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

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 (20mg/kg). Initially at 2h doxorubicin inhibits both complex I- and II-supported respiration and increases H2O2 emission, both of which are partially restored after 24h. The relationship between oxygen consumption and membrane potential (ΔΨ) is shifted to the right at 24h, 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.

Keywords: Chemotherapy; ETS; Metabolism; Mitochondria; PmFBs; ROS; Reactive oxygen species; Skeletal muscle; TPP; electron transport system; mPTP; mitochondrial permeability transition pore; permeabilized fiber bundles; reactive oxygen species; tetraphenylphosponium.

Figure 1. Rats exposed to doxorubicin exhibit decreased whole-body energy expenditure

Indirect metabolic calorimetry and ambulatory activity in rats following doxorubicin administration. (A) Oxygen consumption; (B) carbon dioxide production; (C) ambulatory activity; and (D) calculated energy expenditure. Data are mean ± SEM; p<0.05 for overall differences by repeated-measures ANOVA; *p<0.05 vs. CTRL in dark phase

Figure 2. Doxorubicin inhibits NADH- and FADH₂-supported respiration in skeletal muscle

Permeabilized myofibers were prepared 2-72 h following doxorubicin administration, and mitochondrial respiratory kinetics were measured. State 3 respiration was measured during (A) glutamate/malate or (C) succinate titration in the presence of maximal ADP (4 mM), and during (B) ADP titration in the presence of maximal glutamate/malate (15/2 mM). Data are mean ± SEM; p<0.05 vs. CTRL for 2 h (#) and 72 h (*) for all substrate concentrations unless otherwise denoted by a horizontal line that signifies differences for concentrations 3-16 mM.

Figure 3. Doxorubicin effects mitochondrial H₂O₂ emission

Mitochondrial H₂O₂ emitting potential of permeabilized myofibers from control (open) and doxorubicin (closed) treated rats (A) 2-72 h and (B) 2 h (+ 1 mM DTT) following injection. Substrate conditions were in the presence of maximal succinate. (C) Maximal state 3 respiration with glutamate/malate of fibers from 2 h experimental group exposed to DTT for 15 min in wash. Dotted line represents previous data from 2 h doxorubicin-treated group (Fig 2A). Data are mean ± SEM; *p<0.05 vs. CTRL.

Figure 4. Doxorubicin impairs mitochondrial calcium retention in skeletal muscle

(A) Representative fluorescence trace of calcium uptake in control (black) and doxorubicin (red) myofibers from 72 h experimental group. (B) Total mitochondrial calcium uptake of permeabilized fibers from control (open) and doxorubicin (closed) treated rats 2-72 h following administration. Substrate conditions were in the presence of maximal malate, glycerol-3-phosphate, glutamate, succinate, pyruvate, and 25 μM ADP. Data are mean ± SEM; *p<0.05 vs. CTRL.

Figure 5. Fluctuations of mitochondrial membrane potential (Δψ) in skeletal muscle following doxorubicin administration

Respiration (JO₂) and membrane potential (Δψ_m) monitored simultaneously during ADP-supported respiration in the presence of maximal glutamate, pyruvate, malate, glycerol-3-phosphate, and succinate. Data are mean ± SEM.

Figure 6. Doxorubicin does not alter mitochondrial content in skeletal muscle

Mitochondrial content evaluated in permeabilized myofibers isolated 72 h following doxorubicin exposure by (A) maximal FCCP-response in the presence of State 3 respiration supported with maximal glutamate, pyruvate, malate, glycerol-3-phosphate, and succinate and by (B) western blot of mitochondrial complexes. Data are mean ± SEM