The purpose of this thesis was to examine changes in the expression of translation regulatory proteins following both an acute bout of endurance exercise and chronic muscle contractile activity. In experiment 1, female Sprague-Dawley rats ran for 2 h at 15 m/min followed by an increase in speed of 5 m/min every 5 min until volitional fatigue. Red gastrocnemius muscle was harvested from non-exercised animals (control), immediately following cessation of exercise (0 h) and after 3 hours of recovery (3 h). Compared to control, rpS6 mRNA was elevated (p < .05) at both 0 h (+32%) and 3 h (+47%). Both eIF2Bε (+127%) and mTOR mRNA (+44%) were higher than control at 3 h, while eIF4E decreased (-24%) immediately following exercise (p < .05). Phosphorylation of mTOR (+40%) and S6K1 (+266%) also increased immediately post-exercise (p < .05). In experiment 2, female Sprague-Dawley rats underwent chronic stimulation of the peroneal nerve continuously for 7 days. The red gastrocnemius muscle was removed 24 h following cessation of the stimulation. Chronic muscle stimulation up-regulated (P < .05) mTOR protein (+74%), rpS6 (+31%), and eIF2α (+44%, P < .07), and this was accompanied by an increase in cytochrome C (+31%). Phosphorylation of rpS6 (Ser235/Ser236) was increased (+51%, P < .05), while mTOR (Ser2448) and 4E-BP1 (Thr37/46) did not change. These experiments demonstrate that acute and chronic endurance contractile activity up-regulate the mTOR signalling pathway and mitochondrial content in murine skeletal muscle. This up-regulation of the mTOR pathway may increase translation efficiency and may also represent an important control point in exercise mediated mitochondrial biogenesis. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2012-10-02 13:35:04.072

Efficient muscle regeneration is essential in mammals in order to overcome daily stress such as wounds, exercise and pathologic processes. This regeneration relies on muscle stem cells, the satellite cells. After a lesion, satellite cells are activated, proliferate and differentiate in fonctionnal muscle fibers. Our laboratory has previously shown that the transcription factor GATA-6 is expressed in the satellite cells. The present thesis confirms the expression of this factor in this cell type. Also, it seems that GATA-6 could be implicated in the maintaining of quiescence of these cells. The GATA-6 heterozygous mouse muscle is characterized by an increase level of Myf5 and Pax7+ cells. Moreover, suppression of one copy of the GATA-6 gene in a muscular dystrophy model mouse, the mdx mice, alleviates its phenotype. Further experiments on a muscle-specific GATA-6 null mouse will allow a better understanding of the role of GATA-6 in muscle regeneration. / Keywords. GATA-6, muscle regeneration, mdx, satellite cells

Galingumas yra ryšys tarp jėgos ir greičio. Galingumo ugdymo pratimai skirti skatinti neuroraumenų sistemą, kad per trumpą laiko tarpą raumuo spėtų išsitempti (ekscentrinis susitraukimas) ir susitraukti (koncentrinis susitraukimas) (Foran, Pound, 2007). Galingumui reikalinga maksimalioji jėga ir didelis judesio greitis (Табачник, 1988; Matthew et al., 2011). Wilson G.J. su bendraautoriais (1993) nustatė, kad galingumas po jėgos treniruočių pagerėjo 5 %; po pliometrijos treniruočių – 10 %; po balistinių (greitumo jėgos) pratimų – 18 %. Tiriant tokias sporto šakas kaip krepšinis ir tinklinis buvo pastebėtos svarbios sąsajos tarp didžiausios jėgos ir didžiausio galingumo (Peterson et al., 2006). Vyksta dideli ginčai dėl to, kuri iš šių savybių turėtų būti svarbiausia treniruočių metu, siekiant išvystyti didžiausią galingumą (Haff et al., 2012). Darbo objektas - skirtingos griaučių raumenų adaptacijos įtaka galingumui. Keliame hipotezę, kad adaptuoti jėgos fiziniams krūviams tiriamieji greičiau bėgs 30 m distancijos pradžią, o adaptuoti maksimaliajam bėgimo greičiui – greičiau bėgs nuotolio pabaigoje. Tikslas — nustatyti skirtingos griaučių raumenų adaptacijos įtaką galingumui. Uždaviniai: 1. Nustatyti skirtingos griaučių raumenų adaptacijos įtaką maksimaliai valingai kojų raumenų, santykinei jėgai bei greitumui. 2. Nustatyti 30 m bėgimo rezultatus, 10 m bėgimo atkarpose, priklausomai nuo griaučių raumenų adaptacijos skirtingoms fizinėms ypatybėms. Tyrimo metodai: 1)... [toliau žr. visą tekstą] / Power is the relationship between force and velocity. Power exercise are made to promote neuromuscular system in a short period of time to make a meaningful muscle stretch (eccentric contraction), and to contract (concentric contraction) (Foran, Pound, 2007). Maximal strength and high speed of motion are necessary for peak power (Tabačnik, 1988; Matthew et al., 2011). Wilson G.J. with co-authors (1993) found the improvement of power after strength training - 5%, after plyometric training - 10%, and after balistic (speed - strength) training - 18%. Investigating such sports as basketball and volleyball it was observed significant correlation between the maximum force and maximum power evelopment (Peterson et al., 2006). There are undergoing some controversy research as to which of these properties should be the most important during training to develop maximum power (Haff et al., 2012). The object of the study - the influence of different skeletal muscle adaptations on muscular power. Hypothesis: subjects having a larger MVC will run faster 30 m distance field, and having a higher maximal running speed - will run faster at the end of range. The aim of this study: to determine the influence of different skeletal muscle adaptations on muscular power. Objectives: 1. To determine the influence of different skeletal muscle adaptations on maximum voluntary strength of leg muscles, the relative strength and quickness. 2. To determine the influence of different skeletal muscle... [to full text]

