Inhibiting Receptor of Advanced Glycation End Products Attenuates Pressure Overload-Induced Cardiac Dysfunction by Preventing Excessive Autophagy

The receptor for advanced glycation end products (RAGE) is involved in heart failure (HF) by mediating diverse pathologic processes, including the promotion of inflammation and autophagy. However, the role of RAGE in pressure overload-induced HF is not well understood. We found that stimulation of RAGE triggered the death of neonatal rat ventricular myocytes (NRVMs), while cell death was alleviated by ATG5 knockdown. Using transverse aortic constriction (TAC) in mice as a model of pressure overload-induced HF, we demonstrated that RAGE knockout or RAGE blockade attenuated cardiac hypertrophy and fibrosis as well as cardiac dysfunction at 8 weeks after TAC. Importantly, RAGE knockout reversed upregulation of autophagy related proteins (LC3BII/I and Beclin 1) and reduced cardiomyocyte death, indicating that excessive autophagy after TAC was inhibited. Moreover, RAGE knockout or blockade reduced the upregulation of pp65-NFκB and BNIP3, which mediate autophagy. Taken together, these results suggest that RAGE plays an important role in the progression of HF by regulating autophagy. Therefore, inhibition of the RAGE-autophagy axis could be a promising new strategy for treatment of heart failure.


INTRODUCTION
Heart failure (HF) is a complicated clinical syndrome that represents the final stage of many cardiac disorders, and it results in multiple organ dysfunction and premature death. Although, treatments targeting neurohormonal signaling have somewhat improved the survival of patients with HF, it remains a major cause of morbidity and mortality worldwide. HF is classified into two major categories, which are heart failure with a reduced ejection fraction (HFrEF) and heart failure with a preserved ejection fraction (HFpEF). Both HFpEF and HFrEF share similar clinical features, but their etiology and treatment are different. In particular, HFpEF is usually associated with myocardial stiffness, whereas HFrEF is associated with impaired myocardial contractility and ventricular dilation (Borlaug and Redfield, 2011). Thus, understanding the mechanisms that lead to these different types of HF is essential for development of new therapies.
Pressure overload is a common cause of HF and cardiac remodeling. Stress on the heart promotes pathologic remodeling, with an increase of cardiomyocyte size, interstitial fibrosis, and inflammation, eventually leading to HF (van Berlo et al., 2013). Modulation of the signaling pathways that regulate these cellular processes could possibly prevent the progression of HF. A role of the advanced glycation end products (AGE)receptor for AGE (RAGE) signaling pathway has recently been identified in the pathogenesis of diverse process, such as inflammation, malignancy, diabetes/diabetic complications, and neurodegeneration. In addition, the AGE-RAGE pathway has an essential role in heart disease. Accumulation of AGE is a major cause of asymptomatic diastolic and systolic cardiac dysfunction in patients with diabetes (Berg et al., 1999;Willemsen et al., 2011), and AGE may also be involved in the development and progression of HF in non-diabetic patients (Hartog et al., 2007). The level of circulating soluble RAGE was reported to be correlated with the severity of cardiac dysfunction (Raposeiras-Roubin et al., 2010;Wang et al., 2011). In addition, administration of a RAGE ligand (HMGB1) exacerbates ischemia/reperfusion injury, whereas RAGE-deficient mice show less myocardial damage after ischemia and reperfusion (Andrassy et al., 2008). Moreover, RAGE has been identified as a prognostic factor for patients with HF (Koyama et al., 2008). However, the mechanisms by which RAGE contributes to HF are not well understood.
Autophagy is an evolutionarily conserved process in which organelles and proteins undergo degradation, followed by recycling to maintain cellular homeostasis (Mizushima et al., 2008;Yang and Klionsky, 2010). There is evidence that basal autophagy plays a role in quality control and housekeeping in cardiomyocytes (Wang et al., 2008;Singh and Cuervo, 2011), while induction of autophagy by cardiac stress has a cytoprotective benefit (Yan et al., 2005;Nakai et al., 2007). However, excessive autophagic activity induced by pressure overload or reperfusion injury has been found to accentuate cardiac remodeling (Matsui et al., 2007;Zhu et al., 2007), indicating that autophagy can be detrimental in HF. Thus, modulation of autophagic activity could be a potential target for the treatment of patients with HF.
It has been reported that the AGE-RAGE pathway is involved in autophagic death of cardiomyocytes (Tsoporis et al., 2010;Hou et al., 2014), and that RAGE blockade improves cardiomyocyte viability through inhibiting the induction of autophagy by AGE (Hou et al., 2014). However, the role of RAGE-mediated autophagy in HF has not been investigated in detail. Therefore, we performed this study to explore the role of RAGE-mediated autophagy in HF induced by transverse aortic constriction (TAC), which leads to cardiac hypertrophy followed by ventricular dilatation and HFrEF over time. We examined the effect of inhibiting autophagy by RAGE blockade and demonstrated that it improved cardiac function after TAC. Importantly, we found that RAGE is a critical factor for regulation of autophagy in the heart, suggesting that treatment which targets RAGE-mediated autophagy could be promising for HF.

