Mechanism of myostatin action during satellite cell activation and muscle wasting.

Myostatin, a Transforming Growth Factor-beta (TGF-β) superfamily member, has been well characterised as a negative regulator of muscle growth and development. In support, inactivation or mutation of the myostatin gene results in a dramatic increase in skeletal muscle mass, however excess myostatin inhibits myogenesis. Recently, myostatin has also been shown to have a role in post-natal muscle growth. Myostatin regulates activation, proliferation and self-renewal of the muscle satellite cell pool. Moreover, loss of myostatin results in enhanced skeletal muscle regeneration in response to injury, whereas increased post-natal myostatin expression is associated with many skeletal muscle wasting conditions. Furthermore, myostatin has been shown to directly induce cachexia following subcutaneous injection of Myostatin over-expressing cells into mice. Despite studies implicating myostatin in the regulation of post-natal skeletal muscle growth, little is known about the processes through which myostatin activity is regulated or the mechanisms through which myostatin functions. Thus this thesis examines regulation of myostatin activity through proteolytic processing, and signaling mechanisms through which myostatin acts to regulate the satellite cell pool and to promote skeletal muscle proteolysis. In this thesis it is demonstrated that processing and secretion of Myostatin is relatively reduced in differentiated myotubes as compared to proliferating myoblasts. Furthermore, processing of Myostatin is developmentally regulated, with decreased Myostatin processing occurring during foetal muscle development when compared to post-natal adult muscle. It is also demonstrated that mature Myostatin negatively regulates furin promoter activity. Furin protease is critical for the processing of several members of the TGF-β superfamily, thus a mechanism is proposed whereby myostatin negatively auto-regulates its proteolytic processing during development to facilitate the process of myoblast differentiation. It was further demonstrated that over-expression of Pax7 in C3H10T1/2 multipotent cells enhances myogenic conversion in these cells. However, over-expression of Pax7 in C2C12 myoblasts delays the onset of differentiation, concomitant with an increase in the population of quiescent, satellite cell-like reserve cells. Furthermore, treatment with Myostatin down-regulates Pax7 expression, while Pax7 expression was higher in myostatin-null myoblasts as compared to wild-type myoblast cultures. Furthermore, absence of myostatin alters cell heterogeneity, whereby an increase in Pax7+/MyoD- reserve cell populations is observed. Pax7 expression persists longer through differentiation in cultured primary myoblasts from myostatin-null animals when compared to wild-type counterparts. Reserve cell populations were also measured, and consistent with increased expression of Pax7, there is an increased pool of quiescent self-renewed reserve cells in differentiated cultures from myostatin-null mice as compared with wild-type cultures. Taken together, these results suggest that increased expression of Pax7 regulates the self-renewal process of satellite cells, and furthermore, growth factors such as myostatin signal through Pax7 to regulate the self-renewed pool of satellite cells. In this thesis it is shown that myostatin induces cachexia through a mechanism independent of NF-κB. Myostatin treatment results in a cachexia phenotype with a reduction in myotube number and size in vitro, as well as a loss of body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 are reduced, while NF-κB localisation and expression remains unchanged. Expression of the ubiquitin-associated genes Atrogin-1, MuRF-1 and E214k are shown to be up-regulated following Myostatin treatment. The mechanism behind myostatin-mediated cachexia was further investigated. It is shown that myostatin antagonises the IGF-1/PI3-K/AKT hypertrophy pathway by inhibiting AKT phosphorylation, thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. In addition, microarray analysis resulted in the identification of a potentially interesting downstream target gene of myostatin during the induction of cachexia. Initial characterisation of CXXC5, or MM1 as it was renamed, was also performed. Over-expression of MM1 in vitro results in the up-regulation of components of the ubiquitin-proteasome pathway including Atrogin-1, E214k, E220k and RC2. It was further demonstrated that increased expression of MM1 enhances the level of protein ubiquitin-conjugation. Furthermore, over-expression of MM1 results in myotube collapse and the formation of multinucleated myosacs. It was also demonstrated that the MM1-induced myotube collapse results in disruption of the typical myotube microtubule structure. Therefore, these data suggest that MM1 is a muscle wasting-inducing gene which functions in the regulation of myostatin-mediated cachexia. Therefore data presented in this thesis highlights a mechanism through which myostatin is regulated, and further delineates the role of myostatin in controlling several key processes during post-natal myogenesis, namely satellite cell replenishment and skeletal muscle wasting

Anisotropic effects following inner shell ionization.

