The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ?? 4 g: X ?? S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ?? 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ?? 7 min) compared to PLAC (115 ?? 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo.

Le muscle squelettique adulte est un tissu avec une grande plasticité étant donné qu’il adapte sa taille suite à la surcharge fonctionnelle et il régénère suite à une lésion. La base de cette plasticité est la myofibre et les cellules souches associées, les cellules satellites (CS). Suite aux stimuli, les CS sortent de la quiescence, elles s’activent, proliférent, s’engagent dans la voie myogénique et fusionnent entre elles ou bien avec la fibre pre-éxistante. Une partie des CS retourne à la quiescence afin de maintenir le « pool » de progéniteurs. Ce projet a pour objectif de mieux caractériser des voies de signalisation responsables des adaptations des CS au cours de la régénération et le l’hypertrophie compensatoire. Srf est un facteur de transcription, particulièrement exprimé dans les muscles. Les gènes cibles de Srf sont des gènes qui participent à la régulation de la prolifération cellulaire et des gènes codant des protéines sarcomériques du muscle ou bien des gènes ayant un rôle dans l’adhésion cellulaire, la migration et l’organisation du cytosquelette. Il a été montré que la perte de fonction de Srf dans la lignée de cellules musculaire C2C12 inhibe leur prolifération et leur différenciation et que Srf contrôle l’expression de MyoD qui est un gène de détermination myogénique. Aucune donnée n’est disponible à ce jour concernant la fonction de Srf dans les CS in vivo. Nous avons généré des souris dépourvues de Srf spécifiquement dans les CS adultes. Les CS ont été recruitées par l’hypertrophie et la régénération musculaire. En parallèle des études ex vivo ont été menées afin de préciser si les phénotypes observés sont cellule-autonomes et afin de disséquer les mécanismes sous-jacents. Nous montrons que la perte de Srf dans les CS affecte fortement les processus de régénération et d’hypertrophie suggérant un rôle de Srf dans le contrôle du destin cellulaire de CS. Nos études montrent que la perte le Srf dans les SC n’affecte pas leur prolifération et leur engagement dans la différenciation myogénique. Par contre, leur motilité et leur capacité de fusion sont fortement réduites. Afin d’identifier les effecteurs de Srf impliqués dans la motilité et le défaut de fusion des CS mutantes, nous avons réalisé des études transcriptomiques et identifié le set de gènes dont l’expression est altérée par la perte de Srf dans des conditions de prolifération et de différenciation. L’analyse des fonctions altérées nous a indiqué que la voie de signalisation du cytosquelette d’actine était perturbée. En effet les CS dépourvues de Srf expriment moins d’actine et présentent une organisation du cytosquelette d’actine perturbée. Des expériences de sauvetage utilisant un modèle de souris permettant la surexpression inductible d’actine alpha dans les CS dépourvues de Srf ont montré que la surexpression d’actine chez les mutants Srf était suffisante pour rétablir partiellement l’organisation du cytosquelette et améliorer les capacités de fusion des CS. De manière intéressante, seule la fusion hétérotypique (entre une cellule contrôle et une cellule mutante), et pas la fusion homotypique (entre deux cellules mutantes), est rétablie par l’expression de l’actine. In vivo, le rétablissement de la fusion hétérotypique restaure la croissance hypertrophique des muscles alors que l’altération de la régénération chez les mutants Srf n’est que faiblement améliorée par la surexpression de l’actine. Cette étude nous a permis d’avoir une vision d’ensemble et mécanistique de la contribution du facteur de transcription Srf dans la biologie des CS et de mettre en évidence l’importance structurale du maintien du cytosquelette d’actine pour la fusion des cellules musculaires. / The adult skeletal muscle is a high plastic tissue as it adapts its size upon overload and it is capable of regeneration upon muscle lesion. The skeletal muscle is composed of a specialized syncytium, the myofiber, which is the functional unit of the muscle and a small population of myogenic progenitors, residing adjacent to the myofibers, termed as satellite cells (SCs). SCs are the muscle-specific stem cells which endow the skeletal muscle with its remarkable capacity to repair and to maintain homeostasis during muscle turnover. In resting adult muscles, SCs are quiescent but they activate upon exposure to stimuli. The activated SCs (myoblasts) proliferate extensively and subsequently differentiate and fuse between them or pre-existing myofibers, a series of cellular events called myogenesis. In parallel to the myogenesis, a reserve population of SCs escapes the myogenic program and self-renews to replenish the SC pool. The current project aims to further characterize the signalling pathways involved in SC functions during muscle regeneration and compensatory hypertrophy (CH). Srf is a muscle-enriched transcription factor with Srf-target genes implicated in cell proliferation, differentiation (sarcomeric proteins), adhesion, migration and cellular cytoskeleton. Studies in C2C12 mouse myogenic cell line showed that Srf loss prevent the myoblast proliferation and differentiation by down-regulating the expression of the myogenic determinant MyoD gene. We used a genetic murine model for adult SC-specific Srf-loss in order to conduct in vivo and ex vivo studies for the Srf role in SCs. Compensatory hypertrophy and regeneration are the two means by which SCs were recruited. We show that loss of Srf in SCs affects the regeneration process and the CH suggesting the Srf role in the SC fate. Srf-depleted SCs display probably no defect in their proliferation and differentiation but reduced capacity in motility and fusion. Transcriptomic analysis revealed altered actin cytoskeleton and signalling. Srf-depleted SCs show reduced actin expression and altered actin cytoskeleton. Rescue of actin expression in Srf-depleted SCs partially restored the cytoskeleton organization and the fusion process. Interestingly by actin overexpression only the heterotypic/asymmetric fusion was established but not the homotypic/symmetric fusion. Therefore actin overexpression restored the hypertrophic growth in the CH (in vivo model of heterotypic fusion) but failed to do so in the regeneration (in vivo model of homotypic fusion). This study contributed to the in vivo investigation of the Srf mechanistic role in adult SCs and underlined the importance of actin cytoskeleton maintenance in the fusion of myogenic cells.

