SHP-2 specific deletion in macrophages accelerates pathological cardiac hypertrophy through promoting IRE1α-XBP1s pathway regulated by IL-6 secretion

doi:10.21203/rs.3.rs-1596871/v1

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SHP-2 specific deletion in macrophages accelerates pathological cardiac hypertrophy through promoting IRE1α-XBP1s pathway regulated by IL-6 secretion

https://doi.org/10.21203/rs.3.rs-1596871/v1

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SHP-2 is a pervasively Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTP) and its specific deletion in macrophages can promote the IL-6 secretion in the pathophysiological process of cardiac hypertrophy. To elucidate the mechanism, AngII infused Lyz-2-Cre/SHP-2^flox/flox mice and SHP-2 MφCKO wild-type (SHP-2^+/+) mice were subjected to transverse aortic constriction (TAC) to induce cardiac hypertrophy. Echocardiographic scans showed that left ventricular (LV) end-diastolic dimension and left ventricular systolic volume were significantly increased in SHP-2^flox/flox group, implying that TAC-induced SHP-2^flox/flox can induce LV hypertrophy and damage cardiac function. In addition, IHC analysis and Western blotting demonstrated that SHP-2^flox/flox obviously elevated the expression of α-SMA, collagen, circulating macrophage numbers and positive areas and expression level of IL-6 in vitro. Moreover, SHP-2^flox/flox positively regulated IRE1α-XBP-1 activation, whereas the expression levels of NF-κB did not differ between SHP-2^flox/flox and SHP-2^+/+ mice. It was also confirmed that the increased expression levels of IL-6, p-IRE1α, and XBP1s in SHP-2^flox/flox mice were markedly reversed by 3,6-DMAD hydrochloride (an inhibitor of the IRE1α-XBP1s pathway). These results demonstrated that SHP-2^flox/flox promotes IL-6 secretion via the IRE1α-XBP1s pathway rather than NF-κB signaling pathway in the development of cardiac hypertrophy.

Cardiac hypertrophy

IRE1α-XBP1s

Lyz-2-Cre/SHP-2flox/flox mice

SHP-2

transverse aortic

Cardiac hypertrophy and resulting sudden death characterize the principal contributors to diseases in the Western world [1, 2]. Cardiac hypertrophy can be defined as extreme growth of the heart, which is associated with cardiomyocyte (CM) hyperplasia and proliferation of cardiac fibroblast (CF) [3]. It is a consensus that an enlarged workload caused by intrinsic and extrinsic stimulation in pathological or physiological processes may induce cardiac hypertrophy, an adaptive response to protect cardiac function and reduce myocardial wall pressure and depletion [4, 5]. Several factors are implicated in the pathological or physiological process of cardiac hypertrophy, of which inflammation plays a critical intermediating role [6]. Additionally, various studies have confirmed that macrophages are implicated in the development of cardiac hypertrophy [1, 4]. Macrophages are well-known inflammatory cells in heart diseases, which are polarized into two main subpopulations of classical M1 macrophage that mediates pro-inflammatory reaction and alternative M2 macrophage that exercises anti-inflammatory function [7]. IL-6 as a key pro-inflammatory cytokine can activate M1 macrophage, thus contributing to the pro-inflammatory effect [8]. Remarkably, it has been demonstrated recently that SHP-2 specific deletion in macrophages positively regulates the increase of IL-6 [9]. However, the mechanism by which SHP-2 deletion in macrophages promotes the IL-6 secretion has yet to be clearly elucidated. In this experimental study, four generations of the Lyz-2-Cre/SHP-2^flox/flox mice were established by breeding Lyz-2-Cre^+/− mice with the SHP-2^flox/flox mice, and it was found that the expression of IL-6 was significantly enhanced in macrophages with SHP-2 specific deletion. There are conflicts in the regulation of IL-6 by SHP-2 specific deletion in macrophages [9, 10]. Therefore, a range of in vivo and in vitro experiments were designed and completed in this present study to explore the mechanism by which SHP-2 specific deletion in macrophages promotes the IL-6 secretion in the development of cardiac hypertrophy.

