Cyclophosphamide leads to persistent deficits in physical performance and in vivo mitochondria function in a mouse model of chemotherapy late effects

Abstract

Fatigue is the symptom most commonly reported by long-term cancer survivors and is increasingly recognized as related to skeletal muscle dysfunction. Traditional chemotherapeutic agents can cause acute toxicities including cardiac and skeletal myopathies. To investigate the mechanism by which chemotherapy may lead to persistent skeletal muscle dysfunction, mature adult mice were injected with a single cyclophosphamide dose and evaluated for 6 weeks. We found that exposed mice developed a persistent decrease in treadmill running time compared to baseline (25.7±10.6 vs. 49.0±16.8 min, P = 0.0012). Further, 6 weeks after drug exposure, in vivo parameters of mitochondrial function remained below baseline including maximum ATP production (482.1 ± 48.6 vs. 696.2 ± 76.6, P = 0.029) and phosphocreatine to ATP ratio (3.243 ± 0.1 vs. 3.878 ± 0.1, P = 0.004). Immunoblotting of homogenized muscles from treated animals demonstrated a transient increase in HNE adducts 1 week after exposure that resolved by 6 weeks. However, there was no evidence of an oxidative stress response as measured by quantitation of SOD1, SOD2, and catalase protein levels. Examination of mtDNA demonstrated that the mutation frequency remained comparable between control and treated groups. Interestingly, there was evidence of a transient increase in NF-ĸB p65 protein 1 day after drug exposure as compared to saline controls (0.091±0.017 vs. 0.053±0.022, P = 0.033). These data suggest that continued impairment in muscle and mitochondria function in cyclophosphamide-treated animals is not linked to persistent oxidative stress and that alternative mechanisms need to be considered.

Fig 1. Transient weight loss after exposure to a single Cy dose.

Percent body weight change by group (mean and SD) compared to Day 0 (dosing day) for saline and Cy groups, N = 10 per group.

Fig 2. Persistent exercise intolerance in adult mice exposed to a single Cy dose.

(A) Saline control group treadmill running capacity in minutes, with no difference between baseline and 6 week timepoints. (B) Cy group treadmill running capacity in minutes, with significant decline in running time between baseline and 6 week timepoints. (C) Fraction change in running capacity between the 2 groups. Data presented as box plot showing min, median, and max data point. N = 10 for each group.

Fig 3. Lower gastrocnemius muscle weight in Cy exposed mice.

Gastrocnemius weight relative to tibia length was descriptively smaller in the Cy group than the saline group 6 weeks after treatment, but the difference did not reach P<0.050. Data presented as box plot showing min, median, and max data point. N = 8 for each group.

Fig 4. In vivo mitochondria function is impaired after exposure to a single Cy dose.

(A) Maximal mitochondrial ATP production (ATPmax) was reduced in the hindlimb skeletal muscles of Cy treated mice compared 1 day and 6 weeks after treatment. In the 1 day dataset one data point that was outside the typical range of values and that failed the outlier test was excluded from the analysis. Excluding this point makes data significantly depressed at P = 0.036. (B) PCr/ATP ratio was calculated from mean PCr and ATP concentrations (S1 Table). Cy treatment led to a reduced PCr/ATP ratio at both timepoints. (C) There was no effect of Cy treatment on coupling of oxidative phosphorylation (P/O). Data presented as box plot showing min, median, and max data point. (N = 5 for baseline and 1 day timepoints, N = 8 for 6 week timepoint) (NS, not significant).

Fig 5. No differences for in vitro mitochondria function 6 weeks after exposure to Cy.

(A) Flux control ratios relative to fully uncoupled respiration for state 4, state 3 for complex I (CI) and complexes I+II (CI+II) substrates in permeabilized EDL did not differ between saline and Cy treated mice, (N = 8 for each group). (B) Citrate synthase activity from homogenized gastrocnemius muscle was not different between saline and Cy groups (N = 8 for each group). Data presented as box plot showing min, median, and max data point. P>0.05 for all.

Fig 6. Skeletal muscle of mice exposed to Cy have evidence of mild oxidative damage at 1 week after exposure.

SDS-PAGE separation of homogenized EDL muscle followed by immunobloting with specific HNE antiserum. Data presented as box plot showing min, median, and max data point. N = 4 for 1 day and 1 week timepoints and N = 8 for 6 week timepoint.