Original Research Cardiology 2015;130:249–259 DOI: 10.1159/000371489

Received: July 8, 2014 Accepted after revision: December 11, 2014 Published online: March 21, 2015

ß3-Integrin Inhibits Lipopolysaccharide-Induced Autophagy in Cardiomyocytes via the Akt Signaling Pathway Ying Zhu Li Li Shijin Gong Yihua Yu Haiwen Dai Guolong Cai Jing Yan Intensive Care Unit and Zhejiang Provincial Key Laboratory of Geriatrics, Zhejiang Hospital, Hangzhou, PR China

Abstract Objective: To investigate the role of β3-integrin in lipopolysaccharide (LPS)-induced autophagy in cardiomyocytes and its underlying mechanism. Methods: β3-Integrin expression in cardiomyocytes was up- or downregulated by adenovirus transfection or cyclic arginine-glycine-aspartic acid (cRGD) peptide treatment before LPS stimulation. The expression of autophagy-associated proteins (LC3-II, Beclin-1 and Bcl-2) and the activation of Akt were determined using Western blotting. Autophagosomes and autophagic vacuoles were observed using monodansylcadaverine (MDC) dye and transmission electron microscopy, respectively. Results: Downregulation of β3-integrin with cRGD peptide resulted in enhanced LC3-II and Beclin-1 and decreased Bcl-2 expression. Low Beclin-1 levels were detected after LPS stimulation in adenovirus β3-integrin-transfected cardiomyocytes. There was no significant difference in LC3-II levels between control and adenovirus β3-integrin-transfected cardiomyocytes. Enhanced accumulation of MDC dye and autophagosomes, which were inhibited by β3-integrin overexpression, were

© 2015 S. Karger AG, Basel 0008–6312/15/1304–0249$39.50/0 E-Mail [email protected] www.karger.com/crd

detected after LPS treatment. The increased phosphorylation of Akt after LPS stimulation was inhibited by cRGD and enhanced by β3-integrin overexpression. Furthermore, the Akt inhibitor triciribine inhibited the negative effect of β3integrin on autophagy, as shown by LC3-II and Beclin-1 upregulation. Conclusions: β3-Integrin inhibits LPS-induced autophagy in cardiomyocytes. The inhibition of Akt signaling might be an important mechanism in this process. © 2015 S. Karger AG, Basel

Introduction

Severe sepsis and septic shock are the leading causes of morbidity and mortality in hospitalized patients [1]. Approximately 20–50% of patients with severe sepsis and septic shock exhibit various degrees of cardiac dysfunction [2]. Compared to patients without heart impairment, the mortality rate of patients with sepsis-associated cardiac dysfunction may be increased by 20–70% [3]. However, the precise mechanism of septic heart injury remains unknown. Previous studies have suggested that cardiomyocyte autophagy is involved in the pathogenesis of sepsis-related cardiac dysfunction [4].

Li Li or Jing Yan Intensive Care Unit, Zhejiang Hospital 12 Lingyin Road Hangzhou 310013 (PR China) E-Mail lilihbch @ 163.com or yanjing2012 @ 163.com

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Key Words Akt signaling pathway · Autophagy · Cardiomyocytes · β3-Integrin · Lipopolysaccharides · Sepsis

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[19, 20]. The focal adhesion complex, which is the downstream signaling component of integrin, directly controls the activation of Akt in response to cell-matrix adhesion [21]. Therefore, β3-integrin might modulate autophagy through activation of the Akt signaling pathway. Therefore, this study aimed to investigate the role of β3-integrin in LPS-induced autophagy in cardiomyocytes. Furthermore, we examined the underlying mechanism involving the Akt signaling pathway.

