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Article type: Original Article
Multisite phosphorylation of Bcl-2 via PKCδ facilitates apoptosis of hypertrophic cardiomyocytes
Juan-Juan Sheng*, Yan Chen*, Hui Chang, Yun-Ying Wang, Bo Jiao, Zhi-Bin Yu
Department of Aerospace Physiology, Fourth Military Medical University, 169# Changlexi Road, Xi’an 710032, China
Running head: Mechanisms of apoptosis in hypertrophic cardiomyocytes
*These authors contributed equally to this study.
Corresponding author: Dr. Zhi-Bin Yu Department of Aerospace Physiology, Fourth Military Medical University, 169# Changlexi Road, Xi’an 710032, China. Tel: 86-29-84774807, Fax: 86-29-84774387. E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12295 This article is protected by copyright. All rights reserved.
Accepted Article
Multisite phosphorylation of Bcl-2 via PKCδ facilitates apoptosis of hypertrophic cardiomyocytes
The activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to mitochondria, and subsequently triggers the apoptotic signaling pathway. The mechanisms remain elusive. The aim of this study was to investigate mitochondrial translocation of PKCδ might phosphorylate multiple sites of Bcl-2 which induced an imbalance between Bcl-2 and Bak or Bax, and then enhanced apoptotic susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII). Chronic pressure overload was induced by transverse aortic constriction model (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. AngII-treated hearts exhibited a higher rate of TUNEL positive cells, while PKCδ inhibition prevented the increase in AngII-induced apoptosis. The expression of PKCδ increased, but the expression of Bcl-2 decreased in the hypertrophied myocardium compared with the synchronous control. AngII-treatment activated PKCδ and induced translocation of PKCδ to mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl-2 at serine-87 and serine-70 sites. Bcl-2 with multisite phosphorylation released from mitochondria, meanwhile reduced its affinity to Bak and Bax. The imbalance between Bcl-2 and Bak/Bax induced the release of mitochondrial cytochrome c, and then activated the caspase-3 apoptotic pathway during AngII stimulation in hypertrophied cardiomyocytes. PKCδ inhibition reduced these effects induced by AngII. These results suggest that PKCδ can counteract the anti-apoptosis effect of Bcl-2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl-2 in
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hypertrophied cardiomyocytes.
Keywords: cardiomyocyte; apoptosis; PKCδ; Bcl-2; multisite phosphorylation
1. Introduction Patients with heart failure are characterized by deteriorated cardiac function, impaired labor ability, and unacceptably high mortality; thus heart failure has made a serious health problem which threats the public society.1 Hypertension is a major pathogenic factor which leads to heart failure. The chronic pressure overload induces compensatory cardiac hypertrophy, and then transit to decompensated heart failure. Apoptosis of cardiomyocytes plays a critical role in this transition. But the mechanisms underlying the highly apoptotic rate of hypertrophic cardiomyocytes remain elusive. Protein kinase C (PKC) is a pivotal point promoting cardiac hypertrophy signal pathway.2 There are expressions of PKCα, PKCβI, PKCβII, PKCε, and PKCδ in the rat myocardium.3 PKCα, PKCβI, PKCβII, or PKCε are independently involved in cardiac hypertrophy under different stimulations.2 PKCε predominantly mediates cardiac hypertrophy under chronic pressure overload of the heart. PKCδ, as a stress protein, is activated at the early stage of pressure overload and the phase of heart failure.2 Kwatra and colleagues reported that PKCε activation via the G-protein coupled receptor (GPCR) mediated cardiomyocyte hypertrophy; but the activated PKCδ under the GPCR
over-activation
condition
translocated
to
mitochondria
and
sarcolemma.4
Mochly-Rosen and colleagues found that the activated PKCδ translocated to mitochondria and promoted apoptosis of cardiomyocytes in response to cardiac ischemia and reperfusion damage.5 Our study showed that a high dose of angiotensin II induced not only much This article is protected by copyright. All rights reserved.
