Basic Res Cardiol (2015) 110:28 DOI 10.1007/s00395-015-0484-7

ORIGINAL CONTRIBUTION

Sialyltransferase7A, a Klf4-responsive gene, promotes cardiomyocyte apoptosis during myocardial infarction Dongmei Zhang1 • Liang Zhu1 • Chunmei Li2 • Jingzhou Mu1 • Yuanshan Fu3 Qiong Zhu1 • Zhenzhen Zhou1 • Pixu Liu4 • Chuanchun Han4

•

Received: 4 November 2014 / Revised: 15 March 2015 / Accepted: 31 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Myocardial infarction (MI) is one major cause of heart failure through its induction of cardiomyocyte death. However, the molecular mechanisms associated with MI-induced cardiomyocyte apoptosis in the context of sialylation of heart are not yet understood. In this study, we found that sialyltransferase7A (Siat7A), one of the members of sialyltransferase family, was significantly increased in the ischemic myocardium, as well as in the human cardiomyocyte cell line AC16 under hypoxic condition. The Sialyl-Tn antigen (Neu5Aca2–6GalNAc-O-Ser/Thr) synthesized by Siat7A also increased in the AC16 cardiomyocytes following hypoxic stimulus. Increased Siat7A promoted cardiomyocyte apoptosis. The knockdown of Siat7A expression reduced cardiomyocyte apoptosis in both of vivo and vitro. Furthermore, the decreased extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2)

activity was involved in the Siat7A-induced cardiomyocyte apoptosis. Notably, we showed that Kru¨ppel-like factor 4 (Klf4), one of the transcription factors, specifically bound to the Siat7A promoter by ChIP assays. Deletion and mutagenesis analysis identified that Klf4 could transactivate the Siat7A promoter region (nt -655 to -636 bp). The upregulated Siat7A expression, which was paralleled by the increased Klf4 in the ischemic myocardium, contributed to cardiomyocyte apoptosis. Our study suggests Siat7A could be a valuable target for developing treatments for MI patients. Keywords Myocardial infarction  Sialyltransferase7A  ERK1/2  Klf4

Introduction D. Zhang and L. Zhu contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00395-015-0484-7) contains supplementary material, which is available to authorized users. & Dongmei Zhang [email protected] & Pixu Liu & Chuanchun Han 1

Department of Physiology, Dalian Medical University, Dalian, People’s Republic of China

2

Pathology Department of Dalian Medical University, Dalian, People’s Republic of China

3

Anatomy Department of Dalian Medical University, Dalian, People’s Republic of China

4

Institute of Cancer Stem Cell, Dalian Medical University, Dalian, People’s Republic of China

Myocardial infarction (MI) is the most common reason for heart failure and is one of the most prevalent causes of morbidity and mortality worldwide [26, 47]. The cardiomyocyte apoptosis and necrosis that follow MI lead to the loss of cardiomyocytes, which substantially decreases the pumping of the heart and results in congestive heart failure [18, 43]. Experimental studies have shown that the amount of cardiomyocyte apoptosis is significantly higher than that of cardiomyocyte necrosis. Clinical studies have reported that the rate of cardiomyocyte apoptosis at the infarction site is significantly associated with persistent infarct-related artery occlusion [1]. These studies suggest that cardiomyocyte apoptosis is a critical cellular process leading to heart failure following MI. Previous studies have also shown that cellular molecules involved in transcription or protein glycosylation, such as transcription factors or glycotransferases, may be

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responsible for cardiomyocyte apoptosis [15, 16, 25]. Glycosylation is a post-translational modification that specifically determines the configuration and composition of carbohydrate sequences attached to glycoproteins. Disregulation of glycosylation may change the functions of glycoproteins, and as such is involved in many diseases, including cancer, cardiovascular disease, and certain immunological disorders [5, 25, 29, 33]. Glycotransferases conduct the attachment of carbohydrate sequences to the polypeptide portions of glycoproteins, and they are specifically selected as the therapeutic target for correlated diseases [3, 13, 19]. In particular, sialyltransferases catalyze the transfer of a sialic acid residue from cytidine 50 -monophosphono-Nacetylneuraminic acid (CMP-Neu5Ac) to oligosaccharide chains of glycoproteins and glycolipids, known as the terminal glycosylation process [12]. The sialyltransferase family consists of 20 members. All members are type II transmembrane proteins with a short NH2-terminal signal anchor domain in the cytoplasm and a large COOH-terminal catalytic domain in the Golgi apparatus. Each sialyltransferase catalyzes the transfer of CMP-Neu5Ac to a specific substrate and forms only one linkage. The heart is heavily glycosylated, specifically sialylated [12, 32, 40]. Aberrant sialylation caused by different sialyltransferases in the heart has been shown to alter the function of voltagegated K? and Na? channels and cause arrhythmias [32, 40, 41]. Sialyltransferase7A (Siat7A), one of the CMPNeu5Ac:GalNAc-R a2,6-sialyltransferases for synthesizing the Sialyl-Tn antigen (Neu5Aca2–6GalNAc-O-Ser/ Thr), has been found consistently overexpressed at the mRNA level in the hypertrophic ventricles of three hypertensive rat models independently of genetic background [4, 12, 28]. However, the specific roles and the molecular mechanisms of Siat7A in heart disease are still unknown. Kru¨ppel-like factors (Klfs), a class of transcription factors that contains three highly conserved C2H2 zinc finger motifs, are characterized by the ability to bind CACCC- or GC-rich sites in promoter and enhancer regions of the target genes, thereby regulating fundamental cell processes [20, 31]. Klf4 has been identified as the most highly implicated in apoptosis among the seventeen members of the mammalian Klf family [31, 42]. Klf4 regulates apoptosis in a damage-dependent manner, and has been shown to increase in induced pathologic hypertrophy. Deletion of Klf4 in mice cardiomyocytes sensitized the cells to induction of pathologic hypertrophy following transverse aortic constriction. Klf4 expression has also been shown to inhibit pathologic cardiac hypertrophy through modulating the expression and activity of fetal cardiac genes [27, 44, 46]. These studies strongly indicate that Klf4 plays a critical role in cardiac hypertrophy. However, despite evidence of increased Klf4 mRNA expression in rat cardiomyocytes

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under oxidative stress, the mechanism of Klf4 in cardiomyocyte damage following MI has not been clarified [6]. In this study, we found increased expression of Siat7A in the ischemic ventricle myocardium. This elevation of Siat7A, coinciding with its substrate, the Sialyl-Tn antigen, induced cardiomyocyte apoptosis following MI. In parallel to Siat7A expression increase, Klf4 was also elevated in the ischemic ventricle myocardium and was found to transactivate Siat7A expression in cardiomyocytes. We also demonstrated that Klf4 upregulated Siat7A expression and promoted cardiomyocyte apoptosis initiated by MI through inhibition of the MAPK/ERK1/2 signaling pathway.