Materials and Methods The experimental procedures involving animals were performed in accordance with the institutional guidelines of the National Research Council. These guidelines conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Primary Cell Culture Neonatal rat ventricular cardiomyocytes were prepared using 1- to 2-day-old Sprague-Dawley pups as described previously [22]. Cells were plated at a density of 2 × 106 cells/60-mm dish and maintained for 48 h in DMEM/F12 containing 0.3 g/l glutamine, 4.5 g/l glucose and 10% calf serum before LPS exposure. LPS (Sigma, St. Louis, Mo., USA) was administered to cells in culture (1 μg/ml) in serum-containing media. Blockade of β3-integrin with cyclic arginine-glycine-aspartic acid (cRGD; EMD Bioscience, Darmstadt, Germany) was achieved by incubation with this compound (10 μg/ ml) for 2 h prior to LPS treatment. Time zero represents cells that were incubated prior to the addition of LPS. Adenovirus Transfection The recombinant adenovirus vector was purchased from Shanghai SunBio Biomedical Technology (Shanghai, PR China). Cardiomyocytes were seeded into 60-mm dishes at a density of 2 × 106 cells. Cells were then incubated at different multiplicities of infection (MOIs) of the GFP adenovirus vector. The amount of gene transfer was determined after 24 h of incubation with the adenovirus vector using a fluorescence microscope. SDS-PAGE and Western Blotting The protein concentrations were determined by the Bradford method. A total of 10 μl of protein was loaded in each lane, subjected to SDS-PAGE (10–12%) and then transferred to a polyvinylidene difluoride membrane. The blots were probed with one of the following antibodies: anti-light chain (LC3; Sigma), anti-Beclin-1, anti-Bcl-2, anti-phospho-Akt (Cell Signaling Technology, Boston, Mass., USA), anti-β3-integrin (Abcam, Cambridge, Mass., USA) or anti-β-actin (Sigma). The membrane was then rinsed and incubated with the corresponding secondary horseradish peroxidase-conjugated anti-mouse IgG (Roche, Indianapolis, Ind., USA) or anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) antibody. The membranes were then rinsed, and bound antibodies were detected using an ECL chemiluminescence detection system (Roche, Mannheim, Germany) and quantified by scanning densitometry. β-Actin was used as a loading control.

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Autophagy is an important catabolic mechanism that controls cellular homeostasis. It can be induced in a variety of circumstances. In addition to classical signals, such as starvation and energy exhaustion, many pathogen-associated molecular patterns have been shown to promote autophagic activation [5]. Bacterial lipopolysaccharide (LPS) is a mediator of host responses that can lead to sepsis and ultimately multiorgan dysfunction, as well as a stimulator of autophagic signaling in cultured cardiomyocytes and other cells [6–8]. Autophagosome accumulation has been observed in liver biopsies in patients with sepsis and cecal ligation and puncture models in rodents [9, 10]. Other investigators have reported increased formation of autophagosomes in septic myocardium and cardiomyocytes incubated with LPS [4, 7, 8]. These findings suggest that autophagy is an important process during sepsis-induced cardiac suppression. However, the underlying mechanism of autophagy in relation to sepsisinduced cardiac suppression remains unknown. Integrins, which serve as a bridge between the extracellular matrix and the intracellular cytoskeleton, conduct a variety of signals from the extracellular matrix that affect cell survival and differentiation [11]. β3-Integrin is the main integrin heterodimer on the surface of cardiomyocytes [12]. Previous studies have shown the protective effect of β3-integrin in cardiovascular diseases. It prevents programmed cell death of cardiomyocytes in pressureoverloaded myocardium and activates survival signaling during myocardial hypertrophy [11–13]. However, little is known regarding the role of β3-integrin during sepsisinduced cardiac injury. In our previous study, we observed that β3-integrin expression levels were increased to approximately eight times the normal level in the myocardium of septic rats [14]. There are also many studies which suggest that integrins are involved in the regulation of autophagy [15]. The effect of integrins on cellular autophagy might be different under various experimental conditions. Therefore, we speculate that increased levels of β3-integrin might be involved in the regulation of the intensity and duration of autophagy in cardiomyocytes upon LPS stimulation. At present, the mechanism underlying β3-integrin involvement in the regulation of autophagy is unknown. Integrins can activate various signaling pathways related to autophagy. The IκB kinase/nuclear factor-κB, mitogen-activated protein kinase and AMP-activated protein kinase pathways activate autophagy [16–18], while only the Akt pathway has inhibitory potential. β3-Integrin can also activate Akt signaling in macrophages when LPS is added and in isolated cardiomyocytes simulated by H2O2

Visualization of Monodansylcadaverine-Labeled Autophagic Vacuoles Autophagic vacuoles were detected with monodansylcadaverine (MDC; Sigma) according to the method of Biederbick et al. [23]. The cells were washed with PBS to remove the culture medium and then incubated with 0.05 mM MDC in PBS at 37 ° C for 45 min. After incubation, the cells were washed four times with PBS and immediately analyzed by fluorescence microscopy.  

