We have shown that administration of four cycles of doxorubicin and dexamethasone induces a progressive inhibition of growth that becomes irreversible after the fourth cycle of treatment in non-tumor-bearing mice. Three months after the last treatment was given, treated animals demonstrated the typical spinal kyphosis seen in muscle dystrophies and premature aging models28,29 and had lower muscle mass and a global reduction in muscle fiber cross-sectional area (Fig 1). In light of prior data showing that anthracycline toxicity in cardiac muscle is closely linked to mitochondrial dysfunction, we studied the effects of the dexamethasone and doxorubicin treatment on mitochondrial function in skeletal muscle. Chemotherapy treatment led to a severe impairment in mitochondrial energetics, as shown by the marked reduction in muscle respiratory capacity and increased mitochondrial ROS production per unit oxygen consumption without any change in mitochondrial sensitivity to permeability transition (Fig 2). The low muscle respiratory capacity observed is not the result of a reduction in mitochondrial content, as there was no reduction in mRNA expression of key transcription factors for mitochondrial biogenesis nor protein components of mitochondrial outer and inner membranes (VDAC and OXPHOS, respectively; Fig 3). Furthermore our studies did not reveal any differences in mtDNA copy number nor evidence of mtDNA deletions in chemotherapy-treated mice. This is in contrast to prior studies in cardiac muscle tissue, where anthracycline toxicity leads to long-term mitochondrial dysfunction by a mechanism that involves increases in mtDNA deletions and reduced mitochondrial volume.

In fact our observations of a marked reduction in skeletal muscle mitochondrial function in the absence of corresponding changes in quantity of key mitochondrial components are consistent with the small amount of published data on skeletal muscle from prior studies of long-term effects of anthracyclines. Yamada et al described an anthracycline-induced reduction in Complex I activity in rat diaphragm muscle15 while Lebrecht et al showed no change in gastrocnemius mtDNA-encoded COX1 or nuclear-encoded COX4 protein levels11. Our data are also consistent with prior data showing that, in contrast to cardiac muscle, anthracycline-containing chemotherapy does not cause a reduction in mtDNA content or increased levels of mtDNA deletions in skeletal muscle11.

It is impossible to use experimental rodent models to mimic current therapy of childhood ALL with precision, due to both changes in clinical treatment regimes over time and important species-specific differences in patterns of normal growth, development and metabolism. One other study in mice focused on evolution of chemo-resistance in ALL, used a more complex regime containing doxorubicin, dexamethasone, asparaginase and vincristine to mimic ALL treatment30. However, in the current study we were interested in longer term effects of chemotherapy treatment on host muscle tissue and chose to use a simplified regime combining doxorubicin with a lower dose of dexamethasone per cycle. Prolonged continuous use of higher doses of corticosteroids is a well-known cause of proximal myopathy, but the long-term impact of corticosteroids on mitochondrial function after treatment has not been well studied. In one report muscle biopsies from patients who had been taking an oral corticosteroid for months or years for a variety of chronic medical conditions19, revealed that very long-term steroid treatment was associated with an isolated reduction in muscle mitochondrial complex I activity and increased oxidative nuclear and mtDNA damage in patients, compared with steroid-naïve diseased and healthy controls19. However, our experiments used four cycles of 5-day treatments with dexamethasone at three-week intervals (i.e. a total of 20 days of treatment spread over three months), rather than continuous daily use. It is noteworthy that there is no prior data on the expected impact that this limited, intermittent steroid treatment might have on skeletal muscle mitochondria many weeks or months later. Further studies are needed to determine whether the observed long-term effects on skeletal muscle mitochondria are due to either doxorubicin or dexamethasone alone or an additive or even synergistic toxic effect of the combination treatment. There is good reason to believe that both may contribute to the observed deficits in skeletal muscle but the nature of any interaction is not yet clear and this information will obviously be important for future potential preventative measures.