This thesis looks into the possibility that X radiation following inner shell ionization by electron impact might be polarized. There has been some speculation on this point: one published conclusion (Cooper and Zare, 1968) is that the polarization must be zero; another conclusion (Mehlhorn, 1968) is that the polarization need not be Jero and can be substantially polarized. By application of the Bethe and Born collision theories (Chapters 4 and 5), it will be shown that both these assertions are wrong: a non-zero polarization can exist, but will be extremely small, even in the region of high impact energies. This work (McFarlane, 1972) has been indirectly confirmed by measurements of the related phenomenon of the angular distribution of Auger electrons following inner shell ionization by electrons (Cleff and Mehlhorn, 1971). By extending the Bethe theory to include relativistic corrections after the manner of Miller (1932) it is shown (Chapter 6) that the polarization approaches its high energy limit only very slowly. The thesis also looks at other anisotropic processes following electron and photon impact. Chapter 7 deals 1 the related problem of Auger electron angular distributions following inner shell photoionization. Chapter 8 postulates a directional correlation between photoelectrons and Auger electrons. Chapter 9 shows that the spin of a photoelectron is correlated with its direction of ejection, if there is significant fine structure interaction in the bound state. An appendix is concerned with the high energy limit of the form of anisotropies, and shows that this limit is more subtle than has been realized. An analytic, compact expression for the line polarization is hence derived and tested successfully against experiment and a more complicated theory

G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in β-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle

Required Assets for a Nuclear Energy Applied R&D Program

This report is one of a set of three documents that have collectively identified and recommended research and development capabilities that will be required to advance nuclear energy in the next 20 to 50 years. The first report, Nuclear Energy for the Future: Required Research and Development Capabilities—An Industry Perspective, was produced by Battelle Memorial Institute at the request of the Assistant Secretary of Nuclear Energy. That report, drawn from input by industry, academia, and Department of Energy laboratories, can be found in Appendix 5.1. This Idaho National Laboratory report maps the nuclear-specific capabilities from the Battelle report onto facility requirements, identifying options from the set of national laboratory, university, industry, and international facilities. It also identifies significant gaps in the required facility capabilities. The third document, Executive Recommendations for Nuclear R&D Capabilities, is a letter report containing a set of recommendations made by a team of senior executives representing nuclear vendors, utilities, academia, and the national laboratories (at Battelle’s request). That third report can be found in Appendix 5.2. The three reports should be considered as set in order to have a more complete picture. The basis of this report was drawn from three sources: previous Department of Energy reports, workshops and committee meetings, and expert opinion. The facilities discussed were winnowed from several hundred facilities that had previously been catalogued and several additional facilities that had been overlooked in past exercises. The scope of this report is limited to commercial nuclear energy and those things the federal government, or more specifically the Office of Nuclear Energy, should do to support its expanded deployment in order to increase energy security and reduce carbon emissions. In the context of this report, capabilities mean innovative, well-structured research and development programs, a viable work force, and well-equipped specialized facilities

Required Assets for a Nuclear Energy Applied R&D Program

This report is one of a set of three documents that have collectively identified and recommended research and development capabilities that will be required to advance nuclear energy in the next 20 to 50 years. The first report, Nuclear Energy for the Future: Required Research and Development Capabilities—An Industry Perspective, was produced by Battelle Memorial Institute at the request of the Assistant Secretary of Nuclear Energy. That report, drawn from input by industry, academia, and Department of Energy laboratories, can be found in Appendix 5.1. This Idaho National Laboratory report maps the nuclear-specific capabilities from the Battelle report onto facility requirements, identifying options from the set of national laboratory, university, industry, and international facilities. It also identifies significant gaps in the required facility capabilities. The third document, Executive Recommendations for Nuclear R&D Capabilities, is a letter report containing a set of recommendations made by a team of senior executives representing nuclear vendors, utilities, academia, and the national laboratories (at Battelle’s request). That third report can be found in Appendix 5.2. The three reports should be considered as set in order to have a more complete picture. The basis of this report was drawn from three sources: previous Department of Energy reports, workshops and committee meetings, and expert opinion. The facilities discussed were winnowed from several hundred facilities that had previously been catalogued and several additional facilities that had been overlooked in past exercises. The scope of this report is limited to commercial nuclear energy and those things the federal government, or more specifically the Office of Nuclear Energy, should do to support its expanded deployment in order to increase energy security and reduce carbon emissions. In the context of this report, capabilities mean innovative, well-structured research and development programs, a viable work force, and well-equipped specialized facilities