 

Electron Microscopy The cells were fixed with 2.0% glutaraldehyde for at least 2 h, postfixed with 2% osmium tetroxide for 2 h, and finally incubated with 0.5% uranyl acetate and sucrose overnight. The specimens were then dehydrated and embedded in Spurr’s resin. Ultrathin sections (70–80 nm thick) were cut on an ultramicrotome, placed onto copper grids, and stained for contrast with 1% uranyl acetate and 1% lead citrate. The specimens were examined with a transmission electron microscope (TEM). Statistical Analysis All data are presented as means ± SD. Statistical evaluation of the data was performed with Student’s t test for paired or unpaired observations, and by analysis of variance. Scheffe’s test for multiple comparisons was used to identify differences among groups. Values were considered to be significantly different when p < 0.05.

Results

β3-Integrin Expression Is Increased in Cardiomyocytes after LPS Stimulation Following treatment with 1.0 μg/ml of LPS, β3-integrin protein levels in cardiomyocytes gradually increased from 2 to 8 h, reaching a plateau between 8 and 16 h. Subsequently, β3-integrin protein levels decreased (from 16 to 24 h; fig. 1a). Immunohistochemistry showed that β3integrin was present in the membrane of cardiomyocytes and was upregulated after LPS stimulation (fig. 1b). LPS Induces Autophagy in Cardiomyocytes The protein levels of LC3-total and LC3-II increased in a time-dependent manner following LPS treatment, peaking at 16 h and then declining to 24 h (fig. 2a). Deβ3-Integrin Inhibits LPS-Induced Autophagy

creased Bcl-2 protein levels were observed in the late phase at 16 h (p < 0.05) and 24 h (p < 0.05; fig. 2b). Therefore, the time points of 8 and 16 h were chosen for further experiments. The Adenovirus Vector Facilitates Efficient β3-Integrin Delivery in Cardiomyocytes We measured the efficiency of gene transduction by observing green fluorescence under microscopic magnification. A total of 95% of the cells were transfected after 24 h at an MOI of 80 (fig. 3a). Western blot analysis showed that β3-integrin protein levels were significantly upregulated in cardiomyocytes transfected with adenovirus β3-integrin compared to control cells (p < 0.05; fig. 3b). Effects of β3-Integrin on LPS-Induced Autophagy in Cardiomyocytes Cardiomyocytes were preincubated with cRGD (10 μg/ml), an inhibitor of αVβ3 integrin, for 2 h or transfected with adenovirus β3-integrin for 24 h before being treated without or with LPS for 8 and 16 h. We observed that blockade of β3-integrin by cRGD led to enhanced expression of LC3-II (p < 0.05; fig. 4a) together with upregulation of Beclin-1 (p < 0.05) and downregulation of Bcl-2 (p < 0.05; fig. 4b, c). LC3-II protein levels were significantly lower in β3-integrin-overexpressing cells than in LPS-treated cells at 16 h (p < 0.05; fig. 4a). Bcl-2 protein levels were significantly increased in β3-integrin-overexpressing cells compared to LPS-treated cells at 16 h (p < 0.05; fig. 4c). However, LC3-II and Bcl-2 protein levels in cells transfected with adenovirus β3-integrin were unchanged compared to LPS-treated cells at 8 h (p > 0.05; fig. 4a, b). Beclin-1 protein levels were significantly lower in β3-integrin-overexpressing cells than in LPS-treated cells at 8 h (p < 0.05) and 16 h (p < 0.05; fig. 4b, c). Autophagic vacuoles were observed by TEM. Cardiomyocytes in the control group showed a normal structure and distribution of mitochondria (fig.  5a). LPS-treated cells exhibited higher vacuolization than control cells (fig. 5b). The number of autophagosomes was significantly lower in adenovirus β3-integrin-transfected cells than in LPS-treated cells (p < 0.05). However, there was no significant difference in the number of autophagosomes between LPS-treated cells and cRGD-preincubated cells (fig. 5c, d). Only basal levels of autophagy were observed in control cells, while accumulation of MDC dye was detected after LPS treatment, which was inhibited in cells preincubated with cRGD (fig. 6).