Materials and methods Establishment of the MI animal model and siRNA delivery Wistar rats weighing between 180 and 220 g were purchased from the Animal Medicine Center, Dalian Medical University. Twenty-two rats were randomly divided into two groups: a sham group (n = 6) and an acute MI (AMI) group (n = 16). Additional thirty rats were randomly divided into the following four groups to receive different treatments, respectively: sham-operated rats treated with the injection of scramble siRNA (sham-siNC, n = 6), sham-operated rats treated with the injection of Siat7A siRNA (sham-siSiat7A, n = 6), MI-operated rats treated with the injection of scramble siRNA and the ligation of the left anterior descending coronary (LAD) (MI-siNC, n = 9) and MI-operated rats treated with the injection of Siat7A siRNA and the LAD ligation (MI-siSiat7A, n = 9). All the rats were anesthetized by intraperitoneal administration of pentobarbital (50 mg/kg), and then endotracheally intubated and mechanically ventilated (Jiangxi Teli, China) with supplemental oxygen. The heart was exposed through a thoracotomy at the left fourth intercostal space, and the LAD was identified. During the siRNA injection treatment, a total of 60 ll solution composed of 30 ll siRNA (1 lg scramble siRNA or Siat7A siRNA/1 ll, GenePharma, Shanghai, China), 15 ll EntransterTM in vivo transfection reagent (Engreen Biosystem, Beijing, China) and 15 ll 20 % glucose was injected into three regions of the myocardium of anterior left ventricular wall nearby the LAD using a syringe with 29 gage needle (BD, Insulin Syringe). This treatment was followed by closing the chest cavity with sutured muscles and skin in three layers. Only AMI group rats were treated with LAD ligation with a 6-0 silk suture 1–2 mm below the tip of the left atrial appendage 48 h after the injection operation. To prevent dehydration during this procedure, physiological saline was

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continuously infused at a rate of 5 ml/kg/h with a syringe pump (CFV-3200; Nihon Kohden). According to the published literature, we killed the rats 3 h after the LAD ligation and collected myocardial tissue in the anteroapical left ventricular wall for analysis [7, 8, 38].

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antigen–antibody complex. At least three fields from each tissue section were photographed under light microscopy at 2009 or 4009. The sections were also stained with hematoxylin–eosin for morphological examination. Cell culture and reagents

Echocardiography and hemodynamic assessment A polyethylene tubing (PE-10, Becton–Dickinson) filled with saline and connected to a fluid-filled transducer (AD Instruments PowerLab, Australia) was cannulated into the right carotid artery to measure the blood pressure (BP). A poly-leads physiological detector (AD Instruments PowerLab 8/35, Australia) was used to display BP and echocardiography (ECG). Selection of human samples We compared six hearts collected during autopsy from the subjects died after MI. The cause of death was acute MI in four cases, and congestive heart failure in the other cases. Tissue specimens were obtained at sites of infarction and in the remote regions of the left ventricle, and then stained for histological and immunohistochemical examination. Informed consent was obtained from the families of the subjects as approved by the Institutional Review Board protocol from Dalian Medical University. Hematoxylin–eosin (HE) and immunohistochemistry analysis The heart tissue specimens were fixed in 10 % phosphatebuffered formalin for 48 h. The ventricles were sectioned from apex to base in a plane parallel to the atrioventricular groove. Slices with 2–2.5 mm in thickness were taken from each tissue specimen in the anteroapical left ventricular wall. Tissue slices from each heart were paraffin embedded after dehydration and clearing. 5-lm paraffin-embedded sections were cut and mounted on glass slides. The sections were deparaffinized in xylene twice, rehydrated through a series of gradient ethanol washes, and rinsed in water. Antigen retrieval was performed by immersing the slides in 0.1 M citrate buffer solution (pH 6.0), and heating the slides in an autoclave at 120 °C for 5 min. The sections were allowed to cool and washed in PBS for 20 min. Endogenous peroxidase activity was blocked by incubating sections in 3 % hydrogen peroxide for 15 min at room temperature. The slides were washed in water and in PBS for 20 min. After incubation in a blocking solution containing 10 % normal goat serum for 15 min at room temperature, the slides were incubated in antibody and 3,30 diaminobenzidine solution. Sections were counterstained with hematoxylin. Brown deposits indicate the sites of

AC16, a human cardiomyocyte-like cell line, was obtained from the American Type Culture Collection (Manassas, VA, USA). The AC16 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM/F12) (Invitrogen), supplemented with 12.5 % fetal calf serum, 50 U/ml penicillin, and 50 lg/ml streptomycin. The cells were maintained at 37 °C under 5 % CO2 in humidified air. For hypoxia experiments, we added CoCl2 (Sigma-Aldrich, No. 232696) at 200, 400, 600 and 800 lmol/l into the medium and incubated the cells in the presence of CoCl2 for the indicated times [2]. In additional hypoxia experiments, we transferred cells grown in serum-deprived DMEM/F12 into an airtight incubator and maintained the cells at a humidified hypoxic atmosphere of 1 % O2 with nitrogen and 5 % CO2 for 36 h. The following antibodies were used in this study: anti-Siat7A (Abcam Int, Ab82821), anti-SialylTn (Abcam Int, Ab115957), anti-GAPDH (Santa Cruz Biotechnology, SC-32233), anti-ERK (Cell Signaling, 4372S), anti-phospho-ERK1/2 (Cell Signaling, 4370S), anti-connexin43 (71-0700, Zymed), anti-PARP (Santa Cruz Biotechnology, SC-8007), anti-cleaved-caspase-3 (Cell Signaling, 9661), anti-Bcl-2 (Abcam Int, Ab137751), anti-Bax (Abcam Int, Ab10813) and anti-KLF4 (Millipore 09-821). U0126 (Cell Signaling 9903) was selected as the MAPK/ERK inhibitor. Lentivirus transfection The lentiviral transduction particles for shRNA-mediated knockdown of Siat7A were purchased from GenePharma (Shanghai, China). The shRNA sequences targeting Siat7A were 5-GGAAGAGCCAGGACACAAAGA-3 (Siat7A-1) and 5-GCTACACGATGAAGGGATAAT-3 (Siat7A-2). The KLF4 shRNA was cloned using the PLKO.1 vector and the targeting sequence was 5-GCTCCATTACCAAGAGCTCAT-3. Stable knockdown cells were established as previously described [11]. The primers 5-GCGAATTCATGAGGCAGCCACCTG GCGA-3 and 5-GCGGATCCTTAAAAATGCCTCTTC ATGT-3 were used to generate plasmids encoding fulllength KLF4. The KLF4 cDNA was then amplified by RTPCR using total RNA from AC16 cells. To generate lentivirus expressing KLF4, HEK 293T cells grown on a 6-cm dish were transfected with 2 lg of pCDH-Flag-KLF4 or control vector, 1.5 lg of psPax2, and 0.5 lg of pMD2G. 24 h after the transfection, cells were cultured with DMEM

