Doxorubicin induces trans-differentiation and MMP1 expression in cardiac fibroblasts via cell death-independent pathways

Abstract

Although doxorubicin (DOX)-induced cardiomyopathy causes lethal heart failure (HF), no early detection or effective treatment methods are available. The principal mechanisms of cardiotoxicity are considered to involve oxidative stress and apoptosis of cardiomyocytes. However, the effect of DOX on cardiac fibroblasts at non-lethal concentrations remains unknown. The aim of this study was to investigate the direct effect of doxorubicin on the activation of cardiac fibroblasts independent of cell death pathways. We first found that DOX induced α-SMA expression (marker of trans-differentiation) at a low concentration range, which did not inhibit cell viability. DOX also increased MMP1, IL-6, TGF-β and collagen expression in human cardiac fibroblasts (HCFs). In addition, DOX promoted Akt and Smad phosphorylation. A Smad inhibitor prevented DOX-induced α-SMA and IL-6 protein expression. An PI3K inhibitor also prevented MMP1 mRNA expression in HCFs. These findings suggest that DOX directly induces fibrotic changes in HCFs via cell death-independent pathways. Furthermore, we confirmed that these responses are organ- and species-specific for HCFs based on experiments using different types of human and murine fibroblast cell lines. These results suggest potentially new mechanisms of DOX-induced cardiotoxicity from the viewpoint of fibrotic changes in cardiac fibroblasts.

Fig 1. Microarray analysis of gene expression induced by DOX in HCFs, NHDFs and mCFs.

Human cardiac fibroblasts (HCFs), normal human dermal fibroblasts (NHDFs) and murine cardiac fibroblasts (mCFs) were exposed to 0.1 μM DOX, and mRNA expression levels were analyzed with a microarray. A, Cytokine-associated gene expression in HCFs, NHDFs and mCFs at 6 (black) and 24 (gray) hours. B, Fibrosis-associated and MMP gene expression in HCFs, NHDFs and mCFs at 6 (black) and 24 (gray) hours. (n = 1).

Fig 2. DOX induced trans-differentiation of HCFs but not NHDFs.

A, ACTA2 mRNA expression in the presence of DOX for 24 hours in HCFs (left) and NHDFs (right) (n = 5–6; *p < 0.05; ns: no significant difference). B, α-SMA protein expression in the presence of DOX (0.1 μM) for 24 to 72 hours in HCFs (left) and NHDFs (right) (n = 4–6; **p < 0.01, and *p < 0.05). C, α-SMA protein expression in the presence of DOX (0.005 to 0.1 μM) for 48 hours in HCFs (left) and NHDFs (right) (n = 4; **p < 0.01; ns: no significant difference). The original immunoblot image is shown in S2 Fig.

Fig 3. DOX induced MMP1 expression and production in HCFs but not in NHDFs.

A, MMP1 mRNA expression in the presence of DOX (0.1 μM) for 24 hours in HCFs (left) and NHDFs (right) (n = 4–5; *p < 0.05; ns: no significant difference). B, MMP1 protein expression in HCFs (left) and NHDFs (right) stimulated by DOX (0.1 μM) for 48 hours (n = 4–6; *p < 0.05; ns: no significant difference). The original immunoblot image is shown in S2B Fig. C, Activity of MMP1 in supernatant from HCFs exposed to DOX (0.1 μM) for 48 hours (n = 4; **p<0.01).

Fig 4. DOX induced IL-6, TGF-β and collagen expression in HCFs.

A, IL-6 mRNA expression in HCFs with or without DOX treatment (0.1 μM) for 3 to 6 hours (n = 4; *p < 0.05). B, TGF-β mRNA expression in HCFs with or without DOX treatment (0.1 μM) for 3 to 6 hours (n = 4; **p<0.01, ***p<0.001). C, Production of IL-6 in supernatant from HCFs with DOX treatment (0.1 μM) for 24 to 48 hours (n = 4; ***p<0.001).

Fig 5. DOX phosphorylated Smad and Akt in HCFs.

A, The time course of protein phosphorylation (p-S6K-t389, p-Akt-s473 and p-Smad2-s465/467) in HCFs stimulated by DOX (0.1 μM) for 0 to 60 min. The original immunoblot image is shown in S8 Fig. B, A TGF-β/Smad inhibitor (SB431512) decreased the DOX-induced IL-6 and α-SMA expression. A PI3K inhibitor (LY294002) decreased DOX-induced protein expression and production of MMP1 in HCFs (n = 4–6; *p < 0.05, **p < 0.01, ***p<0.001). The original immunoblot image is shown in S9 Fig.

Fig 6. A proposed schematic diagram of DOX-induced pro-fibrotic changes in HCFs.

DOX activated Smad and Akt signaling and increased IL-6, α-SMA and MMP1 expression in HCFs.