Skeletal muscle injury results in functional deficits that can take several weeks to fully recover. Ultimate recovery of function is dependent on the muscle’s ability to regenerate, a highly coordinated process that involves transient muscle inflammation and the replacement of damaged myofibers. Instrumental in the inflammatory response, is the pro-inflammatory cytokine TNF-α. Expression of TNF-α is thought to be regulated, in part, by the stress sensing 70 kDa heat shock protein (Hsp70). However, it remains unclear how Hsp70 alters TNF-α following injury, and if so, how these changes affect skeletal muscle repair. Therefore, we up-regulated Hsp70 expression using 17-allylamino-17-demethoxygeldanamycin (17-AAG) prior to and following BaCl2-induced injury, and assessed TNF-α and myogenin content. Regenerating fiber cross-sectional area (CSA) and in vivo isometric torque were also analyzed in the weeks following the injury. Treatment of 17-AAG resulted in a ~5 fold increase in Hsp70 of the uninjured muscle, but did not affect any other biochemical, morphological or functional variables compared to controls. In the days following the injury, TNF-α and myogenin were elevated and directly correlated. At these earlier time points (≤7 days), treatment of 17-AAG increased TNF-α above that of the injured controls and resulted in a sustained increase in myogenin. However, no differences were observed in regenerating fiber CSA or in vivo torque production between the groups. Together, these data suggest that Hsp70 induction increases TNF-α and myogenin content following BaCl2-induced injury, but does not appear to alter skeletal muscle regeneration or attenuate functional deficits in otherwise healthy young mice.

<p>The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence. </p><p>Major results of the dissertation are as follows. Culture conditions of 100 &#956;g/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.</p> / Dissertation

Muscle biopsies were taken at baseline, post eccentric exercise, post aerobic training, and after training followed by eccentric exercise from adults with different health status. In Cell Western analysis of pAkt/Akt ratio suggests that muscle cells isolated from baseline biopsies respond to insulin in a dose dependent manner that tracks with sensitivity to insulin of the host; however, this is uncoupled from glucose disposal in vitro. Nitrotyrosine (NY), a marker of free radical damage, was employed to assess the efficacy of the exercise paradigm. NY immunohistochemistry on muscle cross-sections revealed that eccentric exercise significantly increased damage in older (>55 years of age), but not middle aged (age) subjects, and that training reversed the post eccentric damage significantly in the younger, but not the older group, suggesting distinct adaptation to eccentric exercise. Assessment of total macrophage content by CD68 immunohistochemistry showed that macrophage abundance increased in response to training in the >55 years age group, but not in the training, macrophages increased in response to eccentric exercise in middle aged and decreased in older subjects, showing a disconnect from NY damage. Macrophage phenotypes were assessed in these groups via the M1 marker CD11b, and the M2 marker, CD206. Two dominant populations of macrophages were identified, one of which co-expressed CD11b and CD206, and another which only expressed CD11b. These two populations of macrophages showed the same trends in expression in response to exercise observed with CD68, but did not achieve statistical significance. Bivariate analysis revealed that CD11b/CD206 macrophage densities were correlated with gene activities associated with fibrosis and angiogenesis, whereas CD11b macrophages correlated with gene activities associated with proteostasis and cellular turnover. Lastly, an in vitro model of skeletal muscle cell and macrophage integration was developed to study how macrophage phenotype influences insulin responsiveness. Data suggest that M1 macrophages inhibit insulin stimulated glucose disposal, whereas M2 macrophages enhance this response. Taken together these results suggest a functional distinction between inflammatory (M1) and alternative macrophages (M2) in exercise and insulin resistance that is altered with age.

