Elsevier

Biomedicine & Pharmacotherapy

Volume 93, September 2017, Pages 1197-1204
Biomedicine & Pharmacotherapy

Role of GAB1/PI3K/AKT signaling high glucose-induced cardiomyocyte apoptosis

https://doi.org/10.1016/j.biopha.2017.07.063Get rights and content

Abstract

Gestational diabetes mellitus (GDM) is a risk factor for abnormal heart development. Previous work showed that a decrease of myocardial cells and an increase of apoptotic cells leading to heart defects under hyperglycemia, and many genes and protein have been found to play important roles in cardiomyocyte apoptosis. However, there are still many blind nodes in HG-induced cardiac apoptosis. Our study showed that down-regulation of GAB1 occurred concurrently with HG-induced cardiomyocytes apoptosis and in the heart tissues of offspring of diabetic rats in vitro and in vivo. MTT and apoptosis assay showed GAB1 played a key role in mediating HG-induced apoptosis of cardiomyocytes. Down-regulation of XIAP and increased activities of Caspase3/7 was associated with GAB1-mediated cardiomyocyte apoptosis in response to HG treatment. Further study showed that the phosphorylation levels of AKT (Ser473) decreased after HG treatment. Over-expression of GAB1 resisted the reduction in AKT phosphorylation in response to HG. LY294002, which is an effective inhibitor of the PI3 K/AKT signaling pathway, partly inhibited GAB1 to suppress apoptosis induced by HG in cardiomyocytes, and partly suppressed GAB1 to resist the decrease of XIAP in response to HG, indicating AKT signaling, XIAP, and Caspase3/7 participated in GAB1-mediated cardiomyocyte apoptosis in response to HG. Generally, we demonstrate a novel role of GAB1 and its down-stream signaling PI3 K/AKT for modulating cardiomyocyte apoptosis in response to high glucose in vitro and vivo.

Graphical abstract

This study analyzed the role and mechanism of GAB1/AKT signaling in HG-induced apoptosis. The down-regulation of GAB1 occurred concurrently with HG-induced cardiomyocytes apoptosis and in the heart tissues of offspring of diabetic rats in vitro and in vivo. Down-regulation of XIAP and increased activities of Caspase3/7 was associated with GAB1-mediated cardiomyocyte apoptosis in response to HG treatment. Further study showed that the phosphorylation levels of AKT (Ser473) decreased after HG treatment. Over-expression of GAB1 resisted the reduction in AKT phosphorylation in response to HG. LY294002, which is an effective inhibitor of the PI3K/AKT signaling pathway, partly inhibited GAB1 to suppress apoptosis induced by HG in cardiomyocytes, and partly suppressed GAB1 to resist the decrease of XIAP in response to HG, indicating AKT signaling, XIAP and Caspase3/7 participated in GAB1-mediated cardiomyocyte apoptosis in response to HG.

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Introduction

Congenital heart disease (CHD) composes the largest group of congenital anomalies and is also an important cause of mortality and morbidity in children. The worldwide prevalence of CHD is estimated to be eight to ten per 1000 live births, but the prevalence greatly varies between regions [1], [2]. Postnatal studies showed many of the abnormalities were congenital heart diseases (CHDs) in infants with diabetic mothers [3], [4], [5]. GDM is one of the maternal diseases which cause congenital anomalies in infants [6], [7], [8]. structural defects in infants with diabetic mothers increases than those with normal mothers [9], [10], [11]. Now study on the molecular basis of pathogenesis of CHDs in pregestational diabetes was few and factors responsible for the high incidence of CHDs in pregestational diabetes are still obscure. Several reports and our previous results based on animal studies have suggested that a decrease of myocardial cells and an increase of apoptotic cells may play a role in pathogenesis of CHDs [12], [13], [14], [15], [16]. While much work still needs to be done to elucidate this important mechanism of how maternal diabetes influence cell apoptosis and cardiac development.