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Immunocytochemistry The cells were fixed with 4% paraformaldehyde followed by treatment with 10% hydrogen peroxide to quench the endogenous peroxidase. The cells were treated with 1% Triton X-100 for 30 min to unmask the antigens and 4% bovine serum albumin to block nonspecific binding. The sections were incubated with primary antibodies against β3-integrin (1: 100) followed by appropriate HRP-conjugated secondary antibodies (1:1,000). The antigen-antibody complex was visualized using a DAB kit (Invitrogen, Grand Island, N.Y., USA). The controls consisted of staining without a primary antibody or with normal serum instead of a primary antibody.

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Effects of β3-Integrin on the Activation of the Akt Signaling Pathway LPS activates multiple molecular pathways in cardiomyocytes. As early as 15 min after the addition of LPS, upregulation of Akt phosphorylation was detected (p < 0.05; fig. 7a). However, upregulation of Akt phosphorylation by LPS was eliminated when the cells were treated with cRGD in advance, and overexpression of β3-integrin resulted in continuous and enhanced Akt phosphorylation (p < 0.05; fig. 7a). The Akt inhibitor triciribine re252

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duced the levels of Akt phosphorylation. In cardiomyocytes, the inhibitory function of a concentration of 2.5 μM of triciribine was already detectable  15 min before LPS treatment (p < 0.05; fig. 7b). Triciribine reduced the level of Akt phosphorylation, which was upregulated in β3integrin-overexpressing cells (p < 0.05; fig.  7c). Triciribine also inhibited the negative effect of β3-integrin on autophagy by upregulating the LC3-II (p < 0.05) and Beclin-1 protein levels (p < 0.05; fig. 7d, e).

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Fig. 1. LPS stimulation increases β3-integrin expression in cardiomyocytes. Neonatal rat cardiomyocytes, which were pretreated with LPS (1 μg/ml), were harvested at the indicated time periods, and the levels of β3-integrin in cell extracts were determined. a The top panel contains representative immunoblots showing that LPS simulation increases β3-integrin expression in cardiomyocytes. β-Actin was assayed to verify equal loading of cell lysates. The bottom panel shows quantification of the bands by densitometry. Results are shown from three independent experiments compared with the control values (at 0 h). * p < 0.05 vs. control. b Immunodetection of β3integrin in cardiomyocytes pretreated with LPS (1 μg/ml). Scale bar: 1 mm.

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LPS stimulation. Neonatal rat cardiomyocytes, which were pretreated with LPS (1 μg/ml), were harvested at the indicated time periods, and LC3 (a) and Bcl-2 (b) protein levels in the cell extracts

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Discussion

In the present study, we confirmed the findings from previous studies, which have shown that LPS induces autophagy [4, 7, 8], as evidenced by the presence of autophagy-associated proteins, phagosomes and autophagic vacuoles. Importantly, we showed that β3-integrin was upregulated after LPS treatment in cardiomyocytes. Furthermore, β3-integrin inhibited autophagy by deactivat254

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ing phosphorylation of Akt. To the best of our knowledge, these findings are the first to demonstrate the role of β3integrin in LPS-induced autophagy in cardiomyocytes and the related signaling pathway. Accumulating evidence has shown that autophagy plays an important role in heart diseases, including ischemia/reperfusion injury, ventricular hypertrophy and septic cardiomyopathy [4, 24, 25]. Complete induction of autophagy protects the septic heart by improving the Zhu/Li/Gong/Yu/Dai/Cai/Yan

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mitochondria and cytoplasmic material can be observed after LPS treatment (arrows). e The graph represents quantitation of the number of autophagosomes per cross section of a cell. Data represent the means ± SEMs from three different sections. # p < 0.05 vs. control (Con), * p < 0.05 vs. LPS treatment group.

