Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Application of a new method in the study of pelvic floor muscle passive properties in continent women

The aim of this study was to present a new methodology for evaluating the pelvic floor muscle (PFM) passive properties. The properties were assessed in 13 continent women using an intra-vaginal dynamometric speculum and EMG (to ensure the subjects were relaxed) in four different conditions: (1) forces recorded at minimal aperture (initial passive resistance); (2) passive resistance at maximal aperture; (3) forces and passive elastic stiffness (PES) evaluated during five lengthening and shortening cycles; and (4) percentage loss of resistance after 1 min of sustained stretch. The PFMs and surrounding tissues were stretched, at constant speed, by increasing the vaginal antero-posterior diameter; different apertures were considered. Hysteresis was also calculated. The procedure was deemed acceptable by all participants. The median passive forces recorded ranged from 0.54 N (interquartile range 1.52) for minimal aperture to 8.45 N (interquartile range 7.10) for maximal aperture while the corresponding median PES values were 0.17 N/mm (interquartile range 0.28) and 0.67 N/mm (interquartile range 0.60). Median hysteresis was 17.24 N∗mm (interquartile range 35.60) and the median percentage of force losses was 11.17% (interquartile range 13.33). This original approach to evaluating the PFM passive properties is very promising for providing better insight into the patho-physiology of stress urinary incontinence and pinpointing conservative treatment mechanisms.

Le phénomène d'escalier mesuré par phonomyographie : réaction musculaire et influence du type de contraction en anesthésie

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Proprioception in ankle sprain from exercises and bandages

Ankle sprains are the most common injuries in sports, usually causing damage to the lateral ligaments. Recurrence has as usual result permanent instability, and thus loss of proprioception. This fact, together with residual symptoms, is what is known as chronic ankle instability, CAI, or FAI, if it is functional. This problem tries to be solved by improving musculoskeletal stability and proprioception by the application of bandages and performing exercises. The aim of this study has been to review articles (meta-analisis, systematic reviews and revisions) published in 2009-2015 in PubMed, Medline, ENFISPO and BUCea, using keywords such as “sprain instability”, “sprain proprioception”, “chronic ankle instability”. Evidence affirms that there does exist decreased proprioception in patients who suffer from CAI. Rehabilitation exercise regimen is indicated as a treatment because it generates a subjective improvement reported by the patient, and the application of bandages works like a sprain prevention method limiting the range of motion, reducing joint instability and increasing confidence during exercise. As podiatrists we should recommend proprioception exercises to all athletes in a preventive way, and those with CAI or FAI, as a rehabilitation programme, together with the application of bandages. However, further studies should be generated focusing on ways of improving proprioception, and on the exercise patterns that provide the maximum benefit.

Locomotor Muscle Fatigue Does Not Alter Oxygen Uptake Kinetics during High-Intensity Exercise

The V˙O2 slow component (V˙O2sc) that develops during high-intensity aerobic exercise is thought to be strongly associated with locomotor muscle fatigue. We sought to experimentally test this hypothesis by pre-fatiguing the locomotor muscles used during subsequent high-intensity cycling exercise. Over two separate visits, eight healthy male participants were asked to either perform a non-metabolically stressful 100 intermittent drop-jumps protocol (pre-fatigue condition) or rest for 33 min (control condition) according to a random and counterbalanced order. Locomotor muscle fatigue was quantified with 6-s maximal sprints at a fixed pedaling cadence of 90 rev·min−1. Oxygen kinetics and other responses (heart rate, capillary blood lactate concentration and rating of perceived exertion, RPE) were measured during two subsequent bouts of 6 min cycling exercise at 50% of the delta between the lactate threshold and V˙O2max determined during a preliminary incremental exercise test. All tests were performed on the same cycle ergometer. Despite significant locomotor muscle fatigue (P = 0.03), the V˙O2sc was not significantly different between the pre-fatigue (464 ± 301 mL·min−1) and the control (556 ± 223 mL·min−1) condition (P = 0.50). Blood lactate response was not significantly different between conditions (P = 0.48) but RPE was significantly higher following the pre-fatiguing exercise protocol compared with the control condition (P < 0.01) suggesting higher muscle recruitment. These results demonstrate experimentally that locomotor muscle fatigue does not significantly alter the V˙O2 kinetic response to high intensity aerobic exercise, and challenge the hypothesis that the V˙O2sc is strongly associated with locomotor muscle fatigue.

