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Creatine monohydrate is prolifically used as an ergogenic aid but is less commonly used for its antioxidant properties. The use of creatine, as an antioxidant, has been studied in multiple disease domains as an intervention to improve skeletal muscle quality for Duchenne muscular dystrophy by impacting calcium ion sensitivity in myocytes, Huntington’s disease by facilitating regulation of succinate dehydrogenase in the citric acid cycle and amyotrophic lateral sclerosis by positively impacting muscular atrophy. Reactive oxygen species (ROS) impact both physiological and pathophysiological roles in aerobic life such that an imbalance to the redox environment of the cell can cause intracellular eustress or distress. Doxorubicin (DOX), a highly effective anti-tumor antibiotic chemotherapeutic, is incorporated into more than half of all breast cancer and two thirds of pediatric cancer treatment plans but is highly cardiotoxic and negatively impacts cancer survivorship. Although the mechanistic pathophysiology of anthracycline-induced cardiotoxicity is not completely understood, there is evidence of doxorubicin-induced reactive oxygen species distress in cardiomyocytes leading to either acute and or chronic left ventricular dysfunction. Creatine monohydrate’s potential to act as a direct antioxidant underpins the purpose of this study to investigate global doxorubicin-induced hydrogen peroxide-mediated cysteine sulfenylation (S-sulfenylation) of left ventricle muscle tissue of Sprague-Dawley rats with dietary creatine monohydrate supplementation and doxorubicin treatment using a dimedone-based chemoselective labeling technique.

Ten-week-old male Sprague-Dawley rats were randomly assigned to one of two, four-week creatine feeding groups or a control, four-week standard rat chow group. Creatine group one (Cr1) were fed rodent chow with two percent creatine monohydrate ad libitum for a duration of four weeks and creatine feeding group two (Cr2) and were fed rodent chow supplemented with four percent creatine monohydrate ad libitum for one week, followed by three weeks of two percent creatine monohydrate-supplemented chow. At the completion of the four-week nutritional intervention; Cr1 and Cr2 group animals received 15 mg/kg DOX injections (Cr1+Dox, Cr2+Dox) and control group animals were randomly assigned into a 15 mg/kg DOX injection group (Con+Dox) or a sterile saline injection group (Con+Sal) and continued their respective dietary feeding protocols. Animals were sacrificed at five-days post-injection, the heart was excised, and tissues were analyzed by Western Blotting to quantify global cysteine sulfenylation. One-way ANOVA results showed that there were no significant differences in global S-sulfenylation between groups (F= 1.146, p>0.05). There was a significant difference between a prominent single protein band and Tukey’s post hoc analysis revealed that the Control+Sal group had significantly higher S-sulfenylation compared to the Control+DOX group (F=3.259, p<0.05). A marked increase in S-sulfenylation was also observed among the single dark protein band such that the Control+DOX group had the lowest S-sulfenylation with an increase observed in Cr1+DOX and higher still in Cr2+DOX group. The main finding of this study is that there was not a statistically significant difference from a maladaptive to adaptive hydrogen peroxide-mediated reactive oxygen species stress response as measured in the form of global cysteine sulfenylation; however, there was a protein band identified that exhibited significantly higher S-sulfenylation in Control+Sal compared to Control+DOX. In comparison to Control+DOX, there was a minor and marked increase in S-sulfenylation in both Cr1+DOX and Cr2+DOX (respectively). These results indicate that the post-translational sulfenome of rat cardiac tissues supplemented with dietary creatine monohydrate and/or treated with DOX did not exhibit significant differences in global cysteine sulfenylation.