ejection fraction and ATP levels [4], while suppression of autophagy ameliorates LPS-induced contractile dysfunction of cardiomyocytes [26]. These findings suggest that the autophagic balance is disturbed by sepsis and that incomplete or excessive autophagy might be harmful [27]. Therefore, the balance of autophagy in the septic heart needs to be regained. In our study, we found that β3integrin acted as a suppressor in LPS-induced autophagy. We propose that elevated expression of β3-integrin after LPS stimulation might help to prevent autophagic apoptosis. Integrins are associated with a reduction in apoptosis as well as decreased oxidative stress and an accumulation of autophagic vacuoles in a dilated cardiomyopathy rodent model [28]. Future studies should focus on changes in heart function and the regulation of integrin expres-

sion, as well as other molecules downstream from integrin that might regulate autophagy upon activation. We observed that the upregulated LC3-II expression and downregulated Bcl-2 expression occurred 16 h after LPS stimulation, which is in contrast to some previous studies that have shown that autophagy occurs earlier after LPS stimulation [7, 26]. The reason for this difference between studies might be related to the method of induction and the different cell types used. To investigate the effects of β3-integrin on LPS-induced autophagy, we modified either the levels of β3-integrin expression or the functional area of β3-integrin. cRGD is a synthetic peptide containing the RGD sequence, which has been shown to compete with adhesive proteins that bind to β3-integrin receptors. cRGD peptide abolishes

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αVβ3 ligation to the extracellular matrix and the effects on the secretion of TNF-α in macrophages and interrupts the subsequent signal transduction upon β3-integrin ligation [19, 29]. Our experiments showed that autophagy-related proteins were increased, and autophagosomes formed and accumulated in cardiomyocytes in which β3-integrin was blocked by cRGD. Autophagy-related protein levels were unchanged, and autophagosome formation and accumulation were decreased in cardiomyocytes overexpressing β3-integrin. This evidence indicates that β3integrin inhibits autophagy during LPS stimulation. Autophagy is regulated by various types of immune signaling, which function in both the induction and suppression of autophagy [5]. A key regulator or gatekeeper of autophagic induction is the Akt/mTOR pathway. However, many signaling pathways are involved in the regulation of cardiomyocyte autophagy. The Akt signaling pathway exerts inhibitory effects on autophagy; however, autophagy could be induced through other signaling pathways, including the AMPK, MAPK, STAT and JNK signaling pathways [5, 27]. Moreover, these signaling pathways might produce cross talk. In addition, after LPS stimulation, cardiomyocytes produced TNF-α, reactive oxygen species and misfolded proteins, which might be involved in the regulation of β3-integrin [7, 8]. Therefore, β3-integrin overexpression via adenoviral transfection might fail to completely inhibit LPS-induced autophagy. In the present 256

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study, β3-integrin overexpression led to significantly decreased protein levels of LC3-II and Beclin 16 h after LPS treatment, while the protein levels of LC3-II and Beclin-1 were significantly increased in cardiomyocytes in which β3-integrin was blocked with cRGD 16 h after LPS treatment. Beclin-1 is part of the complex that recruits effectors and translocates to the autophagosome formation site [5]. LC3-II participates in the elongation of the double membrane [5]. It is consistent with our observation that the downregulation of Beclin-1 occurs before the decrease in LC3-II expression 8 h after LPS stimulation. Our findings suggest that β3-integrin may inhibit the process of autophagy in cardiomyocytes that are exposed

Fig. 7. Effects of β3-integrin on the activation of the Akt signaling pathway. Cardiomyocytes were treated as described in the legend to figure 6. The phospho (P)-Akt levels in cell extracts were analyzed by Western blot at the indicated times (a). The phospho-Akt levels in cardiomyocytes that were preincubated with triciribine (T) at different concentrations for 30 min or with triciribine (2.5 μm) for different time intervals are shown (b). Cells transfected with adenovirus β3 and control (Con) cells were preincubated in the absence or presence of triciribine before LPS stimulation. The protein levels of phospho-Akt (b), LC3-II (d) and Beclin-1 (e) were measured. Actin was used as a loading control. The measurements represent the mean of four separate experiments. # p < 0.05 vs. control, * p < 0.05 between the indicated groups. (For figure see next page.)

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mation of LPS-induced autophagosomes in cardiomyocytes. Cardiomyocytes were transfected with adenovirus β3-integrin for 24 h or pretreated with cRGD for 2 h in the absence or presence of LPS (1 μg/ml) at the indicated times. The cells were then stained with MDC dye to visualize autophagosomes. Scale bar: 1 mm.

