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No description available.

Although lymphatic vessel behavior is analogous to hearts (e.g. systole and diastole) and blood vessels (e.g. basal tone), hearts and blood vessels have fundamentally different contractile properties. While summation during contraction is minimized in the heart, summation is necessary for tonic contraction in blood vessels. Because lymphatic vessel behavior mimics cardiac and vascular behavior, we hypothesized that above a critical contraction frequency there is significant summation, evidenced by significantly increased diastolic active tension (i.e. basal tone). We used an isovolumic, controlled-flow preparation to examine the interaction of contraction cycle-time with contraction frequency. Using segments of isolated lymphatic vessels (~1 cm in length and 3-4 mm in diameter) from bovine mesentery, we measured transmural pressure and diameter for end-diastole and end-systole during spontaneous contractions for 10 volume steps. We found time between contractions (beat-to-beat period) decreases with increasing diameter, and total contraction time (vessel twitch length, 11.08 ± 1.54 s) slightly increases with increasing diameter. At the intersection of these relationships, there is a critical period, below which the vessel does not have time to fully relax. Above the diameter at the critical period, diastolic active tension (end-diastolic minus passive vessel tension) significantly increases with increases in diameter (309 to 562% change in slope, p<0.0001), and, below the critical period, diastolic active tension increases with decreases in beat-to-beat period (712 to 2208% change in slope, p<0.0014). Because this transition occurs within a physiological range, it suggests summation may be crucial for lymphatic vessel function as a pump and a conduit.

Although lymphatic vessel behavior is analogous to hearts (e.g. systole and diastole) and blood vessels (e.g. basal tone), hearts and blood vessels have fundamentally different contractile properties. While summation during contraction is minimized in the heart, summation is necessary for tonic contraction in blood vessels. Because lymphatic vessel behavior mimics cardiac and vascular behavior, we hypothesized that above a critical contraction frequency there is significant summation, evidenced by significantly increased diastolic active tension (i.e. basal tone). We used an isovolumic, controlled-flow preparation to examine the interaction of contraction cycle-time with contraction frequency. Using segments of isolated lymphatic vessels (~1 cm in length and 3-4 mm in diameter) from bovine mesentery, we measured transmural pressure and diameter for end-diastole and end-systole during spontaneous contractions for 10 volume steps. We found time between contractions (beat-to-beat period) decreases with increasing diameter, and total contraction time (vessel twitch length, 11.08 ± 1.54 s) slightly increases with increasing diameter. At the intersection of these relationships, there is a critical period, below which the vessel does not have time to fully relax. Above the diameter at the critical period, diastolic active tension (end-diastolic minus passive vessel tension) significantly increases with increases in diameter (309 to 562% change in slope, p<0.0001), and, below the critical period, diastolic active tension increases with decreases in beat-to-beat period (712 to 2208% change in slope, p<0.0014). Because this transition occurs within a physiological range, it suggests summation may be crucial for lymphatic vessel function as a pump and a conduit.

BACKGROUND: Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM. METHODS: To test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured. RESULTS: No effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested. CONCLUSIONS: These findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders.

The ability of an individual to perform a functional movement is determined by a range of mechanical properties including the force and power producing capabilities of muscle, and the interplay of force and power outputs between different muscle groups (intermuscular coordination). Cycling presents an ideal experimental model to investigate these factors as it is an ecologically valid multi-joint movement in which kinematics and resistances can be tightly controlled. The overall goal of this thesis was thereby to investigate mechanical muscle properties and intermuscular coordination during maximal and submaximal cycling. The specific research objectives were (a) to determine the contribution of these factors to maximal and submaximal cycling, and (b) to determine the extent to which these factors set the limit of performance in maximal cycling. The contribution of mechanical muscle properties and intermuscular coordination were investigated by observing joint kinetics and joint kinematics across variations in crank lengths and pedalling rates during maximal and submaximal cycling. The extent to which these factors set the limit of performance in maximal cycling was assessed by observing joint-level kinetics of world-class track sprint cyclists. The findings of this investigation formed the rationale for the fourth study which used an ankle brace intervention to investigate the effects of a fixed ankle on joint biomechanics and performance during maximal cycling. Sophisticated intermuscular coordination strategies were observed in both submaximal and maximal cycling, supporting the generalised notion that high levels of intermuscular coordination are required to perform functional multi-joint movement tasks. Furthermore, it was found that the maximal cycling task is governed by the interaction of the force-velocity relationship and excitation-relaxation kinetics, suggesting that task-specific mechanical muscle properties are the dominant contributing factor in maximal movements. In terms of the extent to which these factors limit performance in maximal cycling, it was demonstrated that world-class track sprint cycling performance is governed by the ability to generate higher joint moments at the ankle and knee, and that these joint moments are facilitated by enhanced muscular strength about these joints. These findings allow us to speculate that the limits of performance in maximal human movements lie in extraordinary muscular strength in task-specific joint actions. These findings give an insight into the mechanisms that underpin maximal and submaximal cycling, and provide a theoretical framework with which to understand sprint cycling performance. This knowledge has significant applied relevance for athletes and coaches seeking to improve sprint cycling performance.