The overall objective of this thesis was to examine mechanisms involved in the acute regulation of sarcoplasmic reticulum (SR) Ca2+-handling properties by second messenger signaling pathways in skeletal and cardiac muscle. The aim of the first study (Chapter Two) was to characterize changes in the kinetic properties of sarco(endo)-plasmic reticulum Ca2+-ATPase (SERCA) proteins in cardiac and skeletal muscles in response to b-adrenergic, Ca2+-dependent calmodulin kinase II (CaMKII) and protein kinase C (PKC) signaling. The aim of the second study (Chapter Three) was to determine if insulin signaling could acutely regulate SERCA kinetic properties in cardiac and skeletal muscle. The aim of the final study (Chapter Four) was to determine if alterations in plasma glucose, epinephrine and insulin concentrations during exercise are able to influence SR Ca2+-handling properties in contracting human skeletal muscle. Data collected in Chapter Two and Chapter Three were obtained using tissue prepared from a group of 28 male Sprague-Dawley rats (9 weeks of age; mass = 280 ± 4 g: X ± S.E). Crude muscle homogenates (11:1 dilution) were prepared from selected hind limb muscles (soleus, SOL; extensor digitorum longus, EDL; the red portion of gastrocnemius, RG; and the white portion of gastrocnemius, WG) and the left ventricle (LV). Enriched SR membrane fractions, prepared from WG and LV, were also analyzed. A spectrophotometric assay was used to measure kinetic properties of SERCA, namely, maximal SERCA activity (Vmax), and Ca2+-sensitivity was characterized by both the Ca50, which is defined as the free Ca2+-concentration needed to elicit 50% Vmax, and the Hill coefficient (nH), which is defined as the relationship between SERCA activity and Ca2+f for 10 to 90% Vmax. The observations made in Chapter Two indicated that b-adrenergic signaling, activated by epinephrine, increased (P<0.05) Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50), without altering Vmax in LV and SOL but had no effect (P<0.05) on EDL, RG, or WG. Further analysis using a combination of cAMP, the PKA activator forskolin, and/or the PKA inhibitor KT5270 indicated that the reduced Ca50 in LV was activated by cAMP- and PKA-signaling mechanisms. However, although the reduced Ca50 in SOL was cAMP-dependent, it was not influenced by a PKA-dependent mechanism. In contrast to the effects of b-adrenergic signaling, CaMKII activation increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 and increased nh, without altering SERCA Vmax in LV but was without effect in any of the skeletal muscles examined. The PKC activator PMA significantly reduced SERCA Ca2+-sensitivity, by inducing a right-shift in Ca50 and decreased nH in the LV and all skeletal muscles examined. PKC activation also reduced Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG and WG), but did not alter Vmax in LV or SOL. The results of Chapter Three indicated that insulin signaling increased SERCA Ca2+-sensitivity, as shown by a left-shift in Ca50 (i.e. reduced Ca50) and an increased nH, without altering SERCA Vmax in crude muscle homogenates prepared from LV, SOL, EDL, RG, and WG. An increase in SERCA Ca2+-sensitivity was also observed in enriched SERCA1a and SERCA2a vesicles when an activated form of the insulin receptor (A-INS-R) was included during biochemical analyses. Co-immunoprecipitation experiments were conducted and indicated that IRS-1 and IRS-2 proteins bind SERCA1a and SERCA2a in an insulin-dependent manner. However, the binding of IRS proteins with SERCA does not appear to alter the structural integrity of the SERCA Ca2+-binding site since no changes in NCD-4 fluorescence were observed in response to insulin or A-INS-R. Moreover, the increase in SERCA Ca2+-sensitivity due to insulin signaling was not associated with changes in the phosphorylation status of phospholamban (PLN) since Ser16 or Thr17 phosphorylation was not altered by insulin or A-INS-R in LV tissue. The data described in Chapter Four was collected from 15 untrained human participants (peak O2 consumption, VO2peak= 3.45 ± 0.17 L/min) who completed a standardized cycle test (~60% VO2peak) on two occasions during which they were provided either an artificially sweetened placebo (PLAC) or a 6% glucose (GLUC) beverage (~1.00 g CHO per kg body mass). Muscle biopsies were collected from the vastus lateralis at rest, after 30 min and 90 min of exercise and at fatigue in both conditions to allow assessment of metabolic and SR data. Glucose supplementation increased exercise ride time by ~19% (137 ± 7 min) compared to PLAC (115 ± 6 min). This performance increase was associated with elevated plasma glucose and insulin concentrations and reduced catecholamine concentrations during GLUC compared to PLAC. Prolonged exercise reduced (p<0.05) SR Ca2+-uptake, Vmax, Phase 1 and Phase 2 Ca2+-release rates during both PLAC and GLUC. However, no differences in SR Ca2+-handling properties were observed between conditions when direct comparisons were made at matched time points between PLAC and GLUC. In summary, the results of the first study (Chapter Two) indicate that b-adrenergic and CaMKII signaling increases SERCA Ca2+-sensitivity in