- Email: [email protected]
P38 MAPK signalling in mice
Accepted Manuscript Loss of TRADD attenuates pressure overload-induced cardiac hypertrophy through regulating TAK1/P38 MAPK signalling in mice Lianpin Wu, Zhiyong Cao, Liqin Mei, Ling Ji, Qike Jin, Jingjing Zeng, Jiafeng Lin, Maoping Chu, Lei Li, Xiangjun Yang PII:
S0006-291X(16)32164-7
DOI:
10.1016/j.bbrc.2016.12.104
Reference:
YBBRC 36978
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 9 December 2016 Accepted Date: 16 December 2016
Please cite this article as: L. Wu, Z. Cao, L. Mei, L. Ji, Q. Jin, J. Zeng, J. Lin, M. Chu, L. Li, X. Yang, Loss of TRADD attenuates pressure overload-induced cardiac hypertrophy through regulating TAK1/P38 MAPK signalling in mice, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2016.12.104. 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 Loss of TRADD attenuates pressure overload-induced cardiac hypertrophy through regulating TAK1/P38 MAPK signalling in mice
Jiafeng Lin2, Maoping Chu2, Lei Li2# & Xiangjun Yang1#
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Lianpin Wu1,2*, Zhiyong Cao3*,Liqin Mei4, Ling Ji5, Qike Jin2, Jingjing Zeng2,
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Department of Cardiology, The First Affiliated Hospital of Suzhou Medical University. 188 Shizi Road, Suzhou, Jiangsu, 215006, China 2 Department of Cardiology, The Second Affiliated Hospital of Wenzhou Medical University ＆ Yuying Children Hospital. 109 Xueyuan Road, Wenzhou, Zhejiang, 325027, China 3 Department of Cardiology, Huazhong University of Science and Technology, No 411 Hospital of People's Liberation Army, 15 East Jiangwan Road, Shanghai, 200081, China 4 Department of Oral Prophylaxis and Hygiene, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China 5 Department of Laparoscopic Surgery, The First Hospital Affiliated to Wenzhou Medical College, Wenzhou, Zhejiang, 325027, China
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*Equal contribution to this work. The co-corresponding author of this paper.
#
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Corresponding author: * Correspondence to Lei Li & Xiangjun Yang Phone：+86-139-0663-6339；Fax：+86-577-88002252 Email: [email protected]; [email protected]
ACCEPTED MANUSCRIPT Abstract We investigated the role of tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) on pressure overload-induced cardiac hypertrophy and the underlying molecular mechanisms by using a TRADD deficiency mice model. 6-8
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weeks wild-type and TRADD knockout mice were performed to transverse aorta constriction (TAC) or sham operation (6-8 mice for each group). 14 days after TAC, cardiac function was measured by echocardiography, as well as by pathological and molecular analyses of heart samples. The expressions of cardiac hypertrophic and
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fibrotic markers were detected by qPCR. Phosphorylated and total TAK1, Akt, and p38 MAPK levels were examined by Western blotting. The ratios of lung or
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heart/body weight, wall thickness/ chamber diameter of left ventricular and cross area of cardiomyocyte were significantly reduced in TRADD knockout (KO) mice than those of wild-type mice after TAC. Moreover, cardiac hypertrophic and fibrotic markers were downregulated in TRADD knockout mice than those of wild-type mice following TAC. Protein expression analysis showed phosphorylated TAK1, p38
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MAPK and AKT were upregulated after TAC in both wild-type and TRADD KO mice, phosphorylation of TAK1 and p38 MAPK was reduced more remarkablely after TRADD deficiency, while phosphorylated AKT expression was similar between
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TRADD KO and wild-type mice following TAC. Our data suggest that TRADD KO blunts pressure overload-induced cardiac hypertrophy through mediating TAK1/p38
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MAPK but not AKT phosphorylation in mice.
Key words: TAK1, pressure overload, TRADD, cardiac hypertrophy
ACCEPTED MANUSCRIPT 1. Introduction Cardiac hypertrophy is a common response to many forms of heart failure[1], of which molecular and cellular mechanism keep largely unclear. After a long term of compensatory adaptation, cardiac hypertrophy is associated with functional and
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structure deterioration of the myocardium, fibrosis, inflammation, and altered cardiac gene expression [2-3]. Cardiac hypertrophy is a physiopathological process to chronic pressure. The hypertrophic heart has developed expression of proto-oncogenes and increased rate of protein synthesis [4]. The process is initially beneficial in terms of
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compensatory adaptation through increasing left ventricular wall thickness, while cardiac dysfunction will ultimately be developed after long-term chronic pressure
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overload stimulation [5].
Several pathways are implicated with the pathogenesis of cardiac hypertrophy [4, 6-7]. Transforming growth factor beta-activated kinase 1 (TAK1) signalling plays an important role in regulating cardiac hypertrophy [8-9]. Including our investigation, numerous studies show that the TAK1 signaling system is a crucial regulator of this
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process [8-9]. Moreover, modulation of nuclear factor κB (NF-κB) signaling in the heart may provide a novel approach to attenuate the development of heart failure after cardiac hypertrophy[8]. TAK1 exerts a node function in cell survival and death
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through NF-κB signaling [8-9]. Activated TAK1 results in activation of p38 mitogen-activated protein kinase (p38 MAPK) and JNK signaling pathways, enhances hypertrophic markers expression, and subsequently leads to cardiac hypertrophy[3,
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10]. Further, activation of TAK1 leads to signaling through the IKK/IB/NF-κB pathway,
promoting
proinflammatory
cytokine
expression
and
leading
to
inflammation [8].
Tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) is upstream molecule, which is a central adaptor in the TNFR1 signalling complex that mediates both cell death and inflammatory signals[11-12], TRADD mediates both cell death and pro-inflammatory signals[13]. Although TRADD is usually considered a cytoplasmic protein, it may also have a function in the nucleus [11]. Given the role of TRADD in pro-inflammatory TNFR1 signalling and tumour suppression, it has been
ACCEPTED MANUSCRIPT postulated that TRADD functions should abrogate cell growth. TRADD was shown to be an important protective factor in a variety of cardiac injuries, including endoplasmic reticulum (ER) stress, ischemia reperfusion (I/R) injury [14-15]. The role of TRADD on pressure-overload-induced cardiac hypertrophy has not been fully
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determined. In this study, we investigated the function of TRADD in pressure overload-induced adaptive cardiac hypertrophy using TRADD knockout mice.
2. Methods
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Materials
The antibodies against TAK1/p-TAK1, p38/p38 MAPK, AKT/p-AKT, ANP, BNP,
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β-MHC, Collagen I, Collagen III and transforming growth factor (TGF)-activated kinase β1 (TGF β1) were purchased from Cell Signaling Technology. Acryl-amide, bisacrylamide, glycine, HRP-ECL luminescent substrate and other reagents were purchased from Sigma. Animals
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All protocols were approved by the institutional guidelines. All surgeries and subsequent analyses were performed in a fashion blinded for genotype. TRADD mice were produced as described previously. 8-10 week-old male mice with TRADD
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Knockout (KO) and their control littermates were used in the study. Genotyping was performed by polymerase chain reaction (PCR) as described previously [11]. Mice were maintained on a 12:12 h light-dark cycle at 21-23°C with free access to water
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and mouse chow.
Transverse aortic constriction (TAC) TAC was performed as described previously [9, 16]. Briefly, Age- and sex-matched Wild-type (WT) and KO mice were anesthetized with isoflurane inhalation (1.5%), endotracheally
intubated
and
ventilated
(Type
7025;
Harvard
Apparatus,
March-Hugstetten, Germany). Parasternal thoracotomy was performed in the second intercostal space. After isolation of the transverse aorta, A 7.0 nylon suture was banded against a 27-gauge needle at the transverse aorta to produce a 70-75% constriction after removal of the needle. Doppler view was performed to ensure that
ACCEPTED MANUSCRIPT constriction of the aorta was succeeded. Mice were sacrificed, hearts and lungs were harvested and weighed to compare lung, heart weight/body weight (LW, HW/BW, mg/g) in KO and sham mice. Echocardiography
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Echocardiography was performed as described in our previous study [17]. We performed transthoracic echocardiography to detect the mice heart with a high-frequency ultrasound system Vevo770 (VisualSonics Inc, Toronto, ON, Canada) with a 30-MHz central frequency scanhead [16]. End-systole and end-diastole views
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were defined as the sections in which the smallest and largest areas of the left ventricle (LV), respectively, were obtained. Heart’s geometrical values including
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interventricular septum depth at end-diastolic stage (IVSd), left ventricular posterior wall diastolic stage (LVPWd), fractional shortening (FS) were measured from the LV M-mode at the mid-papillary muscle level. Pressure gradients (mmHg) were calculated from the peak blood velocity (Vmax) (m/s) obtained by Doppler across the tranverse constriction site, which was unchanged in all surgery mice. Hearts, lungs,
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and tibiae of the mice were isolated, weighed and calculated the heart weight (HW)/body weight (BW) (mg/g), and HW/tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios in the different groups. Echocardiographic assessment
studies.
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had good intraobserver and interobserver agreement as reported in our previous
RNA isolation, reverse transcriptase and quantitative Real-time polymerase chain
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reaction (qPCR)
qPCR was performed as described in our previous study[17]. Total RNA was isolated from fresh-frozen left ventricular myocardium using by TRIzol (Invitrogen, USA). 500 ng RNA from per sample was reverse-transcribed for Real-time PCR assay. The quantitative PCR was performed on a Bio-Rad IQ5 examination system by employing 1.5 µg of template complemental DNA. Hypertrophic or fibrosis biomarker of heart were detected using primer below: For atrial natriuretic peptide(ANP,) Forward , GGTGTCCAACACAGATCTGA, Reverse, CCACTAGACCAC TCATCTAC; Brain
natriuretic peptide(BNP), Forward , TATAAAAGGCAGAGGCACCG, Reverse, ATCA
ACCEPTED MANUSCRIPT TCTGGGACAGCACCT; β-myosin heavy chain(β-MHC), Forward , TCGATTTGGGAAA TTCATCC, Reverse, CGCATAATCGTAGGGGTTGT; Collagen I, Forward , CCTGGTAA AG ATGGTGCC, Reverse, CACCAGGTTCACCTTTCGCACC; Collagen III, Forward , GTTCT AGAGGATGGCTGTACT, Reverse, TTGCCTTGCGTGTTTGATATTC; Transforming
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growth factor β1(TGFβ1), Forward , TTGCTTCAGCTCCACAGAGA, Reverse, TGGTTGTAGAGGGCAAGGAC; glyceraldehyde-3- phosphate dehydrogease(GAPDH),
Forward , CCACTCTTCCACCTTCGATG, Reverse, TCCACCACCCTGTTGCTGTA. A
comparative cycle threshold method was used to determine the relative quantification
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of RNA expression, which involves comparing the cycle threhold values of the
samples of target genes with that of sham-operated samples. All Real-time PCR
Western Blotting (WB)
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reactions were performed in triplicate.
