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. 2010 Oct 19;107(42):18121-6.
doi: 10.1073/pnas.1009700107. Epub 2010 Oct 4.

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Min Zhang  1 , Alison C Brewer, Katrin Schröder, Celio X C Santos, David J Grieve, Minshu Wang, Narayana Anilkumar, Bin Yu, Xuebin Dong, Simon J Walker, Ralf P Brandes, Ajay M Shah
Affiliations

NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis

Min Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROS sources remains unclear. Here we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level, which increases in cardiomyocytes under stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte hypoxia inducible factor 1 and the release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a unique inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of nonspecific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cardiac Nox4 induction during stress and generation of Nox4-null mice. (A) Changes in Nox4 expression after pressure overload (Band), myocardial infarction (MI), or in vitro hypoxia (Hyp; 24 h) compared with respective controls (Ctl). **P < 0.01; n = 4–6/group. (B) Nox4 expression in 3-mo (3m)- and 12-mo (12m)-old mice compared with fetal heart. **P < 0.01; n = 5/group. (C) Western blots showing loss of Nox4 protein in heart and kidney of homozygous Nox4-KO mice (Top) and lack of change in cardiac Nox2 and p22phox levels (Bottom). (D) H2O2 production in heart and kidney of Nox4-KO compared with WT. *P < 0.05; n = 6/group.
Fig. 2.
Fig. 2.
Nox4-null mice have exaggerated load-induced dysfunction. (A) Echocardiography of WT and KO mice subjected to 6 wk of chronic pressure overload. IVSD: interventricular septal diameter; LVESV: LV end-systolic volumes; LVEDV: LV end-diastolic volumes; EF: ejection fraction. (B) Representative H&E-stained longitudinal sections of WT and KO hearts. Scale bar: 2 mm. (C and D) Representative sections for cardiomyocyte area (WGA-stained) and interstitial fibrosis (Picrosirius red-stained), respectively. Scale bars: 20 μm. Mean data are shown at the right. **P < 0.01; *P < 0.05 for band vs. respective sham; ##P < 0.01 for KO band vs. WT band; n = 12–15/group. LV/BW: LV/body weight.
Fig. 3.
Fig. 3.
Cardiomyocyte-targeted Nox4 overexpression protects against load-induced dysfunction. (A) Protein expression in hearts of Nox4-transgenic mice (TG) and wild-type littermates (WT). (B) H2O2 production in TG compared with WT myocardium. **P < 0.01; n = 6/group. (C) Contractile assessment of pressure-overloaded Nox4-transgenic and WT mice by LV pressure–volume analysis. End-systolic pressure volume relation (ESPVR) and preload-recruitable stroke work (PRSW) are measures of systolic function. LV end-diastolic pressure (LVEDP) and dP/dtmin are measures of diastolic function. Representative pressure–volume curves are shown at the right. **P < 0.01; *P < 0.05 for band vs. respective sham; ##P < 0.01; #P < 0.05 for TG band vs. WT band; n = 15/group. (D–F) Mean data for heart hypertrophy (n > 20/group), cardiomyocyte cross-sectional area (n = 6/group), and interstitial fibrosis (n = 6/group), respectively. Statistics in D–F as in C.
Fig. 4.
Fig. 4.
Nox4-dependent maintenance of myocardial capillary density. (A) Representative LV sections from Nox4-null mice and WT littermates stained with isolectin B4 to label myocardial capillaries (yellow) and WGA to outline cardiomyocytes (red). 1, WT sham; 2, KO sham; 3, WT band; 4, KO band. Mean data at the right (n = 7–12/group). (B) Increased myocardial capillary density in Nox4-transgenic mice. Representative LV sections at the left. 1, WT sham; 2, TG sham; 3, WT band; 4, TG band. Mean data to the right (n = 6/group). **P < 0.01 for band vs. respective sham; ##P < 0.01; #P < 0.05 for TG or KO band vs. WT band. Scale bars: 20 μm.
Fig. 5.
Fig. 5.
Nox4-dependent increase in VEGF and Hif1α levels in heart. (A) Western blots for VEGF in LV of Nox4-transgenic mice (Upper left) and Nox4-null mice (Upper right) and respective WT littermates subjected to pressure overload or control surgery. Mean data are shown below. ##P < 0.01; #P < 0.05 for TG or KO vs. respective WT; n = 4/group. (B) Hif1α protein levels in LV of Nox4-transgenic mice (Upper left) or Nox4-null mice (Upper right) compared with respective WT. #P < 0.05 for TG vs. WT band; n = 4/group. #P < 0.05 for KO vs. WT band; n = 8/group.
Fig. 6.
Fig. 6.
Enhancement of cardiomyocyte Hif1α and VEGF by Nox4. Nox4-dependent regulation of Hif1α protein levels in (A) cardiomyocytes and (B–D) nuclear extracts of H9c2 cells. Doses were CoCl2 (200 μmol/L), diphenylene iodonium (DPI; 20 μmol/L), and PEG-catalase (catalase; 125 U/mL). Scb: scrambled siRNA. (E) Effect of β-gal control, Nox4, and a Hif1 prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG; 1 mmol/L) on Hif1α hydroxylation using anti-hydroxylated-Pro402 and -Pro564 antibodies. Representative blots at the left and mean data at the right. 1, β-gal; 2, Nox4; 3, β-gal hypoxia; 4, Nox4 hypoxia; 5, DMOG. *P < 0.05 for all groups vs. β-gal control; #P < 0.05 vs. β-gal hypoxia. Hydroxylated Hif1α was expressed relative to total HIF1α. All blots are representative of three or more independent experiments.
Fig. 7.
Fig. 7.
Nox4-dependent enhancement of angiogenic response. Immunoblotting for VEGF in (A) cardiomyocytes and (B) culture supernatants of cardiomyocytes overexpressing Nox4 or a β-gal control (Ctl). Actin and Coomassie blue (CB) staining were used to confirm equal protein loading. Mean data are shown below. **P < 0.01; *P < 0.05 for Nox4 hypoxia vs. normoxia; ##P < 0.01 for Nox4 vs. β-gal; n = 3/group. (C) Mean data showing effects of conditioned media on tube formation. **P < 0.01 vs. β-gal control (Ctl) or vs. Nox4 overexpression + VEGF antibody; n = 4/group.
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References

    1. Mudd JO, Kass DA. Tackling heart failure in the twenty-first century. Nature. 2008;451:919–928. - PubMed
    1. Catalucci D, Latronico MV, Ellingsen O, Condorelli G. Physiological myocardial hypertrophy: How and why? Front Biosci. 2008;13:312–324. - PubMed
    1. Heineke J, Molkentin JD. Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol. 2006;7:589–600. - PubMed
    1. Giordano FJ. Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest. 2005;115:500–508. - PMC - PubMed
    1. Anilkumar N, Sirker A, Shah AM. Redox sensitive signaling pathways in cardiac remodeling, hypertrophy and failure. Front Biosci. 2009;14:3168–3187. - PubMed

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