the LV and SOL; while PKC signaling reduces SERCA Ca2+-sensitivity in all tissues. PKC activation also reduces Vmax in the fast-twitch skeletal muscles (i.e. EDL, RG, and WG) but has no effect on Vmax in the LV and SOL. The results of the second study (Chapter Three) indicate that insulin signaling acutely increases the Ca2+-sensitivity of SERCA1a and SERCA2a in all tissues examined, without altering the Vmax. Based on our observations, it appears that the increase in SERCA Ca2+-sensitivity may be regulated, in part, through the interaction of IRS proteins with SERCA1a and SERCA2a. The results of the final study (Chapter Four) indicate that alterations in plasma glucose, epinephrine and insulin concentrations associated with glucose supplementation during exercise, do not alter the time course or magnitude of reductions in SERCA or Ca2+-release channel (CRC) function in working human skeletal muscle. Although glucose supplementation did increase exercise ride time to fatigue in this study, our data does not reveal an association with SR Ca2+-cycling measured in vitro. It is possible that the strength of exercise signal overrides the hormonal influences observed in resting muscles. Additionally, these data do not rule out the possibility that glucose supplementation may influence E-C coupling processes or SR Ca2+-cycling properties in vivo.

Tourniquet mediated Ischemia Reperfusion (I/R) injury causes damage to skeletal muscle, often resulting in prolonged functional impairment. The current study utilizes immunohistochemistry (IHC) to determine whether the controlled release of the anabolic factor, insulin-like growth factor-I (IGF-I), from the biodegradable PEGylated fibrin gel matrix can facilitate the recovery of skeletal muscle from I/R. Treatment groups following a 2-hour tourniquet applied to the limb of 6-9 month rats, included intramuscular injections of saline, PEGylated fibrin gel (PEG-Fib) only and IGF-I conjugated to PEGylated fibrin gel (PEG-Fib-IGF). Expression of the myogenic regulatory factors MyoD and myogenin detected via IHC in the PEG-Fib-IGF group was significantly lower compared to the saline group, showing a 1.4±0.8% nuclear co-localization for MyoD and a 2.0±0.8% nuclear co-localization for myogenin at 14 days of recovery. The saline group showed higher values, 31.4±4.4% and 44.1±7.3% for MyoD and myogenin nuclear co-localization respectively. A significantly greater percentage, 88.8±3.7% of Desmin positive myofibers was seen at 14 days of recovery, while a lower percentage of fibers expressing neonatal myosin, 7.7±2.7% was seen in the PEG-Fib-IGF group compared to the saline treatment group. These results indicate that IGF-I delivered intramuscularly via PEGylated fibrin gel, functions therapeutically in skeletal muscle recovery, from I/R mediated damage. In a separate injury model that deals with volumetric muscle loss, IHC analyses were performed to test the efficacy of a novel tissue engineering strategy utilizing extracellular matrix (ECM) as a scaffold. In this model, also called the defect model, a 1.0 X 1.0 cm piece of the lateral gastrocnemius was removed and replaced with a muscle-derived ECM. The constructs were then seeded with bone marrow derived cells (BMSCs), adipose derived stem cells (ADSCs) or the peroneal nerve was relocated to the area of the ECM implant. 42 days post recovery IHC analysis was performed on the ECM implants. The quantification of desmin-positive regenerating myofibers bearing centrally located nuclei, showed significantly greater values in the top, middle and bottom region of the ECM implants that received peroneal nerve relocation, when compared to the experimental group that received the ECM implant alone. Blood vessel density increases were seen within the middle region of the ECM implant groups that received BMSC+Nerve treatment and the bottom region of the ECM implant groups that received ADSC+Nerve treatment. Thus, these results corroborate the therapeutic effect of peroneal nerve relocation, which stimulated an increase in myofiber regeneration and vascular maintenance within the construct. / text

Master of Science / Department of Kinesiology / Thomas J. Barstow / Near infrared spectroscopy is currently routinely used to assess tissue (muscle) oxygenation at rest and during exercise. While most investigators assume that hemoglobin ([Hb]) is the major contributor to the responses seen during exercise, the relative contribution of myoglobin ([Mb]) to the NIRS signals remains controversial. PURPOSE: a) To calculate the range of light absorbing potential (LAP) of hemoglobin and myoglobin in mammalian skeletal muscle at rest based on analysis of published chemical and morphometric data in humans and other mammals (Part 1), and b) use the information in a) to interpret changes in total [Hb+Mb] from NIRS during exercise (Part 2). METHODS: Part 1: Information was retrieved from five published studies with regard to capillary density (#caps/mm2) and [Mb] in skeletal muscle of human, horse and rat. Preference was given to studies in which both measurements were provided for the same muscles. [Hb] in skeletal muscle was estimated as a function of capillary density, [Hb] in