashi-1 has been suggested to be a cancer stem cell-related marker in gliomas, and high levels of Musashi-1 have been associated with high tumor grades and hence poor prognosis. Samples of 241 gliomas diagnosed between 2005 and 2009 were stained with an anti-Musashi-1 antibody using a fluorescent staining protocol followed by automated image acquisition and processing. Musashi-1 area fraction and intensity in cytoplasm and in nuclei were quantified by systematic random sampling in 2 % of the vital tumor area. In WHO grade III tumors high levels of Musashi-1 were associated with poor survival in multivariate analysis (HR 3.39, p = 0.02). We identified a sub-population of glioblastoma (GBM) patients with high levels of Musashi-1 and a superior prognosis (HR 0.65, p = 0.038). In addition patients with high levels of Musashi-1 benefitted most from post-surgical treatment, indicating that Musashi-1 may be a predictive marker in GBMs. In conclusion, our results suggest that high levels of Musashi-1 are associated with poor survival in patients with WHO grade III tumors and that Musashi-1 may be a predictive marker in GBMs, although further validation is needed. We find the combination of immunofluorescence and automated quantitation to be a feasible, robust, and reproducible approach for quantitative biomarker studies.

Myopathy as a cause of Long COVID fatigue: Evidence from quantitative and single fiber EMG and muscle histopathology.

OBJECTIVE: To describe neurophysiological abnormalities in Long COVID and correlate quantitative electromyography (qEMG) and single fiber EMG (sfEMG) results to clinical scores and histopathology. METHODS: 84 patients with non-improving musculoskeletal Long COVID symptoms were examined with qEMG and sfEMG. Muscle biopsies were taken in a subgroup. RESULTS: Mean motor unit potential (MUP) duration was decreased in ≥ 1 muscles in 52 % of the patients. Mean jitter was increased in 17 % of the patients in tibialis anterior and 25 % in extensor digitorum communis. Increased jitter was seen with or without myopathic qEMG. Low quality of life score correlated with higher jitter values but not with qEMG measures. In addition to our previously published mitochondrial changes, inflammation, and capillary injury, we show now in muscle biopsies damage of terminal nerves and motor endplate with abundant basal lamina material. At the endplate, axons were present but no vesicle containing terminals. The post-synaptic cleft in areas appeared atrophic with short clefts and coarse crests. CONCLUSIONS: Myopathic changes are common in Long COVID. sfEMG abnormality is less common but may correlate with clinical scores. sfEMG changes may be due to motor endplate pathology. SIGNIFICANCE: These findings may indicate a muscle pathophysiology behind fatigue in Long COVID.

Long-term blocking of calcium channels in mdx mice results in differential effects on heart and skeletal muscle.

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD). Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.

Endoscopic Injections of Botulinum Toxin Type A in the Piglet Esophagus Is Safe and Feasible but Did Not Result in any Significant Structural Changes 3 Days after Injection.

INTRODUCTION: Treatment for long-gap esophageal atresia (LGEA) aims at achieving primary anastomosis with minimal tension. Previous studies have shown that intramural injections with botulinum toxin type-A (BTX-A) from the adventitial side can increase the elongation of the piglet and rat esophagus before bursting, and that this effect is dose and time dependent. Our aim was to determine if endoscopic injections would be feasible, safe, and with an effect on the mechanical properties of the esophagus. METHODS: Twenty-two male piglets (5.15 kg) were randomized into two groups, one receiving 2 units/kg BTX-A, the other equal volume 0.9% NaCl. On day 3, the esophagus was harvested and tested in a stretch-tension machine to evaluate elongation and maximum load, followed by histological examination. RESULTS: No adverse effects to the procedure were observed. No statistically significant difference in elongation or maximum load before bursting between the treatment and placebo group was found. In histopathological analysis, inflammation and abscess formation were observed with no statistically significant difference between the two groups. CONCLUSION: Endoscopic placement of BTX-A injections in the piglet esophagus was safe and feasible but did not result in any difference in the mechanical properties or histology of the esophagus.

Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type.

Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, VO2 max = 68 ± 5 ml kg-1 min-1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. .

Oxaliplatin Neuropathy: Predictive Values of Skin Biopsy, QST and Nerve Conduction.

