Sarcolipin (SLN) is a small sarcoplasmic reticulum (SR) integral membrane protein that regulates the SR Ca2+-ATPase (SERCA). Previous studies indicate that the functional interaction between SLN and SERCA is thermogenic in nature. Recently, SLN knock-out (SLNKO) mice have been shown to develop excessive obesity and glucose intolerance when placed on a high-fat diet (HFD; 42% kcal derived from fat) relative to wild-type (WT) littermates, implicating SLN in diet-induced obesity. The purpose of this thesis was two-fold: 1) to determine whether an excessively obese phenotype persists when SLNKO mice are given access to voluntary exercise, and 2) to determine if SLN ablation results in a deficit in skeletal muscle oxidative capacity, given the integral role cellular Ca2+ plays in mitochondrial metabolism. Mice were fed either standard chow or a HFD for 8 weeks, and remained sedentary or given access to voluntary running wheels during this period. Glucose tolerance was assessed pre- and post-diet, along with weight gain and adiposity. Skeletal muscle succinate dehydrogenase (SDH), citrate synthase (CS), cytochrome c oxidase (COX), and 3-hydroxyacyl CoA dehydrogenase (ß-HAD) activities were measured in the soleus (SOL) and extensor digitorum longus (EDL) of both chow- and high-fat fed sedentary mice. Both average daily running distance and total exercise volume were not different between WT and SLNKO mice given voluntary running wheels. As before, sedentary SLNKO mice gained more mass following the HFD relative to WT counterparts (P < 0.05); however, no difference in mass gain existed between genotype for voluntary exercising mice on a HFD. Despite this, SLNKO animals were more obese and glucose intolerant following high-fat feeding, regardless of activity status (P < 0.05). Under chow-fed conditions COX activity was higher in the EDL of SLNKO mice (P < 0.05), while no differences in SDH, CS, or ß-HAD existed between genotype in either muscle group. Following the HFD, no changes in mitochondrial enzyme activities within the SOL existed. COX activity in the EDL remained elevated in SLNKO mice post-HFD (P < 0.001), while ß-HAD increased in both WT and SLNKO animals relative to chow-fed controls (P < 0.05). These findings suggest that increasing energy expenditure through voluntary activity cannot compensate for increased basal SERCA Ca2+-pumping efficiency during caloric excess. Additionally, ablation of SLN does not result in a metabolic deficit within skeletal muscle, nor does it limit the adaptive enzymatic response of SLNKO mice to high-fat feeding. Thus, the findings of this study provide further support of the view that SLN’s thermogenic role is the primary mechanism of diet-induced obesity in SLNKO mice.

Changes in intracellular Ca2+ ([Ca2+]f) and high-energy phosphates are known to induce adaptive changes in skeletal muscle during endurance exercising training, including mitochondrial biogenesis. Levels of [Ca2+]f are regulated by sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) which are further regulated by sarcolipin (SLN), through a reduction in the apparent affinity of SERCAs for Ca2+. Furthermore, SLN reduces the efficiency of Ca2+ transport by SERCAs supporting a thermogenic role for SLN in skeletal muscle. Thus, it is possible SLN ablation could reduce Ca2+ and metabolic signaling during exercise training and attenuate increases in mitochondrial content. To investigate the potential role of SLN in the exercise-induced adaptive response of skeletal muscle, mice devoid of SLN (SLNKO) underwent endurance training for 8 weeks and were compared to WT controls. Maximal oxygen uptake (V̇O2 max) was measured with an exercise stress test while mitochondrial content was assessed through measurement of protein expression and maximal enzyme activities of several mitochondrial enzymes in soleus and extensor digitorum longus (EDL) muscles, which express high and low levels of SLN, respectively. All data were analyzed using a two-way analysis of variance (ANOVA) and student t-tests were conducted on enzyme data. V̇O2 max was found to not be significantly altered with exercise training in either genotype. Exercise training significantly increased the contents of adenine nucleotide translocase (ANT), cytochrome-c (cyt-c) and cytochrome-c oxidase subunit IV (COXIV) in soleus independent of genotype. Likewise, exercise training significantly increased cyt-c and COXIV expression (P<0.04), while increases in ANT expression were not significant (P=0.13) in the EDL. Two-way ANOVAs of mitochondrial enzymes in soleus revealed an interaction existed for succinate dehydrogenase (SDH) where its activity was increased only in the SLNKO mice (P<0.02). In comparison, exercise training significantly elevated activities of cytochrome c oxidase (COX) and citrate synthase (CS) activities (P<0.02) but not β-hydroxyacyl-CoA dehydrogenase (β-HAD; P=0.08), independent of genotype. Upon closer examination using student t-tests, it was determined that exercise training induced greater increases in COX and CS activity in SLNKO compared to WT controls (P<0.02), similar to and consistent with SDH data. In EDL, only SDH activity increased following exercise training, an effect that was independent of genotype. In conclusion, these data suggest that SLN ablation does not attenuate exercise-induced mitochondrial adaptations and may increase mitochondrial enzyme adaptations to exercise training in slow-twitch muscle. Further examination of the effects of SLN on Ca2+ and metabolic signaling may provide mechanisms explaining the results of this thesis.

