No description available.

No description available.

No description available.

No description available.

Skeletal muscle associated disorders are correlated with significant morbidity, including frailty, fatigue, reduced mobility and poor resistance to treatments as well as mental health repercussions resulting from a loss of independence. Thus, conditions affecting skeletal muscle put considerable pressure on the health care system. In response to injury, skeletal muscle can regenerate and the molecular mechanisms underlying this unique process has been the subject of intense research with the goal of developing better treatment modalities for muscle-related diseases. Our laboratory has previously demonstrated that C/EBPβ is a negative regulator of postnatal myogenic differentiation. Expressed in muscle satellite cells (MuSCs), the primary source of regenerative potential in skeletal muscle, C/EBPβ inhibits entry into the myogenic differentiation program and is required for MuSC self-renewal after injury. Despite the important role of C/EBPβ in muscle homeostasis, little is known about the genes it regulates. To better understand how C/EBPβ regulates these processes, I used both a candidate-based approach to identify the inhibitor of DNA binding and differentiation protein ID3 as a C/EBPβ target gene that mediates inhibition of myogenic differentiation, and an unbiased approach using RNA-seq. I compared gene expression profiles from C2C12 myoblasts overexpressing C/EBPβ to control cells under growth and differentiation conditions. I observed that more than 20% of the molecular signature found in quiescent MuSCs is regulated by C/EBPβ. Caveolin- 1 was implicated as a direct target of C/EBPβ and part of the molecular mechanism by which C/EBPβ maintains MuSCs quiescence. Interestingly, the RNA-seq data identified numerous C/EBPβ-regulated secreted proteins including growth factors and cytokines. Co-culture experiments indicate that secreted proteins mediate the inhibition of cell differentiation and fusion, suggesting that C/EBPβ functions in an autocrine and paracrine fashion to influence activation of myoblasts in the absence of cell-to-cell contact. Given the role of C/EBPβ in regulating secretory proteins that inhibit myogenic differentiation, I examined the requirement of C/EBPβ in the expression of anti myogenic proteins secreted by cancer cells that affect MuSCs function and drive the development of cancer cachexia. Indeed, the expression of C/EBPβ in cancer cells was found to be required for the production of a cachexia-inducing secretome by tumours in vitro and in vivo. Furthermore, C/EBPβ was found to be sufficient to convert non-cachectic tumours into cachexia-inducing ones. In comparing differentially expressed C/EBPβ-regulated secreted protein transcripts from our RNA-seq data to that from 27 different types of human cancers revealed an ~18% similarity between C/EBPβ-regulated secreted proteins and those enriched in cachectic tumours including pancreatic, gastric and brain cancers. Three of these C/EBPβ-regulated secreted proteins (SERPINF1, TNFRSF11B and CD93) were tested further and found to be inducers of muscle atrophy. This work provides molecular insight into the role of C/EBPβ in the regulation of MuSC function and the regulation of cachexia-inducing factors by tumours, placing C/EBPβ as a novel therapeutic target for the treatment of cancer cachexia and other muscle-related diseases.

Muscle fatigue is an intrinsic myoprotective process that prevents damaging ATP depletion during intense or prolonged exercise by limiting ATP demand when ATP production becomes insufficient. One mechanism of fatigue involves a reduction in membrane excitability with the opening of ATP-sensitive K+ (KATP) and ClC-1 Cl- channels, resulting in submaximal sarcoplasmic reticulum Ca2+ release and reduced force generation, but the intracellular signalling pathways for this process is unknown. As a first step toward understanding this process, the objective of this study was to test the hypothesis that adenosine receptors (ARs) and AMPK trigger fatigue when a metabolic stress occurs during muscular activity. Compared to control conditions, a pan-activation of ARs with 10 µM adenosine and NECA initially reduced the fatigue rate during the first 60 s of a 3 min fatigue bout triggered with 1 tetanic contraction every s. An activation of the A1 adenosine receptor (A1R) with 10 and 20 µM ENBA resulted in faster rate of fatigue; an effect blocked by 5 µM DPCPX, an A1R antagonist. At 10 and 20 µM, adenosine, NECA, and ENBA activated AMPK via an increased in T172 phosphorylation. At 10 µM, MK8722, an AMPK agonist, initially caused a reduction in fatigue rate during the first 60 s followed by an increased fatigue rate during the last 2 min of the fatigue bout. Co-activation of ARs and AMPK did not give rise to either an additive or synergistic effect. FDB from AMPK α1-/- and α2-/- mice had faster fatigue rate and greater increased in unstimulated force compared to FDB from AMPK α1+/+ and α2+/+ mice. It is suggested that ARs and AMPK play a role in the mechanism of fatigue when a metabolic stress develops during muscle activity.