GAB1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules [17], [18], which recruits phosphatidylinositol-3 kinase (PI3 K) and other effector proteins in response to a broad range of growth factor [19], [20], [21]. Some reports showed that GAB1 is required for fibroblast cell survival and maintaining cardiac function, and its overexpression reduces Apoptosis in rat neonatal cardiomyocytes following hypoxic stress [22], [23]. These data inspired us to speculate that Gab1 may participate in HG-induced cardiomyocyte apoptosis and developmental defects of the heart.

The PI3 K/Akt pathway is one of the strongest intracellular prosurvival signaling systems. For example, in a study of four downstream effectors of growth factor receptors, PI3 K, Ras, Raf, and Src, PI3 K was the only one to inhibit apoptosis after serum withdrawal [24], [25]. A number of downstream targets that regulate apoptosis are modulated by Akt including members of the Bcl-2/CED9 family Bax, Bak, and BAD [26], [27], [28], XIAP, caspase-9, NF-B families, and so on [29], [30], [31].

XIAP is one of the strongest apoptotic-inhibiting factors, which can inhibit activities of caspase-3, 7 and 9 directly, thus protecting the cell from apoptosis [32], [33], [34]. Meanwhile, XIAP is a critical downstream element of AKT, the activity of which can be promoted by AKT-mediated phosphorylation at Ser87. After phosphorylation, XIAP cannot be degraded by ubiquitin, so the level of XIAP intracellular expression increases, inhibiting apoptosis [32], [35]. However, whether or not the AKT/XIAP signaling pathway involved in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced CHDs have received little attention so far.

In the present study, we aimed to investigate whether the GAB1 and its down-stream signaling PI3 K/AKT play important role in maternal diabetes-induced CHDs and to explore the role and mechanisms of cardiomyocyte apoptosis in response to high glucose in vitro and vivo.

Section snippets

Cell culture

H9C2 cells were maintained in cardiac myocyte medium and Dulbecco's modified Eagle's medium (DMEM) supplemented with10% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C.

Down-regulation of GAB1 was associated with HG-induced apoptosis in cardiomyocytes in vivo and in vitro

Several reports and our previous results based on animal studies have suggested that a decrease of myocardial cells and an increase of apoptotic cells may play a role in pathogenesis of CHDs [12], [13], [14], [15], [16]. In this study, Host33342 staining result showed that many cardiomyocytes cells in ventricular wall, from diabetic offspring exhibited the apoptotic characteristics including cell nuclei shrinkage and chromatin condensation. Immunofluorescence staining showed that decrease

Discussion

Several reports based on animal studies have suggested that cell apoptosis may play a role in pathogenesis of CHDs [12], [13], [14]. Though our previous works have showed that some pathways or genes play important roles in the cardiomyocyte apoptosis [15], [16], much work still needs to be done to elucidate this important mechanism of cardiomyocyte apoptosis in response to HG. Here, we found that the protein and mRNA levels of GAB1 decreased in the fetal heart tissue of diabetic rats (Fig. 1

Conclusion

In conclusion, we analyzed the role and mechanism of GAB1/AKT signaling in HG-induced apoptosis. The down-regulation of GAB1 occurred concurrently with HG-induced cardiomyocytes apoptosis and in the heart tissues of offspring of diabetic rats in vitro and in vivo. MTT and apoptosis assay showed GAB1 played a key role in mediating HG-induced apoptosis of cardiomyocytes. Down-regulation of XIAP and increased activities of Caspase3/7 was associated with GAB1-mediated cardiomyocyte apoptosis in

Conflict of interest

We declare that there are no conflicts of interests.

Funding

This research was supported by grants from the National Key Research and Development Program of China (Grant No.  2016 YFC1000300, 2016 YFC1000307, 2016 YFC1000307-4), the National Natural Science Foundation of China (Grant No.  31301145) and the Central Public interest Scientific Institution Basal Research Fund (Grant No.2016GJZ05).

Acknowledgements

We are grateful to everyone who helped complete this research successfully.

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