NOD2 contributes to myocardial ischemia/reperfusion injury by regulating cardiomyocyte apoptosis and inflammation Yu Liu, Hui Yang, Li-Xin Liu, Wen Yan, Huo-Jie Guo, Wen-Jun Li, Cui Tian, Hui-Hua Li, Hong-Xia Wang PII: DOI: Reference:
S0024-3205(16)30089-3 doi: 10.1016/j.lfs.2016.02.039 LFS 14724
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
14 August 2015 5 January 2016 9 February 2016
Please cite this article as: Liu Yu, Yang Hui, Liu Li-Xin, Yan Wen, Guo Huo-Jie, Li Wen-Jun, Tian Cui, Li Hui-Hua, Wang Hong-Xia, NOD2 contributes to myocardial ischemia/reperfusion injury by regulating cardiomyocyte apoptosis and inflammation, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
NOD2
contributes
to
myocardial
ischemia/reperfusion
injury
by
regulating
cardiomyocyte apoptosis and inflammation
a
a
b
b
c
b
b
b
IP
b
SC R
, Hong-Xia Wang *
a
Department of Pathology, School of Basic Medical Sciences, Capital Medical University,
Beijing, China.
Department of Physiology and Physiopathology, School of Basic Medical Sciences, Capital
NU
b
Medical University, Beijing, China.
Department of Vascular Surgery, Peking University First Hospital, Beijing, China.
*Correspondence to: H.-X. Wang
MA
c
T
Yu Liu , Hui Yang , Li-Xin Liu , Wen Yan , Huo-Jie Guo , Wen-Jun Li , Cui Tian , Hui-Hua Li
D
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital
TE
Medical University, Beijing 100069, China
AC
CE P
E-mail address: [email protected]
ACCEPTED MANUSCRIPT AB ST RACT Aims: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, which plays an important role in the innate immunity and
IP
T
inflammation. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. In this study, we sought to determine the role of NOD2 on cardiac I/R injury.
SC R
Main methods: Mice were induced 30 min ischemia followed by 24 h of reperfusion. Histological examinations were performed on heart sections with Evans blue and triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry and
NU
immunefluorescence staining. The messenger RNA (mRNA) expression and protein levels were detected by real-time polymerase chain reaction (RT-PCR) and western blot analysis
MA
respectively.
Key findings: I/R injury markedly upregulated NOD2 expression in heart tissue. Treatment of
D
WT mice with NOD2 ligand (MDP) significantly increased infarct size, the number of
TE
apoptotic cells and inflammatory cells, as compared with wild-type mice after I/R injury. Furthermore, MDP enhanced I/R-induced cardiomyocyte apoptosis and inflammation in vitro,
CE P
and these effects were attenuated by NOD2-siRNA. The mechanism of NOD2 on cardiac I/R injury is partly associated with JNK, p38 MAPK and NF-κB signaling pathways. Significance: NOD2 aggravates myocardial I/R injury by inducing cardiomyocyte apoptosis and
AC
inflammation through JNK, p38 MAPK and NF-κB signaling pathways. This study provides insight into better understanding the molecular mechanism of NOD2, which may be served as a potential target for the treatment of myocardial I/R injury.
Keywords:
NOD2 · Ischemia/reperfusion injury · Apoptosis · Inflammation
ACCEPTED MANUSCRIPT
Introduction
T
Myocardial ischemia/reperfusion (I/R) injury is a common cardiovascular problem and one
IP
of the major causes of death in the world. It is widely held that the mechanisms contributing to
SC R
I/R injury are complicated. The innate immune system is an essential component of the acute inflammatory response and plays an essential role in the pathogenesis of I/R injury [1, 2]. As the primary recognition receptors of the innate immunity, Toll-like receptors (TLRs) have been
NU
identified as the key mediators in the heart diseases [3, 4]. Researches have shown that the activation of TLR4 have been detected in cardiac cells and shown to mediate myocardial I/R
MA
injury [5-7]. Besides TLRs, other receptors of the innate immunity such as nucleotide-binding oligomerization domain-like receptors (NLRs) have been involved in cardiovascular disease (CVD) [8, 9].
D
NOD2, which is one of members of NLR family, is an intracellular pathogen sensor
TE
through the recognition of muramyl dipeptide (MDP). The activation of transcription factor
CE P
mediated by NOD2 can increase production of inflammatory cytokines. NOD2 can regulate innate immunity and is essential for host defense and inflammation [10-12]. An increasing number of clinical and animal model studies have implicated that the association of NOD2 with
AC
the pathogenesis of atherosclerosis, Alzheimer disease, diabetes mellitus and renal ischemia reperfusion injury, et al [13-15]. Recently, Li et al. demonstrate that NOD2 is a critical component of a signal transduction pathway that links cardiac injury by exacerbation of inflammation and MMP-9 activity in myocardial infarction (MI) [16]. All of the studies indicate that NOD2 may have an important role in cardiovascular disease. However, the role of NOD2 signaling in myocardial I/R has not been elucidated. In the present study, we investigated both in vitro and in vivo the role of NOD2 in regulating myocardial I/R injury. We provide evidences that NOD2 activation by MDP enhanced I/R-induced myocardial injury by aggravating apoptosis and inflammation. These effects were mediated by activating JNK, p38 MAPK and NF-κB signaling pathways. Conversely, knockdown of NOD2 attenuated these effects. These findings suggest that NOD2 could be a novel strategy for treatment of myocardial I/R.
ACCEPTED MANUSCRIPT
Materials and methods
IP
T
Animal model of myocardial I/R
SC R
Male C57/B6J mice were intraperitoneally administered either vehicle (NaCl 0.9%) or MDP (10 mg/kg; San Diego, CA) 30min before the operation [12]. The mouse model of myocardial I/R was prepared as previously described [17]. In brief, male C57BL/6 (WT) mice,
NU
8-10 weeks of age, were used for the study. To generate myocardial I/R, the mice were anaesthetized with isoflurane (1.5%), and was induced by left coronary artery (LCA) ligation
MA
for 30 min and reperfusion for 24h. In sham-operated animals, the suture was placed under the LCA and removed without ligating the artery. In MDP (San Diego, CA)-treated animals, 10mg/kg MDP was injected intraperitoneally 30 min before coronary artery occluded . The
D
study was performed according to the Guide for the Care and Use of Laboratory Animals
TE
published by the US National Institutes of Health, and the protocol was consistent with the
CE P
Animal Care and Use Committee of Capital Medical University.
AC
Evaluation of infarct size
Myocardial infarct size was assessed by Evans Blue and triphenyltetrazolium chloride (TTC) staining method as described [18]. The method is usually used as current “gold standard” to quantitate the area at risk (AAR) and degree of completed infarct. After completion of experiment, the LCA was reoccluded, 1% Evans blue dye was injected via the aorta and coronary arteries to differentiate ischemic (non-blue) and normal (blue) tissue. Then, the heart was quickly frozen for 1 h at -80 ℃. The frozen heart was sliced into uniform sections about 2mm thickness. The slices were incubated for 20 min in phosphate-buffered 1% TTC at 37 ℃ to determine the infarct area (INF, pale white) and the area at risk (AAR, brick red). At last, the INF appears pale white, the AAR stains brick red and the normal area is blue. The INF, the AAR, and the left ventricular (LV) area from each section were analyzed by use of NIH Image software (US National Institutes of Health, Bethesda, MD), multiplied by the weight
ACCEPTED MANUSCRIPT of the section, and then totaled from all sections. The ratio of INF to AAR and AAR to LV were calculated [18].
IP
T
Histological analysis, Immunohistochemistry and Immunofluorescence
morphology,
H&E
staining
was
used
on
SC R
The mice hearts were fixed with 10% formalin and embedded in paraffin. To evaluate the
heart
sections
(5μm)
[19].
For
immunohistochemistry, anti-MAC-2 antibody (Santa Cruz, CA,1:200 dilution) and anti-IL-6
NU
antibody (Santa Cruz, CA, 1:200 dilution) was used as the primary antibody, and the positive cells were detected by 3,30-Diaminobenzidine (DAB) staining. Negative control sections were
MA
treated with IgG antibodies. For immunofluorescence assays, staining was performed as previously reported [17, 20]. Frozen sections were stained with anti-ICAM-1 (Santa Cruz, CA,1:200 dilution) and MCP-1 (Santa Cruz, CA,1:200 dilution) overnight at 4°C. Then, the
D
sections were washed and incubated with Hoechst 33342 (1:1000) for 5 min to stain the nuclei.
TE
The positive areas were calculated from 6 random fields at 200 magnification (Olympus BX-63
AC
Cell culture
CE P
JAPAN).