A forward dynamic model was developed to predict the performance of Spinal Cord Injured (SCI) individuals cycling an isokinetic ergometer using Neuromuscular Electrical Stimulation (NMES) to elicit contractions of the quadriceps, hamstring and gluteal muscles. Computer simulations were performed using three inter-connected models: a kinematic model of segmental linkages, a muscle model predicting forces in response to stimulation, and a kinetic model predicting ergometer pedal forces resulting from muscle stimulation. Specific model parameters for SCI individuals were determined through measurements from isometric and isokinetic contractions of the quadriceps muscles elicited using surface stimulation. The muscle model was fitted to data resulting from these isolated experiments in order to tailor the model's parameters to characteristics of muscles from SCI individuals. Isometric data from a range of knee angles were used to fit tendon slack lengths to the rectus femoris and vastus muscles. Adjustments to the quadriceps moment arm function were not able to improve the match between measured and modelled knee extension torques beyond those using moment arms taken from available literature. Similarly, literature values for constants from the muscle force - velocity relationship provided a satisfactory fit to the decline in torque with angular velocity, and parameter fitting did not improve this fit. Passive visco-elastic resistance remained constant for all velocities of extension except the highest (240 deg/s). Since knee angular velocities this high were not experienced during cycling, a visco-elastic dampener was not included within the present cycling model. The rise and fall in torque following NMES onset and cessation were used to fit constants to match the rate of change in torque. Constants for the rise in torque following NMES onset were significantly altered by changes in knee angle, with more extended angles taking longer for torque to rise. This effect was small, however, within the range of angles used during cycling, and consequently was not included within the cycling model. The decline in torque after NMES cessation was not affected by knee angle. A period of five minutes cyclical isometric activity of the quadriceps resulted in torque declining by more than 75% from rested levels. The activation time constants were largely unaffected by this fatigue, however, with only a small increase in the time for torque to decline, and no change in rise time or the delay between stimulation changes and resulting torque changes. The cycling model, therefore, did not incorporate any effect for changes in activation timing with fatigue. Performance of the full model was evaluated through measurements taken from SCI individuals cycling a constant velocity ergometer using NMES elicited contractions of the quadriceps, hamstring and gluteal muscles. Pedal transducers measured forces applied to the pedals for comparison between measured and modelled values. A five minute period of continuous cycling using just the quadriceps muscles produced similar results to those found for isolated knee extension. External power output dropped by 50% over the five-minute period, however there was no change in the pattern of torque production with fatigue. Cycling experiments were conducted using single muscle groups across a range of NMES firing angles. Experimental protocols were designed to seek the firing angles for each muscle that maximised power output by that group. Changes in power output in response to firing angle changes were not large, however, in comparison to the effects of cumulative fatigue and inconsistent power output between trials. This lead to large uncertainties in the determination of those firing angles that maximised power output by each muscle. Results suggest that NMES firing angles to maximise power output by the quadriceps muscles were relatively similar for each subject. For the hamstring muscles, however, substantial differences were observed in the range of firing angles that maximised power output. Results for the gluteal muscles were variable, with some subjects not applying any measurable torque to the cranks, even with maximal stimulation applied. The model produced a good match to experimental data for the quadriceps muscles, both in the shape of pedal force curves and the firing angles that maximised external power output. The individual variability in hamstring responses was not, however, predicted by the model. Modification of the relative size of the hamstrings' moment arms about the hip and knee substantially improved the match between measured and modelled data. Analysis of results suggests that individual variability in the relative size of these moment arms is a major cause of variation in individual's response to hamstring stimulation. There were apparent limitations in the model's ability to predict the shape of crank torques resulting from stimulation of the gluteus maximus muscle. It is suggested that further research be conducted to enable modelling of this muscle using a range of fibre lengths and moment arms.