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hypertrophy, but also a high apoptotic rate through mitochondrial translocation of PKCδ in the hypertrophic cardiomyocytes.6 However, precise mechanisms underlying PKCδ-induced apoptosis are still being debated. Pressure overload activates a wide variety of cardiovascular bioactive substances in cardiac tissue, including angiotensin II (AngII), endothelin-1, and several peptide growth factors.7 Locally produced AngII, perhaps more than circulating AngII, is a potent stimulator of cardiac hypertrophy.8 Local AngII concentration is further elevated at the end stage of compensated hypertrophy.9 High dose of AngII activates mitochondrial translocation of PKCδ.6 The mitochondrial translocation of activated PKCδ might phosphorylate Bcl-2 located on the outer membrane of mitochondria. Prosurvival Bcl-2 can inhibit the activity of proapoptotic Bak via the interaction between Bcl-2 and Bak on the outer membrane of mitochondria. The balance between Bcl-2 and Bak modulates mitochondrial permeability transition pores.10 Bcl-2 contains at least three phosphorylation sites.11 The phosphorylation of the serine-70 site is required for potent survival phenotype of Bcl-2. In contrast, the phosphorylation of the serine-87 site may somehow reduce survival function even if serine-70 is intact. Translocation of PKCα to mitochondrial outer membranes has been shown to promote Bcl-2 phosphorylation at the serine-70 site in REH leukemic cells.12 These studies give a strong hint that mitochondrial translocation of PKCδ may phosphorylate serine-87 site of Bcl-2 and then reduce the inhibitory effects of Bcl-2 on Bak. Finally the hypertrophic cardiomyocytes will be susceptibility to apoptotic stimulations. In order to test our research hypothesis, a hypertension-induced myocardial hypertrophy rat model with transverse aortic constriction (TAC) has been established. Furthermore, we
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investigated the relationship between the mitochondrial translocation of PKCδ and multisite phosphorylation of Bcl-2, and then observed the balance between Bcl-2 and Bax on mitochondria in hypertrophic cardiomyocytes.
2. Results 2.1 AngII induces a higher apoptotic rate in hypertrophic cardiomyocytes As compared with the synchronous control group (CON), the mean blood pressure of carotid artery was significantly higher in 20 weeks of TAC group (Fig. 1A); the length and width of cardiomyocytes were increased (Fig. 1B); and the number of TUNEL-positive myonuclei of left ventricular myocardium was higher in 20 weeks of TAC group (Fig. 1C and 1D). Apoptotic rate of cardiomyocytes increased significantly in both TAC and CON groups after AngII treatment, the increment of apoptotic rate was bigger in the TAC group. The AngII-induced cardiomyocyte apoptosis was blocked by PKCδ inhibitor in the TAC and CON groups (Fig. 1D). Therefore, hypertrophic cardiomyocytes were susceptibility to AngII-induced apoptosis. 2.2 The increased and activated PKCδ translocates to mitochondria in hypertrophic cardiomyocytes PKCδ and p-PKCδ expression was higher in the TAC group. AngII alone or AngII plus PKCδ inhibitor treatment did not affect PKCδ and p-PKCδ expression (Fig. 2A, 2B, and 2C). Mitochondrial lysates were immunoprecipitated with PKCδ antibody and then blotted for Bcl-2 (Fig. 2D). Mitochondrial Bcl-2 was found to slightly interact with PKCδ in both CON and TAC hearts. Moreover, the amount of PKCδ co-precipitating with the mitochondrial
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Bcl-2 was strikingly increased in AngII-treated TAC hearts (Fig. 2E). PKCδ showed a striate distribution on sarcolemma by an immunofluorescent cytochemistry staining and MitoTracker green stain showed a crystal-like pattern of mitochondria in cardiomyocytes from TAC and CON rats (Fig. 2F and 2G). Pearson’s correlation coefficients which determined the amount of PKCδ and mitochondria colocalization were less than +0.7 in CON and TAC cardiomyocytes (Fig. 2H). AngII induced a subsarcolemma mitochondrial translocation of PKCδ in the CON cardiomyocytes, but an intermyofibrillar mitochondrial translocation of PKCδ in the hypertrophic cardiomyocytes. Pearson’s correlation coefficient was more than +0.7 only in the AngII-treated TAC group. In contrast, PKCδ inhibitor blocked these translocations of PKCδ (Fig. 2F and 2G). 2.3 Expression and phosphorylation of Bcl-2 in the left ventricular myocardium and mitochondria Total Bcl-2 expression was decreased (Fig. 3A and 3B), but Bcl-2 phosphorylated at serine-70 was increased in the left ventricular myocardium of TAC group compared with the CON group. AngII induced a further increase in serine-70 phosphorylation of Bcl-2 in both TAC and CON groups, but PKCδ inhibitor blocked these increase (Fig. 3C). Phosphorylation of Bcl-2 at serine-87 was significantly enhanced in the TAC group (Fig. 3D). AngII promoted more serine-87 phosphorylation of Bcl-2 in both TAC and CON groups, but PKCδ inhibitor reduced AngII-induced serine-87 phosphorylation of Bcl-2 (Fig. 3D). Bcl-2 located at mitochondria was significantly decreased in the TAC group. AngII induced a release of Bcl-2 from mitochondria in the TAC group, but PKCδ inhibitor blocked
