Inflammation and altered metabolism impede efficacy of functional electrical stimulation in critically ill patients.

BACKGROUND: Critically ill patients suffer from acute muscle wasting, which is associated with significant physical functional impairment. We describe data from nested muscle biopsy studies from two trials of functional electrical stimulation (FES) that did not shown improvements in physical function. METHODS: Primary cohort: single-centre randomized controlled trial. Additional healthy volunteer data from patients undergoing elective hip arthroplasty. Validation cohort: Four-centre randomized controlled trial. INTERVENTION: FES cycling for 60-90min/day. ANALYSES: Skeletal muscle mRNA expression of 223 genes underwent hierarchal clustering for targeted analysis and validation. RESULTS: Positively enriched pathways between healthy volunteers and ICU participants were "stress response", "response to stimuli" and "protein metabolism", in keeping with published data. Positively enriched pathways between admission and day 7 ICU participants were "FOXO-mediated transcription" (admission = 0.48 ± 0.94, day 7 = - 0.47 ± 1.04 mean log2 fold change; P = 0.042), "Fatty acid metabolism" (admission = 0.50 ± 0.67, day 7 = 0.07 ± 1.65 mean log2 fold change; P = 0.042) and "Interleukin-1 processing" (admission = 0.88 ± 0.50, day 7 = 0.97 ± 0.76 mean log2 fold change; P = 0.054). Muscle mRNA expression of UCP3 (P = 0.030) and DGKD (P = 0.040) decreased in both cohorts with no between group differences. Changes in IL-18 were not observed in the validation cohort (P = 0.268). Targeted analyses related to intramuscular mitochondrial substrate oxidation, fatty acid oxidation and intramuscular inflammation showed PPARγ-C1α; (P  0.05). CONCLUSIONS: Intramuscular inflammation and altered substrate utilization are persistent in skeletal muscle during first week of critical illness and are not improved by the application of Functional Electrical Stimulation-assisted exercise. Future trials of exercise to prevent muscle wasting and physical impairment are unlikely to be successful unless these processes are addressed by other means than exercise alone

Arginine protects muscle cells from wasting in vitro in an mTORC1-dependent and NO-independent manner

Amino acids are potent regulators of muscle protein synthesis and breakdown and have received considerable attention for the treatment of muscle wasting conditions. Arginine is critically involved in numerous physiological functions including providing substrate for the production of creatine, urea and nitric oxide (NO) and in the synthesis of new proteins. However, little is known about the direct effects of arginine on skeletal muscle protein synthesis during catabolic conditions. The aims of this study were to determine whether exogenous arginine could protect skeletal muscle cells from wasting directly and whether this effect was dependent on production of NO and/or activation of the rapamycin-sensitive mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. To explore these aims, we deprived mature C2C12 myotubes from nutrients and growth factors by incubating them in HEPES buffered saline with arginine or equimolar concentrations of alanine (control). Our results show that arginine: increased the ratio of phosphorylated to total mTOR (146 %), S6 (40 %) and 4EBP1 (69 %); increased protein synthesis (69 %) during the first hour of treatment; and increased myotube diameter by ~15 %. Experiments using the NO synthase inhibitor L-NG-Nitroarginine Methyl Ester showed a NO-independent protection from muscle wasting. On the other hand, the mTORC1 inhibitor rapamycin prevented increases in phosphorylated S6, protein synthesis and myotube diameter. The activation of mTORC1 and protein synthesis by arginine was not associated with changes in the phosphorylation status of Akt, but rather increased the expression of the amino acid-sensitive type III PI3-kinase Vps34 signalling protein. These data support a direct role for arginine in the regulation of mTORC1 in skeletal muscle

Glycine metabolism in skeletal muscle: implications for metabolic homeostasis

PURPOSE OF REVIEW: The review summarizes the recent literature on the role of glycine in skeletal muscle during times of stress. RECENT FINDINGS: Supplemental glycine protects muscle mass and function under pathological conditions. In addition, mitochondrial dysfunction in skeletal muscle leads to increased cellular serine and glycine production and activation of NADPH-generating pathways and glutathione metabolism. These studies highlight how glycine availability modulates cellular homeostasis and redox status. SUMMARY: Recent studies demonstrate that supplemental glycine effectively protects muscles in a variety of wasting models, including cancer cachexia, sepsis, and reduced caloric intake. The underlying mechanisms responsible for the effects of glycine remain unclear but likely involve receptor-mediated responses and modulation of intracellular metabolism. Future research to understand these mechanisms will provide insight into glycine's therapeutic potential. Our view is that glycine holds considerable promise for improving health by protecting muscles during different wasting conditions

