Myocardium lacks the ability to regenerate following injury. This is in contrast to skeletal muscle (SKM), in which capacity for tissue repair is attributed to the presence of satellite cells. It was hypothesized that SKM satellite cells multiplied in vitro could be used to repair injured heart muscle. Fourteen dogs underwent explantation of the anterior tibialis muscle. Satellite cells were multiplied in vitro and their nuclei were labelled with tritiated thymidine 24 h prior to implantation. The same dogs were then subjected successfully to a myocardial injury by the application of a cryoprobe. The cells were suspended in serum-free growth medium and autotransplanted within the damaged muscle. Medium without cells was injected into an adjacent site to serve as a control. Endpoints comprised histology using standard stains as well as Masson trichrome (specific for connective tissue), and radioautography. In five dogs, satellite cell isolation, culture, and implantation were technically satisfactory. In three implanted dogs, specimens were taken within 6-8 wk. There were persistence of the implantation channels in the experimental sites when compared to the controls. Macroscopically, muscle tissue completely surrounded by scar tissue could be seen. Masson trichrome staining showed homogeneous scar in the control site, but not in the test site where a patch of muscle fibres containing intercalated discs (characteristic of myocardial tissue) was observed. In two other dogs, specimens were taken at 14 wk postimplantation. Muscle tissue could not be found. These preliminary results could be consistent with the hypothesis that SKM satellite cells can form neo-myocardium within an appropriate environment. Our specimens failed to demonstrate the presence of myocyte nuclei. It is therefore further hypothesized that in the late postoperative period, the muscle regenerate failed to survive.

Magnetic resonance elastography (MRE) Is capable of non-invasively quantifying the mechanical properties of skeletal muscles in vivo. This information can be clinically useful to understand the effects of pathologies on the mechanical properties of muscle and to quantify the effects of treatment. Advances in inversion algorithms quantify muscle anisotropy in two-dimensional (2D) and three-dimensional (3D) imaging. Databases of the shear stiffness of skeletal muscle have been presented in the relaxed and contracted states in the upper extremity (biceps brachii, flexor digitorum profundus, and upper trapezius), distal leg muscles (tibialis anterior, medial gastrocnemius, lateral gastrocnemius, and trapezius), and proximal leg muscles (vastus lateralis, vastus medialis, and sartorius). MRE measurements have successfully validated a mathematical model of skeletal muscle behavior in the biceps brachii, correlated to electromyographic data in the distal leg muscles and quantified the effects of pathologies on the distal and proximal leg muscles. Future research efforts should be directed toward improving one-dimensional (1D) and 3D MRE data acquisition and image processing, tracking the effects of treatment on pathologic muscle and correlating the shear stiffness with clinical measurements.

Indiana University-Purdue University Indianapolis (IUPUI) / Insulin-stimulated glucose uptake into skeletal muscle cells requires translocation of the glucose transporter-4 (GLUT4) from the cell interior to the plasma membrane. Insulin-stimulated GLUT4 vesicle translocation is dysregulated in Type 2 diabetes (T2D). The Group I p21–activated kinase (PAK1) is a required element in insulin-stimulated GLUT4 vesicle translocation in mouse skeletal muscle in vivo, although its placement and function(s) in the canonical insulin signaling cascade in skeletal muscle cells, remain undetermined. Therefore, the objective of my project is to determine the molecular mechanism(s) underlying the requirement for PAK1 in the process of insulin-stimulated GLUT4 vesicle translocation and subsequent glucose uptake by skeletal muscle cells. Toward this, my studies demonstrate that the pharmacological inhibition of PAK1 activation blunts insulin-stimulated GLUT4 translocation and subsequent glucose uptake into L6-GLUT4myc skeletal myotubes. Inhibition of PAK1 activation also ablates insulin-stimulated F-actin cytoskeletal remodeling, a process known to be required for mobilizing GLUT4 vesicles to the plasma membrane. Consistent with this mechanism, PAK1 activation was also required for the activation of cofilin, another protein implicated in