Cell Culture
Neonatal rat ventricular cardiomyocyte (NRVMs) were isolated from the left ventricles of Sprague-Dawley rats (1-3 days old). Briefly, ventricular tissues were digested with 1.0 mg/ml Type II Collagenase . Then cells were incubated for 1 h in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) to allow attachment of fibroblasts, after which the floating cardiomyocytes were cultured in DMEM supplemented with 0.1 M BrdU (Sigma,, 10% FBS, and 1% penicillin-streptomycin (Thermo Fisher Scientific, Cat No.15140122

Virus Production and Infection of NRVMs
NRVMs were infected with a recombinant lentivirus expressing rat RAGE or short hairpin RNA (shRNA) targeting rat RAGE (GeneChem, Shanghai, China) according to the manufacturer's instructions. For infection with shRNAs targeting ATG5 and ATG7, the relevant sequences were cloned into the pLKO.1 vector. The lentivirus was packaged by co-transfection of pLKO.1-shATG5/7, psPAX, and pMD.2G using lipofectamine 2000. Virions were harvested at 48 and 72 h after transfection and used for infection.

Animals
Male C57/BL mice weighing 18-22 g were purchased from the Laboratory Animal Center of Guangzhou University of Chinese Medicine. RAGE knockout mice (Myint et al., 2006) were a gift from Kanazawa University, Japan. The protocol for this study was approved by the Animal Care Committee of Guangzhou University of Chinese Medicine and was strictly in accord with the National Institutes of Health guidelines for Care and Use of Laboratory Animals.

Mouse Model of Transverse Aortic Constriction
Surgery was performed to create TAC in mice according to the established protocol (Wagner et al., 2001). Briefly, wild-type mice and RAGE knockout mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg, Sigma), and thoracotomy was done while the mice were connected to a ventilator for small animals. The aorta was ligated between the right innominate and left common carotid arteries using a 27G needle and an 8-0 suture. On postoperative day 1, the mice were randomly assigned to treatment with a RAGE antagonist (FPS-ZM1) at 1 mg/kg/day (TAC + FPS-ZM1 group, n = 10) or treatment with an autophagy inhibitor (3MA dissolved in PBS) at 10 mg/kg/day (TAC + 3MA group, n = 10). The sham group underwent the same thoracotomy procedure without aortic constriction. The wild-type TAC group and the RAGE knockout TAC group were administered 0.9% sodium chloride instead of the above drugs. Mice were sacrificed for analysis at 8 weeks postoperatively.

Echocardiography
At 8 weeks after TAC or sham surgery, echocardiography was performed by using a Vevo 2,100 Imaging System (VisualSonics Inc., Toronto, ON, Canada) in mice under anesthesia with 1% isoflurane (RWD Life Science Co., Guangdong, China). The heart was examined in the short-axis view at the papillary muscle level and the average left ventricular (LV) internal dimension across at least 4 cardiac cycles was determined on M-mode images. Analysis of echocardiographic images was performed in a blinded manner.

