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Grb2-associated binder 1 is essential for cardioprotection against ischemia/reperfusion injury

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Abstract

We have shown recently that endothelial Grb-2-associated binder 1 (Gab1), an intracellular scaffolding adaptor, has a protective effect against limb ischemia via mediating angiogenic signaling pathways. However, the role of Gab1 in cardiac ischemia/reperfusion (I/R) injury remains unknown. In this study, we show that Gab1 is required for cardioprotection against I/R injury. I/R injury led to remarkable phosphorylation of Gab1 in cardiomyocytes. Compared with controls, the mice with cardiomyocyte-specific deletion of Gab1 gene (CGKO mice) exhibited an increase in infarct size and a decrease in cardiac function after I/R injury. Consistently, in hearts of CGKO mice subjected to I/R, the activation of caspase 3 and myocardial apoptosis was markedly enhanced whereas the activation of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK), which are critical for cardiomyocyte survival, was attenuated. Oxidative stress is regarded as a major contributor to myocardial I/R injury. To examine the role of Gab1 in oxidative stress directly, isolated adult cardiomyocytes were subject to oxidant hydrogen peroxide and the cardioprotective effects of Gab1 were confirmed. Furthermore, we found that the phosphorylation of Gab1 and Gab1-mediated activation of Akt and MAPK by oxidative stress was suppressed by ErbB receptor and Src kinase inhibitors, accompanied by an increase in apoptotic cell death. In conclusion, our results suggest that Gab1 is essential for cardioprotection against I/R oxidative injury via mediating survival signaling.

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Acknowledgments

We thank Drs. Xiaocheng Gu and Iain C. Bruce for helpful discussions and critical comments. This study was supported by research grants from National Science Foundation of China (91339111, 81170098, and Project 31221002), the Major State Basic Research Development Programs of China (2007CB512100), and Beijing Municipal Science Funds (5082009) to J.L.; the National Basic Research Program of China (2011CB503903), National Natural Science Foundation of China (81030001) and Projects for International Cooperation and Exchanges NSFC (30910103902) to Y.Z.; the Key Project for Drug Discovery and Development in China (2009ZX09501-027) to X.Y. and a National Institutes of Health Research Grant to G.S.F. (NIHR01HL096125).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Laboratory of Vascular Biology, Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing, 100871, China

    Lulu Sun, Beibei Jiang, Yanli Li, Qiuping Deng, Ying Yang, Rongli Zhang, Yao Lu, Yingqing Huo & Jincai Luo

  2. Third Hospital, Peking University and Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides Ministry of Health, Beijing, China

    Chao Chen, Min Sun, Xiangbo An & Youyi Zhang

  3. State Key Laboratory of Proteomics, Institute of Biotechnology and Genetic Laboratory of Development and Disease, Beijing, China

    Xiao Yang

  4. Animal Center, Peking University, Beijing, China

    De-Sheng Zhu

  5. Department of Pathology, School of Medicine, University of California, San Diego, CA, 92093-0864, USA

    Gen-Sheng Feng

  6. Institute of Vascular Medicine, Peking University Third Hospital, 49 Huayuan-Bei Rd, Beijing, 100191, China

    Youyi Zhang

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  1. Lulu Sun
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Additional information

L. Sun, C. Chen and B. Jiang are co-first authors.

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395_2014_420_MOESM1_ESM.tif

Online resource 1 The time course of the ratios of phosphorylation levels of Erk and Akt in ischemia area and non-ischemia area in rat hearts subjected to I/R. The relative band intensities of phosphor-Erk/Akt and total Erk/Akt were measured by NIH imaging system and normalized to the values of GAPDH loading control (*p<0.05). n = 3 per group (TIFF 13392 kb)

395_2014_420_MOESM2_ESM.tif

Online resource 2 The activations of Akt and MAPK were significantly reduced in Gab1-deficient hearts exposed to the ischemia (30 min) and reperfusion at early time points. a The phosphorylation of Gab1 and the activation of its downstream signaling pathways in the hearts subject to the ischemia of 30 min and reperfusion at early time points. b The activations of Akt and MAPK were significantly reduced in Gab1-deficient hearts exposed to the ischemia (30 min) and reperfusion at 2.5 min. Statistic analysis of the intensities of phosphor-Erk/Akt and total Erk/Akt was measured by NIH imaging system and normalized to the values of GAPDH loading control. (*p<0.05, **p<0.01) NS, no significant difference. n = 4 per group (TIFF 14442 kb)

395_2014_420_MOESM3_ESM.tif

Online resource 3 CGKO mice displayed normal cardiac vasculatures. a Representative photographs of heart angiography (left) and statistic analysis of the length of left anterior descending artery (red arrows) (right). NS, no significant difference. n = 5 per group. b Representative photographs of section HE staining of the heart. The vessels are stained with the antibody against CD31, an endothelial specific marker. Insets show magnificent pictures. (TIFF 15843 kb)

395_2014_420_MOESM4_ESM.tif

Online resource 4 No difference in the apoptosis of endothelial cells in the hearts of CGKO and CTR mice after I/R (I, 30 min; R, 2 h). a The representative images of CD31 (brown) and TUNEL (green) double staining analysis of non-ischemia area (N) and ischemia area (A) of the hearts from CGKO and CTR mice after I/R. The nuclei were counterstained with hematoxylin (blue). b The percentage of TUNEL-positive endothelial cells among all the apoptotic cells in ischemic area that includes infarct and peri-infarct border zones. NS, no significant difference. Scale bar = 20 μm. n = 4 per group (TIFF 15397 kb)

395_2014_420_MOESM5_ESM.tif

Online resource 5 No apoptotic inflammatory cells with the markers of CD45 (brown, a) and Mac2 (brown, b) in the hearts of CGKO and CTR mice after I/R (I, 30 min; R, 2 h). The representative double staining images show inflammatory cells (brown) and apoptotic cells (TUNEL, green) with hematoxylin (blue) for counterstaining nuclei. Scale bar = 20 μm. n = 4 per group (TIFF 17754 kb)

395_2014_420_MOESM6_ESM.tif

Online resource 6 No difference in the production of oxidative species in the heart of CGKO and CTR mice before and after I/R injury. a Representative photographs of heart horizontal sections stained with Dihydroethidium for oxidative species (DHE, red) and DAPI for nuclei (Blue). N: non-ischemia area; A: ischemia area. b Statistic analysis of relative fluorescence intensity of DHE staining. NS, no significant difference. Scale bar = 50 μm. n = 3 per group (TIFF 14565 kb)

395_2014_420_MOESM7_ESM.tif

Online resource 7 No difference in the survival rate of cardiac endothelial cells, which were isolated from CGKO and CTR mice, under oxidative stress. Representative morphological images (top panel) and quantitative survival rates (bottom panel) of endothelial cells isolated from adult CGKO and CTR mice were treated with or without H2O2 (100 μM) for 8 hrs and subject to TUNEL analysis (green) and CD31 staining (red). NS, no significant difference. n = 4 per group (TIFF 13840 kb)

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Sun, L., Chen, C., Jiang, B. et al. Grb2-associated binder 1 is essential for cardioprotection against ischemia/reperfusion injury. Basic Res Cardiol 109, 420 (2014). https://doi.org/10.1007/s00395-014-0420-2

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