Following contractile activity, fast twitch skeletal muscle exhibits increases in submaximal force known as potentiation. Although there is no consensus on the purpose of potentiation, it is known to enhance power during rapid dynamic contractions and counteract the early stages of peripheral fatigue. Potentiation is primarily attributed to phosphorylation of the myosin regulatory light chain (RLC) through a calcium-mediated process which results in increased calcium-sensitivity of crossbridge formation. However, there is a growing body of evidence showing that potentiation can be achieved in the absence of RLC phosphorylation, albeit to a lesser degree. A secondary characteristic of the potentiated contraction is an acceleration of relaxation properties, which could be teleologically beneficial to enhance the cycling rate of rapid motions (e.g. running). However, accelerated relaxation is inconsistent with elevations in calcium-sensitivity as this would tend to slow the time course and slow relaxation. Therefore there are multiple mechanisms involved in potentiation, some of which enhance crossbridge formation, and some of which enhance crossbridge detachment. A possible explanation for these events involves contraction-induced changes in the intracellular cytosolic calcium signal that triggers muscle contraction. For example, elevations in submaximal force could be achieved by increasing the amplitude of the calcium signal while enhanced relaxation speed could be achieved by a shorter duration of the calcium signal. Thus the main objective of this thesis was to investigate the contribution of changes in cytosolic Ca²⁺ to force potentiation. To achieve this objective, intact lumbrical muscles were extracted from the hind feet of C57BL/6 mice for use as the experimental model. The first study in this thesis examined cytosolic calcium signals during posttetanic potentiation using high (AM-fura-2 and AM-indo-1) and low (AM-furaptra) affinity calcium-sensitive fluorescent indicators to monitor resting and peak calcium respectively, both before and after a potentiating stimulation protocol of 2.5 s of 20 Hz stimulation at 37^oC. This protocol resulted in an immediate 17±3% increase in twitch force (n=10; P<0.05), though this potentiation dissipated quickly, lasting only 30 s. Resting cytosolic Ca²⁺ was also increased following the potentiating stimulus as indicated by increases of 11.1 ± 1.3% and 8.1 ± 1.3% in the fura-2 and indo-1 fluorescence ratios respectively. Like the force potentiation, these increases were short lived, lasting 20-30 s. No changes were detected in either the amplitude or kinetics of the Ca²⁺ transients following the potentiating stimulus. Western blotting analysis of the myosin heavy chain isoforms which determine the contractile phenotype of lumbrical muscle revealed predominance of fast type IIX fibres, while immunohistochemical analysis of proteins important for relaxation, namely parvalbumin, sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) 1a and SERCA2a, revealed that the expression of these proteins in lumbrical moderated those found in the soleus (slow) and EDL (fast) archetypes. Surprisingly, despite the fast phenotype of the lumbrical, it exhibited low expression of the skeletal muscle isoform of myosin light chain kinase, the enzyme responsible for phosphorylating the myosin RLC, and high expression of myosin targeting phosphatase subunit 2, the enzyme responsible for dephosphorylating the myosin RLC. These data were corroborated by a complete lack of myosin RLC phosphorylation in either the rested or potentiated states. It was thus concluded that elevations in resting cytosolic calcium concentration, in the absence of changes in the intracellular calcium transient and RLC phosphorylation, can potentiate twitch force. The next objective of this thesis was to determine if there are changes in the cytosolic calcium transient during staircase potentiation, defined as a stepwise increase in twitch force during low frequency stimulation (<10 Hz). Staircase potentiation has been repeatedly demonstrated to exhibit more robust potentiation than posttetanic potentiation in the absence of RLC phosphorylation. It was hypothesized that while the calcium transient is not altered during posttetanic potentiation, it may be an important potentiating factor in staircase due to the lower rest intervals between successive contractions. The effects of temperature on the intracellular calcium transient during staircase potentiation were also examined as part of this investigation. Here, lumbricals were loaded with AM- furaptra and then subjected to stimulation at 8 Hz for 8.0 s to induce staircase potentiation at either 30 or 37^oC. This stimulation protocol