BACKGROUND: Oxaliplatin-induced peripheral neuropathy negatively affects the quality of life for patients with gastrointestinal cancers and may cause neuropathic pain. Measures of peripheral nerve structure or function, such as intraepidermal nerve fiber density (IENFD) during treatment could reduce neuropathy severity through individualized dose reduction. OBJECTIVE: The aim was to evaluate the predictive values of IENFD, quantitative sensory testing (QST), and nerve conduction studies (NCS) for significant neuropathy and neuropathic pain. METHODS: Fifty-five patients were examined prospectively before, during, and six months following treatment using skin biopsies, QST and NCS. Clinically significant neuropathy six months after treatment was defined as reduced Total Neuropathy Score of more than five and neuropathic pain was assessed according to International Association for the Study of Pain criteria. RESULTS: Thirty patients had a clinically significant neuropathy, and 14 had neuropathic pain. Vibration detection threshold (VDT) before treatment was correlated with clinically significant neuropathy six months after treatment (OR 0.54, p = 0.01) and reductions in cold detection threshold (CDT) after 25% of treatment (OR 1.38, p = 0.04) and heat pain threshold (HPT) after 50% of treatment (OR 1.91, p = 0.03) with neuropathic pain. Cut off values of 5 for baseline VDT and changes of more than -0.05 °C and -0.85 °C in CDT and HPT were estimated. Sensitivity and specificity was low to moderate. There was no correlation between changes in IENFD or NCS and significant neuropathy or neuropathic pain. CONCLUSIONS: Vibration detection thresholds and thermal detection thresholds may be useful for prediction of clinically significant and painful neuropathy, respectively. However, low to moderate sensitivity and specificity may limit the predictive value in clinical practice.

Subcellular localization-dependent decrements in skeletal muscle glycogen and mitochondria content following short-term disuse in young and old men.

Previous studies have shown that skeletal muscle glycogen and mitochondria are distributed in distinct subcellular localizations, but the role and regulation of these subcellular localizations are unclear. In the present study, we used transmission electron microscopy to investigate the effect of disuse and aging on human skeletal muscle glycogen and mitochondria content in subsarcolemmal (SS), intermyofibrillar (IMF), and intramyofibrillar (intra) localizations. Five young (∼23 yr) and five old (∼66 yr) recreationally active men had their quadriceps muscle immobilized for 2 wk by whole leg casting. Biopsies were obtained from m. vastus lateralis before and after the immobilization period. Immobilization induced a decrement of intra glycogen content by 54% (P < 0.001) in both age groups and in two ultrastructurally distinct fiber types, whereas the content of IMF and SS glycogen remained unchanged. A localization-dependent decrease (P = 0.03) in mitochondria content following immobilization was found in both age groups, where SS mitochondria decreased by 33% (P = 0.02), superficial IMF mitochondria decreased by 20% (P = 0.05), and central IMF mitochondria remained unchanged. In conclusion, our findings demonstrate a localization-dependent adaptation to immobilization in glycogen and mitochondria content of skeletal muscles of both young and old individuals. Specifically, this suggests that short-term disuse preferentially affects glycogen particles located inside the myofibrils and that mitochondria volume plasticity can be dependent on the distance to the fiber border.

Increased subsarcolemmal lipids in type 2 diabetes: effect of training on localization of lipids, mitochondria, and glycogen in sedentary human skeletal muscle.

The purpose of the study was to investigate the effect of aerobic training and type 2 diabetes on intramyocellular localization of lipids, mitochondria, and glycogen. Obese type 2 diabetic patients (n = 12) and matched obese controls (n = 12) participated in aerobic cycling training for 10 wk. Endurance-trained athletes (n = 15) were included for comparison. Insulin action was determined by euglycemic-hyperinsulinemic clamp. Intramyocellular contents of lipids, mitochondria, and glycogen at different subcellular compartments were assessed by transmission electron microscopy in biopsies obtained from vastus lateralis muscle. Type 2 diabetic patients were more insulin resistant than obese controls and had threefold higher volume of subsarcolemmal (SS) lipids compared with obese controls and endurance-trained subjects. No difference was found in intermyofibrillar lipids. Importantly, following aerobic training, this excess SS lipid volume was lowered by approximately 50%, approaching the levels observed in the nondiabetic subjects. A strong inverse association between insulin sensitivity and SS lipid volume was found (r(2)=0.62, P = 0.002). The volume density and localization of mitochondria and glycogen were the same in type 2 diabetic patients and control subjects, and showed in parallel with improved insulin sensitivity a similar increase in response to training, however, with a more pronounced increase in SS mitochondria and SS glycogen than in other localizations. In conclusion, this study, estimating intramyocellular localization of lipids, mitochondria, and glycogen, indicates that type 2 diabetic patients may be exposed to increased levels of SS lipids. Thus consideration of cell compartmentation may advance the understanding of the role of lipids in muscle function and type 2 diabetes.

Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke.

