Elsevier

Free Radical Biology and Medicine

Volume 65, December 2013, Pages 988-996
Free Radical Biology and Medicine

Original Contribution
The anticancer agent doxorubicin disrupts mitochondrial energy metabolism and redox balance in skeletal muscle

https://doi.org/10.1016/j.freeradbiomed.2013.08.191Get rights and content

Highlights

  • Skeletal muscle mitochondria demonstrate a biphasic response to a single doxorubicin injection.

  • Doxorubicin increases mitochondrial H2O2 emission in skeletal muscle.

  • Elevated reducing pressure in the ETS following doxorubicin administration.

  • Rats exposed to doxorubicin exhibit decreased whole-body energy expenditure.

Abstract

The combined loss of muscle strength and constant fatigue are disabling symptoms for cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and premature fatigue along with an increase in reactive oxygen species (ROS). As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin could negatively affect mitochondria by inhibiting respiratory capacity, leading to an increase in H2O2-emitting potential. Here we demonstrate a biphasic response of skeletal muscle mitochondria to a single doxorubicin injection (20 mg/kg). Initially at 2 h doxorubicin inhibits both complex I- and II-supported respiration and increases H2O2 emission, both of which are partially restored after 24 h. The relationship between oxygen consumption and membrane potential (ΔΨ) is shifted to the right at 24 h, indicating elevated reducing pressure within the electron transport system (ETS). Respiratory capacity is further decreased at a later time point (72 h) along with H2O2-emitting potential and an increased sensitivity to mitochondrial permeability transition pore (mPTP) opening. These novel findings suggest a role for skeletal muscle mitochondria as a potential underlying cause of doxorubicin-induced muscle dysfunction.

Introduction

Doxorubicin is a potent anthracycline antibiotic used to treat numerous human malignancies [1]. A severe side effect of doxorubicin is cardiotoxicity characterized by a dose-dependent decline in cardiac function with prolonged exposure [2]. Clinicians manage this side effect by limiting the dosage patients receive; however, even on a limited dose patients can experience disabling muscle weakness and fatigue [3], [4]. In the clinic, fatigue is generally documented as perceived fatigue, or a sense of tiredness, which is difficult to distinguish from physiological fatigue [5]. Physiological fatigue involves muscle-specific peripheral fatigue, which includes two components: muscle fatigue and muscle weakness. Our previous work suggests that the decline in muscle function observed in patients could be due to an effect specifically on skeletal muscle. Healthy rodents exposed to a clinical dose of doxorubicin exhibit a decrease in both hindlimb and respiratory muscle strength, along with an accelerated rate of fatigue [6], [7], [8]. The loss of strength in combination with constant fatigue can burden patients, not only during therapy, but up to 10 years following the cessation of therapy [9].

Potential mediators of doxorubicin-induced muscle weakness and fatigue are reactive oxygen species (ROS). In cardiac muscle, doxorubicin is known to increase ROS by localizing to the mitochondria [10] where it is reduced by complex I to form a semiquinone radical [11]. In addition, indirect ROS production can occur with doxorubicin through inhibition of the mitochondrial electron transport system (ETS). Previous reports demonstrate that doxorubicin inhibits the ETS in isolated heart mitochondria, specifically complexes I and II [12], [13]. An overproduction of oxidants due to a block in the ETS can lead to redox modifications of cell macromolecules (e.g., proteins, lipids, DNA) with detrimental downstream effects on whole organ function [14], [15].

In skeletal muscle, cytosolic oxidant activity and markers of protein oxidation are elevated following doxorubicin exposure [7], [16]. Mitochondria represent a primary source of oxidant generation in skeletal muscle [17], [18]. Doxorubicin-induced oxidants are blunted in C2C12 myotubes following incubation with Bendavia (Stealth Peptides, Newton, MA; formerly known as SS31) [19], a cell-permeable peptide that localizes to the mitochondria and lessens ROS production. These findings suggest that doxorubicin could be affecting skeletal muscle mitochondrial function, leading to ROS production.

The objective of this study was to determine the exact nature and extent to which mitochondrial function is impacted by doxorubicin treatment, specifically in skeletal muscle. It was hypothesized that doxorubicin would inhibit skeletal muscle mitochondrial respiration, leading to an increase in ROS-emitting potential. To test this hypothesis mitochondrial function was evaluated in permeabilized fiber bundles (PmFBs) 2, 24, and 72 h following a single doxorubicin injection (20 mg/kg). The results indicated a biphasic response. Doxorubicin initially (2 h) induced an increase in H2O2 emission and membrane potential with a decline in respiratory function that was reversed after 24 h. After 72 h, respiratory capacity was again decreased along with H2O2-emitting potential and membrane potential, indicative of a decline in overall mitochondrial function.

Section snippets

Overview of experimental design

Male Sprague-Dawley rats (Charles River Laboratories) 8–10 weeks old (~250 g) received an intraperitoneal injection of doxorubicin (20 mg/kg in phosphate-buffered saline), a clinically applicable dose that is equivalent to what patients with hematological malignancies receive [20], based on the conversion factor established by Freireich et al. [21]. Control animals received the same volume of vehicle (PBS). Following injection, rats were housed in metabolic chambers for 72 h for indirect open

Whole-body effects of doxorubicin

In agreement with our previous work [6], [7] and other reports [29], a single doxorubicin injection caused a significant decline in body weight 48–72 h following exposure (% change from CTRL: 24 h, −5±5; 48 h, -9±5, P<0.01; 72 h, −15±6, P<0.01). Whole-body fat (21.3±0.8 g CTRL; 18.9±0.9 g DOX, P<0.05) and lean mass (250±5 g CTRL; 207±5 g DOX, P<0.01) were reduced 72 h following doxorubicin exposure.

Fig. 1 illustrates whole-body metabolism and ambulatory activity measured by indirect calorimetry. Just 24 

Discussion

Our novel findings demonstrate that the anticancer agent doxorubicin causes skeletal muscle mitochondrial dysfunction in a time course-dependent manner. Initially, doxorubicin induces a demonstrable inhibition in mitochondrial respiration and a marked increase in H2O2 emission, both of which surprisingly are at least partially restored to control levels within 24 h after doxorubicin exposure. However, the relationship between oxygen consumption and ΔΨ is clearly shifted to the right at 24 h,

Acknowledgments

This project was supported by National Institutes of Diabetes and Digestive and Kidney Diseases (R01-DK073488, P.D.N.) and Arthritis and Musculoskeletal and Skin Diseases (F32-AR061946, L.A.G.) of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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