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.

The inhibitor of apoptosis (IAP) proteins traditionally regulate programmed cell death by binding to and inhibiting caspases. Recent studies have uncovered a variety of alternate cellular roles for several IAP family members. The cellular inhibitor of apoptosis 1 (cIAP1) protein, for instance, regulates different axes of the NF-κB signalling pathway. Given the extensive functions of NF-κB signalling in muscle differentiation and regeneration, I asked if cIAP1 also plays critical roles in skeletal muscle myogenesis. In a primary myoblast cell-culture system, genetic and pharmacological approaches revealed that loss of cIAP1 dramatically increases the fusion of myoblasts into myotubes. NF-κB signalling occurs along a classical and an alternative pathway, both of which are highly active in cIAP1-/- myoblasts. Suppression of the alternative pathway attenuates myotube fusion in wildtype and cIAP1-/- myoblasts. Conversely, constitutive activation of the alternative pathway increases myoblast fusion in wildtype myoblasts. cIAP1-/- mice have greater muscle weight and size than wildtypes, as well as an increased number of muscle stem cells. These results identify cIAP1 as a regulator of myogenesis through its modulation of classical and alternative NF-κB signalling pathways. Loss of the structural protein dystrophin in the mdx mouse model of Duchenne muscular dystrophy leads to chronic degeneration of skeletal muscle. The muscle pathology is strongly influenced by NF-κB signaling. Given the roles demonstrated for cIAP1 in cell culture and in vivo, I asked whether loss of cIAP1 would influence muscle pathology in the mdx mouse. To address this question, double-mutant mice were bred lacking both cIAP1 and dystrophin (cIAP1-/-;mdx). Histological analyses revealed that double-mutant mice exhibited reduced indications of damage on several measures, as compared to single-mutant (cIAP1+/+;mdx) controls. Unexpectedly, these reductions were seen in the “slow-twitch” soleus muscle but not in the “fast-twitch” extensor digitorum longus (EDL) muscle. The improvements in pathology of double-mutant solei were associated with reductions in muscle infiltration by CD68-expressing macrophages. Finally, the double-mutant mice exhibited improved endurance and resistance to damage during treadmill-running exercise. Taken together, these results suggest that loss of cIAP1, through its multiple regulatory functions, acts to improve myogenesis and increase muscle resistance to damage.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

C/EBPβ is a bZIP transcription factor known to be involved in various physiological processes, including adipogenesis, osteogenesis and liver development. Previous studies in this laboratory revealed an inhibition of myogenesis and reduced myogenic protein expression in 5-azacytidine treated mesenchymal stem cells retrovirally transduced to overexpress C/EBPβ. The goal of this thesis was to evaluate the role of C/EBPβ in myogenic differentiation by overexpression in C2C12 myoblasts and primary myoblasts. We demonstrate reduced MyoD protein expression and subsequent downregulation of myogenic proteins during differentiation following C/EBPβ overexpression. We localized C/EBPβ to the quiescent Pax7+ satellite cells associated with the muscle fiber. Upon satellite cell activation, we observed the downregulation of C/EBPβ protein expression prior to MyoD protein expression. Furthermore, the re-expression of C/EBPβ correlated with the loss of MyoD expression later in differentiation. Histological analysis of C/EBPβ-/- mice revealed smaller fibers and a reduced Pax7+ satellite cell population as compared to control animals. In this thesis, we propose that C/EBPβ is a negative regulator of skeletal muscle differentiation by inhibiting the expression of MyoD, thus impairing proper progression through the myogenic program. In addition, we propose a role for C/EBPβ in the maintenance of undifferentiatied satellite cells.