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Basic Res Cardiol (2015) 110:28 b Fig. 1 Siat7A expression was increased in the MI tissue and in the

damaged cardiomyocytes under hypoxic conditions. A Representative example of the recordings for ECG and blood pressure (BP) following the ligation of the left coronary artery (LCA). Inverted T wave and decreased BP were initiated after LCA ligation (n = 7). B Representative immunoblots of homogenate of the anteroapical left ventricular walls from sham and MI rats, probed with polyclonal Siat7A antibody and the immunoblotting analysis to show the significant difference in levels of Siat7A in the ventricles between the sham group (n = 6) and the MI group (n = 6). C Representative HE and IHC images of rat anteroapical left ventricular walls from sham rats (a and b) (n = 6) and MI rats (c and d) (n = 6). a and c show the morphology of rat cardiomyocytes. Positive immunoreactive signals for Siat7A expression are shown by brown deposits in the cell as the arrow indicates (b and d). D Representative HE and IHC images of human left ventricles from MI patients (n = 6). a and c show the morphology of human cardiomyocytes. Positive immunoreactive signals for Siat7A expression are shown by brown deposits in the cell as the arrow indicates (b and d). E AC16 cells were treated with the hypoxiamimetic agent CoCl2 at 200, 400, 600 and 800 lM for 5 and 10 h. The expression of Siat7A mRNA was examined by RT–PCR analysis. F Representative immunoblots of homogenate of AC16 cells treated with CoCl2 at 200, 400, 600 and 800 lM for 5 and 10 h and probed with Siat7A antibody. The uppermost band was exposed for the longest time and the middle band was exposed for a shorter time. G Representative confocal images of AC16 cells treated with saline (a) and CoCl2 (600 lM) for 5 h (b). Positive immunoreactive signals were concentrated in discrete spots in the cell (green). The scale bar in C and D represents 200 lm. The scale bar in G represents 25 lm. The results are shown as the mean ± SD of three independent experiments. All the results are representative of three independent experiments

containing 10 % FBS for an additional 24 h. The culture medium containing lentiviral particles was centrifuged at 1000g for 5 min. Viral particles collected in the supernatant were used for infection. To establish the stable cell line, the puromycin was used as a selection marker for the infected cells. The expression efficiency was evaluated by Western blot analysis. To generate AC16-Tet-on-KLF4 cells, the full-length KLF4 was cloned using the pTRIPZ vector and stable knockdown cells were established as above. RNA extraction and real-time RT-PCR Total RNA was isolated using Trizol (Ambion). One microgram of total RNA was used to synthesize cDNA using the PrimeScriptTM RT reagent kit (Takara, DRR037A) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR premix EX Taq (TaKaRa) and ROX, and analyzed with Stratagene Mx3000p (Agilent Technologies). Real-time PCR primer sequences were as follows: Actin 5-CTCCATCCTGGCC TCGCTGT-3 and 5-GCTGTCACCTTCACCGTTCC-3; KLF4 5-ACCTACACAAAGAGTTCCCATC-3 and 5-TG TGTTTACGGTAGTGCCTG-3; Siat7A for human 5-AGC TCTGTGACCAGGTGAGT-3 and 5-ATCCCTTCATCGT GTAGCCG-3; Siat7A for rat 5-GAGCCAAGCACAA

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GGGTTTC-3 and 5-GTGGGTGTCAGGGTCACGAT-3; Bcl-2 5-CCAAACAAATATGAAAAGGT-3 and 5-TGTG TGTGTTCTGCTTTA; 18 s ribosomal RNA 5-TGAGGCC ATGATTAAGAGGG-3 and 5-AGTCGGCATCGTTTAT GGTC-3. The signal intensities of Bcl-2, Siat 7A and KLF4 mRNA were normalized to intensities of actin or 18 s ribosomal RNA. The data were represented as mean ± SD from three independent experiments. Western blot analysis Total protein extracted from myocardial tissue or AC16 cell lysates was used for immunoblotting. In brief, cell lysates were clarified by centrifugation at 90009g for 10 min, and then the supernatant was collected. Protein concentration was determined using the BCA Protein Assay Kit (Pierce, USA). Total protein (30–60 lg) was separated on an 8 % or 10 % SDS-PAGE mini-gel, followed by transferring to a nitrocellulose (NC) membrane. After blocking with TTBS (50 mM Tris–HCl, 0.15 M NaCl, 0.1 % Tween-20, pH 7.5) containing 5 % fat-free dry milk overnight at 4 °C, the membrane was incubated with antibodies and an enhanced chemiluminescence (ECL) detection system (Amersham) was used to visualize the expression of these target proteins. Three samples from each group were analyzed and the results were quantified using the Gel-Pro 4.0 analyzer software. Flow cytometry assay The treated or untreated AC16 cells were gently trypsinized. After the single cell suspension was collected, the cells were washed with PBS (pH 7.4). Then the cells (1 9 106 cells/tube) were incubated with rabbit anti-SialylTn polyclonal antibody (1:100) for 1 h at 4 °C. Incubation of cells with rabbit IgG (1:100) was used as the isotype control. The unbound antibodies were removed by washing with PBS (pH 7.4) at 4 °C. FITC-conjugated anti-rabbit IgG (1:100) was then applied to incubate with the cells for 45 min at 4 °C. The unbound secondary antibody was discarded, and the cell mixture was adjusted to 150 ll with PBS for measurement in a FACScan flow cytometer. All the experiments were performed three times in duplicate. ChIP assay The AC16 cells treated with or without CoCl2 were crosslinked with 1 % formaldehyde for 10 min at room temperature. The ChIP assay was performed according to the manufacturer’s instructions using anti-KLF4 and the ChIP assay kit (Millipore, Merck KGaA, Darmstadt,

