Cardiac-specific Traf2 overexpression enhances cardiac hypertrophy through activating AKT/GSK3β signaling Yinqing Huang, Dengyin Wu, Xin Zhang, Minghua Jiang, Chaohui Hu, Jiangfeng Lin, Jifei Tang, Lianpin Wu PII: DOI: Reference:

S0378-1119(13)01729-0 doi: 10.1016/j.gene.2013.12.052 GENE 39356

To appear in:

Gene

Accepted date:

24 December 2013

Please cite this article as: Huang, Yinqing, Wu, Dengyin, Zhang, Xin, Jiang, Minghua, Hu, Chaohui, Lin, Jiangfeng, Tang, Jifei, Wu, Lianpin, Cardiac-specific Traf2 overexpression enhances cardiac hypertrophy through activating AKT/GSK3β signaling, Gene (2013), doi: 10.1016/j.gene.2013.12.052

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ACCEPTED MANUSCRIPT Cardiac-specific Traf2 overexpression enhances cardiac hypertrophy

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through activating AKT/GSK3β signaling

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Yinqing Huang1, Dengyin Wu2, Xin Zhang1, Minghua Jiang1, Chaohui Hu3, Jiangfeng

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Lin1, Jifei Tang1, Lianpin Wu1*

1 Department of cardiology, The Second Affiliated Hospital of Wenzhou Medical

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University, 109 Xueyuan Road ,Wenzhou 25035,Zhejiang,China.

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2 Medical College of Zhejiang University City College, 51 Huzhou Road, Hangzhou, 310000, China.

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3 Department of Cardiology, The Tongji Hospital of Tongji University, 309 Xincun Road,

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Putuo District, Shanghai 200065, China.

Corresponding to

Lianpin Wu MD, PHD Phone：008613806686589；Fax：008657788002252 Email: [email protected]

ACCEPTED MANUSCRIPT Abstract Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal

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through a common scaffolding protein termed tumor necrosis factor receptor-associated

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factor 2 (Traf2); however, Traf2 signaling in the adult mammalian cardiac hypertrophy is not fully understood. This study was aimed to identify the effect of Traf2 on cardiac

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hypertrophy and the underlying mechanisms. A significant up-regulation of Traf2

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expression was observed in mice failing hearts. To further investigate the role of Traf2 in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of

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Traf2 function and cardiac-specific Traf2-overexpressing transgenic (TG) mice. In cultured cardiomyocytes, Traf2 positively regulated angiotensin II (Ang II)-mediated

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hypertrophic growth, as detected by [3H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by

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constriction of transverse aortic (TAC) in TG mice and their wild-type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Traf2 overexpression in the heart remarkably enhanced cardiac hypertrophy, left ventricular dysfunction in mice in response to TAC. Further analysis of the signaling pathway in vitro and in vivo suggested that these adverse effects of Traf2 were associated with the activation of AKT/glycogen synthase kinase 3β (GSK3β). The present study demonstrates that Traf2 serves as a novel mediator that enhanced cardiac hypertrophy by activating AKT/GSK3β signaling. Keywords: Traf2, pressure overload, AKT, cardiomyocyte

ACCEPTED MANUSCRIPT Introduction Cardiac hypertrophy, an increase in cardiocmyocyte size, is the remodeling of the

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myocardium induced by a variety of extrinsic and intrinsic stimuli (Molkentin, 2000;

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Baines and Molkentin, 2005). Although hypertrophic response is initially compensatory

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elicited by an increased workload, prolonged cardiac hypertrophy eventually leads to functional and histological deterioration of the myocardium, fibrosis, inflammation, and

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altered cardiac gene expression and then congestive heart failure, arrhythmia, and sudden

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death (Molkentin, 2004; Heineke and Molkentin, 2006; Maillet et al., 2013). Numerous molecules and signaling pathways have been involved to regulate the process of cardiac

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hypertrophy and heart failure(van Berlo et al., 2013). Increasing evidence suggests that

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the nuclear factor κB (NFκB) signaling system plays a critical role on this process (Santos et al., 2010; Leychenko et al., 2011). Regulation of NF-κB signaling in the heart

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may provide a novel approach to attenuate the development of heart failure elicited by cardiac hypertrophy.

