Nox4-derived reactive oxygen species mediate cardiomyocyte injury in early type 1 diabetes

Abstract

Oxidative stress contributes to diabetic cardiomyopathy. This study explored the role of the NADPH oxidase Nox4 as a source of reactive oxygen species (ROS) involved in the development of diabetic cardiomyopathy. Phosphorothioated antisense (AS) or sense (S) oligonucleotides for Nox4 were administered for 2 wk to rats made diabetic by streptozotocin. NADPH oxidase activity, ROS generation, and the expression of Nox4, but Nox1 or Nox2, were increased in left ventricular tissue of the diabetic rats. Expression of molecular markers of hypertrophy and myofibrosis including fibronectin, collagen, α-smooth muscle actin, and β-myosin heavy chain were also increased. These parameters were attenuated by the administration of AS but not S Nox4. Moreover, the impairment of contractility observed in diabetic rats was prevented in AS- but not S-treated animals. Exposure of cultured cardiac myocytes to 25 mM glucose [high glucose (HG)] increased NADPH oxidase activity, the expression of Nox4, and molecular markers of cardiac injury. These effects of HG were prevented in cells infected with adenoviral vector containing a dominant negative form of Nox4. This study provides strong evidence that Nox4 is an important source of ROS in the left ventricle and that Nox4-derived ROS contribute to cardiomyopathy at early stages of type 1 diabetes.

Fig. 1.

Echocardiographic measurements. A: representative figure of an M-mode echocardiography done in control rats, type 1 diabetic rats (DM1), and type 1 diabetic Nox4 sense (DM1/S)- and antisense (DM1/AS)-treated rats. B and C: fractional shortening (B) and ejection fraction (C) exhibited improved cardiac function in AS Nox4 diabetes mellitus type 1-treated rats compared with diabetic rats (n = 4). *P < 0.05 vs. control rats; #P < 0.05 vs. diabetic rats.

Fig. 2.

Diabetes upregulates Nox4 protein expression and induces oxidative stress assessed by reactive oxygen species (ROS) production and NADPH oxidase activity in the heart of streptozotocin (STZ)-induced type 1 diabetic rats. A: representative Western blot of Nox1, Nox2, Nox4, and β-actin levels. B–D: histograms showing quantitation of the Western blot; results from 4 rats. E–G: Nox4 protein expression (E), superoxide production assessed by dihydroethidium (DHE) staining (F), and NADPH-dependent superoxide generation (G) were significantly higher in the left ventricle (LV) of diabetic rats compared with controls. These parameters were prevented in AS but not S-treated animals. *P < 0.05 vs. control rats; #P < 0.05 vs. diabetic rats.

Fig. 3.

Nox4 mediates diabetes-induced increase in the expression of the molecular markers of hypertrophy and fibrosis in the heart of STZ-induced type 1 diabetic rats. A: representative Western blot of fibronectin, α-smooth muscle actin (α-SMA), β-myosin heavy chain (β-MHC), and β-actin levels. B–D: histograms showing quantitation of the Western blot; results from 4 rats. Expression of these proteins was increased in LV lysates of diabetic rats compared with control rats. AS but not S-treatment for Nox4 significantly reduced the levels of these proteins. E: representative histopathological analysis of LV sections with Masson trichrome staining (fibrosis: blue color) from control, DM1, and DM1/S- and DM1/AS-treated rats. DM1 and DM1/S show high intensity of collagen blue staining in the heart tissue that was decreased in DM1/AS rats. F: histogram showing quantitation of trichrome staining; results from 4 rats. *P < 0.05 vs. control rats; #P < 0.05 vs. diabetic rats.

Fig. 4.

High glucose (HG) induces ROS production, NADPH oxidase activity, and Nox4 expression in cultured cardiac myocytes. Cardiac myocytes were exposed to high glucose (25 mmol/l) for the indicated time points. High glucose concentration resulted in increased ROS generation as measured by 2′,7′-dichlorodihydrofluorescin (DCF) fluorescence (A and B), enhanced superoxide production assessed by NADPH oxidase activity (C), and upregulated Nox4 protein expression as assessed by Western blotting (D). NG, normal glucose; RLU, relative light units. *P < 0.05.

Fig. 5.

Nox4 mediates high glucose-induced increase in the expression of the molecular markers of hypertrophy and fibrosis in cultured cardiac myocytes. Transfection of cultured cardiac myocytes with dominant negative (DN) Nox4 in the presence of high glucose (24 h) markedly reduced high glucose-induced NADPH oxidase activity (A) and protein expression of the cardiac injury markers β-MHC, α-SMA, and fibronectin (B). Ad, adenovirus. *P < 0.05 vs. NG + green fluorescent protein (GFP); #P < 0.05 vs. HG + GFP.