Cell culture was conducted as described previously
[18]
. Cardiomyocytes were isolated
from 1 d old neonatal SD rats and were seeded and incubated in DMEM containing 10% FCS at 37℃. Then, cardiomyocytes were transferred to an ischemic buffer that contains 118 mM NaCl, 24 mM NaHCO3, 1.0 mM NaH2PO4, 2.5 mM CaCl2-2H2O, 1.2 mM MgCl2, 20 mM sodium lactate, 16 mM KCl and 10 mM 2-deoxyglucose (pH 6.2) and incubated at 37 °C for 2 h in a humidified atmosphere of 5% CO2 and 95% nitrogen. During ischemia occurring in vivo, the reduced O2 supply causes an increase in concentrations of intracellular Na +
+
2+
and Ca ,
+
increase in extracellular K , decrease in extracellular Na , and decrease in intracellular ATP and PH. The ischemic buffer including the approximate concentrations of potassium, hydrogen, lactate ions and pH and is designed to simulate the extracellular environment during myocardial ischemia. After 2 h of simulated ischemia, cardiomyocytes were randomly exposed
ACCEPTED MANUSCRIPT to reperfusion for 30 min or 24 h by replacing the ischemic buffer with normal cell medium. Cardiomyocytes were treated with MDP (10 μg/ml) for 30 min before exposure to ischemia
IP
T
buffer.
SC R
RNA interference
Small interfering RNA (siRNA) against the NOD2 and negative control siRNA were purchased from Invitrogen (San Diego, CA). The final concentration of siRNA was 100 nM
NU
diluted in OPTI-MEM medium. The cells were transfected with siRNA using Lipofectamine 2000 in accordance with the manufacturer’s protocol [21]. After 24-48 h, the mRNA level of
MA
NOD2 was determined by quantitative real-time RT-PCR, and the protein expression of NOD2
D
was measured by western blot.
TE
Assay of apoptosis
CE P
The apoptosis of heart sections and cardiomyocytes were measured by the FragEL™ DNA Fragmentation Detection Kit (Roche, Indianapolis, IN). The staining was performed as described [20]. Fluorescence staining was observed using fluorescence microscopy (Olympus
AC
BX-63, JAPAN). The amount of apoptosis cells was analyzed by counting the number of positive cells in 6 fields within the heart sections and cardiomyocytes using 20 × magnification.
Measurement of caspase-3 activity
Caspase-3 activity was performed using a colorimetric activity assay kit [18]. In brief, Using buffer containing 10 mM dithiothreitol and 5 μl Ac-DEVD-AFC (BIOMOL, PA), the samples were incubated at 37°C for 1.5 h. The detection of caspase-3 activity was evaluated through a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 405 nm.
ACCEPTED MANUSCRIPT Quantitative real-time PCR
For quantitative real-time PCR, total RNAs were purified from hearts using Trizol reagent
IP
T
[22, 23]. RNA was transcribed to cDNA with PrimeScript RT reagent Kit. Then qRT-PCR was performed in a 20 ul reaction mixture containing SYBR Green PCR Master Mix (Warrington,
SC R
UK), cDNA and 0.2 mmol/L of each primer at 95℃ for 10 min, 40 cycles at 95 ℃ for 10 s and 60 ℃ for 45 s. The relative gene expression was normalized to internal control GAPDH. Primer sequences for SYBR Green probes of target genes are as follows: forward,
5’-CCAGCGTCTTTGGCCATTCAACAT-3’;
5’-TTGAGCTCATCCAGTGCTTGGAGT-3;
NU
NOD2:
and
GAPDH:
reverse, forward,
MA
5’-CCCCCAATGTATCCGTTGTG-3’; and reverse, 5’-TAGCCCAGGATGCCCTTTAGT-3’.
TE
D
Western blot analysis
Protein was extracted from the heart tissues and cardiomyocytes. The protein was boiled
CE P
and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The primary antibodies dilutions were 1:500 for NOD2 (Santa Cruz, CA), 1:500 for Bax (Beverly, MA) , 1:500 for Bcl-2 (Beverly, MA),
AC
1:1000 for total- and phospho-JNK (Beverly, MA), 1:1000 for total- and phospho-p38 MAPK (Beverly, MA), 1:800 for total- and phospho-p65 (Beverly, MA) , 1:5000 for GAPDH (Beverly, MA). Secondary antibodies (Santa Cruz, CA) were used at a dilution of 1:5000[22, 23].
Statistical analysis
For statistical analysis, SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used. The results were shown as mean ± SEM. One-way ANOVA or t-test was used to compare differences between groups.
P < 0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT Results
IP
T
NOD2 expression was increased in myocardial I/R injury
To test the involvement of NOD2 in myocardial I/R injury, we measured NOD2 level in the
SC R
mouse heart following I/R. As seen in Fig. 1A, RT-PCR analysis showed myocardial I/R significantly increased NOD2 mRNA expression. Western blot analysis also demonstrated NOD2 protein level was significantly increased in the I/R hearts compared to sham hearts,
NU
(Fig.1B). These data suggest that increased NOD2 expression may
MA
injury.
I/R
NOD2 activation exacerbated myocardial infarction and promoted apoptosis after I/R
D
Using Evans Blue-TTC method, myocardial infarct size was detected to evaluate the
TE
effect of NOD2 on myocardial I/R injury. The representative photographs of heart sections are
CE P
shown in Fig. 2A. The results showed that the ratio of infarct area (INF) to area at risk (AAR) was significantly higher in I/R + MDP group compared with I/R group. No significant difference in the ratio of AAR to left ventricular (LV) was found between I/R + MDP group and I/R
AC
group. It indicated NOD2 activation aggravated infarct size in I/R injury. To elucidate whether NOD2 regulates cardiomyocyte apoptosis in I/R, TUNEL staining was used to detect myocardial apoptotic cells in mice hearts. The results identified I/R injury caused apoptosis in the cardiomyocytes. Moreover, MDP pretreatment further increased the incidences of apoptosis (Fig. 2B). The expression of apoptosis-related proteins were assessed by western blot analysis. The decrease of Bcl-2 expression and increase of Bax expression were observed in IR group. Compared with I/R group, MDP further significantly attenuated the Bcl-2 expression but increased Bax expression (Fig. 2C). These results strongly suggest that NOD2 has significant pro-apoptotic effects.
NOD2 activation facilitates cardiac inflammation after I/R injury
ACCEPTED MANUSCRIPT Inflammation induced by I/R injury is a major cause of cardiac impairment. NOD2 has been reported to promote the inflammation response. We examined the macrophage infiltration in heart specimens by immunohistochemistry using an anti-Mac-2 antibody and the
IP
T
study demonstrated a significantly higher macrophage infiltration in the IR+MDP compared
IL-6, ICAM-1, and MCP-1 are the key mediators
SC R
with I/R group (Fig.3A)
myocardial I/R injury. When these proteins were determined in mice hearts by immunohistochemistry or immunofluorescence assay, I/R significantly increased IL-6, ICAM-1,
NU
and MCP-1 levels compared with sham group. Importantly, these cytokines expression was significantly greater in I/R+MDP group compared to I/R group (Fig. 3A-B). Together, these
MA
data suggest that NOD2 is important in the regulation of IR-associated inflammation.
TE
D
NOD2 activation increases I/R-induced apoptosis in neonatal rat cardiomyocytes.
To further confirm the pro-apoptotic effect of NOD2 on cardiomyocytes in vitro, neonatal
CE P
rat cardiomyocytes were subjected to simulated I/R. TUNEL assay revealed that MDP treatment significantly increased the number of apoptotic cells in I/R injury (Fig. 4A). Moreover, caspase 3 activity was significantly higher in MDP-treated cardiomyocytes than that in
AC
untreated cardiomyocytes after I/R (Fig. 4B). These results further suggest that NOD2 was involved in IR injury by inducing apoptosis.
NOD2 activation aggravates myocardial I/R injury through regulation of JNK1/2, p38MAPK and NF-κB pathway
Recent studies have shown that MAPK and NF- kB pathways were involved in I/R injury. We hypothesized that the function of NOD2 in IR injury is mediated by these signaling pathways. As shown in Fig. 5, JNK1/2, p38MAPK and NF-κB were increased following I/R. Compared with I/R, MDP treatment further stimulated JNK1/2, p38MAPK and NF-κB activation. These results indicated that the damage caused by NOD2 in IR can be attributed in part to the upregulation in JNK1/2, p38MAPK and NF-κB pathways.
