CLS-08375; No of Pages 7 Cellular Signalling xxx (2015) xxx–xxx
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Article history: Received 27 September 2014 Received in revised form 1 December 2014 Accepted 17 December 2014 Available online xxxx
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Keywords: 14-3-3η protein Diabetes mellitus Cardiomyocyte apoptosis Cytochrome c Apoptosis signal-regulating kinase-1 High fat diet
Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX 77030, USA c J.K.K. Nattraja College of Pharmacy, Natarajapuram, Komarapalayam, Namakkal District, 638183 Tamil Nadu, India d Department of Pharmacology, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia e Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan
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There is a definite cardioprotective role for 14-3-3η protein against pressure overload induced cardiac hypertrophy and streptozotocin induced cardiac dysfunction in type 1 diabetes mellitus (DM). But it is not conclusive whether it has any influence on mitochondrial mediated cardiomyocyte apoptosis in type 2 DM. In order to test this hypothesis, we have used C57BL6/J (WT) mice with cardiac specific dominant negative mutation of 14-3-3η protein (DN 14-3-3η). Both WT and DN 14-3-3η mice were fed with high fat diet (HFD) for 12 weeks. Their body weight and blood glucose levels were measured weekly and compared with standard diet (SD) fed mice. By the end of 12 weeks, echocardiography was performed. Frozen myocardial sections were prepared to stain the apoptotic cardiomyocytes using TUNEL staining. DN 14-3-3η mice fed with HFD showed cardiac dysfunction as identified by the decreased fractional shortening and ejection fraction and increased cardiomyocyte apoptosis in TUNEL staining. Western blotting analysis using mitochondrial fraction of the ventricular tissue homogenates showed a significant reduction in the level of cytochrome c suggesting its translocation into cytoplasm, which may be crucial in inducing cardiomyocyte apoptosis. In addition, DN 14-3-3η mice depicted significantly increased levels of NADPH oxidase subunits suggesting oxidative stress, a significant reduction in phospho apoptosis signal-regulating kinase-1 (p-Ask-1) and increase in Ask-1 and phospho c-Jun N-terminal kinase (p-JNK) levels suggesting activation of Ask-1/JNK signaling. These results suggest that 14-3-3η has a protective role against mitochondria mediated cardiomyocyte apoptosis with the involvement of Ask-1/JNK signaling during HFD induced type 2 DM. © 2015 Published by Elsevier Inc.
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N-terminal kinase (JNK) or p38 mitogen activated protein kinase (MAPK) [3,4]. Evidences are available for the relation between oxidative stress, apoptosis and 14-3-3 protein as it has been suggested that oxidative stress activates Ask1 by dissociating its inhibitor, 14-3-3 protein, from Ask1 Ser-967 in Cos7 cells [5]. 14-3-3 protein and thioredoxin are reported to limit Ask1 activity by guarding the C-terminal and N-terminal of Ask1 kinase, respectively [6]. Thus modulation of apoptosis may be the central factor in the cardioprotective role of 14-3-3 proteins. We have previously demonstrated that 14-3-3η protein acts as an endogenous cardioprotector and limits the development of diabetic cardiomyopathy by limiting myocardial apoptosis, hypertrophy, fibrosis, and endothelial dysfunction via inhibition of Ask1 activation after induction of experimental diabetes [7–10]. In addition we have reported some of the regulatory effects of 14-3-3η protein on pressureoverload induced cardiac hypertrophy and streptozotocin-induced type 1 diabetes mellitus (DM) [11]. But it is not conclusive whether 14-3-3η protein can influence cardiomyocyte apoptosis during obesity
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Remya Sreedhar a, Somasundaram Arumugam a, Rajarajan A. Thandavarayan b, Vijayasree V. Giridharan c, Vengadeshprabhu Karuppagounder a, Vigneshwaran Pitchaimani a, Rejina Afrin a, Shizuka Miyashita a, Mayumi Nomoto a, Meilei Harima a, Narasimman Gurusamy d, Kenji Suzuki e, Kenichi Watanabe a,⁎
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Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis
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1. Introduction
The tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteins are a family of molecular chaperones commonly referred to as 14-3-3 proteins. The family consists of seven transcripts in mammals: 14-3-3β (YWHAB), 14-3-3γ (YWHAG), 14-3-3ε (YWHAE), 14-3-3ζ (YWHAZ), 14-3-3η (YWHAH), 14-3-3θ (YWHAQ) and 14-33σ also known as stratifin (SFN) [1]. 14-3-3 proteins are intracellular phosphoserine-binding adapter molecules belonging to a class of highly conserved proteins involved in regulating apoptosis, adhesion, cellular proliferation, differentiation, survival, and signal transduction pathways [2]. Along with apoptosis signal regulating kinase (Ask)-1 it can modulate cellular apoptosis, inflammation, differentiation and survival and most of these responses are mediated possibly via either c-Jun
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⁎ Corresponding author. Tel.: +81 250 25 5267; fax: +81 250 25 5021. E-mail address: [email protected] (K. Watanabe).
http://dx.doi.org/10.1016/j.cellsig.2014.12.021 0898-6568/© 2015 Published by Elsevier Inc.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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Transgenic DN 14-3-3η mice were generated as described previously [8]. Briefly, the coding region of human DN (R56A and R60A) 14-3-3η cDNA with a 5′-Myc-1 epitope tag was subcloned into a vector containing the α-myosin heavy chain promoter and an SV40 polyadenylation site. Linearized DNA was injected into the pronuclei of one-cell C57BL/ 6 XSJL embryos at the Neuroscience Transgenic Facility at Washington University School of Medicine. Progeny were backcrossed into the C57BL6 genetic background and were analyzed by polymerase chain reaction to detect transgene integration using mouse-tail DNA as template. Age matched C57BL6/J mice (obtained from Charles River Japan Inc., Kanagawa, Japan) were used as wild type (WT) controls. Mice were maintained with free access to water and chow throughout the period of study, and animals were treated in accordance with the Guidelines for Animal Experimentation of our institute. All animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.
