The purpose of this thesis project was to elucidate the immune transcriptomic changes in the diaphragm of mdx mice treated with microdystrophin gene therapy with and without running wheel activity. Mdx mice are a model of Duchenne Muscular Dystrophy (DMD). Similar to DMD, mdx pathophysiology is associated with chronic inflammation due to sarcolemma fragility and cellular membrane leakage. Immune modulation has not yet been described when endurance exercise and AAV-microdystrophin gene therapy have been combined in mdx mice. An increase of physical activity in DMD individuals is a potential outcome of current clinical studies investigating microdystrophin treatment; therefore, understanding the impacts of physical activity on the immune system, particularly for the diaphragm, may be important to minimize risk.
Recently, the Grange lab published the endurance and contractile property outcomes of combined microdystrophin gene therapy and running wheel activity in mdx mice.1 Diaphragm RNA-seq transcriptomic data were also collected from this study for gene expression analysis. Using this dataset, I tested the hypothesis that relative to mdxGT (mdx mice treated with gene therapy), transcripts related to the immune response such as immune cell recruitment, activation, and downstream signals that promote fibrosis deposition were unchanged or downregulated in mdxRGT (mdx mice treated with gene therapy and access to running wheel). DEGs (differentially expressed genes) were analyzed with Microsoft Excel, R, and bioinformatic tools such as KEGG and DAVID to explain immune system adaptations in response to combined microdystrophin treatment and running in mdx mice.
Two major inflammatory signaling pathways, the IL-6/JAK/STAT and NF-kB signaling pathways translationally relevant to DMD patients were rescued by gene therapy towards WT expression levels. Although running maintained the majority of the rescued transcriptome profile (691 of 724 genes), some immune response-related gene expressions (33 of 724 genes) were modulated including genes related to chemotaxis and cellular migration. These changes suggested potential signaling for angiogenesis and a fast to slow fiber type shift; however, unbiased analysis with bioinformatic tools did not confirm either of these possibilities. The data from this study revealed inflammatory and fibrotic signaling pathways commonly observed in DMD patients and mdx mice were rescued by the AAV microdystrophin gene therapy and were maintained by voluntary wheel running / Master of Science / Duchenne Muscular Dystrophy (DMD) is an X chromosome-linked muscular dystrophy, a genetic disease that affects around 1 in 14,000 boys globally. DMD is lethal and currently there is no cure. Mutations in the DMD gene results in the absence of the protein dystrophin. The dystrophin protein and other proteins associated with it provide structural support to the skeletal muscle membrane. Without it, muscles are more easily damaged during contraction. This damage promotes recruitment of immune cells which initiates the first stage of muscle repair. Under normal circumstances, this inflammatory reaction caused by immune cells restores the skeletal muscles. However, in DMD patients, repeated breakdown and regeneration of skeletal muscles leads to abnormal inflammation which promotes negative outcomes such as increased fibrosis. Fibrosis impairs muscle function, especially the diaphragm .
Hamm et al., 2021 from the Grange lab investigated the effects of microdystrophin gene therapy and increased physical activity in mdx mice, a mouse model of DMD, with the idea that some of the negative changes with muscular dystrophy could be improved. The results showed a positive increase of endurance capacity in mdx mice treated with gene therapy alone (mdxGT group) and a greater increase if the mice also used a running wheel (mdxRGT group) compared to untreated mdx mice (mdx group). These findings suggested that gene therapy can increase a DMD patient's ability to become more physically active. However, the effects of running and microdystrophin gene therapy on the damaging inflammatory response in the diaphragm were not reported. To address this question, gene expression data from diaphragm muscles of all treatment groups were collected in the Hamm et al., 2021 study for later analysis.
In my study, these diaphragm gene expression data were used to compare inflammatory signals between the various treatment groups. Indicators of skeletal muscle damage, immune cell accumulation and fibrosis deposition were rescued (i.e., returned to healthy mice levels) by microdystrophin gene therapy (mdxGT group). Running did not exert any negative effects on the majority of genes rescued by the microdystrophin therapy (mdxRGT group). These results indicated that voluntary wheel running could maintain the reduced inflammatory signals due to the microdystrophin gene therapy in mdx mice. If the function of the skeletal muscle of dystrophic boys was similarly improved by microdystrophin gene therapy and exercise did not interfere with its positive effects, DMD boys could potentially be physically active similar to normal boys of their age.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/113763 |
| Date | 09 February 2023 |
| Creators | Yuan, Zeyu |
| Contributors | Human Nutrition, Foods and Exercise, Grange, Robert W., Craige, Siobhan, Li, Liwu |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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