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Basic Res Cardiol (2015) 110:28 b Fig. 2 Increased Siat7A expression induced cardiomyocyte apoptosis

after MI in vivo. Scramble siRNA (n = 15) or Siat7A siRNA (n = 15) was injected in three regions of anterior left ventricular wall nearby the LAD and then the chest cavity was closed. The LAD ligation was performed 48 h later. At 3 h after the LAD ligation, the anteroapical left ventricular myocardium tissues of 6 rats in each group (Sham-siNC, Sham-siSiat7A, MI-siNC and MI-siSiat7A) were collected for the analysis. A Representative example of the recordings for ECG and BP following the LCA ligation from MI-siNC group rats (a and b) or MI-siSiat7A group rats (c and d). Elevated ST stage (b) and Inverted T wave (d) in ECG, as well as decreased BP were initiated after LCA ligation. B The analysis of Siat7A mRNA at the infarction site. C The analysis of BcL-2 mRNA at the infarction site. D Representative HE and IHC images at the infarction site of rat anteroapical left ventricular wall from Sham-siNC, Sham-siSiat7A, MI-siNC and MI-siSiat7A group. The uppermost panels display HE staining showing the morphology of cardiomyocytes. The middle and lowest panels display the IHC images for cleaved-caspase-3 and Siat7A, respectively. Positive immunoreactive signals for cleavedcaspase-3 and Siat7A expression are shown by brown deposits in the cell as the arrows indicate. E The number of cleaved-caspase-3positive cells at the infarction site of rat anteroapical left ventricular wall from Sham-siNC, Sham-siSiat7A, MI-siNC and MI-siSiat7A group. Each dot represents one cleaved-caspase-3-positive cell counted from six rats in each group and two random fields (9400) per subject. The scale bar in D represents 50 lm. **p \ 0.01 and ***p \ 0.001, respectively

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were seeded in a 96-well black culture plate and incubated for 24 h at 37 °C. Next, the cells were incubated with CoCl2 as indicated. Then 100 lL of caspase-3 reagent was added to each well following the incubation. Finally, the fluorescence intensity (at 370–425 nm excitation and 490–530 nm emission wavelengths) was measured using a SpectraMAX M5 spectrophotometer (Molecular Devices). Immunofluorescence and confocal microscopy The cells grown on the coverslips were fixed in 4 % paraformaldehyde for 30 min at 4 °C. After blocking with 3 % bovine serum albumin for 2 h at 37 °C, rabbit polyclonal antibody directed against Siat7A (1:100) was incubated with the cells overnight at 4 °C. The cells incubated with mouse IgG (Biolegend, CA, USA 1:100) served as isotype controls for Siat7A. The image was captured using a confocal laser scanning microscope system (Leica, SP5II) after incubating the cells for 1 h with FITC-conjugated goat anti-mouse IgG. Reconstructed projection images were obtained from serial optical sections recorded at an interval of 0.5 lm. Statistical analysis

Germany). Anti-rabbit IgG was used as a control. The bound DNA fragments were eluted and amplified by PCR using the following primer pairs: 5-TAAGCAGTGGATACGAATGC-3 and 5-GCTTGGGGTCCAGATCCAAC3 (binding site 1); 5-CCTTGAGGAAGGTGGTTTAA-3 and 5-GTAGCACCTACGAGAAATCA-3 (binding site 2). PCR products were separated on a 2 % agarose gel by gel electrophoresis. Dual-luciferase reporter assay AC16 or 293T cells were transiently transfected with the indicated luciferase reporter together with either FlagKLF4/Flag-KLFDTA/D DBD or control vector. Renilla plasmid was also included in each transfection to normalize the transfection efficiency. Firefly and Renilla luciferase activities were analyzed by Dual-Luciferase Reporter Assay system according to the manufacturer’s instructions (Promega, Madison, WI, USA). The relative luciferase activities were calculated by normalizing the Firefly luciferase activity to Renilla luciferase activity. The data were represented as the mean ± SD of three independent experiments. Cellular caspase-3 activity assay Caspase-3 activity was analyzed using a Caspase-3 Activity Assay Kit (Roche) according to the manufacturer’s instructions. Briefly, the AC16 cells (5 9 103 cells/well)

All data were presented as mean ± SEM. Statistical evaluations were achieved by ANOVA followed by a post hoc Tukey test. p \ 0.05 was considered statistically significant.

Results Siat7A expression was increased in damaged cardiomyocytes after MI To evaluate the status of Siat7A expression in the heart, we first explored Siat7A expression in the anteroapical left ventricles of a cohort of rats after myocardium infarction (Fig. 1A–C). Figure 1A shows representative changes in ECG and BP following LCA ligation. As shown in this case, ischemia occurred as indicated by the inverted T wave and BP substantially decreased after LCA ligation. Siat7A expression at the protein level increased in the anteroapical left ventricular wall of MI rats compared with sham rats, which was assayed by Western blotting and immunohistochemistry (Fig. 1B, C, respectively). The increase in Siat7A expression was substantially higher in the cytoplasm of cardiomyocytes injured from myocardium infarction than that of normal cardiomyocytes (Fig. 1C). To determine the distribution of Siat7A during myocardial ischemia in humans, HE staining was performed in parallel to immunohistochemical analysis of the left ventricular

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tissues stained with anti-Siat7A antibody. Figure 1D shows images of HE staining and immunohistochemical analysis of the ventricular tissue of two MI patients representatively. Figure 1D-a shows the morphology of the uninjured cardiomyocytes. As shown in Fig. 1D-c, cardiomyocytes