Targeting the PI3K/Akt/mTOR pathway in hepatocellular carcinoma

Despite advances in the treatment of cancers through surgical procedures and new pharmaceuticals, the treatment of hepatocellular carcinoma (HCC) remains challenging as reflected by low survival rates. The PI3K/Akt/mTOR pathway is an important signaling mechanism that regulates the cell cycle, proliferation, apoptosis, and metabolism. Importantly, deregulation of the PI3K/Akt/mTOR pathway leading to activation is common in HCC and is hence the subject of intense investigation and the focus of current therapeutics. In this review article, we consider the role of this pathway in the pathogenesis of HCC, focusing on its downstream effectors such as glycogen synthase kinase-3 (GSK-3), cAMP-response element-binding protein (CREB), forkhead box O protein (FOXO), murine double minute 2 (MDM2), p53, and nuclear factor-κB (NF-κB), and the cellular processes of lipogenesis and autophagy. In addition, we provide an update on the current ongoing clinical development of agents targeting this pathway for HCC treatments

Alterations in SAMD9, AHSG, FRG2C, and FGFR4 Genes in a Case of Late-Onset Massive Tumoral Calcinosis

Background/Objective: Tumoral calcinosis (TC) is a rare, arcane, and debilitating disorder of phosphate metabolism manifesting as hard masses in soft tissues. Primary hyperphosphatemic TC has been shown to be caused by pathogenic variants in the genes encoding FGF23, GALNT3, and KLOTHO. We report a case of massive TC mechanistically associated with phosphatonin resistance associated with heterozygous alterations in the sterile alfa motif domain–containing protein-9 gene (SAMD9), alfa 2-Heremans-Schmid glycoprotein gene (AHSG), FSHD region gene 2-family member-C gene (FRG2C), and fibroblast growth factor receptor-4 gene (FGFR4). Case Report: A middle-aged Malay woman with systemic sclerosis presented with painful hard lumps of her axillae, lower limbs, and external genitalia. She was eucalcemic with mild hyperphosphatemia associated with reduced urinary phosphate excretion. Magnetic resonance imaging revealed calcified soft tissue masses. Paradoxically, the serum intact FGF23 level increased to 89.6 pg/mL, corroborated by Western blots, which also showed overexpression of sFRP4 and MEPE, consistent with phosphatonin resistance. Discussion: Whole genome sequencing identified 2 heterozygous alterations (p.A454T and p.T479M) in SAMD9, 2 heterozygous alterations (p.M248T and p.S256T) in AHSG, a frameshift alteration (p.Arg156fs) in FRG2C, and a heterozygous alteration (p.G388R) in FGFR4, all of which are associated with calcinosis. Nonsynonymous alterations of FRP4 and MEPE were also detected. Conclusion: This highlights that the simultaneous occurrence of alterations in several genes critical in phosphate homeostasis may trigger massive TC despite their heterozygosity. These findings should prompt functional studies in cell and animal models to reveal mechanistic insights in the pathogenesis of such crippling mineralization disorders