Mice

The pressure-overloaded Lyz-2-Cre mice were mated with SHP-2^flox/flox mice to generate macrophage-specific SHP-2 deleted mice (Lyz-2-Cre /SHP-2^flox/flox) [11]; SHP-2 MφCKO wild-type (SHP-2^+/+) mice and controls injected with saline injection were selected in this experiment study. In order to verify the deletion of SHP-2, (59-CAGTTGCAACTTTCTTACCTC-39) and (59-GCAGGAGACTGCAGCTCA GTGATG-39) primer within introns 3 and 4 were used.

TAC induced cardiac hypertrophy models

All mice were subjected to thoracic aorta constriction (TAC) as described previously [12] for 14 days to induce cardiac hypertrophy models through cardiac pressure overload: mice were anesthetized with a mix of pentobarbital sodium (50 mg/kg ip) and ketamine (25 mg/kg ip). Supine and endotracheal intubation was completed quickly and carefully. The cannula was attached to a volume-cycled rodent ventilator with a tidal volume of 0.5 mL room air and a respiratory rate of 110 breaths/ min. The chest cavity was inserted in the second intercostal space at the left upper sternal border through a small incision. The thymus was then slightly swerved out of the field of view to expose the aortic arch. After the transverse aorta was isolated between the carotid arteries, it was constricted by a 7 − 0 silk suture ligature tied firmly against a 27-gauge needle. The latter was promptly removed to yield a constriction of 0.4 mm in diameter. Sham-operated mice underwent a similar surgical procedure without constriction of the aorta. The chest was closed with a 5 − 0 silk suture, and they were allowed to recover from anesthesia with light supply to keep the body temperature at 37°C.

Echocardiographic study

Echocardiography was performed by B-mode and Doppler echocardiography system (Vevo 770, Visual Sonics, Canada). Cardiac hypertrophy was assessed through measuring both systole and diastole, including left ventricular end-systolic volume (ESV), left ventricular (LV) end-diastolic dimensions, left ventricular systolic volume, continuous wave (CW), left ventricular posterior wall thickness in diastole (LVPWD), left ventricular posterior wall thickness in systole (LVPWS), left ventricular internal diameter in diastole (LVIDD) left ventricular internal diameter in systole (LVIDS), left ventricular end-diastolic anterior wall thickness (LVAWD), left ventricular end-systolic anterior wall thickness (LVAWs), and ejection fraction (EF).

Histological and IHC staining analysis

Experimental mice were anesthetized, and LV tissues were harvested, then fixed in 10% formalin and sliced into 6 µm-thick sections for histological analysis. The sections were exposed to Masson staining (Trichrome Stain Kit, Sigma-Aldrich) to detect the collagen integrity. Several sections were subjected to immunohistochemistry (IHC) staining for α-smooth muscle actin (1:100; Dako Cytomation), CD68 and IL-6, then measured by light microscope (Fiji) according to the software instructions.

Western blotting

Samples obtained from cells and LV tissues were homogenized in 0.1% SDS buffer (Life Technologies, USA), rinsed with frozen PBS, and centrifuged using a homogenizer in RIPA buffer at 10,000 rpm and 4°C for 10 min. The supernatant was carefully collected to determine the protein concentration using a BCA protein assay kit (Thermo Fisher Scientific, USA). The isolated protein was separated on SDS-PAGE, and incubated using the primary antibody (Abcam) for 12 h at 5°C. Finally, protein signals were identified using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).

Statistical analysis

Values were expressed as mean ± SD. Statistical differences were determined using Student's t-test between two means, and using one-way analysis of variance (ANOVA) between groups following Bonferroni multiple comparisons with SPSS20.0. p < 0.05 was considered statistically significant.

