Thoroughbred racehorses undergo strenuous exercise which often leads to the occurrence of exercise-induced pulmonary hemorrhage (EIPH), in which capillaries rupture within the alveoli in the lungs causing bleeding. Severe cases of EIPH lead to epistaxis and may result in fatality. Presently, the loop diuretic furosemide is the only medication approved to mitigate the effects of EIPH. Often regarded in the racing industry as "performance enhancing" due to 4% weight loss ensued by its diuretic effect, it is unknown what effects furosemide may have on muscle recovery. Therefore, the objective of this study was to determine the effects various doses of furosemide may have on equine satellite cell (eqSC) myogenesis and metabolism. Mitotic index was increased (P<0.05) as a result of treatment with 100 µg/mL furosemide, a 10-fold pharmacological dose, in comparison to vehicle, but was not different (P>0.05) compared to the physiological dose of 10 µg/mL furosemide. Average cell number decreased (P<0.05) in the excess furosemide group compared to all other groups. Pax7 expression did not differ (P>0.05) between groups. Expression of the differentiation transcription factor myogenin, and embryonic sarcomeric myosin heavy chain decreased (P<0.05) when cells were treated with 100 µg/mL furosemide. Fusion index and myotube area decreased (P<0.05) as a result of treatment with excess furosemide. Glycogen concentration in myotubes was lower (P<0.05) following treatment with 100 µg/mL furosemide, while IGF-1 was unsuccessful in rescuing the effects of furosemide. Excess furosemide decreased expression of muscle creatine kinase while increasing expression of phosphoglucomutase 1, glycogen synthase 1, and glycogen branching enzyme 1 (P<0.05). Excess furosemide decreased basal oxygen consumption rate (OCR) and increased OCR after addition of oligomycin (P<0.05). Excess furosemide did not affect myotube glycolysis rates in vitro. In conclusion, furosemide inhibits muscle differentiation and oxidative metabolism in eqSCs. / Master of Science / Thoroughbred racehorses often bleed from the lungs as a result of high-intensity exercise. This condition can oftentimes be fatal depending on severity. Furosemide, is used in the industry to reduce blood pressure within the lungs during racing to prevent bleeding. Furosemide, a diuretic given four hours prior to a race, causes a horse to excrete up to 4% of its body weight. This effect of furosemide decreases the weight a horse must carry during a race, thus allowing the horse to run faster. Therefore, deemed as a performance enhancing drug due to its effects on the kidney, to our knowledge, no research has been conducted on what effects furosemide might have on muscle generation. High-intensity exercise causes massive muscle damage and therefore must be repaired to prepare for the next bout of exercise. Muscle generation is called myogenesis. Stem cells, or satellite cells, that lie within the muscle become activated, recognizing the need for muscle repair. Satellite cells divide, increasing in cell number and then fuse together, forming new muscle fibers. Satellite cells undergo different types of metabolism depending on their state of development. For example, proliferating cells require glucose for energy, while cells fusing together forming myotubes, require oxidative metabolism for long-lasting energy. Therefore, the objective of this study was to determine the effects furosemide might have on muscle formation and metabolism. The excess furosemide dose (100 µg/mL) decreased cell proliferation. The expression of regulatory factors responsible for forming myotubes at different stages of muscle development are decreased when cells were treated with the defined excess furosemide dose. Furosemide decreased the ability of satellite cells to generate myotubes. Glycogen concentration was also decreased as a result of excess furosemide treatment. Gene expression of enzymes involved in glycogen synthesis were increased from treatment with our excess furosemide dose. No effect of furosemide was seen on glycolysis, whereas oxidative metabolism suffered as a result of treatment with excess furosemide. In conclusion, furosemide does indeed affect muscle generation and oxidative metabolism.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/96608 |
| Date | 29 January 2020 |
| Creators | Helsel, Patricia J. |
| Contributors | Animal and Poultry Sciences, Johnson, Sally E., Rhoads, Robert P., Gerrard, David E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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