No description available.

Type 1 diabetes (T1D) is the result of the autoimmune-mediated destruction of the pancreatic beta-cells leading to the inability to produce insulin sufficiently and, in turn, regulate blood glucose levels. Abnormal levels of blood glucose, specifically hyperglycemia, have been linked to many diabetic complications, with Brownlee proposing decreased GAPDH activity and the resultant increase in four main pathways as the mechanism(s) leading to these complications. Though skeletal muscles play a major role in glucose uptake, they are believed to be relatively protected against these complications as they are able to regulate their glucose uptake. However, evidence is accumulating that skeletal muscles are adversely affected in T1D, particularly with respect to their mitochondrial function. This led us to consider that the skeletal muscles of those with T1D would experience substrate overload (high intracellular lipids and recurrent, high levels of intracellular glucose), which would initiate a negative spiral whereby substrate excess would damage mitochondria - leading to an impaired ability to utilize these substrates - further worsening the substrate overload. Therefore, the objective of this study was to investigate glycogen and intramyocellular lipid (IMCL) content in the muscles of mice and humans with T1D, as well as the potential downstream effects in the form of post-translational modifications (PTMs), mitochondrial content, and lipofuscin accumulation. The Akita T1D mouse model was used to assess substrate overload in uncontrolled diabetes, whereas human participants were used to investigate substrate overload in the presence of insulin therapy. Assessment of glycogen and IMCL content revealed no difference between controls and diabetic cohorts in both the rodent and human study, indicating the lack of substrate overload. Post-translational modifications did not significantly change between Akita and wild-type mice; however, there was a main effect of diabetes on acetylation levels within Akita mice. Lastly, most mitochondrial properties, except for subsarcolemmal pixel density, did not differ either between diabetic and non-diabetic subjects in the human study. Thus, despite mitochondrial complex impairments in diabetic subjects, its extent was not significant enough to cause alterations to the mitochondria as a whole and result in mitochondrial degradation and lipofuscin formation. This study has provided novel insight into the metabolic properties of skeletal muscle during diabetes. Although there was no indication of substrate overload, diabetes still resulted in some changes to PTM levels and mitochondrial pixel density. However, the effects of these changes did not significantly alter the muscle and resulted in pathway impairments of those that were studied. This could be due to an adaptive mechanism in mice, although future studies are needed to confirm this hypothesis. In the human study, healthy, well-controlled individuals could explain why there was hardly any difference seen, suggesting that controlling glycemic levels was imperative in preventing diabetic complications in muscle. / Thesis / Master of Health Sciences (MSc)