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2.2. Experimental design
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Both WT and DN 14-3-3η mice were given high fat diet 32 (HFD) (energy—507.6 Kcal/100 g) (CLEA Japan, Inc.) or standard diet (SD) (energy—342.7 Kcal/100 g) from the start of the study and continued until 12 weeks. Body weight and blood glucose levels (tail vein bleeding using NIPRO blood glucose monitoring system) were measured weekly.
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2.3. Transthoracic echocardiographic analysis
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Two-dimensional echocardiography studies were performed in anesthetized mice (pentobarbital, 50 mg/kg, i.p.) to evaluate cardiac function using an echocardiographic machine with 7.5, 12 and 30 MHz transducers linked to an ultrasound system (VISUALSONICS, Vevo770, Japan). The short-axis view of the left ventricle (LV) was recorded to measure the LV dimension in systole (LVDs) and diastole (LVDd), percent ejection fraction (%EF) as well as the percent fractional shortening (%FS). Hearts were harvested for analysis from control and diabetic mice. The ventricle portion was quickly dissected and cut into two parts. One part was immediately transferred into liquid nitrogen and then stored at −80 °C for protein analysis. The other part was either stored in 10% formalin or stored at −80 °C after the addition of TissueTek OCT compound (Sakura Co. Ltd., Tokyo, Japan) for histopathological analysis [10].
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Protein lysate was prepared from heart tissue as described previously [8]. The total protein concentration in samples was measured by the bicinchonic acid method. For Western blotting experiments, 50 mg of total protein was loaded and proteins were separated by SDS-PAGE (200 V for 40 min) and electrophoretically transferred to nitrocellulose filters (semidry transfer at 10 V for 30 min). Filters were blocked with 5% non-fat dry milk in Tris-buffered saline (20 mM Tris, pH 7.6, 137 mM NaCl) with 0.1% Tween 20, washed, and then incubated with primary antibody. Primary antibodies employed included: rabbit polyclonal anti-apoptosis signal-regulating kinase (Ask-1) and antiphospho (Ser-967) Ask-1, rabbit polyclonal anti-p38 MAPK and antiphospho (Thr-180/Tyr-182) p38 MAPK, rabbit polyclonal anti-JNK and anti-phospho JNK, rabbit polyclonal anti-cytochrome c (Cell Signaling Technology Inc., MA, USA), rabbit polyclonal anti-p22phox, antip47phox, goat polyclonal anti-p67phox and goat polyclonal antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology Inc., CA, USA). After incubation with the primary
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quantified in a blinded manner. Only nuclei that were clearly located in cardiac myocytes were considered [8]. Similar procedure was performed but without counterstaining with diaminobenzidine (DAB) using another set of samples to observe via a fluorescence microscope (LSM700, Carl Zeiss, Japan). Similarly, the paraffin embedded sections were used to stain cleaved caspase 3 positive cells immunohistochemically as described previously [12,13]. Briefly, the slides were deparaffinized and hydrated to proceed with microwave assisted antigen retrieval using citrate buffer. Then the slides were blocked with goat serum and then incubated with polyclonal rabbit anti-cleaved caspase 3 antibody (Cell Signaling Technology Inc., MA, USA). After overnight incubation at 4 °C, the slides were washed and incubated with goat anti-rabbit secondary antibody and then counterstained with DAB and Mayer's hematoxylin to view under a light microscope (Amscope, FMA050, USA).
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induced type 2 DM and if so what will be its role in mitochondria mediated activation of apoptotic signaling. Thus in the present study we have used cardio-specific dominant negative (DN) 14-3-3η mutant strain of C57BL6/J mice and fed them high fat diet to induce obesity mediated type 2 DM so as to identify the role of 14-3-3η protein against the mitochondria mediated activation of apoptotic signaling.
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2.4. Analysis of cardiac apoptosis by terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemistry for cleaved caspase 3 Frozen ventricular tissues embedded in OCT compound were cut into 4 μm-thick sections and fixed in 4% paraformaldehyde (pH 7.4) at room temperature. The TUNEL assay was performed as specified in the instructions for the in situ apoptosis detection kit (Takara Bio Inc., Shiga, Japan) and examined using light microscopy. Digital photomicrographs were obtained by using a color image analyzer (CAI-102, Olympus, Japan) at × 400 magnification, and 100 random fields from each heart were chosen and the number of TUNEL positive nuclei was
Fig. 1. Body weight analysis. Graphs depict the comparison of body weight changes during the course of the study of A, Wild type (C57BL6/J) mice fed with standard diet (SD) or high fat diet (HFD), and B, DN 14-3-3η mice fed with SD or HFD. **P b 0.01, ***P b 0.001 vs C57 + SD; $P b 0.05, $$P b 0.01, $$$P b 0.001 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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antibody, the bound antibody was visualized with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of expression of each protein in control WT mice was taken as one arbitrary unit (AU). For Western blotting analysis, all primary antibodies were used at a dilution of 1:1000 and secondary antibodies were used at a dilution of 1:5000. Films were scanned and band densities were quantified by densitometric analysis using Scion image software (Epson GT-X700; Tokyo, Japan). Mitochondrial fraction was separated from the homogenate using differential centrifugation [14] and similar procedure was followed for the estimation of mitochondrial levels of cytochrome c and voltage dependent anion channel (VDAC)-1 using rabbit polyclonal anticytochrome c (Cell Signaling Technology Inc., MA, USA) and goat polyclonal anti-VDAC-1 primary antibodies.
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3. Results
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There was a significant increase in the body weight of both WT and 190 DN 14-3-3η mice fed HFD when compared with the SD fed mice. In the 191 WT mice significant difference was observed starting from the first 192
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All the values are expressed as means ± SEM. Statistical analysis of differences between the groups was performed by Student's ‘t’ test or one-way analysis of variance, followed by either Dunnett's test or Tukey's multiple comparison test wherever necessary using GraphPad Prism 5 software. A value of p b 0.05 was considered statistically significant.
