Muscle Cultures Research Articles

Factors affecting the structure and maturation of human tissue engineered skeletal muscle

Tissue engineered skeletal muscle has great utility in experimental studies of physiology, clinical testing and its potential for transplantation to replace damaged tissue. Despite recent work in rodent tissue or cell lines, there is a paucity of literature concerned with the culture of human muscle derived cells (MDCs) in engineered constructs. Here we aimed to tissue engineer for the first time in the literature human skeletal muscle in self-assembling fibrin hydrogels and determine the effect of MDC seeding density and myogenic proportion on the structure and maturation of the constructs. Constructs seeded with 4 × 105 MDCs assembled to a greater extent than those at 1 × 105 or 2 × 105, and immunostaining revealed a higher fusion index and a higher density of myotubes within the constructs, showing greater structural semblance to in vivo tissue. These constructs primarily expressed perinatal and slow type I myosin heavy chain mRNA after 21 days in culture. In subsequent experiments MACS® technology was used to separate myogenic and non-myogenic cells from their heterogeneous parent population and these cells were seeded at varying myogenic (desmin +) proportions in fibrin based constructs. Only in the constructs seeded with 75% desmin + cells was there evidence of striations when immunostained for slow myosin heavy chain compared with constructs seeded with 10 or 50% desmin + cells. Overall, this work reveals the importance of cell number and myogenic proportions in tissue engineering human skeletal muscle with structural resemblance to in vivo tissue.

Obstacles for Changes within the (Swedish) Police Force: Professional Motivations, Homosociality, and Ordering Practices

This article discusses how professional motivations and homosociality within the Swedish police (training programme) become obstacles to both a changed police norm and diversity within the police force. This issue is discussed on the basis of two studies: one focused on the professional motivations of police students; the other focused on the issue of norms and normation within the police training programme. The norm regarding how a police officer should be is viewed as a manifestation of ordering practices as a form of continuous, on-going normation processes that emphasize practical, physically demanding, and violent working conditions focused on combating crime. This produces powerful conceptions of the types of body that are suitable for the profession, a normative (male) body. This normative body effects those bodies that are assumed to contribute to diversity in a police force dominated by white, Swedish, heterosexual males. This together moulds a culture founded on the conceptions of physically demanding and action-focused work that promotes a muscle culture that emphasizes the work as practical, rather than moving towards an intellectual and reflective approach to police work.

162 Potentiation of Descending Inhibition to Colonic Circular Smooth Muscle of Mice and Humans by Chronic Stress and Stress Hormones: A Mechanism for Stress-Induced Diarrhea?

Background: A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While they may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo system. Methods: Using organotypic cultures of longitudinal muscle and myenteric plexus (LMMP) from Wnt1-cre;tdTomato (tdT) we performed EdU uptake assays at different time points and EdU incubations. To evaluate stem cell properties, LMMP treated with EdU was digested and flow cytometry performed with antibodies to nestin, CD49b, p75, and GFAP. We also performed a clonogenic sphereforming assay in which single cells were seeded into microwells to assess sphere forming capacity and retention of EdU signal. To evaluate for neurogenesis, LMMP treated with EdU was analyzed by flow cytometry and immunofluorescence with antibodies to neuronal and glial markers including Tuj, PGP9.5, nNOS, VIP, GFAP, and S100B. Protein expression for PTEN, pPTEN, Akt, and pAkt was analyzed by western blot and RT-PCR. Finally, organotypic cultures were treated with inhibitors BpV(phen) and LY294002, and EdU uptake assays were performed. Results: Culturing LMMP with EdU for 48h resulted in EdU uptake in 78% of neural crest cells (tdT+), indicating that the enteric nervous system has tremendous replicative capacity. Administering pulses of EdU at different time points, we found that proliferation occurred after an initial lag phase that lasted slightly over 24h. While undetectable at baseline and 24h, EdU uptake reached nearly 45% of tdT+ cells by 72h (p ,.001). Flow cytometry revealed that .90% of EdU+ neural crest cells expressed enteric neural stem cell markers nestin, CD49b, p75 and GFAP. Tracking single cells we found that EdU+ cells are capable of forming secondary NLBs suggestive of stem cell-like properties. Under differentiating conditions (withdrawal of growth factors), EdU uptake was detected in cells expressing Tuj, PGP9.5, nNOS, VIP, GFAP and S100B. This was supported with flow cytometry where .50% of cells positive for neuronal markers Tuj or PGP9.5 demonstrated EdU uptake (also seen for glial markers GFAP and S100B). Proliferation appears to be dependent on the PTEN/PI3K/Akt pathway with inhibition of PTEN (50% decrease in PTEN expression and 80-fold increase in pPTEN:PTEN (inactivation)) corresponding to activation of Akt (4-fold increase in pAkt:Akt) by 24h. Treatment of LMMP with PI3K inhibitor, LY294002, caused over 95% reduction (p,.01) and PTEN inhibitor, BpV(phen), caused 3fold increase (p,.001) in EdU uptake. Conclusion: This data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.

