Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts.Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot.We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts

Genetic ablation of soluble TNF does not affect lesion size and functional recovery after moderate spinal cord injury in mice

Traumatic spinal cord injury (SCI) is followed by an instant increase in expression of the microglial-derived proinflammatory cytokine tumor necrosis factor (TNF) within the lesioned cord. TNF exists both as membrane-anchored TNF (mTNF) and as cleaved soluble TNF (solTNF). We previously demonstrated that epidural administration of a dominant-negative inhibitor of solTNF, XPro1595, to the contused spinal cord resulted in changes in Iba1 protein expression in microglia/macrophages, decreased lesion volume, and improved locomotor function. Here, we extend our studies using mice expressing mTNF, but no solTNF (mTNFΔ/Δ), to study the effect of genetic ablation of solTNF on SCI. We demonstrate that TNF levels were significantly decreased within the lesioned spinal cord 3 days after SCI in mTNFΔ/Δ mice compared to littermates. This decrease did, however, not translate into significant changes in other pro- and anti-inflammatory cytokines (IL-10, IL-1β, IL-6, IL-5, IL-2, CXCL1, CCL2, or CCL5), despite a tendency towards increased IL-10 and decreased IL-1β, TNFR1, and TNFR2 levels in mTNFΔ/Δ mice. In addition, microglial and leukocyte infiltration, activation state (Iba1, CD11b, CD11c, CD45, and MHCII), lesion size, and functional outcome after moderate SCI were comparable between genotypes. Collectively, our data demonstrate that genetic ablation of solTNF does not significantly modulate postlesion outcome after SCI

Heritability and circulating concentrations of pregnancy-associated plasma protein-A and stanniocalcin-2 in elderly monozygotic and dizygotic twins

IntroductionPregnancy-associated plasma protein-A (PAPP-A) is an IGF-activating enzyme suggested to influence aging-related diseases. However, knowledge on serum PAPP-A concentration and regulation in elderly subjects is limited. Therefore, we measured serum PAPP-A in elderly same-sex monozygotic (MZ) and dizygotic (DZ) twins, as this allowed us to describe the age-relationship of PAPP-A, and to test the hypothesis that serum PAPP-A concentrations are genetically determined. As PAPP-A is functionally related to stanniocalcin-2 (STC2), an endogenous PAPP-A inhibitor, we included measurements on STC2 as well as IGF-I and IGF-II.MethodsThe twin cohort contained 596 subjects (250 MZ twins, 346 DZ twins), whereof 33% were males. The age ranged from 73.2 to 94.3 (mean 78.8) years. Serum was analyzed for PAPP-A, STC2, IGF-I, and IGF-II by commercial immunoassays.ResultsIn the twin cohort, PAPP-A increased with age (r=0.19; P<0.05), whereas IGF-I decreased (r=-0.12; P<0.05). Neither STC2 nor IGF-II showed any age relationship. When analyzed according to sex, PAPP-A correlated positively with age in males (r=0.18; P<0.05) and females (r=0.25; P<0.01), whereas IGF-I correlated inversely in females only (r=-0.15; P<0.01). Males had higher levels of PAPP-A (29%), STC2 (18%) and IGF-I (19%), whereas serum IGF-II was 28% higher in females (all P<0.001). For all four proteins, within-pair correlations were significantly higher for MZ twins than for DZ twins, and they demonstrated substantial and significant heritability, which after adjustment for age and sex averaged 59% for PAPP-A, 66% for STC2, 58% for IGF-I, and 52% for IGF-II.DiscussionThis twin study confirms our hypothesis that the heritability of PAPP-A serum concentrations is substantial, and the same is true for STC2. As regards the age relationship, PAPP-A increases with age, whereas STC2 remains unchanged, thereby supporting the idea that the ability of STC2 to inhibit PAPP-A enzymatic activity decreases with increasing age

Transgenic Overexpression of ADAM12 Suppresses Muscle Regeneration and Aggravates Dystrophy in Aged mdx Mice

Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, α7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12+/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12+) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy

Early-life exposure to perfluoroalkyl substances in relation to serum adipokines in a longitudinal birth cohort

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) exposure has been linked to metabolic health outcomes such as obesity, and changes in adipokine hormones may be one of the underlying biological mechanisms. We prospectively evaluated the associations between prenatal and early childhood exposures to PFASs and adipokines in children.MATERIAL AND METHODS: PFAS concentrations were measured in serum samples collected at birth, 18 months, and 5 and 9 years, and adiponectin, leptin, leptin receptor, and resistin were measured in serum samples collected at birth and 9 years. We used multivariable linear regression models to estimate the percent change in serum-adipokine concentrations for a doubling in serum-PFAS concentrations. The potential sex-specific effect of PFAS was assessed by including an interaction term between PFAS and sex in each model. Bayesian kernel machine regression (BKMR) was implemented to evaluate the overall effect of PFAS mixtures.RESULTS: Significant associations with leptin, leptin receptor, and resistin at age 9 years were observed for serum-PFAS concentrations at 18 months and 5 and 9 years, whereas associations for PFAS concentrations at birth were mostly null. However, we observed a positive association between serum-PFHxS at birth and leptin receptor at birth. We found limited evidence regarding modification effect of sex on serum-PFAS concentrations. BKMR findings were consistent and suggested some significant effects of the overall PFAS mixtures at 18 months and 5 and 9 years on adipokine concentrations at 9 years.CONCLUSIONS: Given the associations of PFAS exposure with both adipokine hormones and metabolic functions, future studies should include assessment of adipokine hormones when examining PFAS-associated metabolic alterations