In order to elucidate the pathophysiological role of SHP-2 specific deletion in macrophages in the development of cardiac hypertrophy, we established pressure overload cardiac hypertrophy models with Lyz-2-Cre/SHP-2^flox/flox mice and SHP-2 MφCKO wild-type (SHP-2^+/+) mice by TAC. Firstly, we performed Doppler ultrasound scans to examine the cardiac function of mice in the three groups. High-quality representative echocardiographic images are shown in Fig. 1A. It was found that LV end-diastolic dimensions and left ventricular systolic volume were significantly increased in both SHP-2^flox/flox and SHP-2^+/+ mice compared with those in controls, implying that the cardiac hypertrophy models were constructed successfully. To further assess the progression of cardiac hypertrophy, continuous wave (CW) ultrasound scans were performed in four-chamber view (4CV). As expected, the blood velocity in SHP-2^flox/flox and SHP-2^+/+ mice was visibly higher than that in controls (Fig. 1A). Furthermore, the quantitative analysis of wave peak velocity demonstrated that SHP-2^flox/flox markedly enhanced the CW peak velocity compared with SHP-2^+/+ (p < 0.05). To sum up, these data confirmed that SHP-2^flox/flox can induce LV hypertrophy and damage cardiac function.

We further investigate the end left ventricular ESV for the three groups by two-dimensional B-mode high-resolution ultrasonographic imaging. Representative echocardiographic images are shown in Fig. 2A, and it was found that the ESV was obviously increased in both SHP-2^flox/flox and SHP-2^+/+ mice, especially in SHP-2^flox/flox mice. In addition, SHP-2^flox/flox mice exhibited much more severe cardiac hypertrophy than SHP-2^+/+ mice, as evaluated by the following echocardiographic parameters: LVPWD, LVPWS, LVIDD, LVIDS, LVAWD, LVAWs, and EF. These levels of echocardiographic parameters were obviously enhanced in both SHP-2^flox/flox and SHP-2^+/+ mice, especially in SHP-2^flox/flox mice (Fig. 2B. a-g). Collectively, these echocardiographic data implied that SHP-2 specific deletion in macrophages aggravates the cardiac hypertrophy.

It is a consensus that cardiac hypertrophy is associated with significant cardiac myocyte (CM) amplification and CF multiplication [13, 14]. Masson staining was conducted to investigate the CM size and fibrosis, and representative images are demonstrated in Fig. 3A. It was found that the tissues from SHP-2^flox/flox mice and SHP-2^+/+ mice adaptively exhibited a significantly enlarged size of CM (Fig. 3A). Additionally, SHP-2^flox/flox mice displayed more severe amplification of CM (Figure.3A.c). Furthermore, SHP-2^flox/flox and SHP-2^+/+ mice had a higher heart-to-body weight (HW/BW) ratio than controls, and it was further higher in SHP-2^flox/flox mice (Fig. 3C.a). Evidence has indicated that fibrosis is another component of cardiac hypertrophy, which is characterized by the excessive deposition of collagen [15]. To further investigate whether SHP-2^flox/flox aggravates cardiac hypertrophy, we further assessed the effect of SHP-2 on cardiac fibrosis by immunofluorescent staining. The results showed that SHP-2^flox/flox and SHP-2^+/+ induced a marked increase of collagen in CF (Fig. 3B). Furthermore, Fig. 3B.b-c showed increased sedimentation of collagen (red and green fluorescence). The fibroblast of SHP-2^flox/flox mice exhibited further increased collagen with clear fibers and an enlarged cell area (Fig. 2B.c). Consistently, immunostaining quantitative analysis also confirmed that SHP-2^flox/flox group exhibited a marked increase in the fibrotic area compared with WT-group and control group (Fig. 2C.b; p < 0.01). Collectively, these data implied that SHP-2 specific deletion in macrophages promotes the hypertensive cardiac hypertrophy with significant CM amplification and CF multiplication.