F-actin remodeling. Interestingly, my studies reveal a novel molecular mechanism involving PAK1 signaling to p41-ARC, a regulatory subunit of the cytoskeletal Arp2/3 complex, and its interactions with another cytoskeletal factor, N-WASP, to elicit the insulin-stimulated F-actin remodeling in skeletal muscle cells. Pharmacological inactivation of N-WASP fully abrogated insulin-stimulated GLUT4 vesicle translocation to the cell surface, coordinate with blunted F-actin remodeling. Furthermore, my studies revealed new insulin-induced interactions amongst N WASP, actin, p41-ARC and PAK1; inactivation of PAK1 signaling blocked these dynamic interactions. Taken together, the above studies demonstrate the significance of PAK1 and its downstream signaling to F-actin remodeling in insulin-stimulated GLUT4 vesicle translocation and glucose uptake, revealing new signaling elements that may prove to be promising targets for future therapeutic design.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19271号 / 医博第4035号 / 新制||医||1011(附属図書館) / 32273 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 松田 秀一, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20993号 / 医博第4339号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 羽賀 博典, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21445号 / 医博第4412号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 篠原 隆司, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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<p>Skeletal muscle is dependent upon its microvasculature to deliver oxygen and substrates to support the metabolic demands of muscle contraction. Skeletal muscle capillary density is determined by a variety of factors including muscle fiber metabolic phenotype and mitochondrial volume as well as prior exercise training status. Additionally, muscle microvascular density and function can diminish with age, contributing to several age-related muscle dysfunctions. Skeletal muscle fibers regulate their surrounding microvasculature through the release of angiogenic and angiostatic signaling factors. A robust increase in angiogenic signaling from skeletal muscle facilitates increases in muscle capillarization following endurance exercise. Extracellular vesicles (EV) are membrane bound signaling factors secreted by every cell type. Skeletal muscle-derived EVs (SkM-EVs) may help facilitate numerous signaling functions of skeletal muscle including between skeletal muscle and its microvasculature.</p> <p>The primary aim of my dissertation research was to determine the signaling roles that SkM-EVs in regulating endothelial cell homeostasis and angiogenesis in states of aging and health. Chapter 1 provides an overview of the relevant literature. Chapter 2 represents an investigation into how age-related cellular senescence impacts the angiogenic potential of skeletal muscle progenitor cells. We found that stress-induced senescence increases release of small EVs and has pro-senescent and angiostatic effects on culture endothelial cells. In Chapter 3 we compared the release, contents, and angiogenic potential of SkM-EVs collected from primarily oxidative or primarily glycolytic skeletal muscle tissue in mice. We found that oxidative muscle tissue secretes more EVs than glycolytic muscle tissue, and the miR contents of EVs differ greatly between the two phenotypes. Additionally, EVs from oxidative tissue enhanced endothelial cell migration and tube formation compared to glycolytic tissue EVs, in a potentially nitric oxide mediated fashion. In Chapter 4, we tested how PGC-1α overexpression effected myotube EV release and angiogenic potential. We found that PGC-1α overexpression did not impact myotube EV release, but increased the angiogenic signaling potential of SkM-EVs. Chapter 5 is a brief summary of the results and limitations of the projects presented in Chapters 2-4, with a short discussion of potential future research directions.</p>

