Increased mitochondrial emission of reactive oxygen species and calpain activation are required for doxorubicin-induced cardiac and skeletal muscle myopathy

Abstract

Although doxorubicin (DOX) is a highly effective anti-tumour agent used to treat a variety of cancers, DOX administration is associated with significant side effects, including myopathy of both cardiac and skeletal muscles. The mechanisms responsible for DOX-mediated myopathy remain a topic of debate. We tested the hypothesis that both increased mitochondrial reactive oxygen species (ROS) emission and activation of the cysteine protease calpain are required for DOX-induced myopathy in rat cardiac and skeletal muscle. Cause and effect was determined by administering a novel mitochondrial-targeted anti-oxidant to prevent DOX-induced increases in mitochondrial ROS emission, whereas a highly-selective pharmacological inhibitor was exploited to inhibit calpain activity. Our findings reveal that mitochondria are a major site of DOX-mediated ROS production in both cardiac and skeletal muscle fibres and the prevention of DOX-induced increases in mitochondrial ROS emission protects against fibre atrophy and contractile dysfunction in both cardiac and skeletal muscles. Furthermore, our results indicate that DOX-induced increases in mitochondrial ROS emission are required to activate calpain in heart and skeletal muscles and, importantly, calpain activation is a major contributor to DOX-induced myopathy. Taken together, these findings show that increased mitochondrial ROS production and calpain activation are significant contributors to the development of DOX-induced myopathy in both cardiac and skeletal muscle fibres.

Figure 1. Hydrogen peroxide release and oxidative damage in heart and skeletal muscles

A–D, rates of hydrogen peroxide release from permeabilized cardiac and skeletal muscle fibres. Values are the mean ± SEM. *DOX significantly different vs. CON, CON + SS31 and DOX + SS31 (P < 0.05). E–H, 4-hydroxynonenal conjugates were analysed as an indicator of lipid peroxidation. Representative western blot images of 4-hydroxynonenal are shown to the right. *DOX significantly different vs. CON and DOX + SS31 (P < 0.05).

Figure 2. Diaphragm force-frequency response in all experimental groups

Values are the mean ± SEM. ^§DOX significantly different vs. CON and CON + SS31 (P < 0.05). *DOX significantly different vs. CON, CON + SS31 and DOX + SS31 (P < 0.05). ^#DOX + SS31 significantly different vs. CON and CON + SS31 (P < 0.05).

Figure 3. Fibre cross-sectional area of diaphragm, plantaris and soleus

A) diaphragm, B) plantaris and C) soleus. Values are the mean ± SEM. *DOX significantly different vs. CON, CON + SS31 and DOX + SS31 (P < 0.05). ^§DOX significantly different vs. CON and CON + SS31 (P < 0.05). ^@DOX significantly different vs. CON (P < 0.05). Representative images are shown at the top. Fibre membranes are highlighted by dystrophin stain (red). Fibre types can be identified as MHC I (blue) and MHC IIa (green), and all remaining fibres were classified as MCH IIb/x (black).

Figure 4. Calpain and caspase-3 activation in heart and skeletal muscles

A–D, calpain activation [autoproteolytic product (75 kDa) of calpain 1] in heart and skeletal muscles. E–H, Caspase-3 activation in heart and skeletal muscles. *DOX significantly different vs. CON and DOX + SS31 (P < 0.05). ^@DOX significantly different vs. CON (P < 0.05). Representative western blot images are shown to the left.

Figure 5. Number of positive TUNEL nuclei in heart and skeletal muscles

A) heart, B) diaphragm, C) plantaris and D) soleus. *DOX significantly different vs. CON and DOX + SS31 (P < 0.05). The arrows indicate TUNEL positive nuclei. Representative images are shown to the right.

Figure 6. Diaphragm force-frequency response

Values are the mean ± SEM. *DOX significantly different vs. CON, CON + SJA and DOX + SJA (P < 0.05). ^#DOX + SJA significantly different vs. CON and CON + SJA (P < 0.05). ^§DOX significantly different vs. CON and CON + SJA (P < 0.05).

Figure 7. Cross-sectional area of diaphragm, plantaris and soleus

A) diaphragm, B) plantaris and C) soleus. Values are the mean ± SEM. *DOX significantly different vs. CON, CON + SJA, and DOX + SJA (P < 0.05). ^@DOX significantly different vs. CON (P < 0.05). Representative images are shown at the top. Fibre membranes are highlighted by dystrophin stain (red). Fibre types can be identified as MHC I (blue) and MHC IIa (green), and all remaining fibres were classified as MCH IIb/x (black).

Figure 8. Calpain and caspase-3 activation in heart and skeletal muscles

A–D, calpain activation [autoproteolytic product (75 kDa) of calpain 1] in heart and skeletal muscles. E–H, Caspase-3 activation in heart and skeletal muscles. *DOX significantly different vs. CON and DOX + SJA (P < 0.05). ^∞DOX significantly different vs. DOX + SJA. Representative Western blot images are shown to the left.

Figure 9. Number of positive TUNEL nuclei in heart and skeletal muscles

A) heart, B) diaphragm, C) plantaris and D) soleus. *DOX significantly different vs. CON and DOX + SJA (P < 0.05). ^$DOX + SJA significantly different than CON (P < 0.05). The arrows indicate TUNEL positive nuclei. Representative images are shown to the right.