WB was performed as described in our previous study [17]. Mouse hearts were homogenized in lysis buffer containing inhibitor cocktail (Merck). Total protein was extracted from homogenized left ventricular tissues and then electrophoresed in 8%
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polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Phospho (p)-Akt and total(t)-Akt proteins were examined with the primary antibodies (Santa Cruz).The secondary antibody linked to Alkaline phosphatase was used to detected
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the immumo precipitation. Measurement of precipitation was calculated by using an automated image analysis system (Image-Pro Plus 5.0, Media Cybernetics, Bethesda, MD, USA). Phosphorylated Akt normalized to total Akt, and ratios between different
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groups were compared. GAPDH was used as the internal control. Histological analysis
Histological analysis was performed as described in our previous study [17]. Hearts were harvested to immerse immediately in normal saline, fixed in 10% formalin, embedded in paraffin, and cut into 4-µm-thick sections in the short axis at the papillary muscle level. Section was stained with haematoxylin-eosin (HE) for histopathology then visualized by magnification of 400 times for myocyte size measurement. To measure the cross-sectional area of myocytes, HE-stained sections were analysis. Fibrosis area was calculated based on Masson Trichrome staining
ACCEPTED MANUSCRIPT (Fig1D). A single myocyte was measured using an image quantitative digital analysis system (Image-Pro plus 6.0). The quantitative analysis of histological images was performed in a blinded fashion. Statistical analyses
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Data are expressed as Mean±SEM. Differences among groups were based on two-way ANOVA followed by a post hoc Tukey test. Comparisons between two groups were performed through an unpaired Student’s t-test. A value of P <0.05 was considered
3.
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significant.
Results
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TRADD KO attenuates pathological cardiac hypertrophy induced by pressure overload
To investigate the role of TRADD in the heart induce by pressure overload, we performed TAC surgery on 8- to 10-week–old TRADD KO and WT mice. As shown in Figure 1, HW/BW and LW/BW ratios were obviously decreased in KO group
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compared to WT mice, as well as cardiac fibrosis (Figure1A-E). Cardiac function was examined by echocardiography after 2 weeks of surgery. The increases in left ventricle chamber dimensions and wall thickness induced by TAC were also markedly
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attenuated during both systole and diastole in KO mice compared to WT littermates (Table 1). Gross heart and HE staining further confirmed the inhibitory effect of TRADD on cardiac remodeling following TAC (Figure 1A).
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Echocardiographic results showed the effects of TRADD on cardiac hypertrophy following 2 weeks TAC surgery To explore the role of TRADD on heart hypertrophy induced by pressure overload, we performed TAC surgery on 8- to 10-week–old mice. From our echocardiographic results, PWs of the mice are significantly higher in TAC-WT and TAC-KO groups than those of sham mice (Table 1). PW has no difference between the TAC-WT and the TAC-KO group. HR was similar among three groups. Cardiac hypertrophy and function was first evaluated by echocardiography. Quantitative analysis showed that both LV end-systolic posterior wall thickness (LVPWs) and LVPWd were
ACCEPTED MANUSCRIPT significantly increased by 2 weeks of TAC but not by sham operation. TRADD ablation significantly reduced the increases in LVPWs and LVPWd by TAC. On the other hand, loss of TRADD attenuated the enlargement in LV internal dimension at end systole and diastole (LVESD and LVEDD) and LV ejection fraction (LVEF)
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dysfunction by TAC (Table 1). TRADD ablation repressed the expression of cardiac hypertrophy markers in response to pressure overload
We examined the expression of several cardiac hypertrophy markers in KO and WT
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littermates after TAC surgery by qPCR and WB analysis. Expression levels of hypertrophic gene including ANP, BNP, and β-MHC were increased to a higher level
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in WT-TAC littermates, and such increases were markedly attenuated in TRADD KO mice (Figure 2). These data suggest that TRADD deletion in mice decreased the expression of cardiac hypertrophy markers induced by pressure overload. The effects of TRADD on the expression of fibrosis markers induced by pressure overload
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Heart sections were stained with Masson staining to detect fibrosis. In both groups, collagen continued to accumulate in the heart after 2 weeks of TAC. As shown in Figure 3, quantitative analysis showed that increased collagen deposition was
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significantly regressed in TRADD KO mice. Reduced fibrosis in KO mice suggested increased collagen degradation or decreased collagen synthesis following TAC. We, next, examined the synthesis of collagen by examining the expression of mRNA and
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protein expression collagen I, collagen III and TGF-β1, known to be involved in the proliferation of cardiac fibroblasts and the biosynthesis of extracellular matrix proteins. The results showed that as for collagen I, collagen III and TGF-β1, mRNA, as well as protein expressions, were significantly lower in TRADD KO compared to in WT mice induced by pressure overload (Figures 3). Loss of TRADD reduced pressure overload-induced TAK1/p38 Signaling To examine the molecular mechanisms of TRADD on cardiac hypertrophy in response to TAC, we investigated activation of p38 MAPK pathway in the TAC mice models. We found that the phosphorylated levels of p38 MAPK and TAK1 were
ACCEPTED MANUSCRIPT significantly increased after TAC stimuli in WT hearts. However, the phosphorylation of TAK1 and p38 MAPK was almost completely attenuated in TRADD KO mice hearts, whereas AKT activation was similar in the two groups after TAC (Figure 4). Although AKT signaling functioned as a crucial role in the mediation of cardiac
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remodeling and function, we did not observe any difference in AKT activation between WT-TAC and KO-TAC mice, as determined by immunoblotting for phosphorylation of AKT (Figure 4A and D). Taken together, these result indicate that TRADD knockout repressed the activation of TAK1 and p38 MAPK although it has
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no inhibitory effects on AKT activation in hearts following TAC.
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4. Discussion
Our data show that TRADD deletion abrogated pressure overload-induced cardiac hypertrophy.
TRADD
KO
is
involved
with
reduced
TAK1/p38
MAPK
phosphorylation while phosphorylation of AKT was unchanged by TRADD. This is the first report investigating the underlying molecular mechanisms of TRADD in
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pressure overload-induced cardiac hypertrophy at a compensatory stage. Myocardial hypertrophy initially develops following short-term or long-term hypertension, which can be defined as early compensatory and late decompensated
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cardiac hypertrophy [5, 18]. During the beginning of hypertension, heart usually increases hypertrophy to keep up with the increased afterload and to hold cardiac output [19-20]. Previous studies have reported that cardiac ejection function (EF)
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maintained normal in mice subjected to 2 week TAC surgery, however, EF reduced and gradually developed into dysfunction after 4 weeks of TAC [21-22]. Cardiac hypertrophy might be a beneficial process to perverse normal EF at the early stage and attenuation of cardiac hypertrophy at this early stage might be more beneficial in TRADD knockout mice [21, 23-24]. Our results demonstrated that the TAK1 signalling might be implicated in pressure overload- induced hypertrophy process. Previous studies have suggested a critical role of TAK1/p38 MAPK pathway in the control of cell size, survival [25-26]. It is known that TAK1 activity is crucial for compensatory (physiological) and maladaptive (pathological) hypertrophy in the heart.
