Dual Roles of CCN5/WISP2 in Cytosol and Secretome for Maintaining Muscle Homeostasis and Preventing Sarcopenia

Background: Sarcopenia is a progressive and systemic skeletal muscle disorder characterized by the decline of muscle mass and function. Despite its early-stage, 'possible sarcopenia' has been emphasized for prompt intervention; there are currently no specific biomarkers for the diagnosis and treatment.

Methods: RNA Sequencing of human skeletal muscle across sarcopenia stages identified CCN5. Adeno-associated virus (AAV) is intramuscularly injected into young C57BL/6J mice to knockdown CCN5 in skeletal muscle. Phenotypic alterations are assessed through behavioural testing, body composition analysis, oil red O staining and transmission electron microscopy. Mechanisms were investigated in C2C12 myotubes using lentiviral Infection, Western blotting, immunofluorescence, cellular electron microscopy and seahorse assays. Finally, aged C57BL/6J mice received intramuscular AAV injections to overexpress CCN5 in skeletal muscle, evaluating its therapeutic efficacy against sarcopenia.

Results: Clinical samples included 56 participants (48.2% female; mean age: 63.21 ± 8.76 years). By comparing gene expression in human skeletal muscle across three stages of sarcopenia, we identified CCN5 as a gene exhibiting decreased protein expression in possible sarcopenia stage (approximately 33% reduction, p = 0.0245), with this decline persisting into sarcopenia stage (p = 0.0093). In young mice, CCN5 knockdown induced a sarcopenia-like phenotype, encompassing skeletal muscle dysfunction and myosteatosis (fat mass: p < 0.01; intramyocellular triglyceride: 66.02 ± 3.798 vs. 104.5 ± 8.542 μg/mg tissue, p < 0.01). CCN5 deficiency also impaired mitochondrial content and function, particularly by reducing lipid droplet-mitochondrial (LD-Mt) interaction (approximately 47% reduction, p < 0.05). In C2C12 cells, CCN5 knockdown disrupted lipid metabolism (particularly reduced lipid oxidation, CPT1A: approximately 56% reduction, p < 0.001), promoted lipid accumulation and compromised mitochondrial content and function. Mechanistically, secreted CCN5 enhanced LD-Mt interaction and stimulated mitochondrial biogenesis by activating nuclear β-catenin translocation to enhance FOXO3A-dependent transcription, while intracellular CCN5 mitigated myosteatosis by inhibiting PPARγ signalling. In aged mice, CCN5 overexpression improved skeletal muscle function, reduced myosteatosis (fat mass: p < 0.001; intramyocellular triglyceride: 175.0 ± 11.18 vs. 92.18 ± 10.53 μg/mg tissue, p < 0.001) and restored mitochondrial function.

Conclusions: CCN5 mitigates myosteatosis and counteracts sarcopenia by promoting mitochondrial biogenesis and enhancing LD-Mt interactions through dual pathways, positioning it as a promising therapeutic target for muscle aging and sarcopenia.