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"Engineered muscle tissue offers a promising solution for the treatment of large muscle defects. Three-dimensional tissue engineered matrices, such as microthreads, can be used to grow new myofibers that will reduce scar formation and integrate easily into native myofibers. We hypothesize that adsorbing growth factors to the surface of braided collagen scaffolds using crosslinking strategies will promote muscle derived fibroblastic cell (MDFC) attachment and growth, which will serve as a platform for delivering cells to large muscle defects for muscle regeneration. To test this hypothesis, self-assembled type I collagen threads were braided and crosslinked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with and without heparin and 5 ng/mL, 10 ng/mL, or 50 ng/mL fibroblast growth factor (FGF-2) bound to the surface. Using immunhistochemistry, braided collagen scaffolds showed the presence of FGF-2 on the surface, and braiding the microthreads increased the mechanical properties compared to single threads. To determine the effect of FGF-2 on MDFC attachment, growth, and alignment, scaffolds were seeded with a MDFC cell suspension for 4 hours using a PDMS mold with a sealed 1 mm by 12 mm channel and cultured for 1, 5, or 7 days. After 1 day of culture, the results show a significant increase in cell attachment on braids crosslinked with EDC/NHS with heparin and no significant difference in attachment between the different concentrations of FGF-2 and EDC/NHS crosslinked scaffolds. After 7 days in culture, the MDFCs responded to FGF-2 with a positive linear correlation between growth rate and concentration of FGF-2 on the surface. Additionally, all control scaffolds showed cellular alignment after 7 days, while MDFCs on FGF-2 modified scaffolds showed limited alignment. These results show braided collagen scaffolds crosslinked with EDC/NHS with heparin delivering a controlled quantity of FGF-2 can support MDFC attachment and growth, which may serve as an exciting new approach to facilitate the growth and ultimately the delivery of cells to large defects in muscle regeneration."

Occurrence of obesity has steadily increased in the human population and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. In mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury, however, the mechanism underlying these changes in satellite cell (SC) biology have yet to be explored. To test the negative impacts of obesity on SC behaviors, we fed C57BL/6 mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks and performed SC proliferation and differentiation assays in vitro. SCs from HFD mice formed colonies with smaller numbers (P < 0.001) compared to those isolated from NC mice, and this observation was confirmed (P < 0.05) by BrdU incorporation. Moreover, in vitro differentiation assays consisting of equally seeded SCs derived from NC and HFD muscles showed that HFD SCs exhibited compromised (P < 0.001) differentiation capacity compared to NC SCs. Immunocytochemical staining of cultured SCs demonstrated that the percentage of Pax7+/MyoD- (self-renewed) SC subpopulation decreased (P < 0.001) with HFD treatment group compared to the control. In single fiber explants, a higher ratio of SCs experienced apoptotic events as revealed by the expression of cleaved caspase 3 (P < 0.001). To investigate further the impact of obesity on SC quiescence and cycling properties in vivo, we used an inducible H2B-GFP mouse model to trace the turnover rate of GFP and thus cell division under normal and obese conditions. Flow cytometric analysis revealed that SCs from HFD treatment cycled faster (P < 0.001) than their NC counterparts, as reflected by the quicker loss of the GFP intensity. To test for SC muscle regenerative capacity in vivo, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC quiescence, proliferation, differentiation, and self-renewal in vitro, ex vivo and in vivo. / MS / The prevalence of obesity in the human population has steadily increased over the past decades and, along with it, associated health complications such as systemic insulin resistance, which can lead to the development of type 2 diabetes mellitus. Obesity is a complex metabolic disorder that often leads to chronic inflammation and an overall decline in human and animal health. Along with the multitude of health disorders associated with obesity, in mouse skeletal muscle, obesity has been shown to impair muscle regeneration after injury. The mechanisms underlying the impairment in muscle regeneration as seen in obesity are unknown. To better understand how obesity affects skeletal muscle, we looked at satellite cells (SC). Satellite cells, or muscle stem cells, are skeletal muscle resident cells that play a vital role in muscle repair after damage. To test the negative impacts of obesity on SC behaviors, we fed mice normal chow (NC, control) or high-fat diet (HFD) for 10 wks to obtain an obesogenic mouse model. Our first experiments involved culturing the SCs derived from the HFD and NC mouse muscles and growing them in an artificial environment. These experiments showed SCs derived from HFD mice had a decreased ability to replicate and divide compared to those isolated from NC mice. Moreover, the SCs from the HFD mice exhibited compromised capacity to form myotubes in culture, an essential part in muscle regeneration after damage. Our next set of experiments conducted looked at individual muscle fibers isolated from mouse muscle. In these experiments the SCs on the HFD muscle fibers had a higher ratio of SCs experiencing cell death in comparison to the control. To test the SC cycling properties in the living mouse we used a mouse model to trace the activity and cell division of SCs under normal and obese conditions. Using this model revealed that SCs from HFD treatment cycled faster than their control counterparts, even in the absence of notable muscle damage. To test for SC muscle regenerative capacity after muscle damage, we used cardiotoxin (CTX) to induce wide-spread muscle damage in the tibialis anterior muscle (leg muscle) of the living mouse. After analysis we found that HFD leads to a compromised, though mild, impairment in muscle regeneration. Taken together, these findings suggest that obesity negatively affects SC behaviors and function.

