Cellular dysfunctions caused by dystrophin deficiency and the interaction of diet-induced insulin resistance

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2023-05
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Krishna, Swathy
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Selsby, Joshua T
Valentine, Rudy J
McNeill, Elizabeth M
Kim, Jinoh
Walley, Justin W
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Animal Science
Abstract
Duchenne muscular dystrophy (DMD), caused by the absence of a functional dystrophin protein, is a devastating degenerative muscle disease. The absence of dystrophin leaves the muscle vulnerable to injuries, especially during eccentric contractions, and causes metabolic dysfunctions and dysregulation of cellular processes including, but not limited to inflammation, mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. The commonly used mouse model to study these cellular dysfunctions and pathophysiology is the mdx mouse model. However, this model has a mild disease phenotype compared to humans. Given this, an emerging mouse model, the D2-mdx mouse, was developed, which has a more severe pathology than mdx mice. Previously, we identified impaired autophagosomal degradation in mdx mice. Given the D2-mdx model’s expanding use, we evaluated how markers of autophagy are modified in skeletal muscles from 11-mo-old D2-mdx mice. We discovered that autophagosomal degradation was impaired in the diaphragm but not gastrocnemius. Further analysis in gastrocnemius muscles by evaluation of markers of autophagy in lysosomal and cytosolic fractions suggested increased lysosomal abundance so as to compensate for impaired lysosomal function. The findings from this study, together with the wide range of cellular dysfunctions identified in dystrophic muscles, point to an accumulation of unfolded proteins in the intracellular environment. This led us to the second research chapter, where we explored how the markers of ER stress and the unfolded protein response (UPR) are altered by dystrophin deficiency. In support of the findings from the first research chapter, we identified increased ER stress and activation of the UPR in diaphragm from D2-mdx mice. To add further clarity to these findings, we also considered how ER stress and the UPR may be impacted by DMD by probing a publicly available human Affymetrix data set. We discovered increased transcript abundance of ER stress and UPR-related transcripts and also predicted transcription factors that regulate the identified upregulation profile. Dystrophin deficiency results in a broad array of cellular dysfunctions, including discoveries made in the first two research chapters. DMD is also frequently accompanied by metabolic complications such as obesity, insulin resistance, hyperglycemia, hyperinsulinemia, and metabolic syndrome. Independent of dystrophin deficiency, these metabolic alterations can also cause a range of cellular dysfunctions raising the possibility of an additive or even synergistic interaction of DMD and obesity. In chapter three, we explored changes associated with a high-fat high sucrose diet (HFHSD) in mdx mice. We discovered diet-induced insulin resistance, glucose intolerance, hyperglycemia, and dyslipidemia in HFHSD-fed C57 (control) and, for the first time, in HFHSD-fed mdx mice. Interestingly, mdx mice on a control diet were inherently insulin resistant, raising the possibility that this may be a fundamental consequence of dystrophin deficiency. Metabolomic and lipidomic analyses suggested unique and common consequences of diet-induced insulin resistance with dystrophin deficiency. To further understand the molecular consequences of a HFHSD in dystrophic skeletal muscles, in the fourth research chapter, we performed and analyzed proteomics and phosphoproteomics on skeletal muscle from these mice. We discovered that the HFHSD resulted in some common changes in the muscle proteome and phosphoproteome in muscle from C57 and mdx mice, but importantly, dystrophin deficiency also caused some unique molecular consequences. Further, using these datasets we identified key transcription factors predicted to regulate these unique changes in the obese, dystrophic proteome. Data produced in support of this dissertation provide substantially new information regarding the fundamental consequences of dystrophin deficiency as well as new information regarding the complexities of obesity and insulin resistance.
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