Effects of short-term endurance exercise training on acute doxorubicin-induced FoxO transcription in cardiac and skeletal muscle

Abstract

Doxorubicin (DOX) is a potent antitumor agent used in cancer treatment. Unfortunately, DOX can induce myopathy in both cardiac and skeletal muscle, which limits its clinical use. Importantly, exercise training has been shown to protect against DOX-mediated cardiac and skeletal muscle myopathy. However, the mechanisms responsible for this exercise-induced muscle protection remain elusive. These experiments tested the hypothesis that short-term exercise training protects against acute DOX-induced muscle toxicity, in part, due to decreased forkhead-box O (FoxO) transcription of atrophy genes. Rats (n = 6 per group) were assigned to sedentary or endurance exercise-trained groups and paired with either placebo or DOX treatment. Gene expression and protein abundance were measured in both cardiac and skeletal muscles to determine the impact of DOX and exercise on FoxO gene targets. Our data demonstrate that DOX administration amplified FoxO1 and FoxO3 mRNA expression and increased transcription of FoxO target genes [i.e., atrogin-1/muscle atrophy F-box (MaFbx), muscle ring finger-1 (MuRF-1), and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)] in heart and soleus muscles. Importantly, exercise training protected against DOX-induced increases of FoxO1 and MuRF-1 in cardiac muscle and also prevented the rise of FoxO3, MuRF-1, and BNIP3 in soleus muscle. Furthermore, our results indicate that exercise increased peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) in both the heart and soleus muscles. This is important because increased PGC-1α expression is known to suppress FoxO activity resulting in reduced expression of FoxO target genes. Together, these results are consistent with the hypothesis that exercise training protects against DOX-induced myopathy in both heart (FoxO1 and MuRF-1) and skeletal muscles (FoxO3, MuRF-1, and BNIP3).

Keywords: doxorubicin; exercise; reactive oxygen species; skeletal muscle.

Fig. 1.

FoxO1 and FoxO3 mRNA levels in heart and soleus. A significant interaction effect was present for heart FoxO1 mRNA (A) where *SED DOX was higher (P < 0.05) compared with EXE DOX. Significant main effects for drug were observed for heart FoxO3 mRNA (B) and soleus FoxO1 mRNA (C) where #DOX increased (P < 0.05) these levels regardless of activity level. Significant main effects for drug were observed for soleus FoxO3 mRNA (D) where #DOX increased (P < 0.05) these levels only in the SED animals. Data are mean ± SD and fold difference is expressed relative to SED PLA. DOX, doxorubicin; EXE, short-term exercise training; FoxO, forkhead-box O; PLA, placebo; SED, sedentary.

Fig. 2.

Atrogin-1/MaFbx, MuRF-1, and BNIP3 mRNA levels in heart and soleus. Significant main effects for drug were observed for heart atrogin-1/MaFbx mRNA (A) where #DOX increased (P < 0.05) mRNA expression regardless of activity level. A significant interaction effect was present for heart MuRF-1 mRNA (B) and soleus MuRF-1 mRNA (E) where &SED DOX was higher (P < 0.05) compared with both SED PLA and EXE DOX. A significant interaction effect was present for heart BNIP3 mRNA (C) and soleus atrogin-1/MaFbx mRNA (D) where @DOX increased (P < 0.05) the expression of these genes regardless of activity level. Significant main effects for drug and activity were observed for soleus BNIP3 mRNA (F) where SED DOX was higher (P < 0.05) than #SED PLA and ΨEXE DOX. Data are mean ± SD and fold difference is expressed relative to SED PLA. MaFbx, muscle atrophy F-box (also called atrogin-1); MuRF-1, muscle ring finger-1; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3.

Fig. 3.

Ratio of pAMPK/AMPK protein, PGC-1α mRNA, and PGC-1α protein levels in heart and soleus. Significant main effects for activity were observed for heart pAMPK/AMPK (A) and soleus PGC-1α mRNA (E) where ψEXE PLA was higher (P < 0.05) than SED PLA. Significant main effects for activity were observed for heart PGC-1α mRNA (B), cardiac PGC-1α protein levels (C), and soleus PGC-1α protein (F) where ΨEXE increased (P < 0.05) these levels regardless of drug. Significant main effects were observed for soleus pAMPK/AMPK (D) where SED DOX was lower (P < 0.05) than #SED PLA and ΨEXE DOX. Representative images for protein levels are shown below the histograms. Data are mean ± SD. Fold difference is expressed relative to SED PLA. Protein expression is expressed relative to SED PLA (percent control). AMPK, AMP-activated protein kinase; pAMPK, phosphorylated AMP-activated protein kinase; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha.

Fig. 4.

Mitofusin-2 (Mfn2) protein levels in heart and soleus. Significant main effects for activity were observed for heart (A) and soleus (B) Mfn2 where ΨEXE increased (P < 0.05) these levels regardless of drug. Representative images for protein levels are shown below the histograms. Data are mean ± SD. Protein expression is expressed relative to SED PLA (percent control).

Fig. 5.

Myostatin mRNA levels in heart and soleus. Significant main effects for drug were observed for heart myostatin mRNA (A) where #DOX increased (P < 0.05) these levels only in the SED animals. Significant main effects for drug and activity were observed for soleus myostatin mRNA (B) where SED DOX was higher (P < 0.05) than #SED PLA and ΨEXE DOX. Data are mean ± SD and fold difference is expressed relative to SED PLA.

Fig. 6.

Potential mechanisms of exercise-induced protection from DOX-induced myopathy. DOX can increase expression of FoxO1, or FoxO3, or both leading to increased transcription of target proteolytic genes (e.g., atrogin-1/MaFbx, MuRF-1, BNIP3) and myostatin resulting in myopathy. Exercise training upregulates PGC-1α, which can inhibit FoxO1/FoxO3 DNA binding and can lead to increased Mfn2 and improved mitochondria function that can protect from DOX-induced myopathy. In our experiments, exercise training prior to DOX treatment protected against DOX-induced increases of FoxO1, MuRF-1, and myostatin in cardiac muscle and FoxO3, MuRF-1, BNIP3, and myostatin in soleus muscle, but did not protect against atrogin-1/MaFbx in either heart or soleus muscles. See text for additional details.