ACCEPTED MANUSCRIPT Loss of TAK1 impaired the development of adaptive ventricular remodeling following pressure overload [8-9]. Consistent with other studies [18], our study showed loss of TRADD deactivated TAK1, p38 MAPK and improved EF and left ventricular remodeling following TAC. However, TRADD ablation did not affect the
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activation of AKT, but it obviously reduced TAK1 and p38 MAPK activation induced by TAC.
Conclusion: our results suggested that TRADD deletion may benefit adaptive cardiac hypertrophy through TAK1/p38 MAPK pathway. Considering that the initially
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adaptive cardiac hypertrophy might be a protective process, the decrement of cardiac hypertrophy following TAC in TRADD KO mice might be beneficial to pressure
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overload-induced heart failure in the long term. Further studies are needed to investigate the potential pathways on how TRADD ablation affected TAK1 and p38 MAPK activation following TAC.
Disclosures
Acknowledgements
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All authors have no conflicts of interest to declare.
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This work was supported by grants from Fund of National Natural Science Foundation of China (81570238), Project of Science and Technology Department of Zhejiang Province (2015C33163), Scientific Research Foundation of Wenzhou
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(Y20150015), Zhejiang Provincial Medical and Health Science and Technology plan (2016KYB197).
ACCEPTED MANUSCRIPT Table 1 WT-sham
KO-sham
WT-TAC
KO-TAC
Number
6
8
7
7
HR (beats/min)
511±54
515±58
521±51
510±53
PW (mmHg)
2.23±0.69
2.53±0.52
60±6.71*
57±57*
HW/BW (mg/g)
4.53±0.63
4.45±0.56
8.71±0.82* 6.25±0.67*#
EF (%）
75±6
76±8
41±6*
FS (%)
33±3
31±3
IVSd (mm)
0.81±0.03
0.87±0.07
LVESD (mm)
1.70±0.08
LVEDd (mm)
3.53±0.52
LVPWd (mm)
0.84±0.11
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Parameters
16±2*
56±5*#
28±2#
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1.34±0.07* 1.07±0.05*#
1.69±0.07
3.47±0.11* 2.64±0.06*#
3.61±0.42
4.93±0.47* 3.81±0.37#
0.79±0.10
1.29±0.06* 0.95±0.08#
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*P<0.01 V.S the sham group, respectively; # P<0.01 V.S for WT-TAC group, All values are mean ± SEM.HR=heart rate; BW=body weight; HW=heart weight; PW=pressure gradient; IVSd= interventricular septum depth at end-diastolic stage; LVEDd=left ventricular end-diastolic diameter; LVESD=left ventricular end-systolic
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diameter; LVPWd=left ventricular posterior wall, diastolic. FS=fractional shortening.
ACCEPTED MANUSCRIPT Figure legend Figure 1. Loss of TRADD attenuates pathological cardiac remodeling induced by pressure overload. A, representative picture of whole hearts, haematoxylin-eosin staining and cardiomyocyte cross-sectional area (CSA) in the wild type (WT) and
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TRADD knockout (KO) mice at 2weeks after TAC. Whole heart view of heart (HE staining, scale bar: 40 µm) and microscopic view of cardiomyocyte (HE staining, scale bar: 10 µm). B, CSA was increasing significantly in WT-TAC group, but not in TRADD KO-TAC group at two weeks. C-D, Heart and lung to body weight ratio
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(LW/BW; HW/BW, mg/g). HW/BW and LW/BW increased significantly in WT-TAC group, but the increase of this index was blocked in TRADD KO group after two
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weeks of surgery. E, Fibrotic areas from histological sections was quantified using an image-analyzing system. F, TRADD were detected by WB. *P<0.01 vs WT-sham. #
P<0.01 vs WT-TAC.
Figure 2. TRADD knockout attenuates the expression of cardiac hypertrophy markers in vivo.Total RNA was extracted from hearts of mice, and the expression of ANP,
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BNP and β-MHC induced by TAC were detected by qPCR or WB. Data represent typical results of 3-4 different experiments as mean±SEM. *P<0.01 vs WT-sham. #
P<0.01 vs WT-TAC.
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Figure 3. The effects of TRADD on the expression of fibrous markers in vivo. Western blot and qPCR analyses of collagen I, collagen III and TGF-β1 were performed to determine protein expression levels in indicated groups. GAPDH was
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used as the normalization control. Data represent typical results of 3 different experiments as mean±SEM. *P<0.01 vs WT-sham. #P<0.01 vs WT-TAC. Figure 4. TRADD deficiency attenuates pressure overload-induced kinase activation. The level of total and phosphorylated TAK1, P38, and AKT in hearts tissues of mice in indicated groups. A, Representative blots. C-D, quantitative results. Values are mean ± SEM. *P<0.01 vs WT-sham. #P<0.01 vs WT-TAC.
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Figure 3
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Figure 4
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MAPK pathway sensitises human colon cancer cells to 5-fluorouracil treatment, Int J Oncol, 38 (2011) [26] D. Khiem, J.G. Cyster, J.J. Schwarz, B.L. Black, A p38 MAPK-MEF2C pathway regulates B-cell
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proliferation, Proc Natl Acad Sci U S A, 105 (2008) 17067-17072.