Ribosomes are the essential machinery for cellular protein synthesis. Ribosome content is hypothesized to support muscle growth and is suggested that those with more ribosomes may better respond to resistance training. Aerobic training also elicits distinct physiological adaptations; however, no direct measures of ribosome content following aerobic training have been measured. Ribosomes interact with mitochondria for mitochondrial protein synthesis and import. Mitochondria may also provide cellular energy to ribosomes. We hypothesized that aerobic and resistance training would increase ribosome content and that ribosome content following aerobic training would correspond to changes in mitochondrial-related protein content and gene expression. Fourteen young men and women performed 6 weeks of single-legged aerobic followed by 10 weeks of bilateral resistance training. Muscle biopsies were taken following aerobic (Pre RT) and resistance training (Post RT) in the aerobically trained (EX) and control (CTL) legs. Pre RT, EX had greater COXIV staining intensity in Type 1 (1.17-fold; p=0.020) and Type 2 (1.22-fold; p=0.015) fibres compared to CTL; however, no differences in whole-muscle mitochondrial-related protein content or gene expression were observed (p>0.05). No differences in regulatory (UBF, Cyclin D1, TIF-1A, POLR-1B), cytosolic (45S, 5.8S, 18S, 28S rRNAs) or mitochondrial (12S rRNA) ribosome-related gene expression were observed (p>0.05), except for c-Myc (CTL>EX; p=0.034) and 5S rRNA (Pre RT CTL<Pre RT EX; p=0.076). When stratified for leg-lean soft tissue mass (LLSTM), legs with greater LLSTM had lower expression in 3/13 ribosome-related genes (p<0.10). When stratified for ΔLLSTM following resistance training, legs with the greatest ΔLLSTM had lower expression in 11/13 ribosome-related genes prior to (p<0.10) and less change or decrease in expression in 9/13 genes following resistance training (p<0.05). These results indicate that baseline ribosome content was sufficient to support aerobic adaptations (capillarization, VO2 peak) that were previously observed and that ribosome’s efficiency, rather than content, is likely more important to support increases in muscle hypertrophy following resistance training. / Thesis / Master of Science in Kinesiology / Ribosomes are essential in making proteins within the cell, and their content has been hypothesized to support the adaptive responses observed with exercise training. Ribosome content has previously been shown to increase following resistance training likely to support skeletal muscle growth. However as aerobic training also influences cellular adaptations, it is plausible that ribosome content also supports these training adaptations. We hypothesized that both aerobic and resistance training would increase ribosome content. Contrary to our hypotheses, no changes in ribosome content were observed following aerobic or resistance training despite previously observing adaptations characteristic of each respective training stimulus. However, those with the greatest increases in muscle mass had lower baseline ribosome content and less change in content following resistance training. These results suggest that baseline ribosome content is sufficient for aerobic adaptations and that ribosome’s efficiency is likely more important than content to elicit resistance training adaptations.

Stretch activation (FSA) is the delayed increase in fiber specific tension (force per cross-sectional area) following a rapid stretch and can improve muscle performance during repetitive cyclical contractions. Historically considered minimal in skeletal muscle, our recent work showed the ratio ofstretch- to calcium-activated specific tension (FSA/F0) increased from 10 to 40% with greater inorganic phosphate (Pi) levels in soleus muscle fibers (Straight et al., 2019). Given Pi increases with muscle fatigue, we hypothesize that FSA helps maintain force generation during fatigue. To test this, FSA, induced by a stretch of 0.5% fiber length, was examined during Active (pCa 4.5 (pCa = -log([Ca2+]), pH 7.0, Pi 5 mM), High Ca2+ Fatigue (pCa 4.5, pH 6.2, Pi 30 mM) and Low Ca2+ Fatigue (pCa 5.1, pH 6.2, Pi 30 mM) in fibers expressing myosin heavy chain (MHC) I, IIA, IIX and IIB isoforms from soleus and extensor digitorum longus muscles of C57BL/6NJ mice. F0 of all MHC isoforms decreased from Active to High Ca2+ Fatigue to Low Ca2+ Fatigue, as expected. In MHC IIX and IIB fibers, FSA occurred under all conditions and FSA/F0 increased from Active (17-20%) to High Ca2+ Fatigue (32-35%) to Low Ca2+ Fatigue (42-44%). In MHC IIA fibers, FSA/F0 increased similarly to MHC IIX and IIB fibers from Active (14%) to High Ca2+ Fatigue (32%) but stayed elevated under Low Ca2+ Fatigue (35%). For MHC I fibers, no discernable FSA was apparent in either High – or Low Ca2+ Fatigue, leaving an FSA/F0 value in Active only ( 4%). These results show that FSA is a significant modulator of specific tension production under fatiguing conditions in fast-contracting muscle fibers. This mechanism could play an important physiological role during cyclical contractions, when the antagonistic muscle rapidly stretches the agonist muscle, by reducing the effect of fatigue on specific tension production.