Resistance and endurance exercise training improves muscle mass and the inflammatory/fibrotic transcriptome in a rhabdomyosarcoma model

Abstract

Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that most often develops in children. Chemoradiation therapy is a standard treatment modality; however, the detrimental long-term skeletal muscle consequences of this therapy in juvenile cancer survivors include muscle atrophy and fibrosis resulting in decreased physical performance. Using a novel model of murine resistance and endurance exercise training, we investigate its role in preventing the long-term effects of juvenile RMS plus therapy.

Methods: Four-week-old male (n = 10) and female (n = 10) C57Bl/6J mice were injected with M3-9-M RMS cell into the left gastrocnemius with the right limb serving as an internal control (CON). Mice received a systemic vincristine injection and then five doses of 4.8 Gy of gamma radiation localized to the left hindlimb (RMS + Tx). Mice were then randomly divided into either sedentary (SED) or resistance and endurance exercise training (RET) groups. Changes in exercise performance, body composition, myocellular adaptations and the inflammatory/fibrotic transcriptome were assessed.

Results: RET improved endurance performance (P < 0.0001) and body composition (P = 0.0004) compared to SED. RMS + Tx resulted in significantly lower muscle weight (P = 0.015) and significantly smaller myofibre cross-sectional area (CSA) (P = 0.014). Conversely, RET resulted in significantly higher muscle weight (P = 0.030) and significantly larger Type IIA (P = 0.014) and IIB (P = 0.015) fibre CSA. RMS + Tx resulted in significantly more muscle fibrosis (P = 0.028), which was not prevented by RET. RMS + Tx resulted in significantly fewer mononuclear cells (P < 0.05) and muscle satellite (stem) cells (MuSCs) (P < 0.05) and significantly more immune cells (P < 0.05) than CON. RET resulted in significantly more fibro-adipogenic progenitors (P < 0.05), a trend for more MuSCs (P = 0.076) than SED and significantly more endothelial cells specifically in the RMS + Tx limb. Transcriptomic changes revealed significantly higher expression of inflammatory and fibrotic genes in RMS + Tx, which was prevented by RET. In the RMS + Tx model, RET also significantly altered expression of genes involved in extracellular matrix turnover.

Conclusions: Our study suggests that RET preserves muscle mass and performance in a model of juvenile RMS survivorship while partially restoring cellular dynamics and the inflammatory and fibrotic transcriptome.

Keywords: cachexia; cancer; chemotherapy; exercise; fibro-adipogenic progenitors; fibrosis; inflammation; muscle satellite cells; radiation.

Figure 1

Resistance and endurance exercise training (RET) improves endurance performance, body composition and muscle weight following rhabdomyosarcoma (RMS) plus therapy. (A) Study design. Four‐week‐old male (n = 10) and female (n = 10) C57Bl/6 mice received M3‐9‐M RMS cell injections into the left gastrocnemius to generate the tumours with the left limb serving injected with phosphate‐buffered saline (PBS) as an internal control (CON). After 3 days, all mice received systemic vincristine treatment administered by intraperitoneal (i.p.) injection. Two days after chemotherapy, mice were administered with five doses of 4.8 Gy of gamma radiation localized to the left hindlimb only (RMS + Tx). Following treatment, mice were randomly divided into either sedentary (SED) or resistance and endurance exercise training (RET) groups for 8 weeks. Figure created with Biorender.com. (B) Endurance performance assessed by total distance run (m) until volitional exhaustion. (C) Body weight (g). (D) Body fat percentage and (E) lean mass (g). ^*** P < 0.001 and ^**** P < 0.0001, SED versus RET. In (C), α = significantly different than baseline, Pre‐Tx and Pre‐RET; β = significantly different than 2‐week RET; and σ = significantly different than 5‐week RET. In (E), α = significantly different than baseline and Pre‐Tx and β = significantly different than Pre‐RET. Three‐way analysis of variance (ANOVA) or two‐way ANOVA (E), Sidak post hoc test. n = 7–9 per group.

