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. 2009 Nov;47(5):730-42.
doi: 10.1016/j.yjmcc.2009.08.010. Epub 2009 Aug 18.

Extracellular superoxide dismutase regulates cardiac function and fibrosis

Corrine R Kliment  1 , Hagir B Suliman, Jacob M Tobolewski, Crystal M Reynolds, Brian J Day, Xiaodong Zhu, Charles F McTiernan, Kenneth R McGaffin, Claude A Piantadosi, Tim D Oury
Affiliations

Extracellular superoxide dismutase regulates cardiac function and fibrosis

Corrine R Kliment et al. J Mol Cell Cardiol. 2009 Nov.

Abstract

Extracellular superoxide dismutase (EC-SOD) is an antioxidant that protects the heart from ischemia and the lung from inflammation and fibrosis. The role of cardiac EC-SOD under normal conditions and injury remains unclear. Cardiac toxicity, a common side effect of doxorubicin, involves oxidative stress. We hypothesize that EC-SOD is critical for normal cardiac function and protects the heart from oxidant-induced fibrosis and loss of function. C57BL/6 and EC-SOD-null mice were treated with doxorubicin, 15 mg/kg (i.p.). After 15 days, echocardiography was used to assess cardiac function. Left ventricle (LV) tissue was used to assess fibrosis and inflammation by staining, Western blot, and hydroxyproline analysis. At baseline, EC-SOD-null mice have LV wall thinning and increases in LV end diastolic dimensions compared to wild-type mice but have normal cardiac function. After doxorubicin, EC-SOD-null mice have decreases in fractional shortening not apparent in WT mice. Lack of EC-SOD also leads to increases in myocardial apoptosis and significantly more LV fibrosis and inflammatory cell infiltration. Administration of the metalloporphyrin AEOL 10150 abrogates the loss of cardiac function, and potentially fibrosis, associated with doxorubicin treatment in both wild-type and EC-SOD KO mice. EC-SOD is critical for normal cardiac morphology and protects the heart from oxidant-induced fibrosis, apoptosis, and loss of function. The antioxidant metalloporphyrin AEOL 10150 effectively protects cardiac function from doxorubicin-induced oxidative stress in vivo. These findings identify targets for the use of antioxidant agents in oxidant-induced cardiac fibrosis.

