Skeletal muscle atrophy is a devastating and defining feature of cancer cachexia that reduces quality of life, treatment tolerance, and survival, but cannot be prevented or reversed by current management strategies. Ursolic acid is a natural dietary compound that has been shown to inhibit atrophy-associated changes in skeletal muscle mRNA expression in rodents and dogs, leading to beneficial changes in skeletal muscle structure and function. We hypothesized that dietary supplementation with ursolic acid might help support skeletal muscle mass and function during cancer. To test this hypothesis, we investigated ursolic acid's effects in five in vivo mouse models of cancer cachexia that are driven by pancreatic, colon, and lung cancer cells of mouse and human origin. We found that dietary supplementation with ursolic acid has broad-spectrum effects toward cancer-induced skeletal muscle atrophy, significantly preserving muscle mass in all five cancer cachexia models. Ursolic acid's positive effects on muscle mass and muscle fiber size led to significant improvements in grip strength and muscle tetanic force, persisted in the presence of chemotherapy, and were not associated with discernible changes in food intake or tumor growth. Ursolic acid appeared to generate its beneficial effects in skeletal muscle by acting directly on muscle cells, inhibiting catabolic effects of tumor-derived secreted factors, and inhibiting >90% of cancer-induced changes in skeletal muscle mRNA expression. These results strongly nominate ursolic acid as a promising potential nutritional approach for supporting muscle mass and function in individuals with cancer. NEW & NOTEWORTHY Cancer-induced muscle wasting affects many people with cancer, reducing treatment tolerance and survival. We identified a natural dietary compound, ursolic acid, that attenuates muscle atrophy across five preclinical cancer models spanning pancreatic, colon, and lung cancer. Ursolic acid inhibits cancer-induced changes in muscle mRNA expression, preserves muscle strength, and remains protective during chemotherapy, without affecting food intake or tumor burden. These results identify ursolic acid as a promising, translatable dietary supplement for supportive cancer care.

BACKGROUND:Hepatic stellate cells (HSCs) play a pivotal role in driving fibrosis during chronic liver injury. HSCs produce vast amounts of fibrotic proteins, causing endoplasmic reticulum (ER) stress and initiating the unfolded protein response (UPR). While the UPR is important for fibrogenesis, how signaling through UPR transducer Protein Kinase R-like ER Kinase (PERK) and its effectors impact HSC activation and fibrogenesis is unclear. Here, we sought to uncover the role of PERK and its effector GADD45A in liver fibrosis. METHODS:PERK-GADD45A signaling was assessed in primary and immortalized HSCs treated with TGFβ, and mouse models of fibrosis. Genetic and pharmacological disruption of PERK or GADD45A was used to assess the role of PERK or GADD45A in HSC activation, proliferation, and fibrogenesis. HSC-specific Gadd45a-null mice were utilized to investigate the role of GADD45A on CCl4-induced fibrosis. RESULTS:We found that TGFβ-induction of collagen I drives activation of PERK signaling in HSCs. Furthermore, loss or inhibition of PERK limits long-term HSC activation as illustrated by reduced levels of collagen I and fibronectin, impaired collagen I deposition, and reduced cell proliferation in vitro. Next, we show that PERK signaling induces expression of GADD45A during HSC activation, and loss of GADD45A disrupts HSC activation and expression of proliferation and cell-cycle-associated genes in immortalized and primary HSCs. Finally, HSC-specific GADD4Aa loss limits CCl4-driven fibrogenesis in vivo. CONCLUSION:PERK signaling is critical for HSC activation, and loss of the PERK downstream effector GADD45A limits fibrosis progression. Disruption of HSC activation and proliferation, coupled with dysregulation of cell-cycle-associated genes upon PERK or GADD45A loss, suggests that PERK-GADD45A signaling impacts multiple facets of HSC pathophysiology.

Sarcopenia increases the risk of frailty, morbidity, and mortality in older adults. Resistance exercise training improves muscle size and function; however, the response to exercise training is variable in older adults. The objective of our study was to determine both the age-independent and age-dependent changes to the transcriptome following progressive resistance exercise training. Skeletal muscle biopsies were obtained before and after 12 weeks of resistance exercise training in 8 young (24 ± 3.3 years) and 10 older (72 ± 4.9 years) men. RNA was extracted from each biopsy and prepared for analysis via RNA sequencing. We performed differential mRNA expression, gene ontology, and gene set enrichment analyses. We report that when comparing post-training vs pre-training 226 mRNAs and 959 mRNAs were differentially expressed in the skeletal muscle of young and older men, respectively. Additionally, 94 mRNAs increased, and 17 mRNAs decreased in both young and old, indicating limited overlap in response to resistance exercise training. Furthermore, the differential gene expression was larger in older skeletal muscle. Finally, we report three novel findings: 1) resistance exercise training decreased the abundance of ATF4-activated and senescence-associated skeletal muscle mRNAs in older men; 2) resistance exercise-induced increases in lean mass correlate with increased mRNAs encoding mitochondrial proteins; and 3) increases in muscle strength following resistance exercise positively correlate with increased mRNAs involved in translation, rRNA processing, and polyamine metabolism. We conclude that resistance exercise training elicits a differential gene expression response in young and old skeletal muscle, including reduced ATF-4 activated and senescence-associated gene expression.