systemic blood, and the ratio of capillary-to-systemic hematocrit at rest and during exercise. Part 2: Changes in total [Hb] + [Mb] (as t[Hb+Mb]) from published NIRS data obtained from human subjects performing cycling or knee extension exercise were interpreted in the context of the results of Part 1. RESULTS: Part 1: Individual group mean values for skeletal muscle [Mb] in the literature ranged from 0.25-0.67 mM in human samples, with a similar range for muscles of the rat hindlimb; horse limb muscles tended to be higher (up to 1.0 mM). Capillary densities ranged from ~200 to 600 caps/mm2 in human and rat muscles, and up to 800 caps/mm2 in horse muscle. Assuming a resting capillary hematocrit of 22% and 4 fold greater LAP for each mole [Hb] vs [Mb], the resulting estimation of capillary [Hb] ranged from ~0.03 to 0.09 mM in human and rat muscles, and up to ~0.13 mM in horse muscles. The results suggest that [Mb] could contribute ~50-70% of the total LAP at rest in human skeletal muscle. Part 2: With exercise, total heme by NIRS can increase ≥ 30% in individual human subjects. Assuming this increase reflects only increased [Hb], this fits well with the observed increase in capillary hematocrit with exercise. CONCLUSIONS: 1) In skeletal muscle at rest, [Mb] is likely to be at least as significant a light absorbing heme as is [Hb] in most mammalian muscles, including the human leg. 2) Observed increases in t[Hb+Mb] with NIRS during exercise can be explained by an increase in capillary hematocrit, even in the presence of significant [Mb].

Doctor of Philosophy / Department of Animal Sciences and Industry / Bradley J. Johnson / Zilpaterol (ZIL) is a β-adrenergic receptor (β-AR) agonist that has been recently approved for use in feedlot cattle to improve production efficiencies and animal performance. One of the mechanisms through which this occurs is increased skeletal muscle growth. Therefore, two experiments were conducted to determine the effects of ZIL both in vivo and in vitro. In the first experiment, ZIL addition to bovine satellite cells resulted in a tendency to increase IGF-I mRNA and increased myosin heavy chain IIA (MHC) mRNA with 0.001 [micro symbol]M and decreased MHC mRNA with 0.01 and 10 [micro symbol]M. There were no effects of ZIL on protein synthesis or degradation. In myoblast cultures, there was a decrease in all three β-AR mRNA, and this was also reported in western blot analysis with a reduction in β2-AR expression due to ZIL treatment. In myotubes, there was an increase in β2-AR protein expression. In the second and third experiment, ZIL improved performance and carcass characteristics of feedlot steers and heifers. Additionally, ZIL decreased MHC IIA mRNA in semimembranosus muscle tissue collected from both steers and heifers. An additional part of the third study was conducted to determine the effects of melengestrol acetate (MGA) on bovine satellite cell and semimembranosus muscle gene expression. There were no effects of MGA on the expression of genes analyzed from semimembranosus muscle tissue collected. However, the addition of MGA to cultured bovine satellite cells resulted in increased β1 and β2-AR mRNA. These experiments aid in our understanding of the mechanism of action of MGA in heifers, as well as the effects of ZIL on both steers and heifers. Furthermore, they increase our knowledge and understanding of the mechanism of action of ZIL, as well as other β-agonists used to promote growth and efficiency in feedlot animals.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Background: Understanding the contribution of divergent individual response patterns remains a key objective in identifying mechanisms of inflammation and potential factors limiting the resolution of inflammation. The purpose of this research project was to investigate downstream effects of inflammation following exercise-induced muscle damage in human subjects. Methods: For three different studies, a total of 53 untrained healthy male participants were recruited and divided into a non-exercising control (n=13) and exercise-induced muscle damage groups (n=40). The study design for the three studies was the same (with few exceptions): Downhill running (DHR) (12 x 5min bouts, 10% decline, 15 km.h-1) with blood samples taken pre, post, after 2 and 4 hours post-exercise (2h, 4h) and on days 1, 2, 3, 4 and 7 (d1-d7). Serum was analysed for creatine kinase activity (CK), myoglobin (Mb), cortisol, cytokine (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokine (G-CSF, MIP-1β) and adhesion factor (sICAM-1, sP-selectin) concentrations. Tissue degradation was assessed by serum matrix metalloprotease (MMP-9) and myeloperoxidase (MPO) content. White blood cell differential count was determined and the surface expression of various cluster of differentiation factors (CD11b, CD163, CD68, CD88, CD34) as well as intracellular MPO were assessed in whole bood using flow cytometry. Nuclear localization of the inflammatory mediator NFĸB in isolated perhipheral blood mononuclear cells (PBMCs) was determined using immunofluorescence microscopy. Muscle biopsies (vastus lateralis) taken at baseline, 4h, d1 and d2 were analysed for fibre type, inflammatory and stress-induced pathways (STAT3, IĸBα, p38MAPK), myogenic factors (MyoD, myogenin), neutrophil activity (MPO) and satellite cell number (Pax7). Results: Participants in the DHR group were subdivided into those with a normal recovery (DHR1) and those who developed secondary damage (DHR2). CK peaked on d1 in both subgroups (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) and again on d4 only in the DHR2 group (1110 ± 184 u.L-1). A similar IL-6 and IL-10 response was evident immediately post DHR in all individuals. Additional IL-6 was released in the DHR2 subgroup peaking at 4h (10.3 ± 4.2 pg.mL-1) whereas IL-10 had returned to baseline. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) and CD163+ (3%) monocytes were significantly higher in the DHR2 subgroup. Neutrophil count at 2h (DHR1: 8.6 ± 0.8 x109 cells.L-1, DHR2: 11.4 ± 1.8 x109 cells.L-1) was significantly (p<0.02) correlated to CK activity on d4. PBMC NFĸB p65 nuclear localization was slightly less at 2h in the DHR2 compared to the DHR1 and control groups. Intramuscular STAT3 signalling and MPO were significantly higher in the DHR2 compared to the DHR1 subgroup at 4h and d2 respectively. The progenitor cell response was similar for all DHR individuals with an increase in Pax7+ SC observed at 4h (0.06 ± 0.01 Pax+ SCs/fibre) and d1 (0.07 ± 0.02 Pax+ SCs/fibre). Conclusion: Healthy young men can be divided into those with a adequate and those with a less efficient capacity to control the post damage inflammatory response. The early cytokine response, especially IL-6, seems to be a key role player in the cascade of events leading to late secondary skeletal muscle damage. / AFRIKAANSE OPSOMMING: Agtergrond: Die begrip van uiteenlopende individuele reaksie patrone, is belangrik in die identifisering van faktore asook meganismes betrokke in die ontwikkeling en resolusie van inflammasie. Die doel van hierdie navorsingsprojek was om die gevolge van oefening-geïnduseerde spierskade en inflammasie te ondersoek in menslike proefpersone. Metodiek: ‘n Totaal van 53 gesonde mans is tydens drie verskillende studies, gegroepeer in ’n kontrole (geen oefening) (n=13) en oefening geinduseerde spier skade (DHR) groep (n=40). Die uitleg van de studies was eenders (met min uitsonderings): Afdraende hardloop (12 x 5min hardloop sessies, 10% afdraende, 15km.h-1) met bloed monsters geneem voor, na, 2 ure, 4 ure (pre, post, 2h, 4h) en op dag 1, 2, 3, 4 en 7 (d1-7). Serum is ontleed vir die volgende: kreatien kinase aktiwiteit (CK), kortisol, sitokiene (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokien (G-CSF, MIP-1β) en adhesie molekuul (sICAM-1, sP-selectin) konsentrasies. Weefsel degradasie is vasgestel deur die teenwoordigheid van matriks metalo-protease-9 (MMP-9) en miëloperoksidase (MPO) in serum te meet. Differensiële witbloed sel (WBC) telling asook die teenwoordigheid van sekere differensiasie faktore (CD11b, CD163, CD68, CD88, CD34) op die sel oppervlak asook intrasellulêre MPO vlakke is bepaal deur gebruik te maak van vloeisitometrie. Die lokalisering van NFĸB in die selkerne van geïsoleerde bloed mononukleêre selle (PBMC) is bepaal deur fluoriserende mikroskopie. Spierbiopsies (vastus lateralis) geneem tydens rus (basislyn), na 4h, en op d1 en d2 is ontleed vir veseltipe, inflammatoriese en stresverwante faktore (STAT3, IĸBα, p38 MAPK), miogeniese faktore (myoD, myogenin), neutrofiel aktiwiteit (MPO) en aantal satelliet selle (Pax7). Resultate: Deelnemers in die DHR-groep is onderverdeel in twee groepe. Persone wat normaalweg herstel het is saam gegroepeer (DHR1) en diegene wat sekondêre skade ontwikkel het is saam gegroepeer (DHR2). CK aktiwiteit in serum het hoogtepunte bereik op d1 in beide subgroepe (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) en weer op d4 in die DHR2 groep (1110 ± 184 u.L-1). 'n Soortgelyke IL-6 en IL-10 reaksie is onmiddellik na oefening (in al die proefpersone) waargeneem. Addisionele IL-6 is vrygestel in die DHR2 subgroep en het ’n hoogtepunt bereik na 4h (10.3 ± 4.2 pg.mL-1), terwyl IL-10 reeds teruggekeer het na rustende waardes. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) en CD163+ (3%) monosiete was aansienlik hoër in die DHR2 subgroep. Neutrofieltelling na 2h (DHR1: 8.6 ± 0.8 x109cells.L-1, DHR2: 11.4 ± 1.8 x109cells.L-1) het verband (p <0,02) gehou met CK-aktiwiteit op d4. In vergelyking met die DHR1 en kontrole groep was die lokalisering van NFĸB p65 in PBMC selkerne na 2h effens minder in die DHR2 subgroep. STAT3- en MPO-vlakke in die spiere was aansienlik hoër in die DHR2 subgroep as in die DHR1 subgroep na 4h en op d2 onderskeidelik. Die spierherstel proses was eenders vir alle individue wat aan die oefening deelgeneem het; 'n toename in Pax7+ Satelietselle (SC) is waargeneem na 4h (0.06 ± 0.01 Pax+ SC/spiervesel) en op d1 (0.07 ± 0.02 Pax+ SC/spiervesel). Gevolgtrekking: Gesonde jong mans kan verdeel word in diegene met 'n bevoegde en diegene met 'n minder doeltreffende vermoë om oefenings-geïnduseerde spierskade en die inflammatoriese reaksie te beheer. Die sitokien-reaksie, veral IL-6, blyk om 'n belangrike rolspeler in die ontwikkeling van sekondêre skeletspierskade te wees.