ACCEPTED MANUSCRIPT
Suppressing NOD2 with NOD2 siRNA decreases apoptosis induced by I/R in neonatal cardiomyocytes
IP
T
To further assess the impact of NOD2 on cardiomyocyte apoptosis, cardiomyocytes were isolated and transfected with siRNA-control and siRNA-NOD2 and then exposed to mimetic
SC R
I/R. The decrease of NOD2 protein expression was observed after transfection with siRNA-NOD2. Cardiomyocyte apoptosis, caspase-3 activity were increased on mimetic I/R
NU
treatment, whereas siRNA-NOD2 treatment ameliorated these effects (Fig. 6B and Fig. 6C).
neonatal rat cardiomyocytes after I/R
MA
Suppressing NOD2 with NOD2-siRNA attenuates NF-κB, p38MAPK and JNK1/2 expression in
D
Since above results have showed NOD2 can activate JNK1/2, p38MAPK and NF-κB
TE
signaling pathways (Fig. 5), we further examined whether down-regulation of NOD2 inhibited activation of these pathways. Then, we observed that down-regulation of NOD2 by siRNA
CE P
markedly suppressed the I/R-induced activation of JNK1/2, p38MAPK and NF-κB pathways, Thus, taking all these results together, we conclude that NOD2 prompts cardiomyocytes
AC
apotosis and inflammation via these pathways (Fig. 7).
Discussion
In this study, we presented first evidence that NOD2 expression was increased during the processes of myocardial I/R Injury. Activation of NOD2 by MDP aggravated infarct size, cardiomyocyte apoptois and inflammation in the heart tissues or cardiomyocytes after I/R. In contrast, down-regulation of NOD2 by NOD2-siRNA attenuated these effects. During this process, JNK1/2, p38MAPK and NF-κB signaling pathway were activated by NOD2 and accounted for inflammatory responses and apoptosis. It has been well demonstrated that apoptosis play a critical role in I/R-induced injury [24]. NOD2 has been causally linked to the pathogenesis of apoptosis [25]. It is reported that NOD2 may enhance caspase-9-induced apoptosis by interaction with the CARD domain [26].
ACCEPTED MANUSCRIPT In our study, treatment with MDP resulted in significant increasement in cardiomyocyte apoptosis in vivo, indicating that the pro-apoptotic effect of NOD2 in myocardial I/R. Caspase-3, a member of the cysteine proteases, is implicated in apoptosis. Activation of
IP
T
caspase-3 is associated with a series of signal transduction cascades that include Bcl-2 and Bax proteins. Bcl-2 can form a heterodimer with Bax, thereby prevent Bax homodimerization
SC R
and the sequential activation of caspase-3 [27]. The present study revealed that I/R markedly up-regulated expression of Bax and down-regulated expression of Bcl-2 in the myocardium of I/R mice, consistent with the previous researchers [18, 28]. Meanwhile, treatment with MDP
NU
exacerbated I/R-induced increase Bax and decrease of Bcl-2 expression in myocardium. Furthermore, these results were verified in vitro. It is suggesting that NOD2 may exert its
MA
pro-apoptotic effects by regulating Bax and Bcl-2, thus promoted the activation of caspase-3. Several investigations have shown that inflammatory responses such as the accumulation
D
of inflammatory cells and pro-inflammatory cytokines participate in the development of I/R
TE
injury [29]. Recent studies have implicated ischemia promotes rapid recruitment of inflammatory cells into the myocardium [30, 31]. The cytokines and chemokines released by
CE P
inflammatory cells further attract macrophage infiltration and promote inflammatory reactions [32, 33]. It has been reported that NOD2 is important to regulate inflammation in some diseases [34-36]. In the present study, the macrophage infiltration and generation of IL-6,
AC
ICAM-1, and MCP-1 were all increased significantly in the mice hearts following I/R injury. Each of these changes was promoted by MDP pretreatment. This suggests that NOD2 activates the myocardium inflammation during I/R injury. Moreover, several signaling pathways involved in the apoptosis and inflammation are related to myocardial I/R injury. The c-Jun NH2-terminal kinase (JNK), p38 MAPK are the major groups of MAPK subfamilies. Activation of JNK as well as p38 MAPK induced myocardial I/R injury by regulating cardiomyocyte apoptosis and proinflammatory cytokine production [37]. Moreover, NF-kB is a key mediator of inflammation in myocardial injury caused by I/R. Recently, several studies demonstrate that NOD2 stimulation induces the activation of MAPK and NF-kB pathway [38-40]. Consistently, we found that JNK, p38 MAPK and NF-kB pathway were activated after myocardial I/R injury in WT mice, while activation of NOD2 by MDP significantly increased the levels of JNK, p38 MAPK and NF-kB. To further
ACCEPTED MANUSCRIPT confirm the mechanisms of NOD2 in I/R-induced myocardial injury, we cultured neonatal rat cardiomyocytes in vitro, and found that the effects of NOD2 on JNK, p38 MAPK and NF-kB pathway were all blocked by NOD2-siRNA. Taken together, these data indicate that, by
IP
T
powerfully activating JNK, p38 MAPK and NF-kB, NOD2 has special detrimental effect on myocardium.
SC R
In summary, the present study demonstrated that NOD2 exacerbates myocardial infarct size following I/R injury. The mechanisms involve activating I/R-activated apoptotic signaling including JNK1/2, p38MAPK and NF-κB activity, resulting in increase of I/R-induced
MA
management of ischemic heart disease.
NU
inflammation and apoptosis. NOD2 may be an attractive target for the treatment and
D
Acknowledgements
TE
This work was supported by grants from China National Natural Science Funds (No. 81330003, 81000343, 81500320), Beijing Higher Education Young Elite Teacher Project (No.
CE P
YETP1668)，and The Importation and Development of High Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD201404180). Author contributions: H.X.W. were responsible for conception and design. Y.L. analyzed
AC
the data and wrote the article. H.H.L. revised the article. H.Y., L.X. L., W.Y., H.J. G., W.J. L. and C.T collected the data. All authors have read and approved the final version of the manuscript.
References
[1] A. Linde, D. Mosier, F. Blecha, T. Melgarejo.Innate immunity and inflammation--New frontiers in comparative cardiovascular pathology, Cardiovasc. Res. 73 (1) (2007) 26-36. [2] S. Epelman, P. P. Liu, D. L. Mann.Role of innate and adaptive immune mechanisms in cardiac injury and repair, Nat. Rev. Immunol. 15 (2) (2015) 117-129. [3] S. Frantz, G. Ertl, J. Bauersachs.Mechanisms of disease: Toll-like receptors in cardiovascular disease, Nat. Clin. Pract. Cardiovasc. Med. 4 (8) (2007) 444-454.
ACCEPTED MANUSCRIPT [4] R. Spirig, J. Tsui, S. Shaw.The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease, Cardiol. Res. Pract. 2012 (2012) 181394. [5] J. Cha, Z. Wang, L. Ao, et al.,Cytokines link Toll-like receptor 4 signaling to cardiac dysfunction
IP
T
after global myocardial ischemia, Ann. Thorac. Surg. 85 (5) (2008) 1678-1685.
[6] J. Oyama, C. Blais, Jr., X. Liu, et al.,Reduced myocardial ischemia-reperfusion injury in toll-like
SC R
receptor 4-deficient mice, Circulation. 109 (6) (2004) 784-789.
[7] P. Zhao, J. Wang, L. He, et al.,Deficiency in TLR4 signal transduction ameliorates cardiac injury and cardiomyocyte contractile dysfunction during ischemia, J. Cell. Mol. Med. 13 (8A) (2009)
NU
1513-1525.
[8] L. Franchi, N. Warner, K. Viani, G. Nunez.Function of Nod-like receptors in microbial recognition
MA
and host defense, Immunol. Rev. 227 (1) (2009) 106-128.
[9] T. D. Kanneganti, M. Lamkanfi, G. Nunez.Intracellular NOD-like receptors in host defense and
D
disease, Immunity. 27 (4) (2007) 549-559.
TE
[10] A. Rehman, C. Sina, O. Gavrilova, et al.,Nod2 is essential for temporal development of intestinal microbial communities, Gut. 60 (10) (2011) 1354-1362.
CE P
[11] R. G. Correa, S. Milutinovic, J. C. Reed.Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases, Biosci. Rep. 32 (6) (2012) 597-608. [12]
E.
Perez-Chanona,
M.
intestinal
injury
C.
Jobin.The
through
microbiota
nucleotide-binding
protects
against
oligomerization
AC
ischemia/reperfusion-induced
Muhlbauer,
domain-containing protein 2 (NOD2) signaling, Am. J. Pathol. 184 (11) (2014) 2965-2975. [13] P. Du, B. Fan, H. Han, et al.,NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy, Kidney. Int. 84 (2) (2013) 265-276. [14] H. Han, Y. Wang, X. Li, et al.,Novel role of NOD2 in mediating Ca2+ signaling: evidence from NOD2-regulated podocyte TRPC6 channels in hyperhomocysteinemia, Hypertension. 62 (3) (2013) 506-511. [15] M. Y. Kwon, X. Liu, S. J. Lee, et al.,Nucleotide-binding oligomerization domain protein 2 deficiency enhances neointimal formation in response to vascular injury, Arterioscler. Thromb. Vasc. Biol. 31 (11) (2011) 2441-2447. [16] X. Li, F. Li, Y. Chu, et al.,NOD2 deficiency protects against cardiac remodeling after myocardial infarction in mice, Cell. Physiol. Biochem. 32 (6) (2013) 1857-1866.