The family with sequence similarity 65, member B (Fam65b) protein is thought to facilitate fusion of myocytes and formation of myotubes during the differentiation of human myogenic cells. Fam65b and histone deacetylase 6 (HDAC6) co-immunoprecipitate and together regulate the levels of acetylated tubulin, which might control microtubule stability in myogenic cells. In this thesis, to gain further insight on the role of Fam65b in the differentiation pathway and motility of myogenic cells, we characterized a Fam65b knock-out (KO) mouse model. Genotyping and transcriptional analysis revealed that a thirteen exons-long region of the Fam65b gene has been successfully ablated and the mRNA amplicons within the deleted segment are not transcribed. Nevertheless, mRNA products corresponding to genomic regions downstream of the deleted area are still detected. Furthermore, analysis of skeletal muscle lysates via western blot (WB) does not show a complete loss of Fam65b expression, but only reduced translation of some isoforms. Nevertheless, WBs of myogenic cells that have been directly isolated from Fam65b KO mice and expanded in vitro revealed the absence of a 120 Kd band, which putatively corresponds to the long isoform of Fam65b. Finally, our data show that Fam65b KO mice are significantly heavier than wild type (WT) mice, and that this phenotype is consistently observed across both genders during the first seven months of age. While functional and molecular analyses of the KO mouse model are still ongoing, future work might include generating a new KO model via the CRISPR-Cas9 technology to ablate all isoforms of Fam65b.

Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

IRF2BP2 is a corepressor of IRF2, a transcription factor involved in the immune response. IRF2BP2 is also a coactivator of the VGLL4/TEAD4 complex in muscle. Given its functional duality, we asked how IRF2BP2 deletion would affect mouse development and adult muscle regeneration. Most Irf2bp2-/- mice die prior to birth, those that survive develop lymphoma in adulthood. Microarray profiling of Irf2bp2 knockout liver, heart, and skeletal muscle revealed a shared program of upregulated genes involved in inflammation and immunity. The function of IRF2BP2 in adult skeletal muscle recovery from cardiotoxin-induced injury was evaluated. Compared to WT mice, mice with macrophage-specific ablation of IRF2BP2 (Irf2bp2flox/LysMCre) or muscle-specific ablation of Irf2bp2 (Irf2bp2flox/MckCre) mice showed increased inflammation and impaired muscle regeneration. Global deletion of Irf2bp2 in mice results predominantly in embryonic death or lymphoma in adults. Irf2bp2 suppresses genes that mediate inflammation in mouse liver, heart, and in skeletal muscle, where IRF2BP2 promotes regeneration.

