Browsing by Subject "Muscle Fibers, Skeletal"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item Muscle Function Improvement in Injured Mice with Combination Treatment(2017-01-17) Kulangara, Rohan G.; Sehat, Alvand, J.; Maredia, Navin; Maxwell, Christian; Liu, Ming-Mei; DeSpain, Kevin; Wolf, Steven E.; Song, JuquanINTRODUCTION: Loss of skeletal muscle from direct injury presents debilitating effects to an individual. Current treatments addressing muscle loss are limited by insufficient reconstitution of functioning muscle. Novel regenerative medicine technologies include the application of Urinary Bladder Matrix (UBM) and mesenchymal stem cells (MSCs) to restore functional muscle tissue. In our previous studies, we found that UBM increased muscle myoblast cell proliferation. Therefore, we examined whether co-treatment with MSCs would further augment regeneration as compared to individual treatments. METHODS: Twenty C57BL/6 male adult mice received bilateral laceration injuries on the gastrocnemius muscle under anesthesia, and were randomly grouped to a designed treatment applied 14 days after injury. Treatment groups were 1) DMEM culture medium, 2) UBM only (150μg), 3) MSCs only (1 million mouse derived cells), and 4) UBM+MSCs. 4 additional mice served as a control baseline not receiving injury. Efficacy of treatment was analyzed through isometric muscle force testing as well as histomorphologic examination at 50 days after injury. Two-way ANOVA was applied for statistical analysis. RESULTS: Isometric muscle force was measured, including twitch (Pt), tetanic (Po), and fatigue isometric functions with the muscle stretched to optimal length (Lo). Muscle twitch (Pt) significantly decreased in the DMEM group compared to the non-injured group at day 50 (p < 0.05). Furthermore, twitch significantly increased with UBM treatment, but not with MSC treatment. Regenerating myofiber nuclei were counted and myofiber cross sectional area was measured with histology. New myotubes were identified as having centrally located nuclei. Further, Ki-67 nuclear immunofluorescence staining was performed to demonstrate proliferating satellite cells. The myofiber cross sectional area and the number of Ki-67/DAPI overlapping stained nuclei significantly increased in the DMEM group compared to the non-injured group (p < 0.05). No differences were observed with other treatments in injured mice at day 50. CONCLUSION: We observed a significant improvement in muscle function with combination treatment and single UBM treatment applied 50 days post-injury. The current animal model provides a tool to study muscle regeneration, and is feasible for clinical translation to address impairment in skeletal muscle function after burn injury.Item A Role for POU3F3 in Myocyte Differentiation: Exploring New Frontier in Alveolar Rhabdomyosarcoma Development(2015-01-26) Denegre, Amelia; Granados, Valerie; Avirneni, Usha; Galindo, ReneSUMMARY: Children who are diagnosed with pediatric rhabdomyosarcoma (RMS), a mesenchymal-derived soft tissue cancer that comprises 3.5% of childhood cancers, are often delivered a bleak prognosis with little hope of a future. Despite significant advances illuminating transcription factor signaling in RMS onset and progression, research is still needed to precisely understand RMS pathogenesis on a molecular level in order to develop targeted treatment options. OBJECTIVE: The goal of this project is to explore the role of POU3F3 in myogenesis, particularly in relation to cell fusion and myocyte differentiation. METHODS: Immunofluorescence: POU3F3 knockdown cells were differentiated in 2% horse serum. On day 2 of differentiation, cells were probed with POU3F3 primary and red immunofluorescent secondary antibody, allowing for imaging of POU3F3 localization during myoblast differentiation. Western Blot: Three knock-down shPOU3F3 C2C12 cell-line constructs were tested. Western blots were performed that compared C2C12 control, POU3F3 overexpression, and POU3F3 knockdown cells. Crystal Violet: After differentiation, POU3F3 knock-down and control cell lines were stained with crystal violet stain to visualize the effect of POU3F3 knockdown on differentiation. RESULTS: Immunofluorescence: We confirmed that in knockdown C2C12 cells, POU3F3 localizes like control C2C12, in the periphery. Previous research has shown that in overexpressed POU3F3 C2C12 cell lines, POU3F3 localizes to the nucleus. The implication of the disconnect between POU3F3 location in controls and knock-downs versus overexpressed cell lines is an area that is an opportunity for further research. Western Blot: Western Blot analysis confirmed that POU3F3 knockdown was successful, and provides a platform for further POU3F3 interrogation. Crystal Violet: Crystal violet staining suggests that POU3F3 participates in a myoblast differentiation, as the control cells fuse into myotubes, while POU3F3-silenced cells do not. CONCLUSION: These initial results suggest that POU3F3 participates in muscle differentiation. Next, the Galindo lab will be probing POU3F3 function in myogenesis in greater depth, insights they will next apply to RMS.Item Role of NPHS Gene in Mammalian Myogenesis and Rhabdomyosarcoma(2019-03-27) Lawson, Kelly; Galindo, Rene; Castrillon, Diego H.; Le, Lu Q.BACKGROUND: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma comprised histologically of neoplastic skeletal muscle-lineage precursors that fail to terminally differentiate or fuse into syncytial muscle. The underlying RMS pathogenetic mechanisms that impede differentiation and promote neoplastic transformation remain largely unclear. The lab has shown misregulated myoblast fusion activity as a molecular underpinning of RMS pathogenesis. Data point to Immunoglobulin Superfamily transmembrane Receptors family members KIRREL and NPHS as fusion receptors. OBJECTIVE: We hypothesize that KIRREL and NPHS are essential for muscle development, with alterations underlying RMS pathogenesis. METHODS: Here, we look at pattern of expression of NPHS throughout myogenesis and its effects on differentiation and fusion. We use a C2C12 murine myoblast cell line to evaluate the role of NPHS in muscle development. Pattern of expression is evaluated through a qPCR analysis to quantify levels of NPHS RNA throughout the six-day mammalian myogenesis process. Loss-of- function studies are achieved by transfecting C2C12 cells with vectors containing sequences necessary to express shRNA directed against NPHS RNA, effectively knocking down the NPHS gene product in these cells. Gain-of-function studies are achieved by transfecting C2C12 cells with vectors containing the NPHS gene. The myoblasts with NPHS knockdown and overexpression are observed throughout the six-day myogenesis process to assess for alteration in differentiation, fusion, or both differentiation and fusion. To better visualize the results, crystal violet stains and immunofluorescent stains are used. RESULTS: Quantification of NPHS RNA illustrates the precisely regulated expression of NPHS during myogenesis. Specifically, upregulation of the gene is seen during key points in differentiation and fusion, with a dramatic increase in NPHS expression on days three and six of the six-day myogenesis process. NPHS loss-of-function studies show a decrease in myoblast differentiation and fusion. When viewed on day four, myoblasts with reduced expression of NPHS showed a significant decrease (p < 0.01) in differentiation and fusion when compared to controls. NPHS gain-of-function studies show enhanced differentiation and fusion. Day four and day six myoblasts with increased expression of NPHS had significantly increased levels of differentiation and fusion when compared to the control myoblasts (p < 0.01). CONCLUSION: These results lead us to conclude that NPHS is critical for proper muscle development, and potentiates RMS, making it a prospective treatment target. With this knowledge as a foundation, the pathways of myogenesis and the RMS pathogenesis will continue to be elucidated. This information will allow for novel targeted treatments of RMS that will not carry the morbidity and mortality of traditional treatments such as surgery, radiation, and chemotherapy.