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were seriously injured following MI. This is indicated in multiple areas where cardiomyocytes were replaced with the formation of fibrosis. Siat7A expression was significantly stronger in the cytoplasm of the injured cardiomyocytes (Fig. 1D-d), compared to the uninjured

Basic Res Cardiol (2015) 110:28 b Fig. 3 Siat7A enhanced cardiomyocyte apoptosis by inhibiting ERK

phosphorylation under hypoxic conditions. A Representative immunoblots of homogenate of AC16 cells treated with CoCl2 at 200, 400, 600 and 800 lM for 5 and 10 h and probed with PARP antibody. Arrows indicate position of cleaved PARP. B AC16 cells were treated with CoCl2 at 200, 400, 600 and 800 lM for 5 and 10 h, after which Caspase-3 activity was assayed with a spectrophotometer. C Siat7Adeleted AC16 cells were generated by transduction of lentivirus carrying Siat7A interfering RNAs (shRNAs). The cells were then treated with CoCl2 (600 lM) for 10 h, and the level of Siat7A mRNA was measured using RT–PCR analysis. D Representative immunoblots of homogenate of Siat7A-deleted AC16 cells treated with CoCl2 (600 lM) for 10 h and probed with Siat7A antibody. E Representative immunoblots of homogenate of Siat7A-deleted AC16 cells treated with CoCl2 (600 lM) for 10 h and probed with PARP antibody. F Caspase-3 activity was assayed with Siat7Adeleted AC16 cells treated by CoCl2 (600 lM) for 10 h. G Representative immunoblots of homogenate of Siat7A-deleted AC16 cells treated with CoCl2 (600 lM) for 10 h and probed with ERK1/2 and phosphorylated ERK1/2 antibodies. H Siat7A-deleted AC16 cells were treated with U0126 (MAPK/ERK12 inhibitor), and the levels of cleaved PARP and phosphorylated ERK1/2 were measured by immunoblotting. The results are shown as the mean ± SD of three independent experiments. All the results are representative of three independent experiments. **p \ 0.01 and ***p \ 0.001, respectively

cardiomyocytes (Fig. 1D-b). Given that MI induces a shortage in the oxygen supply to the cardiomyocytes [14, 34], we examined the effect of hypoxia on Siat7A expression in vitro. Real-time PCR and immunoblotting as well as confocal microscopy analysis were performed using the cultured human cardiomyocyte cell line, AC16 (Fig. 1E–G). As shown in Fig. 1E, F, Siat7A expression increased significantly at both the mRNA and protein levels in a time- and concentration- dependent manner under the chemically induced hypoxic conditions achieved through CoCl2 treatment. Also shown in Fig. 1G, Siat7A expression increased specifically in the cytoplasm of the AC16 cells after the treatment with 600 lM CoCl2 for 5 h, in contrast to the Siat7A baseline expression after the treatment with physiological saline for 5 h. Similar Siat7A increase and hypoxia-inducible transcription factor-1a (HIF-1a) expression were identified in the AC16 cells through incubating with 1 % oxygen for 36 h (Supplementary Figure 1A, B). Together, these results indicate that Siat7A expression increased in the damaged cardiomyocytes following MI. Siat7A expression induced cardiomyocyte apoptosis after MI To determine the effects of increased Siat7A expression on the cardiomyocytes after MI in vivo, we locally delivered siRNA against Siat7A into the myocardium and first checked the effects of increased Siat7A on

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connexin43 expression in ischemic myocardium. It was reported that connexin43 is the predominant protein responsible for composing gap-junction channels and implicated in the electrical coupling of excitable ventricular tissue. Immunodetectable connexin43 is obviously reduced in the damaged cardiomyocytes caused by acute myocardial infarction [2, 22]. Thereby, after delivery of Siat7A siRNA into the anterior left ventricular wall, which was followed by the treatment of LAD ligation for 3 h, we identified Siat7AmRNA was significantly depressed (Fig. 2B) and then detected the content of connexin43 in the myocardium of anteroapical left ventricular wall. As shown in Supplementary Figure 2A, connexin43 was remarkably reduced in the ischemic myocardium without the delivery of Siat7A siRNA. The knockdown of Siat7A by injection of Siat7A siRNA into the myocardium 48 h prior to the LAD ligation could inhibit the depression of connexin43. To further verify whether or not siRNA against Siat7A could inhibit cardiomyocyte apoptosis after LAD ligation, the expressions of BcL-2 mRNA and cleaved-caspase 3 protein in the myocardium tissue of anteroapical left ventricular wall were analyzed. According to the report that characteristic signs of apoptosis are identified in the ischemic myocardium after 2.75 h of LAD ligation [7], we checked the expression of cleaved-caspase 3 in the anteroapical left ventricular wall after 0, 1, 2.5 and 3 h of LAD ligation. As shown in Supplementary Figure 2B, the expression of cleaved-caspase 3 could be clearly found in the ischemic myocardium after 3 h of LAD ligation. Figure 2A-a, b shows representative changes in ECG and BP following LCA ligation in the MI-siNC group rats. Figure 2A-c, d shows representative changes in ECG and BP following LCA ligation in the MI-siSiat7A group rats. As shown in this case, ischemia, indicated by the ST elevation or inverted T wave, occurred and BP was decreased after LCA ligation. Figure 2B and C show that Siat7A was significantly higher and BcL-2 was significantly lower in the MI-siNC group rats than the MI-siSiat7A group rats. In addition, 2–4 myocardiocytes expressing cleaved-caspase3 positivity paralleling to the evidently positive Siat7A expression per field (4009) at light microscopy could be detected at the infarction site in the MI-siNC group rats. Almost none of the cleaved-caspase-3-positive cells and positive Siat7A expression could be detected at the infarction site in the MI-siSiat7A group rats. Six rats from each group and two random fields per subject (Fig. 2D, E) had been examined in the experiments. These in vivo data have clearly shown that the expression of Siat7A was increased in the infracted myocardium and induced the cardiomyocyte apoptosis.

