BGP-15 Protects against Oxaliplatin-Induced Skeletal Myopathy and Mitochondrial Reactive Oxygen Species Production in Mice

Abstract

Chemotherapy is a leading intervention against cancer. Albeit highly effective, chemotherapy has a multitude of deleterious side-effects including skeletal muscle wasting and fatigue, which considerably reduces patient quality of life and survivability. As such, a defense against chemotherapy-induced skeletal muscle dysfunction is required. Here we investigate the effects of oxaliplatin (OXA) treatment in mice on the skeletal muscle and mitochondria, and the capacity for the Poly ADP-ribose polymerase (PARP) inhibitor, BGP-15, to ameliorate any pathological side-effects induced by OXA. To do so, we investigated the effects of 2 weeks of OXA (3 mg/kg) treatment with and without BGP-15 (15 mg/kg). OXA induced a 15% (p < 0.05) reduction in lean tissue mass without significant changes in food consumption or energy expenditure. OXA treatment also altered the muscle architecture, increasing collagen deposition, neutral lipid and Ca²⁺ accumulation; all of which were ameliorated with BGP-15 adjunct therapy. Here, we are the first to show that OXA penetrates the mitochondria, and, as a possible consequence of this, increases mtROS production. These data correspond with reduced diameter of isolated FDB fibers and shift in the fiber size distribution frequency of TA to the left. There was a tendency for reduction in intramuscular protein content, albeit apparently not via Murf1 (atrophy)- or p62 (autophagy)- dependent pathways. BGP-15 adjunct therapy protected against increased ROS production and improved mitochondrial viability 4-fold and preserved fiber diameter and number. Our study highlights BGP-15 as a potential adjunct therapy to address chemotherapy-induced skeletal muscle and mitochondrial pathology.

Keywords: BGP-15; mitochondria; mitochondrial reactive oxygen species; muscle wasting; oxaliplatin chemotherapy; protein synthesis; skeletal muscle.

Figure 1

Body weight and food consumption over the treatment period. OXA significantly reduced final post treatment body weights, with weight loss plateauing from D8 of OXA treatment (A). Body weight of OXA treated mice (including OXAB) was significantly reduced from VEH from D10 onwards. No differences were detected in (B) Food consumption or (C) Water consumption between the groups. D#, treatment day number; Significance, ^†p < 0.005 OXA and OXAB different from VEH. n = 6–8.

Figure 2

BGP-15 protects against Oxaliplatin-induced lean tissue but not fat mass loss. (A) BGP-15 protected against the OXA-induced reduction in LMI. (B) FMI was reduced by OXA treatment by 15% with no protection afforded by BGP-15. (C) Hydration Index calculated by [(echo derived total water – echo derived free water)/echo derived lean mass]. (D) Heart weight indexed against body weight showed OXAB treatment significantly increased heart size in relation to body weight. (E) Absolute wet tissue weights showed no significant difference either as raw or indexed against body weight, however OXA treatment reduced liver size with adjunct BGP-15 therapy further exacerbating this reduction (Table significance: A = p < 0.05 OXA to VEH. B = p < 0.05 OXA to OXAB. C = p < 0.0001 OXAB to VEH). BW, Body weight; EDL, Extensor Digitorum Longus; TID. ANT, Tibialis Anterior. Significance: ^*p < 0.05, ^**p<0.005; Trend: ^#p = 0.076. n = 6–8.

Figure 3

BGP-15 reduces basal energy expenditure with OXA treatment not affecting exercise capacity. (A) There was no significant effect of treatment on the respiratory quotient. (B) Energy expenditure at rest was reduced by BGP-15 adjunct therapy. (C) Oxygen consumption was not effected by treatment. (D,F) Time budgets of activities of daily living: OXA treatment reduced time spent engaged in long lounges (p < 0.05) while OXAB treatment reduced the time spent engaged in voluntary wheel running. (E) There was no effect of treatment on total meters covered (combination of wheel and pedestrian meters). CHO, Carbohydrate; VO₂, Volume Oxygen. Table significance: (A) p < 0.05 OXA vs. VEH, (B) p < 0.05 OXA vs. OXAB, (C) trend (p = 0.088) OXA vs. OXAB, (D) trend (p = 0.057) OXA vs. VEH. Significance: ^*p < 0.05. n = 6–8.