3.1. Body weight and blood glucose level changes Fig. 2. Blood glucose analysis. Graphs depict the comparison of blood glucose level changes during the course of the study of A, Wild type (C57BL6/J) mice fed with SD or HFD, and B, DN 14-3-3η mice fed with SD or HFD. *p b 0.05, **p b 0.01, ***p b 0.001 vs C57 + SD; $ p b 0.05, $$p b 0.01, $$$p b 0.001 vs 14-3-3 + SD.
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Fig. 3. Echocardiography. A&B, Left ventricular internal dimension in diastole (LVIDd) and systole (LVIDs), C&D, left ventricular percent fractional shortening (%FS) and ejection fraction (%EF). C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎⁎⁎p b 0.001 vs C57 + SD; ###p b 0.001 vs C57 + HFD; $$p b 0.01, $$$p b 0.001 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
3.2. Echocardiography
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In order to reveal the effect of DN mutation of cardiac 14-3-3η protein on cardiac function in HFD induced type 2 diabetic mice, we have performed transthoracic echocardiography on the 12th week of the study, which showed that the DN 14-3-3η mice fed HFD diet had a significant increase in their LVIDs when compared with other groups. Similarly %EF and %FS were also significantly reduced in this group when compared with all other groups. But the WT mice fed HFD did not show any worsening of these parameters (Fig. 3). They showed
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TUNEL staining is the method to detect the apoptotic nuclei, as it labels the exposed 3′ hydroxyl termini of the apoptotic DNA and makes it visible under a microscope. In the present study, TUNEL staining assay revealed that the myocardial sections of HFD fed DN 14-3-3η mice had a significantly increased number of apoptotic cardiomyocytes when compared with other groups, whereas HFD fed WT mice showed a slight insignificant increase in apoptotic cell count compared with SD fed mice (Fig. 4A and A1). Increased apoptotic cells were also confirmed with the fluorescence stained images of the DN 14-3-3η mice (Fig. 4B). Further the immunohistochemical analysis revealed extensive staining for cleaved caspase 3 in the DN 14-3-3η mice fed with HFD and thereby confirming the increased apoptosis in this group of mice (Fig. 4C). Thus the cardiac dysfunction observed in DN 14-3-3η mice may be due to the significant reduction in cardiomyocytes number. Thus to confirm the
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week of the HFD diet (Fig. 1A) whereas DN 14-3-3η mice showed significant elevation after 7 weeks of HFD (Fig. 1B). Similarly blood glucose levels were also raised in both the groups fed HFD with significance after second week and first week for WT (Fig. 2A) and DN 14-3-3η HFD fed mice (Fig. 2B) respectively. This increase was consistent over the duration of the experiment. There was an elevation in the heart weight of the mice belonging to both groups but did not show increment when compared with their body weight (data not shown).
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Fig. 4. Immunohistochemical identification of apoptotic cells. A, TUNEL staining of ventricular tissue slices depicting apoptotic nuclei (×400) and the bar graph showing the average number of TUNEL positive cells/field. B, Fluorescence stained TUNEL positive apoptotic cells (×400). C, Cleaved caspase 3 positive cells demonstrated by brown staining (×400). C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎⁎⁎p b 0.001 vs C57 + SD; ##p b 0.01 vs C57 + HFD; $$$p b 0.001 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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role of mitochondria in the increased apoptosis in DN 14-3-3η mice we have performed Western blotting to identify specific proteins responsible for apoptosis.
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There is a definite relation between the cardiac 14-3-3η signaling and oxidative stress. In order to identify the role of DN 14-3-3η on cardiac oxidative stress status, we have measured the myocardial levels of NADPH oxidase subunits such as p22phox, p47phox and p67phox in ventricular tissue homogenates of all the groups of mice. The levels of p22phox and p67phox were increased in both the HFD fed mice but significance was observed only in the mice of DN 14-3-3η group. There was no difference observed in the levels of p47phox in any of the groups (Fig. 5).
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3.5. Mitochondrial cytochrome c level
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Cytochrome c plays an important role in cardiomyocyte apoptosis as its leakage from mitochondria during cellular stress leads to apoptosis and followed by cell death. In the present study we have performed Western blotting of the mitochondrial fraction of ventricular tissue homogenates of the DN 14-3-3η mice and identified that their hearts showed significant reduction in its cytochrome c level when compared with SD fed mice (Fig. 6), but there was no significant difference in the total cytochrome c present in the total homogenates of all the groups. HFD fed WT mice also showed reduction in mitochondrial cytochrome c level but it did not reach any significance.
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3.6. Changes in the levels of apoptosis signaling proteins
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In order to identify the involvement of various apoptosis signaling proteins during HFD induced type 2 DM, we have measured the levels of Ask-1, p38 MAPK and JNK and their respective phosphorylated proteins in the ventricular tissue homogenates of different groups of mice. Ask-1 level was significantly increased in the DN 14-3-3η mice hearts whereas its activated form phospho Ask-1 level was decreased significantly (Fig. 7). Similarly the levels of phospho JNK Swas
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significantly increased in the DN 14-3-3η mice heart when compared 259 with both groups of SD fed mice (Fig. 8). But the activation of p38 260 MAPK was not observed in both groups of HFD fed mice (Fig. 8). 261 4. Discussion
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14-3-3 proteins are intracellular, dimeric, phosphoserine binding molecules and they play a critical role in signal transduction, apoptotic, and checkpoint control pathways by binding to many signaling proteins such as Raf1, protein kinase C, Ask-1, cdc25c, BAD, and forkhead transcription factor1 (FKHRL1). Via enzymatic binding with Ask-1, an MAPKKK, 14-3-3 inhibits the activation JNK and p38 MAPK pathways (4). As belonging to the family of 28–33-kDa signaling molecules, its primary function is to inhibit apoptosis via the regulation of B-cell lymphoma (Bcl)-2 proteins and MAPK cascades [15]. Thus most of the research on 14-3-3 proteins suggests its protective role against apoptotic cell death. We have also previously identified the role of 14-3-3η protein against cardiac dysfunction induced by pressure overload or
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Fig. 5. Western blotting analysis for determining the myocardial protein levels of NADPH oxidase subunits; p22phox, p47phox and p67phox. Graphs depict their mean density values expressed as ratios relative to that of GAPDH. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD; $p b 0.05 vs 14-3-3 + SD.