Glycine Protects Muscle Cells From Wasting in vitro via mTORC1 Signaling

Glycine supplementation can protect skeletal muscles of mice from cancer-induced wasting, but the mechanisms underlying this protection are not well-understood. The aim of this study was to determine whether exogenous glycine directly protects skeletal muscle cells from wasting. C2C12 muscle cells were exposed to non-inflammatory catabolic stimuli via two models: serum withdrawal (SF) for 48 h; or incubation in HEPES buffered saline (HBS) for up to 5 h. Cells were supplemented with glycine or equimolar concentrations of L-alanine. SF- and HBS-treated myotubes (with or without L-alanine) were ~20% and ~30% smaller than control myotubes. Glycine-treated myotubes were up to 20% larger (P < 0.01) compared to cells treated with L-alanine in both models of muscle cell atrophy. The mTORC1 inhibitor rapamycin prevented the glycine-stimulated protection of myotube diameter, and glycine-stimulated S6 phosphorylation, suggesting that mTORC1 signaling may be necessary for glycine's protective effects in vitro. Increasing glycine availability may be beneficial for muscle wasting conditions associated with inadequate nutrient intake

Early myogenic responses to acute exercise before and after resistance training in young men

To enable dynamic regulation of muscle mass and myofiber repair following injury, a satellite cell precursor population exists to supply additional nuclei. Activated satellite cells express many genes and associated proteins necessary for maturation and incorporation into the damaged fiber. There is little knowledge about the response of these markers following whole-body resistance exercise training. We investigated the impact of 12 weeks of progressive whole-body resistance training on the expression of MRFs, PAX7, NCAM, and FA1, incorporating both acute and chronic resistance exercise components. Ten young recreationally active males (21.2 ± 3.5 years) performed 12 weeks of whole-body resistance training at 70-85% of their predetermined one-repetition maximum (1RM). At the initiation and completion of the training period, muscular strength was assessed by RM and dynamometer testing, and vastus lateralis samples were obtained prior to and 3 h following an acute resistance exercise test (both whole-body and isometric exercises). Increased mRNA expression of PAX7 (threefold), NCAM (threefold), MYF5 (threefold), MYOD (threefold) and MYOGENIN (twofold) was observed 3 h after the acute resistance exercise test, both pre and posttraining. Similarly, PAX7 (11-fold) and FA1 (twofold) protein abundance increased after acute exercise, while resting NCAM (eightfold) and FA1 (threefold) protein abundance increased following 12 weeks of resistance training. It is possible that these molecular changes are primarily due to the preceding exercise bout, and are not modified by long-term or whole-body exercise training

L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism

Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H2O2) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions

Deletion of suppressor of cytokine signaling 3 (SOCS3) in muscle stem cells does not alter muscle regeneration in mice after injury

Muscles of older animals are more susceptible to injury and regenerate poorly, in part due to a persistent inflammatory response. The janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway mediates inflammatory signaling and is tightly regulated by the suppressor of cytokine signaling (SOCS) proteins, especially SOCS3. SOCS3 expression is altered in the muscle of aged animals and may contribute to the persistent inflammation and impaired regeneration. To test this hypothesis, we performed myotoxic injuries on mice with a tamoxifen-inducible deletion of SOCS3 specifically within the muscle stem cell compartment. Muscle stem cell-specific SOCS3 deletion reduced muscle mass at 14 days post-injury (-14%, P < 0.01), altered the myogenic transcriptional program, and reduced myogenic fusion based on the number of centrally-located nuclei per muscle fiber. Despite the delay in myogenesis, muscles with a muscle stem cell-specific deletion of SOCS3 were still able to regenerate after a single bout or multiple bouts of myotoxic injury. A reduction in SOCS3 expression in muscle stem cells is unlikely to be responsible for the incomplete muscle repair in aged animals

Metric precision of flat panel volumetric computed tomography and different orthopantomographic procedures in pre-implantological diagnostics