Traf2, a member of tumor necrosis factor receptor-associated factor (Traf) family, plays a central role in many biological activities, such as the regulation of immune and inflammatory responses and control of apoptosis (Jono et al., 2004; Ermolaeva et al., 2008; Shih et al., 2011). Overexpression of Traf2 inhibited cell apoptosis by activating NFκB nuclear transcription in breast cancer cells (Jang et al., 2011; Zhang et al., 2013). Recent studies showed that Traf2 expression protects various cell types from TNFmediated apoptosis (Zheng et al., 2006; Shih et al., 2011; Khan et al., 2013). Traf2 is highly expressed in the heart (Burchfield et al., 2010; Divakaran et al., 2013), suggesting a possible function for Traf2 in cardiac disease. The role of Traf2 in cardiac

ACCEPTED MANUSCRIPT hypertrophy, however, has not yet been fully studied. Given that Traf2 is an important signing protein in NFκB signaling, which are all key players in cardiac hypertrophy. We

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hypothesized that Traf2 may be involved in the cardiac hypertrophic process. In the

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present study, we used cultured neonatal rat cardiomyocytes with gain and loss of Traf2 function, and cardiac-specific Traf2-overexpressing transgenic (TG) mice to investigate

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the role of Traf2 in cardiac hypertrophy induced by pressure overload stimulation and the

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related molecular mechanisms. We show that Traf2 functions as a novel hypertrophic protein that enhanced maladaptive remodeling and the transition to heart failure by

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activating AKT/glycogen synthase kinase 3β (GSK3β) signalling.

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2.1 Animal models

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2. Methods

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All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee at our Hospital, China. All surgeries and subsequent analyses were performed in a blinded fashion. Full-length human Traf2 cDNA was cloned downstream of the cardiac myosin heavy chain (MHC) promoter. TG mice were then produced by microinjection of the α-MHC-Traf2 construct into fertilized mouse embryos (C57BL/6 background). Three independent transgenic lines were established and studied. TG mice were identified by PCR analysis of tail genomic DNA. Functional data and gene expression levels were analysed in pairs of α-MHC-Traf2 (TG) and wild type (WT) male littermates in age ranging from 8 to 9 weeks.

ACCEPTED MANUSCRIPT Male TG and WT littermate control mice aged 8–10 weeks were subjected to transverse aortic constriction (TAC) or a sham operation as described previously (Li et al., 2010).

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Briefly, mice were anesthetized by intraperitoneal injection of a cocktail of ketamine (100mgkg-1) and xylazine (5mg kg-1), and respiration was artificially controlled with a

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tidal volume of 0.2 ml and a respiratory rate of 110 breaths min-1. The TAC was

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constricted with 7-0 nylon strings by ligating the aorta with a blunted 27-gauge needle to

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yield a narrowing of 0.4 mm in diameter when the needle was pulled out later. 2.2 Echocardiographic evaluation

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Echocardiography was performed on anaesthetized (1.5% isoflurane) mice, using a 30MHz high-frequency scanhead (VisualSonics Vevo770, VisualSonics, Toronto, ON,

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Canada). End-systole and end-diastole were defined as the phases in which the smallest and largest areas of the LV, respectively, were obtained. LV end-systolic diameter

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(LVESD) and LV end-diastolic diameter (LVEDD) were measured from the LV M-mode at the mid-papillary muscle level. Mice were euthanized by cervical dislocation 4 weeks post-operatively. Hearts, lungs, and tibiae of the mice were dissected and weighed or measured to compare the heart weight (HW)/body weight (BW) (mg/g), HW/tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios in the different groups. 2.3 Histological analysis Hearts were excised, placed immediately in 10% potassium chloride solution to ensure that they were stopped in diastole, washed with pre-cold phosphate-buffered saline (PBS) solution, and placed in 10% formalin. Hearts were sectioned transversely close to the apex to visualize the left and right ventricles. Several sections (4–5 mm thick) were

ACCEPTED MANUSCRIPT prepared and stained with haematoxylin-eosin (HE) for histopathology then visualized by light microscopy. To determine the cross-sectional area of myocytes, HE-stained sections

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were used. A single myocyte was measured using an image quantitative digital analysis

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system (Image-Pro Plus 6.0). Between 100 and 200 myocytes in the LVs were outlined in each group. The quantitative analysis of histological images was performed in a blinded

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fashion.

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2.4 Recombinant adenoviral vectors, cultured neonatal rat cardiac myocytes, and fibroblasts

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To overexpression of Traf2, we used replication-defective adenoviral vectors encompassing the full coding region of the Traf2 gene under the control of the

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cytomegalovirus promoter. A similar adenoviral vector encoding the GFP gene was used as a control. To knock down Traf2 expression, three rat shTraf2 constructs were obtained.