Evidence from a number of studies has suggested an important role for IL-6 as a pro-inflammatory cytokine in the development of cardiac hypertrophy [16–19]. In order to investigate whether SHP-2^flox/flox affected cardiac macrophage accumulation and phenotype, IHC analysis for CD68 in cardiac macrophages was performed in the three groups. As shown in Fig. 4A, there were significantly increased circulating macrophages (red), as seen in SHP-2^flox/flox and SHP-2^+/+ mice compared with control mice, whereas the number of circulating macrophages was much larger in SHP-2^flox/flox mice than SHP-2^+/+ mice. It is a consensus that macrophages exist in two major subsets of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. To further investigate the phenotype of macrophages, we investigated the macrophage marker IL-6 using IHC analysis. As shown in Fig. 4B, the positive areas of IL-6 (red) were much larger in SHP-2^flox/flox mice, implying that SHP-2^flox/flox induces a pro-inflammatory phenotype. Consistent with IHC data, the results of immunostaining quantitative analysis confirmed that the number of circulating macrophage (red) and IL-6 positive areas (red) were obviously increased in SHP-2^flox/flox mice compared with those in SHP-2^+/+ mice (p < 0.05). In addition, Western blotting was also performed to investigate the expression levels of IL-6 in tissues. As shown in Fig. 4D, the results of Western blotting and quantitative analysis both demonstrated that the expression levels of IL-6 were markedly enhanced in SHP-2^flox/flox mice compared with SHP-2^+/+ mice (p < 0.05). Besides, quantitative analysis was performed for IL-6 mRNA Fold, and the results showed that IL-6 mRNA Fold levels were obviously increased in SHP-2^flox/flox mice compared with SHP-2^+/+ mice (p < 0.05) (Fig. 4C). Collectively, these results illustrated that SHP-2^flox/flox can increase circulating macrophages, IL-6 expression level and mRNA Fold level.

It is increasingly clear that activated IRE1α can catalyze the X-box-binding protein 1 (XBP1) through the unconventional mRNA splicing that creates an activated XBP1 protein (XBP1s) to trigger a critical UPR program [20, 21]. To validate the association of SHP-2 and IRE1α-XBP1s with cardiac hypertrophy, Western blotting and quantitative analysis were conducted in SHP-2^flox/flox and SHP-2^+/+ mice tissues. In Western blotting, it was found that the expression levels of p-IRE1α and XBP1s were markedly enhanced in SHP-2^flox/flox mice compared with SHP-2^+/+ mice (Fig. 5A). In apparent agreement with this, the quantitative analysis of p-IRE1α and XBP1s expression levels also confirmed that SHP-2^flox/flox could markedly enhance p-IRE1α and XBP1s expression compared with SHP-2^+/+ (p < 0.05). To sum up, SHP-2^flox/flox can promote p-IRE1α and XBP1s expression levels in the progression of cardiac hypertrophy. Additionally, it is a consensus that NF-κB is a well-known signaling pathway through which SHP-2 promotes IL-6 secretion [22, 23]. However, in this present study, it was found by Western blotting and quantitative analysis that the expression levels of NF-κB did not differ between SHP-2^flox/flox and SHP-2^+/+ mice. Collectively, these results illustrated that SHP-2^flox/flox is a positive regulator for the IRE1α-XBP1s signaling pathway in the development of myocardial hypertrophy.

It has been previously shown that XBP1s is an important regulator for various inflammatory cytokines such as IL-6 in macrophages [24–26], and IL-6 can be activated through the combination of IL-6 promoter and XBP1 [25]. In this study, SHP-2^flox/flox was confirmed to be a positive regulator for IRE1α-XBP1s signaling. However, whether SHP-2^flox/flox regulates IL-6 production in the development of cardiac hypertrophy via the IRE1α-XBP1s pathway has not yet been investigated. As such, an inhibitor of the IRE1α-XBP1s pathway, 3,6-DMAD hydrochloride was used to investigate whether IL-6 is closely related to the regulation of SHP-2^{flox/flox via} the IRE1α-XBP1ssignaling pathway in the development of cardiac hypertrophy. As expected, it was found in Western blotting that the increased expression levels of IL-6, p-IRE1α and XBP1s in SHP-2^flox/flox mice were markedly reversed by 3,6-DMAD hydrochloride (Fig. 5A). The quantitative analysis of the expression levels of p-IRE1α, XBP1s and IL-6 clearly demonstrated that SHP-2^flox/flox markedly enhanced p-IRE1α, XBP1s and IL-6 expression levels compared with SHP-2^+/+ (p < 0.05). Additionally, it is a consensus that NF-κB is a well-known signaling pathway through which SHP-2 promotes IL-6 secretion, whereas our data demonstrated that SHP-2^flox/flox was not involved in the NF-κB signaling pathway. Together, these results illustrated that SHP-2^flox/flox indeed promotes IL-6 secretion via the IRE1α-XBP1s pathway rather than the NF-κB signaling pathway in the development of myocardial hypertrophy.

