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. 2010 Aug;59(8):2033-42.
doi: 10.2337/db09-1800. Epub 2010 Jun 3.

Deficiency of rac1 blocks NADPH oxidase activation, inhibits endoplasmic reticulum stress, and reduces myocardial remodeling in a mouse model of type 1 diabetes

Jianmin Li  1 , Huaqing Zhu, E Shen, Li Wan, J Malcolm O Arnold, Tianqing Peng
Affiliations

Deficiency of rac1 blocks NADPH oxidase activation, inhibits endoplasmic reticulum stress, and reduces myocardial remodeling in a mouse model of type 1 diabetes

Jianmin Li et al. Diabetes. 2010 Aug.

Abstract

Objective: Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes.

Research design and methods: Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose.

Results: In diabetic hearts, NADPH oxidase activation, its subunits' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose-induced ER stress was inhibited by blocking Rac1 or NADPH oxidase.

Conclusions: Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy.

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Figures

FIG. 1.
FIG. 1.
Effects of Rac1 knockout on NADPH oxidase and ROS production. Rac1-ko mice (KO) and their WT littermates were injected with STZ. Two months later, NADPH oxidase activation and expression and ROS production in heart tissues were measured. A: Translocalization of Rac1 and p67phox to the membrane. The protein levels of Rac1 (mRac1) and p67phox (mp67phox) were decreased in the membrane fractions of Rac1 KO compared with WT diabetic hearts. The top panel is the representative Western blot for membrane mRac1, mp67phox, and gp91phox from three out of five to six different hearts in each group, and the lower panel is the quantification of mRac1, mp67phox, and gp91phox. NADPH oxidase activity (B), superoxide production (C), and H2O2 production (D) were decreased in diabetic Rac1 KO compared with WT hearts. C is the representative DHE staining (Red signal) for superoxide production from five to six different hearts in each group. E: Rac1, p67phox, and gp91pho protein expression. The protein levels of Rac1 and p67pho were decreased in Rac1 KO compared with WT diabetic hearts. The top panel is the representative Western blot for Rac1, p67phox, and gp91phox from three out of five to six different hearts in each group and the lower panel is the quantification of Rac1, p67phox, and gp91phox. F: Mitochondrial superoxide production was increased in WT diabetic hearts, which was significantly decreased in Rac1 KO hearts. G: Thioredoxin reductase activity was preserved in Rac1 knockout diabetic hearts. Magnification ×40. Data are means ± SD, n = 5–8. *P < 0.05 vs. sham; #P < 0.05 vs. STZ in WT. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
NADPH oxidase and ROS production. Wild-type mice were rendered diabetic by STZ injection, and apocynin was administrated in the drinking water for 2 months. Apocynin treatment significantly reduced NADPH oxidase activity (A) and H2O2 production (B) in diabetic heart tissues. Data are means ± SD, n = 6 – 8. *P < 0.05 vs. nondiabetes (ND) in vehicle; #P < 0.05 vs. diabetes (DM) in vehicle. Cultured adult rat cardiomyocytes were transfected with gp91phox siRNA, Nox4 siRNA, or a scrambled siRNA as a control and then incubated with normal glucose (NG, 5.5 mmol/l) or high glucose (HG, 33 mmol/l) for 24 h. NADPH oxidase activity (C and E) and superoxide production (D and F) were measured in cardiomyocytes. Data are means ± SD, n = 3–4. *P < 0.05 vs. scrambled siRNA in NG; #P < 0.05 vs. scrambled siRNA in HG.
FIG. 3.
FIG. 3.
Role of Rac1/NADPH oxidase in cardiac hypertrophy. Diabetes was induced by injection of STZ in Rac1-ko and their WT littermates. In a separate experiment, WT diabetic mice were treated with vehicle or apocynin in drinking water for 2 months. A and D: The hearts were fixed, embedded, and sectioned. Sections were stained for membranes with fluorescein isothiocyanate (FITC)-WGA and for nuclei with DAPI. Cardiomyocyte cross-sectional area was measured with an image quantitative digital analysis system. The outline of 200 cardiomyocytes was traced in each section. The mRNA levels of β-MHC (B and E) and ANP (C and F) were determined by real-time RT-PCR in Rac1-ko and WT hearts. Data are means ± SD, n = 6–8. *P < 0.05 vs. nondiabetes in WT or vehicle; #P < 0.05 vs. diabetes in WT or vehicle. O.D., optical density.
