The Sirtuin-1 Agonist SRT2104 Mitigates Redox Signaling and nNOS Translocation in Unloaded Skeletal Muscle

Background: Skeletal muscles adapt their mass (e.g., hypertrophy, atrophy) due to changes in loading, or mechanotransduction, as well as in response to nutrient and energy sensing. There are multiple load sensing pathways that could trigger unloading-induced muscle atrophy. Over the past 15 years, multiple laboratories have reported that post-translational translocation of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the cytosol flips the role of nNOS from anabolic to catabolic. Loss of nNOS during mechanical unloading appears to be regulated by oxidative stress (e.g., NADPH oxidase-2, mitochondria), stress response proteins, and proteoglycans. In addition, Receptor Activator of Nuclear factor Kappa beta Ligand (RANKL), historically linked to osteoporosis in bone, could contribute to myopathies including unloading-induced atrophy. The NAD-dependent deacetylase sirtuin-1 (SIRT1) has numerous cell protective roles in metabolism, mitochondrial function, and stress protection. SIRT1 has recently been linked with muscle hypertrophy. Objective/Hypothesis: We hypothesized that a SIRT1 agonist (SRT2104) would significantly attenuate unloading-induced upregulation of Nox2 and RANKL, linked to preservation of sarcolemmal nNOS and muscle fibers cross-sectional area. Methods: In this experiment, 5-month-old F344 rats were divided into three groups (n=6 each): ambulatory controls (CON), hindlimb unloaded to 10 days (HU), and hindlimb unloaded + SIRT1 (25 mg/kg/day: HUS). Immunoblots, immunofluorescence (IF), and ELISA protein analysis was conducted on muscle samples. Results: We first confirmed that SRT2104 increased SIRT1 protein levels and activity in unloaded gastrocnemius muscle. The Nox2 subunit (p67phox), upstream regulators of Nox2 (e.g., cyclophilin A, acid sphingomyelinase), and RANKL were elevated by 2 - 4 fold by hindlimb unloading; however, unloading-induced elevation of Nox2-RANKL signaling was prevented by SRT2104. Further, loss of sarcolemmal nNOS was also found in HU muscle, while translocation of nNOS was largely prevented by SRT-2104 per both Western blotting and IFs. The sarcolemmal scaffolding protein dystrobrevin suffered loss of membrane localization with HU. In contrast, SRT2104 mitigated disruption of dystrobrevin. Sarcolemmal and blood serum Klotho levels were increased with unloading, which SRT2104 prevented. SRT2104 attenuated upregulation of Klotho and reduction in Akt phosphorylation (Ser308) in unloaded muscle. Conversely, p62 levels decreased with HU and was prevented with HU + SRT-2104. Conclusions: Our findings suggest that targeting SIRT1 could alleviate elevation in redox signaling (Nox2, RANKL), which appear to protect sarcolemmal nNOS. Furthermore, SRT2104 downregulated Klotho signaling and preserved anabolic/catabolic balance. A potential mechanism for SIRT1-induced protection could be through upstream regulators of nNOS and protection of the scaffolding protein dystrobrevin. Supported by NASA (80NSSC19K0432, NNX80NSSC17K0118, NNX13AE45G), NSBRI, Huffines Institute, Fight DMD This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.