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Figures

Figure 1
Figure 1
EC-SOD localization was determined by immunohistochemical staining of paraffin-embedded heart sections using a Nova Red substrate. Images were captured at 40X magnification. A) EC-SOD localizes to myocardial cells and endothelial cells of the vasculature. The lack of EC-SOD results in changes in left ventricular morphology/dimensions in un-injured EC-SOD-null (KO) animals compared to wild type (decreases posterior wall thickness and increased intraventricular area) and after doxorubicin injury. B) LV posterior wall thickness determined by echocardiography (LV PWT, mm), *p<0.05 compared to Control WT; **p<0.05 compared to both WT treatment groups. C) Relative posterior wall thickness (LV PWT/ LVEDD), *p<0.05 compared to both WT treatment groups. D) LV end diastolic dimensions (LVEDD, mm), *p<0.05 compared to both WT treatment groups. All data are reported as mean ± SEM and analyzed by 2-way ANOVA with Bonferroni post-test.
Figure 2
Figure 2
Lack of EC-SOD results in a significant decrease in cardiac function after doxorubicin treatment. A) Percent fractional shortening in wild type and EC-SOD KO mice. *p<0.05 compared to control treatment; n=7-8; B) Percent ejection fraction of the left ventricle, *p<0.05 compared to control treatment; n=7-8; C) Representative images for wild type and EC-SOD KO groups depicting 4μm H&E-stained cardiac sections at 2X magnification and B- and M-mode images of the left ventricle from echocardiography, n=7-8.
Figure 3
Figure 3
EC-SOD KO mice have significantly more left ventricular fibrosis after doxorubicin. Fibrosis is evident in paraffin tissue sections by increased Sirius Red within the LV wall tissue of doxorubicin treated wild type and EC-SOD KO mice. Sirius Red staining: A) Control WT mouse; B) Control EC-SOD KO; C) Doxorubicin-treated WT; D) Doxorubicin-treated EC-SOD KO; Increased collagen content is also evident by Trichrome staining (blue staining) in the LV posterior wall: E) Control WT; F) Control EC-SOD KO; G) Doxorubicin-treated WT; H) Doxorubicin-treated EC-SOD KO; I) Collagen deposition (blue staining) at higher magnification in the LV tissue of a doxorubicin-treated EC-SOD KO mouse. J) Cellular ultra-structural pathology can be appreciated by cytoplasmic vacuolization (arrows) in cardiac sections from a doxorubicin-treated EC-SOD KO mouse. These are also noted in section I. Hydroxyproline: K) Significant increase in hydroxyproline content after doxorubicin injury (μg/mg LV tissue); *p<0.05 WT dox or KO dox compared to respective control mice. Bar with *p<0.05, EC-SOD KO doxorubicin compared to WT doxorubicin, n=6-8.
Figure 4
Figure 4
Lack of EC-SOD and doxorubicin treatment results in increased markers of oxidative stress and causes changes in antioxidant responses. Carbonyl modifications and CuZnSOD (SOD1), MnSOD (SOD2) and EC-SOD (SOD3) proteins were detected by western blot analysis. Lane densities were determined and reported as mean ± SEM. A) Increased carbonyl content in the membrane protein fractions of left ventricle tissue homogenate by Oxyblot analysis. A non-derivatized sample (ND) was included as a negative control. Densitometry was completed on each lane, *p<0.05. Superoxide dismutase proteins were analyzed in the soluble protein fractions of left ventricle (LV) homogenates or serum (indicated) after doxorubicin treatment, *p<0.05: B) Serum EC-SOD; C) LV homogenate EC-SOD; D) LV homogenate CuZnSOD; p=0.12 for EC-SOD KO control compared to KO doxorubicin; E) LV homogenate MnSOD. Band net intensity was normalized to ponceau red stain or β-actin.
Figure 5
Figure 5
Lack of EC-SOD exacerbates apoptotic cell death in left ventricular tissue. Apoptosis was analyzed in the soluble protein fraction of LV homogenate samples by western blot for caspase-3 and TUNEL staining on LV tissue sections, 4μm thick. A) Caspase-3, inactive (37kDa pro-form) and active (12-17kDa form), in the soluble protein fractions of LV homogenates. Grey bars: Control group, Black bars: doxorubicin group. Normalized to coomassie blue staining, *p<0.05, n=3-5. B) Relative active caspase-3 (active to inactive form). *p<0.05. C) Quantification of TUNEL positive cells from left ventricle tissue sections (n=3 per group and strain), *p<0.05. Data are reported as the mean percent TUNEL-positive cells ± SEM. D) Representative TUNEL staining of LV sections from each treatment group. Top panel: TUNEL (red), middle panel: DAPI nuclear stain (blue), bottom panel: overlay of TUNEL and DAPI.
Figure 6
Figure 6
Antioxidant AEOL 10150 prevents decreases in fractional shortening and ejection fraction due to doxorubicin-induced oxidative injury. AEOL was administered by twice daily sub-cutaneous injections at 5mg/kg/injection for 15 days after doxorubicin. Female wild type and EC-SOD KO mice were used at 8-10 weeks old (n=6-8 per treatment group). Cardiac function was assessed by echocardiography. A) Percent fractional shortening, *p<0.05. B) Percent ejection fraction, *p<0.05. 2-way statistical analysis with Bonferroni post-test was used. C) Hydroxyproline quantification of left ventricle/intraventricular septum tissues. *p<0.05, mean hydroxyproline ± SEM.
Figure 7
Figure 7
Lack of EC-SOD and doxorubicin treatment results in increased inflammation. Left ventricle tissue sections were stained for CD45, a marker of inflammatory cells, and DAPI nuclear stain. A) Doxorubicin treatment causes an increase in CD45-positive inflammatory cells in the left ventricle of both wild type and EC-SOD KO mice, with enhanced inflammatory cell infiltration in EC-SOD KO mice. (B-C) Blood smears, from wild type and EC-SOD KO mice treated with doxorubicin with or without the antioxidant agent AEOL10150, were stained with Diff Quick. Inflammatory cell differentials (macrophages, neutrophils and lymphocytes) were counted for twenty high power fields (HPFs) at 40x magnification on a bright field microscope, n=6-9 mice per treatment group. Blood smear cell counts: mean cell count ± SEM B) Macrophages, *p<0.05; C) Neutrophils, *p<0.05, ^p=0.09; D) Lymphocytes, *p<0.05.
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References

    1. Chen EP, Bittner HB, Davis RD, Folz RJ, Van Trigt P. Extracellular superoxide dismutase transgene overexpression preserves postischemic myocardial function in isolated murine hearts. Circulation. 1996 Nov 1;94(9 Suppl):II412–7. - PubMed
    1. Lu Z, Xu X, Hu X, Zhu G, Zhang P, van Deel ED, et al. Extracellular superoxide dismutase deficiency exacerbates pressure overload-induced left ventricular hypertrophy and dysfunction. Hypertension. 2008 Jan;51(1):19–25. - PMC - PubMed
    1. Fattman CL, Schaefer LM, Oury TD. Extracellular superoxide dismutase in biology and medicine. Free radical biology & medicine. 2003 Aug 1;35(3):236–56. - PubMed
    1. Folz RJ, Crapo JD. Extracellular superoxide dismutase (SOD3): tissue-specific expression, genomic characterization, and computer-assisted sequence analysis of the human EC SOD gene. Genomics. 1994 Jul 1;22(1):162–71. - PubMed
    1. Folz RJ, Guan J, Seldin MF, Oury TD, Enghild JJ, Crapo JD. Mouse extracellular superoxide dismutase: primary structure, tissue-specific gene expression, chromosomal localization, and lung in situ hybridization. American journal of respiratory cell and molecular biology. 1997 Oct;17(4):393–403. - PubMed

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