Preclinical and clinical proof-of-concept studies have suggested the effectiveness of pharmacological modulation of inflammatory cytokines in ischemic stroke. Experimental evidence shows that targeting tumor necrosis factor (TNF) and interleukin (IL)-1 holds promise, and these cytokines are considered prime targets in the development of new stroke therapies. So far, however, information on the cellular expression of TNF and IL-1 in the human ischemic brain is sparse.We studied 14 cases of human post-mortem ischemic stroke, representing 21 specimens of infarcts aged 1 to > 8 days. We characterized glial and leukocyte reactions in the infarct/peri-infarct (I/PI) and normal-appearing tissue (NAT) and the cellular location of TNF, TNF receptor (TNFR)1 and TNFR2, IL-1α, IL-1ß, and IL-1 receptor antagonist (IL-1Ra). The immunohistochemically stained tissue sections received a score reflecting the number of immunoreactive cells and the intensity of the immunoreactivity (IR) in individual cells where 0 = no immunoreactive cells, 1 = many intermediately to strongly immunoreactive cells, and 2 = numerous and intensively immunoreactive cells. Additionally, we measured blood TNF, TNFR, and IL-1 levels in surviving ischemic stroke patients within the first 8 h and again at 72 h after symptom onset and compared levels to healthy controls.We observed IL-1α and IL-1ß IR in neurons, glia, and macrophages in all specimens. IL-1Ra IR was found in glia, in addition to macrophages. TNF IR was initially found in neurons located in I/PI and NAT but increased in glia in older infarcts. TNF IR increased in macrophages in all specimens. TNFR1 IR was found in neurons and glia and macrophages, while TNFR2 was expressed only by glia in I/PI and NAT, and by macrophages in I/PI. Our results suggest that TNF and IL-1 are expressed by subsets of cells and that TNFR2 is expressed in areas with increased astrocytic reactivity. In ischemic stroke patients, we demonstrate that plasma TNFR1 and TNFR2 levels increased in the acute phase after symptom onset compared to healthy controls, whereas TNF, IL-1α, IL-1ß, and IL-1Ra did not change.Our findings of increased brain cytokines and plasma TNFR1 and TNFR2 support the hypothesis that targeting post-stroke inflammation could be a promising add-on therapy in ischemic stroke patients.

Secreted protein acidic and rich in cysteine (SPARC) in human skeletal muscle.

Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15-16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment.

Lipid droplets interact with mitochondria using SNAP23.

Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.

Non-cultured adipose-derived CD45- side population cells are enriched for progenitors that give rise to myofibres in vivo.

Side population (SP) cells are highly able to exclude the Hoechst 33342 dye through membrane transporters, a feature associated with cell immaturity and therefore proposed as a marker of stem cells. Herein we demonstrate that the adipose tissue derived stromal vascular fraction (SVF) contains a novel population of non-haematopoietic "side population" (SPCD45(-)) cells. Simultaneous qRT-PCR of 64 genes revealed that the freshly isolated SPCD45(-) was highly enriched for cells expressing genes related to stem cells, the Notch pathway, and early vascular precursors. Notably, the expression of smooth muscle actin, C-met and Cd34 together with Angpt2, Flk1, VE-cadherin, and Cd31 suggested a phenotypic resemblance to pericytes and aorta-derived mesoangioblasts. Recent evidence suggests that cells residing within the vascular niche may participate in regeneration of skeletal muscle and although skeletal muscle repair mainly relies on the satellite cell, several reports have shown that vessel-associated cells may adopt a myogenic phenotype when exposed to a muscle environment. In accordance with these findings, we also observed in vitro myogenic specification of SPCD45(-) cells when cocultured with myoblasts. Furthermore, immediate intramuscular engraftment of non-cultured SPCD45(-) cells gave rise to myofibres and cells lining blood vessels, whereas the SVF only provided donor derived mononuclear cells. We therefore conclude that the SPCD45(-) fraction of adipose-derived SVF is enriched for cells expressing vascular associated markers and that the myogenic differentiation potential of these cells does not depend on prior in vitro expansion.

Marker Expression of Interstitial Cells in Human Skeletal Muscle: An Immunohistochemical Study.

There is a growing recognition that myogenic stem cells are influenced by their microenvironment during regeneration. Several interstitial cell types have been described as supportive for myoblasts. In this role, both the pericyte as a possible progenitor for mesenchymal stem cells, and interstitial cells in the endomysium have been discussed. We have applied immunohistochemistry on normal and pathological human skeletal muscle using markers for pericytes, or progenitor cells and found a cell type co-expressing CD10, CD34, CD271, and platelet-derived growth factor receptor α omnipresent in the endomysium. The marker profile of these cells changed dynamically in response to muscle damage and atrophy, and they proliferated in response to damage. The cytology and expression profile of the CD10+ cells indicated a capacity to participate in myogenesis. Both morphology and indicated function of these cells matched properties of several previously described interstitial cell types. Our study suggests a limited number of cell types that could embrace many of these described cell types. Our study indicate that the CD10+, CD34+, CD271+, and platelet-derived growth factor receptor α+ cells could have a supportive role in human muscle regeneration, and thus the mechanisms by which they exert their influence could be implemented in stem cell therapy.