Doxorubicin causes diaphragm weakness in murine models of cancer chemotherapy

Abstract

Doxorubicin is a chemotherapeutic agent prescribed for a variety of tumors. While undergoing treatment, patients exhibit frequent symptoms that suggest respiratory muscle weakness. Cancer patients can receive doxorubicin chemotherapy through either intravenous (IV) or intraperitoneal (IP) injections. We hypothesized that respiratory muscle function would be depressed in a murine model of chemotherapy. We tested this hypothesis by treating C57BL/6 mice with a clinical dose of doxorubicin (20 mg/kg) via IV or IP injection. Three days later we measured contractile properties of muscle fiber bundles isolated from the diaphragm. Doxorubicin consistently depressed diaphragm force with both methods of administration (P < 0.01). Doxorubicin IP exaggerated the depression in diaphragm force and stimulated tissue inflammation and muscle fiber injury. These results suggest that clinically relevant doses of doxorubicin cause respiratory muscle weakness and that the loss of function depends, in part, on the route of administration.

Figure 1

Intravenous (i.v.) doxorubicin administration causes contractile dysfunction of the diaphragm 72 hrs following injection. Panels show changes in (A) absolute force, (B) specific force, and (C) relative force. Data are means ± SE; n = 9/group; for specific and relative force panels, p<0.01 for overall difference by repeated-measures ANOVA; *p<0.01 by Bonferroni test.

Figure 2

Doxorubicin depresses diaphragm contractile function 72 hrs following intraperitoneal (i.p.) injection: (A) absolute force, (B) specific force, and (C) relative force. Data are means ± SE; n = 11/group; for all panels, p<0.01 for overall difference by repeated-measures ANOVA; *p<0.01 by Bonferroni test.

Figure 3

Doxorubicin increases DCFH oxidation in diaphragm fibers following i.p. injection. (A) Fluorescence images of vehicle (top) and doxorubicin (bottom) diaphragms 72 hrs following injection. (B) Averaged data for DCF fluorescence. Data are means ± SE; n = 13/group; *p<0.05 by Student’s t-test.

Figure 4

Doxorubicin i.p. increases nitrotyrosine and 4-hydroxynonenal (HNE) residues on myofibrillar proteins. Myofibrillar proteins isolated from diaphragms exposed to i.p. doxorubicin or vehicle. For original blots, left panels are stained for total protein, and right panels are stained for nitrotyrosine (A) or HNE (B). Averaged densitometry data depict nitrotyrosine- or HNE-conjugated protein-to-total-protein ratios. Data are means ± SE; n = 3/group; *p<0.05 by Student’s t-test.

Figure 5

Doxorubicin i.p. increases myeloperoxidase (MPO) content and disrupts sarcolemmal integrity. In histologic sections, arrows denote abdominal surface of diaphragm. (A) H & E stain of murine diaphragm 72 hrs after i.p. injection of vehicle or doxorubicin (left). Averaged data for MPO protein to total protein in diaphragm (right). Data are means ± SE; n = 4/group; *p<0.05 by Student’s t-test. Scale bar = 20 μm. (B) Evans blue (red) following i.p. injection 72 hrs post vehicle (left) or doxorubicin (right); DAPI used to stain nuclei (blue).

Figure 6

Doxorubicin i.v. does not alter MPO content or sarcolemmal integrity. In histologic sections, arrows denote abdominal surface of diaphragm. (A) H & E stain of murine diaphragm 72 hrs after i.v. injection of vehicle or doxorubicin. Averaged data for MPO-protein-to-total-protein in diaphragm (right). Data are means ± SE; n = 4/group. Scale bar = 20 μm. (B) Evans blue (red) following i.v. injection 72 hrs post vehicle (left) or doxorubicin (right); nuclei stained using DAPI (blue).