The goal of this study was to examine benefit of omega-3 polyunsaturated fatty acids (PUFA) derived from fish oil or resolvin D1, metabolite of docosahexaenoic acid, on skeletal muscle disuse atrophy. Atrophy was induced of the left leg of C57Bl6/J male mice for 1 week.Three different treatment experimental protocols were used by feeding a diet enriched with menhaden oil or daily injections with resolvin D1 before or after immobilization. Evaluation of muscle atrophy was performed by determining the difference in weight of the tibialis anterior muscle between the immobilized (left) and mobile contralateral side (right) and by immunohistochemistry. A 2week prevention protocol with menhaden oil enriched diet or daily injections of 2 ng/g resolvin D1 provided the greatest protection from muscle atrophy.Significant protection was also observed when dietary treatment of the mice was initiated at time of immobilization. This study demonstrated that omega-3 PUFA and metabolites provide protection toward skeletal muscle disuse atrophy.

The loss of skeletal muscle mass during aging is a significant health concern linked to adverse outcomes in older individuals. Understanding the molecular basis of age-related muscle loss is crucial for developing strategies to combat this debilitating condition. Long noncoding RNAs (lncRNAs) are a largely uncharacterized class of biomolecules that have been implicated in cellular homeostasis and dysfunction across a many tissues and cell types. To identify lncRNAs that might contribute to skeletal muscle aging, we screened for lncRNAs whose expression was altered in vastus lateralis muscle from older compared to young adults. We identified FRAIL1 as an aging-induced lncRNA with high abundance in human skeletal muscle. In healthy young and older adults, skeletal muscle FRAIL1 was increased with age in conjunction with lower muscle function. Forced expression of FRAIL1 in mouse tibialis anterior muscle elicits a dose-dependent reduction in skeletal muscle fiber size that is independent of changes in muscle fiber type. Furthermore, this reduction in muscle size is dependent on an intact region of FRAIL1 that is highly conserved across non-human primates. Unbiased transcriptional and proteomic profiling of the effects of FRAIL1 expression in mouse skeletal muscle revealed widespread changes in mRNA and protein abundance that recapitulate age-related changes in pathways and processes that are known to be altered in aging skeletal muscle. Taken together, these findings shed light on the intricate molecular mechanisms underlying skeletal muscle aging and implicate FRAIL1 in age-related skeletal muscle phenotypes.

Muscle atrophy and weakness are prevalent and debilitating conditions in dogs that cannot be reliably prevented or treated by current approaches. In non-canine species, the natural dietary compound ursolic acid inhibits molecular mechanisms of muscle atrophy, leading to improvements in muscle health. To begin to translate ursolic acid to canine health, we developed a novel ursolic acid dietary supplement for dogs and confirmed its safety and tolerability in dogs. We then conducted a randomized, placebo-controlled, proof-of-concept efficacy study in older beagles with age-related muscle atrophy, also known as sarcopenia. Animals received placebo or ursolic acid dietary supplements once a day for 60 days. To assess the study's primary outcome, we biopsied the quadriceps muscle and quantified atrophy-associated mRNA expression. Additionally, to determine whether the molecular effects of ursolic acid might have functional correlates consistent with improvements in muscle health, we assessed secondary outcomes of exercise participation and T-maze performance. Importantly, in canine skeletal muscle, ursolic acid inhibited numerous mRNA expression changes that are known to promote muscle atrophy and weakness. Furthermore, ursolic acid significantly improved exercise participation and T-maze performance. These findings identify ursolic acid as a natural dietary compound that inhibits molecular mechanisms of muscle atrophy and improves functional performance in dogs.