The time course of this increase in muscle n-3 PUFA composition and anabolic protein expression is currently unknown. In Chapter 2 of this thesis ten healthy male participants consumed 5g.d-1 of n-3 PUFA-enriched fish oil for 4 weeks. Muscle biopsies samples were collected in the fasted, rested state 2 weeks prior, immediately before (Week 0), at Week 1, Week 2 and Week 4 after initiation of fish oil supplementation for assessment of changes in lipid composition and expression of anabolic signalling proteins over time. Muscle lipid profile, (% total n-3 PUFA/total fatty acids) increased from W0 to W2 (3.8 ± 0.2 to 5.1 ± 0.3 %) and continued to rise at W4 (6.7 ± 0.4 %). Total protein content of FAK increased from W0 to W4 (3.9 ± 1.5 fold) whereas total mTOR was increased from W0 at W1 (2.4 ± 0.6 fold) with no further significant increases at W2 and W4. For the first time this study demonstrates that oral fish oil consumption results in an increase of n-3 PUFA levels in human skeletal muscle that is associated with increases in the expression of anabolic signalling proteins. Our understanding of the anabolic signalling process that underpins muscle protein synthesis has been advanced by the application of the WB technique. However, the semi-quantitative nature and poor dynamic range associated with the WB technique may lead to incongruence regarding the molecular response of skeletal muscle to anabolic stimulation. Chapter 3 of this thesis developed and applied a quantitative in vitro [γ-32P] ATP kinase assay (KA) alongside a traditional WB methodology to assess p70S6K1 signalling responses in human skeletal muscle to RE and protein feeding. Following validation in tissue culture with rapamycin and optimization of the assay in human skeletal muscle, this methodology was tested in a physiologically relevant context. In this regard, six males performed unilateral resistance exercise (RE) followed by the consumption of 20 g of protein. Skeletal muscle biopsies were obtained at pre-RE, at 1 h and 3 h post-RE. In response to RE and protein consumption, p70S6K1 activity was significantly increased from pre-RE at 1 h and 3 h post-RE (8.84 ± 0.78 to 17.18 ± 2.62 and 15.62 ± 3.12 µU/mg). However, phosphorylated p70S6K1thr389 was not significantly elevated. To assess if a combined stimulus of RE and feeding can influence AMPK activity we directly measured AMPK activity. AMPK activity was suppressed from pre-RE at 3 h post-RE (24.15 ± 1.6 to 15.64 ± 1.07 mU/mg), whereas phosphorylated ACCser79 was unchanged. These data therefore highlight the utility of the KA to study skeletal muscle plasticity. Previous studies have shown that ingestion of n-3 PUFA potentiates the phosphorylation of mTORC1 and associated kinases in response to nutrition. However, no study has identified whether n-3 PUFA supplementation potentiates anabolic kinase activity when RE is performed prior to nutrient provision. In Chapter 4 of this thesis, twenty healthy males consumed 5g.d-1 of either fish oil (FO) or coconut oil (CO) capsules for 8 weeks. Muscle biopsy samples were collected in the fasted, rested state before and after 8 weeks of supplementation for assessment of changes in lipid composition. Following 8 weeks of supplementation muscle samples also were obtained at rest (Rest), post RE in both the exercise leg (Post-RE) and the rested leg (Pre-FED) and also at 3 h post RE and protein feeding from both the exercise leg (3 h post-REF) and rested leg (3 h post-FED). There was a 2-fold increase in muscle (5.53 ± 0.3 to 11.16 ± 0.45 % of total fatty acids) n-3 PUFA composition after supplementation in the FO group but no change in the CO group. Following supplementation there was an increase in p70S6K1 activity at 3 h post-REF from Rest in the CO group (5.6 ± 1.4 to 12.2 ± 2.1 µU/mg) but no change in the FO group. In the CO group, AMPKα2 was significantly increased at Post-RE from Rest (3.7 ± 0.7 to 9.9 ± 2.0 mU/mg). These data show that 8 weeks of n-3 PUFA enriched fish oil supplementation suppresses the activity of p70S6K1 in response to RE and protein feeding.

