Lipid hydroperoxides promote sarcopenia through carbonyl stress

**Background:** Reactive oxygen species (ROS) accumulation is a hallmark of skeletal muscle atrophy, but the specific downstream mediators responsible for muscle mass and strength loss are unclear. Lipid hydroperoxides (LOOH) are a class of ROS generated from polyunsaturated fatty acid (PUFA) peroxidation, and they form secondary reactive lipid aldehydes such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), which induce carbonyl stress. Glutathione peroxidase 4 (GPx4) is the primary enzyme that reduces LOOH to non-toxic metabolites. This study investigated whether LOOH accumulation drives sarcopenia (age- and disuse-related muscle atrophy and weakness).

**Methods:** The authors examined LOOH levels in skeletal muscle from aged humans and mice, and in mice subjected to hindlimb unloading (HU) to model disuse atrophy. They used C2C12 myotubes with genetic (GPx4 knockdown [KD], LPCAT3 KD, ATG3 KD) and pharmacological (erastin, RSL3, ferrostatin-1, L-carnosine, N-acetylcarnosine, bafilomycin A1, MG132) interventions. In vivo, they studied young (4 months) and old (20 months) mice with global GPx4 haploinsufficiency (Gpx4+/-), muscle-specific tamoxifen-inducible GPx4 knockout (GPx4-MKO), muscle-specific ATG3 knockout (ATG3-MKO), and global GPx4 overexpression (GPx4Tg), with or without HU. They also conducted preclinical trials of L-carnosine and N-acetylcarnosine in drinking water (80 mM). Outcomes included muscle mass, fiber cross-sectional area (CSA), force-generating capacity, and LOOH markers (oxidized phospholipids via mass spectrometry, 4-HNE and MDA via immunoblotting and assays).

**Key Results:** Aging reduced GPx4 expression in human and mouse skeletal muscle. Comprehensive oxidolipidomics in mouse gastrocnemius detected over 300 oxidized lipid species, with oxidized phosphatidylethanolamine (PE) species showing the most robust age-related increases (six of the top ten most increased species were oxidized PE). 4-HNE and MDA were elevated with age. HU robustly elevated muscle LOOH levels before atrophy onset. In C2C12 myotubes, GPx4 KD increased LOOH and ferroptosis markers (Chac1, Ptgs2) and reduced myotube diameter. LPCAT3 KD (which prevents PUFA incorporation into phospholipids) completely rescued the reduction in myotube diameter induced by GPx4 KD. Erastin-induced myotube atrophy was prevented by the lysosomal inhibitor bafilomycin A1 (BafA1) but not by the proteasomal inhibitor MG132. ATG3 KD completely rescued GPx4 KD-induced myotube atrophy. In vivo, Gpx4+/- mice showed augmented HU-induced soleus atrophy in young but not old mice. GPx4-MKO mice were more prone to disuse-induced atrophy with reduced CSA across all fiber types. ATG3-MKO mice were protected from HU-induced atrophy and weakness. Unexpectedly, inhibition of the autophagy-lysosome axis (ATG3 deletion or BafA1) also prevented LOOH accumulation induced by GPx4 deletion, erastin, or RSL3, and 4-HNE was highly co-localized with LAMP2. GPx4Tg mice were resistant to HU-induced atrophy and weakness in both young and old mice, with complete suppression of HU-induced 4-HNE. Ferrostatin-1 suppressed LOOH and protected from myotube atrophy. L-carnosine and N-acetylcarnosine suppressed 4-HNE and cell death in vitro. In vivo, N-acetylcarnosine treatment (80 mM in drinking water) suppressed muscle 4-HNE and ameliorated HU-induced atrophy in soleus and weakness in both soleus and EDL in young and old mice, with protection of myofiber CSA regardless of fiber type.

**Clinical Implications:** This study identifies LOOH as a critical downstream mediator of oxidative stress-induced muscle atrophy and weakness, revealing a novel mechanism linking lipid peroxidation to sarcopenia. The finding that N-acetylcarnosine, a compound with a longer half-life than L-carnosine, effectively prevents disuse-induced muscle dysfunction in both young and old mice has strong translational potential, as N-acetylcarnosine may be more suitable for human use. The data also suggest that LOOH contributes to muscle weakness independently of atrophy, possibly through carbonyl stress on contractile or excitation-contraction coupling proteins. The authors note that while GPx4 overexpression rescued age-associated decreases in muscle strength, short-term N-acetylcarnosine treatment did not, warranting long-term studies. The study supports the potential for clinical trials testing N-acetylcarnosine to prevent muscle atrophy in humans, particularly in conditions of disuse (e.g., bed rest, surgery recovery) and aging.