Myostatin, a TGF-β superfamily member, is a key negative regulator of embryonic and postnatal muscle growth. In order to further elucidate the role of myostatin during postnatal growth, several lines of investigation were undertaken in mice. Analysis of myostatin downstream target genes identified several known and unknown genes. From these, the regulation of an androgen receptor binding co-factor, ARA70, was selected for further investigation. Reverse Northern analysis on the differentially expressed cDNA library indicated an increased expression of ARA70 in myostatin-null muscles, which was later confirmed by Northern blot and semi-quantitative PCR analysis. In corroboration, treatment of myoblast cultures with exogenous myostatin resulted in the down-regulation of ARA70, confirming that myostatin is a negative regulator of ARA70 gene expression. The role of myostatin during sarcopenia, a progressive age-related loss of skeletal muscle mass and strength, was also investigated. The atrophy associated with sarcopenia is frequently correlated with insufficient muscle regeneration, resulting from an impaired propensity of satellite cells to activate and a subsequent decline in myogenesis. Myostatin is a known inhibitor of postnatal satellite cell activation and muscle regeneration, thus muscle mass and regeneration, and satellite cell behaviour were examined in young and aged myostatin-null mice. Myostatin-null mice had increased individual muscle weights, as a consequence of massive fibre hypertrophy and hyperplasia, and an increased proportion of type IIB fibres. Aging induced oxidative fibre type changes and atrophy in the wild-type muscle while no fibre type switching was observed in the myostatin-null muscle and atrophy was minimal. No decrease in satellite cell numbers was observed with aging in both genotypes; though a gradual decline in the number of activated satellite cells was noted during aging. However, both young and aged myostatin-null mice displayed increased satellite cells and activation compared to wild-type mice, suggesting a greater myogenic potential in the myostatin-null satellite cells. Consistent with this, aged myostatin-null myoblasts proliferated faster and displayed a higher fusion index during differentiation than the aged wild-type myoblasts, confirming that the reduced sarcopenia in the myostatin-null mice was due to a preserved increase in the myoblast myogenic activity. An increase in a Pax7-only myoblast population from myostatin-null muscle indicated an enhanced satellite cell self-renewal process, consistent with the increased satellite cell number observed on the myostatin-null muscle fibres. Additionally, muscle regeneration of aged myostatin-null muscle following notexin injury was accelerated, and fibre hypertrophy and type were recovered with regeneration, unlike the aged wild-type muscle. Testing the therapeutic value of a myostatin antagonist, Mstn-ant1, indicated that a short term blockade of myostatin by the antagonist significantly enhanced muscle regeneration in aged mice after injury and during sarcopenia. Antagonism of myostatin led to satellite cell activation, increased Pax7 and MyoD protein levels, and greater myoblast and macrophage cell migration culminating in enhanced muscle regeneration in the aged mice. In conclusion, the hypertrophic phenotype associated with myostatin-null mice may in part result from increased androgen receptor (AR) activity due to the up-regulation of ARA70, given that increased expression of the AR leads to hypertrophy. Additionally, the increased muscle mass in myostatin-null mice is likely to result from an augmented myogenic potential and self-renewal process. Overall, a prolonged absence of myostatin reduced sarcopenia and the associated loss of muscle regenerative capacity. Furthermore, the antagonism of myostatin displayed significant therapeutic potential in the alleviation of sarcopenia, through the restoration of the myogenic and inflammatory responses in the aged environment. Thus, the research work clearly demonstrates the role of myostatin in sarcopenia, and documents for the first time a valid therapeutic for alleviating sarcopenia.