Figure 4

OXA and BGP-15 treatment effect fiber size distribution in TA muscle. (A) Representative images of H&E stained TA and fiber size frequency histogram (B) OXA treatment significantly increased the frequency of fibers in the 600-899 μm² and >4,900 μm² size bins whilst reducing the frequency of fibers in the 1,500–1,799 μm² size bin. Significance: ^Φp < 0.0005 VEH compared to OXA and p < 0.05 VEH from OXAB; ^‡p < 0.005 VEH and OXAB compared to OXA, ^†p < 0.05 OXA and OXAB compared to VEH. n = 4.

Figure 5

BGP-15 protects against OXA-induced increases in intracellular Ca²⁺, collagen deposition and fat infiltration in TA muscle. OXA treatment increased (A) intracellular Ca²⁺ content (B) fibrotic tissue and collagen deposition and (C) fat infiltration with OXAB protecting against all these parameters in TA muscles. A.U., Arbitrary units; ORO, Oil Red O. Significance: ^*p < 0.05, ^**p < 0.005.

Figure 6

OXA penetrates the nuclear and mitochondrial fractions of TA muscle homogenates and increases succinate dehydrogenase (SDH) content. (A) OXA penetrated the nuclear and (B) mitochondrial fractions as quantified by Pt detection (n = 3–4). (C) SDH staining was significantly increased with OXA treatment but OXAB had no effect on this measure (n = 6–8). [Pt], Platinum concentration; SDH, SDH Succinate dehydrogenase; ppm, parts per million. Significance: ^*p = <0.05, ^**p = <0.005, ^***p = <0.0005.

Figure 7

OXA and BGP-15 treatment does not alter PARP expression in mouse TA. No changes in (A) total PARylation (B) PARP1 or (C) PARP2 were detected following OXA treatment. Adjunct treatment with the PARP inhibitor BGP-15 (OXAB) did not alter PARP expression. (D) Representative stainfree Western blot images are digitally cut together to remove blots of other chemotherapies not presented in this manuscript, no other alteration was performed.

Figure 8

BGP-15 protects against OXA-induced mitochondrial superoxide production and fiber diameter loss and improves mitochondrial viability in FDB fibers. OXA treatment induced (A) A 4-fold increase in mitochondrial density, with OXAB protecting against this increase. (B) OXAB protected against a 25% increase in MitoSOX fluorescence induced by OXA treatment. (C) OXA treatment reduced mitochondrial viability while OXAB treatment protected against the effects of OXA and improved viability 4-fold from VEH. Representative images of MitoSOX and MitoTracker stained FDB fibers (D,E). OXA treatment also induced a (F) 25% reduction in FDB Fiber diameter with OXAB protecting against this reduction. FDB, Flexor Digitorum Longus; Mito, Mitochondria; SOX, Superoxide. Significance: ^*p < 0.05, ^**p < 0.005, ^***p < 0.0005, ^****p < 0.0001. Trend: ^#p = 0.073, ^##p = 0.077. n = 4–6.

Figure 9

OXA treatment reduces protein synthesis and concentration within TA muscle. (A) OXA treatment showed a trend to reduce total protein concentration, and reduced (B) total p70S6K and (C) total rpS6 when compared to VEH suggesting a suppression of protein synthesis. (D) Apoptosis initiation marker total Bax was also supressed in TA muscle. Contrastingly no change in (E) total ubiquitin-proteasome marker MuRF1 or (F) autophagy marker p62 expression was noted. (G) Western blot representative images are digitally cut together to remove blots of other chemotherapies not presented in this manuscript, no other alteration was performed. Significance: ^*p < 0.05, ^**p < 0.005 Trend: ^#p = 0.081. n = 6–8.