<p>The research presented here represents a quest to understand and address limitations in the field of skeletal muscle tissue engineering, with hopes to better understand the factors involved in producing viable engineered skeletal muscle tissue. The driving force behind this research was to address two of the many factors important in muscle cell proliferation and differentiation, toward developing mature and functional bioartificial skeletal muscles (BAMs). Our work focused on understanding the individual effects of mechanical stimulation and microRNAs (miRNAs), as well as the synergistic relationship between the two factors. We hypothesized that (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via temporally regulated miRNAs and that (2) modulating these miRNAs can enhance skeletal muscle function in a 3D tissue-engineered system.</p><p>We first established a BAM system using C2C12 mouse myoblasts in a collagen gel, showing that these cells were able to produce mature sarcomeres when cultured under steady, passive tension for up to 36 days. Staining muscle-specific proteins and electron microscopy showed distinct striations and myofiber organization as early as 6 days, post-differentiation. At 33 days, cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming.</p><p>We then focused on the effects of mechanical stimulation on C2C12 myoblasts in a more simple, 2D system. In particular, we assessed miRNA and muscle-specific gene expression over time and in response to two cyclic stretch regimens using miRNA microarray technology and quantitative real time RT-PCR. Both miRNAs and certain genes, such as SRF and Mef2c, had differential responses to the two regimens. Over-expression and inhibition studies of one muscle-specific miRNA, miR-1, abrogated the stretch response and suggest that a balancing mechanism is in place to avoid large fluctuations in miRNA levels. </p><p>Finally, since miRNA modulation quenched the stretch-mediated response in myoblasts, we chose to examine 3D BAM function when miRNA levels were altered to promote differentiation. Using the same collagen gel model established previously, a muscle-specific miRNA, miR-133, known to promote proliferation, was transiently inhibited (anti-miR-133) to encourage differentiation. Forces in the anti-miR-133 BAMs were, on average, 20% higher over the negative control. Further, myofiber diameters were significantly greater and striations were more organized in the anti-miR-133 BAMs, suggesting that transient, exogenous delivery of miRNAs may be a viable approach to create a more fully differentiated muscle.</p> / Dissertation

The risk of injury, inflammation, and oxidative stress increases in skeletal muscle with aging. It has been postulated that pro-oxidant signaling, including upregulation of inducible nitric oxide synthase (iNOS) contributes to inflammation, pathology, and aging in the brain, liver and heart. Exercise training reduces the risk of injury and inflammation. The purpose of this study was: 1) to identify the mechanisms that upregulate iNOS, pro-oxidant and pro-inflammatory signaling in skeletal muscle, and 2) to identify the mechanisms by which exercise training reduces pro-oxidant signaling. Protein levels and activity of iNOS were measured in 4 groups of male Fischer-344 rats (5 mo and 24 mo, n=10/group), old-control (OC), old-trained (OT), young-control (YC), and young-trained (YT). Exercise training protocol was 60 min at 15 m/min at 15° incline for 5 d/wk for 12 wk. Both iNOS protein expression and activity were significantly higher in OC compared to YC, but exercise training reversed the elevation of iNOS levels lower than OC in tibialis anterior. Surprisingly, NF-&#954;B DNA binding activity was significantly lower in OC than YC, while increased with exercise training in white and red gastrocnemius in both OT and YT. In contrast, protein expression of p65, a regulatory subunit of NF-&#954;B was significantly greater in OC than YC, while exercise training significantly reduced p65 in OT compared to OC from the white gastrocnemius. These data indicate that regulation of NF-&#954;B activity with aging is post-translational and alterations in iNOS expression may result from alternative NF-&#954;B pathways. As decreased NF-&#954;B activity with aging could result in downstream increase in pro-apoptotic signaling, we tested follow-up hypotheses that aging would increase pro-apoptotic regulator Bax and decrease the anti-apoptotic regulator Bcl-2. Bax increased while Bcl-2 decreased in OC in white gastrocnemius when compared to YC. In contrast, exercise training resulted in a dramatic upregulation of Bcl-2 and downregulation of Bax protein expression in OT when compared to OC. These novel results indicate that alterations in pro-inflammatory and pro-apoptotic signaling occur in skeletal muscle during the aging process. Importantly, our findings strongly support the hypothesis that exercise training reverses age-induced changes in pro-inflammatory and pro-apoptotic signaling.

