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Sodium nitrate treatment escalates doxorubicin-induced cachexia in mice

D.G. Campelj,1,2 B.M. Butcher,1,2 D.A. Debruin,1,2 C.A. Timpani,1,2 A. Hayes1,2 and E. Rybalka,1,2 1Institute of Sport, Exercise & Active Living (ISEAL), College of Health & Biomedicine, Victoria University, Melbourne, VIC 8001, Australia and 2Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, Melbourne, VIC 8001, Australia.

Introduction: Anthracycline chemotherapy instigates skeletal muscle pathology and wasting, which is independent of cancer-induced cachexia – this is best exemplified for the highly toxic agent, doxorubicin (DOX; reviewed in Sorensen et al., 2006). Since anthracyclines are effective anti-cancer agents, investigating potential co-therapies to clinically address the skeletal muscle-specific side effects of chemotherapy treatment is important, particularly since lean mass is a predictor of patient morbidity and mortality. Sodium Nitrate (SN) treatment has been shown to afford protection against DOX-induced cardiomyopathy in mice (Zhu et al.,2011; Xi et al., 2012). SN increases endogenous nitric oxide (NO) production, which is thought to increase anti-oxidative defence against the oxygen radicals produced during DOX metabolism by oxidases. As such, we aimed to investigate whether SN could also protect against DOX-induced skeletal myopathy.

Methods: Six-week old male Balb/C mice were treated with three intraperitoneal injections of either vehicle (0.9% NaCl, n=12), DOX (4mg/kg, n=12). To test the hypothesis that sodium nitrate treatment could protect the skeletal muscle against doxorubicin-induced myopathy, a group of mice (n=12) were treated with 1mmol.l-1 NaNO2 in drinking water during DOX (4mg/kg) treatment. Mice were assessed for body composition using echoMRI before and after the treatment regimen. Thereafter, mice were anaesthetized with isoflurane (4% induction; 2% maintenance) and m. extensor digitorum longus (EDL) and m. soleus (SOL) were excised to investigate contractile properties. M. flexor digitorum brevis (FDB) was also excised, and fibres were isolated and cultured overnight to assess mitochondrial functional parameters.

Results: DOX treatment induced cachexia in mice as evidenced by a reduction in the % change in whole body composition (P<0.0001), lean mass (P<0.005) and fat mass (P<0.005) from pre- to post-treatment. SN treatment exacerbated the DOX-induced reduction in total body composition (P<0.05), yet not significantly so for the individual measures of lean and fat mass. There was no effect of DOX or DOX+SN treatment on specific force production in the EDL or SOL muscles, or on whole body grip strength. With respect to mitochondrial function, there was a strong trend for DOX treatment to increase the spare respiratory capacity (P=0.08) and the oxidative metabolic potential (P=0.06) in response to chemical uncoupling with FCCP. DOX+SN significantly increased the spare respiratory capacity (P<0.05).

Conclusions: We have demonstrated DOX-induced cachexia in mice which appears to be exacerbated by SN co-treatment. Thus, while SN appears to protect the cardiac muscle from DOX-induced myopathy, it affords no benefit to the skeletal musculature, but rather, exacerbates cachexia.

Sorensen, J.C., Cheregi, B.D., Timpani, C.A., Nurgali, K, Hayes, A. & Rybalka, E. (2016) Mitochondria: Inadvertent targets in chemotherapy-induced skeletal muscle toxicity and wasting? Cancer Chemotherapy & Pharmacology 78: 673-83.

Xi L, Zhu, S-G, Das A, Chen Q, Durrant D, Hobbs, DC, Lesnefsky EJ, Kukreja RC. (2012). Dietary Inorganic Nitrate Alleviates Doxorubicin Cardiotoxicity: Mechanisms and Implications. Nitric Oxide 26: 274–284.

Zhu S-G, Kukreja RC, Das A, Chen Q, Lesnefsky EJ, & Xi L. (2011). Dietary nitrate supplementation protects against doxorubicin-induced cardiomyopathy by improving mitochondrial function. Journal of the American College of Cardiology57: 2181–2189.