161 Mirtazapine Improves Early Satiation, Nutrient Intake, Weight Recovery and Quality of Life in Functional Dyspepsia With Weight Loss: A Double-Blind, Randomized, Placebo-Controlled Pilot Study

Background: A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While they may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo system. Methods: Using organotypic cultures of longitudinal muscle and myenteric plexus (LMMP) from Wnt1-cre;tdTomato (tdT) we performed EdU uptake assays at different time points and EdU incubations. To evaluate stem cell properties, LMMP treated with EdU was digested and flow cytometry performed with antibodies to nestin, CD49b, p75, and GFAP. We also performed a clonogenic sphereforming assay in which single cells were seeded into microwells to assess sphere forming capacity and retention of EdU signal. To evaluate for neurogenesis, LMMP treated with EdU was analyzed by flow cytometry and immunofluorescence with antibodies to neuronal and glial markers including Tuj, PGP9.5, nNOS, VIP, GFAP, and S100B. Protein expression for PTEN, pPTEN, Akt, and pAkt was analyzed by western blot and RT-PCR. Finally, organotypic cultures were treated with inhibitors BpV(phen) and LY294002, and EdU uptake assays were performed. Results: Culturing LMMP with EdU for 48h resulted in EdU uptake in 78% of neural crest cells (tdT+), indicating that the enteric nervous system has tremendous replicative capacity. Administering pulses of EdU at different time points, we found that proliferation occurred after an initial lag phase that lasted slightly over 24h. While undetectable at baseline and 24h, EdU uptake reached nearly 45% of tdT+ cells by 72h (p ,.001). Flow cytometry revealed that .90% of EdU+ neural crest cells expressed enteric neural stem cell markers nestin, CD49b, p75 and GFAP. Tracking single cells we found that EdU+ cells are capable of forming secondary NLBs suggestive of stem cell-like properties. Under differentiating conditions (withdrawal of growth factors), EdU uptake was detected in cells expressing Tuj, PGP9.5, nNOS, VIP, GFAP and S100B. This was supported with flow cytometry where .50% of cells positive for neuronal markers Tuj or PGP9.5 demonstrated EdU uptake (also seen for glial markers GFAP and S100B). Proliferation appears to be dependent on the PTEN/PI3K/Akt pathway with inhibition of PTEN (50% decrease in PTEN expression and 80-fold increase in pPTEN:PTEN (inactivation)) corresponding to activation of Akt (4-fold increase in pAkt:Akt) by 24h. Treatment of LMMP with PI3K inhibitor, LY294002, caused over 95% reduction (p,.01) and PTEN inhibitor, BpV(phen), caused 3fold increase (p,.001) in EdU uptake. Conclusion: This data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.

Welander distal myopathy is caused by a mutation in the RNA‐binding protein TIA1

A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy. The genetic linkage was confirmed and defined by microsatellite and single nucleotide polymorphism haplotyping. The whole linked genomic region was sequenced with targeted high-throughput and Sanger sequencing, and coding transcripts were sequenced on the cDNA level. WDM muscle biopsies were studied by Western blotting and immunofluorescence microscopy. Splicing of TIA1 and its target genes in muscle and myoblast cultures was analyzed by reverse transcriptase polymerase chain reaction. Mutant TIA1 was characterized by cell biological studies on HeLa cells, including quantification of stress granules by high content analysis and fluorescence recovery after photobleaching (FRAP) experiments. The linked haplotype at 2p13 was narrowed down to <806 kb. Sequencing by multiple methods revealed only 1 segregating coding mutation, c.1362 G>A (p.E384K) in the RNA-binding protein TIA1, a key component of stress granules. Immunofluorescence microscopy of WDM biopsies showed a focal increase of TIA1 in atrophic and vacuolated fibers. In HeLa cells, mutant TIA1 constructs caused a mild increase in stress granule abundance compared to wild type, and showed slower average fluorescence recovery in FRAP. WDM is caused by mutated TIA1 through a dominant pathomechanism probably involving altered stress granule dynamics.