Fig. 6. Western blotting analysis for determining the mitochondrial protein levels of cytochrome c and voltage dependent anion channel-1 (VDAC-1). Graph depicts the mean density values of mitochondrial cytochrome c expressed as ratios relative to that of mitochondrial VDAC-1. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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preventing cardiomyocyte apoptosis during HFD induced type 2 DM. This was in accordance with the previous reports on 14-3-3η against apoptotic cell death during cardiovascular abnormalities such as pressure overload and type 1 DM [10,16]. Mitochondrion is one of the important cellular organelles, which can activate apoptotic pathways following cellular stress. In the mitochondria-mediated apoptotic pathway, release of cytochrome c activates a cascade of death inducing proteins, a key and irreversible point in the development of apoptosis [17]. Thus to identify any possible involvement of mitochondria in the increased apoptosis of cardiomyocytes of DN 14-3-3η mice during type 2 DM, we have measured the level of mitochondrial cytochrome c. In the present study, there was a significant reduction in its level indicating that its translocation might be an influence for the activation of apoptotic pathways. Compared with the controls, HFD in DN 14-3-3η mice might have induced serious damage to the mitochondrial membranes, resulting in
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streptozotocin induced type 1 DM; however its specific role and possible molecular mechanism involved in type 2 DM are not clear. Thus we have carried out this study to dissect any potential influence of 14-3-3η against apoptosis during type 2 DM. Induction of type 2 DM was achieved by feeding mice with HFD for 12 weeks. Both WT and DN 14-3-3η mice exhibited significant and sustained increase in their body weight as well as blood glucose level until sacrifice. By the end of 12 weeks we have performed echocardiography, which confirmed the cardiac malfunction in the DN 14-3-3η mice as their LVIDs was significantly increased and both %EF and %FS were significantly decreased. After confirmation of cardiac functional abnormalities, we have carried out TUNEL staining of the ventricular tissue sections to identify the number of apoptotic cells present in the mice of all the groups. There was a significant increase in the apoptotic cells in the DN14-3-3η mice hearts when compared with all other group mice. These data confirm the involvement of 14-3-3 protein in
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Fig. 7. Western blotting analysis for determining the myocardial protein levels of apoptosis signal-regulating kinase-1 (Ask-1) and its phosphorylated form (p-Ask-1). Graphs depict their mean density values expressed as ratios relative to that of GAPDH. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD; ##p b 0.01 vs C57 + HFD; $$p b 0.01 vs 14-3-3 + SD.
Fig. 8. Western blotting analysis for determining the myocardial protein levels of c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK) and their respective phosphorylated forms (p-JNK and P-p38 MAPK). Graphs depict the mean density values of phospho proteins expressed as ratios relative to their unphosphorylated forms. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD; $p b 0.05 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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The present study has confirmed the influence of 14-3-3η protein against the cardiomyocyte apoptosis during HFD-induced type 2 DM. We have identified three possible modes for this cardioprotective role; 1) preventing the activation of Ask-1/JNK signaling cascade, 2) prevention of mitochondrial cytochrome c mediated apoptotic signaling pathways and 3) prevention of oxidative stress mediated apoptosis. There is a definite increase in the oxidative stress in the DN 14-3-3η
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mice with type 2 DM and oxidative stress leads to mitochondria mediated apoptosis, but here in the present study increased oxidative stress in DN 14-3-3η mice leads to apoptosis or whether it's a consequence of cardiomyocyte loss must be studied in detail in future.
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the release of cytochrome c from mitochondria. Thus following its release into the cytosol, cytochrome c activates various caspases leading to apoptosis of cardiomyocytes [18,19]. To rule out the possible mode of mitochondrial dysfunction in HFD fed DN 14-3-3η mice, we have detected the level of oxidative stress, mitochondrial cytochrome c and Ask-1 signaling proteins. Oxidative stress is a major mediator of mitochondrial dysfunction causing activation of mitochondria mediated apoptotic pathways [20,21] and increased expression of NADPH oxidase subunits can be a predictor of oxidative stress [22]. Thus in the present study, we have measured the levels of NADPH oxidase subunits such as p22phox, p47phox and p67phox in the ventricular tissue homogenates. There was a significant increase in the levels of p22phox and p67phox in the hearts of HFD fed DN 14-3-3η mice suggesting that these mice suffered from oxidative stress. Thus oxidative stress mediated mitochondrial dysfunction may be one of the possible modes for the increased apoptosis in HFD fed DN 14-3-3η mice. There are several reports suggesting that 14-3-3 protein binding to BAD prevents its translocation into mitochondria to induce cytochrome c leakage into cytosol causing activation of apoptotic signaling in the cortical neurons [17,23,24]. Thus in the present study, DN mutation of 14-3-3η protein might be responsible for the increased cardiomyocyte apoptosis possibly via enhanced BAD translocation into mitochondria and thereby causing cytochrome c leakage into cytosol leading to activation of apoptotic cell death. Thus 14-3-3η protein, by preventing mitochondrial cytochrome c leakage into cytosol possibly via the involvement of BAD might be another mode of preventing cardiomyocyte apoptosis. In addition to the above-mentioned mechanism, there is another possible mode for the increased apoptosis observed in the present study. As reported earlier binding of 14-3-3 protein to phospho Ask-1 can prevent the apoptotic role of Ask-1. Dephosphorylation of Ask-1 by increased oxidative stress can release Ask-1 from 14-3-3 complex and causes activation of p38MAPK or JNK leading to activation of apoptotic signaling cascade [25]. In accordance with this report, DN 14-3-3η mice in the present study when fed with HFD showed increased cardiomyocyte apoptosis along with increased levels of Ask-1 and phospho JNK suggesting the activation of Ask-1/JNK signaling cascade, leading to cardiomyocyte apoptosis. Thus by binding to Ask-1 and thereby preventing the activation of JNK might be another cellular mechanism of 14-3-3η in preventing cardiomyocyte apoptosis during HFD induced type 2 DM.