Histological Examination
Hearts were harvested and perfused with cold phosphatebuffered saline (PBS), followed by fixation in 4% paraformaldehyde overnight. Then the hearts were dehydrated and embedded in paraffin. Next, 5 µm thick sections were cut at the papillary muscle level for hematoxylin-eosin (H&E) and Masson's trichrome staining. Each section was completely scanned by using a Caseviewer 2.0 (Panoramic 250/MIDI, 3D HISTECH, Hungary). For morphometric analysis, photographs were observed at 1× magnification to view the whole heart, and at 400× magnification to determine the single cardiomyocyte area. The relative fibrotic area was calculated by comparing the fibrotic area ratio (the ratio of the stained fibrotic area to the left ventricular area) in each treated group to that in the control group. Quantification of all data was done with Image J software.

Immunohistochemistry and TUNEL Staining
Immunohistochemical staining was performed by using a Vectastain Elite ABC HRP Kit according to the manufacturer's protocol. Briefly, sections were deparaffinized and rehydrated, incubated with blocking serum for 1 h, and then incubated with antibodies targeting RAGE (1:100, Abcam) or Beclin 1 (1:200, Abcam) for 60 min at room temperature. After being washed 3 times in PBS, the sections were incubated with the biotinylated secondary antibody for 30 min, and detection was performed with ABC reagent. Cell death was detected by using paraffin sections and the TMR Red In situ Death Detection Kit (Roche, Indianapolis, IN, United States) according to the manufacturer's instructions. Nuclei were counterstained with DAPI.

Quantitative Real-Time PCR
Total RNA was extracted from LV tissue by using an RNAprep Pure Tissue Kit (Tiangen, Beijing, China), after which first-strand cDNA was synthesized with a FastKing RT Kit (Tiangen, Beijing, China) and quantitative PCR (qPCR) was performed using SYBR R Premix Ex Taq TM II (Takara, Beijing, China). Target gene expression was normalized for that of GAPDH and compared among the groups. Details of the primers are provided in Table 1.

Statistical Analysis
Results are expressed as the mean ± SEM. Analyses were performed by two-way ANOVA, followed by the multiple

The AGE-RAGE Pathway Triggers Autophagic Death of Cardiomyocytes
To study the potential influence of AGE-RAGE on cardiomyocyte growth, we examined the viability of NRVMs after AGE treatment by using the CCK8 assay. This assay showed that AGE reduced NRVMs viability in a dose-dependent manner ( Figure 1A).  Figure S1). Importantly, we observed that Beclin 1 (a protein related to autophagy) was increased after AGE treatment, a finding consistent with a previous report that AGE can trigger autophagy (Hou et al., 2014). Furthermore, Beclin 1 expression was further elevated by AGE treatment of RAGE-overexpressing NRVMs, while the response of Beclin 1 to AGE treatment was reduced in NRVMs with RAGE knockdown. We then hypothesized that the AGE-RAGE pathway could trigger autophagic death of NRVMs. To test this hypothesis, we studied the effect of ATG5/ATG7 knockdown on NRVM viability after AGE treatment. While ATG5/ATG7 knockdown did not cause either death or proliferation of NRVMs in the absence of AGE treatment, ATG5/ATG7 knockdown led to reduction of cell death after AGE treatment (Figures 1F-H). Taken together, our results suggested that the AGE-RAGE pathway triggered autophagic death of cardiomyocytes.

