X-ray diffraction studies of muscle : observations on the anterior byssus retractor muscle of Mytilus edulis and the insect flight muscle from Sarcophaga bullata

The interfilament spacing of the anterior byssus retractor muscle from Mytilus edulis was studied as the muscle was extended. It was found that variations in this spacing were very small and consistent with the hypothesis that the interfilament spacing was independent of the extension of the muscle. It was observed that the interfilament spacing was dependent on the osmolarity of the bathing medium. In concentrated solutions of the artificial seawater, the interfilament spacing decreased; while in dilute solutions of artificial seawater, it was observed that the interfilament spacing was increasing. X-ray diffraction patterns were obtained from fresh, and glutaraldehyde fixed, specimens of insect flight muscle from Sarcophaga bullata. There patterns were in general agreement with previous X-ray diffraction studies of insect flight muscle. A reflexion G at 93A was observed and interpreted as arising from diffraction in the mitochondria. Specimens of dried insect flight muscle produced a diffraction pattern consisting of arc and ring reflexions. This was interpreted as suggesting an ordered arrangement of cristae, in the mitochondria from these muscles.

Carbohydrate refeeding rapidly reverses the adaptive upregulation of human skeletal muscle pyruvate dehydrogenase kinase following a high fat diet

The time course for the reversal of the adaptive increase in pyruvate dehydrogenase kinase (PDK) activity following a 6d high fat diet (HP: 4.2 ± 0.2 % carbohydrate; 75.6 ± 0.4 % fat; 19.5 ± 0.8 % protein) was investigated in human skeletal muscle (vastus lateralis). HF feeding increased PDK activity by 44% (from 0.081 ± 0.025 min"' to 0.247 ± 0.025 mm\p < 0.05). Following carbohydrate re-feeding, (88% carbohydrate; 5% fat; 7% protein), PDK activity had returned to baseline (0.111 ± 0.014 min"') within 3h of re-feeding. The active fraction of pyruvate dehydrognease (PDHa) was depressed following 6d of the HF diet (from 0.89 ± 0.21 mmol/min/kg WW to 0.32 ± 0.05 mmol/min/kg ww,p <0.05) and increased to pre-HF levels by 45 min of post re-feeding (0.74 ±0.19 mmol/min/kg ww) and remained elevated for 3h. Western blotting analysis of the PDK isoforms, PDK4 and PDK2, revealed a 31% increase in PDK4 protein content following the HF diet, with no change in PDK2 protein. This adaptive increase in PDK4 protein content was reversed with carbohydrate re-feeding. It was concluded that the adaptive up-regulation in PDK activity and PDK4 protein content was fiilly reversed by 3h following carbohydrate re-feeding.

Modulation of muscle contraction by a FMRFamide-related peptide

A FMRFamide-like neuropeptide with the sequence "DRNFLRF-NH2" was recently isolated from pericardial organs of crayfish (Mercier et aI., Peptides, 14, 137-143, 1993). This neuropeptide, referred to as "DF2'" has already been shown to elicit cardioexcitation and to enhance synaptic transmission at neuromuscular junctions. Possible effects ofDF2 on muscle were investigated using superficial extensor muscles of the abdomen of the crayfish, Procambarus clar/ai. These muscles are of the tonic type and generate slow contractions that affect posture. DF2, at concentrations of 10-8 M or higher, increased muscle tonus and induced spontaneous, rhythmic contractions. These effects were antagonized by 5 rnM Mn2+ but not by lO-7M tetrodotoxin (TTX). Thus, they represent direct actions on muscle cells (rather than effects on motor neurons) and are likely to involve calcium influx. In contrast, deep abdominal extensor muscles, responsible for rapid swimming movements, and superficial flexor muscles do not generate contractions in response to the peptide. 2 Spontaneous contractions were also induced in the superficial extensor muscles by decreasing the temperature to II-13°C. Such contractions were also TTX-insensitive and they were antagonized by adding calcium channel blockers (Mn2+, Cd2+ or Ni2+) or by removing calcium from the bathing solution. This suggests that the spontaneous contractions depend on an influx of calcium from the extracellular solution. N-type and L-type voltage dependent calcium channel blockers did not reduce the effect of the peptide or the spontaneous contractions suggesting that calcium influx is not through N- or L-type calcium channels.