One of the strengths of this study is that prior studies of the effects of chemotherapy on mitochondrial function in muscle have frequently used only a few selected surrogate indices. In contrast, we performed comprehensive mitochondrial functional testing and employed a technique using saponin-permeabilized muscle fibers that avoids some of the bias inherent in using mechanically isolated mitochondria31. Our results provide the most complete functional assessments of the effects of chemotherapy treatment on muscle mitochondria to date. Moreover, our results include a detailed analysis of potential causes of mitochondrial dysfunction. Rather than being spared of any long-term toxic effects of anthracycline-containing chemotherapy, our data show that the mitochondria from skeletal muscle of mice treated with four cycles of doxorubicin and dexamethasone have substantial and persistent functional impairment, even 3 months after treatment was completed. The magnitude of mitochondrial respiratory impairment observed here (a 36% reduction in intrinsic mitochondrial respiratory capacity), equals or exceeds the most severe respiratory impairments reported in the literature for any disease or condition using the saponin-permeabilized myofiber preparation32,33,34, underscoring the persistent negative impact of the chemotherapy treatment.

Undoubtedly the cumulative long-term effects of doxorubicin and dexamethasone treatment may include effects on other organs or tissues. It is possible that the observed impairment of muscle mitochondrial function is related to indirect mechanisms rather than direct muscle-specific drug toxicity. However, the profile of muscle mitochondrial dysfunction in treated mice is distinct and does not correspond to patterns described for other putative indirect mechanisms such as premature aging-related changes, reduced food intake or reduced contractile activity levels. Thus, in aging muscle there is an increased sensitivity to mPTP opening, modest uncoupling and increased mitochondrial-derived proapoptotic signaling21,34, with fibre-type dependent reduction in mitochondrial content34. Similarly, long-term calorie restriction leads unchanged or elevated mitochondrial respiratory capacity and reduced ROS emission35. Finally, muscle disuse induces wasting with reduced muscle mitochondrial biogenesis and content36,37,38, increased absolute ROS production and oxidative damage of cytosolic proteins39.

Given the strong evidence for the involvement of oxidative damage in anthracycline-induced cardiac muscle dysfunction and the increase in ROS production per unit of respiration in muscle from treated mice of the current study (Fig 2), we looked for evidence of accumulation of oxidative damage. However, levels of the lipid peroxidation product 4-HNE were unchanged in whole muscle homogenates (Fig 4), demonstrating that despite causing persistent long-term mitochondrial dysfunction, chemotherapy treatment did not induce a parallel, sustained increase in oxidative damage at the whole tissue level. However, we recognize that the lack of evidence for increased oxidative damage at the whole muscle level after chemotherapy treatment, does not rule out accumulation of oxidative damage targeting specific mitochondrial proteins that are more sensitive to oxidative stress e.g. aconitase40 and mitochondrial adenine nucleotide translocase41.

In healthy tissues, overall mitochondrial functional integrity is maintained by a quality control process involving regular degradation (mitophagy) and replacement35. Impaired mitophagy alone can drive deterioration in mitochondrial function by disrupting normal quality control mechanisms42 and consistent with this idea, we observed a marked reduction in levels of Parkin (Fig 4), a protein which binds to VDAC on the outer mitochondrial membrane and triggers mitophagy26. This finding points to an acquired impairment of mitochondrial turnover and we propose that the persistent defect mitochondrial respiration observed in the late group after chemotherapy is primarily due to a reduction in the rate of removal of mitochondria through mitophagy. Indeed, impaired mitophagic signaling may be a common mechanism contributing to long-term persistence of diverse patterns of mitochondrial dysfunction in muscle as a similar reduction in mitophagic potential (i.e. the Parkin-to-VDAC ratio) has also been reported in ageing muscle21.

To conclude, repeated administration of a combination of dexamethasone and doxorubicin leads to a profound impairment of skeletal muscle mitochondrial respiratory capacity and an increase in ROS production per unit respiration that persists three months after treatment has been completed. Further studies are needed to elucidate the molecular mechanisms underpinning this novel form of mitochondrial dysfunction more fully. In addition, it will be important to verify that similar changes are observed in adult survivors of ALL and even in other cancer survivors who received similar anti-cancer treatments. Confirming the presence of persistent muscle mitochondrial dysfunction will provide the mechanistic groundwork to guide intervention studies aimed at preserving muscle function and improving the long-term health status of these cancer survivors.