RAGE Knockout or Blockade Attenuates Cardiac Dysfunction at 8 Weeks After TAC
We then hypothesized that RAGE might play an important role in HF, since AGE-RAGE induced autophagic cell death and cardiomyocyte death are essential for progression of HF (van Empel et al., 2005). To test this hypothesis, we examined cardiac function in mice with RAGE knockout (Myint et al., 2006; Supplementary Figure S2) and mice with RAGE blockade mice after TAC. Compared to sham mice, echocardiography showed significant reduction of the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in TAC mice, as well as an increase of left ventricular enddiastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), the end-diastolic left ventricular internal dimension (LVIDd), the end-systolic left ventricular internal dimension (LVIDs), and left ventricular mass (LV mass) (Figure 2 and Supplementary Figure S3). In contrast, mice with RAGE knockout (TAC + RAGE−/− group) or RAGE blockade (TAC + FPS-ZM1 group) showed a significant increase of LVEF and LVFS, as well as a significant decrease of LVEDV, LVESV, LVIDd, LVIDs, and LV mass, when compared to control TAC mice with HF assessed at 8 weeks. These results indicated that RAGE knockout and RAGE blockade could improve cardiac dysfunction.

RAGE Knockout or Blockade Suppresses Cardiac Hypertrophy in TAC Mice
To investigate whether RAGE knockout or blockade could ameliorate cardiac hypertrophy after TAC, we analyzed cardiomyocyte size by examining H&E-stained cardiac sections. We also calculated the heart weight/body weight ratio and the lung weight/body weight ratio, as well as determining the expression of hypertrophy marker gene mRNAs by qPCR. Histological examination of cardiac sections showed that TAC mice had a larger total heart size and larger single cardiomyocyte area compared to sham mice (Figures 3A,B). TAC mice also had a significantly larger heart weight/body weight ratio (HW/BW, Figure 3C) and lung weight/body weight ratio (LW/BW, Figure 3D) than sham mice. Furthermore, expression of the pro-hypertrophic genes ANP ( Figure 3E) and BNP ( Figure 3F) was upregulated in TAC mice, confirming that TAC caused cardiac hypertrophy. Both RAGE knockout and RAGE blockade significantly reduced the heart size and the single cardiomyocyte area after TAC. Consistent with these findings, the HW/BW, LW/BW, and expression of ANP and BNP were all significantly decreased by RAGE knockout or RAGE blockade. These findings indicated that RAGE knockout or blockade could prevent induction of cardiac hypertrophy by pressure overload.

RAGE Knockout or Blockade Suppresses Cardiac Fibrosis in TAC Mice
To investigate whether RAGE knockout or blockade suppressed the development of cardiac fibrosis in response to pressure overload, we analyzed the fibrosis area by histological examination of sections with Masson's trichrome staining and we also assessed the expression of fibrosis-related genes by qPCR. The fibrosis area was significantly increased in TAC mice compared with sham mice. In contrast, there was significant reduction of the fibrosis area in both RAGE knockout mice and RAGE blockade mice compared with TAC mice (Figures 4A,B).
In agreement with these results, expression of pro-fibrotic genes [type I collagen, fibronectin, and connective tissue growth factor (CTGF)] showed marked upregulation in TAC mice, while upregulation of these genes was reduced in RAGE knockout or RAGE blockade mice (Figures 4C-E). These results  demonstrated that RAGE was essential for progression of cardiac fibrosis.

RAGE Knockout or Blockade Inhibits Excessive Autophagy After TAC
Because RAGE induced autophagic cell death in vitro (Figure 1), we hypothesized that RAGE promoted cardiac dysfunction and hypertrophy in HF by inducing excessive autophagy. To investigate this idea, we first analyzed the expression of RAGE and other proteins related to autophagy in mice. Western blotting showed that RAGE expression was upregulated in TAC mice (Figures 5A,B and Supplementary Figure S4), along with a significant increase in the expression of proteins related to autophagy, LC3BII/I and Beclin 1 (Figures 5A,C,D). Notably, the LC3BII/I ratio and Beclin 1 protein expression were significantly decreased in RAGE knockout mice and RAGE blockade mice compared with TAC mice (Figures 5A,C,D). In agreement with this finding, immunostaining showed that upregulation of RAGE and Beclin 1 expression in the heart after TAC was  reversed by inhibition of RAGE ( Figure 5E). To investigate whether inhibition of RAGE attenuated cardiac dysfunction after TAC by reducing cell death in vivo, we performed the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. We found that TAC significantly increased cell death (Figures 5E,F), while cell death after TAC was significantly reduced in mice with RAGE knockout or RAGE blockade (Figures 5E,F). Moreover, inhibition of autophagy by 3-MA improved cardiac dysfunction and remodeling after TAC (Figures 2-4), in association with reduction of the LC3BII/I ratio and Beclin 1 expression as well as a decrease of cell death (Figures 5A-F). These results suggested that RAGE knockout or blockade improved cardiac function by reducing cell death through suppression of excessive autophagy after TAC.