Attenuation of skeletal muscle FFA-induced insulin resistance by the polyphenol resveratrol. Elucidation of the mechanisms involved

Excess plasma free fatty acids (FFA) are correlated with insulin resistance and are a risk factor for the development of type 2 diabetes. In this study we examined the effect of the polyphenol resveratrol on FF A-induced insulin resistance in skeletal muscle cells and the mechanisms involved. Incubation of L6 myotubes with the FF A palmitate significantly decreased the insulin-stimulated glucqse uptake. Importantly, the effect of palmitate was ameliorated by resveratrol. Palmitate significantly increased serine phosphorylation of IRS..; 1 and reduced insulin-stimulated Akt phosphorylation, an effect that was abolished by resveratrol. We then investigated the effect of palmitate and resveratrol on the expression and phosphorylation of JNK, mTOR, p70-S6K, and AMPK kinases. The results demonstrated that our treatments had no effect on the expression of these proteins. However, palmitate increased the phosphorylation of mTOR and p70- S6K, whereas resveratrol abolished this effect and increased the phosphorylation of AMPK. Furthermore, all effects of resveratrol were abolished with sirtuin inhibitors, sirtinol and nicotinamide. These results indicate that resveratrol ameliorated FF A-induced insulin resistance by regulating mTOR and p70-S6K phosphorylation in skeletal muscle cells, through a mechanism involving sirtuins.

Human skeletal muscle pyruvate dehydrogenase phosphatase activity and expression : the effect of aerobic capacity

Activation of pyruvate dehydrogenase (PDH), which converts pyruvate into acetyl-CoA, is accomplished by a pair of specific phosphatases (PDP 1 & 2). A cross-sectional study investigating the effect of aerobic capacity on PDP activity and expression found that: 1) PDP activity and PDP! protein expression were positively correlated with most aerobic capacity measures in males (n=lS), but not females (n=12); 2) only males showed a positive correlation between PDP activity and PDPl protein expression (r=0.47; p=O.05), indicating that the increase in PDP activity in males is largely explained by increased PDPl protein expression, but that females rely on another level for PDP activity regulation; and 3) PDP} and Ela protein expression increase in unison when expressed relative to the E2 core. These data suggest that with increased aerobic capacity there is an increased capacity for carbohydrate oxidation through PDH, via El a, and an increased ability to activate PDH, via PDP, when exercising maximally.

Muscle strength and activation characteristics of power- trained and non-athlete boys and men

During maturation, muscle strength is enhanced through muscle growth, although neuro-muscular factors are also believed to be involved. In adults, training for power sports has been shown to enhance muscle strength and activation. The purpose of this study was to examine muscle strength and activation in power-trained athletes (POW) compared with non-athletes (CON), in boys and in adults. After familiarization subjects performed ten 5-s explosive maximal voluntary contractions for elbow and knee flexion and extension. The adults were stronger then the boys and the adult POW were stronger then the adult CON, even after correction for muscle size. Normalized rate of torque development was higher in the adults then in the boys and higher in the POW then CON boys. The rate of muscle activation was higher in the adults and POW groups. The results suggest that maturation and power-training have an additive effect on muscle activation.

Mode of action of FMRFamide-like peptide on drosophila body wall muscle conractions

Neuropeptides are the largest group of signalling chemicals that can convey the information from the brain to the cells of all tissues. DPKQDFMRFamide, a member of one of the largest families of neuropeptides, FMRFamide-like peptides, has modulatory effects on nerve-evoked contractions of Drosophila body wall muscles (Hewes et aI.,1998) which are at least in part mediated by the ability of the peptide to enhance neurotransmitter release from the presynaptic terminal (Hewes et aI., 1998, Dunn & Mercier., 2005). However, DPKQDFMRFamide is also able to act directly on Drosophila body wall muscles by inducing contractions which require the influx of extracellular Ca 2+ (Clark et aI., 2008). The present study was aimed at identifying which proteins, including the membrane-bound receptor and second messenger molecules, are involved in mechanisms mediating this myotropic effect of the peptide. DPKQDFMRFamide induced contractions were reduced by 70% and 90%, respectively, in larvae in which FMRFamide G-protein coupled receptor gene (CG2114) was silenced either ubiquitously or specifically in muscle tissue, when compared to the response of the control larvae in which the expression of the same gene was not manipulated. Using an enzyme immunoassay (EIA) method, it was determined that at concentrations of 1 ~M- 0.01 ~M, the peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. In addition, the physiological effect of DPKQDFMRFamide at a threshold dose was not potentiated by 3-lsobutyl-1-methylxanthine, a phosphodiesterase inhibitor, nor was the response to 1 ~M peptide blocked or reduced by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. The response to DPKQDFMRFamide was not affected in the mutants of the phosholipase C-~ (PLC~) gene (norpA larvae) or IP3 receptor mutants, which suggested that the PLC-IP3 pathway is not involved in mediat ing the peptide's effects. Alatransgenic flies lacking activity of calcium/calmodul in-dependent protein kinase (CamKII showed an increase in muscle tonus following the application of 1 JlM DPKQDFMRFamide similar to the control larvae. Heat shock treatment potentiated the response to DPKQDFMRFamide in both ala1 and control flies by approximately 150 and 100 % from a non heat-shocked larvae, respectively. Furthermore, a CaMKII inhibitor, KN-93, did not affect the ability of peptide to increase muscle tonus. Thus, al though DPKQDFMRFamide acts through a G-protein coupled FMRFamide receptor, it does not appear to act via cAMP, cGMP, IP3, PLC or CaMKl1. The mechanism through which the FMRFamide receptor acts remains to be determined.

