Doxorubicin-induced p53 interferes with mitophagy in cardiac fibroblasts

Abstract

Anthracyclines are the critical component in a majority of pediatric chemotherapy regimens due to their broad anticancer efficacy. Unfortunately, the vast majority of long-term childhood cancer survivors will develop a chronic health condition caused by their successful treatments and severe cardiac disease is a common life-threatening outcome that is unequivocally linked to previous anthracycline exposure. The intricacies of how anthracyclines such as doxorubicin, damage the heart and initiate a disease process that progresses over multiple decades is not fully understood. One area left largely unstudied is the role of the cardiac fibroblast, a key cell type in cardiac maturation and injury response. In this study, we demonstrate the effect of doxorubicin on cardiac fibroblast function in the presence and absence of the critical DNA damage response protein p53. In wildtype cardiac fibroblasts, doxorubicin-induced damage correlated with decreased proliferation and migration, cell cycle arrest, and a dilated cardiomyopathy gene expression profile. Interestingly, these doxorubicin-induced changes were completely or partially restored in p53-/- cardiac fibroblasts. Moreover, in wildtype cardiac fibroblasts, doxorubicin produced DNA damage and mitochondrial dysfunction, both of which are well-characterized cell stress responses induced by cytotoxic chemotherapy and varied forms of heart injury. A 3-fold increase in p53 (p = 0.004) prevented the completion of mitophagy (p = 0.032) through sequestration of Parkin. Interactions between p53 and Parkin increased in doxorubicin-treated cardiac fibroblasts (p = 0.0003). Finally, Parkin was unable to localize to the mitochondria in wildtype cardiac fibroblasts, but mitochondrial localization was restored in p53-/- cardiac fibroblasts. These findings strongly suggest that cardiac fibroblasts are an important myocardial cell type that merits further study in the context of doxorubicin treatment. A more robust knowledge of the role cardiac fibroblasts play in the development of doxorubicin-induced cardiotoxicity will lead to novel clinical strategies that will improve the quality of life of cancer survivors.

Fig 1. DOX alters cardiac fibroblast function.

Growth curves over 72 hours of (A) WT and (B) p53^-/- after standard DOX exposure. (C) demonstrates the mask used to measure confluence. (D) Twenty-four hours after standard DOX exposure cells were plated on a modified Boyden chamber to assess migration. (E), (F) trypan blue exclusion was used to assess cell viability. (G), (J) Relative genetic content was assessed with PI dye 3 hrs after standard treatment. Samples were fixed and sorted via flow cytometry. (H), (I) Three hrs after standard treatment, cellular protein content was labeled with SRB dye and absorbance was measured to assess relative quantity. (K), (L) Changes from baseline metabolic activity were assessed with an MTT assay. Cell cycle distribution was analyzed with a Chi² test. Graphs are an average of 3 biological replicates (with 8 technical replicates each) ± SEM. Scale bar (C) is 200 μm.

Fig 2. DOX stimulates cardiac fibroblast mitochondrial ROS production and membrane depolarization in WT cells.

(A) DCFDA-stained cells were exposed to 1, 3, or 5 μL for 4 hrs. Fluorescence from ROS-reduced DCFDA was measured to determine relative ROS production. After standard treatment, cells were incubated with (B) MitoSOX Red, (D) JC-1, and (F) Mitotracker Green. Fluorescent signal for MitoSox Red and Mitotracker Green was measured via flow cytometry, while the two wavelengths for JC-1 were assessed using a spectrophotometer. (C), (E), and (G) are representative images of each stain, MitoSox, JC-1, and Mitotracker Green, respectively. Quantification includes at least 3 biological replicates and is represented as average ± SEM.

Fig 3. DOX-induced mitochondrial dysfunction is partially ameliorated in p53^-/- fibroblasts.

Oxygen consumption (A), (B), and ECAR via pH (E), (F) was measured before and after the addition of mitochondrial stressors in CFs previously exposed to DOX. (C), (D) demonstrate the metabolic potential of oxidative respiration in WT and p53^-/- cells, respectively. Metabolic potential is a ratio of OCR after mitochondrial stressors over OCR at baseline. Metabolic potential of glycolysis was similarly calculated (G), (H).Quantification included 3 biological replicates and is represented as average ± SEM.

Fig 4. Mitophagy and biogenesis balance.

(A) Western blot demonstrates relative protein content of p53 in WT control, Dox-exposed, and FCCP-treated cells with standard treatment. (B) Genotpying confirmed mice without the p53 allele and (C) null protein expression was verified via WB. (F), (G) PGC1α protein expression was measured using WBs. Densitometry of WB bands was obtained using image J and all samples were normalized to total protein content. Quantification includes 3+ biological replicates and is represented as average ± SEM.

Fig 5. Parkin and p53 exhibit different staining patterns in response to FCCP and DOX.

After standard treatment, WT (A) and p53^-/- were stained with fluorescent p53 and Parkin antibodies. Parkin expression in p53^-/- cells was upregulated and exposure time had to be decreased compared to WT cells to avoid oversaturation. 40X Magnification.

Fig 6. DOX increases Parkin: p53 interactions.

(B, C) A proximity ligation assay demonstrates the increased interactions between the Parkin and p53 proteins in WT cells exposed to DOX. (A) Quantification of fluorescent signal. Quantification consists of 3–4 biological replicates with at least 10 fields of view acquired at 20X magnification. Samples were normalized to nuclear stain. Average ± SEM.

Fig 7. Parkin/p53 interference.

DOX induced a deleterious phenotype in WT CFs. Due to the cell’s inability to respond to multiple stressors, an early DCM gene profile was adopted. This included increased cardiac remodeling genes and increased inflammatory signaling. The phenotype also removes the cell’s ability to respond to injury by inhibiting proliferation and migration. Lastly, the cell is open to increased damage as it is unable to respond to metabolic stressors due to mitochondrial dysfunction and deficits in clearing the dysfunctional mitochondria.