Musculoskeletal Health Theses and Dissertations
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Item Purine Nucleotide Regulation in Mitochondrial Dysfunction and Muscle Wasting(2024-11) Law, Andrew S.; Brault, Jeff; Bonewald, Lynda; Welc, Steven; Elmendorf, JeffreyPurine nucleotides are critical to cellular energetics and information storage. Changes in the relative amounts of purine (adenine and guanine) nucleotides are mutagenic for DNA in simple model organisms. In mammals, long-standing theories have attributed the onset of mitochondrial DNA (mtDNA) mutations that occur with aging to reactive oxygen species (ROS) based DNA damage, but this has not been shown experimentally. This investigation tested the hypothesis that changes in purine nucleotide content during skeletal muscle atrophy in mice is mutagenic for mtDNA. Our investigation required new analytical techniques to precisely quantitate purine nucleotides. We developed a new ultra performance liquid chromatography (UPLC) method compatible with mass spectrometry (MS). Using this method, we measured guanine and adenine nucleotides after overexpression of AMPD3 and IMPDH2 in C2C12 myotubes and showed substantial changes in the GTP:ATP ratios. Having demonstrated these two enzymes coordinate to control relative nucleotide pools, we then demonstrated using wild-type and AMPD3-deficient C57Bl/6J mice that denervation-induced muscle atrophy results in an AMPD3-dependent increase in GTP. Thus, we established denervation as model of muscle atrophy capable of purine nucleotide dysregulation. To accurately determine the rate to which muscle atrophy was mutagenic to mtDNA, we utilized young POLG mutator mice, which lack the ability to repair mtDNA insults because of a mutation in the mitochondrially restricted DNA polymerase. We demonstrated that denervation of muscles of POLO mice exacerbated mitochondrial dysfunction as measured by decreased NAD+: NADH ratio. This was associated with an increased number of unique mtDNA variants, demonstrating an increased mutational burden of the denervated leg compared to sham. Critically, the vast majority of the variants predicted to be most deleterious were localized in genes encoding NADHdehydrogenase. This investigation demonstrates that AMPD3 and IMPDH2 coordinately control adenine and guanine nucleotide pools in skeletal muscle. Denervation atrophy results in an upregulation of these enzymes, resulting in a shift of the purine nucleotide pool, favoring OTP. Finally, denervation atrophy induces mtDNA mutations in skeletal muscle. Ultimately, this suggests an alternative framework to explain mtDNA-based ageing in skeletal muscle whereby atrophying muscle is a mutagenic environment for mitochondria, leading to accelerated mtDNA insults.Item Association of Tryptophan and NAD+ Metabolites with Brain and Skeletal Muscle Function in Critical Care Patients and Survivors(2024-07) Yates, Brandon Alston; Coggan, Andrew; Khan, Babar; Kroenke, Kurt; Liu, Ziyue; Newman, JohnBackground: Patients who survive Intensive Care Unit (ICU) acquired delirium will likely experience new or worsened physical, mental, and/or cognitive impairments (termed Post-Intensive Care Syndrome (PICS)). Although advances in critical care treatments have reduced mortality rates among older adult ICU survivors, roughly 67% suffer PICS. The most vulnerable to long-term physical and cognitive impairments are older adults or those who exhibit accelerated aging because of pre-existing physical frailty or cognitive frailty. Yet, identification of at-risk patients during admittance is likely difficult because of the 1) homogeneity in the clinical presentation of patients with pre-existing age-related physical frailty and critical illness compared to those suffering from only critical illness and 2) many patients arrive severely debilitated making administration of physical function or other volitional assessments difficult. Therefore, it is essential that new biomarkers to guide early diagnosis, prognosis, and disease monitoring are identified. To this point, tryptophan derivatives, particularly kynurenines and nicotinamide family (e.g., NAD+), have been shown to mediate the relationship between chronic inflammation and physical impairment or signal accelerated aging, respectively. However, it remains unknown if similar associations exist in ICU patients and the prognostic utility of elevated neurotoxic tryptophan metabolites relative to neuroprotective tryptophan metabolites to predict adverse health outcomes while in the ICU. Methods: A secondary analysis of pooled data from three randomized control trials was used to investigate the following aims. To address Aims 1 and 2, blood samples from patients with ICU acquired delirium were analyzed for kynurenine and salvage pathway metabolites. To address Aim 3, blood samples from patients who survived an ICU stay, experienced ICU delirium, and completed both objective and subjective physical function assessments within in month of ICU discharge and before the COVID-19 pandemic. Results: Delirium duration was significantly (p< 0.05) associated with elevated circulating kynurenine and lower NAD+. Delirium severity was significantly associated with elevated circulating lower NAD+ but not kynurenine. Post-ICU physical function performance was significantly associated with elevated circulating kynurenine but not NAD+. Conclusion: Elevated concentration of frailty biomarkers are associated with delirium severity and duration in the ICU and post-ICU physical function.Item The Effects of FNDC5/Irisin on Osteocyte Functions(2023-07) Shimonty, Anika; Plotkin, Lilian I.; Bonewald, Lynda F.; Brault, Jeff; Robling, Alexander G.; White, Kenneth E.Irisin is a myokine generated when Fibronectin type III Domain Containing protein 5 (FNDC5) is proteolytically cleaved during exercise. While irisin has been shown to be beneficial in the functions of the brain, heart, and adipose tissue, its effect on bone cells remains contradictory. Osteocytes are the most abundant and longest-living bone cells, with different transcriptomes based on sex and age. One of the major functions of osteocytes is osteocytic osteolysis, the removal of their perilacunar matrix. A previous study showed that irisin deletion protects bone from ovariectomy-induced loss due to less osteoclastic resorption and less osteocytic osteolysis. Therefore, we hypothesized that FNDC5/irisin modulates the osteocyte function of osteocytic osteolysis in a sex-dependent manner. Under normal conditions, there was no difference in bone parameters between wildtype and FNDC5-null adult female mice starting from 5 to 20 months of age. However, 5-month-old null male mice had higher bone mass, but weaker bone compared to wildtype males, which persisted up to 20 months. Both 5-month-old female and male null mice had significantly lower TRAP-positive osteocytes, suggesting a role of irisin in priming the osteocytes for bone resorption. Osteocytes from female wildtype mice show higher lacunar area and upregulated resorption genes compared to wildtype males. However, null females and null males do not have significant differences in the lacunar area or resorption genes. Under calcium-deficient conditions, both 5 and 18-month-old female null mice lost less bone compared to their wildtype counterparts. In contrast, both 5 and 18-month-old null male mice lost more bone than age-matched wildtype males. Additionally, the percentage of bone loss was greater in the aged null male mice compared to 5-month-old null males. In summary, in female osteocytes, irisin works to release calcium from bone during lactation, ensuring offspring survival; however, with aging and hypocalcemia, irisin exerts a negative effect on bone mass. In contrast, irisin works to maintain bone mass and strength by modulating male osteocyte function. This study is the first to demonstrate a sex and age-specific irisin effect on the osteocyte function of osteocytic osteolysis, which has implications for the development of osteoporosis treatment.