Myonuclear mispositioning and ferroptosis-related changes in skeletal muscle during cancer cachexia.

Cancer cachexia is a multifactorial syndrome characterized by severe skeletal muscle wasting. While the major molecular mechanisms driving cancer cachexia have been extensively investigated in animal models, translating these findings into successful human therapies still remains a challenge. Previous studies performed on human patients with gastrointestinal cancers, have shown for the first time that this condition is linked to neuromuscular alterations including neuromuscular junction instability, muscle fiber denervation and myonuclear mispositioning, as well as to an imbalanced mitochondrial network dynamics shifted towards fragmentation. These findings suggest that cachectic muscle is exposed to chronic neuromuscular and mitochondrial stress, which may contribute to structural disorganization of muscle fibers and impaired muscle function. To 1) characterize the nature of mispositioned nuclei observed in cachectic muscles, and to 2) investigate whether ferroptosis-like vulnerability may contribute to the myopathic phenotype, we analysed intraoperative rectus abdominis muscle biopsies from either weight stable precachectic and cachectic oncologic patients and healthy controls, using muscle morphometry, immunofluorescence and western blot techniques. The morphometrical and histological analyses confirmed muscle atrophy in oncologic patients, along with an increased number of fibers displaying mispositioned and centrally located nuclei. Immunofluorescence was used to evaluate the expression of markers specific for mature myonuclei, and myogenic transcription factors, revealing that mispositioned nuclei are non-regenerative and indicative of structural failure of nuclear anchoring, likely linked to denervation and mitochondrial network remodelling. Western blot analyses, performed to evaluate susceptibility to ferroptotic stress, revealed a significant downregulation of glutathione peroxidase 4 (GPX4) in cachectic muscle, indicating a diminished protection against lipid peroxidation and a potential induction of ferroptosis. However, histochemical staining for iron deposition did not show massive iron accumulation within the muscle fibers; instead, it revealed a reticular pattern consistent with the accumulation of labile mitochondrial iron. This reduced GPX4 expression, together with the reticular pattern of iron staining, supports the presence of ferroptosis-related alterations and mitochondrial vulnerability in skeletal muscle cells. These findings suggest a model in which denervation-driven mitochondrial dysfunction disrupts nuclear mispositioning and increases oxidative susceptibility, contributing to progressive muscle atrophy in cancer cachexia and identify potential targets for future therapeutic strategies. Further investigation of oxidative stress pathways, mitochondrial redox imbalance, and iron handling will be essential to identify therapeutic targets capable of preventing or reversing cachexia-associated myopathy.