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Fig. 4 Klf4 upregulated Siat7A expression in the cardiomyocytes. A, B AC16- Tet-on-KLF4 cells were generated and the levels of Klf4 and Siat7A were analyzed by RT-PCR and immunoblotting. C AC16 cells were treated with CoCl2 (600 lM) for 5 and 10 h, and the mRNA levels of Klf4 and Siat7A were then measured by RT-PCR analysis. D AC16 cells were treated with CoCl2 (600 lM) for 5 and 10 h, and the protein levels of Klf4 and Siat7A were then measured by immunoblotting. E AC16 cells were transfected by shRNAKlf4

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and then treated with CoCl2 (600 lM) for 10 h. The mRNA levels of Klf4 and Siat7A were measured by RT-PCR analysis. F AC16 cells were transfected by shRNAKlf4 and then treated with CoCl2 (600 lM) for 10 h. The protein levels of Klf4 and Siat7A were measured by immunoblotting. The results are shown as the mean ± SD of three independent experiments. All the results are representative of three independent experiments. **p \ 0.01 and ***p \ 0.001, respectively

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Siat7A promoted cardiomyocyte apoptosis via inhibition of ERK1/2 activity under hypoxic conditions To determine the mechanisms for Siat7A-involved cardiomyocyte apoptosis after MI, we measured the effect of changes in Siat7A expression on cardiomyocyte apoptosis under hypoxic conditions in vitro. As shown in Fig. 3A, B, poly(ADP-ribose)polymerase (Parp) cleavage and caspase 3 activity were increased in the AC16 cells following the treatment with CoCl2 in a concentration- and time- dependent manner. Coincidentally, B cell lymphoma 2 (Bcl2) was significantly decreased and Bcl-2-associated X protein (Bax) was significantly increased in the AC16 cells following the treatment with CoCl2 (Supplementary Figure 3A, B). Transfection of shRNA Siat7A resulted in the knockdown of Siat7A (Fig. 3C, D) and significantly inhibited not only the Parp cleavage and caspase 3 activity (Fig. 3E, F) but also the depression of Bcl-2 and the elevation of Bax (Supplementary Figure 3C, D) induced by the hypoxic treatment. To identify the effect of increased Siat7A expression on its activity in ischemic cardiomyocytes, we detected the induction of the Sialyl-Tn antigen which is the special substrate of Siat7A in the AC16 cells under hypoxic conditions. As shown in Supplementary Figure 4A, B, the expression of Sialyl-Tn antigen was significantly increased in the AC16 cells following the treatment with CoCl2, which was significantly inhibited due to the knockdown of Siat7A in the AC16 cells. These data indicate that Siat7A might exert its function via the Sialyl-Tn antigen. Several studies have reported that the extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2) cascade participates in cardiomyocyte apoptosis following MI [23, 37, 45]. Therefore, we examined the impact of the increased Siat7A expression on ERK activity in the ischemic cardiomyocytes. We found that the knockdown of Siat7A increased the phosphorylation of ERK 1/2 in the AC16 cells and decreased cardiomyocyte apoptosis under hypoxic conditions. When the knockdown of Siat7A was combined with pharmacological ERK inhibition via U0126 treatment, the phosphorylation of ERK 1/2 was inhibited and increased Parp cleavage was present in the hypoxic AC16 cells (Fig. 3G, H). Klf4 upregulated Siat7A expression in the cardiomyocytes After examining multiple transcription factors identified as potential candidates in the regulation of Siat7A expression, we found that Klf4 is associated with Siat7A expression (Supplementary Figure 5A–C). Transfection of Klf4 significantly increased Siat7A expression in AC16 cells under

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normoxic conditions. Moreover, overexpression of Klf4 resulted in increased Siat7A expression in a time-dependent manner (Fig. 4A, B). The cardiomyocyte cell line AC16 showed consistently increased Klf4 expression and Siat7A expression at both the mRNA and protein levels under hypoxic conditions (Fig. 4C, D). The knockdown of Klf4 through transfection of shRNA Klf4 significantly decreased Klf4 expression, and therefore significantly decreased Siat7A expression, in AC16 cells, even under hypoxic conditions (Fig. 4E, F). In summary, these data indicate that Klf4, as a transcription factor, may regulate Siat7A expression in cardiomyocytes. Klf4 transactivated Siat7A gene expression by binding to the Siat7A promoter region (nt 2655 to 2636 bp) Since Klf4 was found to regulate Siat7A expression, we constructed two Siat7A promoter segments for Klf4 binding sites using a transcription factor search website (TFSEARCH) (Fig. 5A). To determine that Klf4 binds to the putative Klf4 element at the Siat7A promoter in vivo, we performed chromatin immunoprecipitation (ChIP) to detect the relevant DNA–protein interactions. As shown in Fig. 5B, the Siat7A promoter in the nt -655 to -636 region was detected using an anti-Klf4 antibody. A luciferase assay showed that mutagenesis of either the Klf4 or the Siat7A promoter sequence (Fig. 5C), or the knockdown of Klf4, substantially decreased Siat7A promoter activity under both normal (Fig. 5D, E) and hypoxic conditions (Fig. 5F, G). These results show that Klf4 transactivated Siat7A gene expression by binding to the Siat7A promoter region (nt -655 to -636 bp). Klf4 was increased in ischemic myocardium and promoted cardiomyocyte apoptosis via regulation of Siat7A expression under hypoxic conditions To investigate the role of Klf4 in cardiomyocyte apoptosis induced by MI, we transfected Klf4 into AC16 cells and treated the cells with 600 lM CoCl2 for 10 h. After the treatment with CoCl2, we found that Parp cleavage was significantly increased, while phosphorylation of ERK1/2 was significantly decreased in the cardiomyocytes overexpressing Klf4 under hypoxic conditions (Fig. 6B). However, when Klf4 was transfected into Siat7A-deficient AC16 cells, both the Parp cleavage and the phosphorylation of ERK1/2 showed no changes comparing to the Siat7A-deficient AC16 cells co-transfected by a vector gene (Fig. 6C). Knockdown of Klf4 increased the phosphorylation of ERK1/2 and inhibited Parp cleavage, but when combined with the ERK inhibitor U0126, the knockdown

