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- Unloading-induced losses of bone and muscle occur in those that experience disuse due to weightlessness, aging, prolonged bed rest, neurological disease, or trauma. Together, osteopenia and sarcopenia result in decreased strength and function, increasing the risk for falls and fractures, which in turn increase the risk of morbidity and mortality. However, the interaction between these tissues and how osteopenia and sarcopenia affect each other is unclear. With the rate of musculoskeletal limitations being twice that of any other disability, including circulatory disorders, a better understanding of the bone-muscle relationship is necessary to develop more effective countermeasures and treatments to unloading-induced loss.The objective of this dissertation is to characterize the relationship between muscle and bone and determine their interdependence in response to unloading situations. To address these objectives, we utilized a ground-based murine model of simulated microgravity known as hindlimb suspension (HLS). Our central hypothesis is that unloading-induced osteopenia is at least partially-dependent on unloading-induced sarcopenia, and unloading-induced sarcopenia is at least partially dependent on unloading-induced osteopenia.To test our central hypothesis, we examined two specific aims: (1) Determine if exaggerating unloading-induced muscle atrophy during HLS would also exaggerate unloading-induced bone loss by casting one limb of a HLS mouse; (2) Determine if exaggerating unloading-induced bone loss during HLS would also exaggerate unloading-induced muscle loss by administering exogenous receptor activator of nuclear factor kappa-B ligand (RANKL) to HLS mice.For specific aim 1, we unilaterally casted one limb of HLS mice to exaggerate muscle loss in an unloading situation. This minimized any passive muscle contraction that would exert force on the bones and allowed us to examine what effect this muscle atrophy has on bone. HLS was carried out for 14 days, at which point tissues were harvested for analysis. Results showed that HLS produced muscle atrophy in both the gastrocnemius and quadriceps that was likely due to decreased protein synthesis, a probable result of decreased mTOR activity, evidenced by decreased S6K1 and 4E-BP1 phosphorylation. HLS + casted mice lost even more muscle mass than HLS alone; however, we observed an increase of protein synthesis in HLS + casted mice compared to HLS alone, albeit still lower than ground control. While expression of atrogin1 and MuRF1 did not differ among any groups, HLS + casted limbs experienced an increase in autophagy-related proteins LC3, Atg7, and Atg5-12 complex. While we did not investigate this further, these data suggest a possible muscle atrophy threshold that must be met during unloading to activate the autophagy degradation pathway. Trabecular bone loss in HLS mice was marked by decreased bone volume fraction, trabecular number, trabecular thickness, bone mineral density, and increased trabecular spacing. Cortical bone was more variable. However, the added stress of casting did not exacerbate these changes. These data would suggest a temporal disconnect between muscle and bone in response to unloading.In specific aim 2, we administered exogenous soluble RANKL to HLS mice to induce osteoclastogenesis and exaggerate unloading-induced bone loss. Mice received 3 doses at 24 hours intervals of either RANKL or vehicle control starting on Day 1 of suspension. HLS was again carried out for 14 days and tissues collected. We found that ground control mice receiving RANKL and HLS mice had approximately the same decrease in bone volume, and density in trabecular bone of tibias and femurs. Decreases in bone volume, density, and strength in cortical bone of femurs of ground control + RANKL and HLS mice were also similar. An exception to this generalization was that HLS decreased trabecular thickness with RANKL having no effect, while RANKL decreased trabecular number and increased trabecular separation with no effect of HLS. The combination of HLS and RANKL exacerbated the losses in bone volume, density, and strength in trabecular bone and in cortical bone, but to a lesser extent. There was no difference in markers of bone degradation across any groups. Interestingly, mice receiving RANKL injections had increased markers of bone formation, suggesting a negative feedback loop or reverse signaling occurring with RANKL. Muscle atrophy also occurred as a result of HLS and, again, this was observed to be due to decreased protein synthesis and markers of mTOR activity. The double treatment of HLS and RANKL had no further detrimental effects on muscle mass or protein synthesis in either the gastrocnemius or quadriceps. Proteasome activity and expression of E3 ligases did not differ among any groups. These results also represent a level of disconnect in muscle and bone in response to unloading.In summary, we were able to successfully exaggerate both unloading-induced muscle loss and unloading-induced bone loss without effects on the other tissue. Together, our results indicate that muscle and bone are not as integrally connected as originally believed in response to acute unloading and countermeasures to protect one tissue may not necessarily have an effect on the other tissue.
- Dissertation Note:
- Ph.D. Pennsylvania State University 2019.
- Technical Details:
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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