Cardiomyocyte Specific Ablation of p53 Is Not Sufficient to Block Doxorubicin Induced Cardiac Fibrosis and Associated Cytoskeletal Changes

Doxorubicin (Dox) is an anthracycline used to effectively treat several forms of cancer. Unfortunately, the use of Dox is limited due to its association with cardiovascular complications which are manifested as acute and chronic cardiotoxicity. The pathophysiological mechanism of Dox induced cardiotoxicity appears to involve increased expression of the tumor suppressor protein p53 in cardiomyocytes, followed by cellular apoptosis. It is not known whether downregulation of p53 expression in cardiomyocytes would result in decreased rates of myocardial fibrosis which occurs in response to cardiomyocyte loss. Further, it is not known whether Dox can induce perivascular necrosis and associated fibrosis in the heart. In this study we measured the effects of acute Dox treatment on myocardial and perivascular apoptosis and fibrosis in a conditional knockout (CKO) mouse model system which harbours inactive p53 alleles specifically in cardiomyocytes. CKO mice treated with a single dose of Dox (20 mg/kg), did not display lower levels of myocardial apoptosis or reactive oxygen and nitrogen species (ROS/RNS) compared to control mice with intact p53 alleles. Interestingly, CKO mice also displayed higher levels of interstitial and perivascular fibrosis compared to controls 3 or 7 days after Dox treatment. Additionally, the decrease in levels of the microtubule protein α-tubulin, which occurs in response to Dox treatment, was not prevented in CKO mice. Overall, these results indicate that selective loss of p53 in cardiomyocytes is not sufficient to prevent Dox induced myocardial ROS/RNS generation, apoptosis, interstitial fibrosis and perivascular fibrosis. Further, these results support a role for p53 independent apoptotic pathways leading to Dox induced myocardial damage and highlight the importance of vascular lesions in Dox induced cardiotoxicity

Dox treatment induces ROS and 3-nitrotyrosine generation independent of p53 gene status in the heart.

<p>(A–C) Detection of superoxide/ROS after 3 day Dox treatment in frozen sections of wild type or p53 CKO hearts using dihydroethidium (DHE) staining. (<b>A, B</b>) DHE positive endothelial or smooth muscle cells in saline (S) or Dox (D) treated wild type (WT) hearts are indicated by arrows. (<b>C</b>) Examples of DHE positive cardiomyocytes are indicated by arrowheads. Bar, 50 µm, A–C. (<b>D–F</b>) Immunostaining of 3-nitrotyrosine (3-NT) in sections derived from saline (S) or Dox (D) treated WT (D, E) and p53 CKO (F) hearts at 3 day time point. Note the increase in 3-NT staining in cardiomyocytes after DOX treatment in both WT and p53 CKO hearts. Bar, 50 µm, D–F.</p

Quantification of cardiac fibrosis in tissue sections after 3 or 7 days of Dox treatment.

<p>Percent fibrosis values in PSFG-stained heart sections were calculated using Image processing Tool Kit software and image analysis was performed on a minimum of ten slides per animal for each treatment group (n = 3–5 mice per group). Dox (D) treatment significantly increased cardiac fibrosis in wild type (WT) and p53 CKO hearts compared to saline (S) treated hearts after 3 day (A) or 7 day (B) treatment periods. 3 day Dox time point: *<i>P</i><0.05 compared to WT(S), **<i>P</i><0.05 compared to WT(D), ANOVA, Tukey's multiple comparisons test; 7 day Dox time point: #<i>P</i><0.05 compared to WT(S), ANOVA, Tukey's multiple comparison test.</p

Expression of p53 in the heart after Dox treatment.

<p>(<b>A</b>) Very low levels of p53 immunostaining were observed in the control untreated heart. (<b>B and C</b>) Increased expression of p53 was evident in cytoplasmic and nuclear compartments of cardiomyocytes in hearts treated with Dox for 3 or 6 hrs. Bar, 20 µm (A–C). (<b>D</b>) Western blot analysis of p53 protein levels in wild type (WT) and p53 conditional knockout (MC⁺ and p53^F/F or ^F/+) hearts treated with saline (S) or Dox (D) for 3 days (3 d) or 7 days (7 d). A 25 kD band from Naphthol Blue Black stained blots was used as a loading control (LC) for protein loading.</p

Generation and characterization of p53 conditional knockout mice.