TIME COURSE OF SIGNALING PROCESSES INVOLVED WITH EXTRACELLULAR OSMOTIC - INDUCED GLUCOSE UPTAKE IN MAMMALIAN SKELETAL MUSCLE

Extracellular hyper-osmotic (HYPER) stress increases glucose uptake to defend cell volume, when compared to iso-osmotic (ISO) conditions in skeletal muscle. The purpose of this study was to determine a time course for changes in common signaling proteins involved in glucose uptake during acute hyper-osmotic stress in isolated mammalian skeletal muscle. Rat extensor digitorum longus (EDL) muscles were excised and incubated in a media formulated to mimic ISO (290 ± 10 mmol/kg) or HYPER (400 ± 10 mmol/kg) extracellular condition (Sigma Media-199). Signaling mechanisms were investigated by determining the phosphorylation states of Akt, AMPK, AS160, cPKC and ERK after 30, 45 and 60 minutes of incubation. AS160 was found to be significantly more phosphorylated in HYPER conditions compared to ISO after 30 minutes (p<0.01). It is speculated that AS160 phosphorylation increases glucose transporter 4 (GLUT4) content at the cell surface thereby facilitating an increase in glucose uptake under hyper-osmotic stress.

Sarcolemmal lipid analysis from mechanically skinned rat muscle fibres.

Membrane lipid composition, which includes phospholipid (PL) headgroup, and fatty acid (FA) saturation, has been shown to affect cellular function. The sarcolemma (SL) membrane is integral to skeletal muscle function and health. Previous studies assessing SL lipid composition are limited as they have 1) restricted analysis to a PL level and neglected FA composition and 2) relied on aggressive membrane isolation procedures resulting in t-tubule and sarcoplasmic reticulum contamination and unknown levels of nuclear and mitochondrial contamination. Thus, to overcome these limitations, this study assessed a method of individually skinned skeletal muscle fibres as an alternative to analyze complete sarcolemmal membrane lipid composition. The major findings of this study were 1) complete SL lipid composition can be obtained 2) the SL had higher sphingomyelin content than previous studies and 3) the SL membrane had minimal nuclear and mitochondrial contamination and was void of contamination from sarcoplasmic reticulum and t-tubules.

Protein Turnover in Rat Skeletal Muscle During In Vitro Hypo-Osmotic Stress

Hypo-osmolality influences tissue metabolism, but research on protein turnover in skeletal muscle is limited. The purpose of this investigation was to examine the effects of hypo-osmotic stress on protein turnover in rat skeletal muscle. We hypothesized increased protein synthesis and reduced degradation following hypo-osmotic exposure. EDL muscles (n=8/group) were incubated in iso-osmotic (290 Osm/kg) or hypo-osmotic (190 Osm/kg) modified medium 199 (95% O2, 5% CO2, pH 7.4, 30±2 °C) for 60 min, followed by 75 min incubations with L-U[14C]phenylalanine or cycloheximide to determine protein synthesis and degradation. Immunoblotting was performed to assess signalling pathways involved. Phenylalanine uptake and incorporation were increased by 199% and 169% respectively in HYPO from ISO (p < 0.05). This was supported by elevated phosphorylation of mTOR Ser2448 (+12.5%) and increased Thr389 phosphorylation on p70s6 kinase (+23.6%) (p < 0.05). Hypo-osmotic stress increased protein synthesis and potentially amino acid uptake. Future studies should examine the upstream mechanisms involved.

