Prevention of chemotherapy-induced cachexia by ACVR2B ligand blocking has different effects on heart and skeletal muscle

Abstract

Background: Toxicity of chemotherapy on skeletal muscles and the heart may significantly contribute to cancer cachexia, mortality, and decreased quality of life. Doxorubicin (DOX) is an effective cytostatic agent, which unfortunately has toxic effects on many healthy tissues. Blocking of activin receptor type IIB (ACVR2B) ligands is an often used strategy to prevent skeletal muscle loss, but its effects on the heart are relatively unknown.

Methods: The effects of DOX treatment with or without pre-treatment with soluble ACVR2B-Fc (sACVR2B-Fc) were investigated. The mice were randomly assigned into one of the three groups: (1) vehicle (PBS)-treated controls, (2) DOX-treated mice (DOX), and (3) DOX-treated mice administered with sACVR2B-Fc during the experiment (DOX + sACVR2B-Fc). DOX was administered with a cumulative dose of 24 mg/kg during 2 weeks to investigate cachexia outcome in the heart and skeletal muscle. To understand similarities and differences between skeletal and cardiac muscles in their responses to chemotherapy, the tissues were collected 20 h after a single DOX (15 mg/kg) injection and analysed with genome-wide transcriptomics and mRNA and protein analyses. The combination group was pre-treated with sACVR2B-Fc 48 h before DOX administration. Major findings were also studied in mice receiving only sACVR2B-Fc.

Results: The DOX treatment induced similar (~10%) wasting in skeletal muscle and the heart. However, transcriptional changes in response to DOX were much greater in skeletal muscle. Pathway analysis and unbiased transcription factor analysis showed that p53-p21-REDD1 is the main common pathway activated by DOX in both skeletal and cardiac muscles. These changes were attenuated by blocking ACVR2B ligands especially in skeletal muscle. Tceal7 (3-fold to 5-fold increase), transferrin receptor (1.5-fold increase), and Ccl21 (0.6-fold to 0.9-fold decrease) were identified as novel genes responsive to blocking ACVR2B ligands. Overall, at the transcriptome level, ACVR2B ligand blocking had only minor influence in the heart while it had marked effects in skeletal muscle. The same was also true for the effects on tissue wasting. This may be explained in part by about 18-fold higher gene expression of myostatin in skeletal muscle compared with the heart.

Conclusions: Cardiac and skeletal muscles display similar atrophy after DOX treatment, but the mechanisms for this may differ between the tissues. The present results suggest that p53-p21-REDD1 signalling is the main common DOX-activated pathway in these tissues and that blocking activin receptor ligands attenuates this response, especially in skeletal muscle supporting the overall stronger effects of this treatment in skeletal muscles.

Keywords: Activins; Ccl21; Doxorubicin; Myostatin; Transcriptome; p53.

Figure 1

(A) Experimental design. The study included acute as well as 2 and 4 week experiments. (B) Adjusted mass (control = 1, adjusted to tibial length) of skeletal muscle and the heart after 4 weeks of cumulative 24 mg/kg doxorubicin administration (mean ± SD). Skeletal muscle mass is a sum of tibialis anterior, gastrocnemius, and soleus masses. n = 15, 16, and 17 in skeletal muscle and n = 14, 16, and 16 in heart in CTRL, DOX, and in DOX + sACVR2B, respectively. General linear model analysis of variance with Bonferroni post hoc test was used. * or *** = significant (P < 0.05 or P < 0.001, respectively) difference to respective CTRL. ### = significant (P < 0.001) difference to respective DOX.

Figure 2

Top 10 genes with largest response to doxorubicin in skeletal muscle (A and B) and in the heart (C and D). Top 10 genes with largest change by sACVR2B‐Fc in skeletal muscle (E and F). C = CTRL, D = DOX, A = DOX + sACVR2B. *, **, or *** = significant (P < 0.05, P < 0.01, or P < 0.001, respectively) adjusted difference (false discovery rate). n = 5 per group.

Figure 3

Doxorubicin alters the gene expression of (A) Cdkn1a (p21), while sACVR2B‐Fc increases (B) Tceal7 in skeletal muscle and attenuates (C) Ccl21 mRNA levels. (D) Doxorubicin decreases transferrin receptor (Tfrc) mRNA in both tissues while its mRNA and protein (E) levels are increased by sACVR2B‐Fc. Tceal7 was expressed in heart only very weakly, and thus, it was not analysed. The values are presented as fold changes compared with the control group. For multiple group comparisons, general linear model analysis of variance with Bonferroni post hoc test (A and B) or Kruskal‐Wallis test with Holm‐Bonferroni corrected Mann–Whitney U post hoc test (C–E) were used. For two‐group comparisons, the Student's t‐test (A–C) or non‐parametric Mann–Whitney U‐test (E) were used. *, **, or *** = significant (P < 0.05, P < 0.01, or P < 0.001, respectively) difference to respective CTRL. # or ### = significant (P < 0.05 and P < 0.001, respectively) difference to respective DOX. n = 7–9 per group in the doxorubicin experiment and n = 5–6 per group in the sACVR2B‐Fc alone vs. PBS experiment.

