Work focusing on the combination of EMG and fMRI in skeletal muscle. / Two commonly used diagnostic techniques for examining muscle function in vivo are functional magnetic resonance imaging (fMRI) and electromyography (EMG). EMG allows for examination of the functional, electrical activity of muscle during force production. Comparatively, fMRI or more specifically blood oxygen level dependant imaging can be applied to visualize muscle activation and recovery post-exercise. It is a combination of oxygenation, metabolism, blood flow and blood volume. The proposed method combines both techniques in simultaneous data acquisition to provide greater muscle physiological information during exercise. Additionally, both techniques are non-invasive making repeated measurements feasible. EMG hardware filtering was designed and constructed to facilitate EMG measurements alongside MRI scans during simultaneous acquisition. Next, a complex artifact subtraction method called fMRI artifact slice template removal (FASTR) was implemented. With custom scripts and small adaptations to FASTR, it was modified for use with EMG/fMRI, specifically, with a echo planar imaging (EPI) BOLD sequence. Several experiments were then performed to test it's capabilities improving the signal-to-noise ratio (SNR) of the EMG data from 2.8 to 46 in one case. After EMG hardware and software were developed and implemented, a simple exercise protocol was developed to investigate changes in concurrent BOLD/EMG, recording before, during and following exercise. A linear correlation analyses was performed to compare EMG and BOLD results. A strong correlation between the EMG root-mean-square (RMS) peak amplitude and the length of time to recover back to baseline was noted (r=0.681, n=3). For future studies, multiple EMG measurements should be applied to improve the amount of information collected during voluntary exercise. Lastly, this technique may have usage with not just BOLD MRI scans, but with various other techniques such as near infrared spectroscopy (NIRS), and diffusion tensor imaging (DTI) in order to further probe muscle physiology. / Thesis / Master of Applied Science (MASc) / Two commonly used methods for detecting disease and injury in muscle are magnetic resonance imaging (MRI), and electromyography (EMG). EMG provides information about the electrical activity of muscle during exercise, while MRI scans give two or three dimensional images of the body. Using these two techniques at the same time, provides the opportunity to obtain greater physiological information of muscle during and after exercise. The goal of this work was to design and create an EMG system that functioned alongside MRI scans. However, combining these two techniques presented several challenges that needed to be solved before this was possible. These issues were resolved and diminished by utilizing specific hardware and software solutions alongside rigorous testing. Additionally, results from the combination of these two techniques have demonstrated there is great potential for future studies. In conclusion, using EMG and MRI together is feasible, and allows for further investigation into muscle physiology.

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Over the past two decades, a vast body of research has demonstrated the importance of G protein-coupled receptor kinase 2 (GRK2) in the physiology and pathophysiology of the heart. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 following cardiac insult exacerbates injury and speeds progression to heart failure. In this dissertation we turned our attention towards two novel aspects of GRK2 biology. Firstly, despite the importance of this GRK2 activity in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In the first study of this dissertation, we generated a novel skeletal muscle specific GRK2 knockout (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle, yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared to wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, this study provides the first insights into the role of GRK2 in skeletal muscle physiology, and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2-adrenergic receptor-induced hypertrophy. In the second part of this dissertation, we report surprising novel metabolic phenotypes that arise from modulating GRK2 activity exclusively in the heart. We show that transgenic βARKct (TgβARKct) mice (cardiac specific expression of a GRK2 inhibitory peptide) are more susceptible to high-fat diet (HFD) induced obesity. TgβARKct mice exhibit marked increase in adiposity on HFD relative to control animals. Conversely transgenic GRK2 mice (TgGRK2) mice (cardiac specific overexpression of GRK2) show resistance to weight gain on a HFD and decrease in adipose tissue mass relative to control animals. Furthermore, conditioned media from βARKct expressing neonatal rat ventricular myocytes enhances adipocyte differentiation in vitro. These results suggest that the heart produces a secreted factor to control whole body metabolism, and that GRK2 is a regulator of this mechanism. / Biomedical Sciences