Fadiga e conforto do quadricípite femoral gerados por estimulação eléctrica neuromuscular com modulação da forma de onda

Mestrado em Fisioterapia

The role of intracellular calcium perturbations in muscle damage and dysfunction in mouse models of muscular dystrophy

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. DMD is clinically characterized by severe, progressive and irreversible loss of muscle function, in which most patients lose the ability to walk by their early teens and die by their early 20’s. Impaired intracellular calcium (Ca2+) regulation and activation of cell degradation pathways have been proposed as key contributors to DMD disease progression. This dissertation research consists of three studies investigating the role of intracellular Ca2+ in skeletal muscle dysfunction in different mouse models of DMD. Study one evaluated the role of Ca2+-activated enzymes (proteases) that activate protein degradation in excitation-contraction (E-C) coupling failure following repeated contractions in mdx and dystrophin-utrophin null (mdx/utr-/-) mice. Single muscle fibers from mdx/utr-/- mice had greater E-C coupling failure following repeated contractions compared to fibers from mdx mice. Moreover, protease inhibition during these contractions was sufficient to attenuate E-C coupling failure in muscle fibers from both mdx and mdx/utr-/- mice. Study two evaluated the effects of overexpressing the Ca2+ buffering protein sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) in skeletal muscles from mdx and mdx/utr-/- mice. Overall, SERCA1 overexpression decreased muscle damage and protected the muscle from contraction-induced injury in mdx and mdx/utr-/- mice. In study three, the cellular mechanisms underlying the beneficial effects of SERCA1 overexpression in mdx and mdx/utr-/- mice were investigated. SERCA1 overexpression attenuated calpain activation in mdx muscle only, while partially attenuating the degradation of the calpain target desmin in mdx/utr-/- mice. Additionally, SERCA1 overexpression decreased the SERCA-inhibitory protein sarcolipin in mdx muscle but did not alter levels of Ca2+ regulatory proteins (parvalbumin and calsequestrin) in either dystrophic model. Lastly, SERCA1 overexpression blunted the increase in endoplasmic reticulum stress markers Grp78/BiP in mdx mice and C/EBP homologous protein (CHOP) in mdx and mdx/utr-/- mice. Overall, findings from the studies presented in this dissertation provide new insight into the role of Ca2+ in muscle dysfunction and damage in different dystrophic mouse models. Further, these findings support the overall strategy for improving intracellular Ca2+ control for the development of novel therapies for DMD.

A study of the electrical basis for the inhibition and excitation in autonomically innervated smooth muscle

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.

Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient.

Spatiotemporal neuromodulation therapies engaging muscle synergies improve motor control after spinal cord injury.

Electrical neuromodulation of lumbar segments improves motor control after spinal cord injury in animal models and humans. However, the physiological principles underlying the effect of this intervention remain poorly understood, which has limited the therapeutic approach to continuous stimulation applied to restricted spinal cord locations. Here we developed stimulation protocols that reproduce the natural dynamics of motoneuron activation during locomotion. For this, we computed the spatiotemporal activation pattern of muscle synergies during locomotion in healthy rats. Computer simulations identified optimal electrode locations to target each synergy through the recruitment of proprioceptive feedback circuits. This framework steered the design of spatially selective spinal implants and real-time control software that modulate extensor and flexor synergies with precise temporal resolution. Spatiotemporal neuromodulation therapies improved gait quality, weight-bearing capacity, endurance and skilled locomotion in several rodent models of spinal cord injury. These new concepts are directly translatable to strategies to improve motor control in humans.