We evaluated the efficacy of treating eccentric muscle damage with IGF-1 PEGylated to a fibrin biomatrix. With one injection, delivered one hour after the induction of eccentric muscle damage we saw an attenuation of force loss early in recovery, maintenance of muscle weight, and progression to the repair/regeneration of the damaged fibers at a greater speed and magnitude in the first week of recovery. As opposed to introducing an unbound bolus of IGF-1, we believe the ability of the PEGylated-fibrin to stabilize and sustain delivery of the molecule results in significantly better recovery. Coupling IGF-1, which has multiple beneficial effects in tissue repair, with this system of delivery provides a simple and easy to administer treatment for eccentric muscle damage. With this form of damage being the most prevalent of all skeletal muscle damage types, since it is underlies all muscle strain, a simple and effective treatment is important for increasing functional recovery after injury. / text

The purpose of this study was to investigate the effects of 6 weeks of hydroxytyrosol (HT) supplementation on markers of muscle damage in healthy, recreationally active men before and throughout acute aerobic exercise bouts. Using a randomized, double-blind, repeated-measures, placebo-controlled design, sixty-one (n = 61) subjects (21.46 ± 0.22 yrs, 179.46 ± 0.79 cm, 78.91 ± 1.19 kg) consumed either a high dose (HI) HT supplement (150 mg HT), a low dose (LO) HT supplement (50 mg HT), or a placebo (PLA) every day for 6 weeks. Throughout the course of the study, the subjects performed four time trial rides (TT1-TT4) on a cycle ergometer. TT1 occurred before supplementation, TT2 halfway through the supplementation period, and TT3 and TT4 occurred in the sixth week and final two days of supplementation. Blood was drawn prior to (pre) and just before termination (end) of each time trial to measure markers of muscle damage during exercise. We observed that endurance exercise increased indicators of muscle damage, CPK and myoglobin, but an association between HT treatment and reduced muscle damage indicators during exercise were not demonstrated. However, the HT supplementation for 6 weeks in recreationally-active males improved time trial performance in the HT treatment groups over the course of the study and this improvement was accompanied by a lower increase in myoglobin concentration in blood in the HI treatment group than in the LO treatment group. Also, performance was improved after 6 weeks in the PLA group. This improvement was associated with an increase in rating of perceived exertion (RPE). RPE was not increased in either the LO and HI treatment groups, although time trial performance was significantly improved. It is possible that HT can improve performance by altering perception of effort. We conclude that chronic and acute HT supplementation did not reduce markers of muscle damage in this population at rest, during, or following exercise, but improved aerobic performance. / text

Skeletal muscle possesses remarkable plasticity in responses to altered mechanical load. An established murine model used to increase mechanical load on a muscle is the surgical removal of the gastrocnemius and soleus muscles, thereby placing a functional overload on the plantaris muscle. As a consequence, there is hypertrophic growth of the plantaris muscle. We used this model to study the molecular mechanisms regulating skeletal muscle hypertrophy. Aged skeletal muscle demonstrates blunted hypertrophic growth in response to functional overload. We hypothesized that an alteration in gene expression would contribute to the blunted hypertrophic response observed with aging. However, the difference in gene expression was modest, with cluster analysis showing a similar pattern of expression between the two groups. Despite ribosomal protein gene expression being higher in the aged group, ribosome biogenesis was significantly lower in aged compared with young skeletal muscle in response to the hypertrophic stimulus (50% versus 2.5-fold, respectively). The failure to fully up-regulate pre-47S ribosomal RNA (rRNA) expression in old skeletal muscle undergoing hypertrophy indicated ribosomal DNA transcription by RNA polymerase I was impaired. Contrary to our hypothesis, the findings of the study suggest that impaired ribosome biogenesis was a primary factor underlying the blunted hypertrophic response observed in old skeletal muscle rather than dramatic differences in gene expression. As it appears ribosomal biogenesis may limit muscle hypertrophy, we assessed the dynamic changes in global transcriptional output during muscle hypertrophy, as the majority of global transcription is dedicated to ribosome biogenesis during periods of rapid growth. Metabolic labeling of nascent RNA using 5-ethynyl uridine permitted the assessment of cell type specific changes in global transcription and how this transcription is distributed within the myofiber. Using this approach, we demonstrate that myofibers are the most transcriptionally active cell-type in skeletal muscle, and furthermore, myonuclei are able to dramatically upregulate global transcription during muscle hypertrophy. Interestingly, the myonuclear accretion that occurs with hypertrophy actually results in lower transcriptional output across nuclei within the muscle fiber relative to sham conditions. These findings argue against the notion that nuclear accretion in skeletal muscle is necessary to increase the transcriptional capacity of the cell in order to support a growth response.