<p> We hypothesized that acute inhibition of the contraction-induced expansion of the muscle TCA cycle intermediate (TCAI) pool via would not adversely effect metabolism or contractile function. Forty rats were anaesthetized and the gastrocnemius muscle (GAS) from one leg was vascularly isolated and perfused with saline (CON) or a red cell media containing DL-cycloserine (CYCLO; Sigma C-7005; dose=0.05 mg/g), an inhibitor of alanine aminotransferase (AAT). After 1h of perfusion, the GAS muscle was either snap frozen (CON-Rest, n=11; CYCLO-Rest, n=9) or stimulated to contract for 10 min (1Hz, 0.3 ms, 2 V) with blood flow fixed at 30 ml min-1 100g-1 and then snap frozen (CON-Stim, n=10; CYCLO-Stim, n=10). The maximal activity of AAT was lower (P≤0.05) at both CYCLO-Rest (0.61±0.02 mmol·kg-1w.w./min; mean± SEM) and CYCLO-Stim (0.63±0.01 mmol·kg-1w.w./min) vs CON-Rest (3.56±0.16 mmol·kg-1w.w./min) and CON-Stim (3.92±0.29 mmol·kg-1w.w./min). Consistent with lower net flux through AAT, muscle [alanine] was lower (P≤0.05) after CYCLO-Stim (6.97±0.26 mmol·kg-1 dw) compared to CON-Stim (8.55±0.56 mmol·kg-1 dw) and not different vs CON-Rest (6.79±0.41 mmol·kg-1 dw). The sum of five measured TCAI (malate, fumarate, citrate, isocitrate, and 2-oxoglutarate) was higher (P≤0.05) at both CON-Rest (2.10± 0.09 mmol·kg-1 dw) and CON-Stim (2.48± 0.11 mmol·kg-1 dw) vs CYCLO-Rest (1.56± 0.11 mmol·kg-1 dw) and CYCLO-Stim (1.88± 0.15 mmol·kg-1 dw) respectively. Despite the reduction in [TCAI] following CYCLO treatment, there was no difference between conditions in muscle lactate accumulation or phosphocreatine degradation after 10 min of stimulation. Contractile function was not different (P≤0.05) between conditions at either rest or stimulation and the decline in force production over ten minutes of stimulation was identical (~60%) between CON-Stim and CYCLO-Stim respectively. We conclude that flux through AAT was reduced after cycloserine treatment, however the acute inhibition of TCAI expansion did not compromise aerobic energy provision. These data support the hypothesis that the contraction-induced increase in muscle [TCAI] is not causally linked to oxidative energy delivery.</p> / Thesis / Master of Science (MSc)

The purpose of this study was to determine the effects of short-term (14d) unilateral leg immobilization using a simple knee brace (60° flexion)/crutchmediated model on muscle function and morphology in men (M, N=13) and women (W, N=14). Isometric and isokinetic (concentric SLOW, 0.52 rad•s-1 and FAST, 5.24 rad•s-1) knee extensor peak torque was determined at three time points (PRE, DAY -2, and DAY -14). At the same time points, magnetic resonance imaging was used to measure the cross-sectional area of the quadriceps femoris and DEXA scanning was used to calculate leg lean mass. Muscle biopsies were taken from vastus lateralis at PRE and DAY-14 for myosin ATPase and myosin heavy chain analysh Women showed greater decreases (PRE vs. DAY-14) compared to men in specific strength (N•cm-2) for isometric (M=3.1±13.3, W=17.1±15.9%; p= 0.055, [mean±SD]) and concentric SLOW (M=4.7±11.3, W=16.6±18.4%; p<0.05) contractions. There were no immobilization-induced sex-specific differences in the decrease in quadriceps femoris cross-sectional area (M=5.7±5.0, W=5.9±:5.2%) or leg lean mass (M=3.7±4.2, W=2.7±2.8%). There were no fiber type transformations, and the decrease in Type I (M=4.8±5.0, W=5.9±3.4%), IIa (M=7.9±9.9, W=8.8±8.0%) and Ilx (M=10.7±10.8, W=10.8±12.1 %) fiber areas was similar between sexes. These findings indicate that immobilization-indt: ced loss of knee extensor muscle strength is greater in women compared tc men in spite of a similar extent of atrophy at the myofiber and whole muscle levels after only 14d of unilateral leg immobilization. Furthermore, we have described an effective and safe method of knee immobilization that results in significant reductions in quadriceps muscle strength and fiber size. / Thesis / Master of Science (MS)

