SUMMARY Skeletal muscle atrophy is a common and debilitating condition that lacks a pharmacologic therapy. To develop a potential therapy, we identified 63 mRNAs that were regulated by fasting in both human and mouse muscle, and 29 mRNAs that were regulated by both fasting and spinal cord injury in human muscle. We used these two unbiased mRNA expression signatures of muscle atrophy to query the Connectivity Map, which singled out ursolic acid as a compound whose signature was opposite to those of atrophy-inducing stresses. A natural compound enriched in apples, ursolic acid reduced muscle atrophy and stimulated muscle hypertrophy in mice. It did so by enhancing skeletal muscle insulin/IGF-I signaling, and inhibiting atrophy-associated skeletal muscle mRNA expression. Importantly, ursolic acid’s effects on muscle were accompanied by reductions in adiposity, fasting blood glucose and plasma cholesterol and triglycerides. These findings identify a potential therapy for muscle atrophy and perhaps other metabolic diseases.

Background:In skeletal muscle, diverse stresses induce the transcription factor ATF4, which promotes muscle atrophy by an unknown mechanism. Results: ATF4 causes muscle atrophy by inducing Gadd45a, which reprograms myonuclear gene expression to repress antiatrophy mechanisms and induce pro-atrophy mechanisms. Conclusion: Gadd45a is a critical stress-induced mediator of muscle atrophy. Significance: The ATF4/Gadd45a pathway is a potential therapeutic target in atrophic muscle.

Prolonged fasting alters skeletal muscle gene expression in a manner that promotes myofiber atrophy, but the underlying mechanisms are not fully understood. Here, we examined the potential role of activating transcription factor 4 (ATF4), a transcription factor with an evolutionarily ancient role in the cellular response to starvation. In mouse skeletal muscle, fasting increases the level of ATF4 mRNA. To determine whether increased ATF4 expression was required for myofiber atrophy, we reduced ATF4 expression with an inhibitory RNA targeting ATF4 and found that it reduced myofiber atrophy during fasting. Likewise, reducing the fasting level of ATF4 mRNA with a phosphorylation-resistant form of eukaryotic initiation factor 2alpha decreased myofiber atrophy. To determine whether ATF4 was sufficient to reduce myofiber size, we overexpressed ATF4 and found that it reduced myofiber size in the absence of fasting. In contrast, a transcriptionally inactive ATF4 construct did not reduce myofiber size, suggesting a requirement for ATF4-mediated transcriptional regulation. To begin to determine the mechanism of ATF4-mediated myofiber atrophy, we compared the effects of fasting and ATF4 overexpression on global skeletal muscle mRNA expression. Interestingly, expression of ATF4 increased a small subset of five fasting-responsive mRNAs, including four of the 15 mRNAs most highly induced by fasting. These five mRNAs encode proteins previously implicated in growth suppression (p21(Cip1/Waf1), GADD45alpha, and PW1/Peg3) or titin-based stress signaling [muscle LIM protein (MLP) and cardiac ankyrin repeat protein (CARP)]. Taken together, these data identify ATF4 as a novel mediator of skeletal myofiber atrophy during starvation.

Skeletal muscle Akt activity stimulates muscle growth and imparts resistance to obesity, glucose intolerance and fatty liver disease. We recently found that ursolic acid increases skeletal muscle Akt activity and stimulates muscle growth in non-obese mice. Here, we tested the hypothesis that ursolic acid might increase skeletal muscle Akt activity in a mouse model of diet-induced obesity. We studied mice that consumed a high fat diet lacking or containing ursolic acid. In skeletal muscle, ursolic acid increased Akt activity, as well as downstream mRNAs that promote glucose utilization (hexokinase-II), blood vessel recruitment (Vegfa) and autocrine/paracrine IGF-I signaling (Igf1). As a result, ursolic acid increased skeletal muscle mass, fast and slow muscle fiber size, grip strength and exercise capacity. Interestingly, ursolic acid also increased brown fat, a tissue that shares developmental origins with skeletal muscle. Consistent with increased skeletal muscle and brown fat, ursolic acid increased energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis. These data support a model in which ursolic acid reduces obesity, glucose intolerance and fatty liver disease by increasing skeletal muscle and brown fat, and suggest ursolic acid as a potential therapeutic approach for obesity and obesity-related illness.

Fox DK, Ebert SM, Bongers KS, Dyle MC, Bullard SA, Dierdorff JM, Kunkel SD, Adams CM. p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization. Am J Physiol Endocrinol Metab 307: E245-E261, 2014. First published June 3, 2014; doi:10.1152/ajpendo.00010.2014.-Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we demonstrate that 3 days of muscle immobilization induces muscle atrophy and increases expression of p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization-induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote atrophy, and coexpression of p53 and ATF4 induces more atrophy than either transcription factor alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4, and p21 is required for atrophy induced by immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy and discover p21 as a critical downstream effector of the p53 and ATF4 pathways.

Bongers KS, Fox DK, Ebert SM, Kunkel SD, Dyle MC, Bullard SA, Dierdorff JM, Adams CM. Skeletal muscle denervation causes skeletal muscle atrophy through a pathway that involves both Gadd45a and HDAC4. Am J Physiol Endocrinol Metab 305: E907-E915, 2013.

Background: Skeletal muscle atrophy is a common and serious condition that lacks a pharmacologic therapy.Results: We used a systems-based strategy to identify tomatidine, a natural compound from tomato plants, as a novel small molecule inhibitor of muscle atrophy.Conclusion: Tomatidine may have utility as a therapeutic agent or lead compound for muscle atrophy.Significance: These results suggest new therapeutic strategies for muscle atrophy.

Background: Aging reduces skeletal muscle strength and mass.Results: The transcription factor ATF4 is required for age-related muscle weakness and atrophy, and the small molecules ursolic acid and tomatidine reduce ATF4 activity, weakness, and atrophy in aged skeletal muscle.Conclusion: ATF4 is an essential mediator of muscle aging.Significance: These results identify new strategies for reducing weakness and muscle loss during aging.