The Effect of Creatine and Creatinine on Myocellular Injury in Doxorubicin-Treated Skeletal Muscle Myoblasts

Abstract

Doxorubicin (DOX) is a powerful chemotherapy agent that is associated with a number of deleterious side effects including skeletal muscle dysfunction and atrophy. Although the exact mechanisms behind the observed myotoxicity are not fully understood, DOX treatment has been shown to result in the generation of reactive oxygen species and changes in short-term energy metabolism. Conversely, creatine (Cr) supplementation has been shown to have a therapeutic role in several disease states characterized by muscle atrophy, which is a hallmark of DOX treatment. PURPOSE: To examine the ability of Cr and CrN to attenuate the decline in Cr metabolism and minimize DOX-induced apoptosis and necrosis in skeletal muscle myoblasts. METHODS: Rat skeletal muscle myoblasts were cultured until they reached 85-90% confluency using rat skeletal muscle growth media (GM). Cells were subcultured and treated with one of the following for 12 hours: normal GM (control); Cr (10mM Cr+GM); CrN (10mM CrN+GM); DOX (25μM DOX+GM); DOX+Cr+GM; and DOX+CrN+GM. After incubation, protein analysis was performed using western blotting and rates of apoptosis and necrosis were assessed using an Annexin V apoptosis detection kit and high contrast staining. A one-way ANOVA with Tukey’s post-hoc testing was used to detect significance. RESULTS: There was a significant change relative to GAPDH in creatine kinase (CK) expression between the control and DOX-treated cells (15±18.2% vs. 90±8.7%, p=0.03). In addition,13.2±7.5% of DOX-treated cells were undergoing apoptosis, which was significantly higher than the 3.26±5.5% in the control cells (p=0.04). No significant differences in rates of apoptosis were found between control samples and cells treated with DOX+Cr or DOX+CrN. CONCLUSION: These findings suggest that CK expression is significantly altered in skeletal muscle myoblasts treated with DOX relative to control cells and that DOX treatment results in higher rates of necrosis and apoptosis. Treatment with Cr or CrN minimized the DOX-induced change in CK expression, as well as the rate of apoptosis and necrosis. These findings suggest that Cr and CrN may attenuate the degree of skeletal muscle dysfunction and atrophy during chemotherapy with DOX.