Resistance exercise training (RET) can lead to muscle hypertrophy; however, the relative contribution that exogenous (protein supplementation and specific training variables) versus endogenous (biology inherent to the individual) factors have on RET-induced muscle hypertrophy is controversial. In Study 1, we provided an evidence-based conclusion that protein supplementation during periods of RET results in a small but statistically significant increase in RET-induced muscle hypertrophy. In Study 2, we corroborate previous research and observed that the amount of mass lifted per repetition (load) did not determine RET-induced muscle hypertrophy in resistance-trained men when RET was performed to volitional fatigue. In Study 4, we observed similar muscle fibre activation following resistance exercise with lighter versus heavier loads when both were lifted until volitional fatigue. In Studies 2 and 3, we observed no relationship between circulating anabolic hormones (e.g., testosterone) and RET-induced muscle hypertrophy. Nonetheless, in Study 3, we found significantly greater muscle androgen receptor content in the top versus the bottom quintile of respondents for muscle hypertrophy following 12 weeks of RET indicating that androgen receptor content, and not circulating androgen concentration, may be an important determinant of hypertrophy. Finally, in Study 5, we observed that RET-induced muscle hypertrophy was an consistent within an individual (independent of load and limb) but considerably different between participants. Together, these data suggest that the exogenous factors we studied – protein supplementation and load (when RET was performed to volitional fatigue) – had a relatively small influence on RET-induced muscle hypertrophy. In contrast, we found that endogenous variables, such as intramuscular androgen receptor content and likely other genetic influences, appear to contribute more to the significant heterogeneity seen in RET-induced muscle hypertrophy. Future research in this area should prioritize understanding the biology that underpins the individual variability in RET-induced muscle hypertrophy. / Thesis / Doctor of Philosophy (PhD) / Resistance exercise training (RET) increases muscle size (hypertrophy); however, the relative influence that protein supplementation, specific training variables, and individual (genetic) variation have on the RET-induced hypertrophy is controversial and largely unknown. Broadly, data in this thesis show that protein supplementation slightly augments RET-induced hypertrophy, and that the magnitude of RET-induced hypertrophy may be related to the number of androgen (e.g., testosterone) receptors inside an individual’s muscle. In contrast, we found that neither load nor hormones affect RET-induced hypertrophy. Interestingly, data in this thesis also show that RET-induced hypertrophy is consistent within an individual but varies considerably between people, which illustrates the greater influence that individual variation has on RET-induced hypertrophy. We conclude that when RET is performed with a high degree of effort, protein supplementation and specific training variables confer a relatively small benefit on RET-induced hypertrophy compared to the influence of biological variability between people.

This thesis considered three potential factors that may influence skeletal muscle responses to interval exercise training in healthy adults, with a focus on mitochondrial remodelling: biological sex, fibre type and contraction pattern. Study 1 assessed the acute response to a sprint interval training (SIT) protocol that involved three, 20-second ‘all-out’ cycling efforts interspersed with 2 minutes of recovery. It found similar exercise-induced increases in the mRNA expression of genes linked to mitochondrial biogenesis in men and women. Study 2 showed that a single session of SIT and moderate-intensity continuous training (MICT) induced similar increases in the phosphorylation of signalling proteins linked to mitochondrial biogenesis in type I and IIa fibres when compared within the same individuals. It also assessed responses to 12 weeks of training in two different groups and found MICT elicited greater increases in markers of mitochondrial content, including cytochrome c oxidase subunit IV (COXIV) protein content, in type I fibres, whereas the increase in type IIa fibres was similar compared to SIT. Study 3 utilized single-leg cycling as a within-subject model to compare interval and continuous training protocols that were matched for exercise intensity and total training volume, but the pattern of contraction differed. Measurements of mitochondrial content, including subsarcolemmal mitochondrial area, the maximal activity of citrate synthase and COXIV protein content, increased after 4 weeks of continuous training but were unchanged after interval training. Overall, this thesis advances our understanding of the influence of biological sex, muscle fibre type and contraction pattern on skeletal muscle mitochondrial responses to exercise. Major findings include: the acute response to SIT was similar between sexes, acute responses to interval and continuous exercise were similar but training elicited some divergent fibre type-specific responses, and mitochondrial content increased following continuous training but was unchanged following work- and intensity-matched interval training. / Dissertation / Doctor of Philosophy (PhD) / This thesis considered the role of biological sex, fibre type and contraction pattern on skeletal muscle responses to exercise. It focused on mitochondria, which are important components of the cell that affect health and performance. The responses of genes that regulate the formation of new mitochondria to a single session of brief, “sprint” interval exercise were similar between men and women. Acute sprint interval exercise also activated molecular pathways similar to traditional endurance exercise in the two main types of muscle fibres; however, 12 weeks of endurance training induced greater increases in mitochondrial content in type I (“slow-twitch”) fibres, whereas the increases in type II (“fast-twitch”) fibres were similar compared to interval training. Lastly, mitochondrial content increased after continuous training but not interval training when the intensity and total amount of exercise was matched. Our findings improve our understanding of the mechanisms by which exercise elicits mitochondrial remodelling in humans.

