ATF4-Mediate Changes in Protein Turnover During Age-Related Skeletal Muscle Atrophy
ABSTRACT: Age-related skeletal muscle atrophy is a debilitating condition that has significant negative impacts on health and quality of life. Despite broad clinical impact, the molecular basis of age-related skeletal muscle atrophy is not well understood. Here, we determined protein turnover rates in skeletal muscle of 22-month-old control mice and 22-month-old muscle-specific ATF4 knockout (ATF4 mKO) mice, which are partially resistant to age-related muscle atrophy. All samples were analyzed by DDA TripleTOF 6600 mass spectrometer for downstream analysis of protein turnover. Quantitative MS1 peak areas were extracted from DDA raw files in the Skyline-Daily software platform. ProteinPilot search results were imported into the Skyline software to build a spectral library, followed by import of raw MS files for extraction of chromatographic peak areas. A custom skyline report containing all peptide and protein characteristics, annotations, and quantitative information including isotopologue peak areas was exported and used for downstream analysis and calculation of protein turnover rates in R using in-house R scripts (TurnoveR tool). Precursor-pool corrected protein turnover rates were calculated using the same approach employed in previous studies using the Topograph software platform. For calculation of protein abundance changes, DIA acquisitions from six samples (3 ATF4 KO and 3 WT samples) were quantitatively processed using Spectronaut v14. Analysis of protein turnover indicates that most proteins with altered turnover rates in ATF4 mKO muscles have decreased half-lives and are components of the mitochondrion. These proteins include subunits of the ATP Synthase (Atp5b, Atp5o), Ubiquinol-Cytochrome C Reductase (Uqcrc1, Uqcrh) and Cytochrome C Oxidase (Cox6b1) complexes, among others. These findings implicate increased rates of mitochondrial protein turnover as a mechanism that underlies, at least in part, the protection from age-induced muscle atrophy in ATF4 mKO mice.
Project description:The turnover of cytochrome c was determined in the three skeletal-muscle fibre types of adult male rats by a kinetic analysis that followed the time course of cytochrome c content change. Confirming evidence was obtained with double-labelling studies using delta-aminolaevulinate. Cytochrome c turnover was most rapid in the low-oxidative fast-twitch white fibre [t1/2 (half-life) about 4 days], slowest in the high-oxidative fast-twitch red fibre (t1/2 9-10 days) and relatively rapid in the high-oxidative slow-twitch red fibre (t1/2 5-6 days). Thus cytochrome c turnover does not strictly conform to either the appearance (i.e. red or white) or the contractile characteristics (i.e. fast or slow) of the muscle fibres. The synthesis rates needed to maintain the corresponding cytochrome c concentrations, however, were similarly high in the two mitochondria-rich red fibre types. These data illustrate that both the synthesis and degradation processes are important in establishing the cytochrome c concentrations that distinguish the different skeletal-muscle fibre types.
Project description:Measurement of rates of synthesis of skeletal-muscle proteins in adult rats shows that the faster overall rate of turnover in diaphragm and soleus muscles compared with several other, more glycolytic, muscles is also exhibited by the myofibrillar proteins, since the ratio of sarcoplasmic to myofibrillar protein synthesis is similar for all muscles. Further, throughout postnatal development, when the overall turnover rate falls with age, parallel changes occur for the myofibrillar proteins, as indicated by a constant ratio of sarcoplasmic to myofibrillar protein synthesis (2.06) in the steady state after overnight starvation. Only in the youngest (4 weeks old) rats is a slightly lower ratio observed (1.72). These results indicate that, when changes in the overall turnover rate of muscle proteins occur, the relative turnover of the two major protein fractions stays constant. However, measurements in the non-steady state during growth and after starvation for 4 days show that the relative synthesis rates of the two fractions change as a result of a disproportionate increase in myofibrillar protein synthesis during growth and decrease during starvation. Thus the synthesis rate of the slower-turning-over myofibrillar protein fraction is more sensitive to nutritional state than is that of the sarcoplasmic protein. It is suggested that such responses may help to maintain constant tissue composition during non-steady-state conditions of growth and atrophy.