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of Klf4 inhibited phosphorylation of ERK1/2 and increased cleaved Parp in the hypoxic AC16 cells (Fig. 6A). To further evaluate the association of Klf4 with Siat7A expression in vivo, we examined the status of Klf4 and Siat7A expression in the same area of cardiomyocytes from both MI rats and patients through immunohistochemical analysis. As shown in Fig. 6D and E, Klf4 expression was also increased in the injured cardiomyocytes of both MI rats (Fig. 6D-e, f) and MI patients (Fig. 6E-e, f), it is consistent with increased Siat7A expression. These data suggest that Klf4 and Siat7A expressions were increased in parallel in the ischemic ventricles, and Klf4 promoted cardiomyocyte apoptosis through Siat7A expression under hypoxic conditions. The above effects were exerted through the ERK 1/2 signaling pathway. To further clarify if the inhibition of ERK1/2 activity affects the Klf4 transcription activity or Siat7A expression, we inhibited the ERK1/2 activity by adding U0126 into the medium prior to treating AC16 cells with 600 lM CoCl2 for the indicated time. As shown in Supplementary Figure 6, although pharmacological ERK inhibition inhibited the phosphorylation of ERK 1/2, it did not affect the induction of both KLF4 and Siat7A in the AC16 cardiomyocytes following hypoxic treatment. Therefore, it is approved that there was no effect of inhibition of ERK1/2 activity on the Klf4-regulated Siat7A expression.

Discussion In this study, we demonstrated that Klf4 and Siat7A were both increased in the damaged cardiomyocytes following MI. The knockdown of Siat7A reduced cardiomyocyte apoptosis due to MI both in vivo and in vitro. Increased Klf4 and Siat7A expression promoted cardiomyocyte apoptosis via the inhibition of the MAPK/ERK1/2 signaling pathway under hypoxic conditions. Klf4, as a transcription factor, directly bound to the Siat7A promoter and upregulated Siat7A expression. The knockdown of Klf4 suppressed the Siat7A expression and cardiomyocyte apoptosis by increasing the MAPK/ERK1/2 activation under hypoxic conditions. The overexpression of Klf4 increased cardiomyocyte apoptosis and decreased the MAPK/ERK1/2 activation under hypoxic conditions, which was reversed by deletion of Siat7A. These findings as shown in the schematic representation (Fig. 7) provide the basis for a therapeutic strategy of inhibiting cardiomyocyte apoptosis following MI through the Klf4Siat7A-MAPK/ERK1/2 pathway. Given that Siat7A mRNA has been found to be consistently overexpressed in the left ventricles of the spontaneously hypertensive rats regardless of genetic background, we examined the Siat7A expression in the

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Basic Res Cardiol (2015) 110:28 Fig. 5 Klf4 transactivated Siat7A gene expression by binding to the c Siat7A promoter region (nt -655 to -636 bp). A Schematic illustration of the putative KLF4-binding region located at 1225–1214 and 655–635 bp upstream of the Siat7A translational start site. B ChIP analysis showed binding of KLF4 to the putative binding sites. Input and immunoprecipitate from AC16 cells with or without CoCl2 treatment were amplified by PCR with primer pairs complementary to the Siat7A promoter. C Schematic illustration of PGL3-basic-based reporter constructs used in luciferase assays to examine the transcriptional activity of the binding site 1. The red sequences indicate the mutated nucleotide residues. D 293T cells were transfected with pGL3-Siat7A WT as well as flag-KLF4, flagKLF4DTA (deletion of the transcription activity domain), or flagKLF4DDBD (deletion of the DNA binding domain). 24 h after transfection, transcription activity was determined with dual-luciferase assays. E 293T cells were transfected with pGL3-Siat7A WT or pGL3-Siat7A mut as well as increasing amounts of Flag-KLF4. 24 h after transfection, transcription activity was determined by luciferase assays. F AC16 cells were co-transfected with the indicated reporter constructs and Renilla luciferase plasmid. 24 h after transfection, cells were treated with CoCl2 for the indicated times, and the transcription activity was determined by luciferase assays. G AC16 cells with and without KLF4 knockdown by shRNA were cotransfected with the indicated reporter constructs and Renilla luciferase plasmid. 24 h after transfection, cells were treated with CoCl2 for the indicated times and the transcription activity was determined by luciferase assays. All the results are representative of three independent experiments. **p \ 0.01 and ***p \ 0.001, respectively

ischemic left ventricles of both rats and humans, and in the cardiomyocyte cell line AC16 under hypoxic conditions [4]. It was found that the Siat7A expression was consistently increased in damaged cardiomyocytes following MI or chemically induced hypoxic conditions after CoCl2 treatment. In addition, our supplementary data showed that the Siat7A expression was also increased in the cardiomyocytes with physically induced hypoxia by applying 1 % oxygen for 36 h. Moreover, both chemical and physical hypoxic treatments can similarly induce HIFa expression, which confirmed that CoCl2 can act as a hypoxiamimetic agent and recapitulate the hypoxic response in the ischemic cardiomyocyte [9]. It has been reported that MI induces a shortage in the oxygen supply to the cardiomyocytes, therefore, cultured cardiomyocytes with hypoxic treatment in vitro were selected to mimic the cardiomyocyte damage caused by MI in vivo [2, 14, 34, 39]. It is well known that multifactors, such as neurohumoral factors, are involved in the pathophysiological process of cardiomyocyte death after MI, we will study the effects of some neurohumoral factors on the Siat7A-induced cardiomyocyte apoptosis in our future research [21]. The increasing Siat7A expression was consistent with the increasing extent of cardiomyocyte apoptosis induced by different concentrations of CoCl2. Downregulation of Siat7A expression significantly suppressed cardiomyocyte apoptosis resulting from MI. These results indicated that increasing Siat7A accelerated cardiomyocyte apoptosis

