Hypoxia Impairs Muscle Function and Reduces Myotube Size in Tissue Engineered Skeletal Muscle

Contemporary tissue engineered skeletal muscle models display a high degree of physiological accuracy compared with native tissue, and therefore may be excellent platforms to understand how various pathologies affect skeletal muscle. Chronic obstructive pulmonary disease (COPD) is a lung disease which causes tissue hypoxia and is characterized by muscle fiber atrophy and impaired muscle function. In the present study we exposed engineered skeletal muscle to varying levels of oxygen (O₂; 21–1%) for 24 h in order to see if a COPD like muscle phenotype could be recreated in vitro, and if so, at what degree of hypoxia this occurred. Maximal contractile force was attenuated in hypoxia compared to 21% O₂; with culture at 5% and 1% O₂ causing the most pronounced effects with 62% and 56% decrements in force, respectively. Furthermore at these levels of O₂, myotubes within the engineered muscles displayed significant atrophy which was not seen at higher O₂ levels. At the molecular level we observed increases in mRNA expression of MuRF‐1 only at 1% O₂ whereas MAFbx expression was elevated at 10%, 5%, and 1% O₂. In addition, p70S6 kinase phosphorylation (a downstream effector of mTORC1) was reduced when engineered muscle was cultured at 1% O₂, with no significant changes seen above this O₂ level. Overall, these data suggest that engineered muscle exposed to O₂ levels of ≤5% adapts in a manner similar to that seen in COPD patients, and thus may provide a novel model for further understanding muscle wasting associated with tissue hypoxia. J. Cell. Biochem. 118: 2599–2605, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Glutamate Increases Glycogen Content and Reduces Glucose Utilization in Primary Astrocyte Culture

The glycogen content of primary cultured astrocytes was approximately doubled by incubation with 1 mM L-glutamate or L-aspartate. Other amino acids and excitatory neurotransmitters were without effect. The increase in glycogen level was not blocked by the glutamate receptor antagonist kynurenic acid but was completely blocked by the glutamate uptake inhibitor threo-3-hydroxy-D,L-aspartate and by removal of Na+ from the medium. Incubation with radiolabeled glucose and glutamate revealed that the increased glycogen content was derived almost entirely from glucose. Glutamate at 1 mM was also found to cause a 53 +/- 12% decrease in glucose utilization and a 112 +/- 69% increase in glucose-6-phosphate levels. These results suggest that the glycogen content of astrocytes is linked to the rate of glucose utilization and that glucose utilization can, in turn, be affected by the availability of alternative metabolic substrates. These relationships suggest a mechanism by which brain glycogen accumulation occurs during decreased neuronal activity.     

Skeletal Muscle Cellularity and Glycogen Distribution in the Hypermuscular Compact Mice

The TGF-beta member myostatin acts as a negative regulator of skeletal muscle mass. The Compact mice were selected for high protein content and hypermuscularity, and carry a naturally occurring 12-bp deletion in the propeptide region of the myostatin precursor. We aimed to investigate the cellular characteristics and the glycogen distribution of the Compact tibialis anterior (TA) muscle by quantitative histochemistry and spectrophotometry. We have found that the deficiency in myostatin resulted in significantly increased weight of the investigated hindlimb muscles compared to wild type. Although the average glycogen content of the individual fibers kept unchanged, the total amount of glycogen in the Compact TA muscle increased two-fold, which can be explained by the presence of more fibers in Compact compared to wild type muscle. Moreover, the ratio of the most glycolytic IIB fibers significantly increased in the Compact TA muscle, of which glycogen content was the highest among the fast fibers. In summary, myostatin deficiency caused elevated amount of glycogen in the TA muscle but did not increase the glycogen content of the individual fibers despite the marked glycolytic shift observed in Compact mice.Key words: Compact mice, fiber-type, GDF-8, glycogen, muscle, myostatin     

EGFR Mimotope Reduces the Expression of LYVE-1 in the Intestine from the M3R Immunized Mice

International Journal of Peptide Research and Therapeutics - Primary Sjögren’s syndrome (pSS) is a prototypic autoimmune disease characterized by immune injury to the salivary and...     

Insulin Resistance is Associated with MCP1-Mediated Macrophage Accumulation in Skeletal Muscle in Mice and Humans

Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D). However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFα expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.     

Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans

Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.     

A Glyoxalase-1 Variant Associated with the t-Complex in House Mice

A quantitative variant of glyoxalase-1 associated with the t-complex in house mice is described. GLO-1C in red cell lysates from mice heterozygous for complementing t-haplotypes and from mice homozygous for the tw8-haplotype had less than one-third the GLO-1 activity of NZB/BlNJ, the inbred strain with the lowest activity previously reported. GLO-1C appeared to be determined by the structural locus Glo-1 and, together with two partial t6-haplotypes, was used to map Glo-1 to the telomeric portion of the t6-haplotype. Glo-1c was associated with all t-haplotypes tested and has not been found in mice that lack a t-complex. Thus, this variant of Glo-1c provides both a further example of gametic disequilibrium between the t-complex and linked loci and a readily identifiable marker for the t-complex.     

Functional Hypermethylation of ALDH1L1 , PLCL2 , and PPP2R3A in Colon Cancer

DNA hypermethylation and mutations are key mechanisms for the downregulation of tumor suppressor genes. NotI-microarrays allowed us to detect hypermethylation and/or deletions in 180 NotI sites associated with 188 genes of human chromosome 3, in 24 paired (tumor/normal) colon samples. The most frequent aberrations (in more than 20% of tumor samples) were detected in the promoter regions of 20 genes. Expression and promoter methylation of these genes were analyzed using the data for paired colon samples from The Cancer Genome Atlas project. Three genes—ALDH1L1, PLCL2, and PPP2R3A—revealed a more than two-fold average decrease in expression and a negative correlation between mRNA level and promoter hypermethylation. The expression of these three genes was then evaluated in 30 paired colon samples by quantitative PCR. Frequent (in more than 60% of cases) and significant (5–9-fold on average) mRNA level decrease was found for each of the genes in the tumor samples. The results indicate a suppressor role of the ALDH1L1, PLCL2, and PPP2R3A genes in colon cancer, as well as functional significance of hypermethylation in the downregulation of these genes.     

Assessment of genomic imprinting of PPP1R9A, NAP1L5 and PEG3 in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it's expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

A prevalent variant in PPP1R3A impairs glycogen synthesis and reduces muscle glycogen content in humans and mice

Background

Stored glycogen is an important source of energy for skeletal muscle. Human genetic disorders primarily affecting skeletal muscle glycogen turnover are well-recognised, but rare. We previously reported that a frameshift/premature stop mutation in PPP1R3A, the gene encoding R_GL, a key regulator of muscle glycogen metabolism, was present in 1.36% of participants from a population of white individuals in the UK. However, the functional implications of the mutation were not known. The objective of this study was to characterise the molecular and physiological consequences of this genetic variant.

Methods and Findings

In this study we found a similar prevalence of the variant in an independent UK white population of 744 participants (1.46%) and, using in vivo ¹³C magnetic resonance spectroscopy studies, demonstrate that human carriers (n = 6) of the variant have low basal (65% lower, p = 0.002) and postprandial muscle glycogen levels. Mice engineered to express the equivalent mutation had similarly decreased muscle glycogen levels (40% lower in heterozygous knock-in mice, p < 0.05). In muscle tissue from these mice, failure of the truncated mutant to bind glycogen and colocalize with glycogen synthase (GS) decreased GS and increased glycogen phosphorylase activity states, which account for the decreased glycogen content.

Conclusions

Thus, PPP1R3A C1984ΔAG (stop codon 668) is, to our knowledge, the first prevalent mutation described that directly impairs glycogen synthesis and decreases glycogen levels in human skeletal muscle. The fact that it is present in ∼1 in 70 UK whites increases the potential biomedical relevance of these observations.