There is strong evidence that skeletal muscle health is compromised in persons with type 1 diabetes mellitus (T1D). These impairments include reduced strength, mitochondrial dysfunction, and decreased satellite cell (SC) content. Maintaining healthy muscle requires successful muscle repair. Preclinical models of T1D consistently show impaired muscle regeneration. To date, the impact of T1D on human skeletal muscle repair has not been established; however, attenuated repair would account for the reduced functional capacity and premature institutionalization that often characterizes those with diabetes. The purpose of this study was to determine the impact of T1D on the recovery of skeletal muscle function, morphology, and ultrastructure after 300 unilateral eccentric contractions (90°/s) of the knee extensors. Eighteen men and women (18-30 years old) with (n=9) and without (n=9) T1D performed the exercise protocol. Pre-damage, and at 48- and 96-hours post-damage, subjects gave a blood sample and vastus lateralis biopsy, and performed a maximal isometric knee extension. Given the sex-specific differences in muscle damage, control and T1D men and women were analyzed together and separately. Force production and recovery were comparable between control and T1D men and women. Exercise-related increases in creatine kinase activity and ultrastructural damage were also comparable between groups. There was a trend towards T1D men having more type 2 fast-twitch muscle fibers than T1D women (p=0.055). While baseline SC content was not different between groups, proliferating SC content was trending lower at 48-, and higher at 96-hours post-damage in T1D women compared to controls (p=0.07). In those with T1D, there was no correlation between muscle damage and HbA1c, but HbA1c was strongly correlated with vigorous physical activity (r=0.881, p=0.002). Contrary to preclinical studies, our data is the first to show that skeletal muscle repair is largely unaltered in otherwise healthy young adults with T1D. We attribute these differences to glycemic control and speculate that muscle repair is unaffected if individuals are optimally managing their diabetes. Considering the exercise-related dysglycemia seen in T1D, our results emphasize a need to define the dose of physical activity required for those with diabetes to properly regulate their blood glucose levels. We expect that this would in turn, improve skeletal muscle health and ultimately, extend the healthy lifespan of those living with T1D. / Thesis / Master of Science in Medical Sciences (MSMS) / Type 1 diabetes mellitus (T1D) is a chronic disease where the body does not make enough insulin to control blood glucose levels. Overtime, unstable blood glucose levels can damage major organ systems, including skeletal muscle. Skeletal muscle plays a pivotal role in regulating our physical and metabolic capacities. In those with T1D, exercise-mediated improvements in muscle health have been shown to delay health complications. However, we do not know how diabetic skeletal muscle repairs from exercise in humans. In this thesis, we investigated the ability of skeletal muscle to recover from damaging exercise in young adults with T1D. For the first time, we showed that skeletal muscle repair was similar between otherwise healthy young adults with T1D and those without diabetes. Our findings suggest that persons with T1D can engage in high levels of physical activity without compromising their muscle health. Further studies are needed to understand how exercise type, intensity, and duration impact glycemic control in men and women with T1D.

In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling. After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously supported SR Ca2+-uptake due to glycogen extraction affected the source of ATP synthesis (either PK or CK), the effectiveness of energy utilization for Ca2+ transport (SERCA), or altered the metabolic channeling properties. / Ph. D.