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after MI. Previous studies have identified the activity of Siat7A in carcinoma cells and have shown that the overexpression of Siat7A increases gastric carcinoma cell apoptosis [28, 36]. Siat7A is responsible for mediating the transfer of sialic acid via an a2,6-linkage to the N-acetylgalactosamine residue on O-linked oligosaccharides of glycoproteins or glycolipids [28]. It has been previously reported that the level of sialic acid is significantly increased in the heart tissue and serum of patients with heart failure secondary to MI. Thus, the level of sialic acid is relevant to the risk of MI [24]. We found that the Sialyl-Tn antigen synthesized by Siat7A was significantly increased in the cardiomyocytes under hypoxic condition. The knockdown of Siat7A effectively inhibited the expression of the Sialyl-Tn antigen in the hypoxia-treated cardiomyocytes. Our data supported that Siat7A is responsible for the synthesis of the Sialyl-Tn antigen in the cardiomyocytes. Klf4 is a transcription factor associated with diverse and cell type-specific responses to divergent pathophysiological stresses. In the hypoxic lung or endothelial cell, Klf4 expression is decreased [10, 17]. Conversely, hypoxic stimulus increases Klf4 expression in human embryonic stem cells and astrocytes [30, 35]. Regarding the effects of different pathophysiological stresses on Klf4 expression in the heart, it has been shown that Klf4 is induced by either hypertrophic stimulus or by oxidative stress in cardiomyocytes [6, 27, 44]. In the present study, we also found that Klf4 expression was increased in cardiomyocytes either following MI or under hypoxic conditions. Interestingly, Siat7A increased in parallel to Klf4 specifically in damaged cardiomyocytes after MI. Similar to the role of Siat7A in the hypoxic cardiomyocyte, overexpressed Klf4 also accelerated cardiomyocyte apoptosis under hypoxic conditions. It has been reported that glycosyltransferases are regulated by multiple transcriptional networks. The transcription factors have major impacts on glycosyltransferase expression, structure, and activity [33]. We transfected Klf4 into unstressed cardiomyocytes, and surprisingly found that Siat7A expression was significantly upregulated. In the case of Klf4 knockdown, induction of Siat7A was significantly inhibited in cardiomyocytes under hypoxic conditions. Additional results from ChIP and luciferase assays compellingly suggested that Siat7A was directly bound and transactivated by Klf4. To clarify the association of Klf4 with Siat7A in the process of cardiomyocyte apoptosis following MI, we transfected Klf4 into Siat7Adeficient cardiomyocytes and then subjected the cells to hypoxic conditions. We found that cardiomyocyte apoptosis was suppressed due to the deletion of Siat7A. These

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Basic Res Cardiol (2015) 110:28 Fig. 6 KLF4 was increased in ischemic myocardium and promoted c cardiomyocyte apoptosis by regulating Siat7A expression under hypoxic conditions. A Klf4-deleted AC16 cells were treated with MAPK/ERK inhibitor (U0126) and then treated with CoCl2 (600 lM) for 10 h. The levels of cleaved PARP and phosphorylated ERK were measured by immunoblotting. B KLF4-overexpressed AC16 cells were generated by transduction of lentiviruses expressing KLF4 and then treated with CoCl2 (600 lM) for 10 h. The levels of cleaved PARP, phosphorylated ERK, and Klf4 were measured by immunoblotting. C Lentiviruses expressing KLF4 were transfected into the Siat7A-deleted AC16 cells, and the cells were then treated with CoCl2 (600 lM) for 10 h. The levels of cleaved PARP, phosphorylated ERK, Siat7A and Klf4 were measured by immunoblotting. D Representative HE and IHC images of rat anteroapical left ventricular wall from sham rats (a, b and c) (n = 6) and MI rats (d, e and f) (n = 6). a and d show the morphology of cardiomyocytes. Positive immunoreactive signals for Klf4 and Siat7A expression are shown by brown deposits in the cell as the arrows indicate (e and f, respectively). E Representative HE and IHC images of human left ventricles from MI patients. a and d show the morphology of cardiomyocytes from MI and non-MI regions, respectively. Positive immunoreactive signals for Klf4 and Siat7A expression are shown by brown deposits in the cell as the arrows indicate (e and f, respectively). The scale bar in D and E represents 100 lm. All the results are representative of three independent experiments

results show that Klf4 promoted cardiomyocyte apoptosis through transactivating Siat7A expression in ischemic myocardium. The ERK1/2 signaling cascade plays a key anti-apoptotic regulatory role in stress-induced cardiomyocyte apoptosis [23, 37, 45]. In the present study, we found that inhibition of ERK1/2 activity aggravated cardiomyocyte apoptosis under hypoxic conditions, which indicates that elevated phosphorylation of ERK1/2 protected cardiomyocytes from apoptosis induced by MI. Moreover, we found that deletion of either Siat7A or Klf4 increased the expression of phosphorylated ERK1/2 and inhibited cardiomyocyte apoptosis under hypoxic conditions. On the other hand, overexpression of Klf4 resulted in decreased expression of phosphorylated ERK1/2 and increased cardiomyocyte apoptosis under hypoxic conditions, which was reversed by the knockdown of Siat7A. However, inhibition of the ERK1/2 signaling pathway did not affect the expressions of Klf4 and Siat7A. Notably, our results show that Klf4 upregulated Siat7A and promoted cardiomyocyte apoptosis via inhibition of ERK1/2 activity under hypoxic conditions. Our present study identified a novel route that contributes to cardiomyocyte apoptosis initiated by MI. Klf4 expression and activity were increased under hypoxia condition, which then bound the promoter of Siat7A and directly transactivated Siat7A expression. The increasing Siat7A suppressed the activation of the downstream kinase

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7. 8.

9. Fig. 7 Schematic representation showing the mechanism for the Siat7A-induced cardiomyocyte apoptosis following MI. Myocardial infarction (MI) results in the shortage in oxygen supply to myocardium, under which condition the transcriptional activity of Klf4 was increased. Binding to the Siat7A promoter region, Klf4 transactivated Siat7A gene. The Siat7A expression and activity was elevated, thus resulting in the increased Sialyl-Tn antigen specially synthesized by Siat7A. Thereby ERK1/2 signaling pathway was depressed, which induced the cardiomyocyte apoptosis indicated by the decrease in anti-apoptotic protein Bcl-2 expression, simultaneously the increase in caspase-3 activity and the cleavage of Parp

ERK1/2 and resulted in cardiomyocyte apoptosis. These findings have the potentials that may lead to a therapeutic target for regulating MI-induced cardiomyocyte loss. Acknowledgments This work is supported by National Natural Science Foundation of China Research Grant (No. 81200155 to Dongmei Zhang, No. 81402260 to Chuanchun Han, and No. 81372853 to Pixu Liu), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) to Pixu Liu, Liaoning Provincial Climbing Scholars Supporting Program of China to Pixu Liu, and Liaoning Provincial Natural Science Foundation of China (No. 2014023011) to Pixu Liu. Conflict of interest The authors have nothing to disclose. On behalf of all authors, the corresponding author states that there is no conflict of interest.

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