A relationship exists between replicative senescence and cardiovascular health

A growing body of evidence demonstrates that the accumulation of senescent cells is a plausible ageing mechanism. It has been proposed that the senescence of vascular cells plays a causal role in the development of cardiovascular pathologies. A key prediction arising from this hypothesis is that cultures of cells derived from donors with cardiovascular disease will show reduced in vitro replicative capacities compared to those derived from disease-free controls. Accordingly, we carried out a formal review of the relationship among donor age, cardiovascular health status and maximum population doubling level attained in vitro by cultures of vascular smooth muscle and endothelial cells. Data were available to us on a total of 202 independent cell cultures. An inverse relationship was found to exist between replicative capacity and donor age in both endothelial and vascular smooth muscle cells. Cultures derived from donors with cardiovascular disease showed a lower overall replicative potential than age-matched healthy controls. In general the replicative potential at the start of the lifespan was found to be higher in those individuals without disease than those with disease and the difference in average cumulative population doublings (CPDs) in age-matched individuals in the two groups remained roughly constant throughout the lifetime. These results are consistent with the model in which the inherited replicative capacity of vascular cells is a stronger determinant of the onset of cardiovascular disease later in life, than wear-and-tear throughout the life course.

MT-ND5Mutation Causing Exercise Intolerance Displays Intercellular Heteroplasmy and Rapid Shifts Between Generations

We studied the inheritance and cellular segregation of a maternally inherited, heteroplasmic MT-ND5 mutation, m.13271T>C, previously shown to cause only exercise intolerance despite being present in multiple tissues. The mutation was present at low levels in early passage, bulk muscle culture, but on subcloning, only homoplasmic clones were found. Studies of transmission showed that the mutation expanded from very low levels in the patient's mother to higher levels in the patient, particularly skeletal muscle, but was not found in the placenta and umbilical cord blood of her child. Our study suggests that the m.13271T>C is either already strictly segregated (intercellular heteroplasmy), or moves rapidly to this state in cultured cells. Transmission studies suggest that intercellular heteroplasmy may also be present in the patient's germline. Although rapid shifts in heteroplasmic mitochondrial DNA mutations reflect a bottleneck in the female germline, complete segregation will accentuate the effects of this and further complicate genetic counseling.

Unique acyl-carnitine profiles are potential biomarkers for acquired mitochondrial disease in autism spectrum disorder

Autism spectrum disorder (ASD) has been associated with mitochondrial disease (MD). Interestingly, most individuals with ASD and MD do not have a specific genetic mutation to explain the MD, raising the possibility of that MD may be acquired, at least in a subgroup of children with ASD. Acquired MD has been demonstrated in a rodent ASD model in which propionic acid (PPA), an enteric bacterial fermentation product of ASD-associated gut bacteria, is infused intracerebroventricularly. This animal model shows validity as it demonstrates many behavioral, metabolic, neuropathologic and neurophysiologic abnormalities associated with ASD. This animal model also demonstrates a unique pattern of elevations in short-chain and long-chain acyl-carnitines suggesting abnormalities in fatty-acid metabolism. To determine if the same pattern of biomarkers of abnormal fatty-acid metabolism are present in children with ASD, the laboratory results from a large cohort of children with ASD (n=213) who underwent screening for metabolic disorders, including mitochondrial and fatty-acid oxidation disorders, in a medically based autism clinic were reviewed. Acyl-carnitine panels were determined to be abnormal if three or more individual acyl-carnitine species were abnormal in the panel and these abnormalities were verified by repeated testing. Overall, 17% of individuals with ASD demonstrated consistently abnormal acyl-carnitine panels. Next, it was determined if specific acyl-carnitine species were consistently elevated across the individuals with consistently abnormal acyl-carnitine panels. Significant elevations in short-chain and long-chain, but not medium-chain, acyl-carnitines were found in the ASD individuals with consistently abnormal acyl-carnitine panels—a pattern consistent with the PPA rodent ASD model. Examination of electron transport chain function in muscle and fibroblast culture, histological and electron microscopy examination of muscle and other biomarkers of mitochondrial metabolism revealed a pattern consistent with the notion that PPA could be interfering with mitochondrial metabolism at the level of the tricarboxylic-acid cycle (TCAC). The function of the fatty-acid oxidation pathway in fibroblast cultures and biomarkers for abnormalities in non-mitochondrial fatty-acid metabolism were not consistently abnormal across the subgroup of ASD children, consistent with the notion that the abnormalities in fatty-acid metabolism found in this subgroup of children with ASD were secondary to TCAC abnormalities. Glutathione metabolism was abnormal in the subset of ASD individuals with consistent acyl-carnitine panel abnormalities in a pattern similar to glutathione abnormalities found in the PPA rodent model of ASD. These data suggest that there are similar pathological processes between a subset of ASD children and an animal model of ASD with acquired mitochondrial dysfunction. Future studies need to identify additional parallels between the PPA rodent model of ASD and this subset of ASD individuals with this unique pattern of acyl-carnitine abnormalities. A better understanding of this animal model and subset of children with ASD should lead to better insight in mechanisms behind environmentally induced ASD pathophysiology and should provide guidance for developing preventive and symptomatic treatments.