SHP-2, acts as a pervasively SH2 domain-containing PTP, plays a momentous role in cellular function and heart disease [27, 28]. In order to elucidate the pathophysiological role of SHP-2 specific deletion in macrophages in the development of cardiac hypertrophy, pressure overload cardiac hypertrophy models were established with Lyz-2-Cre/SHP-2^flox/flox and SHP-2 MφCKO wild-type (SHP-2^+/+) mice by TAC [12]. Interestingly, in this study, we found that SHP-2^flox/flox could lead to cardiac hypertrophy. Firstly, we performed Doppler ultrasound scans to examine the cardiac function of mice in the three groups. We found that LV end-diastolic dimensions and left ventricular systolic volume were significantly increased in SHP-2^flox/flox mice, suggesting that SHP-2^flox/flox can induce LV hypertrophy and damage cardiac function (Fig. 1). In addition, SHP-2^flox/flox mice exhibited higher ESV and much more severe cardiac hypertrophy than SHP-2^+/+ mice, as evaluated by several echocardiographic parameters (Fig. 2). It is widely accepted that progressive pressure overload causes cardiac hypertrophy and, as a result, heart failure, which is becoming increasingly common and is a deadly condition with high death rates [29–31]. Cardiac interstitial fibrosis and CM expansion are main features of cardiac hypertrophy [32–34]. Moreover, cardiac interstitial fibrosis is one of the characteristic features of maladaptive cardiac hypertrophy [35, 36]. In the present study, it was illustrated that SHP-2^flox/flox indeed accelerated CM expansion and fibrosis. As shown in Fig. 3, SHP-2^flox/flox significantly enhanced cardiac fibrosis in vivo and increased collagen creation in vitro. Moreover, it is well known that α-SMA and fibronectin and collagen are vital factors involved in the process of myofibroblast differentiation [37, 38]. In our study, it was also found that SHP-2^flox/flox obviously elevated the expression of α-SMA, collagen and fibronectin in SHP-2^flox/flox mice (Fig. 3). Evidence from a number of studies has suggested an important role of IL-6 as a pro-inflammatory cytokine in the development of cardiac hypertrophy [18, 39]. In our study, in order to investigate whether SHP-2^flox/flox affected cardiac macrophage accumulation and macrophage phenotype, IHC analysis for CD68 in cardiac macrophages was performed in the three groups. As shown in Fig. 4A, there were significantly increased circulating macrophages (red) in SHP-2^flox/flox mice. To further investigate the phenotype of macrophages, we investigated the macrophage marker IL-6 using IHC analysis, and it was found that the positive areas of IL-6 (red) were much larger in SHP-2^flox/flox mice, implying that SHP-2^flox/flox induces M1 macrophage pro-inflammatory phenotype. In addition, we also performed Western blotting to investigate the expression levels of IL-6 in mice tissues in the three groups. As shown in Fig. 4D, the results of Western blotting and quantitative analysis both demonstrated that the expression levels of IL-6 were markedly increased in SHP-2^flox/flox mice compared with SHP-2^+/+ mice (p < 0.05). Besides, quantitative analysis was performed for IL-6 mRNA Fold, and the results showed that IL-6 mRNA Fold levels were obviously increased in SHP-2^flox/flox mice compared with SHP-2^+/+ mice (p < 0.05) (Fig. 4C). To sum up, these results illustrated that SHP-2^flox/flox can increase circulating macrophage, IL-6 expression level and mRNA Fold level.