ACCEPTED MANUSCRIPT [17] C. W. Xu, T. P. Zhang, H. X. Wang, H. Yang, H. H. Li.CHIP enhances angiogenesis and restores cardiac function after infarction in transgenic mice, Cell. Physiol. Biochem. 31 (2-3) (2013) 199-208. [18] Y. Zhang, Y. Zeng, M. Wang, et al.,Cardiac-specific overexpression of E3 ligase Nrdp1 increases
[19]
H.
H.
Li,
V.
Kedar,
C.
Zhang,
et
IP
T
ischemia and reperfusion-induced cardiac injury, Basic. Res. Cardiol. 106 (3) (2011) 371-383. al.,Atrogin-1/muscle
atrophy F-box inhibits
SC R
calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex, J. Clin. Invest. 114 (8) (2004) 1058-1071.
[20] K. Yang, T. P. Zhang, C. Tian, L. X. Jia, J. Du, H. H. Li.Carboxyl terminus of heat shock protein
NU
70-interacting protein inhibits angiotensin II-induced cardiac remodeling, Am. J. Hypertens. 25 (9) (2012) 994-1001.
MA
[21] H. Yang, X. J. Zeng, H. X. Wang, et al.,Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor, Peptides. 32 (10) (2011) 2108-2115.
D
[22] H. M. Jiang, H. X. Wang, H. Yang, et al.,Role for granulocyte colony stimulating factor in
(2013) 1224-1233.
TE
angiotensin II-induced neutrophil recruitment and cardiac fibrosis in mice, Am. J. Hypertens. 26 (10)
CE P
[23] H. X. Wang, H. Yang, Q. Y. Han, et al.,NADPH oxidases mediate a cellular "memory" of angiotensin II stress in hypertensive cardiac hypertrophy, Free. Radic. Biol. Med. 65 (2013) 897-907. [24] C. Lu, D. Ren, X. Wang, et al.,Toll-like receptor 3 plays a role in myocardial infarction and
AC
ischemia/reperfusion injury, Biochim. Biophys. Acta. 1842 (1) (2014) 22-31. [25] A. A. Shigeoka, A. Kambo, J. C. Mathison, et al.,Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury, J. Immunol. 184 (5) (2010) 2297-2304. [26] G. Rogler.The effects of NOD2/CARD15 mutations on the function of the intestinal barrier, J. Crohns. Colitis. 1 (2) (2007) 53-60. [27] J. Guo, S. B. Wang, T. Y. Yuan, et al.,Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation, Atherosclerosis. 231 (2) (2013) 384-391. [28] L. Song, H. Yang, H. X. Wang, et al.,Inhibition of 12/15 lipoxygenase by baicalein reduces myocardial ischemia/reperfusion injury via modulation of multiple signaling pathways, Apoptosis. 19 (4) (2014) 567-580.
ACCEPTED MANUSCRIPT [29] K. Kitano, S. Usui, H. Ootsuji, et al.,Rho-kinase activation in leukocytes plays a pivotal role in myocardial ischemia/reperfusion injury, PLoS. One. 9 (3) (2014) e92242. [30] L. Formigli, L. I. Manneschi, C. Nediani, et al.,Are macrophages involved in early myocardial
IP
T
reperfusion injury?, Ann. Thorac. Surg. 71 (5) (2001) 1596-1602.
[31] T. Kakio, A. Matsumori, K. Ono, H. Ito, K. Matsushima, S. Sasayama.Roles and relationship of
SC R
macrophages and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in the ischemic and reperfused rat heart, Lab. Invest. 80 (7) (2000) 1127-1136.
Med. 15 (5) (2005) 163-169.
NU
[32] N. G. Frangogiannis, M. L. Entman.Chemokines in myocardial ischemia, Trends. Cardiovasc.
[33] N. G. Frangogiannis, C. W. Smith, M. L. Entman.The inflammatory response in myocardial
MA
infarction, Cardiovasc. Res. 53 (1) (2002) 31-47.
[34] O. Gutierrez, C. Pipaon, N. Inohara, et al.,Induction of Nod2 in myelomonocytic and intestinal
D
epithelial cells via nuclear factor-kappa B activation, J. Biol. Chem. 277 (44) (2002) 41701-41705.
TE
[35] J. T. Tigno-Aranjuez, D. W. Abbott.Ubiquitination and phosphorylation in the regulation of NOD2 signaling and NOD2-mediated disease, Biochim. Biophys. Acta. 1823 (11) (2012) 2022-2028.
CE P
[36] A. Negroni, L. Stronati, M. Pierdomenico, et al.,Activation of NOD2-mediated intestinal pathway in a pediatric population with Crohn's disease, Inflamm. Bowel. Dis. 15 (8) (2009) 1145-1154. [37] E. Tamagno, G. Robino, A. Obbili, et al.,H2O2 and 4-hydroxynonenal mediate amyloid
AC
beta-induced neuronal apoptosis by activating JNKs and p38MAPK, Exp. Neurol. 180 (2) (2003) 144-155.
[38] Y. J. Jeong, M. J. Kang, S. J. Lee, et al.,Nod2 and Rip2 contribute to innate immune responses in mouse neutrophils, Immunology. 143 (2) (2014) 269-276. [39] L. Zhao, J. Y. Lee, D. H. Hwang.The phosphatidylinositol 3-kinase/Akt pathway negatively regulates Nod2-mediated NF-kappaB pathway, Biochem. Pharmacol. 75 (7) (2008) 1515-1525. [40] S. Maeda, L. C. Hsu, H. Liu, et al.,Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing, Science. 307 (5710) (2005) 734-738.
ACCEPTED MANUSCRIPT
Figure legends:
IP
T
Fig.1 Expression of NOD2 was increased in myocardial I/R injury. A. NOD2 mRNA levels were
analysis and summarized data (bottom) (n=3). *P < 0.05 versus sham group,
SC R
detected by RT-PCR (n=5). B. NOD2 protein (top) expression was measured by western blot
NU
Fig.2 NOD2 stimulation exacerbated I/R injury and improved cardiomyocyte apoptosis. A. Representative photographs staining with triphenyl tetrazolium chloride (TTC) and Evans blue.
MA
(n=5). B. Representative images of TUNEL-stained heart sections from different groups. Scale bar: 50 mm. Quantification of all TUNEL+ nuclei showed as percentage of total number of
D
cardiomyocyte (n=5). C. Western blot determined protein level of Bcl-2 and Bax in ischemia
TE
myocardium. The results were expressed as ratio of Bax/Bcl-2 (n=3). *P < 0.05 versus sham #
CE P
group, P < 0.05 versus I/R group.
Fig.3 NOD2 stimulation facilitates inflammation following I/R. A. Histological evaluation of heart tissue and immunohistochemical staining for Mac-2 and IL-6 in mice myocardium. Mac-2
AC
and IL-6 expression were quantified by digital image analysis (n=5). B. Immunofluorescence #
imaging of MCP-1 and ICAM-1(n=5). Scale bar: 50 mm. *P < 0.05 versus sham group, P < 0.05 versus I/R group.
Fig.4 NOD2 stimulation increases IR-induced apoptosis in primary cultured cardiomyocytes. Cultured cardiomyocytes were incubated in the presence or absence MDP and then subjected to mimetic I/R. A. Apoptotic cells were detected by TUNEL assay in cultured cardiomyocytes and the representative field was shown for each group. Percentages of TUNEL-positive nuclei over total number of nuclei were measured (n=5). B. Caspase 3 activity was assessed #
after I/R and the values were normalized to sham (n=5). *P < 0.05 versus control group, P < 0.05 versus I/R group.
ACCEPTED MANUSCRIPT
Fig.5 NOD2 stimulation mediated myocardial I/R injury through regulation of JNK1/2, p38MAPK and NF-κB pathway in vivo. A. Western blots showing JNK1/2, p38MAPK, NF-κB
IP
T
phosphorylation and expressions of total JNK1/2, p38MAPK, NF-κB in heart tissues. B. Quantitative analysis of protein expression was seen in histograms (n=3). *P < 0.05 versus #
SC R
sham group; P < 0.05 versus I/R group.
Fig.6 NOD2 deficiency reduced apoptosis after myocardial ischemia-reperfusion injury in vitro.
NU
A. Expression of NOD2 protein was determined by western blot (n=3). B. The percentage of TUNEL-positive nuclei to total nuclei was counted (n=3). C. The measurement of caspase-3 #
NOD2
deficiency
attenuates
NF-κB,
p38MAPK
and
JNK1/2
expression
in
D
Fig.7
MA
activity (n=3). *P < 0.05 versus control group; P < 0.05 versus I/R group.
TE
cardiomyocytes after I/R. Representative Western blot gel documents and summarized data showing the protein levels of NF-κB, p38MAPK and JNK1/2 expression in cardiac #
AC
CE P
cardiomyocytes (n=3). *P < 0.05 versus control group; P < 0.05 versus I/R group.