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Next, we generated three Ad-shTraf2 adenoviruses and selected the one that produced a significant decrease inTraf2 levels for further experiments. Ad-shRNA was the nontargeting control. We infected cardiac myocytes with Ad-Traf2, Ad-green fluorescent protein (AD-GFP), Ad-shTraf2, or Ad-shRNA, which resulted in transgenic expression without toxicity in 80-100% of the cells. Neonatal (1–3-day-old) Sprague-Dawley rats were killed by swift decapitation and their hearts were used for the isolation and cultures of neonatal rat cardiac myocytes, as described previously (Golden et al., 2012; Gerilechaogetu et al., 2013). Briefly, hearts from newborn 1-3-day-old Sprague-Dawley were minced, and cells were isolated by trypsinization incubation at 37°C. Non-cardiomyocytes were separated from the cardiomyocytes by differential preplating. Mild trituration was used to dissociate the

ACCEPTED MANUSCRIPT digested tissue mechanically, and single-cell suspensions were obtained by filtering this digested material through 70 µm sterile mesh filters. The cells were collected by low-

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speed centrifugation (1500 rpm for 10 min at room temperature). The supernatant was

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discarded and the cell pellet resuspended in DMEM (high glucose) culture medium

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containing 10% FCS (Hyclone Laboratories, USA). Dispersed cells were preplated for 90 min to remove fibroblasts and other proliferation cells, and unattached cells counted

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and seeded onto 6-well culture plates at a density of 1106/well. The media was

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changed every 48 h beginning the day after seeding. Bromodeoxyuridine (0.1 mM) was added to the culture media for the first 72 h to minimize contamination from fibroblasts.

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Using this method, we routinely obtained primary cultures with >95% myocytes, as

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assessed by microscopic observation of spontaneous contraction and by

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immunocytochemical staining with a monoclonal anti-cardiac α-myosin heavy chain antibody. Cell viability was determined by measuring the cell number, frequency of contractions, cellular morphology, and trypan blue exclusion. For cell infection, cardiac myocytes were cultured at a density of 1 X 106 cells/well in 6well plates and exposed to 1 X 108 pfu each of virus in 1 mL of serum-free medium for 24 h. The cells were then washed and incubated in serum-containing medium for 24 h. Additional treatments are described in the figure legends. 2.5 [3H]-Leucine incorporation and surface area [3H]-Leucine incorporation was measured as described below. Briefly, cardiac myocytes were infected with different adenoviruses for 24 h and subsequently stimulated with Ang II (1 mM, Sigma) and co-incubated with [3H]-Leucine (1 mCi/mL, MP Biomedical) for

ACCEPTED MANUSCRIPT the indicated time. At the end of the experiment, the cells were washed with Hanks’ solution, scraped from the well, and treated with 10% trichloroacetic acid at 40C for 60

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min. The precipitates were then dissolved in NaOH (1 N) and subsequently counted with a scintillation counter. For surface area measurements, the cells were fixed with 4%

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formaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS, and stained with α-

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actinin (Sigma) at a dilution of 1:100 using standard immunofluorescence staining

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tecnhiques. 2.6 Real-time quantitative RT-PCR

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Total RNA was prepared from the heart tissues of mice by using TRIzol reagent (15596018, Gibco BRL, Carlsbad, CA, USA); the reverse transcription PCR was performed by

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using TOYOBO ReverTra Ace-a-RT-PCR kit according to the manufacturer’s instruction. The real-time PCR was performed on a Bio-Rad IQ5 multicolor detection system by

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using 2 mg of synthesized cDNA. Atrial natriuretic peptide (ANP), B-type natriuretic peptides (BNP), β-myosin heavy chain (β-MHC) and glyceraldehyde-3-phosphate

dehydrogenase were amplified using their specific primers. The primers used for PCR analysis are as follows: for ANP gene, forward 5’-ACGCAGCTTGGTCACATTGC-3’ and reverse 5’-CCACTAGACCACTCATCTAC-3’; for BNP gene, Forward: 5′-GGGC TGTGACGGGCTGAGGTT-3′and Reverse: 5′-AGTTTGTGCTGGAAGTAAGA-3′; for β-MHC, forward 5’- CACCAACC TGTCCAAGTTCC-3’ and reverse 5’-GGAGCTGG GTAGCACAAGAG-3’; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)gene, 5’-CCACTCTTCCACCTTCGATG-3’ and 5’-CCACCACCCTG TTGCTGTA-3’. A comparative CT method was used to determine the relative quantification of RNA expression, which involves comparing the CT values of the samples of interest with a

ACCEPTED MANUSCRIPT control or calibrator such as a non-treated sample or RNA from normal tissue. All realtime PCR reactions were performed in triplicate.

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2.7 Western blotting

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Western blotting was conducted to determine protein levels of Traf2, hypertrophic

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markers (ANP, BNP, β-MHC), and the activation state of AKT/GSK3β signaling. Quantification of western blots were measured by using an automated image analysis

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system (Image-Pro Plus 5.0, Media Cybernetics, Bethesda, MD, USA). The specific

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protein expression levels were normalized to either GAPDH for the total cell lysate. 2.8 statistical analysis

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Data are expressed as mean+SEM. Differences among groups were determined by two-

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way ANOVA followed by a post hoc Tukey test. Comparisons between two groups were performed using an unpaired Student’s t-test. A value of P