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Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
Contents lists available at ScienceDirect
Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig
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Article history: Received 27 September 2014 Received in revised form 1 December 2014 Accepted 17 December 2014 Available online xxxx
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Keywords: 14-3-3η protein Diabetes mellitus Cardiomyocyte apoptosis Cytochrome c Apoptosis signal-regulating kinase-1 High fat diet
Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX 77030, USA c J.K.K. Nattraja College of Pharmacy, Natarajapuram, Komarapalayam, Namakkal District, 638183 Tamil Nadu, India d Department of Pharmacology, College of Pharmacy, King Khalid University, Abha, Kingdom of Saudi Arabia e Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan
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There is a definite cardioprotective role for 14-3-3η protein against pressure overload induced cardiac hypertrophy and streptozotocin induced cardiac dysfunction in type 1 diabetes mellitus (DM). But it is not conclusive whether it has any influence on mitochondrial mediated cardiomyocyte apoptosis in type 2 DM. In order to test this hypothesis, we have used C57BL6/J (WT) mice with cardiac specific dominant negative mutation of 14-3-3η protein (DN 14-3-3η). Both WT and DN 14-3-3η mice were fed with high fat diet (HFD) for 12 weeks. Their body weight and blood glucose levels were measured weekly and compared with standard diet (SD) fed mice. By the end of 12 weeks, echocardiography was performed. Frozen myocardial sections were prepared to stain the apoptotic cardiomyocytes using TUNEL staining. DN 14-3-3η mice fed with HFD showed cardiac dysfunction as identified by the decreased fractional shortening and ejection fraction and increased cardiomyocyte apoptosis in TUNEL staining. Western blotting analysis using mitochondrial fraction of the ventricular tissue homogenates showed a significant reduction in the level of cytochrome c suggesting its translocation into cytoplasm, which may be crucial in inducing cardiomyocyte apoptosis. In addition, DN 14-3-3η mice depicted significantly increased levels of NADPH oxidase subunits suggesting oxidative stress, a significant reduction in phospho apoptosis signal-regulating kinase-1 (p-Ask-1) and increase in Ask-1 and phospho c-Jun N-terminal kinase (p-JNK) levels suggesting activation of Ask-1/JNK signaling. These results suggest that 14-3-3η has a protective role against mitochondria mediated cardiomyocyte apoptosis with the involvement of Ask-1/JNK signaling during HFD induced type 2 DM. © 2015 Published by Elsevier Inc.
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N-terminal kinase (JNK) or p38 mitogen activated protein kinase (MAPK) [3,4]. Evidences are available for the relation between oxidative stress, apoptosis and 14-3-3 protein as it has been suggested that oxidative stress activates Ask1 by dissociating its inhibitor, 14-3-3 protein, from Ask1 Ser-967 in Cos7 cells [5]. 14-3-3 protein and thioredoxin are reported to limit Ask1 activity by guarding the C-terminal and N-terminal of Ask1 kinase, respectively [6]. Thus modulation of apoptosis may be the central factor in the cardioprotective role of 14-3-3 proteins. We have previously demonstrated that 14-3-3η protein acts as an endogenous cardioprotector and limits the development of diabetic cardiomyopathy by limiting myocardial apoptosis, hypertrophy, fibrosis, and endothelial dysfunction via inhibition of Ask1 activation after induction of experimental diabetes [7–10]. In addition we have reported some of the regulatory effects of 14-3-3η protein on pressureoverload induced cardiac hypertrophy and streptozotocin-induced type 1 diabetes mellitus (DM) [11]. But it is not conclusive whether 14-3-3η protein can influence cardiomyocyte apoptosis during obesity
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Remya Sreedhar a, Somasundaram Arumugam a, Rajarajan A. Thandavarayan b, Vijayasree V. Giridharan c, Vengadeshprabhu Karuppagounder a, Vigneshwaran Pitchaimani a, Rejina Afrin a, Shizuka Miyashita a, Mayumi Nomoto a, Meilei Harima a, Narasimman Gurusamy d, Kenji Suzuki e, Kenichi Watanabe a,⁎
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1. Introduction
The tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation proteins are a family of molecular chaperones commonly referred to as 14-3-3 proteins. The family consists of seven transcripts in mammals: 14-3-3β (YWHAB), 14-3-3γ (YWHAG), 14-3-3ε (YWHAE), 14-3-3ζ (YWHAZ), 14-3-3η (YWHAH), 14-3-3θ (YWHAQ) and 14-33σ also known as stratifin (SFN) [1]. 14-3-3 proteins are intracellular phosphoserine-binding adapter molecules belonging to a class of highly conserved proteins involved in regulating apoptosis, adhesion, cellular proliferation, differentiation, survival, and signal transduction pathways [2]. Along with apoptosis signal regulating kinase (Ask)-1 it can modulate cellular apoptosis, inflammation, differentiation and survival and most of these responses are mediated possibly via either c-Jun
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⁎ Corresponding author. Tel.: +81 250 25 5267; fax: +81 250 25 5021. E-mail address: [email protected] (K. Watanabe).
http://dx.doi.org/10.1016/j.cellsig.2014.12.021 0898-6568/© 2015 Published by Elsevier Inc.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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Transgenic DN 14-3-3η mice were generated as described previously [8]. Briefly, the coding region of human DN (R56A and R60A) 14-3-3η cDNA with a 5′-Myc-1 epitope tag was subcloned into a vector containing the α-myosin heavy chain promoter and an SV40 polyadenylation site. Linearized DNA was injected into the pronuclei of one-cell C57BL/ 6 XSJL embryos at the Neuroscience Transgenic Facility at Washington University School of Medicine. Progeny were backcrossed into the C57BL6 genetic background and were analyzed by polymerase chain reaction to detect transgene integration using mouse-tail DNA as template. Age matched C57BL6/J mice (obtained from Charles River Japan Inc., Kanagawa, Japan) were used as wild type (WT) controls. Mice were maintained with free access to water and chow throughout the period of study, and animals were treated in accordance with the Guidelines for Animal Experimentation of our institute. All animals were handled according to the approved protocols and animal welfare regulations of the Institutional Review Board at Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.