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
S0024-3205(16)30089-3 doi: 10.1016/j.lfs.2016.02.039 LFS 14724
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
14 August 2015 5 January 2016 9 February 2016
Please cite this article as: Liu Yu, Yang Hui, Liu Li-Xin, Yan Wen, Guo Huo-Jie, Li Wen-Jun, Tian Cui, Li Hui-Hua, Wang Hong-Xia, NOD2 contributes to myocardial ischemia/reperfusion injury by regulating cardiomyocyte apoptosis and inflammation, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
NOD2
contributes
to
myocardial
ischemia/reperfusion
injury
by
regulating
cardiomyocyte apoptosis and inflammation
a
a
b
b
c
b
b
b
IP
b
SC R
, Hong-Xia Wang *
a
Department of Pathology, School of Basic Medical Sciences, Capital Medical University,
Beijing, China.
Department of Physiology and Physiopathology, School of Basic Medical Sciences, Capital
NU
b
Medical University, Beijing, China.
Department of Vascular Surgery, Peking University First Hospital, Beijing, China.
*Correspondence to: H.-X. Wang
MA
c
T
Yu Liu , Hui Yang , Li-Xin Liu , Wen Yan , Huo-Jie Guo , Wen-Jun Li , Cui Tian , Hui-Hua Li
D
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital
TE
Medical University, Beijing 100069, China
AC
CE P
E-mail address: [email protected]
ACCEPTED MANUSCRIPT AB ST RACT Aims: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular pattern recognition receptor, which plays an important role in the innate immunity and
IP
T
inflammation. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. In this study, we sought to determine the role of NOD2 on cardiac I/R injury.
SC R
Main methods: Mice were induced 30 min ischemia followed by 24 h of reperfusion. Histological examinations were performed on heart sections with Evans blue and triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, immunohistochemistry and
NU
immunefluorescence staining. The messenger RNA (mRNA) expression and protein levels were detected by real-time polymerase chain reaction (RT-PCR) and western blot analysis
MA
respectively.
Key findings: I/R injury markedly upregulated NOD2 expression in heart tissue. Treatment of
D
WT mice with NOD2 ligand (MDP) significantly increased infarct size, the number of
TE
apoptotic cells and inflammatory cells, as compared with wild-type mice after I/R injury. Furthermore, MDP enhanced I/R-induced cardiomyocyte apoptosis and inflammation in vitro,
CE P
and these effects were attenuated by NOD2-siRNA. The mechanism of NOD2 on cardiac I/R injury is partly associated with JNK, p38 MAPK and NF-κB signaling pathways. Significance: NOD2 aggravates myocardial I/R injury by inducing cardiomyocyte apoptosis and
AC
inflammation through JNK, p38 MAPK and NF-κB signaling pathways. This study provides insight into better understanding the molecular mechanism of NOD2, which may be served as a potential target for the treatment of myocardial I/R injury.
Keywords:
NOD2 · Ischemia/reperfusion injury · Apoptosis · Inflammation
ACCEPTED MANUSCRIPT
Introduction
T
Myocardial ischemia/reperfusion (I/R) injury is a common cardiovascular problem and one
IP
of the major causes of death in the world. It is widely held that the mechanisms contributing to
SC R
I/R injury are complicated. The innate immune system is an essential component of the acute inflammatory response and plays an essential role in the pathogenesis of I/R injury [1, 2]. As the primary recognition receptors of the innate immunity, Toll-like receptors (TLRs) have been
NU
identified as the key mediators in the heart diseases [3, 4]. Researches have shown that the activation of TLR4 have been detected in cardiac cells and shown to mediate myocardial I/R
MA
injury [5-7]. Besides TLRs, other receptors of the innate immunity such as nucleotide-binding oligomerization domain-like receptors (NLRs) have been involved in cardiovascular disease (CVD) [8, 9].
D
NOD2, which is one of members of NLR family, is an intracellular pathogen sensor
TE
through the recognition of muramyl dipeptide (MDP). The activation of transcription factor
CE P
mediated by NOD2 can increase production of inflammatory cytokines. NOD2 can regulate innate immunity and is essential for host defense and inflammation [10-12]. An increasing number of clinical and animal model studies have implicated that the association of NOD2 with
AC
the pathogenesis of atherosclerosis, Alzheimer disease, diabetes mellitus and renal ischemia reperfusion injury, et al [13-15]. Recently, Li et al. demonstrate that NOD2 is a critical component of a signal transduction pathway that links cardiac injury by exacerbation of inflammation and MMP-9 activity in myocardial infarction (MI) [16]. All of the studies indicate that NOD2 may have an important role in cardiovascular disease. However, the role of NOD2 signaling in myocardial I/R has not been elucidated. In the present study, we investigated both in vitro and in vivo the role of NOD2 in regulating myocardial I/R injury. We provide evidences that NOD2 activation by MDP enhanced I/R-induced myocardial injury by aggravating apoptosis and inflammation. These effects were mediated by activating JNK, p38 MAPK and NF-κB signaling pathways. Conversely, knockdown of NOD2 attenuated these effects. These findings suggest that NOD2 could be a novel strategy for treatment of myocardial I/R.
ACCEPTED MANUSCRIPT
Materials and methods
IP
T
Animal model of myocardial I/R
SC R
Male C57/B6J mice were intraperitoneally administered either vehicle (NaCl 0.9%) or MDP (10 mg/kg; San Diego, CA) 30min before the operation [12]. The mouse model of myocardial I/R was prepared as previously described [17]. In brief, male C57BL/6 (WT) mice,
NU
8-10 weeks of age, were used for the study. To generate myocardial I/R, the mice were anaesthetized with isoflurane (1.5%), and was induced by left coronary artery (LCA) ligation
MA
for 30 min and reperfusion for 24h. In sham-operated animals, the suture was placed under the LCA and removed without ligating the artery. In MDP (San Diego, CA)-treated animals, 10mg/kg MDP was injected intraperitoneally 30 min before coronary artery occluded . The
D
study was performed according to the Guide for the Care and Use of Laboratory Animals
TE
published by the US National Institutes of Health, and the protocol was consistent with the
CE P
Animal Care and Use Committee of Capital Medical University.
AC
Evaluation of infarct size
Myocardial infarct size was assessed by Evans Blue and triphenyltetrazolium chloride (TTC) staining method as described [18]. The method is usually used as current “gold standard” to quantitate the area at risk (AAR) and degree of completed infarct. After completion of experiment, the LCA was reoccluded, 1% Evans blue dye was injected via the aorta and coronary arteries to differentiate ischemic (non-blue) and normal (blue) tissue. Then, the heart was quickly frozen for 1 h at -80 ℃. The frozen heart was sliced into uniform sections about 2mm thickness. The slices were incubated for 20 min in phosphate-buffered 1% TTC at 37 ℃ to determine the infarct area (INF, pale white) and the area at risk (AAR, brick red). At last, the INF appears pale white, the AAR stains brick red and the normal area is blue. The INF, the AAR, and the left ventricular (LV) area from each section were analyzed by use of NIH Image software (US National Institutes of Health, Bethesda, MD), multiplied by the weight
ACCEPTED MANUSCRIPT of the section, and then totaled from all sections. The ratio of INF to AAR and AAR to LV were calculated [18].
IP
T
Histological analysis, Immunohistochemistry and Immunofluorescence
morphology,
H&E
staining
was
used
on
SC R
The mice hearts were fixed with 10% formalin and embedded in paraffin. To evaluate the
heart
sections
(5μm)
[19].
For
immunohistochemistry, anti-MAC-2 antibody (Santa Cruz, CA,1:200 dilution) and anti-IL-6
NU
antibody (Santa Cruz, CA, 1:200 dilution) was used as the primary antibody, and the positive cells were detected by 3,30-Diaminobenzidine (DAB) staining. Negative control sections were
MA
treated with IgG antibodies. For immunofluorescence assays, staining was performed as previously reported [17, 20]. Frozen sections were stained with anti-ICAM-1 (Santa Cruz, CA,1:200 dilution) and MCP-1 (Santa Cruz, CA,1:200 dilution) overnight at 4°C. Then, the
D
sections were washed and incubated with Hoechst 33342 (1:1000) for 5 min to stain the nuclei.
TE
The positive areas were calculated from 6 random fields at 200 magnification (Olympus BX-63
AC
Cell culture
CE P
JAPAN).