RAGE Regulates Autophagy Through p65-NFκB and BNIP3
To investigate the mechanism underlying RAGE-mediated autophagy in HF, we examined the expression of several regulators of autophagy (p65-NFκB, phophos-p65-NFκB (pp65-NFκB), and BNIP3) by western blotting. We found significant upregulation of both p65-NFκB and pp65-NFκB expression in TAC mice compared with sham mice (Figures 6A-C and Supplementary Figure S5). Importantly, RAGE knockout and RAGE blockade both prevented the increase of p65-NFκB and pp65-NFκB expression after TAC. Because p65-NFκB activates the autophagy related gene Beclin 1 (Copetti et al., 2009), it is likely that deletion of RAGE suppressed autophagic activity through downregulation of p65-NFκB. Interestingly, RAGE knockout or blockade also prevented an increase in expression of the autophagy regulator BNIP3 in TAC mice (Figures 6A,D).
Considering that NFκB regulates BNIP3, our findings suggested that RAGE knockout or RAGE blockade suppress excessive autophagy in this mouse model of HF possibly by modulating the NFκB/BNIP3/Beclin 1 pathway.

DISCUSSION
RAGE plays an essential role in various processes causing cell damage, such as oxidative stress and inflammation (Ray et al., 2016), and RAGE blockade has been shown to have a beneficial effect on heart disease (Bangert et al., 2016). In the present study, we demonstrated that RAGE blockade ameliorated TACinduced cardiac dysfunction by inhibiting excessive autophagy. In addition, RAGE knockout inhibited the development of cardiac hypertrophy and cardiac fibrosis in TAC mice, as well as enhancing cardiac function. We also showed that RAGE is a critical regulator of autophagy in HF, since RAGE knockout reduced the expression of BNIP3 and p65-NFκB to prevent overexpression of proteins related to autophagy (LC3BII/I and Beclin 1) after TAC. Thus, RAGE-mediated autophagy regulated the progression of HF in the present TAC model and RAGE could be a potential target for treating HF induced by pressure overload.
It has been unclear whether RAGE directly regulates excessive autophagy or autophagy occurs as a consequence of TAC-induced HF. The present study provided evidence that RAGE induces cardiac autophagy during HF. We demonstrated that treatment with AGE or overexpression of RAGE induced the expression of Beclin 1 (a gene related to autophagy) in NRVMs, while RAGE knockout reduced Beclin 1 expression. Moreover, RAGE knockout or blockade significantly decreased the myocardial expression of autophagy related proteins (LC3BII/I and Beclin 1) after TAC, suggesting that autophagic activity was reduced in mice with knockout or blockade of RAGE. Consistent with this finding, previous studies have shown that RAGE mediates autophagy in other cellular process (Kang et al., 2011a,b;Hou et al., 2014). Therefore, our results suggest that RAGE may directly regulate autophagic activity both in vitro and in vivo.
An important finding of this study was that activation of RAGE may induce excessive autophagy, leading to autophagic cell death and eventually contributing to progression of HF. Previous study shows that treatment of FPS-ZM1 attenuated cardiac remodeling after TAC via enhancing phosphorylation of AMPK and reducing phosphorylation of mTOR (Liu et al., 2016). In our study, we further show that deletion of RAGE could attenuate the HF progression, which could be more directly characterizing the role of RAGE after TAC. Importantly, our study has identified a new mechanism that targeting RAGE mediated autophagy exerts the benifical effect after TAC. We showed that exposure of NRVMs to AGE caused a dosedependent increase of cell death, while this could be prevented by RAGE knockout. Consistently, expression of the autophagy related protein Beclin 1 was decreased by RAGE knockout, suggesting that the AGE-RAGE pathway could trigger autophagic death of cultured NRVMs. Because RAGE is known to promote autophagic activity in vivo, it seems likely that RAGE induces excessive autophagy that leads to cardiomyocyte death during progression of HF.