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Both WT and DN 14-3-3η mice were given high fat diet 32 (HFD) (energy—507.6 Kcal/100 g) (CLEA Japan, Inc.) or standard diet (SD) (energy—342.7 Kcal/100 g) from the start of the study and continued until 12 weeks. Body weight and blood glucose levels (tail vein bleeding using NIPRO blood glucose monitoring system) were measured weekly.
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Two-dimensional echocardiography studies were performed in anesthetized mice (pentobarbital, 50 mg/kg, i.p.) to evaluate cardiac function using an echocardiographic machine with 7.5, 12 and 30 MHz transducers linked to an ultrasound system (VISUALSONICS, Vevo770, Japan). The short-axis view of the left ventricle (LV) was recorded to measure the LV dimension in systole (LVDs) and diastole (LVDd), percent ejection fraction (%EF) as well as the percent fractional shortening (%FS). Hearts were harvested for analysis from control and diabetic mice. The ventricle portion was quickly dissected and cut into two parts. One part was immediately transferred into liquid nitrogen and then stored at −80 °C for protein analysis. The other part was either stored in 10% formalin or stored at −80 °C after the addition of TissueTek OCT compound (Sakura Co. Ltd., Tokyo, Japan) for histopathological analysis [10].
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Protein lysate was prepared from heart tissue as described previously [8]. The total protein concentration in samples was measured by the bicinchonic acid method. For Western blotting experiments, 50 mg of total protein was loaded and proteins were separated by SDS-PAGE (200 V for 40 min) and electrophoretically transferred to nitrocellulose filters (semidry transfer at 10 V for 30 min). Filters were blocked with 5% non-fat dry milk in Tris-buffered saline (20 mM Tris, pH 7.6, 137 mM NaCl) with 0.1% Tween 20, washed, and then incubated with primary antibody. Primary antibodies employed included: rabbit polyclonal anti-apoptosis signal-regulating kinase (Ask-1) and antiphospho (Ser-967) Ask-1, rabbit polyclonal anti-p38 MAPK and antiphospho (Thr-180/Tyr-182) p38 MAPK, rabbit polyclonal anti-JNK and anti-phospho JNK, rabbit polyclonal anti-cytochrome c (Cell Signaling Technology Inc., MA, USA), rabbit polyclonal anti-p22phox, antip47phox, goat polyclonal anti-p67phox and goat polyclonal antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology Inc., CA, USA). After incubation with the primary
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quantified in a blinded manner. Only nuclei that were clearly located in cardiac myocytes were considered [8]. Similar procedure was performed but without counterstaining with diaminobenzidine (DAB) using another set of samples to observe via a fluorescence microscope (LSM700, Carl Zeiss, Japan). Similarly, the paraffin embedded sections were used to stain cleaved caspase 3 positive cells immunohistochemically as described previously [12,13]. Briefly, the slides were deparaffinized and hydrated to proceed with microwave assisted antigen retrieval using citrate buffer. Then the slides were blocked with goat serum and then incubated with polyclonal rabbit anti-cleaved caspase 3 antibody (Cell Signaling Technology Inc., MA, USA). After overnight incubation at 4 °C, the slides were washed and incubated with goat anti-rabbit secondary antibody and then counterstained with DAB and Mayer's hematoxylin to view under a light microscope (Amscope, FMA050, USA).
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induced type 2 DM and if so what will be its role in mitochondria mediated activation of apoptotic signaling. Thus in the present study we have used cardio-specific dominant negative (DN) 14-3-3η mutant strain of C57BL6/J mice and fed them high fat diet to induce obesity mediated type 2 DM so as to identify the role of 14-3-3η protein against the mitochondria mediated activation of apoptotic signaling.
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2.4. Analysis of cardiac apoptosis by terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunohistochemistry for cleaved caspase 3 Frozen ventricular tissues embedded in OCT compound were cut into 4 μm-thick sections and fixed in 4% paraformaldehyde (pH 7.4) at room temperature. The TUNEL assay was performed as specified in the instructions for the in situ apoptosis detection kit (Takara Bio Inc., Shiga, Japan) and examined using light microscopy. Digital photomicrographs were obtained by using a color image analyzer (CAI-102, Olympus, Japan) at × 400 magnification, and 100 random fields from each heart were chosen and the number of TUNEL positive nuclei was
Fig. 1. Body weight analysis. Graphs depict the comparison of body weight changes during the course of the study of A, Wild type (C57BL6/J) mice fed with standard diet (SD) or high fat diet (HFD), and B, DN 14-3-3η mice fed with SD or HFD. **P b 0.01, ***P b 0.001 vs C57 + SD; $P b 0.05, $$P b 0.01, $$$P b 0.001 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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antibody, the bound antibody was visualized with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of expression of each protein in control WT mice was taken as one arbitrary unit (AU). For Western blotting analysis, all primary antibodies were used at a dilution of 1:1000 and secondary antibodies were used at a dilution of 1:5000. Films were scanned and band densities were quantified by densitometric analysis using Scion image software (Epson GT-X700; Tokyo, Japan). Mitochondrial fraction was separated from the homogenate using differential centrifugation [14] and similar procedure was followed for the estimation of mitochondrial levels of cytochrome c and voltage dependent anion channel (VDAC)-1 using rabbit polyclonal anticytochrome c (Cell Signaling Technology Inc., MA, USA) and goat polyclonal anti-VDAC-1 primary antibodies.
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3. Results
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All the values are expressed as means ± SEM. Statistical analysis of differences between the groups was performed by Student's ‘t’ test or one-way analysis of variance, followed by either Dunnett's test or Tukey's multiple comparison test wherever necessary using GraphPad Prism 5 software. A value of p b 0.05 was considered statistically significant.
3.1. Body weight and blood glucose level changes Fig. 2. Blood glucose analysis. Graphs depict the comparison of blood glucose level changes during the course of the study of A, Wild type (C57BL6/J) mice fed with SD or HFD, and B, DN 14-3-3η mice fed with SD or HFD. *p b 0.05, **p b 0.01, ***p b 0.001 vs C57 + SD; $ p b 0.05, $$p b 0.01, $$$p b 0.001 vs 14-3-3 + SD.