Cell culture was conducted as described previously
[18]
. Cardiomyocytes were isolated
from 1 d old neonatal SD rats and were seeded and incubated in DMEM containing 10% FCS at 37℃. Then, cardiomyocytes were transferred to an ischemic buffer that contains 118 mM NaCl, 24 mM NaHCO3, 1.0 mM NaH2PO4, 2.5 mM CaCl2-2H2O, 1.2 mM MgCl2, 20 mM sodium lactate, 16 mM KCl and 10 mM 2-deoxyglucose (pH 6.2) and incubated at 37 °C for 2 h in a humidified atmosphere of 5% CO2 and 95% nitrogen. During ischemia occurring in vivo, the reduced O2 supply causes an increase in concentrations of intracellular Na +
+
2+
and Ca ,
+
increase in extracellular K , decrease in extracellular Na , and decrease in intracellular ATP and PH. The ischemic buffer including the approximate concentrations of potassium, hydrogen, lactate ions and pH and is designed to simulate the extracellular environment during myocardial ischemia. After 2 h of simulated ischemia, cardiomyocytes were randomly exposed
ACCEPTED MANUSCRIPT to reperfusion for 30 min or 24 h by replacing the ischemic buffer with normal cell medium. Cardiomyocytes were treated with MDP (10 μg/ml) for 30 min before exposure to ischemia
IP
T
buffer.
SC R
RNA interference
Small interfering RNA (siRNA) against the NOD2 and negative control siRNA were purchased from Invitrogen (San Diego, CA). The final concentration of siRNA was 100 nM
NU
diluted in OPTI-MEM medium. The cells were transfected with siRNA using Lipofectamine 2000 in accordance with the manufacturer’s protocol [21]. After 24-48 h, the mRNA level of
MA
NOD2 was determined by quantitative real-time RT-PCR, and the protein expression of NOD2
D
was measured by western blot.
TE
Assay of apoptosis
CE P
The apoptosis of heart sections and cardiomyocytes were measured by the FragEL™ DNA Fragmentation Detection Kit (Roche, Indianapolis, IN). The staining was performed as described [20]. Fluorescence staining was observed using fluorescence microscopy (Olympus
AC
BX-63, JAPAN). The amount of apoptosis cells was analyzed by counting the number of positive cells in 6 fields within the heart sections and cardiomyocytes using 20 × magnification.
Measurement of caspase-3 activity
Caspase-3 activity was performed using a colorimetric activity assay kit [18]. In brief, Using buffer containing 10 mM dithiothreitol and 5 μl Ac-DEVD-AFC (BIOMOL, PA), the samples were incubated at 37°C for 1.5 h. The detection of caspase-3 activity was evaluated through a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 405 nm.
ACCEPTED MANUSCRIPT Quantitative real-time PCR
For quantitative real-time PCR, total RNAs were purified from hearts using Trizol reagent
IP
T
[22, 23]. RNA was transcribed to cDNA with PrimeScript RT reagent Kit. Then qRT-PCR was performed in a 20 ul reaction mixture containing SYBR Green PCR Master Mix (Warrington,
SC R
UK), cDNA and 0.2 mmol/L of each primer at 95℃ for 10 min, 40 cycles at 95 ℃ for 10 s and 60 ℃ for 45 s. The relative gene expression was normalized to internal control GAPDH. Primer sequences for SYBR Green probes of target genes are as follows: forward,
5’-CCAGCGTCTTTGGCCATTCAACAT-3’;
5’-TTGAGCTCATCCAGTGCTTGGAGT-3;
NU
NOD2:
and
GAPDH:
reverse, forward,
MA
5’-CCCCCAATGTATCCGTTGTG-3’; and reverse, 5’-TAGCCCAGGATGCCCTTTAGT-3’.
TE
D
Western blot analysis
Protein was extracted from the heart tissues and cardiomyocytes. The protein was boiled
CE P
and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The primary antibodies dilutions were 1:500 for NOD2 (Santa Cruz, CA), 1:500 for Bax (Beverly, MA) , 1:500 for Bcl-2 (Beverly, MA),
AC
1:1000 for total- and phospho-JNK (Beverly, MA), 1:1000 for total- and phospho-p38 MAPK (Beverly, MA), 1:800 for total- and phospho-p65 (Beverly, MA) , 1:5000 for GAPDH (Beverly, MA). Secondary antibodies (Santa Cruz, CA) were used at a dilution of 1:5000[22, 23].
Statistical analysis
For statistical analysis, SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) was used. The results were shown as mean ± SEM. One-way ANOVA or t-test was used to compare differences between groups.
P < 0.05 was considered statistically significant.
ACCEPTED MANUSCRIPT Results
IP
T
NOD2 expression was increased in myocardial I/R injury
To test the involvement of NOD2 in myocardial I/R injury, we measured NOD2 level in the
SC R
mouse heart following I/R. As seen in Fig. 1A, RT-PCR analysis showed myocardial I/R significantly increased NOD2 mRNA expression. Western blot analysis also demonstrated NOD2 protein level was significantly increased in the I/R hearts compared to sham hearts,
NU
(Fig.1B). These data suggest that increased NOD2 expression may
MA
injury.
I/R
NOD2 activation exacerbated myocardial infarction and promoted apoptosis after I/R
D
Using Evans Blue-TTC method, myocardial infarct size was detected to evaluate the
TE
effect of NOD2 on myocardial I/R injury. The representative photographs of heart sections are
CE P
shown in Fig. 2A. The results showed that the ratio of infarct area (INF) to area at risk (AAR) was significantly higher in I/R + MDP group compared with I/R group. No significant difference in the ratio of AAR to left ventricular (LV) was found between I/R + MDP group and I/R
AC
group. It indicated NOD2 activation aggravated infarct size in I/R injury. To elucidate whether NOD2 regulates cardiomyocyte apoptosis in I/R, TUNEL staining was used to detect myocardial apoptotic cells in mice hearts. The results identified I/R injury caused apoptosis in the cardiomyocytes. Moreover, MDP pretreatment further increased the incidences of apoptosis (Fig. 2B). The expression of apoptosis-related proteins were assessed by western blot analysis. The decrease of Bcl-2 expression and increase of Bax expression were observed in IR group. Compared with I/R group, MDP further significantly attenuated the Bcl-2 expression but increased Bax expression (Fig. 2C). These results strongly suggest that NOD2 has significant pro-apoptotic effects.
NOD2 activation facilitates cardiac inflammation after I/R injury
ACCEPTED MANUSCRIPT Inflammation induced by I/R injury is a major cause of cardiac impairment. NOD2 has been reported to promote the inflammation response. We examined the macrophage infiltration in heart specimens by immunohistochemistry using an anti-Mac-2 antibody and the
IP
T
study demonstrated a significantly higher macrophage infiltration in the IR+MDP compared
IL-6, ICAM-1, and MCP-1 are the key mediators
SC R
with I/R group (Fig.3A)
myocardial I/R injury. When these proteins were determined in mice hearts by immunohistochemistry or immunofluorescence assay, I/R significantly increased IL-6, ICAM-1,
NU
and MCP-1 levels compared with sham group. Importantly, these cytokines expression was significantly greater in I/R+MDP group compared to I/R group (Fig. 3A-B). Together, these
MA
data suggest that NOD2 is important in the regulation of IR-associated inflammation.
TE
D
NOD2 activation increases I/R-induced apoptosis in neonatal rat cardiomyocytes.
To further confirm the pro-apoptotic effect of NOD2 on cardiomyocytes in vitro, neonatal
CE P
rat cardiomyocytes were subjected to simulated I/R. TUNEL assay revealed that MDP treatment significantly increased the number of apoptotic cells in I/R injury (Fig. 4A). Moreover, caspase 3 activity was significantly higher in MDP-treated cardiomyocytes than that in
AC
untreated cardiomyocytes after I/R (Fig. 4B). These results further suggest that NOD2 was involved in IR injury by inducing apoptosis.
NOD2 activation aggravates myocardial I/R injury through regulation of JNK1/2, p38MAPK and NF-κB pathway
Recent studies have shown that MAPK and NF- kB pathways were involved in I/R injury. We hypothesized that the function of NOD2 in IR injury is mediated by these signaling pathways. As shown in Fig. 5, JNK1/2, p38MAPK and NF-κB were increased following I/R. Compared with I/R, MDP treatment further stimulated JNK1/2, p38MAPK and NF-κB activation. These results indicated that the damage caused by NOD2 in IR can be attributed in part to the upregulation in JNK1/2, p38MAPK and NF-κB pathways.
ACCEPTED MANUSCRIPT
Suppressing NOD2 with NOD2 siRNA decreases apoptosis induced by I/R in neonatal cardiomyocytes
IP
T
To further assess the impact of NOD2 on cardiomyocyte apoptosis, cardiomyocytes were isolated and transfected with siRNA-control and siRNA-NOD2 and then exposed to mimetic
SC R
I/R. The decrease of NOD2 protein expression was observed after transfection with siRNA-NOD2. Cardiomyocyte apoptosis, caspase-3 activity were increased on mimetic I/R
NU
treatment, whereas siRNA-NOD2 treatment ameliorated these effects (Fig. 6B and Fig. 6C).
neonatal rat cardiomyocytes after I/R
MA
Suppressing NOD2 with NOD2-siRNA attenuates NF-κB, p38MAPK and JNK1/2 expression in
D
Since above results have showed NOD2 can activate JNK1/2, p38MAPK and NF-κB
TE
signaling pathways (Fig. 5), we further examined whether down-regulation of NOD2 inhibited activation of these pathways. Then, we observed that down-regulation of NOD2 by siRNA
CE P
markedly suppressed the I/R-induced activation of JNK1/2, p38MAPK and NF-κB pathways, Thus, taking all these results together, we conclude that NOD2 prompts cardiomyocytes
AC
apotosis and inflammation via these pathways (Fig. 7).