ESC/iPSC-derive somatic cells may be ideal for treating disorders caused by cellular deficiency or dysfunction. To form a lineage-specific cell population, ESCs/iPSCs undergo a multi-step process that recapitulates embryonic development. ESC/iPSC differentiation protocols are hampered by the limitation of our understanding in development. Zebrafish embryos are fertilized and developed externally, a feature facilitates the observation and manipulation of embryonic development. To explore the zebrafish as a system to study cell lineage determination, in this thesis, I 1) identified an ortholog of the key pluripotency regulator Nanog in zebrafish and examined its role in early cell fate determination; 2) developed a high-throughput image-based chemical screening system in zebrafish blastomere cell culture that is very similar to, but much faster than, ESC/iPSC differentiation screens. Specifically, in an effort to examine the role of Nanog in vivo, I identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. I examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. The study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction. In the second part of the thesis, I developed a system that allows high-throughput image-based chemical screening using cultured zebrafish blastomere cells. To demonstrate its potential, this system is utilized to study skeletal muscle development. I screened 2,400 chemicals, finding 11 chemicals that block mature muscle cell differentiation and 17 chemicals that block skeletal muscle progenitor formation. The subsequent studies of these hits illustrate an RTK-PI3K-mTOR- GSK3 signaling cascade that is critical for skeletal muscle development. Preliminary data in mouse Skeletal Muscle Precursors (SMPs) suggest the pathway is conserved in murine adult muscle stem cells. This system, which can be modified for any cell lineage, promises to enhance our understanding of fundamental biology and to identify chemicals for cell-based therapies for many diseases.

Amino acids are important modulators of skeletal muscle metabolism, but their impact on glucose uptake by skeletal muscle remains unclear. To address the effect of an amino acid (AA) mixture consisting predominately of isoleucine on glucose uptake we first conducted a dose-response experiment, investigating how different concentrations of the AA mixture affect glucose uptake by isolated rat epitrochlearis muscle. In a subsequent experiment we examined how the AA mixture affects insulin-stimulated glucose uptake by isolated rat epitrochlearis muscle. It was found that the AA mixture with as little as 0.5 mM Ile increases [H3]2-deoxy-D-glucose (2-DG) uptake by 76% compared to basal glucose uptake. The AA mixtures with 1, 2 or 4 mM Ile provided no significant additional effect. Next we combined the AA mixture consisting of 2 mM Ile, 0.012 mM Cys, 0.006 mM Val and 0.014 mM Leu with physiological levels (75 μU/ml, sINS) and maximally-stimulating levels (2 mU/ml, mINS) of insulin. The AA mixture only, sINS and mINS significantly increased 2-DG uptake compared to basal by 63, 79 and 298%, respectively. When the AA mixture was combined with sINS and mINS 2-DG uptake was further increased significantly by 26 and 14%, respectively. Western blotting analysis revealed that compared to basal the AA mixture increased AS160 phosphorylation, while phosphorylation of Akt and mTOR did not change. Combining the AA mixture with sINS resulted in no additional phosphorylation compared to sINS alone. Interestingly, addition of the AA mixture to mINS resulted in increased phosphorylation of mTOR, Akt and AS160 compared to mINS alone. Our results suggest that certain AAs (1) increase glucose uptake in the absence of insulin and (2) augment insulin-stimulated glucose uptake in an additive manner. These effects on glucose uptake appear to be mediated via a molecular pathway that is partially independent from the canonical insulin signaling cascade. / text

Background: Patients with heart failure (HF) are characterized by exercise intolerance, breathlessness, fatigue and excessive neurohormonal activation associated with premature mortality. Recently, inflammatory activation has been described as an important factor in the progression of HF. Increased levels of certain pro-inflammatory cytokines (e.g., TNF-ɑ, IL-6) have been related to increased severity of left ventricular dysfunction, the activation of the sympathetic and renin-angiotensin systems and the catabolism of skeletal muscle. Although exercise training is important in the management of HF, acute bouts of exercise may lead to increases in proinflammatory cytokines. It is believed that the skeletal muscle abnormalities associated with HF may increase the risk of damage to skeletal muscle, (i.e., exercise-induced muscle injury (EIMI) with associated inflammatory activation) especially following unaccustomed exercise training. Recently, several training methods have been proposed for patients with HF that challenge the traditional “steady-state” (SS) training model, including interval training (IT). Interval training methods employ greater muscular loading than SS and therefore may increase the risk of inflammatory system activation EIMI, and/or reduced muscle function. There is no study that has examined the effects of IT on EIMI, muscle function and/or inflammatory markers. Material and Methods: Fourteen male participants with HF (mean age: 59 +/- 7.8 yrs; mean VO2 peak: 13.64 +/- 4.5 ml/kg/m-1; EF < 45%) were matched (for body mass and aerobic fitness) and randomized into SS or IT for 20 minutes. The IT involved 2 minute work:recovery phases of 90% and 40% of heart rate reserve, respectively. The SS involved continuous exercise at 65% of heart rate reserve. Biochemical markers of muscle damage and acute inflammation, concentric and eccentric isokinetic muscle torque, and subjective indicators of delayed onset muscle soreness (DOMS) and lower extremity function were evaluated at baseline, and then immediately following the training bout, and at 6, 24, and 48 hours post. Results: There were no significant differences between the IT and the SS training group for markers of skeletal muscle injury or inflammatory activation. Conclusions: The findings from the present study suggest that IT or SS do not result in excessive inflammatory system activation or skeletal muscle injury. These results have important implications for clinicians prescribing exercise regimes for HF patients who may be starting back into activity after a prolonged sedentary period. Additionally, results from this study indicate that there is a need for future research looking at the actual and perceived effect of even a single about of exercise on lower extremity function.