ACCEPTED MANUSCRIPT Dear Editor-In-Chief,
Of course, there has been no duplicate publication or submission elsewhere of any part of the work (excluding abstracts), all authors have read and approved the
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manuscript, and there are no financial or other relations that could lead to a conflict of interest.
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Sincerely, Lei Li, PhD
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Institute of cardiovascular development and translational medicine, the Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, Zhejiang China. China.
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Tel: 86(0577) 88002581, Fax: 86(0577) 88832693, E-mail: [email protected]
S0006-291X(16)32164-7
DOI:
10.1016/j.bbrc.2016.12.104
Reference:
YBBRC 36978
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 9 December 2016 Accepted Date: 16 December 2016
Please cite this article as: L. Wu, Z. Cao, L. Mei, L. Ji, Q. Jin, J. Zeng, J. Lin, M. Chu, L. Li, X. Yang, Loss of TRADD attenuates pressure overload-induced cardiac hypertrophy through regulating TAK1/P38 MAPK signalling in mice, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2016.12.104. 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 Loss of TRADD attenuates pressure overload-induced cardiac hypertrophy through regulating TAK1/P38 MAPK signalling in mice
Jiafeng Lin2, Maoping Chu2, Lei Li2# & Xiangjun Yang1#
1
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Lianpin Wu1,2*, Zhiyong Cao3*,Liqin Mei4, Ling Ji5, Qike Jin2, Jingjing Zeng2,
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Department of Cardiology, The First Affiliated Hospital of Suzhou Medical University. 188 Shizi Road, Suzhou, Jiangsu, 215006, China 2 Department of Cardiology, The Second Affiliated Hospital of Wenzhou Medical University ＆ Yuying Children Hospital. 109 Xueyuan Road, Wenzhou, Zhejiang, 325027, China 3 Department of Cardiology, Huazhong University of Science and Technology, No 411 Hospital of People's Liberation Army, 15 East Jiangwan Road, Shanghai, 200081, China 4 Department of Oral Prophylaxis and Hygiene, School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China 5 Department of Laparoscopic Surgery, The First Hospital Affiliated to Wenzhou Medical College, Wenzhou, Zhejiang, 325027, China
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*Equal contribution to this work. The co-corresponding author of this paper.
#
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Corresponding author: * Correspondence to Lei Li & Xiangjun Yang Phone：+86-139-0663-6339；Fax：+86-577-88002252 Email: [email protected]; [email protected]
ACCEPTED MANUSCRIPT Abstract We investigated the role of tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) on pressure overload-induced cardiac hypertrophy and the underlying molecular mechanisms by using a TRADD deficiency mice model. 6-8
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weeks wild-type and TRADD knockout mice were performed to transverse aorta constriction (TAC) or sham operation (6-8 mice for each group). 14 days after TAC, cardiac function was measured by echocardiography, as well as by pathological and molecular analyses of heart samples. The expressions of cardiac hypertrophic and
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fibrotic markers were detected by qPCR. Phosphorylated and total TAK1, Akt, and p38 MAPK levels were examined by Western blotting. The ratios of lung or
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heart/body weight, wall thickness/ chamber diameter of left ventricular and cross area of cardiomyocyte were significantly reduced in TRADD knockout (KO) mice than those of wild-type mice after TAC. Moreover, cardiac hypertrophic and fibrotic markers were downregulated in TRADD knockout mice than those of wild-type mice following TAC. Protein expression analysis showed phosphorylated TAK1, p38
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MAPK and AKT were upregulated after TAC in both wild-type and TRADD KO mice, phosphorylation of TAK1 and p38 MAPK was reduced more remarkablely after TRADD deficiency, while phosphorylated AKT expression was similar between
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TRADD KO and wild-type mice following TAC. Our data suggest that TRADD KO blunts pressure overload-induced cardiac hypertrophy through mediating TAK1/p38
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MAPK but not AKT phosphorylation in mice.
Key words: TAK1, pressure overload, TRADD, cardiac hypertrophy
ACCEPTED MANUSCRIPT 1. Introduction Cardiac hypertrophy is a common response to many forms of heart failure[1], of which molecular and cellular mechanism keep largely unclear. After a long term of compensatory adaptation, cardiac hypertrophy is associated with functional and
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structure deterioration of the myocardium, fibrosis, inflammation, and altered cardiac gene expression [2-3]. Cardiac hypertrophy is a physiopathological process to chronic pressure. The hypertrophic heart has developed expression of proto-oncogenes and increased rate of protein synthesis [4]. The process is initially beneficial in terms of
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compensatory adaptation through increasing left ventricular wall thickness, while cardiac dysfunction will ultimately be developed after long-term chronic pressure
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overload stimulation [5].
Several pathways are implicated with the pathogenesis of cardiac hypertrophy [4, 6-7]. Transforming growth factor beta-activated kinase 1 (TAK1) signalling plays an important role in regulating cardiac hypertrophy [8-9]. Including our investigation, numerous studies show that the TAK1 signaling system is a crucial regulator of this
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process [8-9]. Moreover, modulation of nuclear factor κB (NF-κB) signaling in the heart may provide a novel approach to attenuate the development of heart failure after cardiac hypertrophy[8]. TAK1 exerts a node function in cell survival and death
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through NF-κB signaling [8-9]. Activated TAK1 results in activation of p38 mitogen-activated protein kinase (p38 MAPK) and JNK signaling pathways, enhances hypertrophic markers expression, and subsequently leads to cardiac hypertrophy[3,
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10]. Further, activation of TAK1 leads to signaling through the IKK/IB/NF-κB pathway,
promoting
proinflammatory
cytokine
expression
and
leading
to
inflammation [8].
Tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) is upstream molecule, which is a central adaptor in the TNFR1 signalling complex that mediates both cell death and inflammatory signals[11-12], TRADD mediates both cell death and pro-inflammatory signals[13]. Although TRADD is usually considered a cytoplasmic protein, it may also have a function in the nucleus [11]. Given the role of TRADD in pro-inflammatory TNFR1 signalling and tumour suppression, it has been
ACCEPTED MANUSCRIPT postulated that TRADD functions should abrogate cell growth. TRADD was shown to be an important protective factor in a variety of cardiac injuries, including endoplasmic reticulum (ER) stress, ischemia reperfusion (I/R) injury [14-15]. The role of TRADD on pressure-overload-induced cardiac hypertrophy has not been fully
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determined. In this study, we investigated the function of TRADD in pressure overload-induced adaptive cardiac hypertrophy using TRADD knockout mice.
2. Methods
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Materials
The antibodies against TAK1/p-TAK1, p38/p38 MAPK, AKT/p-AKT, ANP, BNP,
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β-MHC, Collagen I, Collagen III and transforming growth factor (TGF)-activated kinase β1 (TGF β1) were purchased from Cell Signaling Technology. Acryl-amide, bisacrylamide, glycine, HRP-ECL luminescent substrate and other reagents were purchased from Sigma. Animals
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All protocols were approved by the institutional guidelines. All surgeries and subsequent analyses were performed in a fashion blinded for genotype. TRADD mice were produced as described previously. 8-10 week-old male mice with TRADD
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Knockout (KO) and their control littermates were used in the study. Genotyping was performed by polymerase chain reaction (PCR) as described previously [11]. Mice were maintained on a 12:12 h light-dark cycle at 21-23°C with free access to water
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and mouse chow.
Transverse aortic constriction (TAC) TAC was performed as described previously [9, 16]. Briefly, Age- and sex-matched Wild-type (WT) and KO mice were anesthetized with isoflurane inhalation (1.5%), endotracheally
intubated
and
ventilated
(Type
7025;
Harvard
Apparatus,
March-Hugstetten, Germany). Parasternal thoracotomy was performed in the second intercostal space. After isolation of the transverse aorta, A 7.0 nylon suture was banded against a 27-gauge needle at the transverse aorta to produce a 70-75% constriction after removal of the needle. Doppler view was performed to ensure that
ACCEPTED MANUSCRIPT constriction of the aorta was succeeded. Mice were sacrificed, hearts and lungs were harvested and weighed to compare lung, heart weight/body weight (LW, HW/BW, mg/g) in KO and sham mice. Echocardiography
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Echocardiography was performed as described in our previous study [17]. We performed transthoracic echocardiography to detect the mice heart with a high-frequency ultrasound system Vevo770 (VisualSonics Inc, Toronto, ON, Canada) with a 30-MHz central frequency scanhead [16]. End-systole and end-diastole views
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were defined as the sections in which the smallest and largest areas of the left ventricle (LV), respectively, were obtained. Heart’s geometrical values including
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interventricular septum depth at end-diastolic stage (IVSd), left ventricular posterior wall diastolic stage (LVPWd), fractional shortening (FS) were measured from the LV M-mode at the mid-papillary muscle level. Pressure gradients (mmHg) were calculated from the peak blood velocity (Vmax) (m/s) obtained by Doppler across the tranverse constriction site, which was unchanged in all surgery mice. Hearts, lungs,
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and tibiae of the mice were isolated, weighed and calculated the heart weight (HW)/body weight (BW) (mg/g), and HW/tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios in the different groups. Echocardiographic assessment
studies.
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had good intraobserver and interobserver agreement as reported in our previous
RNA isolation, reverse transcriptase and quantitative Real-time polymerase chain
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reaction (qPCR)
qPCR was performed as described in our previous study[17]. Total RNA was isolated from fresh-frozen left ventricular myocardium using by TRIzol (Invitrogen, USA). 500 ng RNA from per sample was reverse-transcribed for Real-time PCR assay. The quantitative PCR was performed on a Bio-Rad IQ5 examination system by employing 1.5 µg of template complemental DNA. Hypertrophic or fibrosis biomarker of heart were detected using primer below: For atrial natriuretic peptide(ANP,) Forward , GGTGTCCAACACAGATCTGA, Reverse, CCACTAGACCAC TCATCTAC; Brain
natriuretic peptide(BNP), Forward , TATAAAAGGCAGAGGCACCG, Reverse, ATCA
ACCEPTED MANUSCRIPT TCTGGGACAGCACCT; β-myosin heavy chain(β-MHC), Forward , TCGATTTGGGAAA TTCATCC, Reverse, CGCATAATCGTAGGGGTTGT; Collagen I, Forward , CCTGGTAA AG ATGGTGCC, Reverse, CACCAGGTTCACCTTTCGCACC; Collagen III, Forward , GTTCT AGAGGATGGCTGTACT, Reverse, TTGCCTTGCGTGTTTGATATTC; Transforming
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growth factor β1(TGFβ1), Forward , TTGCTTCAGCTCCACAGAGA, Reverse, TGGTTGTAGAGGGCAAGGAC; glyceraldehyde-3- phosphate dehydrogease(GAPDH),
Forward , CCACTCTTCCACCTTCGATG, Reverse, TCCACCACCCTGTTGCTGTA. A
comparative cycle threshold method was used to determine the relative quantification
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of RNA expression, which involves comparing the cycle threhold values of the
samples of target genes with that of sham-operated samples. All Real-time PCR
Western Blotting (WB)
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reactions were performed in triplicate.
