Dissertation
Gadd45a is a molecular mediator of skeletal muscle atrophy
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2023
DOI: 10.25820/etd.006930
Abstract
Skeletal muscle atrophy is a debilitating condition common to many acute and chronic medical afflictions, including muscle disuse, denervation, starvation, aging, critical illness and chronic disease. The effects of skeletal muscle atrophy include decreased independent living and perceived quality of life due to a loss of strength, mobility and the delayed recovery from illness and injury. Over the past several years, our laboratory has identified and begun to characterize a novel signaling pathway that causes skeletal muscle fiber atrophy.
Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via the expression of activating transcription factor 4 (ATF4) within skeletal muscle fibers. However, the direct biochemical mechanism by which ATF4 promotes muscle atrophy is unknown. ATF4 is a member of the basic leucine zipper transcription factor (bZIP) superfamily. Because bZIP transcription factors are obligate dimers, and because ATF4 is unable to form highly stable homodimers, we hypothesized that ATF4 may promote muscle atrophy by forming a heterodimer with another bZIP family member. To test this hypothesis, we biochemically isolated skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo. Interestingly, we found that ATF4 forms at least five distinct heterodimeric bZIP transcription factors in skeletal muscle fibers. Furthermore, one of these heterodimers, composed of ATF4 and CCAAT enhancer-binding protein beta (C/EBPb) beta, mediates muscle atrophy. Within skeletal muscle fibers, the ATF4–C/EBPb heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF–C/EBP composite site in exon 4 of the Gadd45a gene (Growth arrest and DNA-damage-inducible alpha). This three-way interaction between ATF4, C/EBPb, and the ATF–C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy.
Gadd45a importance in muscle atrophy is supported by our preliminary studies which indicate that: 1) Gadd45a expression is strongly associated with skeletal muscle atrophy; 2) RNAi-mediated knockdown of Gadd45a decreases muscle fiber atrophy during a wide range of stress conditions; 3) transfection of a Gadd45a cDNA is sufficient to induce muscle fiber atrophy in young and otherwise healthy muscles. Collectively, these results established Gadd45a as an important mediator of muscle fiber atrophy. However, the higher-level effects of Gadd45a on whole-body skeletal muscle mass, function and metabolism remain unknown. To address these important issues, we generated and studied transgenic mice with skeletal muscle-specific expression of Gadd45a. We found that Gadd45a induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MEKK4, a protein kinase that is directly activated by Gadd45a. By inducing these changes, Gadd45a decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans.
To complement our studies in Gadd45a mTg mice, we generated skeletal muscle-specific Gadd45a knock-out mice (Gadd45a mKO). Here, we use Gadd45a mKO mice to test the hypothesis that Gadd45a expression in skeletal muscle is necessary for some or all of the deleterious consequences of muscle fiber atrophy, namely deteriorations in muscle mass and strength. We found that under basal non-atrophy conditions Gadd45a mKO mice exhibit normal muscle mass and strength. However, after undergoing 7 days of immobilization-induced atrophy, these mice showed partial protection against losses in both muscle mass and muscle strength. Furthermore, compared to littermate controls, the muscles of Gadd45a mKO mice failed to induce mRNAs associated with protein catabolism including those of the ubiquitin-proteasome and the autophagy-lysosome systems. These results elucidate a critical stress-induced pathway that reprograms muscle gene expression to cause atrophy.
Together, these findings indicate the ATF4-C/EBPb-Gadd45a signaling axis is a critical stress-induced pathway implicated in skeletal muscle atrophy and may open future avenues for developing targeted pharmacological agents that treat or limit the progression of muscle atrophy.
Details
- Title: Subtitle
- Gadd45a is a molecular mediator of skeletal muscle atrophy
- Creators
- George Marcotte
- Contributors
- Christopher M Adams (Advisor)Vitor A Lira (Committee Member)Chad E Grueter (Committee Member)Peter M Snyder (Committee Member)Joseph Zabner (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biomedical Science (Molecular Medicine)
- Date degree season
- Autumn 2023
- DOI
- 10.25820/etd.006930
- Publisher
- University of Iowa
- Number of pages
- xix, 165 pages
- Copyright
- Copyright 2023 George Marcotte
- Language
- English
- Date submitted
- 10/31/2023
- Description illustrations
- illustrations, graphs, tables
- Description bibliographic
- Includes bibliographical references (pages 155-165).
- Public Abstract (ETD)
- Skeletal muscle atrophy, also known as muscle wasting, is a debilitating and highly prevalent complication of malnutrition, critical illness, chronic disease, muscle disuse, and aging. At a cellular level, skeletal muscle atrophy is explained by a decrease in size of the major cell type in skeletal muscle, skeletal muscle fibers. Atrophy of skeletal muscle fibers leads to impairments in skeletal muscle mass, function, and metabolism. The molecular mechanisms that cause muscle fibers to undergo atrophy are complex and only beginning to be understood. The Adams laboratory has identified and begun to characterize a novel signaling pathway that causes skeletal muscle fiber atrophy. This pathway is both necessary and sufficient for muscle atrophy during aging, immobilization, and fasting. One component of this pathway is a transcription factor complex composed of ATF4 and C/EBPβ (Activating transcription factor 4 and CCAAT enhancer-binding protein beta, respectively). Conditions that induce muscle atrophy (including fasting, immobilization and aging) result in the formation of an ATF4- C/EBPβ heterodimer. The ATF4-C/EBPβ transcription factor complex activates several genes important in muscle atrophy and one of these genes is the growth arrest and DNA-damage- inducible 45 alpha (Gadd45a). In this thesis, I present our work identifying C/EBPβ as the primary ATF4 interactor responsible for Gadd45a expression and muscle atrophy. We show that the ATF4-C/EBPβ heterodimer binds a regulatory element in the Gadd45a gene, leading to an increase in Gadd45a content. To understand how Gadd45a influences whole-body skeletal muscle mass, function and metabolism, we generated skeletal muscle specific Gadd45a knockout mice (Gadd45a mKO mice) and skeletal muscle specific Gadd45a transgenic mice (Gadd45a mTg mice). Gadd45a mKO mice exhibit normal muscle mass and strength under basal conditions, suggesting Gadd45a ix has no effect in the absences of stress. However, under conditions of immobilization-induced atrophy, Gadd45a mKO exhibit reduced losses of muscle mass, muscle strength and a diminished induction of transcripts associated with muscle atrophy. Conversely, Gadd45a mTg mice exhibit a reduction in skeletal muscle mass, muscle mitochondria content, muscle oxidative capacity, and alterations in muscle fiber type. These cellular changes are at least partly mediated by MEKK4 (mitogen-activated protein kinase kinase kinase 4, Map3k4), a protein kinase that is directly activated by Gadd45a. Together, these findings indicate the ATF4-C/EBPβ-Gadd45a signaling axis is a critical stress-induced pathway implicated in skeletal muscle atrophy.
- Academic Unit
- Biomedical Science Program
- Record Identifier
- 9984546649102771
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