It is increasingly clear that activated IRE1α can catalyze the XBP1 through the unconventional mRNA splicing that creates an activated XBP1s to trigger a critical UPR program [21, 25, 40]. In the present study, Western blotting results indicated that SHP-2^flox/flox might be a positive regulator for cardiac hypertrophy through promoting IL-6 expression and IRE1α-XBP1 activation (Fig. 5).

To further validate the association of SHP-2^flox/flox and IRE1α-XBP1s with cardiac hypertrophy, Western blotting and quantitative analysis were conducted in SHP-2^flox/flox and SHP-2^+/+ mice tissues. As shown in Fig. 5, SHP-2^flox/flox could markedly enhance p-IRE1α and XBP1s expression compared with SHP-2^+/+ (p < 0.05). Additionally, it is a consensus that NF-κB is a well-known signaling pathway through which SHP-2 promotes IL-6 secretion [22, 23, 41]. However, in this present study, it was found by Western blotting and quantitative analysis that the expression level of NF-κB did not differ between SHP-2^flox/flox and SHP-2^+/+ mice (Fig. 5). Collectively, these results illustrated that SHP-2^flox/flox is a positive regulator for the IRE1α-XBP1s signaling pathway in the development of myocardial hypertrophy. Furthermore, it has been previously shown that XBP1s is an important regulator for various inflammatory cytokines such as IL-6 in macrophages, and IL-6 can be activated through the combination of IL-6 promoter and XBP1. However, whether SHP-2 regulates IL-6 production in the development of cardiac hypertrophy via the IRE1α-XBP1s pathway has not yet been investigated. As such, an inhibitor of the IRE1α-XBP1s pathway, 3,6-DMAD hydrochloride was used to investigate whether IL-6 is closely related to the regulation of SHP-2 via the IRE1α-XBP1s signaling pathway in the development of cardiac hypertrophy. In Western blotting, it was found that the increased expression levels of IL-6, p-IRE1α and XBP1s in SHP-2^flox/flox mice were markedly reversed by 3,6-DMAD hydrochloride (Fig. 6A), confirming that SHP-2^flox/flox can promote IL-6 secretion via the IRE1α-XBP1s pathway rather than the NF-κB signaling pathway in the development of cardiac hypertrophy. To our knowledge, our data provided the first evidence for the essential role of SHP-2^flox/flox in accelerating cardiac hypertrophy and collagen synthesis through promoting the IRE1α-XBP1s pathway regulated by IL-6 secretion. Therefore, targeting SHP-2 may be an effective therapeutic strategy for pressure overload-induced cardiac hypertrophy.

Acknowledgments:

This study was supported by Funding for the prevention and control of geriatric diseases in 2018 (N0:201813589-2)

Funding:

This work was supported by grants from the National Natural Science Foundation of China (81703894)

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Conceptualization: Sixuan Wang . methodology: Deshuang Yang, Sixuan Wang . validation: Jiangquan Liao, Xianglun Li. resources: Xiang Xiao .data curation: Yan Wang, Peng Yang. writing-original draft preparation: Deshuang Yang. writing-review and editing: Deshuang Yang, Dongliang Fu. visualization: Lin Li, Jinhang Du. supervision: Yi Liu. project administration: Huan Gu. funding acquisition: Mingjing Shao.

Data Availability

The datasets generated during and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Ethics approval

Approval was granted by the Laboratory Animal Ethical Committee, China-Japan Friendship Hospital.

Consent to participate

Not Applicable

Consent to publish

Not Applicable

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No competing interests reported.

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SHP-2 specific deletion in macrophages accelerates pathological cardiac hypertrophy through promoting IRE1α-XBP1s pathway regulated by IL-6 secretion

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Abstract

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Introduction

Method

Mice

TAC induced cardiac hypertrophy models

Echocardiographic study

Histological and IHC staining analysis

Western blotting

Statistical analysis

Results

Discussion

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References

Additional Declarations

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