Early-life associations between per- and polyfluoroalkyl substances and serum lipids in a longitudinal birth cohort

Background: Exposures to per- and polyfluoroalkyl substances (PFASs) may affect metabolic outcomes, including lipid concentrations in the blood. However, few studies have evaluated potential associations between PFASs and lipids longitudinally. Objectives: We estimated associations between PFAS and lipid concentrations at birth and at several points in childhood. Methods: We measured concentrations of five major PFASs in cord serum and in serum collected at 18 months, five years and nine years in 490 children from a prospective cohort in the Faroe Islands. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) concentrations were measured at birth, 18 months and nine years. We estimated associations between PFAS and lipid concentrations and evaluated possible effect modification by sex. We also tested whether PFAS associations with age-nine lipids varied by exposure period. Results: Serum PFAS concentrations at ages five and nine were positively associated with lipid concentrations at age nine. Cross-sectional associations between PFASs and lipids at age nine were the strongest, with increases in serum concentrations of perfluorodecanoic acid (PFDA), perfluorononanoic acid (PFNA) and perfluorooctanesulfonic acid (PFOS) associated with increases in TC, HDL-C and LDL-C. We found statistically significant differences in estimated PFAS effects by sex, where girls had stronger positive associations between PFASs and TC and LDL-C and boys had stronger positive associations with HDL-C. In repeated measure models, exposure period was a significant modifier of PFAS effects. Conclusions: Our findings suggest that childhood PFAS exposures may be associated with elevated serum lipid concentrations. This is a public health concern, as a detrimental lipid profile in childhood is a risk factor for later development of hyperlipidemia and cardiovascular disease

SPARC is up-regulated during skeletal muscle regeneration and inhibits myoblast differentiation

Skeletal muscle repair is mediated primarily by the muscle stem cell, the satellite cell. Several factors, including extracellular matrix, are known to regulate satellite cell function and regeneration. One factor, the matricellular Secreted Protein Acidic and Rich in Cysteine (SPARC) is highly up-regulated during skeletal muscle disease, but its function remains elusive. In the present study, we demonstrate a prominent yet transient increase in SPARC mRNA and protein content during skeletal muscle regeneration that correlates with the expression profile of specific muscle factors like MyoD, Myf5, Myf6, Myogenin, NCAM, CD34, and M-Cadherin, all known to be implicated in satellite cell activation/proliferation following muscle damage. This up regulation was detected in more cell types. Ectopic expression of SPARC in the muscle progenitor cell line C2C12 was performed to mimic the high levels of SPARC seen in muscle disease. SPARC overexpression almost completely abolished myogenic differentiation in these cultures as determined by substantially reduced levels of myogenic factors (Pax7, Myf5, Myod, Mef2a, Myogenin, and Myostatin) and a lack of multinucleated myotubes. These results demonstrate that there is a delicate temporal regulation of SPARC to which more sources in the micro environment contribute, and that disturbances in this, such as extensive up regulation, may have an adverse effect on muscle regeneration

The loss-of-function disease-mutation G301R in the Na+/K+-ATPase α2 isoform decreases lesion volume and improves functional outcome after acute spinal cord injury in mice

Abstract Background The Na+/K+-ATPases are transmembrane ion pumps important for maintenance of ion gradients across the plasma membrane that serve to support multiple cellular functions, such as membrane potentials, regulation of cellular volume and pH, and co-transport of signaling transmitters in all animal cells. The α2Na+/K+-ATPase subunit isoform is predominantly expressed in astrocytes, which us the sharp Na+-gradient maintained by the sodium pump necessary for astroglial metabolism. Prolonged ischemia induces an elevation of [Na+]i, decreased ATP levels and intracellular pH owing to anaerobic metabolism and lactate accumulation. During ischemia, Na+/K+-ATPase-related functions will naturally increase the energy demand of the Na+/K+-ATPase ion pump. However, the role of the α2Na+/K+-ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice for the loss-of-function disease-mutation G301R in the Atp1a2 gene (α 2 +/G301R ) to study the effect of reduced α2Na+/K+-ATPase expression in a moderate contusion spinal cord injury (SCI) model. Results We found that α 2 +/G301R mice display significantly improved functional recovery and decreased lesion volume compared to littermate controls (α 2 +/+ ) 7 days after SCI. The protein level of the α1 isoform was significantly increased, in contrast to the α3 isoform that significantly decreased 3 days after SCI in both α 2 +/G301R and α 2 +/+ mice. The level of the α2 isoform was significantly decreased in α 2 +/G301R mice both under naïve conditions and 3 days after SCI compared to α 2 +/+ mice. We found no differences in astroglial aquaporin 4 levels and no changes in the expression of chemokines (CCL2, CCL5 and CXCL1) and cytokines (TNF, IL-6, IL-1β, IL-10 and IL-5) between genotypes, just as no apparent differences were observed in location and activation of CD45 and F4/80 positive microglia and infiltrating leukocytes. Conclusion Our proof of concept study demonstrates that reduced expression of the α2 isoform in the spinal cord is protective following SCI. Importantly, the BMS and lesion volume were assessed at 7 days after SCI, and longer time points after SCI were not evaluated. However, the α2 isoform is a potential possible target of therapeutic strategies for the treatment of SCI