Model for uptake of organic chemicals by plants

Uptake, in-plant transport, and local accumulation of organic chemicals by plants are influenced by plant characteristics, properties of the chemical and the soil, and by environmental conditions. Evaluations of plant contamination required by regulatory agencies cannot be made experimentally for the many thousands of xenobiotic chemicals in existence or being developed. A predictive simulator in the form of a mathematical model would provide a valuable tool for such evaluations. For this reason, a mathematical model (UTAB, uptake, Translocation, Accumulation, Biodegradation) was formulated by defining a generic plant as a set of adjacent compartments representing the major pools involved in transport and accumulation of water and solutes. The model consists of one root compartment, three stem compartments, and three leaf compartments. Each compartment is subdivided into two transport compartments, one for xylem and one for phloem, and a storage compartment. In addition, two compartments model the root volume outside the Casparian strip, one for the apparent free space and one for the cell volume. Values for the anatomical dimensions of the compartments and for physical and chemical coefficients were chosen from the literature. The complete system of equations, which describes uptake and accumulation, consists of 24 differential equations which are solved in terms of the chemical mass in each compartment as a function of time. The solution procedure is also developed and presented. For calibration purposes, concentrations measured in roots, stems, and leaves were compared with model predictions, while model parameters were changed until no further improvement in matching model predictions with experimental results was obtained. This exercise revealed important plant behavior that was not accounted for in the original formulation of the model and, as such, showed the value of the model for elucidating plant response. The model satisfactorily predicted the observed uptake and distribution patterns for bromacil in soybean plants, at the stage of growth and under the environmental conditions used in the experiments, involving a range of transpiration rates. This indicates that the model is flexible enough to provide an accurate representation of uptake and the influence of transpiration rate on the uptake and translocation of this chemical. Parameter values used in the model were selected from literature and experimental observation. They functioned well in these simulations and they are appropriately applied in the model. The chemical parameters for storage, mobilization, and diffusion when used in the model also yielded satisfactory results, suggesting that they are also appropriately applied. Finally, the calibration, although of limited scope, showed that the model equations yielded an accurate picture of the actual uptake patterns for bromacil in soybeans used in these experiments. The theoretical exercise of compiling the model is shown to be a constructive step in learning how to predict the fate of xenobiotic contamination in plants. The model shows excellent promise for future use. However, additional testing and validation are needed.Published May 1990. Facts and recommendations in this publication may no longer be valid. Please look for up-to-date information in the OSU Extension Catalog: http://extension.oregonstate.edu/catalo

Irisin treatment improves healing of dystrophic skeletal muscle

Background: Irisin is an exercise induced myokine that is shown to promote browning of adipose tissue and hence, increase energy expenditure. Furthermore, our unpublished results indicate that Irisin improves myogenic differentiation and induces skeletal muscle hypertrophy. Since exercise induced skeletal muscle hypertrophy improves muscle strength, we wanted to investigate if ectopic injection of Irisin peptide improves skeletal muscle function in a mouse model of muscular dystrophy. This utility of Irisin peptide is yet to be studied in animal models. Methods: In order to test this hypothesis, we expressed and purified recombinant murine Irisin peptide from E. coli. Three- to six-week-old male mdx mice were injected IP with either vehicle (dialysis buffer) or Irisin recombinant peptide for two or four weeks, three times-a-week. Results: Irisin injection increased muscle weights and enhanced grip strength in mdx mice. Improved muscle strength can be attributed to the significant hypertrophy observed in the Irisin injected mdx mice. Moreover, Irisin treatment resulted in reduced accumulation of fibrotic tissue and myofiber necrosis in mdx mice. In addition, Irisin improved sarcolemmal stability, which is severely compromised in mdx mice. Conclusion: Irisin injection induced skeletal muscle hypertrophy, improved muscle strength and reduced necrosis and fibrotic tissue in a murine dystrophy model. These results demonstrate the potential therapeutic value of Irisin in muscular dystrophy

Are interactions between epicardial adipose tissue, cardiac fibroblasts and cardiac myocytes instrumental in atrial fibrosis and atrial fibrillation?

Atrial fibrillation is very common among the elderly and/or obese. While myocardial fibrosis is associated with atrial fibrillation, the exact mechanisms within atrial myocytes and surrounding non-myocytes are not fully understood. This review considers the potential roles of myocardial fibroblasts and myofibroblasts in fibrosis and modulating myocyte electrophysiology through electrotonic interactions. Coupling with (myo)fibroblasts in vitro and in silico prolonged myocyte action potential duration and caused resting depolarization; an optogenetic study has verified in vivo that fibroblasts depolarized when coupled myocytes produced action potentials. This review also introduces another non-myocyte which may modulate both myocardial (myo)fibroblasts and myocytes: epicardial adipose tissue. Epicardial adipocytes are in intimate contact with myocytes and (myo)fibroblasts and may infiltrate the myocardium. Adipocytes secrete numerous adipokines which modulate (myo)fibroblast and myocyte physiology. These adipokines are protective in healthy hearts, preventing inflammation and fibrosis. However, adipokines secreted from adipocytes may switch to pro-inflammatory and pro-fibrotic, associated with reactive oxygen species generation. Pro-fibrotic adipokines stimulate myofibroblast differentiation, causing pronounced fibrosis in the epicardial adipose tissue and the myocardium. Adipose tissue also influences myocyte electrophysiology, via the adipokines and/or through electrotonic interactions. Deeper understanding of the interactions between myocytes and non-myocytes is important to understand and manage atrial fibrillation