The Role of Ferroptosis in Mediating Muscle Atrophy under Hyperglycemic Conditions

Objective: This study investigates the potential role of ferroptosis, a recently identified form of cell death mechanism, in diabetic myopathy. We hypothesized that ferroptosis plays a role in modulating the morphology and size of C2C12 myotubes under hyperglycemic conditions, potentially through the regulation of ferroptosis-related genes and the induction of oxidative stress. Methods: C2C12 mouse skeletal muscle myoblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum. To induce myotube differentiation, C2C12 myoblasts were seeded at a density of 50,000 cells per well in 12-well plates. Upon reaching confluence, the medium was replaced with differentiation medium (DMEM supplemented with 2% horse serum). On day 4 of differentiation, myotubes were exposed to either normal (25 mM) or high (100 mM) glucose conditions. RNA was then isolated for cDNA library construction and real-time PCR analysis of ferroptosis-related gene expression. Key biomarkers of ferroptosis, including ACSL4, PTGS2, TFR1, FTL, FTH1, GPX4, and SLC7A11, were analyzed. Intracellular reactive oxygen species (ROS) levels in myotubes were assessed using the H2DCFDA fluorescent probe. Results: Under high glucose conditions, myotubes exhibited altered morphology, being shorter and fatter compared to those under normal glucose conditions. Ferroptosis-related gene expression was significantly upregulated in the high glucose group, with ASCL4 showing the most marked increase. Conversely, PTGS2 and SLC7A11 exhibited a trend toward altered expression, but these changes were not statistically significant. TFR1 expression was significantly higher in the normal glucose group, while FTL levels were significantly lower. FTH1 expression remained similar between the two groups. Additionally, intracellular ROS were elevated in the high glucose condition, suggesting increased oxidative stress. Conclusions: In this study, the morphological differences observed between normal and high glucose conditions suggest that hyperglycemia disrupts myotube integrity. Considering ferroptosis gene expression, this potentially implicates ferroptosis as a contributing factor. Elevated oxidative stress levels under hyperglycemic conditions further link hyperglycemic muscle atrophy to the induction of ferroptosis. The complexity of the pathway necessitates studies with larger sample sizes to better interpret these results. Testing ferroptosis inhibitors in normal versus high glucose conditions will help elucidate the role of ferroptosis in hyperglycemia-induced muscle atrophy during myotube formation. Additionally, further investigation into lipid peroxidation and the antioxidant system is required to clarify their roles in ferroptosis regulation. This research was supported by the NIH STEP UP R25 DK078381 grant. 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.