Figure 4

(A–D) Doxorubicin differentially alters the gene expression of Ppargc1 (PGC‐1) mRNA isoforms in muscle and the heart. Notice that in (C) Ppargc1a exon 1c, the * in S. muscle with lines depicts the doxorubicin effect of both doxorubicin groups pooled when compared with control without treatments. General linear model analysis of variance with Bonferroni post hoc test was used. *, **, or *** = significant (P < 0.05, P < 0.01, or P < 0.001, respectively) difference to respective CTRL. n = 6–9 per group.

Figure 5

(A) Doxorubicin administration and sACVR2B‐Fc treatment did not alter heart protein synthesis. Protein synthesis relative to doxorubicin was analysed with puromycin incorporation method. Representative blot (left) with Ponceau S staining (right). (− = negative control for puromycin). Doxorubicin administration had no effect on the amount of ubiquitinated proteins in the heart (B) despite minor changes in the mRNA expression of E3 ubiquitin ligases MuRF1 and Atrogin1 (C). (D) sACVR2B‐Fc treatment did not alter doxorubicin levels in gastrocnemius muscle at the 20 h timepoint after doxorubicin‐administration, when the RNA and protein samples were also taken. Kruskal‐Wallis test with Holm‐Bonferroni corrected Mann–Whitney U post hoc test (A and B), and general linear model analysis of variance with Bonferroni post hoc test (C and D) were used. Doxorubicin effect in Atrogin1 expression in the heart was evaluated with Student's t‐test. n = 7–9 per group in the doxorubicin experiment. Regarding doxorubicin experiment, muscle results of Murf1 can be found as Supporting Information in earlier publication.4

Figure 6

Gene set enrichment analysis was conducted from the microarray results. Positive and negative enrichment scores denote a large number of genes up‐regulated or down‐regulated, respectively, in the given gene set. Gene sets with false discovery rate < 0.001 are presented for the doxorubicin effects in muscle (A) and the heart (B). Normalized enrichment score (NES)‐values of up‐regulated gene sets in doxorubicin‐treated mice (DOX vs. CTRL) are expressed as red bars and NES‐values of down‐regulated gene sets as blue bars. There was no significant enrichment of gene sets down‐regulated by doxorubicin in the heart (B). For the sACVR2B‐Fc effects, gene sets with false discovery rate < 0.05 are presented (C). NES‐values of up‐regulated gene sets in treated mice (DOX + sACVR2B vs. DOX) are expressed as red bars and NES‐values of down‐regulated gene sets as blue bars. There was no significant enrichment of gene sets altered by sACVR2B‐Fc at false discovery rate < 0.05 in the heart.

Figure 7

mRNA expression changes of genes containing p53 targeted motifs (DOX vs. CTRL fold change > 1.5 and false discovery rate < 0.05) (A) in skeletal muscle and (B) in the heart. C = control, D = DOX, and D + A = DOX + sACVR2B. Sig. designates p‐adjusted false discovery rate of the difference between the doxorubicin and controls (* = false discovery rate < 0.05, ** = false discovery rate < 0.01, *** = false discovery rate < 0.001). Rank = the rank number (all genes in the genome) of likelihood of being a p53 target. Motifs = the number of p53 associated motifs found in regulatory sequence. The table presents results of mean of probes with multiple transcripts. n = 5 per group. (C) The effects of DOX and combined DOX + sACVR2B on p53 protein level. Kruskal‐Wallis test with Holm‐Bonferroni corrected Mann–Whitney U post hoc test was used. n = 8–9 in heart and 4–6 in skeletal muscle.

Figure 8

(A) Activin receptor type IIB and its major ligands are differentially expressed in the heart and skeletal muscle. The effects of doxorubicin and sACVR2B‐Fc administration on mRNA expression of (B) Gdf8 (myostatin), (C) Acvr2b, (D) Activin A, and (E) Gdf11 in skeletal muscle and the heart. n = 12 per group in muscle and heart comparison (A), n = 6–9 per group in the doxorubicin experiment (B–E). For multiple group comparisons, general linear model analysis of variance with Bonferroni post hoc test was used (B–E). For two‐group comparisons, the non‐parametric Mann–Whitney U‐test (A) or Student's t‐test (doxorubicin effect in D) were used.