<p>(<b>A</b>) Schematic representation of transgenic alleles corresponding to MesP1-Cre (MC), floxed p53 (p53^F2-10) and conditional p53 deletion (p53^ΔF2-10) mice. Positions of exons and loxP sites are indicated by numbered black boxes and arrowheads respectively. Positions of primer pairs used for genotyping are indicated by arrows. (<b>B</b>) MC positive (+) and negative (−) mice were identified by PCR amplification of ear punch genomic DNA using NeoAL and GR1 primers. Mice homozygous (F/F) or heterozygous (F/+) for floxed p53 alleles were identified using 10FM and 10RM primers. Wt: wild type; H2O: no template control for PCR. (<b>C</b>) Excision of exons 2–10 of p53 gene in double transgenic hearts (MC⁺ and p53^F/F or ^F/+) was confirmed by PCR amplification of cardiac genomic DNA using 1FA and 10RA primers. Note the absence of PCR amplification product with 1FA/10RA primers in MC⁻ p53^F/F mice. Excision of p53 exons does not occur in MesP1 negative non-cardiacmyocytes. As a result, primers specific for intron 10 (10FA/10RA) and intron 1 (1FA/1RA) can also amplify PCR products from cardiac genomic DNA.</p

Expression of p53 and TUNEL staining in vascular cells after 3 day Dox treatment in MC⁺ p53^F/F conditional knockout hearts.

<p>Cardiac sections were processed for p53 (A), VWF (B) and nuclear (C) staining. Bar, 50 µm (A–C). (<b>D</b>) Examples of TUNEL positive vascular cells in MC⁺ p53^F/+ hearts after 3 day Dox treatment. (<b>E</b>) Examples of TUNEL positive cardiomyocytes in MC⁺ p53^F/F hearts. Bars, 50 µm, D and E.</p

Primers used for genotyping analysis.

<p>Primers used for genotyping analysis.</p

Quantification of α-tubulin levels in the hearts of Dox treated and control mice.

<p>Ventricular lysates were analyzed 3 days (A) and 7 days (B) after saline (S) or Dox (D) treatment. The levels of α-tubulin were normalized to those of CBF-A for each sample and ratios were expressed as fold changes relative to WT(S) (n = 3 hearts per group). (<b>A</b>) Following a 3 day treatment period, α-tubulin/CBF-A ratios were significantly lower in Dox treated hearts (both wild type (WT) and p53 CKO) compared to those ratios in saline treated hearts. *<i>P</i><0.05 compared to WT(S), ANOVA, Tukey's multiple comparison test. (<b>B</b>) Following a 7 day treatment period, α-tubulin/CBF-A ratios were not significantly different between Dox (D) or saline (S) treated hearts.</p

Dox treatment induces myocardial fibrosis independent of p53 gene status in the heart.

<p>(<b>A–D</b>) Representative photomicrographs of PSFG stained cardiac sections from wild type or p53 CKO hearts treated with saline (A) or Dox (B–D) at 7 day time point. Both reparative (C) and perivascular (D) types of fibrosis were evident in Dox treated MC⁺ p53^F/F conditional knockout hearts. Bar, 100 µm A–D. (<b>E</b>) Quantification of the spread of cardiac fibrosis lesions in Dox and Isoporterenol (ISO) treated hearts at 7 day time point. LL: lesion length, LW: lesion width. ISO induced LL was significantly greater (∼2.5 fold) compared to Dox induced LL (n = 3–5 mice per group, *<i>P</i><0.005 compared with other groups. ANOVA, Tukey's multiple comparison test). (<b>F</b>) No significant difference is observed between groups in heart weight and body weight ratios of mice used in these studies at 7 day time point.</p