Skeletal muscle performs essential functions, including movement and posture. Musculoskeletal disorders can disrupt these functions, leading to temporary or permanent impairment. As most muscle abnormalities will cause morphological and physiological changes in skeletal muscle, identifying diseased or injured skeletal muscle relies on having a frame of reference, i.e. a correct characterization of what is considered healthy or 'normal' skeletal muscle. Non-invasive Magnetic Resonance Imaging (MRI) techniques such as 1H Magnetic Resonance Spectroscopy (1H-MRS) to assess the biochemical environment, Magnetization Transfer (MT) to study water dynamics and Blood Oxygen Level Dependent (BOLD) signal to study blood flow and relative (de)oxy-Hb concentration have yet to be extensively explored in skeletal muscle. Therefore, to improve the knowledge of the biochemical environment of skeletal muscle, a series of experiments were performed using these techniques in calf muscles. 1H-MRS investigations showed high repeatability of metabolite quantification within and across scanning sessions despite its challenges due to the high structural organization of skeletal muscle. Furthermore, differences in the metabolic profile between endurance vs. power-oriented participants at rest were found, suggesting 1H-MRS could be used as a non-invasive technique to assess muscle fiber composition. A multimodal MT, and BOLD study were performed on exercised skeletal muscle to complement the metabolic understanding of skeletal muscle. It was shown that high-quality data could be obtained in simultaneous studies of BOLD/EMG. In addition, during a multimodal MT and BOLD acquisition, MT signal showed a decrease after exercise and was linearly correlated to the BOLD signal activation. The ability of MT to distinguish between highly/lowly activated muscle groups during exercise opens the opportunity to non-invasively investigate muscle group recruitment with a higher spatial resolution compared to EMG, and lower scanning times compared to BOLD. Overall, the main purpose of this thesis was to investigate, characterize and provide unique metrics to study the functional and metabolic profile of healthy skeletal muscle at rest and during exercise. / Thesis / Master of Applied Science (MASc) / Skeletal muscle performs vital functions such as movement, heat generation, and posture. The impact of musculoskeletal disorders, which can disrupt these functions and cause temporary or permanent impairment of physical activity and movement, is expected to grow in the future. Correctly characterizing healthy or 'normal' skeletal muscle is necessary to identify diseased or injured skeletal muscle, as most muscle abnormalities cause changes in morphology and physiology. Non-invasive MRI techniques to assess the biochemical environment, water dynamics, blood flow and relative (de)oxy-Hb concentration have yet to be extensively explored in healthy skeletal muscle. Thus, the primary purpose of this thesis was to investigate, characterize and provide unique metrics to study the functional and metabolic profile of healthy skeletal muscle at rest and during exercise. The metrics investigated can be used to establish a baseline to detect abnormal skeletal muscle.

The main purpose of this dissertation was to compare performance and physiological outcomes of between a repetition maximum (RM) and a relative intensity using sets-and-repetitions (RISR) resistance training (RT) program in well-trained lifters. Fifteen subjects underwent RT 3 d·wk-1 for 10-weeks in either a RM group (n=8) or RISR group (n=7). The RM group achieved a relative maximum each day while the RISR group trained based on percentages. Testing included percutaneous needle biopsies of the vastus lateralis, ultrasonography, unweighted (g to assess within and between-group alterations. RISR from pre-to-post yielded statistically significant increases in Type I CSA (p=0.018), Type II CSA (p=0.012), ACSA (p=0.002), unweighted (p=0.009) and 20 kg SJ JH (p=0.012), unweighted (p=0.003) and 20kg SJ PPa (p=0.026), IPF (ppSR increased in unweighted (p=0.023) and 20kg SJ JH (p=0.014), and 20kg SJ PPa (p=0.026) from pre-to-post taper. RM yielded statistically significant increases from only pre-to-post taper for 20kg SJ JH (p=0.003) and CMJ JH (p=0.031). Additionally, RM had a statistically significant pre-to-post decrease in RFD from 0-50ms (p=0.018) and 0-100ms (p=0.014). Between-group effect sizes supported RISR for Type I CSA (g=0.48), Type II CSA (g=0.50), ACSA (g=1.03), all MYH isoforms (g=0.31-0.87), all SJ variables (g=0.64-1.07), unweighted and 20kg CMJ JH (g=0.76-0.97), unweighted CMJ PPa (g=0.35), IPFa (g=0.20), and all RFD (g=0.31-1.25) time-points except 0-200ms; with all other effects being of trivial magnitude (gSR training yielded greater improvements in vertical jump, RFD and maximal strength compared RM training. These performances results may, in part, be explained mechanistically by the superior physiological adaptations observed in the RISR group within the skeletal muscle. Taken together, these data support the use of RISR training in well-trained populations.