In der vorliegenden Studie wurde die metrische Genauigkeit verschiedener röntgenologischer Verfahren zur präimplantologischen Diagnostik untersucht. Das Standardverfahren der Orthopantomographie und das noch im experimentellen Stadium befindliche Verfahren der Flächendetektor-Volumencomputertomographie (engl. flat panel volumetric computed tomography, VCT) wurden zu diesem Zweck mit realen Schnittpräparaten humaner Unterkiefer verglichen. Im Bereich der Orthopantomographie kamen fünf Geräte sowohl konventioneller als auch digitaler Technik zum Einsatz. Das Hauptaugenmerk lag auf der Darstellung des im Unterkiefer verlaufenden Canalis mandibulae und des crestal befindlichen Knochenangebotes. Insbesondere ist dabei die Distanz von dem Oberrand des Canalis mandibulae zum crestalen Alveolarkammrand von Bedeutung. Die Untersuchung wurde an 18 Kopfpräparatehälften durchgeführt. An ihnen wurden röntgenopake Marker fixiert, die an definierten Stellen den Vergleich der röntgenologischen Verfahren mit den realen Schnittpräparaten erlaubten. Die röntgenopaken Marker wurden sowohl in horizontaler als auch vertikaler Dimension vermessen. Zusätzlich wurde der Abstand vom crestalen Alveolarkammrand zum Oberrand des Canalis mandibulae bestimmt. In der vertikalen Dimension variierten bei den fünf Orthopantomographieverfahren die maximalen Vergrößerungsfaktoren von 1,15 bis 1,23. Für jedes der fünf Orthopantomographieverfahren wurde der durchschnittliche vertikale Vergrößerungsfaktor ermittelt. Die Werte lagen zwischen 1,05 und 1,13. In der Horizontalen variierten die maximalen Vergrößerungsfaktoren der fünf Orthopantomographieverfahren von 1,16 bis 1,37. Ebenso wurde der durchschnittliche horizontale Vergrößerungsfaktor für jedes der fünf Orthopantomographieverfahren bestimmt. Die Werte lagen zwischen 1,05 und 1,15. Bei dem Verfahren der VCT unterschieden sich die Werte in der Vertikalen und Horizontalen nur geringfügig von den realen Schnittpräparaten. Der durchschnittliche Vergrößerungsfaktor lag in der Vertikalen bei 1,01, in der Horizontalen bei 1,00. Aufgrund der Abweichung des maximalen Vergrößerungsfaktors zum durchschnittlichen Vergrößerungsfaktor ist zu empfehlen, dass für die präimplantologische Diagnostik und Planung eine metrische Auswertung von Orthopantomogrammen nicht auf der Annahme konstanter Vergrößerungsfaktoren, sondern auf einem Vergleich mit in situ befindlichen Referenzobjekten basieren sollte. Innerhalb der präimplantologischen Diagnostik ist besonders die Abstandsmessung vom crestalen Alveolarkammrand zum Oberrand des Canalis mandibulae zu beachten. Hierbei variierte bei den fünf verschiedenen Orthopantomographieverfahren die Nichtbeurteilbarkeit des Oberrandes von 15,3 bis 25,0 %, bei der VCT hingegen lag sie bei 2,8 %. Bei der Abstandsmessung vom crestalen Alveolarkammrand zum Oberrand des Canalis mandibulae betrug der maximale Vergrößerungsfaktor bei der VCT 1,05. Innerhalb der fünf Orthopantomographieverfahren variierten die maximalen Vergrößerungsfaktoren von 1,34 bis 1,49. Der maximale Vergrößerungsfaktor der Orthopantomographie bei der Abstandsmessung vom crestalen Alveolarkammrand zum Oberrand des Canalis mandibulae ist damit höher als bei den Vermessungen der eindeutig sichtbaren röntgenopaken Marker. Bei dem Umgang mit der bildbearbeitenden Software für die Datensätze der VCT-Aufnahmen war subjektiv eine im Vergleich zu anderen dreidimensionalen Röntgentechniken beeindruckende Detailerkennbarkeit festzustellen, insbesondere bei knöchernen Strukturen. Derzeit ist die VCT im experimentellen Stadium und noch nicht für Untersuchungen am lebenden Menschen zugelassen. Die Größe der zu untersuchenden Objekte ist im Dual-Panel-Mode auf 13,6 cm in der xy-Ebene und 21cm in der z-Richtung beschränkt. Weitere Untersuchungen hinsichtlich der Strahlenbelastung und der Beurteilbarkeit der Knochendichte stehen noch aus, bevor die VCT eventuell zu einer ergänzenden präimplantologischen Röntgendiagnostik herangezogen werden kann.In the present study the metric precision of different radiological procedures is investigated for their application in pre-implantological diagnostics. For this purpose, standard procedures of orthopantomography as well as the procedure of flat panel volumetric computed tomography (VCT), still in the trial phase, were applied and compared with real preparations of human lower jaw. For the orthopantomography a total of five apparatuses of both conventional and digital technology were applied. The main focus was on the presentation of the mandibular canal (lower jaw) and the crestally located bone structures. Of particular significance was the distance between the upper edge of the mandibular canal and the crestal alveolar ridge. The investigation was carried out on 18 preparations of cranial halves. To these radiographical opaque markers were fixed, which allowed for comparison of different radiological procedures with real preparations at defined anatomical sites. Quantification of the radiographical opaque markers occurred in horizontal and vertical dimensions. In addition, the distance between the crestal alveolar ridge and the upper ridge of the mandibular canal were determined. The maximal augmentation factors of the vertical dimension of the five orthopantomograhic procedures varied in values from 1.15-1.23. For each of the five orthopantomographic procedures the average vertical augmentation factor was determined. The values ranged from 1.05-1.13. In the horizontal dimension the maximal augmentation factors of the five orthopantomographies varied in values from 1.16-1.37. Likewise, as carried out for the vertical dimension, the average horizontal augmentation factor was determined for each of the five orthopantomographic procedures. The values were between 1.05-1.15. In the case of the VCT, the values of the vertical and horizontal dimension displayed only minimal discrepancies from the real preparations of human lower jaw. The average of the augmentation factor for the vertical dimension was 1.01, for the horizontal dimension the average was 1.00. For the purpose of preimplantational diagnostics and planning, it is recommended that because of the deviation of the maximal augmentation factor from the average augmentation factor, any metric validation of the orthopantograms not be based on constant augmentation factors, but instead in relation to in situ. Regarding the preimplantological diagnostics, particular attention must be paid to the distance between the crestal alveolar ridge and the upper ridge of the mandibular canal. For all five different orthopantomographies the percentage of cases in which it was not possible to evaluate the upper edge of the mandibular canal varied from 15.3-25 %, whereas for the VCT it was only 2.8 %. The measurement of the distance between the crestal alveolar ridge and the upper edge of the mandibular canal displayed a maximum augmentation factor for the VCT of 1.05. Amongst the five orthopantomographies the values of the maximal augmentation factors varied from 1.34-1.49. The maximal augmentation factor obtained for the measurement of the distance between the crestal alveolar ridge and the upper edge of the mandibular canal was thus higher than that obtained with the clearly visible radiographic opaque markers in the orthopantomographies. In handling the image software for the datasets of the VCT images, the details identified were impressive when subjectively compared to other three-dimensional radiology techniques, in particular with osseous structures. Currently, VCT is in pilot phase and it has, as yet, not been licensed as a diagnostic tool. Its limitations lie in the size of its investigational objects, which are restricted in the dual-pane-mode to 13.6 cm for the xy-plane, and to 21 cm for the z-plane. Further investigations will need to be carried out in the future with regard to the radiation load and assessability of bone density, before VCT can possibly play a part in pre-implantological radiodiagnostics