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Fig. 3. Echocardiography. A&B, Left ventricular internal dimension in diastole (LVIDd) and systole (LVIDs), C&D, left ventricular percent fractional shortening (%FS) and ejection fraction (%EF). C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎⁎⁎p b 0.001 vs C57 + SD; ###p b 0.001 vs C57 + HFD; $$p b 0.01, $$$p b 0.001 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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In order to reveal the effect of DN mutation of cardiac 14-3-3η protein on cardiac function in HFD induced type 2 diabetic mice, we have performed transthoracic echocardiography on the 12th week of the study, which showed that the DN 14-3-3η mice fed HFD diet had a significant increase in their LVIDs when compared with other groups. Similarly %EF and %FS were also significantly reduced in this group when compared with all other groups. But the WT mice fed HFD did not show any worsening of these parameters (Fig. 3). They showed
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TUNEL staining is the method to detect the apoptotic nuclei, as it labels the exposed 3′ hydroxyl termini of the apoptotic DNA and makes it visible under a microscope. In the present study, TUNEL staining assay revealed that the myocardial sections of HFD fed DN 14-3-3η mice had a significantly increased number of apoptotic cardiomyocytes when compared with other groups, whereas HFD fed WT mice showed a slight insignificant increase in apoptotic cell count compared with SD fed mice (Fig. 4A and A1). Increased apoptotic cells were also confirmed with the fluorescence stained images of the DN 14-3-3η mice (Fig. 4B). Further the immunohistochemical analysis revealed extensive staining for cleaved caspase 3 in the DN 14-3-3η mice fed with HFD and thereby confirming the increased apoptosis in this group of mice (Fig. 4C). Thus the cardiac dysfunction observed in DN 14-3-3η mice may be due to the significant reduction in cardiomyocytes number. Thus to confirm the
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week of the HFD diet (Fig. 1A) whereas DN 14-3-3η mice showed significant elevation after 7 weeks of HFD (Fig. 1B). Similarly blood glucose levels were also raised in both the groups fed HFD with significance after second week and first week for WT (Fig. 2A) and DN 14-3-3η HFD fed mice (Fig. 2B) respectively. This increase was consistent over the duration of the experiment. There was an elevation in the heart weight of the mice belonging to both groups but did not show increment when compared with their body weight (data not shown).
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Fig. 4. Immunohistochemical identification of apoptotic cells. A, TUNEL staining of ventricular tissue slices depicting apoptotic nuclei (×400) and the bar graph showing the average number of TUNEL positive cells/field. B, Fluorescence stained TUNEL positive apoptotic cells (×400). C, Cleaved caspase 3 positive cells demonstrated by brown staining (×400). C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎⁎⁎p b 0.001 vs C57 + SD; ##p b 0.01 vs C57 + HFD; $$$p b 0.001 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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role of mitochondria in the increased apoptosis in DN 14-3-3η mice we have performed Western blotting to identify specific proteins responsible for apoptosis.
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There is a definite relation between the cardiac 14-3-3η signaling and oxidative stress. In order to identify the role of DN 14-3-3η on cardiac oxidative stress status, we have measured the myocardial levels of NADPH oxidase subunits such as p22phox, p47phox and p67phox in ventricular tissue homogenates of all the groups of mice. The levels of p22phox and p67phox were increased in both the HFD fed mice but significance was observed only in the mice of DN 14-3-3η group. There was no difference observed in the levels of p47phox in any of the groups (Fig. 5).
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Cytochrome c plays an important role in cardiomyocyte apoptosis as its leakage from mitochondria during cellular stress leads to apoptosis and followed by cell death. In the present study we have performed Western blotting of the mitochondrial fraction of ventricular tissue homogenates of the DN 14-3-3η mice and identified that their hearts showed significant reduction in its cytochrome c level when compared with SD fed mice (Fig. 6), but there was no significant difference in the total cytochrome c present in the total homogenates of all the groups. HFD fed WT mice also showed reduction in mitochondrial cytochrome c level but it did not reach any significance.
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In order to identify the involvement of various apoptosis signaling proteins during HFD induced type 2 DM, we have measured the levels of Ask-1, p38 MAPK and JNK and their respective phosphorylated proteins in the ventricular tissue homogenates of different groups of mice. Ask-1 level was significantly increased in the DN 14-3-3η mice hearts whereas its activated form phospho Ask-1 level was decreased significantly (Fig. 7). Similarly the levels of phospho JNK Swas
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significantly increased in the DN 14-3-3η mice heart when compared 259 with both groups of SD fed mice (Fig. 8). But the activation of p38 260 MAPK was not observed in both groups of HFD fed mice (Fig. 8). 261 4. Discussion
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14-3-3 proteins are intracellular, dimeric, phosphoserine binding molecules and they play a critical role in signal transduction, apoptotic, and checkpoint control pathways by binding to many signaling proteins such as Raf1, protein kinase C, Ask-1, cdc25c, BAD, and forkhead transcription factor1 (FKHRL1). Via enzymatic binding with Ask-1, an MAPKKK, 14-3-3 inhibits the activation JNK and p38 MAPK pathways (4). As belonging to the family of 28–33-kDa signaling molecules, its primary function is to inhibit apoptosis via the regulation of B-cell lymphoma (Bcl)-2 proteins and MAPK cascades [15]. Thus most of the research on 14-3-3 proteins suggests its protective role against apoptotic cell death. We have also previously identified the role of 14-3-3η protein against cardiac dysfunction induced by pressure overload or
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Fig. 5. Western blotting analysis for determining the myocardial protein levels of NADPH oxidase subunits; p22phox, p47phox and p67phox. Graphs depict their mean density values expressed as ratios relative to that of GAPDH. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD; $p b 0.05 vs 14-3-3 + SD.
