TNF/TNFR1 signaling mediates doxorubicin-induced diaphragm weakness

Abstract

Doxorubicin, a common chemotherapeutic agent, causes respiratory muscle weakness in both patients and rodents. Tumor necrosis factor-α (TNF), a proinflammatory cytokine that depresses diaphragm force, is elevated following doxorubicin chemotherapy. TNF-induced diaphragm weakness is mediated through TNF type 1 receptor (TNFR1). These findings lead us to hypothesize that TNF/TNFR1 signaling mediates doxorubicin-induced diaphragm muscle weakness. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via intravenous injection. Three days later, we measured contractile properties of muscle fiber bundles isolated from the diaphragm. We tested the involvement of TNF/TNFR1 signaling using pharmaceutical and genetic interventions. Etanercept, a soluble TNF receptor, and TNFR1 deficiency protected against the depression in diaphragm-specific force caused by doxorubicin. Doxorubicin stimulated an increase in TNFR1 mRNA and protein (P < 0.05) in the diaphragm, along with colocalization of TNFR1 to the plasma membrane. These results suggest that doxorubicin increases diaphragm sensitivity to TNF by upregulating TNFR1, thereby causing respiratory muscle weakness.

Fig. 1.

Doxorubicin exposure in vitro does not alter diaphragm force. Specific force 1 h following doxorubicin (2 μg/ml) exposure. Data are means ± SE; n = 3/group.

Fig. 2.

Etanercept, a soluble TNF receptor, abolishes doxorubicin-induced diaphragm dysfunction 72 h following injection. A: body weight over 3 days following doxorubicin administration. Diaphragm-specific force (B) and relative force (C) 72 h following injection. Control refers to vehicle for doxorubicin group and vehicle + etanercept group. Data are means ± SE; n = 9 (control), 4 (doxorubicin), 5 (doxorubicin + etanercept). For all panels, P < 0.01 for overall difference by repeated-measures ANOVA; *P < 0.01 (control vs. doxorubicin) or #P < 0.05 (control vs. doxorubicin + etanercept) by Bonferroni test.

Fig. 3.

Doxorubicin does not alter diaphragm TNF mRNA or protein. A: TNF mRNA in the diaphragm following doxorubicin administration (24 h: vehicle n = 5, treated n = 6; 48 h: vehicle n = 5, treated n = 5; 72 h: vehicle n = 6, treated n = 6). B: TNF protein with representative lanes (24 h: vehicle n = 3, treated n = 3; 48 h: vehicle n = 3, treated n = 3; 72 h: vehicle n = 3, treated n = 3). Data are expressed as % change from time-matched control values.

Fig. 4.

TNFR1 is elevated in the diaphragm following doxorubicin administration. A: TNFR1 mRNA (24 h: vehicle n = 6, treated n = 6; 48 h: vehicle n = 4, treated n = 8; 72 h: vehicle n = 7, treated n = 8). B: TNFR1 protein (24 h: vehicle n = 6, treated n = 6; 48 h: vehicle n = 5, treated n = 5; 72 h: vehicle n = 6, treated n = 6) in the diaphragm following doxorubicin administration. Data are % change of time-matched vehicles represented as means ± SE. For all panels, P < 0.05 for overall difference by repeated-measures ANOVA. A: *P < 0.05 (48 h); B: *P < 0.05 (55 kDa, 48 kDa) by Bonferroni test.

Fig. 5.

TNFR1 localization after doxorubicin exposure. Panels show representative confocal images of transverse sections from diaphragms of mice treated with vehicle (top) or doxorubicin (bottom); muscle was collected and processed 72 h postinjection. Sections were stained for anti-TNFR1 (green) and counterstained with anti-annexin II (red) and DAPI (blue), a nuclear stain. Scale bar = 10 μm.

Fig. 6.

TNFR1 deficiency protects against doxorubicin-induced diaphragm dysfunction. A: body weight over 3 days following doxorubicin administration. Specific force (B) and relative force (C) measured 72 h following injection. Data are means ± SE; n = 8 (vehicle) or 7 (doxorubicin); for A and C, P < 0.01 for overall difference by repeated-measures ANOVA; *P < 0.01 by Bonferroni test.