WB was performed as described in our previous study [17]. Mouse hearts were homogenized in lysis buffer containing inhibitor cocktail (Merck). Total protein was extracted from homogenized left ventricular tissues and then electrophoresed in 8%
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polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Phospho (p)-Akt and total(t)-Akt proteins were examined with the primary antibodies (Santa Cruz).The secondary antibody linked to Alkaline phosphatase was used to detected
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the immumo precipitation. Measurement of precipitation was calculated by using an automated image analysis system (Image-Pro Plus 5.0, Media Cybernetics, Bethesda, MD, USA). Phosphorylated Akt normalized to total Akt, and ratios between different
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groups were compared. GAPDH was used as the internal control. Histological analysis
Histological analysis was performed as described in our previous study [17]. Hearts were harvested to immerse immediately in normal saline, fixed in 10% formalin, embedded in paraffin, and cut into 4-µm-thick sections in the short axis at the papillary muscle level. Section was stained with haematoxylin-eosin (HE) for histopathology then visualized by magnification of 400 times for myocyte size measurement. To measure the cross-sectional area of myocytes, HE-stained sections were analysis. Fibrosis area was calculated based on Masson Trichrome staining
ACCEPTED MANUSCRIPT (Fig1D). A single myocyte was measured using an image quantitative digital analysis system (Image-Pro plus 6.0). The quantitative analysis of histological images was performed in a blinded fashion. Statistical analyses
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Data are expressed as Mean±SEM. Differences among groups were based on two-way ANOVA followed by a post hoc Tukey test. Comparisons between two groups were performed through an unpaired Student’s t-test. A value of P <0.05 was considered
3.
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significant.
Results
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TRADD KO attenuates pathological cardiac hypertrophy induced by pressure overload
To investigate the role of TRADD in the heart induce by pressure overload, we performed TAC surgery on 8- to 10-week–old TRADD KO and WT mice. As shown in Figure 1, HW/BW and LW/BW ratios were obviously decreased in KO group
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compared to WT mice, as well as cardiac fibrosis (Figure1A-E). Cardiac function was examined by echocardiography after 2 weeks of surgery. The increases in left ventricle chamber dimensions and wall thickness induced by TAC were also markedly
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attenuated during both systole and diastole in KO mice compared to WT littermates (Table 1). Gross heart and HE staining further confirmed the inhibitory effect of TRADD on cardiac remodeling following TAC (Figure 1A).
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Echocardiographic results showed the effects of TRADD on cardiac hypertrophy following 2 weeks TAC surgery To explore the role of TRADD on heart hypertrophy induced by pressure overload, we performed TAC surgery on 8- to 10-week–old mice. From our echocardiographic results, PWs of the mice are significantly higher in TAC-WT and TAC-KO groups than those of sham mice (Table 1). PW has no difference between the TAC-WT and the TAC-KO group. HR was similar among three groups. Cardiac hypertrophy and function was first evaluated by echocardiography. Quantitative analysis showed that both LV end-systolic posterior wall thickness (LVPWs) and LVPWd were
ACCEPTED MANUSCRIPT significantly increased by 2 weeks of TAC but not by sham operation. TRADD ablation significantly reduced the increases in LVPWs and LVPWd by TAC. On the other hand, loss of TRADD attenuated the enlargement in LV internal dimension at end systole and diastole (LVESD and LVEDD) and LV ejection fraction (LVEF)
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dysfunction by TAC (Table 1). TRADD ablation repressed the expression of cardiac hypertrophy markers in response to pressure overload
We examined the expression of several cardiac hypertrophy markers in KO and WT
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littermates after TAC surgery by qPCR and WB analysis. Expression levels of hypertrophic gene including ANP, BNP, and β-MHC were increased to a higher level
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in WT-TAC littermates, and such increases were markedly attenuated in TRADD KO mice (Figure 2). These data suggest that TRADD deletion in mice decreased the expression of cardiac hypertrophy markers induced by pressure overload. The effects of TRADD on the expression of fibrosis markers induced by pressure overload
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Heart sections were stained with Masson staining to detect fibrosis. In both groups, collagen continued to accumulate in the heart after 2 weeks of TAC. As shown in Figure 3, quantitative analysis showed that increased collagen deposition was
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significantly regressed in TRADD KO mice. Reduced fibrosis in KO mice suggested increased collagen degradation or decreased collagen synthesis following TAC. We, next, examined the synthesis of collagen by examining the expression of mRNA and
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protein expression collagen I, collagen III and TGF-β1, known to be involved in the proliferation of cardiac fibroblasts and the biosynthesis of extracellular matrix proteins. The results showed that as for collagen I, collagen III and TGF-β1, mRNA, as well as protein expressions, were significantly lower in TRADD KO compared to in WT mice induced by pressure overload (Figures 3). Loss of TRADD reduced pressure overload-induced TAK1/p38 Signaling To examine the molecular mechanisms of TRADD on cardiac hypertrophy in response to TAC, we investigated activation of p38 MAPK pathway in the TAC mice models. We found that the phosphorylated levels of p38 MAPK and TAK1 were
ACCEPTED MANUSCRIPT significantly increased after TAC stimuli in WT hearts. However, the phosphorylation of TAK1 and p38 MAPK was almost completely attenuated in TRADD KO mice hearts, whereas AKT activation was similar in the two groups after TAC (Figure 4). Although AKT signaling functioned as a crucial role in the mediation of cardiac
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remodeling and function, we did not observe any difference in AKT activation between WT-TAC and KO-TAC mice, as determined by immunoblotting for phosphorylation of AKT (Figure 4A and D). Taken together, these result indicate that TRADD knockout repressed the activation of TAK1 and p38 MAPK although it has
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no inhibitory effects on AKT activation in hearts following TAC.
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4. Discussion
Our data show that TRADD deletion abrogated pressure overload-induced cardiac hypertrophy.
TRADD
KO
is
involved
with
reduced
TAK1/p38
MAPK
phosphorylation while phosphorylation of AKT was unchanged by TRADD. This is the first report investigating the underlying molecular mechanisms of TRADD in
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pressure overload-induced cardiac hypertrophy at a compensatory stage. Myocardial hypertrophy initially develops following short-term or long-term hypertension, which can be defined as early compensatory and late decompensated
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cardiac hypertrophy [5, 18]. During the beginning of hypertension, heart usually increases hypertrophy to keep up with the increased afterload and to hold cardiac output [19-20]. Previous studies have reported that cardiac ejection function (EF)
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maintained normal in mice subjected to 2 week TAC surgery, however, EF reduced and gradually developed into dysfunction after 4 weeks of TAC [21-22]. Cardiac hypertrophy might be a beneficial process to perverse normal EF at the early stage and attenuation of cardiac hypertrophy at this early stage might be more beneficial in TRADD knockout mice [21, 23-24]. Our results demonstrated that the TAK1 signalling might be implicated in pressure overload- induced hypertrophy process. Previous studies have suggested a critical role of TAK1/p38 MAPK pathway in the control of cell size, survival [25-26]. It is known that TAK1 activity is crucial for compensatory (physiological) and maladaptive (pathological) hypertrophy in the heart.
