The capillary supply of human skeletal muscle in health and disease

BACKGROUND: Capillaries function to provide a surface area for nutrient and waste exchange with cells. The capillary supply of skeletal muscle is highly organized, and therefore, represents an excellent choice to study factors regulating diffusion. Muscle is comprised of three specific fibre types, each with specific contractile and metabolic characteristics, which influence the capillary supply of a given muscle; in addition, both environmental and genetic factors influence the capillary supply, including aging, physical training, and various disease processes. OBJECTIVE: The present study was undertaken to develop and assess the functionality of a data base, from which virtual experiments can be conducted on the capillary supply of human muscle, and the adaptations of the capillary bed in muscle to various perturbations. METHODS: To create the database, an extensive search of the literature was conducted using various search engines, and the three key words - "capillary, muscle, and human". This search yielded 169 papers from which the data for the 46 variables on the capillary supply and fibre characteristics of muscle were extracted for inclusion in the database. A series of statistical analyses (ANOVA) were done on the capillary database to examine differences in skeletal muscle capillarization and fibre characteristics between young and old individuals, between healthy and diseased individuals, and between untrained, endurance trained, endurance welltrained, and resistance trained individuals, using SAS. RESULTS: There was a significantly higher capillarization in the young compared to the old individuals, in the healthy compared to the diseased individuals, and in the endurance-trained and endurance well-trained compared to the untrained individuals. CONCLUSIONS: The results of this study support the conclusion that the capillary supply of skeletal muscle is closely regulated by factors aimed at optimizing oxygen and nutrient supply and/or waste removal in response to changes in muscle mass and/or metabolic activity.

Effects of isometric elbow flexion on electromyographic spike analysis

The main objective of this research was to examine the relationship between surface electromyographic (SEMG) spike activity and force. The secondary objective was to determine to what extent subcutaneous tissue impacts the high frequency component of the signal, as well as, examining the relationship between measures of SEMG spike shape and their traditional time and frequency analogues. A total of96 participants (46 males and 50 females) ranging in age (18-35 years), generated three 5-second isometric step contractions at each force level of 40, 60, 80, and 100 percent of maximal voluntary contraction (MVC). The presentation of the contractions was balanced across subjects. The right arm of the subject was positioned in the sagittal plane, with the shoulder and elbow flexed to 90 degrees. The elbow rested on a support in a neutral position (mid pronation/mid supination) and placed within a wrist cuff, fastened below the styloid process. The wrist cuff was attached to a load cell (JR3 Inc., Woodland, CA) recording the force produced. Biceps brachii activity was monitored with a pair of Ag/AgCI recording electrodes (Grass F-E9, Astro-Med Inc., West Warwick, RI) placed in a bipolar configuration, with an interelectrode distance (lED) of 2cm distal to the motor point. Data analysis was performed on a I second window of data in the middle of the 5-second contraction. The results indicated that all spike shape measures exhibited significant (p < 0.01) differences as force increase~ from 40 to 100% MVC. The spike shape measures suggest that increased motor unit (MU) recruitment was responsible for increasing force up to 80% MVC. The results suggested that further increases in force relied on MU III synchronization. The results also revealed that the subcutaneous tissue (skin fold thickness) had no relationship (r = 0.02; P > 0.05) with the mean number of peaks per spike (MNPPS), which was the high frequency component of the signal. Mean spike amplitude (MSA) and mean spike frequency (MSF) were highly correlated with their traditional measures root mean square (RMS) and mean power frequency (MPF), respectively (r = 0.99; r = 0.97; P < 0.01).

The surface electromyography-force relationship during isometric dorsiflexion in males and females

This study evaluated sex-related differences in the tibialis anterior (TA) surface electromyography (EMG) to force relationship. One-hundred participants (50 males and 50 females) performed three isometric contractions at 20, 40, 60, 80, and 100% of maximal voluntary contraction (MVC) in an apparatus designed to isolate the action of the dorsiflexors. The surface EMG signal was amplified (lOOOx), band-pass filtered (10-500Hz), and sampled at 2048 Hz. The load cell signal was low-passed filtered at 100 Hz and sampled at the same rate. Males were stronger than females {P <0.05). However, there was no significant difference in root-mean-square (RMS) values between sexes {P <0.05). Both sexes exhibited a quadratic increase in RMS across force levels (P <0.05). The mean power frequency (MNF) for males was greater than for females {P <0.05). Males and females exhibited a linear increase in both frequency measures up to 80% of MVC (P <0.05). Between 80 and 100% MVC, the frequency values for the females plateaued while males showed a decrease {P <0.05). The magnitude of the difference in MNF between males and females was consistent with sex-specific TA physiology. In general, the pattern of means for RMS and MNF between males and females revealed no sex-related differences in the surface EMG/force relationship. We therefore conclude that there are no sex-related differences in the gradation of muscle force.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