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the release of Bcl-2 (Fig. 4A and 4B). Serine-70 phosphorylation of Bcl-2 was decreased in the TAC group with or without AngII treatment (Fig. 4C). However, serine-87 phosphorylation of Bcl-2 was significantly increased, but PKCδ inhibitor reduced the increase in AngII-treated TAC group (Fig. 4D). 2.4 The amount of Bak and Bax in the mitochondria The amount of Bak in the mitochondria was unchanged in the TAC and CON groups with or without AngII treatment (Fig. 5A and 5B). As compared with the CON group, the amount of Bax in the mitochondria was significantly increased, and AngII-treatment induced a further increase in the TAC group. PKCδ inhibitor resisted the increase in the mitochondrial Bax (Fig. 5C). Mitochondrial lysates were immunoprecipitated with Bcl-2 antibody, and then blotted for Bak or Bax (Fig. 5D). Mitochondrial Bcl-2 had a higher affinity to Bak than to Bax in the CON group (Fig. 5E and 5F). The affinity of mitochondrial Bcl-2 to Bak and Bax was reduced in the TAC group with or without AngII-treatment, however, the reduction in TAC with AngII-treatment was more than that in TAC without AngII-treatment (Fig. 5E and 5F). 2.5 Cytochrome c location and caspase-3 activity in the left ventricular myocardium with or without AngII treatment Cytochrome c showed a co-localization with the mitochondria in the TAC and CON groups (Pearson’s correlation coefficients > +0.7). AngII induced a loss of cytochrome c co-localization with the mitochondria (Pearson’s correlation coefficients < +0.7). PKCδ inhibitor resisted the release of cytochrome c from mitochondria (Pearson’s correlation coefficients returned to +0.7) (Fig. 6A, 6B and 6C).
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Caspase-3 activity was set as 100% in the control group. There were no differences between the CON and TAC groups in caspase-3 activity (Fig. 6D). AngII treatment significantly increased caspase-3 activity in the CON and TAC groups, but the increment of caspase-3 activity in the TAC group was more than that in the CON group. PKCδ inhibitor treatment made caspase-3 activity to return to a similar level of the control group (Fig. 6D).
3. Discussion The principal findings of this study are that the increased and activated PKCδ translocated to mitochondria and further phosphorylated serine-70 and serine-87 residues of Bcl-2 in hypertrophic cardiomyocytes. The multisite phosphorylation of Bcl-2 released from mitochondria, meanwhile reduced the affinity to Bak and Bax. Therefore, mitochondrial apoptosis pathway was easily activated by proapoptotic factors and AngII could induce a higher apoptotic rate in hypertrophic cardiomyocytes. 3.1 The activated PKCδ translocates to mitochondria in hypertrophic cardiomyocytes A lot of researches demonstrate that quite distinct hypertrophic stimuli converge on the level of PKC.2, 13 PKC consists of at least 12 isoforms which are expressed differentially in various species and organs. In adult rat heart the dominant PKC isoforms PKCα, PKCβI, PKCβII, PKCδ and PKCε have been identified on the protein level.3 Each isoform may have distinct subcellular locations, biological substrates, and differential responses to hypertrophic stimuli. An enhanced protein expression of PKCδ and PKCα was reported in both pressure-overload and volume-overload rat cardiac hypertrophy.3, 13 Roman and colleagues showed that PKCβ in PKCβ knockout mice was not necessary for the development of cardiac
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hypertrophy induced by different and independent hypertrophic stimuli such as phenylepinephrine infusion or aortic banding.14 Several studies found that PKCε content was not changed in cardiac hypertrophy;3, 13 but an increased expression of PKCε was found in aortic banding-induced cardiac hypertrophy in rats, guinea pigs and in severe human aortic stenosis.2 In contrast, Murriel and colleagues reported that PKCδ activation and its mitochondrial translocation had a critical proapoptotic role in cardiac responses following ischemia and reperfusion.15 PKCδ inhibition protects against myocardial ischemia and reperfusion injury.5,
16
Therefore, activation of PKCε caused cardioprotection whereas