<p>Electroporation is a biological cell's natural reaction to strong electric fields, where transient pores are created in the cell membrane. While electroporation holds promise of being a safe and effective tool for enhancing molecular delivery in numerous medical applications, it remains largely confined to preclinical research and clinical trials due to an incomplete understanding of the exact mechanisms involved. Muscle fibers are an important delivery target, but traditional theoretical studies of electroporation ignore the individual fiber geometry, making it impossible to study the unique transverse and longitudinal effects from the pulse stimulus. In these long, thin muscle fibers, the total reaction of the fiber to the electric field is due to fundamentally different effects from the constituent longitudinal and transverse components of the electric field generated by the pulse stimulus. While effects from the transverse component have been studied to some degree, the effects from the longitudinal component have not been considered. </p><p>This study develops a model of electroporation and delivery of small molecules in muscle tissue that includes effects from both the transverse and longitudinal components of the electric field. First, an asymptotic model of electric potential in an individual muscle fiber is derived that separates the full 3D boundary value problem into transverse and a longitudinal problems. The transverse and longitudinal problems each have their own respective source functions: the new "transverse activating function" and the well known longitudinal activating function (AF). This separation enhances analysis of the different effects from these two AFs and drastically reduces computational intensity. Electroporation is added to the asymptotic fiber model, and simplified two-compartment mass transport equations are derived from the full 3D conservation of mass equations to allow simulation of molecular uptake due to diffusion and the electric field. Special emphasis is placed on choosing model geometry, electrical, and pulsing parameters that are in accordance with experiments that study electroporation-mediated delivery of small molecules in the skeletal muscle of small mammals.</p><p>Simulations reveal that for fibers close to the electrodes the transverse AF dominates, but for fibers far from the electrodes the longitudinal AF enhances uptake by as much as 2000%. However, on the macroscopic tissue level, the increase in uptake from the longitudinal AF is no more than 10%, given that fibers far from the electrodes contribute so little to the total uptake in the tissue. The mechanism underlying the smaller effect from the longitudinal AF is found to be unique to the process of electroporation itself. Electroporation occurs on the short time scale of polarization via the transverse AF, drastically increases membrane conductance, and effectively precludes further creation of pores from charging of the membrane via the longitudinal AF. The exact value of enhancement in uptake from the longitudinal AF is shown to depend on pulsing, membrane, and tissue parameters. Finally, simulation results reproduce qualitative, and in some cases quantitative, behavior of uptake observed in experiments.</p><p>Overall, percent increase in total tissue uptake from the longitudinal AF is on the order of experimental variability, and this study corroborates previous theoretical models that neglect the effects from the longitudinal AF. However, previous models neglect the longitudinal AF without explanation, while the asymptotic fiber model is able to detail the mechanisms involved. Mechanisms revealed by the model offer insight into interpreting experimental results and increasing efficiency of delivery protocols. The model also rigorously derives a new transverse AF based on individual fiber geometry, which affects the spatial distribution of uptake in tissue differently than predicting uptake based on the magnitude of the electric field, as used in many published models. Results of this study are strictly valid for transport of small molecules through small non-growing pores. For gene therapy applications the model must be extended to transport of large DNA molecules through large pores, which may alter the importance of the longitudinal AF. In broader terms, the asymptotic model also provides a new, computationally efficient tool that may be used in studying the effect of transverse and longitudinal components of the field for other types of membrane dynamics in muscle and nerves.</p> / Dissertation

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The aim of this study was to investigate the powerful muscle regulator, myostatin, and its regulators in response to an acute bout of plyometric training. The participants were recruited and screened by characterization by means of isometric force production tests, baseline blood creatine kinase levels and VO2 max results. The selected individuals (n=15) were subjected to a baseline muscle biopsy for comparative purposes. The study made use of plyometric jumping, as source of eccentric exercise, to serve as an exercise intervention after which muscle biopsies (4 hours post and 24 hours post) and blood draw (4 hours post, 24 hours post and 48 hours post) samples were taken. Maximal voluntary isometric contractions of the knee extensors were also measured immediately after the exercise protocol and after 1 week recovery. Creatine kinase (CK) analysis on the serum samples was used to conclude muscle damage. The muscle biopsy samples were used for protein quantification (Western blot) and gene expression assessment (semi-quantitative and real-time PCR). The