ACCEPTED MANUSCRIPT Loss of TAK1 impaired the development of adaptive ventricular remodeling following pressure overload [8-9]. Consistent with other studies [18], our study showed loss of TRADD deactivated TAK1, p38 MAPK and improved EF and left ventricular remodeling following TAC. However, TRADD ablation did not affect the
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activation of AKT, but it obviously reduced TAK1 and p38 MAPK activation induced by TAC.
Conclusion: our results suggested that TRADD deletion may benefit adaptive cardiac hypertrophy through TAK1/p38 MAPK pathway. Considering that the initially
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adaptive cardiac hypertrophy might be a protective process, the decrement of cardiac hypertrophy following TAC in TRADD KO mice might be beneficial to pressure
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overload-induced heart failure in the long term. Further studies are needed to investigate the potential pathways on how TRADD ablation affected TAK1 and p38 MAPK activation following TAC.
Disclosures
Acknowledgements
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All authors have no conflicts of interest to declare.
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This work was supported by grants from Fund of National Natural Science Foundation of China (81570238), Project of Science and Technology Department of Zhejiang Province (2015C33163), Scientific Research Foundation of Wenzhou
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(Y20150015), Zhejiang Provincial Medical and Health Science and Technology plan (2016KYB197).
ACCEPTED MANUSCRIPT Table 1 WT-sham
KO-sham
WT-TAC
KO-TAC
Number
6
8
7
7
HR (beats/min)
511±54
515±58
521±51
510±53
PW (mmHg)
2.23±0.69
2.53±0.52
60±6.71*
57±57*
HW/BW (mg/g)
4.53±0.63
4.45±0.56
8.71±0.82* 6.25±0.67*#
EF (%）
75±6
76±8
41±6*
FS (%)
33±3
31±3
IVSd (mm)
0.81±0.03
0.87±0.07
LVESD (mm)
1.70±0.08
LVEDd (mm)
3.53±0.52
LVPWd (mm)
0.84±0.11
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Parameters
16±2*
56±5*#
28±2#
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1.34±0.07* 1.07±0.05*#
1.69±0.07
3.47±0.11* 2.64±0.06*#
3.61±0.42
4.93±0.47* 3.81±0.37#
0.79±0.10
1.29±0.06* 0.95±0.08#
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*P<0.01 V.S the sham group, respectively; # P<0.01 V.S for WT-TAC group, All values are mean ± SEM.HR=heart rate; BW=body weight; HW=heart weight; PW=pressure gradient; IVSd= interventricular septum depth at end-diastolic stage; LVEDd=left ventricular end-diastolic diameter; LVESD=left ventricular end-systolic
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diameter; LVPWd=left ventricular posterior wall, diastolic. FS=fractional shortening.
ACCEPTED MANUSCRIPT Figure legend Figure 1. Loss of TRADD attenuates pathological cardiac remodeling induced by pressure overload. A, representative picture of whole hearts, haematoxylin-eosin staining and cardiomyocyte cross-sectional area (CSA) in the wild type (WT) and
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TRADD knockout (KO) mice at 2weeks after TAC. Whole heart view of heart (HE staining, scale bar: 40 µm) and microscopic view of cardiomyocyte (HE staining, scale bar: 10 µm). B, CSA was increasing significantly in WT-TAC group, but not in TRADD KO-TAC group at two weeks. C-D, Heart and lung to body weight ratio
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(LW/BW; HW/BW, mg/g). HW/BW and LW/BW increased significantly in WT-TAC group, but the increase of this index was blocked in TRADD KO group after two
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weeks of surgery. E, Fibrotic areas from histological sections was quantified using an image-analyzing system. F, TRADD were detected by WB. *P<0.01 vs WT-sham. #
P<0.01 vs WT-TAC.
Figure 2. TRADD knockout attenuates the expression of cardiac hypertrophy markers in vivo.Total RNA was extracted from hearts of mice, and the expression of ANP,
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BNP and β-MHC induced by TAC were detected by qPCR or WB. Data represent typical results of 3-4 different experiments as mean±SEM. *P<0.01 vs WT-sham. #
P<0.01 vs WT-TAC.
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Figure 3. The effects of TRADD on the expression of fibrous markers in vivo. Western blot and qPCR analyses of collagen I, collagen III and TGF-β1 were performed to determine protein expression levels in indicated groups. GAPDH was
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used as the normalization control. Data represent typical results of 3 different experiments as mean±SEM. *P<0.01 vs WT-sham. #P<0.01 vs WT-TAC. Figure 4. TRADD deficiency attenuates pressure overload-induced kinase activation. The level of total and phosphorylated TAK1, P38, and AKT in hearts tissues of mice in indicated groups. A, Representative blots. C-D, quantitative results. Values are mean ± SEM. *P<0.01 vs WT-sham. #P<0.01 vs WT-TAC.
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Figure 3
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Figure 4
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ACCEPTED MANUSCRIPT Dear Editor-In-Chief,
Of course, there has been no duplicate publication or submission elsewhere of any part of the work (excluding abstracts), all authors have read and approved the
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manuscript, and there are no financial or other relations that could lead to a conflict of interest.
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Sincerely, Lei Li, PhD
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Institute of cardiovascular development and translational medicine, the Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, Zhejiang China. China.
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Tel: 86(0577) 88002581, Fax: 86(0577) 88832693, E-mail: [email protected]