activation of PKCδ increased damage induced by ischemia in vitro and in vivo. In the present study, abdominal aortic banding not only increased the protein amount of PKCδ and phosphorylated PKCδ, but also induced a mitochondrial translocation of the activated PKCδ in rat hearts. Kilts and colleagues found that stimulation of Gαq-coupled receptors in human atrium activates PKCε and PKCδ; while both translocate to the plasma membrane, PKCδ also redistributes to mitochondria.4 We also demonstrated that AngII stimulation induced mitochondrial translocation of PKCδ in cultured neonatal rat cardiomyocytes.6 However, the role of PKCδ mitochondrial translocation is unclear in hypertrophic cardiomyocytes. 3.2 The translocated PKCδ phosphorylates Bcl-2 at serine-70 and serine-87 residues The Bcl-2 family plays a critical role in mammalian programmed cell death pathways by suppressing or promoting apoptosis. Bcl-2 has a protective effect on cell apoptosis by forming heterodimers with Bax and/or Bak to neutralize the proapoptotic activity of Bax and/or Bak. The antiapoptotic function of Bcl-2 is dependent on its phosphorylation state rather than its expression level.11 Multiple kinases have been reported to phosphorylate Bcl-2.
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The activation of PKC induces a rapid serine-70 phosphorylation of Bcl-2 which suppresses apoptosis in growth factor-dependent cells.12 Further study demonstrates that mitochondrial localization of PKCα in association with Bcl-2 phosphorylation at serine-70 increase the human pre-B acute leukemia cell line REH cell resistance to drug-induced apoptosis.12 A lot of studies involved in agonists or inhibitors of PKC and the mutation of Bcl-2 indicate that serine-70 phosphorylation of Bcl-2 is required for the full and potent survival phenotype of Bcl-2.11 However, Bcl-2 has multiple phosphorylation sites including threonine-69, serine-70, and serine-87.11 Multisite phosphorylation of Bcl-2 results in inhibition of antiapoptotic function in prostate cancer cell lines.17 The study on S87A mutant Bcl-2 indicates that phosphorylation of the serine-87 site may somehow abrogate survival function even if serine-70 is intact.18 PKCα and ERK are able to phosphorylate Bcl-2 at serine-70 in vitro. Bcl-2 phosphorylation at serine-87 by activated p38MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.19 Three sites of Bcl-2 can be phosphorylated by c-Jun N-terminal protein kinase 1 (JNK1).20 Because PKCδ is also a classic stress-activated protein kinase like p38MAPK and JNK1,21 PKCδ located on mitochondria may phosphorylate serine-87 of Bcl-2 in cardiomyocytes. In the present study, Bcl-2 phosphorylation at serine-70 and serine-87 was higher in the hypertrophic myocardium. AngII activated PKCδ which induced a further increase in Bcl-2 phosphorylation at serine-70 and serine-87. Therefore, the amount of Bcl-2 serine-70 and serine-87 phosphorylation was positively related to PKCδ translocation to mitochondria.
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3.3 Serine-87 phosphorylation of Bcl-2 releases from mitochondria and promotes apoptosis in hypertrophic cardiomyocytes All BH3-only proteins in Bcl-2 family initiate apoptosis through the activation of Bax and Bak. The proapoptotic Bax and Bak are maintained in an inactive conformation through direct interactions with antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-XL, and Mcl-1.22 In response to an apoptotic stimulus, proapoptotic BH3-only proteins bind to and neutralize the antiapoptotic Bcl-2 proteins, thereby releasing Bax and Bak which oligomerize to form pores in mitochondrial outer membrane. This leads to permeabilization of mitochondrial outer membrane and release of proapoptotic proteins cytochrome c. Under normal conditions, Bax is localized to the cytosol, but, in response to death stimuli, Bax undergoes a conformational change that triggers its translocation to and insertion into mitochondrial outer membrane. However, Bak is always localized to the mitochondria as an integral membrane protein.23 Thus the balance between Bcl-2 and Bak/Bax is one of important factors to regulate permeability of mitochondrial outer membrane. In this study, the mitochondrial location of total Bcl-2 was considerably reduced in AngII-treated hypertrophic myocardium. The release of Bcl-2 from mitochondria induced a decrease in Bcl-2 with serine-70 phosphorylation and a relative increase in Bcl-2 with serine-87 phosphorylation in the hypertrophic myocardium. The affinity of multisite phosphorylated Bcl-2 to Bak and Bax was reduced in the hypertrophic myocardium. Therefore, except that proapoptotic BH3-only proteins bind to and neutralize the antiapoptotic Bcl-2 proteins, multisite phosphorylation of Bcl-2 also induce a Bcl-2 release from mitochondria. It may be a novel mechanism to induce an imbalance between Bcl-2 and Bak/Bax.