resulted in a 26.8 ± 3.2 % increase in twitch force at 37^oC (P<0.05) and a 6.8 ± 1.9 % decrease in twitch force at 30^oC (P<0.05) at the 8 s mark. Both the peak amplitude and the calcium-time integral of the calcium transient decreased during the first 2.0 s of the protocol (P<0.05), however these decreases were greater at 30^oC than 37^oC (P<0.05 amplitude; P=0.09 area). While peak amplitude remained low throughout the duration of the protocol, the calcium-time integral began to increase after the 2 s time point (P<0.05), a change reflective of the progressive increases in the 50% decay time and full width at half maximum of the calcium transient (P<0.05). Regression analysis of raw furaptra fluorescence ratios revealed a progressive decline in the peak amplitude of the calcium transients throughout the protocol which was not present at 37^oC. The increases in the duration of the calcium transient were mirrored by increases in the half relaxation time of the twitch contractions at both 30 and 37^oC, which had initially been reduced by ~20 and 9 % at 30 and 37^oC during the first 2 s of the protocol. Therefore the degree of staircase potentiation depends, in part, on the magnitude of the decline in the amplitude and the degree of slowing of the cytosolic calcium transient. The declines in calcium transient amplitude noted above occurred simultaneously with increased rates of relaxation and abbreviated contraction times. To determine if there was a causal relationship between the reduced amplitude and the faster contractions, AM-furaptra-loaded lumbrical muscles were stimulated at 8 Hz for 2 s in the presence and absence of caffeine, an agonist of the calcium release channel. Caffeine treatment attenuated the decline of the calcium transient amplitude (P<0.05), and was associated with greater potentiation at 37^oC (P<0.05), and attenuated force loss at 30^oC (P<0.05). Despite the increases in calcium and force, the relaxation times and rates of relaxation exhibited a greater acceleration following caffeine treatment (P<0.05). Therefore the relaxation-enhancing factor during potentiated twitches cannot be attributed to the calcium transient, and must be localized to changes on the myofilament. The case for inorganic phosphate as the effector is made. Similar to the findings of the posttetanic potentiation study, the resting cytosolic calcium concentration was elevated during staircase potentiation, as revealed by fura-2 ratio signals. The largest increase occurring immediately following the first twitch of the protocol. This coincided with the largest increases in force potentiation at both 30 and 37^oC. This finding is in accordance with the initial conclusion that elevations in resting calcium can enhance twitch force and contribute to potentiation, though the mechanism of action is unclear. One possibility is that increases in resting calcium, sub-threshold for force production, can enhance the number of attached but non-force producing crossbridges, thereby accelerating the transition of crossbridges to force-producing states upon calcium-release following stimulation. To test this hypothesis, the resting stiffness, a measure of crossbridge attachment, of lumbrical muscles was examined before and after a potentiating stimulus of 20 Hz 2.5 s. Resting stiffness was assessed using sinusoidal length oscillations, ~0.5 nm per half sarcomere in amplitude and ranging in frequency from 10-200 Hz. Subsequent analysis revealed decreases in the elastic stiffness (P<0.05) that lasted for ~20 s which were greater in magnitude (P<0.05) than increases in viscous stiffness which only lasted for ~5 s. This finding is consistent with the disappearance of short range elastic component (SREC) upon stretch or muscle activation which is commonly attributed to a population of stable, bound crossbridges in resting muscle. Subsequent analysis using imposed length changes to eliminate the SREC prior to contraction had no effect on the amplitude or duration of a subsequent twitch or tetanic contraction, and the changes in elastic and viscous stiffness of resting muscle were identical whether SREC was ablated by a contraction or imposed length change. Therefore it appears that potentiation occurs without an associated increase in bound crossbridges at rest, and may actually occur with fewer bound crossbridges at rest than the unpotentiated state. The lack of effect may be related to the relaxation-enhancing factor discussed above, and be an important feature of skeletal muscle serving to protect against damage via an involuntary eccentric contraction. This thesis describes potentiation as a complex and important biological function which is the sum of factors that serve to enhance and oppose force production.