results showed decreased force production immediately after eccentric exercise (p < 0.05), while returning back to baseline values at 1 week post exercise and CK results showed a significant increases at 4 hours (p<0.05), 24 hours (p<0.001) and 48 hours (p<0.01) after exercise. There were no significant differences in myostatin precursor protein (43 kDa), phosphorylated Smad2,3, Smad7 or activin receptor IIb in response to eccentric exercise. However, the follistatin protein was increased at both 4 hours and 24 hours after exercise (p<0.01). RNA analysis of the extracellular matrix (ECM) protein, decorin, revealed the existence of the splice variants A1 and A2 in human skeletal muscle. The RT-PCR analysis (n=4) of these variants showed no significant difference when comparing pre- to post-exercise. The decorin core protein was also investigated by means of antibody probing and results revealed the need for ABC chondroitinase enzyme treatment before immunoblotting of human skeletal muscle samples. The results concerning knee extensor force reduction and circulating creatine kinase showed the effectiveness of plyometric jumping in producing skeletal muscle damage in the lower limbs of unfit individuals, unaccustomed to eccentric exercise. In conclusion, myostatin, and its associated signalling cascade, are not activated in early muscle regeneration, but follistatin is increased during this phase possibly aiding and initiating the muscle repair process. Future studies: Variants of decorin are expressed in human skeletal muscle, increasing the complexity that should be taken into account in studies concerning the regulation of decorin in a human model. Investigation into myostatin protein at different post-translational levels needs more clarification. Published methods and materials used in different laboratories are not consistent and investigators should attempt to standardise protocols in order to compare results between studies more effectively. Of importance, these results show that the myostatin at protein level report different results compared to mRNA analysis and that more investigation into myostatin regulatory factors, with special reference to follistatin and decorin, is needed in future human models. / AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die kragtige spiere reguleerder, miostatin, en sy reguleerders in reaksie op 'n akute aanval van pliometriese spronge te ondersoek. Die deelnemers is gewerf en gekeur deur karakterisering deur middel van isometriese krag produksie toetse, basislyn bloed kreatien kinase vlakke en VO2maks resultate. Die geselekteerde individue (N = 15) is onderhewig aan 'n basislyn spierbiopsie vir vergelykende doeleindes. Die studie het gebruik gemaak van pliometriese spronge (essentriese spier aksie) as die oefening intervensie waarna spierbiopsie (4 uur na en 24 uur na) en bloed (4 uur na, 24 uur na en 48 uur na) monsters geneem is. Isometriese kontraksies van die knieverlengers is ook gemeet onmiddellik na die oefening protokol en na 1 week se herstel. Kreatine kinase (KK) ontleding van die serum monsters is gebruik om spierskade aftelei. Die spierbiopsie monsters was gebruik vir proteïen kwantifisering (Western klad) en die assessering van geen uitdrukking (semi-kwantitatiewe en real-time PCR). Die resultate het gewys dat krag produksie afgeneem het onmiddellik na essentriese oefening (p <0.05), terwyl dit terugkeer na die oorspronklike waardes 1 week na oefening en KK resultate toon 'n beduidende toename by 4 uur (p <0,05), 24 uur (p <0,001) en 48 uur (p <0,01) na oefening. Daar was geen betekenisvolle verskille in Miostatien voorloper proteïen (43 kDa), gefosforileerde Smad2,3, Smad7 of Activin reseptoor IIb in reaksie op essentriese oefening. Dit is egter die follistatien proteïen wat verhoog by beide 4 uur en 24 uur na oefening (p <0,01). RNS ontleding van die ekstrasellulêre matriks (ESM) proteïen, decorin, het die bestaan van die splitsing variante A1 en A2 in menslike skeletspier, aan die lig gebring. Die RT-PCR analise (n = 4) van hierdie variante het geen betekenisvolle verskille getoon wanneer voor met na-oefening vergelyk is. Die decorin kern proteïen is ook ondersoek deur middel van teenliggaam afhanklike metodes en resultate het die behoefte aan ABC chondroitinase ensiem behandeling voor immunokladding van menslike skeletspier monsters gesteun. Die resultate aangaande knieverlenger krag vermindering en sirkuleerende kreatien kinase het die doeltreffendheid van pliometriese spronge in die vervaardiging van skeletspier skade in die onderste ledemate van individue ongewoond aan essentriese oefening verseker. Ten slotte, Miostatien, en sy verwante sein kaskade, is nie geaktiveer vroeg in spier herstelling, maar follistatien is tydens hierdie fase verhoog en help moontlik met die aanvang van die spier herstel. Toekomstige studies: variante van decorin word uitgedruk in menslike skeletspier, wat die kompleksiteit aangaande decorin verhoog en dit is iets wat in ag geneem moet word in studies wat handel oor die regulering van decorin in mens modelle. Ondersoek na miostatien proteïen op verskillende na-translasie vlakke moet meer duidelikheid verkry. Gepubliseer metodes en materiaal wat gebruik word in verskillende laboratoriums is nie konsekwent en ondersoekbeamptes moet probeer om protokolle te standaardiseer sodat resultate van studies meer effektief kan vergelyk word. Van belang is, die resultate wys dat miostatien op proteïen vlak verskillende resultate vertoon in vergelyking met boodskapper-RNS ontleding en dat meer ondersoek na miostatien regulerende faktore, met spesiale verwysing na follistatien en decorin, nodig is in toekomstige menslike modelle.