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. 2017 Jun;39(6):1409-1420.
doi: 10.3892/ijmm.2017.2979. Epub 2017 May 8.

HO-1/EBP interaction alleviates cholesterol-induced hypoxia through the activation of the AKT and Nrf2/mTOR pathways and inhibition of carbohydrate metabolism in cardiomyocytes

Xiaohan Jin  1 , Zhongwei Xu  2 , Jin Cao  3 , Rui Yan  2 , Ruicheng Xu  3 , Ruiqiong Ran  1 , Yongqiang Ma  1 , Wei Cai  1 , Rong Fan  2 , Yan Zhang  2 , Xin Zhou  1 , Yuming Li  1
Affiliations

HO-1/EBP interaction alleviates cholesterol-induced hypoxia through the activation of the AKT and Nrf2/mTOR pathways and inhibition of carbohydrate metabolism in cardiomyocytes

Xiaohan Jin et al. Int J Mol Med. 2017 Jun.

Abstract

Heme oxygenase-1 (HO-1) is an inducible and cytoprotective enzyme that provides a defense against oxidant damage. The present study screened 137 HO-1/interacting proteins using a profound co-immunoprecipitation (Co-IP) coupled with proteomics, and profiled the global HO-1 interactome network, including oxidative phosphorylation, endoplasmic reticulum and transport vesicle functions. Among these molecules, we observed that a novel interactor, emopamil-binding protein (EBP), is closely related to the cholesterol metabolism process. This study demonstrated that cholesterol promotes excessive oxidative stress and alters the energy metabolism in cardiomyocytes, further triggering numerous cardiovascular diseases. We observed that cholesterol caused the overexpression of EBP and HO-1 by the activation of AKT and Nrf2/mTOR pathways. In addition, HO-1 and EBP performed a myocardial protective function. The overexpression of HO-1 alleviated the cholesterol-induced excessive oxidative stress status by inhibition of the carbohydrate metabolism. Notably, we also confirmed that the loss of partial HO-1 activity aggravated the oxidative damage and cardiac systolic function induced by a high-fat diet in HO-1 heterozygous (HO-1+/-) mice. These findings indicate that the HO-1/EBP interaction plays a protective role in alleviating the dysfunction of oxidative stress and cardiac systolic function induced by cholesterol stimulation.

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Figures

Figure 1
Figure 1
Global profiling and mapping of the heme oxygenase-1 (HO-1)-interacting protein network. (A) The major interactive proteins from two repeats of the MS analysis are shown in the table. (B) The western blot analysis results indicated that HO-1 and emopamil-binding protein (EBP) are interactive. (C) The network of HO-1 interacting proteins using Cytoscape 3.3 software.
Figure 2
Figure 2
Effects of cholesterol on the proliferation ability of H9c2 cells. Cholesterol inhibited the proliferation of H9c2 cells in a time- and dose-dependent manner. Data are presented as means ± SEM of three individual experiments. P values were calculated using one-way ANOVA. *P<0.05 vs. the control group.
Figure 3
Figure 3
Effects of cholesterol on the expression of heme oxygenase-1 (HO-1) and emopamil-binding protein (EBP) in H9c2 cells following cholesterol stimulation. (A) Western blot analysis was used to evaluate the expression levels of HO-1 and EBP. (B and C) The expression levels of HO-1 and EBP quantified by densitometry. β-actin was used as an internal control. Data are presented as means ± SEM of three individual experiments. P-values were calculated using one-way ANOVA. *P<0.05 vs. the control group.
Figure 4
Figure 4
Effects of cholesterol on the expression of heme oxygenase-1 (HO-1)-related molecules. (A) Western blot analysis was used to evaluate the expression levels of nuclear factor erythroid 2-like 2 (Nrf2), AKT, p-AKT, mTOR and p-mTOR proteins (B–D) The expression levels of Nrf2, AKT, p-AKT, mTOR and p-mTOR proteins as quantified by densitometry. β-actin was used as an internal control. Data are presented as means ± SEM of three individual experiments. P-values were calculated using one-way ANOVA. *P<0.05 vs. the control group.
Figure 5
Figure 5
Effects of cholesterol on the expression of nuclear factor erythroid 2-like 2 (Nrf2). (A) Western blot analysis was used to evaluate the expression levels of Nrf2 in the cytoplasm. (B) Western blot analysis was used to evaluate the expression levels of Nrf2 in the nucleus. (C and D) The expression levels of Nrf2 in the cytoplasm and nucleus as quantified by densitometry. β-actin was used as an internal control. Data are presented as means ± SEM of three individual experiments. P values were calculated using one-way ANOVA. *P<0.05 vs. the control group.
Figure 6
Figure 6
Measurements of lipid accumulation and location of heme oxygenase-1 (HO-1) and emopamil-binding protein (EBP) in H9c2 cells following cholesterol stimulation. (A) Oil Red O staining indicated that the concentration of cholesterol was increased in the cholesterol groups. (B) The interaction between HO-1 and EBP was noted in the cell membrane, cytoplasm and nucleus, and both showed the same upregulated expression trend using confocal microscopy (magnification, ×200).
Figure 7
Figure 7
Cholesterol inhibits the expression of LDH and activates the expression of hypoxia inducible factor-1α (HIF-1α) and mitochondrial enzymes. (A) Western blot analysis was used to evaluate the expression levels of each protein. β-actin was used as an internal control. (B–F) The expression level of each protein was quantified by densitometry. Data are presented as means ± SEM of three individual experiments. P-values were calculated using one-way ANOVA. *P<0.05 vs. the control group.
Figure 8
Figure 8
Measurements of left ventricular short axis shortening (LVFS%), left ventricular ejection fraction (LVEF%) and left ventricular (LV) mass in the wild-type (WT) and heme oxygenase-1 (HO-1)+/− mice in 5 individual experiments using cardiac ultrasound. (A) Images of the left ventricle from SAX views illustrating the measurements obtained. (B) Images of the left ventricle from PSLAX views illustrating the measurements obtained. The diameters of mice fed a high-fat diet were decreased compared to the normal diet groups from the images.
Figure 9
Figure 9
Measurements of lipid and the location of heme oxygenase-1 (HO-1) and emopamil-binding protein (EBP) in myocardial tissues. (A) Oil Red O staining indicated that the concentration of lipids was increased in the high-fat diet groups and was more significant in the HO-1+/− groups. (B) HO-1 and EBP had the same location and the expression levels were downregulated in the HO-1+/− group and was upregulated after cholesterol stimulation (magnification, ×200).
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