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Autophagy has been regarded as a double-edged sword. Insufficient autophagy could cause suppression of heart function, while excessive autophagy might lead to autophagic apoptosis. Thus, control of the regulation of autophagy might be manipulated as one strategy for the prevention and therapy of sepsis-related cardiac dysfunction. Autophagy could be induced to protect cardiomyocytes from oxidative stress. On the other hand, inhibition of excessive autophagy of cardiomyocytes could reduce autophagic apoptosis. Therefore, β3-integrin and its upstream activators may be a therapeutic target by weakening the process of autophagy and preventing autophagic apoptosis in patients with sepsis-associated cardiac dysfunction. Our study should be interpreted within the context of its limitations. First, other signaling pathways might be involved in LPS-induced autophagy in cardiomyocytes. These signaling pathways might initiate cross talk with the Akt pathway. Second, our study is restricted to in vitro results. The role of autophagy in cardiomyocytes in the process of sepsis-associated cardiac dysfunction remains unknown. Therefore, more in vivo studies are required. In summary, LPS induces autophagy and upregulates the expression β3-integrin in cardiomyocytes. Furthermore, β3-integrin inhibits autophagy through the Akt signaling pathway.

Acknowledgment This study was partly supported by the National Natural Science Foundation of China (No. 81171784), Zhejiang Province Natural Science Foundation (Nos. Z2100237, Y2110801 and LY14H150006), and the Health and Family Planning Commission of Zhejiang Province (No. 2013KYA002).

References

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to LPS. Consistent with our finding, Zhang et al. [30] observed that knocking down osteopontin, the main ligand of integrin, resulted in downregulation of β3-integrin and elevated the expression of LC3-II and Beclin-1 in MDAMB-231 cells. Inhibiting the function of integrin causes decreased autophagosome formation in starved prostate epithelial cells [31]. Furthermore, cell detachment due to a lack of integrin engagement could also trigger autophagy [32]. However, other reports have shown different roles of integrin in the process of autophagy. Edick et al. [31] found that blocking α3-, α6- or β4-integrin with antibodies inhibited survival and autophagy in primary basal prostate epithelial cells. Stimulation of smooth muscle cells with osteopontin stimulates autophagy-related genes and autophagosome formation, and ultimately cell death occurs [16]. Based on these previous findings, we speculate that the different roles of β3-integrin in the process of autophagy might be due to the use of various cell types and different stimulations. Different β3-integrinmediated signaling pathways might be activated in various circumstances. Therefore, further insight is required to resolve the underlying mechanism regarding β3integrin inhibition of autophagy in cardiomyocytes that are exposed to LPS. Triciribine is highly selective for Akt and inhibits the phosphorylation of all three Akt isoforms in vitro. Some reports have shown that triciribine weakens Akt signaling thus stimulating autophagy in cardiomyocytes and cell lines of T-cell acute lymphoblastic leukemia [33, 34]. Triciribine appears to function primarily by binding to the PH domain of Akt and preventing the recruitment of Akt to the cell membrane, where this protein is normally activated by phosphorylation [35]. In our study, we observed that the effects of β3-integrin overexpression on Akt phosphorylation were partially inhibited by triciribine. Because β3-integrin overexpression is triggered by Akt phosphorylation, triciribine might fail to inhibit Akt that has already been recruited to the cell membrane, thus Akt phosphorylation will be partially inhibited. In our study, cRGD, a β3-integrin inhibitor, abolished the phosphorylation of Akt, which was induced by LPS stimulation, while overexpression of β3-integrin increased the intensity and duration of phospho-Akt expression. Our results suggest that β3-integrin inhibits autophagy through Akt activation in cardiomyocytes stimulated with LPS. In addition, we observed that inhibiting Akt partially reverses the inhibitory function of β3integrin overexpression on autophagy. Other mechanisms that are induced by LPS may be involved in β3integrin inhibition of cardiomyocyte autophagy.

β3-Integrin Inhibits LPS-Induced Autophagy

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ß3-integrin inhibits lipopolysaccharide-induced autophagy in cardiomyocytes via the Akt signaling pathway.

To investigate the role of β 3 -integrin in lipopolysaccharide (LPS)-induced autophagy in cardiomyocytes and its underlying mechanism...
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