CCK, dietary variety, and food intake in rats /

Among the environmental factors that can affect food intake is the extent of dietary variety available in the environment. Numerous studies have demonstrated that variety in a meal can increase the amount of food consumed in humans, rats, and other species. A physiological mechanism that has been demonstrated to affect food intake is the gut peptide cholecystokinin (CCK) which is released from the upper small intestine during the ingestion of food. Peripherally administered CCK has a robust inhibitory effect on the intake of a single-food meal. Thus, dietary variety and CCK both affect meal size, with dietary variety increasing intake and CCK decreasing intake. This raises the question ofhow dietary variety and CCK might interact to affect meal size. Previous studies of CCK's effects have focused on situations in which only one food was available for consumption. However, in an animal's natural environment it would frequently occur that the animal would come across a number of foods either simultaneously or in quick succession, thus providing the animal access to a variety of foods during a meal. Accordingly, the effect ofCCK on food intake in single-food and multiple-food meals was examined. It was found that food intake was greater in multiple-food than in single-food meals provided that foods in the multiplefood meal were presented either simultaneously or in increasing order of preference. When foods in the multiple-food meal were presented in decreasing order of preference, intake was similar to that observed in single-food meals. In addition, it was found that CCK inhibited food intake in a dose-dependent manner, and that its effects on food intake were similar regardless of meal type. Therefore, the inhibitory effects ofCCK were not diminished when a variety of foods were available for consumption. Furthermore, the finding that CCK did not differentially affect the intake of the two types of meals does not provide support for the recent-foods hypothesis which postulates that CCK decreases food intake by reducing the palatability of only recently consumed foods. However, it is consistent with the all-foods hypothesis, which predicts that CCK reduces food intake by decreasing the palatability of all foods. The 600 ng/kg dose of the CCK^-antagonist lorglumide significantly antagonized the inhibitory effect of exogenous CCK on food intake, and the magnitude of this effect was similar for both types of meal. These results suggest that exogenous CCK inhibits food intake through the activation ofCCK^ receptors. However, when administered by itself, the 600^ig/kg dose of lorglumide did not increase food intake in either single-food or multiple-food meals, suggesting that peripheral endogenous CCK may not play a major role in the control of food intake.

Decreased motor unit firing rate in the potentiated tibialis anterior in humans

With repeated activity, force production, rate of force production, and relaxation time are impaired. These are characteristics ofa fatigued muscle (Vandenboom, 2004). However, brief bouts of near maximal to maximal activity results in the increased ability of the muscle to generate force, termed post activation potentiation (P AP)(V andervoort et aI., 1983). The purpose of the present study was to characterize motor unit firing rate (MUFR) in the unfatigued, potentiated tibialis anterior (TA). Using a quadrifilar needle electrode, MUFR was measured during a 5s 50% MVC in which the TA was either potentiated or unpotentiated; monopolar electrodes measured surface parameters. A lOs MVC was used to potentiate the muscle. Firing rate decreased significantly from 20.15±2.9Opps to 18.27±2.99pps, while mean power frequency decreased significantly from 60. 13±7.75 Hz to 53.62±8.56 Hz. No change in root mean square (RMS) was observed. Therefore, in the present study, MUFR decreases in response to a potentiated TA.

The influence of myosin regulatory light chain phosphorylation on the contractile performance of fatigued mammalian skeletal muscle

ABSTRACT The myosm regulatory light chain (RLC) of type II fibres is phosphorylated by Ca2+ -calmodulin dependent myosin light chain kinase (skMLCK) during muscular activation. The purpose of this study was to explore the effect of skMLCK gene ablation on the fatigability of mouse skeletal muscles during repetitive stimulation. The absence of myosin RLC phosphorylation in skMLCK knockout muscles attenuated contractile performance without a significant metabolic cost. Twitch force was potentiated to a greater extent in wildtype muscles until peak force had diminished to ~60% of baseline (37.2 ± 0.05% vs. 14.3 ± 0.02%). Despite no difference in peak force (Po) and shortening velocity (Vo), rate of force development (+dP/dt) and shortening-induced deactivation (SID) were almost two-fold greater in WT muscles. The present results demonstrate that myosin RLC phosphorylation may improve contractile performance during fatigue; providing a contractile advantage to working muscles and protecting against progressive fatigue.