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Bcl-2 binds Bax through two interdependent interfaces to inhibit the proapoptotic oligomerization of Bax. The Bcl-2 homology 3 (BH3) region of Bax and the BH1-3 groove of Bcl-2 form the first BH3-BH1-3 groove interface. Other interaction sites form a second interface that includes helix 6 of Bax and the BH4 region of Bcl-2. Disrupted any interface can abolish Bcl-2-mediated inhibition of Bax oligomerization.24 Multisite phosphorylation of Bcl-2 may interfere in the second interface and thereby regulate the affinity of Bcl-2 to Bax. Bcl-2 phosphorylation at serine-87 seems to inhibit its binding to Bax.17 Korsmeyer and colleagues also demonstrate that phosphorylation of Bcl-2 inhibits its binding to proapoptotic family members.25 In the present study, the amount of Bak unchanged but Bax increased on mitochondria lysates. The imbalance between Bcl-2 and Bak/Bax may easily lead to permeabilization of mitochondrial outer membrane in the hypertrophic cardiomyocytes. Therefore, AngII induced a higher apoptotic rate in the hypertrophic cardiomyocytes.
4. Methods 4.1 Aortic constriction procedure Transverse abdominal aortic constriction (TAC) was performed as described previously.26 Briefly, male Sprague-Dawley rats weighing 150 ~ 200 g were subjected to the abdominal aortic banding procedure. All rats were anesthetized for surgery with sodium pentobarbital (40 mg/kg, i.p.). A midline laparotomy was performed, and a silver clip (0.6 mm ID) was positioned in the transverse aorta just below right renal artery. Sham-operated rats served as controls and were subjected to the same surgeries except for the creation of the aortic band. At the 20th week after surgery, blood pressure of left carotid artery was
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measured by a PE10-catheter connecting to a pressure transducer (SP844, MEMScAP, Norway). Data were recorded and analyzed by Chart 5 software (ADInstruments Inc., Sydney, Australia). All animal procedures were approved by the Animal Care and Use Committee at the Fourth Military Medical University. 4.2 TUNEL assay δV1-1 peptide [δ-PKC inhibitor, composed of amino acids 8 to 17 from δPKC plus a cysteine at the N terminus (C-SFNSYELGSL); AnaSpec, Inc.] was conjugated to TAT carrier peptide [amino acids 47 to 57 plus a cysteine at the N terminus (C-YGRKKRRQRRR); AnaSpec, Inc.] via a cysteine–cysteine S-S bond at their N termini, as previously described.27 There were three groups. The hearts in vehicle or AngII-treated group were balanced at the working heart mode for 30 min and then perfused cyclically with or without 10 nmol/L AngII (Sigma-Aldrich, St. Louis, MO, USA) for 60 min. The hearts in δV1-1 plus AngII-treated group were perfused with 500 nmol/L δV1-1, an inhibitor of PKCδ, at the balancing period for 10 min, and subsequently perfused cyclically with 10 nmol/L AngII plus 500 nmol/L δV1-1 for 60 min. After AngII treatment, all hearts were infused by 4% paraformaldehyde in PBS for 10 min at the Langendorff mode. The heart was then fixed overnight. After paraffin embedding, sections were fixed to glass slides. The 5-μm-thick sections were de-paraffinized by washing in xylene and a descending ethanol series. The sections were incubated with 20 μg/mL proteinase K for 20 min at room temperature. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using dUTP-FITC (FragEL™ DNA Fragmentation Detective Kit, Calbiochem, Darmstadt, Germany) was carried out according to the manufacturer’s protocols. To specifically identify This article is protected by copyright. All rights reserved.