Exon Skipping as a Therapeutic Strategy Applied to a RyR1 Mutation Causing Severe Core Myopathy

Central Core Disease is a myopathy resulting generally from a mutation in the RYR1 gene, encoding the skeletal muscle calcium release channel RyR1. No treatment is currently available for this disease. We studied a pathological situation in which an affected child harbors two recessive mutations, resulting in a massive reduction in the RyR1 amount. The paternal mutation inducing the inclusion of a new in frame exon in the mRNA of RyR1, resulted in the insertion of additional amino-acids and destabilization of the protein. We hypothesized that inducing the skipping of this exon would be sufficient to restore RyR1 expression and normalization of calcium releases. We developed U7-AON lentiviral vectors to induce exon-skipping on affected primary muscle cells. The efficiency of the exon skipping at the mRNA level, at the protein level and at the functional level using calcium imaging were evaluated. We observed in these affected primary muscle culture a reduction in the inclusion of the additional exon, an increase in the RyR1 protein expression, and a restoration of normal calcium releases. This study is the first demonstration of the potential of exon skipping for the therapy of Central Core Disease, from the molecular to the functional level.

Expression profiles of muscle disease-associated genes and their isoforms during differentiation of cultured human skeletal muscle cells

BackgroundThe formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins.MethodsWe investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development.ResultsOur culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development.ConclusionsIn conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.

ENERGY METABOLISM IN SKELETAL MUSCLES IN DOGS DURING TREATMENT OF SHIN FRACTURES WITH ILIZAROV’S TECHNIGUE

Aim - to study the dynamics of the recovery of energy resources in the tibialis anterior (TA) in dogs after shin fractures. Material and methods. The concentration of energy metabolism substrates, the rate of glycolysis and glycogenolysis in TA in 25 dogs during the treatment of tibial fractures by Ilizarov were studied. The values parameters in the experimental animals were compared with values in the intact (non-operated) dogs. Results. The level of glycogen and glucose decreased in TA within 14 days of fixation. On the 28th day of fixing the level of these metabolites corresponded to the level in intact animals. By the end of fixation the rate of glycogen utilization in TA of experimental animals was significantly reduced relative to intact animals, and the rate of glucose utilization was unchanged, though all glucose was almost entirely utilized by aerobic way. A month after treatment there was an increase of glycogen levels in the TA and activity of lactatedehydrogenase MM-isoenzyme. When glycogen solution added to the cell-free culture of muscle the its considerable utilization and the pyruvate accumulation were found. Conclusions. Anaerobic energy metabolism of in the TA of injured segment have prevailed before the 14-th day after the injury, the aerobic energy metabolism - after 28-days. The phenomenon of growth of anaerobic energy metabolism in TA of injured segment within three months after treatment was revealed.