Autophagy can have either a protective or detrimental effect on the heart. For example, excessive autophagic activity in response to pressure overload can be detrimental (Zhu et al., 2007). On the other hand, a recent study showed that mitochondrial autophagy is stimulated after downregulation of general autophagy and can ameliorate pressure overloadinduced cardiac dysfunction (Shirakabe et al., 2016). These contradictory observations suggest that target-specific autophagy has an important role in cardiomyocyte health, while excessive accumulation of autophagosomes may impair cardiomyocyte function. Consistent with this idea, cardiotoxicity of doxorubicin was reported to be due to accumulation of autophagosomes (Li et al., 2016). Therefore, it is critical to consider these discrepancies when targeting autophagy for the treatment of HF. In this study, we showed that RAGE knockout or blockade significantly reduced LV myocardial expression of two potential regulators of autophagy (BNIP3 and p65-NFκB) in TAC mice at 8 weeks after surgery. This decrease of pp65-NFκB/BNIP3 expression following RAGE knockout suggests a cardioprotective effect. Another study has shown a cardioprotective role of NFκB (Misra et al., 2003), which has been reported to suppress BNIP3 expression under pro-apoptotic conditions (Shaw et al., 2008). These differing findings suggest that different disease mechanisms may alter distinct pathways related to NFκB, which is involved in regulation of various molecular pathways, including those for anti-apoptotic and autophagic processes (Baldwin, 2012). It is possible that RAGE mediates an NFκB signaling pathway involved in continuous activation of autophagy in mice with TAC-induced HF. It was previously demonstrated that autophagy is elevated for 3 weeks after induction of pressure overload and that this excessive autophagic response contributes to HF (Zhu et al., 2007). Accordingly, our results suggest that RAGE-NFκB/BNIP3/Beclin 1 may be one of the major contributors to excessive autophagy associated with TAC-induced HF.

CONCLUSION
Our findings demonstrated that RAGE plays a key role in the progression of HF in mice with TAC, and RAGE knockout or RAGE blockade can prevent cardiac remodeling. Importantly, we found that RAGE knockout reduced excessive autophagic activity, suggesting the RAGE autophagy axis could be an attractive target for treatment of HF.

DATA AVAILABILITY
The supporting data and materials presented in this publication are available upon request.

AUTHOR CONTRIBUTIONS
WG was responsible for the in vitro and in vivo experiments as well as data interpretation and manuscript writing. ZZ, BL, YH, LZ, and CY was responsible for generation of the animal model and performed the histological data analysis. JW, LL, and ZW carried out the cell cultures. ZY and YC were responsible for conception and design of the experiments. LW and SX was responsible for the conception and design of the study, edited the manuscript, and provided the funding. All authors read and approved the manuscript for publication.

FUNDING
This work was supported by grants from the National Natural Science Foundation of China (81373570 and 81673920) and the Natural Science Foundation of Guangdong Province (2016A030311030) to LW. This work was also supported by grants from the National Natural Science Foundation of China (81473621 and 81673796) and the Key Lab Foundation of Guangzhou City (201705030006) to SX.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.01333/full#supplementary-material FIGURE S1 | Molecular weight markers to gel data and scans in Figure 1C.    Figure 5A. FIGURE S5 | Molecular weight markers to gel data and scans in Figure 6A.