Aging slowly erodes skeletal muscle strength and mass, eventually leading to profound functional deficits and muscle atrophy. The molecular mechanisms of skeletal muscle aging are not well understood. To better understand mechanisms of muscle aging, we investigated the potential role of ATF4, a transcription regulatory protein that can rapidly promote skeletal muscle atrophy in young animals deprived of adequate nutrition or activity. To test the hypothesis that ATF4 may be involved in skeletal muscle aging, we studied fed and active muscle-specific ATF4 knockout mice (ATF4 mKO mice) at 6 months of age, when wild-type mice have achieved peak muscle mass and function, and at 22 months of age, when wild-type mice have begun to manifest age-related muscle atrophy and weakness. We found that 6-month-old ATF4 mKO mice develop normally and are phenotypically indistinguishable from 6-month-old littermate control mice. However, as ATF4 mKO mice become older, they exhibit significant protection from age-related declines in strength, muscle quality, exercise capacity, and muscle mass. Furthermore, ATF4 mKO muscles are protected from some of the transcriptional changes characteristic of normal muscle aging (repression of certain anabolic mRNAs and induction of certain senescence-associated mRNAs), and ATF4 mKO muscles exhibit altered turnover of several proteins with important roles in skeletal muscle structure and metabolism. Collectively, these data suggest ATF4 as an essential mediator of skeletal muscle aging and provide new insight into a degenerative process that impairs the health and quality of life of many older adults.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle–specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. 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 MAP kinase kinase kinase 4, 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. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software. The computational pipeline of TurnoveR performs critical steps to determine protein turnover rates, including isotopologue demultiplexing, precursor-pool correction, statistical analysis, and generation of data reports and visualizations. This workflow is compatible with many mass spectrometric platforms and recapitulates turnover rates and differential changes in turnover rates between treatment groups calculated in previous studies. We expect that the addition of TurnoveR to the widely used Skyline proteomics software will facilitate wider utilization of protein turnover analysis in highly relevant biological models, including aging, neurodegeneration, and skeletal muscle atrophy.

Activating transcription factor 4 (ATF4) is a multifunctional transcription regulatory protein in the basic leucine zipper superfamily. ATF4 can be expressed in most if not all mammalian cell types, and it can participate in a variety of cellular responses to specific environmental stresses, intracellular derangements, or growth factors. Because ATF4 is involved in a wide range of biological processes, its roles in human health and disease are not yet fully understood. Much of our current knowledge about ATF4 comes from investigations in cultured cell models, where ATF4 was originally characterized and where further investigations continue to provide new insights. ATF4 is also an increasingly prominent topic of in vivo investigations in fully differentiated mammalian cell types, where our current understanding of ATF4 is less complete. Here, we review some important high-level concepts and questions concerning the basic biology of ATF4. We then discuss current knowledge and emerging questions about the in vivo role of ATF4 in one fully differentiated cell type, mammalian skeletal muscle fibers.

Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a-deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.

The protein kinases Unc‐51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2) have evolved from a single yeast gene, autophagy related 1 (Atg1). Although ULK1 and ULK2 are thought to redundantly stimulate autophagy, other functions are emerging. Interestingly, skeletal muscle expresses both ULK1 and ULK2 at high levels (i.e., >2‐fold the median expression of 91 mouse tissues and cell lines) suggesting an important, yet unknown role for their combined elevated expression in muscle. Therefore, to investigate the joint role of ULK1 and ULK2 on muscle homeostasis and whole‐body metabolism we generated mice with skeletal muscle‐specific knockout of these genes (ULK2/1 mKO). Here, we demonstrate that ULK2/1 mKO mice have larger muscles (~12%) that are actually weaker (~21% decrease in relative force) in comparison with wild type littermates. Loss of ULK2/1 in skeletal muscle also affects whole‐body metabolic homeostasis as indicated by decreased body fat mass (~20% at 7wk of age), attenuated fat mass gains with aging (~40% at 21mo. of age), and life‐threatening inability to maintain euglycemia under fasting conditions (~32% reduction of blood glucose compared to WT). Mechanistically, loss of ULK 2/1 not only impairs skeletal muscle autophagy but also AMPK function as indicated by accumulation of LC3, p62, NBR1, and ubiquitinated proteins, as well as reduced phosphorylation of ACC (a surrogate of AMPK activity), respectively. These results identify a previously unknown role for ULK2 and ULK1 in jointly coupling skeletal muscle size with force. They also reveal skeletal muscle ULK2 and ULK1 as fundamental proteins for survival under conditions of reduced nutrient availability.Support or Funding InformationThis work is funded by:AHA 16SDG30360001 (V.A.L.). and NASA Iowa Space Grant Graduate Fellowship Program