A forward dynamic model was developed to predict the performance of Spinal Cord Injured (SCI) individuals cycling an isokinetic ergometer using Neuromuscular Electrical Stimulation (NMES) to elicit contractions of the quadriceps, hamstring and gluteal muscles. Computer simulations were performed using three inter-connected models: a kinematic model of segmental linkages, a muscle model predicting forces in response to stimulation, and a kinetic model predicting ergometer pedal forces resulting from muscle stimulation. Specific model parameters for SCI individuals were determined through measurements from isometric and isokinetic contractions of the quadriceps muscles elicited using surface stimulation. The muscle model was fitted to data resulting from these isolated experiments in order to tailor the model's parameters to characteristics of muscles from SCI individuals. Isometric data from a range of knee angles were used to fit tendon slack lengths to the rectus femoris and vastus muscles. Adjustments to the quadriceps moment arm function were not able to improve the match between measured and modelled knee extension torques beyond those using moment arms taken from available literature. Similarly, literature values for constants from the muscle force - velocity relationship provided a satisfactory fit to the decline in torque with angular velocity, and parameter fitting did not improve this fit. Passive visco-elastic resistance remained constant for all velocities of extension except the highest (240 deg/s). Since knee angular velocities this high were not experienced during cycling, a visco-elastic dampener was not included within the present cycling model. The rise and fall in torque following NMES onset and cessation were used to fit constants to match the rate of change in torque. Constants for the rise in torque following NMES onset were significantly altered by changes in knee angle, with more extended angles taking longer for torque to rise. This effect was small, however, within the range of angles used during cycling, and consequently was not included within the cycling model. The decline in torque after NMES cessation was not affected by knee angle. A period of five minutes cyclical isometric activity of the quadriceps resulted in torque declining by more than 75% from rested levels. The activation time constants were largely unaffected by this fatigue, however, with only a small increase in the time for torque to decline, and no change in rise time or the delay between stimulation changes and resulting torque changes. The cycling model, therefore, did not incorporate any effect for changes in activation timing with fatigue. Performance of the full model was evaluated through measurements taken from SCI individuals cycling a constant velocity ergometer using NMES elicited contractions of the quadriceps, hamstring and gluteal muscles. Pedal transducers measured forces applied to the pedals for comparison between measured and modelled values. A five minute period of continuous cycling using just the quadriceps muscles produced similar results to those found for isolated knee extension. External power output dropped by 50% over the five-minute period, however there was no change in the pattern of torque production with fatigue. Cycling experiments were conducted using single muscle groups across a range of NMES firing angles. Experimental protocols were designed to seek the firing angles for each muscle that maximised power output by that group. Changes in power output in response to firing angle changes were not large, however, in comparison to the effects of cumulative fatigue and inconsistent power output between trials. This lead to large uncertainties in the determination of those firing angles that maximised power output by each muscle. Results suggest that NMES firing angles to maximise power output by the quadriceps muscles were relatively similar for each subject. For the hamstring muscles, however, substantial differences were observed in the range of firing angles that maximised power output. Results for the gluteal muscles were variable, with some subjects not applying any measurable torque to the cranks, even with maximal stimulation applied. The model produced a good match to experimental data for the quadriceps muscles, both in the shape of pedal force curves and the firing angles that maximised external power output. The individual variability in hamstring responses was not, however, predicted by the model. Modification of the relative size of the hamstrings' moment arms about the hip and knee substantially improved the match between measured and modelled data. Analysis of results suggests that individual variability in the relative size of these moment arms is a major cause of variation in individual's response to hamstring stimulation. There were apparent limitations in the model's ability to predict the shape of crank torques resulting from stimulation of the gluteus maximus muscle. It is suggested that further research be conducted to enable modelling of this muscle using a range of fibre lengths and moment arms.

Passive detrusor smooth muscle exhibits both viscoelastic softening and strain softening. Strain softening is a loss of stiffness following a stretch to a longer length and is reversible upon muscle activation. Because of this behavior, steady state passive force in detrusor is not constant for a given muscle length and can be adjusted by an intracellular mechanism. Thus, passive detrusor exhibits adjustable passive stiffness. Existing three-component mechanical models for muscle, the Kelvin and Voigt, are insufficient to display this characteristic. The goal of this thesis is to develop a new biomechanical model for passive force in detrusor by adding additional elements to the Kelvin or Voigt models. Eight mechanical characteristics of detrusor are identified from the literature and with three new experiments, and a novel adjustable passive stiffness model for smooth muscle is proposed. Simulations are performed to demonstrate that the model qualitatively exhibits each of the eight tissue characteristics.