Project description:Preserving skeletal muscle mass and functional capacity is essential for healthy ageing. Transient periods of disuse and/or inactivity in combination with sub-optimal dietary intake have been shown to accelerate the age-related loss of muscle mass and strength, predisposing to disability and metabolic disease. Mechanisms underlying disuse and/or inactivity-related muscle deterioration in the older adults, whilst multifaceted, ultimately manifest in an imbalance between rates of muscle protein synthesis and breakdown, resulting in net muscle loss. To date, the most potent intervention to mitigate disuse-induced muscle deterioration is mechanical loading in the form of resistance exercise. However, the feasibility of older individuals performing resistance exercise during disuse and inactivity has been questioned, particularly as illness and injury may affect adherence and safety, as well as accessibility to appropriate equipment and physical therapists. Therefore, optimising nutritional intake during disuse events, through the introduction of protein-rich whole-foods, isolated proteins and nutrient compounds with purported pro-anabolic and anti-catabolic properties could offset impairments in muscle protein turnover and, ultimately, the degree of muscle atrophy and recovery upon re-ambulation. The current review therefore aims to provide an overview of nutritional countermeasures to disuse atrophy and anabolic resistance in older individuals.
Project description:Skeletal muscle atrophy is associated with a disruption in protein turnover involving increased protein degradation and suppressed protein synthesis. Although it has been well studied that the IGF-1/PI3K/Akt pathway plays an essential role in the regulation of the protein turnover, molecule(s) that triggers the change in protein turnover still remains to be elucidated. TRB3 has been shown to inhibit Akt through direct binding. In this study, we hypothesized that TRB3 in mouse skeletal muscle negatively regulates protein turnover via the disruption of Akt and its downstream molecules. Muscle-specific TRB3 transgenic (TRB3TG) mice had decreased muscle mass and fiber size, resulting in impaired muscle function. We also found that protein synthesis rate and signaling molecules, mTOR and S6K1, were significantly reduced in TRB3TG mice, whereas the protein breakdown pathway was significantly activated. In contrast, TRB3 knockout mice showed increased muscle mass and had an increase in protein synthesis rate, but decreases in FoxOs, atrogin-1, and MuRF-1. These findings indicate that TRB3 regulates protein synthesis and breakdown via the Akt/mTOR/FoxO pathways.
Project description:Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype.
Project description:Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle fiber proteins that associate with Gadd45a as it induces skeletal muscle atrophy in living mice. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, the MAP kinase kinase kinase MEKK4. Furthermore, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into way that skeletal muscle atrophy occurs at the molecular level.
Project description:Mitochondrial dynamics is a conserved process by which mitochondria undergo repeated cycles of fusion and fission, leading to exchange of mitochondrial genetic content, ions, metabolites, and proteins. Here, we examine the role of the mitochondrial fusion protein optic atrophy 1 (OPA1) in differentiated skeletal muscle by reducing OPA1 gene expression in an inducible manner. OPA1 deficiency in young mice results in non-lethal progressive mitochondrial dysfunction and loss of muscle mass. Mutant mice are resistant to age- and diet-induced weight gain and insulin resistance, by mechanisms that involve activation of ER stress and secretion of fibroblast growth factor 21 (FGF21) from skeletal muscle, resulting in increased metabolic rates and improved whole-body insulin sensitivity. OPA1-elicited mitochondrial dysfunction activates an integrated stress response that locally induces muscle atrophy, but via secretion of FGF21 acts distally to modulate whole-body metabolism.
Project description:Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.