The disruption of the survival motor neuron (SMN1) gene leads to the children’s genetic disease spinal muscular atrophy (SMA). SMA is characterized by the degeneration of α-motor neurons and skeletal muscle atrophy. Although SMA is primarily considered a motor neuron disease, the involvement of muscle in its pathophysiology has not been ruled out. To gain a better understanding of the involvement of skeletal muscle pathophysiology in SMA, we have developed three aims: to identify cell-specific Smn-interacting proteins, to characterize postnatal skeletal muscle development in mouse models of SMA, and to assess the functional capacity of muscles from SMA model mice. We have used tandem affinity purification to discover Smn interacting partners in disease relevant cell types. We have identified novel cell-specific Smn interacting proteins of which we have validated myosin regulatory light chain as a muscle-specific Smn associated protein in vivo. We have taken advantage of two different mouse models of SMA, the severe Smn-/-;SMN2 mouse and the less severe Smn2B/- mouse, to study the postnatal development of skeletal muscle. Primary myoblasts from Smn2B/- mice demonstrate delayed myotube fusion and aberrant expression of the myogenic program. In addition, the expression of myogenic proteins was delayed in muscles from severe Smn-/-;SMN2 and less severe Smn2B/- SMA model mice. Muscle denervation and degeneration, however, are not the cause of the aberrant myogenic program. At the functional level, we demonstrate a significant decrease in force production in pre-symptomatic Smn-/-;SMN2 and Smn2B/- mice indicating that muscle weakness is an early event in these mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant levels of developmentally regulated proteins important for muscle function, which may impact muscle force production in skeletal muscle of SMA model mice. The present study demonstrates early and profound intrinsic muscle weakness and aberrant expression of muscle proteins in mouse models of SMA, thus demonstrating how muscle defects can contribute to the disease phenotype independently of and in addition to that caused by motor neuron pathology.

Mitochondrial dysfunction is a common feature in a wide range of disorders and diseases from obesity, diabetes, cancer to cardiovascular diseases. The overall goal of my doctoral research has been to investigate mitochondrial metabolic dysfunction in skeletal and cardiac muscles in the context of chronic disease development. Perinatal nutrition is well known to affect risk for insulin resistance, obesity, and cardiovascular disease during adulthood. The underlying mechanisms however, are poorly understood. Previous research from our lab showed that the in utero maternal undernutrition mouse model is one in which skeletal and cardiac muscle physiology and metabolism is impaired. Here we used this model to study the impact of in utero undernutrition on offspring skeletal primary muscle cells and to determine if there is a cell autonomous phenotype. Metabolic analyses using extracellular flux technologies revealed a shift from oxidative to glycolytic metabolism in primary myotubes. Gene expression profiling identified significant changes in mRNA expression, including an upregulation of cell stress and OXPHOS genes and a downregulation of cell division genes. However, there were no changes in levels of marker proteins for mitochondrial oxidative phosphorylation (OXPHOS). Findings are consistent with the conclusion that susceptibility to metabolic disease in adulthood can be caused at least in part by muscle defects that are programmed in utero and mediated by impaired mitochondrial function. In my second project, the effects of the absence of glutaredoxin-2 (Grx2) on redox homeostasis and on mitochondrial dynamics and energetics in cardiac muscle from mice were investigated. Previous work in our lab established that Grx2-deficient mice exhibit fibrotic cardiac hypertrophy, and hypertension, and that complex I of OXPHOS is defective in isolated mitochondria. Here we studied the role of Grx2 in the control of mitochondrial structure and function in intact cells and tissue, as well as the role of GRX2 in human heart disease. We demonstrated that the absence of Grx2 impacts mitochondrial fusion, ultrastructure and energetics in mouse primary cardiomyocytes and cardiac tissue and that provision of the glutathione precursor, N-acetylcysteine (NAC) did not restore glutathione redox or prevent impairments. Furthermore we used data from the human Genotype-Tissue Expression consortium to show that low GRX2 expression is associated with increased fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, our results indicate that GRX2 plays a major role in cardiac mitochondrial structure and function, and protects against left ventricle pathologies in humans. In my third project, we collaborated with cardiac surgeon, Dr. Calum Redpath, of the Ottawa Heart Institute to study atrial mitochondrial metabolism in atrial fibrillation patients with and without type 2 diabetes (T2DM). T2DM is a major risk factor for atrial fibrillation, but the causes are poorly understood. Atrial appendages from coronary artery bypass graft surgery were collected and analyzed. We showed an impaired complex I respiration in diabetic patients with atrial fibrillation compared to diabetic patients without atrial fibrillation. In addition, and for the first time in atrial fibrillation patients, mitochondrial supercomplexes were studied; results showed no differences in the assembly of the “traditional” complexes but a decrease in the formation of “high oligomeric” complexes. A strong trend for increased protein oxidation was also observed. There were no changes in markers for OXPHOS protein levels. Overall findings reveal novel aspects of mitochondrial dysfunction in atrial fibrillation and diabetes in humans. Overall, our results reveal that in utero undernutrition affects the programming of skeletal muscle primary cells, thereby increasing susceptibility to metabolic diseases. In addition, we show that GRX2 impacts cardiac mitochondrial dynamics and energetics in both mice and humans. Finally, we show impaired mitochondrial function and supercomplex assembly in humans with atrial fibrillation and T2DM. Ultimately, understanding the mechanisms causing mitochondrial dysfunction in muscle tissues during chronic disease development will increase our capacity to identify effective prevention and treatment strategies.

