1 2 3 4 5 6 7 p 53 and ATF 4 Mediate Distinct and Additive Pathways 8 to Skeletal Muscle Atrophy During Limb Immobilization 9 10

24 25 Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well 26 understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two 27 transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we 28 demonstrate that three days of muscle immobilization induces muscle atrophy and increases expression of 29 p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization30 induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the 31 absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote 32 atrophy, and co-expression of p53 and ATF4 induces more atrophy than either transcription factor 33 alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced 34 atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the 35 p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using 36 genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is 37 highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in 38 mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4; and 39 p21 is required for atrophy induced by immobilization, p53 and ATF4. Collectively, these results identify 40 p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy, and 41 discover p21 as a critical downstream effector of the p53 and ATF4 pathways. 42