Skeletal muscle pericyte subtypes differ in their differentiation potential

Neural progenitor cells have been proposed as a therapy for central nervous system disorders, including neurodegenerative diseases and trauma injuries, however their accessibility is a major limitation. We recently isolated Tuj1+ cells from skeletal muscle culture of Nestin–GFP transgenic mice however whether they form functional neurons in the brain is not yet known. Additionally, their isolation from nontransgenic species and identification of their ancestors is unknown. This gap of knowledge precludes us from studying their role as a valuable alternative to neural progenitors. Here, we identified two pericyte subtypes, type-1 and type-2, using a double transgenic Nestin–GFP/NG2–DsRed mouse and demonstrated that Nestin–GFP+/Tuj1+ cells derive from type-2 Nestin–GFP+/NG2–DsRed+/CD146+ pericytes located in the skeletal muscle interstitium. These cells are bipotential as they generate either Tuj1+ cells when cultured with muscle cells or become “classical” α-SMA+pericytes when cultured alone. In contrast, type-1 Nestin–GFP−/NG2–DsRed+/CD146+ pericytes generate α-SMA+pericytes but not Tuj1+ cells. Interestingly, type-2 pericyte derived Tuj1+ cells retain some pericytic markers (CD146+/PDGFRβ+/NG2+). Given the potential application of Nestin–GFP+/NG2–DsRed+/Tuj1+ cells for cell therapy, we found a surface marker, the nerve growth factor receptor, which is expressed exclusively in these cells and can be used to identify and isolate them from mixed cell populations in nontransgenic species for clinical purposes.

Prévention de l’obésité et de l’insulinorésistance par l’activation du récepteur alpha des estrogènes : une action impliquant la fonction de transactivation AF-2

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1−/− mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.

A simplified but robust method for the isolation of avian and mammalian muscle satellite cells

BackgroundCurrent methods of isolation of muscle satellite cells from different animal species are highly variable making inter-species comparisons problematic. This variation mainly stems from the use of different proteolytic enzymes to release the satellite cells from the muscle tissue (sometimes a single enzyme is used but often a combination of enzymes is preferred) and the different extracellular matrix proteins used to coat culture ware. In addition, isolation of satellite cells is frequently laborious and sometimes may require pre-plating of the cell preparation on uncoated flasks or Percoll centrifugation to remove contaminating fibroblasts. The methodology employed to isolate and culture satellite cells in vitro can critically determine the fusion of myoblasts into multi-nucleated myotubes. These terminally differentiated myotubes resemble mature myofibres in the muscle tissue in vivo, therefore optimal fusion is a keystone of in vitro muscle culture. Hence, a simple method of muscle satellite cell isolation and culture of different vertebrate species that can result in a high fusion rate is highly desirable.ResultsWe demonstrate here a relatively simple and rapid method of isolating highly enriched muscle satellite cells from different avian and mammalian species. In brief, muscle tissue was mechanically dissociated, digested with a single enzyme (pronase), triturated with a 10-ml pipette, filtered and directly plated onto collagen coated flasks. Following this method and after optimization of the cell culture conditions, excellent fusion rates were achieved in the duck, chicken, horse and cow (with more than 50% cell fusion), and to a lesser extent pig, pointing to pronase as a highly suitable enzyme to release satellite cells from muscle tissue.ConclusionsOur simplified method presents a quick and simple alternative to isolating highly enriched muscle satellite cell cultures which can subsequently rapidly differentiate into well developed primary myotubes. The use of the same isolation protocol allows better inter-species comparisons of muscle satellite cells. Of all the farm animal species investigated, harvested chicken muscle cells showed the highest percentage of muscle satellite cells, and equine muscle cells presented the highest fusion index, an impressive ≈ 77%. Porcine cells displayed the lowest amount of satellite cells but still achieved a modest fusion rate of ≈ 41%.

Skeletal Muscle Protein Tyrosine Phosphatase 1B Regulates Insulin Sensitivity in African Americans

Protein tyrosine phosphatase 1B (PTP1B) is postulated to modulate insulin action by dephosphorylating the insulin receptor signaling proteins and attenuating insulin signaling. We sought to determine the relationship of skeletal muscle PTP1B to whole-body insulin sensitivity. We studied 17 African Americans with type 2 diabetes mellitus (T2DM) and 16 without diabetes. PTP1B gene expression and protein abundance were determined in the biopsied skeletal muscles at the baseline of a hyperinsulinemic-euglycemic clamp. PTP1B gene expression was significantly higher in subjects with T2DM versus control (P < 0.0001) and remained significantly different after adjusting for age and insulin sensitivity (P = 0.05). PTP1B gene expression was positively related to protein abundance (rs = 0.39; P = 0.03; adjusted for age and insulin sensitivity) and negatively related to insulin sensitivity (rs = −0.52; P = 0.002; adjusted for age). Overexpression and interference RNA of PTP1B were performed in primary human skeletal muscle culture. PTP1B overexpression resulted in reduction of Akt phosphorylation in the control subjects. Moreover, interference RNA transfection downregulated PTP1B expression and enhanced Akt phosphorylation in subjects with T2DM. These data show that skeletal muscle PTP1B gene expression is increased in African American subjects with T2DM, is negatively associated with whole-body insulin sensitivity, and contributes to modulation of insulin signaling.