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [3H]phenylalanine into the protein pool) were investigated in differentiated C2C 12 myotubes and rat diaphragm muscle (DIAm). In C2C 12 myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased ∼30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [3H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

Background. Bisphenol A (BPA) and BPS are environmental pollutants that are associated with the development of insulin resistance and type 2 diabetes (T2D). Although skeletal muscle and adipose tissue dysfunction are involved the development of insulin resistance, there are few studies that have investigated the effects of bisphenols on their metabolism. In this study, we investigated the effects of BPA and BPS exposure on skeletal muscle and adipose tissue metabolism to determine how they contribute to the development of T2D. Methods. L6 muscle cells were treated with BPA during the last 24 hours of differentiation, and mitochondrial function and glucose metabolism was measured. Human subcutaneous adipose tissue was incubated for 24 or 72 hours with BPA or BPS, and adipokine gene expression and glucose metabolism was measured in adipose tissue. Results. L6 muscle cells treated with high concentrations of BPA (10⁵ nM) had mitochondrial dysfunction and a compensatory increase in glucose metabolism; however, there were no effects at environmentally-relevant concentrations. Adipose tissue treated with BPA for 24 hours had reduced expression of proinflammatory cytokines and adipokines, and reduced insulin-stimulated glucose uptake. Conclusions. BPA exposure for 24 hours did not alter L6 muscle cell mitochondrial function and glucose metabolism at environmentally-relevant concentrations; however, adipose tissue had altered proinflammatory expression and glucose metabolism at low concentrations. This has important implications in regulatory guidelines in the use of BPA in the manufacturing of consumer products.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Pulmonary Hypertension (PH) is a devastating disease characterized by pulmonary arterial remodeling, right ventricular dysfunction and ultimately right heart failure. Increased emphasis has been given to skeletal muscle dysfunction in PH, and to its implication in the severe exercise intolerance that is a hallmark of the condition. In this dissertation, skeletal muscle blood flow was measured via the microsphere technique at rest and during exercise (Aim 1), with an acute dose of dietary nitrate via beetroot juice (BRJ) gavage used to determine if supplementation could improve muscle blood flow and alter energetics (Aim 2). VO2max, voluntary running and grip strength tests were used to determine the effect of disease on performance, and to test for an ergogenic effect of BRJ vs. placebo (PL) in healthy and PH rats (Aim 3). Methods: A prospective, randomized, counterbalanced, placebo-controlled trial was used to examine the aforementioned aims across four groups; PH rats (induced with monocrotaline, MCT, 60mg/kg, s.q., 4 weeks) supplemented with BRJ (MCT BRJ, n=9); PH rats supplemented with placebo (MCT PL, n=9); healthy control rats (vehicle, s.q.) supplemented with BRJ (CON BRJ, n=8); healthy control rats supplemented with placebo (CON PL, n=9). Results: Monocrotaline induced a severe PH phenotype evidenced by increased RV wall thickness, RV hypertrophy, RVSP and reduced cardiac output and stroke volume compared to controls (p=<0.001). MCT rats demonstrated lower muscle blood flow at rest, and more prominently during exercise compared to controls (p=0.007-0.047), regardless of supplementation. MCT rats displayed a greater reliance on anaerobic metabolism, demonstrated by increased blood lactate accumulation (p=<0.001), and this was significantly related to reduced blood flow during exercise (r=-0.5879, p=0.001). BRJ supplementation resulted in increased plasma nitrate and nitrite compared to PL (p=<0.001), but at the skeletal muscle level, only nitrate was increased after BRJ. BRJ did not have a significant effect on blood flow, with no improvement during exercise shown vs. PL. Similarly, BRJ did not significantly improve exercise function in MCT or CON rats. Conclusion: MCT rats demonstrated a reduction in muscle blood flow, with BRJ supplementation not resulting in improved flow or exercise performance.