Fig. 6. Western blotting analysis for determining the mitochondrial protein levels of cytochrome c and voltage dependent anion channel-1 (VDAC-1). Graph depicts the mean density values of mitochondrial cytochrome c expressed as ratios relative to that of mitochondrial VDAC-1. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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preventing cardiomyocyte apoptosis during HFD induced type 2 DM. This was in accordance with the previous reports on 14-3-3η against apoptotic cell death during cardiovascular abnormalities such as pressure overload and type 1 DM [10,16]. Mitochondrion is one of the important cellular organelles, which can activate apoptotic pathways following cellular stress. In the mitochondria-mediated apoptotic pathway, release of cytochrome c activates a cascade of death inducing proteins, a key and irreversible point in the development of apoptosis [17]. Thus to identify any possible involvement of mitochondria in the increased apoptosis of cardiomyocytes of DN 14-3-3η mice during type 2 DM, we have measured the level of mitochondrial cytochrome c. In the present study, there was a significant reduction in its level indicating that its translocation might be an influence for the activation of apoptotic pathways. Compared with the controls, HFD in DN 14-3-3η mice might have induced serious damage to the mitochondrial membranes, resulting in
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streptozotocin induced type 1 DM; however its specific role and possible molecular mechanism involved in type 2 DM are not clear. Thus we have carried out this study to dissect any potential influence of 14-3-3η against apoptosis during type 2 DM. Induction of type 2 DM was achieved by feeding mice with HFD for 12 weeks. Both WT and DN 14-3-3η mice exhibited significant and sustained increase in their body weight as well as blood glucose level until sacrifice. By the end of 12 weeks we have performed echocardiography, which confirmed the cardiac malfunction in the DN 14-3-3η mice as their LVIDs was significantly increased and both %EF and %FS were significantly decreased. After confirmation of cardiac functional abnormalities, we have carried out TUNEL staining of the ventricular tissue sections to identify the number of apoptotic cells present in the mice of all the groups. There was a significant increase in the apoptotic cells in the DN14-3-3η mice hearts when compared with all other group mice. These data confirm the involvement of 14-3-3 protein in
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Fig. 7. Western blotting analysis for determining the myocardial protein levels of apoptosis signal-regulating kinase-1 (Ask-1) and its phosphorylated form (p-Ask-1). Graphs depict their mean density values expressed as ratios relative to that of GAPDH. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD; ##p b 0.01 vs C57 + HFD; $$p b 0.01 vs 14-3-3 + SD.
Fig. 8. Western blotting analysis for determining the myocardial protein levels of c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK) and their respective phosphorylated forms (p-JNK and P-p38 MAPK). Graphs depict the mean density values of phospho proteins expressed as ratios relative to their unphosphorylated forms. C57, wild type mice, 14-3-3, Dominant negative 14-3-3η mice; SD, standard diet; HFD, high fat diet. ⁎p b 0.05 vs C57 + SD; $p b 0.05 vs 14-3-3 + SD.
Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
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The present study has confirmed the influence of 14-3-3η protein against the cardiomyocyte apoptosis during HFD-induced type 2 DM. We have identified three possible modes for this cardioprotective role; 1) preventing the activation of Ask-1/JNK signaling cascade, 2) prevention of mitochondrial cytochrome c mediated apoptotic signaling pathways and 3) prevention of oxidative stress mediated apoptosis. There is a definite increase in the oxidative stress in the DN 14-3-3η
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mice with type 2 DM and oxidative stress leads to mitochondria mediated apoptosis, but here in the present study increased oxidative stress in DN 14-3-3η mice leads to apoptosis or whether it's a consequence of cardiomyocyte loss must be studied in detail in future.
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the release of cytochrome c from mitochondria. Thus following its release into the cytosol, cytochrome c activates various caspases leading to apoptosis of cardiomyocytes [18,19]. To rule out the possible mode of mitochondrial dysfunction in HFD fed DN 14-3-3η mice, we have detected the level of oxidative stress, mitochondrial cytochrome c and Ask-1 signaling proteins. Oxidative stress is a major mediator of mitochondrial dysfunction causing activation of mitochondria mediated apoptotic pathways [20,21] and increased expression of NADPH oxidase subunits can be a predictor of oxidative stress [22]. Thus in the present study, we have measured the levels of NADPH oxidase subunits such as p22phox, p47phox and p67phox in the ventricular tissue homogenates. There was a significant increase in the levels of p22phox and p67phox in the hearts of HFD fed DN 14-3-3η mice suggesting that these mice suffered from oxidative stress. Thus oxidative stress mediated mitochondrial dysfunction may be one of the possible modes for the increased apoptosis in HFD fed DN 14-3-3η mice. There are several reports suggesting that 14-3-3 protein binding to BAD prevents its translocation into mitochondria to induce cytochrome c leakage into cytosol causing activation of apoptotic signaling in the cortical neurons [17,23,24]. Thus in the present study, DN mutation of 14-3-3η protein might be responsible for the increased cardiomyocyte apoptosis possibly via enhanced BAD translocation into mitochondria and thereby causing cytochrome c leakage into cytosol leading to activation of apoptotic cell death. Thus 14-3-3η protein, by preventing mitochondrial cytochrome c leakage into cytosol possibly via the involvement of BAD might be another mode of preventing cardiomyocyte apoptosis. In addition to the above-mentioned mechanism, there is another possible mode for the increased apoptosis observed in the present study. As reported earlier binding of 14-3-3 protein to phospho Ask-1 can prevent the apoptotic role of Ask-1. Dephosphorylation of Ask-1 by increased oxidative stress can release Ask-1 from 14-3-3 complex and causes activation of p38MAPK or JNK leading to activation of apoptotic signaling cascade [25]. In accordance with this report, DN 14-3-3η mice in the present study when fed with HFD showed increased cardiomyocyte apoptosis along with increased levels of Ask-1 and phospho JNK suggesting the activation of Ask-1/JNK signaling cascade, leading to cardiomyocyte apoptosis. Thus by binding to Ask-1 and thereby preventing the activation of JNK might be another cellular mechanism of 14-3-3η in preventing cardiomyocyte apoptosis during HFD induced type 2 DM.
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Please cite this article as: R. Sreedhar, et al., Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis, Cell. Signal. (2015), http://dx.doi.org/10.1016/j.cellsig.2014.12.021