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the TUNEL-positive nuclei of cardiomyocytes, sections were concomitantly stained with a rabbit anti-desmin antibody [1:50, Cell Signaling Technology, Inc. (CST), Danvers, MA, USA], followed by incubation with a tetramethyl-rhodamine-labeled (TRITC) secondary antibody (1:400; Molecular Probes, Eugene, OR, USA) for 90 min at room temperature. Tissue sections were also counterstained with 0.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI). Sections were examined at a 60×water objective using a laser-scanning confocal microscope (Olympus FV1000; Olympus Co., Ltd., Tokyo, Japan) equipped with the FV10-ASW system. Ten random fields per section were examined in a blinded fashion. 4.3 Preparation of the isolated cardiomyocytes and quantification of cardiomyocyte length and width Cardiomyocytes were isolated from rat hearts using a previously described technique.28 The heart was perfused in the Langendorff mode with non-circulating Ca2+-free Joklik solution (Sigma-Aldrich) containing 10 mmol/L HEPES and 10 mmol/L NaHCO3 at a constant flow of 8 mL/min for 5 min and then perfused with the circulating digestion Joklik solution containing 0.08% collagenase I (Sigma-Aldrich) plus 0.1% bovine serum albumin (BSA) for 30 min. Finally, the digestion Joklik solution was washed out with Joklik solution for 5 min. The ventricular myocardium was cut into small pieces and gently agitated. The cardiomyocytes were filtered through mesh screens, centrifuged for 3 min at 400×g, and resuspended in part in fresh Joklik solution with 1% BSA at room temperature. CaCl2 was added at 5 min interval to 1.25 mmol/L of Ca2+ concentration. The cardiomyocytes were transferred to a 0.3 mL chamber which was mounted on the stage of an inverted microscope (OlympusX71). The signal from a CCD camera was fed to a video-edge detector enabling This article is protected by copyright. All rights reserved.
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on-line cardiomyocyte data acquisition using software (PTI, Lawrenceville, USA). The length and width of cardiomyocyte were quantified by comparison with a calibrated micrometer on the microscope stage.29 Other cardiomyocytes were resuspended in a warm Dulbecco’s modified Eagle’s medium (Invitrogen Corporation, Carlsbad, CA) containing (in mmol/L) 5 creatine, 2 L-carnitine, 5 taurine, 0.1 insulin, and 0.1% BSA. Cardiomyocytes were plated on culture chamber slides coated with 10 mg/mL laminin (Sigma-Aldrich) at approximately 3×104 cells per well and incubated in a CO2 incubator at 37℃. After settling on the slide, cardiomyocytes were treated with or without 10 nmol/L AngII for 60 min, or pre-incubated with 500 nmol/L δV1-1 for 30 min, and subsequently treated with 10 nmol/L AngII plus 500 nmol/L δV1-1 for 60 min. 4.4 Immunofluorescent cytochemistry and confocal analysis The cardiomyocytes were incubated with MitoTracker green (1:50,000; Invitrogen) for 30 min at 37°C. The treated cardiomyocytes were fixed in 4% paraformaldehyde for 30 min. Cardiomyocytes were permeabilized in 0.1% Triton X-100 in PBS for 30 min, blocked with 1% BSA in PBS for 60 min at room temperature, and then incubated with rabbit polyclonal anti-PKCδ antibody (1:100; Sigma-Aldrich) or anti-cytochrome c (1:50; CST) at 4°C overnight. The slides were rinsed twice in PBS and incubated with TRITC-labeled goat anti-rabbit secondary antibody (1:400; Molecular Probes) for 60 min. The slides were then washed in PBS, incubated in Hoechst 33258 (5 μg/mL; Sigma-Aldrich) for 30 min, and washed twice with PBS. Staining was observed using a laser-scanning confocal microscope equipped with the FV10-ASW system. The TRITC-, MitoTracker green- and Hoechst-labeled