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8h and 48h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30min to 1h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.

Complex interactions between human myoblasts and the surrounding 3D fibrin-based matrix.

Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D) culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05). Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001). Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5±0.8 to 7.4±4.7 kPa during proliferation (p<0.05) and reached 12.2±6.0 kPa during differentiation (p<0.05), thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.

Wnt Signaling Activation in Adipose Progenitors Promotes Insulin-Independent Muscle Glucose Uptake

Adipose tissues provide circulating nutrients and hormones. We present invivo mouse studies highlighting roles for Wnt signals in both aspects of metabolism. β-catenin activation in PPARγ-expressing fat progenitors (PBCA) decreased fat mass and induced fibrotic replacement of subcutaneous fat specifically. In spite of lipodystrophy, PBCA mice did not develop the expected diabetes and hepatosteatosis, but rather exhibited improved glucose metabolism and normal insulin sensitivity. Glucose uptake was increased in muscle independently of insulin, associated with cell-surface translocation of glucose transporters and AMPK activation. Exvivo assays showed these effects were likely secondary to blood-borne signals since PBCA sera or conditioned media from PBCA fat progenitors enhanced glucose uptake and activated AMPK in muscle cultures. Thus, adipose progenitor Wnt activation dissociates lipodystrophy from dysfunctional metabolism and highlights a fat-muscle endocrine axis, which may represent a potential therapy to lower blood glucose and improve metabolism.

Effects of increased preload on the force-frequency response and contractile kinetics in early stages of cardiac muscle hypertrophy

Numerous studies have aimed to elucidate markers for the onset of decompensatory hypertrophy and heart failure in vivo and in vitro. Alterations in the force-frequency relationship are commonly used as markers for heart failure with a negative staircase being a hallmark of decompensated cardiac function. Here we aim to determine the functional and molecular alterations in the very early stages of compensatory hypertrophy through analysis of the force-frequency relationship, using a novel isolated muscle culture system that allows assessment of force-frequency relationship during the development of hypertrophy. New Zealand white male rabbit trabeculae excised from the right ventricular free wall were utilized for all experiments. Briefly, muscles held at constant preload and contracting isometrically were stimulated to contract in culture for 24 h, and in a subset up to 48 h. We found that, upon an increase in the preload and maintaining the muscles in culture for up to 24 h, there was an increase in baseline force produced by isolated trabeculae over time. This suggests a gradual compensatory response to the impact of increased preload. Temporal analysis of the force-frequency response during this progression revealed a significant blunting (at 12 h) and then reversal of the positive staircase as culture time increased (at 24 h). Phosphorylation analysis revealed a significant decrease in desmin and troponin (Tn)I phosphorylation from 12 to 24 h in culture. These results show that even very early on in the compensatory hypertrophy state, the force-frequency relationship is already affected. This effect on force-frequency relationship may, in addition to protein expression changes, be partially attributed to the alterations in myofilament protein phosphorylation.

Proteomic analysis reveals cellular pathways regulating carbohydrate metabolism that are modulated in primary human skeletal muscle culture due to treatment with bioactives from Artemisia dracunculus L.

Insulin resistance is a major pathophysiologic abnormality that characterizes metabolic syndrome and type 2 diabetes. A well characterized ethanolic extract of Artemisia dracunculus L., termed PMI 5011, has been shown to improve insulin action in vitro and in vivo, but the cellular mechanisms remain elusive. Using differential proteomics, we have studied mechanisms by which PMI 5011 enhances insulin action in primary human skeletal muscle culture obtained by biopsy from obese, insulin-resistant individuals. Using iTRAQ™ labeling and LC-MS/MS, we have identified over 200 differentially regulated proteins due to treatment with PMI 5011 and insulin stimulation. Bioinformatics analyses determined that several metabolic pathways related to glycolysis, glucose transport and cell signaling were highly represented and differentially regulated in the presence of PMI 5011 indicating that this extract affects several pathways modulating carbohydrate metabolism, including translocation of GLUT4 to the plasma membrane. These findings provide a molecular mechanism by which a botanical extract improves insulin stimulated glucose uptake, transport and metabolism at the cellular level resulting in enhanced whole body insulin sensitivity.