Iron accumulation in skeletal muscles of old mice is associated with impaired regeneration after ischaemia-reperfusion damage

BACKGROUND: Oxidative stress is implicated in the insidious loss of muscle mass and strength that occurs with age. However, few studies have investigated the role of iron, which is elevated during ageing, in age-related muscle wasting and blunted repair after injury. We hypothesized that iron accumulation leads to membrane lipid peroxidation, muscle wasting, increased susceptibility to injury, and impaired muscle regeneration. METHODS: To examine the role of iron in age-related muscle atrophy, we compared the skeletal muscles of 3-month-old with 22- to 24-month-old 129SvEv FVBM mice. We assessed iron distribution and total elemental iron using laser ablation inductively coupled plasma mass spectrometry and Perls' stain on skeletal muscle cross-sections. In addition, old mice underwent ischaemia-reperfusion (IR) injury (90 min ischaemia), and muscle regeneration was assessed 14 days after injury. Immunoblotting was used to determine lipid peroxidation (4HNE) and iron-related proteins. To determine whether muscle iron content can be altered, old mice were treated with deferiprone (DFP) in the drinking water, and we assessed its effects on muscle regeneration after injury. RESULTS: We observed a significant increase in total elemental iron (+43%, P < 0.05) and lipid peroxidation (4HNE: +76%, P < 0.05) in tibialis anterior muscles of old mice. Iron was further increased after injury (adult: +81%, old: +135%, P < 0.05) and associated with increased lipid peroxidation (+41%, P < 0.05). Administration of DFP did not impact iron or measures of lipid peroxidation in skeletal muscle or modulate muscle mass. Increased muscle iron concentration and lipid peroxidation were associated with less efficient regeneration, evident from the smaller fibres in cross-sections of tibialis anterior muscles (-24%, P < 0.05) and an increased percentage of fibres with centralized nuclei (+4124%, P < 0.05) in muscles of old compared with adult mice. Administration of DFP lowered iron after IR injury (PRE: -32%, P < 0.05 and POST: -41%, P < 0.05), but did not translate to structural improvements. CONCLUSIONS: Muscles from old mice have increased iron levels, which are associated with increased lipid peroxidation, increased susceptibility to IR injury, and impaired muscle regeneration. Our results suggest that iron is involved in effective muscle regeneration, highlighting the importance of iron homeostasis in muscle atrophy and muscle repair