FIG. 4.
FIG. 4.
Role of Rac1/NADPH oxidase in myocardial fibrosis. Diabetes was induced by injection of STZ in Rac1-ko mice and their WT littermates. In a separate experiment, WT diabetic mice were treated with vehicle or apocynin in drinking water for 2 months. The hearts were fixed, embedded, and sectioned. Sections of heart were stained with hematoxylin and eosin and a saturated solution of picric acid containing 1% Sirius red for collagen deposition (see research design and methods). A: Representative staining for collagen deposition is presented for intra-myocardium (IM), small vessel (SV), and big vessel (BV) from each group. Collagen deposition is stained as red color. B and C: Collagen deposition was quantified as percent of cardiac area. Data are means ± SD, n = 6–8. *P < 0.05 vs. nondiabetes in WT or vehicle; #P < 0.05 vs. diabetes in WT or vehicle. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
Effect of Rac1 knockout on pro-fibrotic genes expression. Diabetes was induced by injection of STZ in Rac1-ko (KO) and their WT littermates. Two months after STZ injection, the mRNA levels of Col I (A), Col III (B), osteopontin (C), α-SMA (D), TGF-β1 (E), and TNF-α (F) were quantified in heart tissues by real-time RT-PCR. G and H: Representative immunohistological stainings for TGF-β1 (G) and TNF-α (H) from four to six different hearts in each group (yellow-brown signal). Magnification ×40. Data are means ± SD, n = 6–8. *P < 0.05 vs. nondiabetes in WT; #P < 0.05 vs. diabetes in WT. O.D., optical density. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 6.
FIG. 6.
Effect of apocynin on pro-fibrotic gene expression. Wild-type mice were rendered diabetic by STZ injection, and apocynin was administrated in the drinking water for 2 months. The mRNA levels of Col I (A), Col III (B), osteopontin (C), α-SMA (D), TGF-β1 (E), and TNF-α (F) were quantified in heart tissues by real-time RT-PCR. Data are means ± SD, n = 6–8. *P < 0.05 vs. nondiabetes in vehicle; #P < 0.05 vs. diabetes in vehicle. O.D., optical density.
FIG. 7.
FIG. 7.
Effect of Rac1 knockout on ER stress induction. Diabetes was induced by injection of STZ in Rac1-ko (KO) and their WT littermates. Two months after STZ injection, the mRNA levels of CHOP (A), GRP78 (B), and XBP1 (C) were quantified in heart tissues by real-time RT-PCR. D: GRP78 protein was also determined by immunohistological staining (yellow-brown signal). Magnification ×40. Data are means ± SD, n = 6–8. *P < 0.05 vs. nondiabetes (ND) in WT; #P < 0.05 vs. diabetes (DM) in WT. Cultured adult rat cardiomyocytes were infected with Ad-RacN17 (RacN17) or Ad-gal (gal) and then incubated with normal glucose (NG, 5.5 mmol/l) or high glucose (HG, 33 mmol/l) for 24 h. In a separate experiment, cardiomyocytes were incubated with normal or high glucose in the presence of apocynin (Apo) or vehicle (Veh) for 24 h. Western blot was performed for detection of phosphorylated PERK, cleaved ATF-6, GRP78, and GAPDH protein. Infection of Ad-RacN17 (E) and apocynin administration (F) reduced phosphorylated PERK, cleaved ATF-6, and GRP78 protein. The top panel is the representative blot from at least three different cell cultures and the lower panel is the quantification of phosphorylated PERK, cleaved ATF-6, and GRP78 protein. Data are means ± SD, n = 3–4. *P < 0.05 vs. gal or Veh in NG; #P < 0.05 vs. gal or Veh in HG. O.D., optical density. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 8.
FIG. 8.
Myocardial function in diabetic mice. Wild-type mice were rendered diabetic by STZ injection, and apocynin was administrated in the drinking water for 2 months. Mouse hearts were isolated and perfused in Langendorff system. Contractile function of heart was determined. Changes in heart rate (A), heart work (B), rate of contraction (+ dF/dTmax, C), and relaxation (−dF/dTmin, D) are presented. Data are means ± SD, n = 6–8. *P < 0.05 vs. nondiabetes in WT; #P < 0.05 vs. diabetes in WT.
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