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signals were visualized at 555 nm, 490 nm and 352 nm, respectively. Images were acquired at 60× water objective. The amount of two proteins colocalization was determined using Pearson’s correlation coefficient (r) as described previously.30 Images of cardiomyocytes were analyzed using the Image-Pro Plus software. The degree of colocalization is depicted as the r-values between 0 ~ +0.3 indicate little or no colocalization, +0.3 ~ +0.7 weak positive colocalization, and +0.7~ +1.0 strong positive colocalization.30 4.5 Isolation of mitochondria Mitochondria were isolated as described previously.6 The left ventricular myocardium was homogenized by a glass homogenizer in an ice-cold extraction buffer containing (in mmol/L) 20 Tris-HCl, 330 sucrose, 2.0 EDTA, and 0.2 phenylmethylsulfonyl fluorides (PMSF) at pH 7.4. After the centrifugation of these homogenates at 900×g for 15 min at 4ºC, the supernatants were again centrifuged at 10,000×g for 45 min at 4ºC to obtain the mitochondrial pellet. The supernatants were used as the cytosolic fraction. The mitochondrial pellet was washed and resuspended in a buffer (100 mmol/L KCl, 1 mmol/L EGTA, 20 mmol/L Tris-HCl, pH 7.4) to a concentration of 10 mg protein/mL. 4.6 Immunoprecipitation Immunoprecipitations were made using a Thermo Scientific Pierce Classic IP Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacture protocol. Briefly, mitochondria pellet was lysed in an ice cold IP Lysis/Wash buffer. After an initial preclearing step of 60 min at 4°C, antigens were coupled to 10 μg of purified rabbit polyclonal anti-PKCδ or anti-Bcl-2 antibody (Sigma-Aldrich). Protein-antibody complexes were
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precipitated with a mix of 20 μL Pierce Protein A/G Agarose for 2 hour at 4°C. After 1 washing step with 200 μL of TBS, 3 washing steps with 200 μL of cold IP Lysis/Wash buffer and 1 with 200 μL of conditioning buffer, 50 μL of 2×reducing sample buffer was added to the Agarose spin column, and incubate at 80ºC for 5 minutes. The Agarose spin column was centrifuged to collect eluate. Ten μL of each sample was loaded and separated in a 12% SDS-PAGE. 4.7 Western blot analysis The myocardial protein extracts were resolved by SDS-PAGE using Laemmli gels as described previously.31 Ten percent gel was used for the examination of PKCδ and phosphorylated PKCδ (p-PKCδ); and 12% gel was used for the examination of Bcl-2, β-actin, ANT (adenine nucleotide transporter), and GAPDH (glyceraldehyde
3-phosphate
dehydrogenase). After electrophoresis, proteins were electrically transferred to PVDF membrane using a Bio-Rad semi-dry transfer apparatus. The blotted PVDF membranes were blocked with 1% BSA in Tris-buffered saline (TBS: 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5) and incubated with a rabbit polyclonal anti-PKCδ (1:1,000; Sigma-Aldrich), rabbit polyclonal anti-p Tyr311-PKCδ (1:1,000; CST), rabbit polyclonal anti-Bcl-2 (1:1,000; CST), rabbit polyclonal anti-p-Ser70-Bcl-2 (1:1,000; CST), rabbit polyclonal anti-p-Ser87-Bcl-2 [1:500; Santa Cruz biotechnology(SCB), Inc., CA, USA], rabbit polyclonal anti-ANT (1:200; SCB), mouse monoclonal anti-β-actin (1:4,000; Sigma-Aldrich), or mouse monoclonal anti-GAPDH (1:2,000; SCB) in TBS containing 0.1% BSA at 4°C overnight. The membranes were incubated with IRDye 700DX goat-anti mouse or IRDye 800CW goat-anti rabbit secondary antibodies (1:10,000) for 90 min at room temperature, and visualized using an
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Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). Quantification analysis of blots was performed with the NIH Image J software. 4.8 Caspase-3 activity assay Caspase-3 activity was measured as described previously.32 The left ventricular myocardium was homogenized in an ice-cold lysis buffer containing (in mmol/L) 20 MOPS, 100 NaCl, 1 EDTA, 10% sucrose, 10 dithiothreitol, and 0.1% CHAPS, and 1 PMSF at pH 7.5. The homogenate was centrifuged at 12,000×g for 5 min at 4°C and supernatant was collected. The protein concentration was determined by the Bradford method. For enzyme activity assay, a 96-well microplate was equilibrated to 37°C for 10 min. The cell lysates (1 mg protein/mL) and lysis buffer were added to each well and incubated for 10 min at 37°C before adding the substrate (160 μmol/L DEVD-pNA, Sigma-Aldrich). The absorbance at 405 nm was read using a microtiter reader (EL311s; Bio Tek Instruments, Inc., