Mimicking exercise in vitro: effects of myotube contractions and mechanical stretch on omics.

The number of studies using skeletal muscle (SkM) cell culture models to study exercise in vitro are rapidly expanding. Progressively, more comprehensive analysis methods, such as different omics approaches including transcriptomics, proteomics, and metabolomics have been used to examine the intra- and extracellular molecular responses to exercise mimicking stimuli in cultured myotubes. Among other techniques, exercise-like electrical pulse stimulation (EL-EPS) and mechanical stretch of SkM cells are the two most commonly used methods to mimic exercise in vitro. In this mini-review, we focus on these two approaches and their effects on the omics of myotubes and/or cell culture media. Furthermore, besides traditional two-dimensional (2-D) methods, the use of three-dimensional (3-D) SkM approaches are increasing in the field of in vitro exercise mimicry. Our aim with this mini-review is to provide the reader with an up-to-date overview of the 2-D and 3-D models and the use of omics approaches to study the molecular response to exercise in vitro.

Molecular adaptations of voltage-gated sodium ion channel related proteins after fatiguing stretch-shortening cycle exercise.

Dystrophin associated protein alpha-syntrophin is known to interact with voltage-gated sodium ion channel (NaCh). Dystrophin is known to be sensitive to eccentric muscle actions. For this reason, the function of the NaChs might also be affected. Molecular adaptations of dystrophin, alpha-syntrophin and NaChs were investigated after fatiguing stretch-shortening cycle (SSC) exercise, which consisted of unilateral jumps on a sledge apparatus. Muscle biopsies were taken from the vastus lateralis muscle of eight healthy subjects immediately after (IA) and 2 days after (2D) the exercise to analyze mRNA levels and immunohistochemical staining patterns. SSC exercise resulted in decreased isometric maximal voluntary contraction (IA: -31+/-9%, 2D: -14+/-16%) and a delayed increase of plasma creatine kinase activity (2D: +178+/-211%). Despite muscle soreness (P<0.001), no morphological damage was observed and no changes were found in the mRNA concentrations. However, the relative changes of the mRNA concentrations of alpha-syntrophin and NaChs were highly correlated (r=0.93, P<0.001) 2D after SSC exercise. This consistent pattern of mRNA regulation may imply a functional relationship between these two proteins. In addition, the current experiment emphasises high inter-individual variation in molecular responses to heavy exercise.

Systemic blockade of ACVR2B ligands prevents chemotherapy-induced muscle wasting by restoring muscle protein synthesis without affecting oxidative capacity or atrogenes.

Doxorubicin is a widely used and effective chemotherapy drug. However, cardiac and skeletal muscle toxicity of doxorubicin limits its use. Inhibiting myostatin/activin signalling can prevent muscle atrophy, but its effects in chemotherapy-induced muscle wasting are unknown. In the present study we investigated the effects of doxorubicin administration alone or combined with activin receptor ligand pathway blockade by soluble activin receptor IIB (sACVR2B-Fc). Doxorubicin administration decreased body mass, muscle size and bone mineral density/content in mice. However, these effects were prevented by sACVR2B-Fc administration. Unlike in many other wasting situations, doxorubicin induced muscle atrophy without markedly increasing typical atrogenes or protein degradation pathways. Instead, doxorubicin decreased muscle protein synthesis which was completely restored by sACVR2B-Fc. Doxorubicin administration also resulted in impaired running performance without effects on skeletal muscle mitochondrial capacity/function or capillary density. Running performance and mitochondrial function were unaltered by sACVR2B-Fc administration. Tumour experiment using Lewis lung carcinoma cells demonstrated that sACVR2B-Fc decreased the cachectic effects of chemotherapy without affecting tumour growth. These results demonstrate that blocking ACVR2B signalling may be a promising strategy to counteract chemotherapy-induced muscle wasting without damage to skeletal muscle oxidative capacity or cancer treatment.

Comparison of the behavior of stainless and mild steel manual metal arc welding fumes in rat lung.

The lung retention and clearance of manual metal arc (MMA) stainless steel and mild steel welding fumes were determined in the rat. The exposure simulated the actual welding situation. The duration of exposure in the "nose-only" exposure chamber was 1 h/workday for one, two, three, or four weeks in the retention study and for four weeks in the clearance study. The concentration of exogenous iron was determined by the magnetic measuring method. Instrumental neutron activation analysis was applied to determine the concentration of total iron, chromium, and nickel in the lungs. The results indicated that the lung retention and clearance patterns for the two types of welding fumes were different. A linear relationship was observed between the amount of stainless steel MMA welding fume retained in the lungs and the duration of exposure, whereas the retention of mild steel MMA welding fume in the lung was saturated as a function of the cumulative exposure time rates. The maximum amount of lung-retained contaminants was 880 micrograms for stainless steel MMA welding fume and 220 micrograms for mild steel MMA fume.

Lack of cardiac and high-fat diet induced metabolic phenotypes in two independent strains of Vegf-b knockout mice.

Vascular endothelial growth factor-B (VEGF-B) has been implicated to play a significant role in coronary vessel growth and endothelial uptake and transport of fatty acids in heart and skeletal muscle. Additionally, recent studies have shown that Vegf-b deficiency protects from high-fat diet (HFD)-induced diabetes and insulin resistance. We compared the cardiac function and the effects of HFD on body composition and glucose metabolism in two available Vegf-b knockout (Vegf-b(-/-) strains) mouse strains side by side with their respective littermate controls. We found no differences in HFD-induced weight gain, glucose tolerance or insulin resistance between the Vegf-b(-/-) strains and their littermate control mice. Furthermore, there was no difference in basal cardiac function and cardiac expression of genes involved in glucose or fatty acid metabolism between the Vegf-b(-/-) strains and their littermate control mice. We conclude that VEGF-B is dispensable for normal cardiac function under unstressed conditions and for HFD-induced metabolic changes.

Oxidation of oat ß-glucan in aqueous solutions during processing.

The study investigated carbonyl group formation along the chain and the chain cleavage of cereal ß-glucan during heat treatments, high pressure homogenisation, cold storage and ascorbic acid treatment of aqueous solutions of this soluble dietary fibre. The carbonyl group content and its distribution along the chain were simultaneously determined with the chain cleavage using a HPSEC/labelling method, originally developed for water-insoluble cellulose. Ascorbic acid treatment resulted in a relatively high degree of carbonyl content and extensive degradation of ß-glucan, even in concentrations typically found in foods. The thermal oxidation of the ß-glucan was considerable at 120°C in a ß-glucan solution with co-extracted compounds from oat ingredient, and in the highly purified solutions in presence of ferrous ions. Oxidation also probably contributed to the molecular properties during high pressure homogenisation, even thou the main degradation mechanism is the hydrolysis caused by mechanical energy. In addition to the cleavage of the ß-glucan chain, the formation of compact, high molar mass species or molecule clusters were obtained in the study after ascorbic acid, heat (120°C) and homogenisation treatments.

Effects of exhaustive stretch-shortening cycle exercise on muscle blood flow during exercise.

AIM: The influence of exhaustive stretch-shortening cycle exercise (SSC) on skeletal muscle blood flow (BF) during exercise is currently unknown. METHODS: Quadriceps femoris (QF) BF was measured in eight healthy men using positron emission tomography before and 3 days after exhaustive SSC exercise. The SSC protocol consisted of maximal and submaximal drop jumps with one leg. Needle biopsies of the vastus lateralis muscles were taken immediately and 2 days after SSC for muscle endothelial nitric oxide synthase (eNOS) and interleukin-1-beta (IL-1beta) mRNA level determinations. RESULTS: All subjects reported subjective muscle soreness after SSC (P < 0.001), which was well in line with a decrease in maximal isometric contraction force (MVC) and increase in serum creatine kinase activity (CK) (P = 0.018). After SSC muscle BF was 25% higher in entire QF (P = 0.043) and in its deep and superficial muscle regions, whereas oxygen uptake remained unchanged (P = 0.893). Muscle biopsies revealed increased IL-1beta (30 min: 152 +/- 75%, P = 0.012 and 2 days: 108 +/- 203%, P = 0.036) but decreased or unchanged eNOS (30 min; -21 +/- 57%, P = 0.050 and 2 days: +101 +/- 204%, P = 0.779) mRNA levels after SSC. CONCLUSION: It was concluded that fatiguing SSC exercise induces increased muscle BF during exercise, which is likely to be associated with pro-inflammatory processes in the exercised muscle.

Do antiepileptics phenytoin, carbamazepine, and loreclezole show GABA(A) receptor subtype selectivity in rat brain sections?

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a convulsant site ligand of GABA(A) receptors, was used in autoradiography with rat brain sections to test suggested receptor subtype-selective actions of antiepileptics phenytoin, carbamazepine and loreclezole on native GABA(A) receptors. At maximal 100 microM concentration, both phenytoin and carbamazepine decreased [35S]TBPS binding only by 20%, indicating that their low potency and efficacy prevents their use as alpha1 subunit-identifying compounds. Ten microM loreclezole did not affect the binding, but a further increase in loreclezole concentration strongly decreased it. The action of loreclezole, assumed to reflect beta2/3 subunit-containing receptors, varied from brain region to region, but the effects were unrelated to the regional expression profiles of beta subunit variants. We conclude that in autoradiographic [35S]TBPS binding assay neither carbamazepine, phenytoin nor loreclezole are useful tools in characterizing brain regional heterogeneity of GABA(A) receptors in rats and that only loreclezole exhibits high, pharmacologically relevant efficacy.

Stainless steel manual metal arc welding fumes in rats.

Forty two male Wistar rats were exposed to manual metal arc (MMA) stainless steel (SS) welding fumes generated by an automatic welding device for "nose-only" exposure. The exposure simulated an actual MMA/SS welding environment as closely as possible. For the retention study, the duration of exposure was one hour per workday for one, two, three, of four weeks and for the clearance study four weeks. The retention and clearance of the chromium, nickel, and iron found in MMA/SS welding fumes in the rats' lungs were studied as was the distribution of the metals to other organs. Instrumental neutron activation analysis (INAA) was used for the multi-element chemical activation analyses. The concentrations of chromium and nickel in the blood and the urine were determined by atomic absorption method (AAS). The retention of exogenous iron was determined by a magnetic measuring method. The results indicated that the lungs were the target organs of soluble hexavalent chromates. The half times of lung clearance for Cr, Ni, and Fe were 40 +/- 4 d, 20 +/- d, and 50 +/- 10 d. When the lung clearance curves are compared, the half times of Cr and Fe lung clearance are similar but nickel disappears faster. The distribution and clearance patterns of chromium to other organs differ from those obtained after single intravenous or intratracheal injections of alkaline chromates.

Retention and clearance of stainless steel shieldgas welding fumes in rat lungs.

The use of metal inert gas (MIG) stainless steel (SS) welding techniques is rapidly increasing. The possible health hazards of MIG/SS welding fumes are not known; more attention has been paid to manual metal arc (MMA) welding. In this study, 52 male Wistar rats were exposed to MIG/SS welding fumes generated by an automatic welding device for "nose only" exposure. For the retention study, the duration of exposure was one hour per workday for 1,2,3, and 4 weeks. For the clearance study, the duration was 4 weeks, and the follow-up period was 106 days. The retention and clearance of the alloyed metals of SS (chromium (Cr), manganese (Mn), nickel (Ni] and iron were studied in the rats' lungs, and the results were compared with the corresponding results of MMA/SS welding. The multielement chemical analysis was made using instrumental neutron activation analysis (INAA); the concentration of exogeneous iron (Feex) was determined by a magnetic measuring method. Feex and Cr were retained in the ratio expected; Mn, Ni and total Fe were retained slower than expected. The total Fe did not clear at all even though Feex cleared with the half-time of 50 d which corresponded well with that of pure magnetite. The accumulation of Cr in the lungs was very high: it cleared with the half-time of 240 d. The clearance patterns of Mn and Ni were very similar. They followed a double exponential model with half-times of 2 d and 125 d for Mn, and 3 d and 85 d for Ni. The results indicated that even though the retention patterns for MMA and MIG welding fumes were very similar, the clearances differed very much.