The reliability of an isometric test based on constant perception of effort

To date there is no documented procedure to extrapolate findings of an isometric nature to a whole body performance setting. The purpose of this study was to quantify the reliability of perceived exertion to control neuromuscular output during an isometric contraction. 21 varsity athletes completed a maximal voluntary contraction and a 2 min constant force contraction at both the start and end of the study. Between pre and post testing all participants completed a 2 min constant perceived exertion contraction once a day for 4 days. Intra-class correlation coefficient (R=O.949) and standard error of measurement (SEM=5.12 Nm) concluded that the isometric contraction was reliable. Limits of agreement demonstrated only moderate initial reliability, yet with smaller limits towards the end of 4 training sessions. In conclusion, athlete's na"ive to a constant effort isometric contraction will produce reliable and acceptably stable results after 1 familiarization sessions has been completed.

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.

The effect of a segmental, localized lower limb cooling protocol on muscular strength and balance

The human neuromuscular system is susceptible to changes within the thermal environment. Cold extrinsic temperatures can significantly reduce muscle and nervous system function and communication, which can have consequences for motor performance. A repeated measures design protocol exposed participants to a 12°C cold water immersion (CWI) up to the ankle, knee, and hip to determine the effect that reduced skin and muscle temperature had on balance and strength task execution. Although a linear reduction in the ability to perform balance tasks was seen from the control condition through to the hip CWI, results from the study indicated a significant reduction in dynamic balance (Star Excursion Balance Test reach distance) performance from only the hip CWI (P<0.05). This reduced performance could have been due to an increase in joint stiffness, increased agonist-antagonist co-contraction, and/or reduced isokinetic muscular strength. Reduced physical performance due to cold temperature could negatively impact outdoor recreational athletics.

The role of skeletal muscle PLIN proteins at rest and following lipolytic stimulation

This thesis investigated the subcellular location of skeletal muscle PLIN proteins (PLIN2, PLIN3, and PLIN5) as well as protein interactions with ATGL and HSL at rest and following lipolytic stimulation. In addition, the serine phosphorylation state of PLIN2, PLIN3, and PLIN5 was determined at rest and following lipolytic stimulation. An isolated whole muscle technique was used to study the effects of contraction and epinephrine-induced lipolysis. This method allowed for the examination of the effects of contraction and epinephrine alone and in combination. Further, the soleus was chosen for investigating the role of PLIN proteins in skeletal muscle lipolysis due to its suitability for isolated incubation, and the fact that it is primarily oxidative in nature (~80% type I fibres). It has also been previously shown to have the greatest reliance on lipid metabolism and for this reason is ideal for investigating the role of PLIN proteins in lipolysis. Immunofluorescence microscopy revealed that skeletal muscle lipid droplets are partially co-localized to both PLIN2 and PLIN5 and that contraction does not affect the amount of colocalization, indicating that PLIN5 is not recruited to lipid droplets with contraction (PLIN2 ~65%; PLIN5 ~56%). Results from the immunoprecipitation studies revealed that with lipolysis in skeletal muscle the interaction between ATGL and CGI-58 is increased (study 2: 128% with contraction, p<0.05; study 3: 50% with contraction, 25% epinephrine, 80% contraction + epinephrine, p>0.05). Further PLIN2, PLIN3, and PLIN5 all interact with ATGL and HSL, while only PLIN3 and PLIN5 interact with CGI-58. Among these interactions, the association between PLIN2 and ATGL decreases with lipolytic stimulation (study 2: 21% with contraction, p<0.05). Finally our results demonstrate that PLIN3 and PLIN5 are serine phosphorylated at rest and that the level of phosphorylation remains unchanged in the face of either contractile or adrenergic stimulation. In summary, the regulation of skeletal muscle lipolysis is a complex process involving multiple proteins and enzymes. The skeletal muscle PLIN proteins likely play a role in skeletal muscle lipid droplet dynamics, and the data from this thesis indicate that these proteins may work together in regulating lipolysis by interaction with both ATGL and HSL.

Proprioceptive Neuromuscular Facilitation of the Wrist Flexors

The present study examined a wrist extension-to-flexion contraction pattern that was theorized to result in proprioceptive neuromuscular facilitation. However, the “reversal of antagonists” contraction pattern may have, alternatively, interfered with motor learning-related increases in strength. Participants (N=24) were matched on predicted strength and randomly assigned to either the control or experimental group. Training occurred during three test sessions within a one-week period. Retention and transfer (crossed-condition) tests were administered during a fourth test session two- weeks later. Both groups exhibited comparable increases in strength (20.2%) and decreases in muscle coactivation (35.2%), which were retained and transferred. Decreases in error and variability of the torque traces were associated with parallel decreases in variability of muscle activity. The reversal of antagonists technique did not interfere with motor learning-related increases in strength and decreases in variability. However, the more complex contraction pattern failed to result in proprioceptive neuromuscular facilitation of strength.

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

The action of octopamine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.