Project description:Exacerbations in COPD are often accompanied by pulmonary and systemic inflammation, and associated with increased susceptibility to and prevalence of weight loss and muscle wasting. Muscle mass loss during disease exacerbations may contribute to emphysema-associated muscle atrophy. However, whether pulmonary inflammation in presence of emphysema differentially affects skeletal muscle, including protein synthesis and degradation signaling pathways has not previously been addressed. The aims of this study were to 1) develop a mouse model of disease exacerbation-associated muscle wasting, 2) evaluate whether emphysema and muscle wasting can be monitored non-invasively and 3) assess alterations in muscle protein turnover regulation.Emphysema was induced by three, weekly intra-tracheal (IT) elastase (E) or vehicle control (vc) instillations, followed by one single IT-LPS bolus (L) or vc instillation to mimic pulmonary inflammation-driven disease exacerbation. Consequently, four experimental groups were defined: vc/vc ('C'), E/vc ('E'), vc/LPS ('L'), E/LPS ('E?+?L'). Using micro cone-beam CT-scans, emphysema development and muscle mass changes were monitored, and correlated to muscle weight 48 h after LPS instillation. Protein turnover signaling was assessed in muscle tissue collected 24 h post LPS instillation.Micro-CT imaging correlated strongly with established invasive measurements of emphysema and muscle atrophy. Pulmonary inflammation following LPS instillation developed irrespective of emphysema and body and muscle weight were similarly reduced in the 'L' and 'E?+?L' groups. Accordingly, mRNA and protein expression levels of genes of the ubiquitin-proteasome pathway (UPS) and the autophagy-lysosomal pathway (ALP) were upregulated in skeletal muscle following IT-LPS ('L' and 'E?+?L'). In contrast, mTOR signaling, which controls ALP and protein synthesis, was reduced by pulmonary inflammation ('L' and 'E?+?L') as well as emphysema as a single insult ('E') compared to control.Changes in lung tissue density and muscle mass can be monitored non-invasively to evaluate emphysema and muscle atrophy longitudinally. Acute loss of muscle mass evoked by pulmonary inflammation is similar in control and emphysematous mice. Although muscle atrophy cues in response to pulmonary inflammation are not altered by emphysema, emphysema itself affects protein synthesis and ALP signaling, which may interfere with muscle mass recovery and impair maintenance of muscle mass in emphysema.
Project description:The growth of one smooth and three individual striated muscles was studied from birth to old age (105 weeks), and where possible during the later stages of foetal life also. Developmental changes in protein turnover (measured in vivo) were related to the changing patterns of growth within each muscle, and the body as a whole. Developmental growth (i.e. protein accumulation) in all muscles involved an increasing proportion of protein per unit wet weight, as well as cellular hypertrophy. The contribution of the heart towards whole-body protein and nucleic acid contents progressively decreased from 18 days of gestation to senility. In contrast, post-natal changes in both slow-twitch (soleus) and fast-twitch (tibialis anterior) skeletal muscles remained reasonably constant with respect to whole-body values. Such age-related growth in all four muscle types was accompanied by a progressive decline in both the fractional rates of protein synthesis and breakdown, the changes in synthesis being more pronounced. Age for age, the fractional rates of synthesis were highest in the oesophageal smooth muscle, similar in both cardiac and the slow-twitch muscles, and lowest in the fast-twitch tibialis muscle. Despite these differences, the developmental fall in synthetic rates was remarkably similar in all four muscles, e.g. the rates at 105 weeks were 30-35% of their values at weaning. Such developmental changes in synthesis were largely related to diminishing ribosomal capacities within each muscle. When measured under near-steady-state conditions (i.e. 105 weeks of age), the half-lives of mixed muscle proteins were 5.1, 10.4, 12.1 and 18.3 days for the smooth, cardiac, soleus and tibialis muscles respectively. Old-age atrophy was evident in the senile animals, this being more marked in each of the four muscle types than in the animal as a whole. In each muscle of the senile rats the protein content and composition per unit wet weight, and both the fractional and total rates of synthesis, were significantly lower than in the muscles of younger, mature, animals (i.e. 44 weeks). In the soleus the decreased synthesis rate appeared to be related to a further fall in the ribosomal capacity. In contrast, the changes in synthesis in the three remaining muscles correlated with significant decreases in the synthetic rate per ribosome.(ABSTRACT TRUNCATED AT 400 WORDS)