Abstract
BACKGROUND: Peripheral nerve injury-induced muscle atrophy shares core pathophysiological features with systemic wasting disorders including cachexia and sarcopenia, yet early molecular triggers remain undefined. This study investigates the pathogenic role of receptor-interacting protein kinase 3 (RIPK3) in denervation atrophy. METHODS: Sciatic denervation was induced in rats for initial time-course transcriptomics and in mice for genetic and pharmacological studies. Assessments in wild-type and RIPK3-knockout mice included transcriptomics (RNA-seq, qPCR), muscle morphology (wet weight ratio, cross-sectional area), histological inflammation (H&E, CD68 immunofluorescence), mitochondrial function (complex I/V activity, ultrastructure and biogenesis/fission regulators), STRING analysis to identify downstream effectors, validated key effectors NOX2 and NOX4 (qPCR/Western blotting) and associated redox status (DHE staining), and analysis of myofibrillar protein content and proteolytic markers (Western blotting). Confirmatory studies included RIPK3 overexpression in C2C12 myotubes and its pharmacological inhibition (GSK872) in mice. RESULTS: RIPK3 emerged from transcriptomic analysis as an early upregulated mediator in denervated muscle, with protein levels increasing approximately threefold at 36 h post-injury. Genetic ablation of RIPK3 attenuated muscle atrophy, as shown by improved gastrocnemius wet weight ratio (p = 0.0110). This protective effect was directly evidenced by a 40.7% increase in cross-sectional area (p = 0.04). The morphological preservation was accompanied by markedly suppressed expression of key atrophy markers, including MAFbx, MuRF1 and FoxO3a (all p < 0.01), and preserved MHC levels (p = 0.0278). Mechanistically, RIPK3 knockout reduced inflammation, enhanced oxidative phosphorylation (GSEA FDR < 0.001) and partially restored mitochondrial function, evidenced by significantly increased complex I (p = 0.0438) and complex V (p < 0.001) activity, preserved ultrastructure, upregulated PGC-1α and NRF2 (both p < 0.05) and downregulated mitochondrial fission proteins (p-DRP1, MFF, FIS1; all p < 0.01). STRING analysis predicted NOX4 as a key downstream effector, validated by reduced NOX4 protein (-46.6%, p = 0.0366) and a consequent 52.2% decrease in ROS accumulation (p < 0.001). Consistently, RIPK3 overexpression in C2C12 myotubes elevated NOX4 (p = 0.0046) and atrophy markers, whereas pharmacological inhibition of RIPK3 in mice replicated the protective phenotype, increasing muscle wet weight ratio (p = 0.0277) and suppressing NOX4 (p = 0.0398) and proteolytic markers. CONCLUSIONS: Denervation activates RIPK3 as a master regulator that drives muscle atrophy via NOX4/ROS-induced mitochondrial dysfunction, sustained inflammation and ubiquitin-proteasome activation. Targeting RIPK3 preserves muscle mass and may offer a novel therapeutic strategy for neurogenic muscle atrophy, with possible implications for related wasting disorders.