The Effect of Leucine and Doxorubicin Treatment on Skeletal Muscle Mitochondrial Function

• Published in: The FASEB journal Vol. 36 Suppl 1
• Main Authors: Carlen, Austin E, Marchant, Erik D, Evans, Richard W, Hancock, Chad R
• Format: Journal Article
• Language: English
• Published: United States 01.05.2022
• ISSN: 1530-6860
• DOI: 10.1096/fasebj.2022.36.S1.L8117

Doxorubicin (DOX) is a common, effective chemotherapy drug. However, DOX treatment causes the negative side-effects of skeletal muscle atrophy and weakness. One of the key mechanisms by which DOX can lead to increased atrophy is through increased mitochondrial damage and inhibition of protein synthesis via down-regulation of mTOR. Leucine (LEU), an essential branched-chain amino acid, activates mTOR, increases mitochondrial biogenesis, and muscle protein synthesis. It is not clear if enhanced activation of mTOR by LEU might moderate the damaging effects of DOX in skeletal muscle. The purpose of this study is to determine if LEU supplementation attenuates the loss of mitochondrial function caused by DOX. C2C12 myoblast cells were grown to confluence and then treated with 10 mM LEU, 0.5 μM DOX, or both for 24 hours. High-resolution respirometry was performed on cells using the Oroboros O2K respirometer to assess mitochondrial function in leak, coupled, and uncoupled states. Total cells used for respiration were assessed by aliquoting a portion of cells before respirometry and performing a BCA protein assay on this representative aliquot. Citrate synthase activity was also evaluated on cells used for respirometry as a marker of mitochondrial content. We report that DOX caused reduced leak, maximum coupled respiration, and uncoupled respiration by 33%, 42%, and 38%, respectively (p<0.05). DOX also lowered citrate synthase activity by 44% (p<0.05). Total protein, measured for total cell homogenates was reduced by 63% with DOX treatment (p<0.05). Contrary to our hypothesis that LEU may mitigate the increase in cellular degradation seen with DOX treatment, there was no significant difference between DOX treatment and DOX + LEU treatment. These findings suggest DOX interferes with LEU's ability to upregulate mTOR activity. Interestingly, the total protein content was reduced significantly more by DOX (63%) than either reductions seen in mitochondrial respiration (~40%) and citrate synthase activity (44%). These findings suggest that the effects of DOX on general cellular protein content is far greater than the negative effects observed on mitochondrial content or function.