Exercise interventions alter the DNA methylation profile in skeletal muscle, yet little is known about the role of the DNA methylation machinery in exercise capacity. In this study, we found that in oxidative red muscle, DNMT3A expression increases greatly following a bout of endurance exercise. Mice lacking in skeletal muscle fibers had reduced tolerance to endurance exercise, accompanied by reduced oxidative capacity and reduced mitochondrial counts. Moreover, during exercise, the knockout muscles overproduced reactive oxygen species (ROS), which are major contributors to muscle dysfunction. In mechanistic terms, we demonstrated that is a key target of repression by DNMT3A in red muscles. DNMT3A directly regulated the Aldh1l1 transcription by binding to the promoter region and altering DNA methylation and histone modification. Enforcing ALDH1L1 expression, leading to elevated NADPH, led to overproduction of ROS by the NADPH oxidase complex (NOX) in myotubes, ultimately resulting in mitochondrial defects. Moreover, both genetic inhibition of ALDH1L1 and pharmacological inhibition of NOX rescued oxidative stress and mitochondrial decline in -deficient myotubes, confirming the essential role of ALDH1L1-dependent ROS generation as a downstream effector of DNMT3A loss of function. Together, our results reveal that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress.

Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in 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 ? (C/EBP?), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4?C/EBP? heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF?C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBP?, and the ATF?C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemical mechanism by which ATF4 induces skeletal muscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy.

Skeletal muscle atrophy is a highly-prevalent and debilitating condition that remains poorly understood at the molecular level. Previous work found that aging, fasting, and immobilization promote skeletal muscle atrophy via expression of activating transcription factor 4 (ATF4) in 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. transcription obligate ATF4 is unable to highly-stable homodimers, we that ATF4 muscle atrophy by a bZIP family we skeletal muscle proteins that associate with the dimerization- and DNA-binding domain of ATF4 (the bZIP domain) in mouse skeletal muscle fibers in vivo . we that ATF4 at least five distinct heterodimeric bZIP transcription factors in muscle fibers. one of these heterodimers, of ATF4 and CCAAT enhancer-binding protein (cid:2) (C/EBP (cid:2) ), mediates muscle atrophy. Within skeletal muscle fibers, the ATF4–C/EBP (cid:2) heterodimer interacts with a previously unrecognized and evolutionarily conserved ATF–C/EBP composite site in exon 4 of the Gadd45a gene. This three-way interaction between ATF4, C/EBP (cid:2) , and the ATF–C/EBP composite site activates the Gadd45a gene, which encodes a critical mediator of muscle atrophy. Together, these results identify a biochemicalmechanismbywhichATF4inducesskeletalmuscle atrophy, providing molecular-level insights into the etiology of skeletal muscle atrophy.

Age-related skeletal muscle atrophy is a very common and serious condition that remains poorly understood at the molecular level. Several lines of evidence have suggested that the tumor suppressor p53 may play a central, causative role in skeletal muscle aging, whereas other, apparently contradictory lines of evidence have suggested that p53 may be critical for normal skeletal muscle function. To help address these issues, we performed an aging study in male muscle-specific p53-knockout mice (p53 mKO mice), which have a lifelong absence of p53 expression in skeletal muscle fibers. We found that the absence of p53 expression in skeletal muscle fibers had no apparent deleterious or beneficial effects on skeletal muscle mass or function under basal conditions up to 6 mo of age, when mice are fully grown and exhibit peak muscle mass and function. Furthermore, at 22 and 25 mo of age, when age-related muscle weakness and atrophy are clearly evident in mice, p53 mKO mice demonstrated no improvement or worsening of skeletal muscle mass or function relative to littermate control mice. At advanced ages, p53 mKO mice began to die prematurely and had an increased incidence of osteosarcoma, precluding analyses of muscle mass and function in very old p53 mKO mice. In light of these results, we conclude that p53 expression in skeletal muscle fibers has minimal if any direct, cell autonomous effect on basal or age-related changes in skeletal muscle mass and function up to at least 22 mo of age.NEW & NOTEWORTHY Previous studies implicated the transcriptional regulator p53 as a potential mediator of age-related skeletal muscle weakness and atrophy. We tested this hypothesis by investigating the effect of aging in muscle-specific p53-knockout mice. Our results strongly suggest that p53 activity within skeletal muscle fibers is not required for age-related skeletal muscle atrophy or weakness.

Skeletal muscle atrophy proceeds through a complex molecular signaling network that is just beginning to be understood. Here, we discuss examples of recently identified molecular mechanisms of muscle atrophy and how they highlight an immense need and opportunity for focused biochemical investigations and further unbiased discovery work.