Chronic doxorubicin administration impacts satellite cell and capillary abundance in a muscle-specific manner

Abstract

Anthracycline chemotherapies are effective at reducing disease recurrence and mortality in cancer patients. However, these drugs also contribute to skeletal muscle wasting and dysfunction. The purpose of this study was to assess the impact of chronic doxorubicin (DOX) administration on satellite cell and capillary densities in different skeletal muscles. We hypothesized that DOX would reduce satellite cell and capillary densities of the soleus (SOL) and extensor digitorum longus (EDL) muscles, along with muscle fiber size. Ovariectomized female Sprague-Dawley rats were randomized to receive three bi-weekly intraperitoneal injections of DOX (4 mg∙kg^-1 ; cumulative dose 12 mg∙kg^-1 ) or vehicle (VEH; saline). Animals were euthanized 5d following the last injection and the SOL and EDL were dissected and prepared for immunohistochemical and RT-qPCR analyses. Relative to VEH, CSA of the SOL and EDL fibers were 26% and 33% smaller, respectively, in DOX (P < 0.05). In the SOL, satellite cell and capillary densities were 39% and 35% lower, respectively, in DOX (P < 0.05), whereas in the EDL satellite cell and capillary densities were unaffected by DOX administration (P > 0.05). Proliferating satellite cells were unaffected by DOX in the SOL (P > 0.05). In the SOL, MYF5 mRNA expression was increased in DOX (P < 0.05), while in the EDL MGF mRNA expression was reduced in DOX (P < 0.05). Chronic DOX administration is associated with reduced fiber size in the SOL and EDL; however, DOX appeared to reduce satellite cell and capillary densities only in the SOL. These findings highlight that therapeutic targets to protect skeletal muscle from DOX may vary across muscles.

Keywords: Chemotherapy; Ki67; Pax7; extensor digitorum longus; soleus.

Figure 1

The effect of chronic doxorubicin administration on muscle fiber cross‐sectional area (CSA). Mean fiber CSA of soleus (A) and extensor digitorum longus (C) presented as mean ± SE. Histogram distribution of fiber CSA in soleus (B) and extensor digitorum longus (D). VEH, vehicle group; DOX, doxorubicin group. Soleus (VEH, n = 4; DOX, n = 4); extensor digitorum longus (VEH, n = 4; DOX, n = 6). *P < 0.05 VEH versus DOX.

Figure 2

Representative immunohistochemical image of satellite cell detection in the soleus and extensor digitorum longus. Merged images (A,F) demonstrating laminin (green), Pax7 (red), and 4′,6‐diamidino‐2‐phenylindole (DAPI, blue) with satellite cells denoted by white arrowheads. Images captures at 200×, scale bar represents 50 μm. Merged images (B, G) demonstrating satellite cell (red) location within laminin (green), co‐stained with DAPI (blue; C, H) and denoted by white arrowheads. Single‐channel image (D, I) demonstrating DAPI (blue). Single‐channel image (E, J) demonstrating Pax7 (red).

Figure 3

The effect of chronic doxorubicin administration on satellite cell and myonuclei densities. Mean satellite cell density of soleus (A‐B) and extensor digitorum longus (E‐F) presented as mean (bar) with individual animals represented by an “X”. Mean myonuclei density of soleus (C‐D) and extensor digitorum longus (G‐H). SC, satellite cell; VEH, vehicle group; DOX, doxorubicin group. Soleus (satellite cell: VEH, n = 4; DOX, n = 6; myonuclei: VEH, n = 4; DOX, n = 5); extensor digitorum longus (satellite cell: VEH, n = 4; DOX, n = 6; myonuclei: VEH, n = 4; DOX, n = 6). *P < 0.05 VEH versus DOX.

Figure 4

The effect of chronic doxorubicin administration on proliferating satellite cells. Representative immunohistochemical images of proliferating satellite cells (A‐D). (A) DAPI (blue) and laminin (white); (B) PAX7 (green) and laminin (white); (C), Ki67 (red) and laminin (white); (D) merged image denoting Ki67 (red), PAX7 (green), DAPI (blue), and laminin (white). Images capture at 200×, scale bar represents 50 μm. Percentage of proliferating satellite cells (Ki67+) in the soleus and extensor digitorum longus (E) are presented as mean ± SE. VEH, vehicle group; DOX, doxorubicin group; SOL, soleus, EDL, extensor digitorum longus. Soleus (VEH, n = 4; DOX, n = 7); extensor digitorum longus (VEH, n = 4; DOX, n = 6).

Figure 5

The effect of chronic doxorubicin administration on capillary density. Merged images (A, F) demonstrating CD31 (white) and wheat germ agglutinin (WGA, red). Images captures at 200×, scale bar represents 50 μm. Single‐channel images (B, G) demonstrating CD31 (white). Single‐channel images (C, H) demonstrating WGA (red). Mean capillary density of soleus (D‐E) and extensor digitorum longus (I‐J) presented as mean (bar) with individual animals represented by an “X”. VEH, vehicle group; DOX, doxorubicin group. Soleus (VEH, n = 4; DOX, n = 4); extensor digitorum longus (VEH, n = 4; DOX, n = 6). *P < 0.05 VEH versus DOX.