Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2−/− mice (MDXxLcn2−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

Histology of Muscle Development in Pigs, Epigenetics from Myotubes to Tapered Fibres

Pre-natal muscle development in pigs starts with myotubes (axial nuclei in a tube of myofibrils) and secondary fibres (peripheral nuclei on an axial strand of myofibrils). By the time of birth, the nuclei of myotubes move to a peripheral position like secondary fibres. As pre-natal secondary fibres grow in length, the number of fibres in a transverse section may appear to increase. This stereology may also occur in post-natal muscles that have tapered fibres anchored in endomysial connective tissue around adjacent fibres and with one or both ends not reaching the end of their fasciculus. Up to 100 days gestation, Peroneus longus (no tapered fibres) had larger (P < 0.001) diameter secondary fibres than Longissimus thoracis (with tapered fibres). Up to 100 days gestation, no radial growth of secondary fibres was detected, but myotubes decreased in diameter (P < 0.001). From a curve showing the relative numbers of myotubes and secondary fibres, it was deduced that approximately 80% of muscle fibres in pigs are derived from secondary fibres. In post-natal Sartorius muscle there was an increase (P < 0.005) in the apparent number of muscle fibres attributed to longitudinal growth of tapered fibres. Myotubes located centrally within their fasciculi had the same position as slow-contracting fibres with a high myoglobin content in adult muscle. Post-natal changes in muscle fibre histochemistry were achieved through transitional types, probably neurally regulated rather than by differential longitudinal growth of tapered endings. Secondary fibres are important – they give rise to both the majority of muscle fibres in adult pigs and affect subsurface optical pathways and pork colourimetry.

Robert McNeill Alexander. 7 July 1934—21 March 2016

Neill Alexander graduated in natural sciences at the University of Cambridge in 1955. After a PhD at Cambridge and a lecturership at the University College of North Wales in Bangor, he was appointed to the chair of the Department of Pure and Applied Zoology at the University of Leeds in 1969. At that stage, he switched his research interests abruptly from fishes to the mechanics of legged locomotion. He conducted experiments with a variety of mammals, calculating forces, stresses and strains in muscle fibres, bones and tendons. His speciality became the application of mathematical models to animal locomotion, including repurposing the Froude number, devised by the Victorian engineer William Froude (FRS 1870) for use with ships, to estimate the speed of dinosaurs based on the spacing of their fossil footprints. Subsequent work included modelling the optimization of mammal performance and the minimization of energy costs. In 1992, following an announcement that London Zoo would have to close as a result of shortage of funds, Neill was appointed secretary of the Zoological Society of London. During the period of his secretaryship, the Society's finances recovered, with both its zoos (London and Whipsnade) breaking even in 1993 and the Society returning a surplus in each subsequent year. Neill was awarded the CBE in 2000. The National Portrait Gallery holds his portrait by John Arnison.

Different patterns of chronic hypoxia lead to hierarchical adaptative mechanisms in goldfish metabolism

Some hypoxia-tolerant species, such as goldfish, experience intermittent and severe hypoxia in their natural habitat causing them to develop multiple physiological adaptations. However, in fish, the metabolic impact of regular hypoxic exposure on swimming performance in normoxia is less well understood. Therefore, we experimentally tested whether chronic exposure to constant (30 days at 10% air saturation) or intermittent hypoxia (3hrs in normoxia and 21hrs in hypoxia, 5 days a week) would result in similar metabolic and swimming performance benefits after reoxygenation. Moreover, half of the normoxic and intermittent hypoxic fish were put on a 20-day normoxic training regime. After these treatments, metabolic rate (standard and maximum metabolic rates: SMR and MMR) and swimming performance (critical swimming speed [Ucrit] and cost of transport [COT]) were assessed. In addition, enzyme activities (citrate synthase CS, cytochrome c oxidase COX and lactate dehydrogenase LDH) and mitochondrial respiration were examined in red muscle fibres. We found that acclimation to constant hypoxia resulted in (1) metabolic suppression (-45% SMR, and -27% MMR), (2) increased anaerobic capacity (+117% LDH), (3) improved swimming performance (+80% Ucrit, -71% COT) and (4) no changes at the mitochondrial level. Conversely, the enhancement of swimming performance was reduced following acclimation to intermittent hypoxia (+45% Ucrit, -41% COT), with a 55% decrease in aerobic scope, despite a significant increase in oxidative metabolism (+201% COX, +49% CS). This study demonstrates that constant hypoxia leads to the greatest benefit in swimming performance and that mitochondrial metabolic adjustments only provide minor help in coping with hypoxia.

Acute RyR1 Ca2+ leak enhances NADH-linked mitochondrial respiratory capacity

Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.

V-OG05 Robotic Hellers Myotomy

Background Achalasia is a rare condition affecting less than 1:100,000 patients. Treatment for this rare condition include balloon dilation, botox injection, endoscopic myotomy (POEMS) or surgical myotomy. Laparoscopic surgical myotomy is the “go to” approach for most surgeons; it is tried and tested, can be performed safely and quickly with a low complication rate, minimal pain and a short length of stay. Methods This video presents the technique adopted for robotic oesophageal myotomy in a patient with type II achalasia. A 4 arm technique was adopted with arm 4 on the patients left. The Davinci X system was used in this case. A Nathensen liver retractor was used to retract the liver; robotic instruments included the hook and cadiere forceps x2. Results The procedure was successfully performed; the operative time was 53 minutes, LOS was &lt;24 hours. Check endoscopy revealed a wide open gastro-oesophageal junction and a long myotomy. The patient noted an improvement in symptoms with 24 hours and has had no significant reflux. Conclusion The enhanced magnified 3D view on the robotic platform allows better visualisation of the hiatal structures, vagal nerves and muscle fibres when performing the myotomy. Using the 4th arm to retract the lateral edge of the oesophageal muscle provides a very safe and stable platform to perform a long myotomy. I think the robotic system should be adopted as the standard approach for a hellers myotomy.

Controllable assembly of skeletal muscle-like bundles through 3D bioprinting

3D printing is an effective technology for recreating skeletal muscle tissue in vitro. To achieve clinical skeletal muscle injury repair, relatively large volumes of highly aligned skeletal muscle cells are required; obtaining these is still a challenge. It is currently unclear how individual skeletal muscle cells and their neighbouring components co-ordinate to establish anisotropic architectures in highly homogeneous orientations. Here, we demonstrated a 3D printing strategy followed by sequential culture processes to engineer skeletal muscle tissue. The effects of confined printing on the skeletal muscle during maturation, which impacted the myotube alignment, myogenic gene expression, and mechanical forces, were observed. Our findings demonstrate the dynamic changes of skeletal muscle tissue during in vitro 3D construction and reveal the role of physical factors in the orientation and maturity of muscle fibres.