Regulation of Protein Turnover during Hyper-osmotic Stress in Skeletal Muscle

The purpose of this study was to examine the effect of hyper-osmotic stress on protein turnover in skeletal muscle tissue using an established in-vitro model. Rat EDL muscles were incubated in either hyper-osmotic (400 ± 10 Osm) or isoosmotic (290 ± 10 Osm) custom-modified media (Gibco). L-[14C]-U-phenylalanine (n=8) and cycloheximide (n=8) were used to quantify protein synthesis and degradation, respectively. Western blotting analyses was performed to determine the activation of protein synthesis and degradation pathways. During hyperosmotic stress, protein degradation increased (p<0.05), while protein synthesis was decreased (p<0.05) as compared to the iso-osmotic condition. The decline in protein synthesis was accompanied by a decrease (p<0.05) in p70s6 kinase phosphorylation, while the increase in protein degradation was associated with an increase (p<0.05) in autolyzed calpain. Therefore, hyper-osmotic extracellular stress results in an intracellular catabolic environment in mammalian skeletal muscle tissue.

Reliability of muscle fiber conduction velocity in the tibialis anterior

This document could not have been completed without the hard work of a number of individuals. First and foremost, my supervisor, Dr. David Gabriel deserves the utmost recognition for the immense effort and time spent guiding the production of this document through the various stages of completion. Also, aiding in the data collection, technical support, and general thought processing were Lab Technician Greig Inglis and fellow members of the Electromyographic Kinesiology Laboratory Jon Howard, Sean Lenhardt, Lara Robbins, and Corrine Davies-Schinkel. The input of Drs. Ted Clancy, Phil Sullivan and external examiner Dr. Anita Christie, all members ofthe assessment committee, was incredibly important and vital to the completion of this work. Their expertise provided a strong source of knowledge and went to ensure that this project was completed at exemplary level. There were a number of other individuals who were an immense help in getting this project off the ground and completed. The donation of their time and efforts was very generous and much needed in order to fulfill the requirements needed for completion of this study. Finally, I cannot exclude the contributions of my family throughout this project especially that of my parents whose support never wavers.

Investigating the Biological Effects of Rosemary (Rosmarinus officinalis L.) Extract on Skeletal Muscle Glucose Uptake

Skeletal muscle (SKM) is the most important tissue in maintaining glucose homeostasis and impairments in this tissue leads to insulin resistance (IR). Activation of 5’ AMP-activated kinase (AMPK) is viewed as a targeted approach to counteract IR. Rosemary extract (RE) has been reported to decrease blood glucose levels but its effects on SKM are not known. We hypothesized that RE acts directly on SKM to increase glucose uptake (GU). We found an increase in GU (184±5.07% of control, p<0.001) in L6 myotubes by RE to levels similar to insulin and metformin. Carnosic acid (CA) and rosmarinic acid (RA), major polyphenols found in RE, increased GU. RE, CA, and RA significantly increased AMPK phosphorylation and their effects on GU was reduced by an AMPK inhibitor. Our study is the first to show a direct effect of RE, CA and RA on SKM GU by a mechanism that involves AMPK activation.

Changes in mitochondrial PLIN3 and PLIN5 protein content in rat skeletal muscle following acute contraction and endurance training

Surrounding lipid droplets in skeletal muscle are the perilipin (PLIN2-5) family of proteins, regulating lipid droplet metabolism. During exercise lipid droplets provide fatty acids to the mitochondria for oxidation while increasing their proximity to each other. Whether PLIN3 and PLIN5 associate with mitochondria following contraction has not been examined. To determine whether contraction altered mitochondrial PLIN3 and PLIN5 content, sedentary and endurance trained rats underwent acute contraction. The main outcomes are; 1) mitochondrial PLIN3 content is unaltered while mitochondrial PLIN5 content is increased following an acute contraction 2) mitochondrial PLIN3 content is higher in endurance trained rats when compared to sedentary and mitochondrial PLIN5 content is similar in both conditions 3) only PLIN5 mitochondrial content is increased similarly in both groups following acute contraction. This work highlights the dynamics of these two PLIN proteins, which may have roles not only on the lipid droplet but also on the mitochondria.

Skeletal muscle protein content of lipolytic inhibitor G(0)/G(1) switch gene-2 protein: the effect of endurance training

The first and rate-limiting step of lipolysis is the removal of the first fatty acid from a triglyceride molecule; it is catalyzed by adipose triglyceride lipase (ATGL). ATGL is co-activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 protein (G0S2). G0S2 has also recently been identified as a positive regulator of oxidative phosphorylation within the mitochondria. Previous research has demonstrated in cell culture, a dose dependent mechanism for inhibition by G0S2 on ATGL. However our data is not consistent with this hypothesis. There was no change in G0S2 protein content during an acute lipolytic inducing set of contractions in both whole muscle, and isolated mitochondria yet both ATGL and G0S2 increase following endurance training, in spite of the fact that there should be increased reliance on intramuscular lipolysis. Therefore, inhibition of ATGL by G0S2 appears to be regulated through more complicated intracellular or post-translation regulation.