The neuromuscular junction (NMJ) is a synapse formed between a motoneuron and a muscle fiber which transmits the signals required to initiate muscle contraction. The functional state of the NMJ is intimately tied to the structure and function of the motoneuron, such that reductions in postsynaptic activity retrogradely stimulate sustained reorganization of presynaptic motor terminals in an attempt to maintain normal contractile output. In the adult, these plastic changes occur most notably as regenerative responses following traumatic injury and during the progression of motoneuron diseases (MNDs), and can contribute to a considerable amount of functional repair. However, limitations to the regeneration capacity of motoneurons place an upper limit on the effectiveness of endogenous repair mechanisms and can restrict the extent of functional recovery. Using a combination of immunofluorescence, sharp electrode electrophysiology and live labeling of synaptic vesicle recycling during various forms of synaptic growth and regeneration in vivo and in vitro, I have identified that the neural cell adhesion molecule (NCAM) is a key regulator of the regenerative capacity of motoneurons. In vivo experiments revealed that NCAM influences the maturation and stabilization of regenerated synapses via the recruitment and recycling of synaptic vesicles necessary for effective synaptic transmission. The presence of both pre- and post-synaptic NCAM were necessary to maintain the abundance of recycling synaptic vesicles at regenerated synapses, demonstrating a coordinated influence of these molecules in regenerative synaptic plasticity in vivo. To accurately assess the regenerative potential of motoneurons in vitro, it was necessary to develop a system which could reliably and consistently generate mature NMJs amenable to experimental investigation. Motoneurons differentiated from embryonic stem cells were grown for 3-5 weeks in co-culture with muscle fibers and generated mature NMJs which possessed morphological and functional criteria consistent with NMJs formed in vivo. NMJs formed by NCAM-/- motoneurons did not mature and were found to exhibit deficits consistent with their in vivo counterparts. These studies have revealed that NCAM is a key mediator of regenerative plasticity at the NMJ and may be a target for efforts to enhance endogenous repair following traumatic injury or during the progression of neurodegenerative disease.

Skeletal muscle is a direct target for the group of seco-steroids collectively termed Vitamin D. Skeletal muscle expresses both CYP27A1 and CYP27B1 encoding for the hydroxylases required to convert Vitamin D to 25[OH]D and subsequently the bioactive 1α-25-dihydroxyvitamin D3 (1α-25[OH]2D3) (Girgis et al., 2014b). Crucially, the Vitamin D receptor (VDR) is also present in skeletal muscle (Srikuea, Zhang, Park-Sarge, & Esser, 2012) and upon exposure, binds to its ligand 1α-25[OH]2D3 and initiates genomic and non-genomic rapid signalling responses. At present there is a global prevalence of low serum Vitamin D (25[OH]D) concentrations due to a lack of sun exposure (the primary route for Vitamin D synthesis) as a function of latitude and/or an indoor lifestyle coupled with few dietary sources of Vitamin D (Chen et al., 2007). Accumulating data are now suggestive that low 25[OH]D may be associated with perturbations in contractile activity and the regeneration of human skeletal muscle (Owens, Fraser, & Close, 2014), although a definitive causal relationship is yet to be established. Therefore, this thesis aimed to establish a more precise role for Vitamin D in human skeletal muscle function and regeneration. There were four overarching aims: 1. Explore the role of Vitamin D in human skeletal muscle contractile properties in humans in vivo. 2. Identify the role of Vitamin D in human skeletal muscle contractile properties ex vivo. 3. Investigate the role of Vitamin D in skeletal muscle regeneration following eccentric exercise induced muscle damage in vivo. 4. Elucidate cellular mechanisms of the muscle regeneration process that are responsive to Vitamin D in vitro. The main findings from this work imply that serum 25[OH]D concentrations across a broad range from 18 → 100 nmol.L-1 do not affect skeletal muscle contractile properties. Conversely elevating serum 25[OH]D from < 50 nmol.L-1 