Mitochondria play an essential role in cell metabolism as well as apoptotic signaling. Chronic endurance exercise has been shown to increase mitochondrial content in skeletal muscle. Interestingly, endurance exercise has also been associated with decreased skeletal muscle apoptosis; however, the direct effect of increased skeletal muscle mitochondrial content on apoptotic signaling has not been examined. The purpose of this study was to induce mitochondrial biogenesis in L6 myoblasts and examine the susceptibility of these cells to stress- induced apoptosis. Mitochondrial biogenesis was accomplished using 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and S-nitroso-N-acetylpenicillamine (SNAP), which activate AMPK and donate nitric oxide, respectively. Successful induction of mitochondrial biogenesis was determined by western blot analysis for mitochondrial specific markers. Following SNAP and AICAR treatment, the average increase in the mitochondrial markers was 24% and 38%, respectively. Subsequent exposure of cells to several apoptosis-inducing agents increased apoptosis. Interestingly, SNAP- and AICAR- treated cells had a lower percentage of apoptotic cells as determined by AnnexinV-FITC/PI fluorescent staining, cell cycle analysis, and cell counting/size analysis. In addition, it was shown that SNAP- and AICAR-treated cells had reduced caspase-3 activity following exposure to apoptotic stimuli. Furthermore, treatment with SNAP and AICAR resulted in increased protein content of the antioxidants MnSOD and catalase. Interestingly, mitochondrial ROS production was not significantly altered between groups with total cellular ROS production being increased in the SNAP- and AICAR-treated groups. In summary, this work demonstrates that increasing mitochondrial content in L6 myoblasts provides protection against stress-induced apoptosis. The mechanism for this protective effect remains to be determined; however, it may be mediated by a combination of increased antioxidant capacity and improved mitochondrial calcium buffering capacity.

Excessive abdominal obesity, coupled with a decline in muscle mass and physical function that is exacerbated by sedentary living, contributes substantively to the disease and disability common to our aging population. The Senior Study, a randomized controlled trial designed to investigate the health benefits associated with performing resistance exercise (RE), aerobic exercise (AE), or a combination of both exercise modalities (RAE) for six months in the absence of caloric restriction, was conducted on otherwise healthy but abdominally obese, sedentary men (n=57) and women (n=79) between the ages of 60 and 80. The purpose of the first manuscript (Chapter 3) was to compare the effects of exercise modality on visceral obesity and insulin resistance in the Senior Study. All exercise groups significantly reduced total abdominal and visceral fat (P<0.05) and waist circumference (P<0.001), which measure explained 30% of the variance in total abdominal fat changes (P<0.001). AE and RAE improved insulin sensitivity (P<0.05), but the RE group did not (P>0.1). The greatest insulin sensitivity increase was observed within the RAE group (48% increase, P<0.001). The purpose of the second manuscript (Chapter 4) was to evaluate the effects of exercise modality on cardiorespiratory and functional fitness in the Senior Study. AE and RAE increased cardiorespiratory fitness (P<0.001), whereas RE did not (P>0.1). All exercise groups improved functional fitness performance (P<0.001), but age- and sex-specific percentile ranking improvement within RAE was greater than AE (P<0.01). RE and RAE significantly increased skeletal muscle (P<0.01), predominantly in the upper body, while AE did not (P=1.0). AE and RAE reduced total fat (P<0.001). Both fat loss and muscle gain were independent predictors of improvements in functional fitness (P<0.05). The findings from these studies demonstrate conclusively that a combined resistance and aerobic exercise program without caloric restriction is an optimal strategy for the therapeutic reduction of health risk in abdominally obese men and women. While each exercise modality offers distinct benefits and remains a viable option for needs-based exercise prescription, the combination was associated with the greater simultaneous improvements to body composition, insulin resistance, and cardiorespiratory and functional fitness than either resistance or aerobic exercise alone. / Thesis (Ph.D, Kinesiology & Health Studies) -- Queen's University, 2007-09-13 13:21:31.772