Efeito de um programa de exercícios para prevenção da dor lombar ao nível do endurance e co-ativação bilateral do músculo médio glúteo, sway postural e intensidade da dor

INTRODUÇÃO E OBJETIVOS: A dor lombar (DL) é considerada um dos maiores problemas socioeconómicos dos países industrializados e está associada à manutenção da posição de ereta em atividades ocupacionais e laborais. O objetivo do presente estudo é descrever e analisar o efeito de um programa de exercícios para prevenção da DL ao nível do endurance e co-ativação bilateral do músculo médio glúteo (MG), sway postural e a intensidade da DL em indivíduos que desenvolvem dor (DD) numa tarefa prolongala em pé. É expetável que os indivíduos DD, após o programa de exercícios, reduzam a intensidade subjetiva da dor, o nível de co-ativação bilateral e o sway postural e que aumentem o endurance do músculo MG. MATERIAIS E MÉTODOS: Os participantes (19 homens, 31 mulheres), sem história prévia de DL, foram sujeitos ao teste de indução de DL transitória. Antes do teste foi avaliado o endurance da musculatura lombo-pélvica pelo side-bridge teste (SBT); e durante o teste foram recolhidos dados da atividade do músculo MG através de eletromiografia (EMG) de superfície, avaliado o sway postural com recurso a um acelerómetro 3D e intensidade subjetiva da dor, pela EVA. Um aumento>10mm na EVA durante o teste categorizou os indivíduos como DD. Os participantes foram aleatoriamente distribuídos por sexo e por desenvolver ou não dor (DD/NDD) em grupo experimental (Exp) e controlo (Cont). O grupo Exp realizou um programa e exercícios no domicílio e o grupo Cont manteve as suas atividades. Após 4 semanas procedeu-se à reavaliação. RESULTADOS: Os individuos que constituem a amostra deste estudo foram agrupados de acordo com diversos critérios e avaliados vários aspectos relacionados com a DL. Os resultados revelaram que 48% dos participantes foram classificados como DD. Em muitos aspectos analisados, o comportamento dos indivíduos foi semelhante não existindo diferenças significativas (p≥0,05). No entanto, os resultados mostram que existem diferenças quanto à intensidade subjetiva da dor onde se obteve uma variação significativa entre os dois dias de teste (p<0,05) em indivíduos DDExp. Também se observou um efeito clinicamente relevante na interação da intervenção e sexo enquanto medidas repetidas ao nível do endurance. CONCLUSÕES: O programa de exercícios provou ser benéfico na redução da intensidade da dor durante uma tarefa prolongada em pé. O endurance muscular revelou uma variação positiva ao exercício. Nos participantes em estudo não houve alteração relevante nas medidas de co-ativação muscular e sway postural.

Medial and lateral hamstrings and quadriceps co-activation affects knee joint kinematics and ACL elongation : a pilot study

A review of risk factors for hamstring injury in soccer: a biomechanical approach

The aim has been to review the literature about the risk factors of hamstring injury in soccer from a biomechanical point of view. METHODOLOGY. Data bases of bibliography references were Medline, Scopus and SportDiscuss. RESULTS AND DISCUSSION. Many prospective studies have shown that the previous injury is the greatest risk factor of sustaining the injury. However the primary causes of the injury are unclear in soccer. A lack of hamstring flexibility has been one of the main injury risk factors with controversies on the results. Imbalance of isokinetic force is a risk factor but electrical coactivation of all muscles participating during knee flexion and extension are unknown in football. While the importance of lumbopelvic-hamstrings muscles synchronization during running seems to be crucial for understanding the risk of injury, no research has been developed in this topic in football. CONCLUSIONS. More research using new data recording procedures as Dynamic Scanners, Surface EMG, Inverse Dynamic Analysis are needed. The analysis of more specific movements as running, kicking or jumping is clearly required. Managers, coaches, physical trainers, physiotherapists, sport physicians and researchers should work together in order to improve the injury prevention and rehabilitation programs of football players. Key Words: sports biomechanics, soccer, hamstring injury, risk factors

Otimização da caracterização de fibras musculares estriadas em cão (Canis familiaris) por histoquímica e imunohistoquímica com potencial aplicação ao diagnóstico

Dissertação de Mestrado Integrado em Medicina Veterinária