Discussion
In this study, we presented first evidence that NOD2 expression was increased during the processes of myocardial I/R Injury. Activation of NOD2 by MDP aggravated infarct size, cardiomyocyte apoptois and inflammation in the heart tissues or cardiomyocytes after I/R. In contrast, down-regulation of NOD2 by NOD2-siRNA attenuated these effects. During this process, JNK1/2, p38MAPK and NF-κB signaling pathway were activated by NOD2 and accounted for inflammatory responses and apoptosis. It has been well demonstrated that apoptosis play a critical role in I/R-induced injury [24]. NOD2 has been causally linked to the pathogenesis of apoptosis [25]. It is reported that NOD2 may enhance caspase-9-induced apoptosis by interaction with the CARD domain [26].
ACCEPTED MANUSCRIPT In our study, treatment with MDP resulted in significant increasement in cardiomyocyte apoptosis in vivo, indicating that the pro-apoptotic effect of NOD2 in myocardial I/R. Caspase-3, a member of the cysteine proteases, is implicated in apoptosis. Activation of
IP
T
caspase-3 is associated with a series of signal transduction cascades that include Bcl-2 and Bax proteins. Bcl-2 can form a heterodimer with Bax, thereby prevent Bax homodimerization
SC R
and the sequential activation of caspase-3 [27]. The present study revealed that I/R markedly up-regulated expression of Bax and down-regulated expression of Bcl-2 in the myocardium of I/R mice, consistent with the previous researchers [18, 28]. Meanwhile, treatment with MDP
NU
exacerbated I/R-induced increase Bax and decrease of Bcl-2 expression in myocardium. Furthermore, these results were verified in vitro. It is suggesting that NOD2 may exert its
MA
pro-apoptotic effects by regulating Bax and Bcl-2, thus promoted the activation of caspase-3. Several investigations have shown that inflammatory responses such as the accumulation
D
of inflammatory cells and pro-inflammatory cytokines participate in the development of I/R
TE
injury [29]. Recent studies have implicated ischemia promotes rapid recruitment of inflammatory cells into the myocardium [30, 31]. The cytokines and chemokines released by
CE P
inflammatory cells further attract macrophage infiltration and promote inflammatory reactions [32, 33]. It has been reported that NOD2 is important to regulate inflammation in some diseases [34-36]. In the present study, the macrophage infiltration and generation of IL-6,
AC
ICAM-1, and MCP-1 were all increased significantly in the mice hearts following I/R injury. Each of these changes was promoted by MDP pretreatment. This suggests that NOD2 activates the myocardium inflammation during I/R injury. Moreover, several signaling pathways involved in the apoptosis and inflammation are related to myocardial I/R injury. The c-Jun NH2-terminal kinase (JNK), p38 MAPK are the major groups of MAPK subfamilies. Activation of JNK as well as p38 MAPK induced myocardial I/R injury by regulating cardiomyocyte apoptosis and proinflammatory cytokine production [37]. Moreover, NF-kB is a key mediator of inflammation in myocardial injury caused by I/R. Recently, several studies demonstrate that NOD2 stimulation induces the activation of MAPK and NF-kB pathway [38-40]. Consistently, we found that JNK, p38 MAPK and NF-kB pathway were activated after myocardial I/R injury in WT mice, while activation of NOD2 by MDP significantly increased the levels of JNK, p38 MAPK and NF-kB. To further
ACCEPTED MANUSCRIPT confirm the mechanisms of NOD2 in I/R-induced myocardial injury, we cultured neonatal rat cardiomyocytes in vitro, and found that the effects of NOD2 on JNK, p38 MAPK and NF-kB pathway were all blocked by NOD2-siRNA. Taken together, these data indicate that, by
IP
T
powerfully activating JNK, p38 MAPK and NF-kB, NOD2 has special detrimental effect on myocardium.
SC R
In summary, the present study demonstrated that NOD2 exacerbates myocardial infarct size following I/R injury. The mechanisms involve activating I/R-activated apoptotic signaling including JNK1/2, p38MAPK and NF-κB activity, resulting in increase of I/R-induced
MA
management of ischemic heart disease.
NU
inflammation and apoptosis. NOD2 may be an attractive target for the treatment and
D
Acknowledgements
TE
This work was supported by grants from China National Natural Science Funds (No. 81330003, 81000343, 81500320), Beijing Higher Education Young Elite Teacher Project (No.
CE P
YETP1668)，and The Importation and Development of High Caliber Talents Project of Beijing Municipal Institutions (CIT&TCD201404180). Author contributions: H.X.W. were responsible for conception and design. Y.L. analyzed
AC
the data and wrote the article. H.H.L. revised the article. H.Y., L.X. L., W.Y., H.J. G., W.J. L. and C.T collected the data. All authors have read and approved the final version of the manuscript.
References
[1] A. Linde, D. Mosier, F. Blecha, T. Melgarejo.Innate immunity and inflammation--New frontiers in comparative cardiovascular pathology, Cardiovasc. Res. 73 (1) (2007) 26-36. [2] S. Epelman, P. P. Liu, D. L. Mann.Role of innate and adaptive immune mechanisms in cardiac injury and repair, Nat. Rev. Immunol. 15 (2) (2015) 117-129. [3] S. Frantz, G. Ertl, J. Bauersachs.Mechanisms of disease: Toll-like receptors in cardiovascular disease, Nat. Clin. Pract. Cardiovasc. Med. 4 (8) (2007) 444-454.
ACCEPTED MANUSCRIPT [4] R. Spirig, J. Tsui, S. Shaw.The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease, Cardiol. Res. Pract. 2012 (2012) 181394. [5] J. Cha, Z. Wang, L. Ao, et al.,Cytokines link Toll-like receptor 4 signaling to cardiac dysfunction
IP
T
after global myocardial ischemia, Ann. Thorac. Surg. 85 (5) (2008) 1678-1685.
[6] J. Oyama, C. Blais, Jr., X. Liu, et al.,Reduced myocardial ischemia-reperfusion injury in toll-like
SC R
receptor 4-deficient mice, Circulation. 109 (6) (2004) 784-789.
[7] P. Zhao, J. Wang, L. He, et al.,Deficiency in TLR4 signal transduction ameliorates cardiac injury and cardiomyocyte contractile dysfunction during ischemia, J. Cell. Mol. Med. 13 (8A) (2009)
NU
1513-1525.
[8] L. Franchi, N. Warner, K. Viani, G. Nunez.Function of Nod-like receptors in microbial recognition
MA
and host defense, Immunol. Rev. 227 (1) (2009) 106-128.
[9] T. D. Kanneganti, M. Lamkanfi, G. Nunez.Intracellular NOD-like receptors in host defense and
D
disease, Immunity. 27 (4) (2007) 549-559.
TE
[10] A. Rehman, C. Sina, O. Gavrilova, et al.,Nod2 is essential for temporal development of intestinal microbial communities, Gut. 60 (10) (2011) 1354-1362.
CE P
[11] R. G. Correa, S. Milutinovic, J. C. Reed.Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases, Biosci. Rep. 32 (6) (2012) 597-608. [12]
E.
Perez-Chanona,
M.
intestinal
injury
C.
Jobin.The
through
microbiota
nucleotide-binding
protects
against
oligomerization
AC
ischemia/reperfusion-induced
Muhlbauer,
domain-containing protein 2 (NOD2) signaling, Am. J. Pathol. 184 (11) (2014) 2965-2975. [13] P. Du, B. Fan, H. Han, et al.,NOD2 promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy, Kidney. Int. 84 (2) (2013) 265-276. [14] H. Han, Y. Wang, X. Li, et al.,Novel role of NOD2 in mediating Ca2+ signaling: evidence from NOD2-regulated podocyte TRPC6 channels in hyperhomocysteinemia, Hypertension. 62 (3) (2013) 506-511. [15] M. Y. Kwon, X. Liu, S. J. Lee, et al.,Nucleotide-binding oligomerization domain protein 2 deficiency enhances neointimal formation in response to vascular injury, Arterioscler. Thromb. Vasc. Biol. 31 (11) (2011) 2441-2447. [16] X. Li, F. Li, Y. Chu, et al.,NOD2 deficiency protects against cardiac remodeling after myocardial infarction in mice, Cell. Physiol. Biochem. 32 (6) (2013) 1857-1866.