Figure 2

Rhabdomyosarcoma (RMS) plus therapy induces muscle atrophy and fibrosis. (A) Muscle weight relative to total body weight (g/g). (B) Quantification of trichrome stain for intramuscular fibrosis and collagen content as a % of total muscle area. (C) Representative images of trichrome staining for intramuscular fibrosis and collagen content. Scale = 100 μm. *P < 0.05, SED versus RET. ^# P < 0.05, RMS + Tx versus CON. Two‐way analysis of variance (ANOVA), Sidak post hoc test. n = 7–9 per group.

Figure 3

Rhabdomyosarcoma (RMS) plus therapy reduced Type IIB cross‐sectional area (CSA) and proportion, whereas resistance and endurance exercise training (RET) enlarged myofibre CSA and induced alterations in fibre‐type distribution independent of RMS plus therapy. (A) Representative image of myosin heavy chain stain immunofluorescence (Type IIA: green; Type IIB: orange; Type IIX: black; laminin: red). (B) Total fibre CSA (μm²). (C) Type IIA fibre‐specific CSA (μm²). (D) Type IIB fibre‐specific CSA (μm²). (E) Proportion of Type IIA fibres (% of total fibre number). (F) Proportion of Type IIB fibres (% of total fibre number). *P < 0.05 and ^** P < 0.01, SED versus RET group. ^## P < 0.01, RMS + Tx versus CON. Two‐way analysis of variance (ANOVA), Sidak post hoc test. n = 6–8 per group. Scale = 100 μm.

Figure 4

Resistance and endurance exercise training (RET) enlarged myonuclear domain with no changes in myonuclear accretion followed rhabdomyosarcoma (RMS) plus therapy. (A) Representation of images used for quantifying myonuclei and myonuclear domain. (B) Myonuclear domain of all fibres (μm²). (C) Type IIA fibre‐specific myonuclear domain (μm²). (D) Type IIB fibre‐specific myonuclear domain (μm²). (E) Type IIX fibre‐specific myonuclear domain (μm²). *P < 0.05, ^** P < 0.01 and ^*** P < 0.001. Two‐way analysis of variance (ANOVA), Sidak post hoc test. n = 6–8 per group. Scale = 100 μm.

Figure 5

Rhabdomyosarcoma (RMS) plus therapy and resistance and endurance exercise training (RET) alter cellular dynamic in skeletal muscle. (A) CD45⁺ cells per mg of muscle. (B) CD31⁺ cells per mg of muscle. (C) α7⁺ cells per mg of muscle. (D) Sca1⁺ cells per mg of muscle. *P < 0.05 and ^** P < 0.01, SED versus RET. ^### P < 0.001, RMS + Tx versus CON. Two‐way analysis of variance (ANOVA), Sidak post hoc test. n = 7–9 per group.

Figure 6

Fibrotic and inflammatory signature is induced by rhabdomyosarcoma (RMS) plus therapy and is partially reduced by resistance and endurance exercise training (RET) in skeletal muscle. (A) Volcano plot of log₂‐transformed fold change and −log₁₀‐transformed P values showing up‐regulated (in green) and down‐regulated (in purple) genes in CON‐RET versus CON‐SED and the Top 10 direct enrichment score from the gene set analysis (GSA). (B) Volcano plot of log₂‐transformed fold change and −log₁₀‐transformed P values showing up‐regulated (in green) and down‐regulated (in purple) genes in RMS‐RET versus RMS‐SED and the Top 10 direct enrichment score from the gene set analysis (GSA). (C) Heatmap of differentially expressed genes (DEGs) in RMS‐SED versus CON‐SED, Top 10 (GSA) in RMS‐SED versus CON‐SED and Top 10 biological process Gene Ontology (GO) terms (false discovery rate [FDR], adjusted P value) in RMS‐SED versus CON‐SED. (D) Heatmap of DEGs in RMS‐RET versus CON‐RET, Top 10 GSA in RMS‐RET versus CON‐RET and Top 10 biological process GO terms (FDR, adjusted P value) in RMS‐RET versus CON‐RET. (E) Representative western blot image of MMP‐2, CCR2 and cyclophilin B protein expression. (F) Fold change of pro‐MMP‐2, active MMP‐2 and CCR2 protein expression, normalized by cyclophilin B. *P < 0.05 and ^** P < 0.01. n = 3 per condition.