to > 75 nmol.L-1 resulted in significant improvements in the recovery of maximal voluntary contraction force following a bout of eccentric exercise. Implementing an in vitro model of muscle regeneration also identified potential cellular mechanisms for these observations: Muscle derived cells treated with a total amount of 10 nmol 1α-25[OH]2D3 following a mechanical scrape improved migration dynamics and fusion capability of skeletal muscle derived cells. Taken together, low Vitamin D concentrations are highly prevalent but can be easily corrected with supplementation of Vitamin D3. This may have the advantage of optimising the regenerative capacity of skeletal muscle amongst other health benefits previously characterised by others.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute skeletal muscle injury leads to increases in activin B levels and when selectively neutralized with a monoclonal antibody, there is augmented skeletal muscle repair.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18903号 / 医科博第59号 / 新制||医科||4(附属図書館) / 31854 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 妻木 範行, 教授 山下 潤, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p> Local renin-angiotensin systems (RASs) have been described in many mammalian tissues. However, the role of angiotensin II (Ang II) in skeletal muscle is poorly understood with initial reports suggesting it may function to regulate overload-induced hypertrophy. Therefore, the purpose of this thesis was to 1) investigate the potential that adult skeletal muscle and muscle stem cells possess a local RAS. 2) Describe its role in regulating skeletal muscle regeneration and growth following injury and 3) demonstrate its capacity to regulate muscle stem cell activity and myogenesis. We report that cultured primary and C2C12 myoblasts and myotubes possess a local Ang II signalling system evidenced by the differential expression of angiotensinogen, angiotensin converting enzyme (ACE), and both angiotensin type 1 and 2 (AT1, AT2) receptors. Interestingly, myoblasts demonstrated the capacity to produce Ang II in spite of lacking renin expression. Furthermore, angiotensin receptors demonstrated differential localization with AT1 associated with actin filaments in proliferating myoblasts, and localized to the nucleus in differentiated myotubes. We also report that a local angiotensin system is present in vivo and responsive to myotrauma as cardiotoxin injection (to induce muscle injury) resulted in the increased staining intensity of angiotensinogen and AT1 during myogenesis with a progressive downregulation throughout the regenerative timecourse. </p> <p> To investigate the effects of Ang II signalling blockade on muscle growth and regeneration we induced muscle injury in mice supplemented with captopril (ACE inhibitor) or mice devoid of the AT1 a receptor. Histological analysis revealed that ACE inhibition resulted in a decreased muscle fibre growth, increased proportion of small myofibres, an inability to accrete myonuclei and a robust hyperplasia of muscle fibres. Similarly, AT1 a receptor ablation resulted in decreased muscle fibre growth following injury suggesting that these effects are receptor specific. </p> <p> To investigate the mechanisms underlying these effects we assessed the role of Ang II in regulating muscle satellite cell function. In vitro experiments revealed that Ang II had the ability to regulate the early response of satellite cells to muscle injury by acting as a potent transcriptional activator of quiescent myoblasts and directing their subsequent migration. Furthermore, these migratory effects were mediated through an Ang 11-induced increase in matrix metalloproteinase 2 (MMP2) content and reorganization of the actin cytoskeleton. Interestingly, Ang II may also participate in the fusion of myoblasts as captopril treatment suppressed the expression of markers of differentiation (myogenin) and maintained the expression of markers of proliferation (Pax7, Myf5). In agreement with this, IHC analysis revealed that ACE inhibition also induced a strong trend for a decrease in the proportion of myogenin positive cells following injury. Collectively, these results implicate the activation of local Ang II signalling system as a pleiotropic regulator of skeletal muscle growth. </p> / Thesis / Doctor of Philosophy (PhD)