ACCEPTED MANUSCRIPT [17] C. W. Xu, T. P. Zhang, H. X. Wang, H. Yang, H. H. Li.CHIP enhances angiogenesis and restores cardiac function after infarction in transgenic mice, Cell. Physiol. Biochem. 31 (2-3) (2013) 199-208. [18] Y. Zhang, Y. Zeng, M. Wang, et al.,Cardiac-specific overexpression of E3 ligase Nrdp1 increases
[19]
H.
H.
Li,
V.
Kedar,
C.
Zhang,
et
IP
T
ischemia and reperfusion-induced cardiac injury, Basic. Res. Cardiol. 106 (3) (2011) 371-383. al.,Atrogin-1/muscle
atrophy F-box inhibits
SC R
calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex, J. Clin. Invest. 114 (8) (2004) 1058-1071.
[20] K. Yang, T. P. Zhang, C. Tian, L. X. Jia, J. Du, H. H. Li.Carboxyl terminus of heat shock protein
NU
70-interacting protein inhibits angiotensin II-induced cardiac remodeling, Am. J. Hypertens. 25 (9) (2012) 994-1001.
MA
[21] H. Yang, X. J. Zeng, H. X. Wang, et al.,Angiotensin IV protects against angiotensin II-induced cardiac injury via AT4 receptor, Peptides. 32 (10) (2011) 2108-2115.
D
[22] H. M. Jiang, H. X. Wang, H. Yang, et al.,Role for granulocyte colony stimulating factor in
(2013) 1224-1233.
TE
angiotensin II-induced neutrophil recruitment and cardiac fibrosis in mice, Am. J. Hypertens. 26 (10)
CE P
[23] H. X. Wang, H. Yang, Q. Y. Han, et al.,NADPH oxidases mediate a cellular "memory" of angiotensin II stress in hypertensive cardiac hypertrophy, Free. Radic. Biol. Med. 65 (2013) 897-907. [24] C. Lu, D. Ren, X. Wang, et al.,Toll-like receptor 3 plays a role in myocardial infarction and
AC
ischemia/reperfusion injury, Biochim. Biophys. Acta. 1842 (1) (2014) 22-31. [25] A. A. Shigeoka, A. Kambo, J. C. Mathison, et al.,Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury, J. Immunol. 184 (5) (2010) 2297-2304. [26] G. Rogler.The effects of NOD2/CARD15 mutations on the function of the intestinal barrier, J. Crohns. Colitis. 1 (2) (2007) 53-60. [27] J. Guo, S. B. Wang, T. Y. Yuan, et al.,Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation, Atherosclerosis. 231 (2) (2013) 384-391. [28] L. Song, H. Yang, H. X. Wang, et al.,Inhibition of 12/15 lipoxygenase by baicalein reduces myocardial ischemia/reperfusion injury via modulation of multiple signaling pathways, Apoptosis. 19 (4) (2014) 567-580.
ACCEPTED MANUSCRIPT [29] K. Kitano, S. Usui, H. Ootsuji, et al.,Rho-kinase activation in leukocytes plays a pivotal role in myocardial ischemia/reperfusion injury, PLoS. One. 9 (3) (2014) e92242. [30] L. Formigli, L. I. Manneschi, C. Nediani, et al.,Are macrophages involved in early myocardial
IP
T
reperfusion injury?, Ann. Thorac. Surg. 71 (5) (2001) 1596-1602.
[31] T. Kakio, A. Matsumori, K. Ono, H. Ito, K. Matsushima, S. Sasayama.Roles and relationship of
SC R
macrophages and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in the ischemic and reperfused rat heart, Lab. Invest. 80 (7) (2000) 1127-1136.
Med. 15 (5) (2005) 163-169.
NU
[32] N. G. Frangogiannis, M. L. Entman.Chemokines in myocardial ischemia, Trends. Cardiovasc.
[33] N. G. Frangogiannis, C. W. Smith, M. L. Entman.The inflammatory response in myocardial
MA
infarction, Cardiovasc. Res. 53 (1) (2002) 31-47.
[34] O. Gutierrez, C. Pipaon, N. Inohara, et al.,Induction of Nod2 in myelomonocytic and intestinal
D
epithelial cells via nuclear factor-kappa B activation, J. Biol. Chem. 277 (44) (2002) 41701-41705.
TE
[35] J. T. Tigno-Aranjuez, D. W. Abbott.Ubiquitination and phosphorylation in the regulation of NOD2 signaling and NOD2-mediated disease, Biochim. Biophys. Acta. 1823 (11) (2012) 2022-2028.
CE P
[36] A. Negroni, L. Stronati, M. Pierdomenico, et al.,Activation of NOD2-mediated intestinal pathway in a pediatric population with Crohn's disease, Inflamm. Bowel. Dis. 15 (8) (2009) 1145-1154. [37] E. Tamagno, G. Robino, A. Obbili, et al.,H2O2 and 4-hydroxynonenal mediate amyloid
AC
beta-induced neuronal apoptosis by activating JNKs and p38MAPK, Exp. Neurol. 180 (2) (2003) 144-155.
[38] Y. J. Jeong, M. J. Kang, S. J. Lee, et al.,Nod2 and Rip2 contribute to innate immune responses in mouse neutrophils, Immunology. 143 (2) (2014) 269-276. [39] L. Zhao, J. Y. Lee, D. H. Hwang.The phosphatidylinositol 3-kinase/Akt pathway negatively regulates Nod2-mediated NF-kappaB pathway, Biochem. Pharmacol. 75 (7) (2008) 1515-1525. [40] S. Maeda, L. C. Hsu, H. Liu, et al.,Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing, Science. 307 (5710) (2005) 734-738.
ACCEPTED MANUSCRIPT
Figure legends:
IP
T
Fig.1 Expression of NOD2 was increased in myocardial I/R injury. A. NOD2 mRNA levels were
analysis and summarized data (bottom) (n=3). *P < 0.05 versus sham group,
SC R
detected by RT-PCR (n=5). B. NOD2 protein (top) expression was measured by western blot
NU
Fig.2 NOD2 stimulation exacerbated I/R injury and improved cardiomyocyte apoptosis. A. Representative photographs staining with triphenyl tetrazolium chloride (TTC) and Evans blue.
MA
(n=5). B. Representative images of TUNEL-stained heart sections from different groups. Scale bar: 50 mm. Quantification of all TUNEL+ nuclei showed as percentage of total number of
D
cardiomyocyte (n=5). C. Western blot determined protein level of Bcl-2 and Bax in ischemia
TE
myocardium. The results were expressed as ratio of Bax/Bcl-2 (n=3). *P < 0.05 versus sham #
CE P
group, P < 0.05 versus I/R group.
Fig.3 NOD2 stimulation facilitates inflammation following I/R. A. Histological evaluation of heart tissue and immunohistochemical staining for Mac-2 and IL-6 in mice myocardium. Mac-2
AC
and IL-6 expression were quantified by digital image analysis (n=5). B. Immunofluorescence #
imaging of MCP-1 and ICAM-1(n=5). Scale bar: 50 mm. *P < 0.05 versus sham group, P < 0.05 versus I/R group.
Fig.4 NOD2 stimulation increases IR-induced apoptosis in primary cultured cardiomyocytes. Cultured cardiomyocytes were incubated in the presence or absence MDP and then subjected to mimetic I/R. A. Apoptotic cells were detected by TUNEL assay in cultured cardiomyocytes and the representative field was shown for each group. Percentages of TUNEL-positive nuclei over total number of nuclei were measured (n=5). B. Caspase 3 activity was assessed #
after I/R and the values were normalized to sham (n=5). *P < 0.05 versus control group, P < 0.05 versus I/R group.
ACCEPTED MANUSCRIPT
Fig.5 NOD2 stimulation mediated myocardial I/R injury through regulation of JNK1/2, p38MAPK and NF-κB pathway in vivo. A. Western blots showing JNK1/2, p38MAPK, NF-κB
IP
T
phosphorylation and expressions of total JNK1/2, p38MAPK, NF-κB in heart tissues. B. Quantitative analysis of protein expression was seen in histograms (n=3). *P < 0.05 versus #
SC R
sham group; P < 0.05 versus I/R group.
Fig.6 NOD2 deficiency reduced apoptosis after myocardial ischemia-reperfusion injury in vitro.
NU
A. Expression of NOD2 protein was determined by western blot (n=3). B. The percentage of TUNEL-positive nuclei to total nuclei was counted (n=3). C. The measurement of caspase-3 #
NOD2
deficiency
attenuates
NF-κB,
p38MAPK
and
JNK1/2
expression
in
D
Fig.7
MA
activity (n=3). *P < 0.05 versus control group; P < 0.05 versus I/R group.
TE
cardiomyocytes after I/R. Representative Western blot gel documents and summarized data showing the protein levels of NF-κB, p38MAPK and JNK1/2 expression in cardiac #
AC
CE P
cardiomyocytes (n=3). *P < 0.05 versus control group; P < 0.05 versus I/R group.
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
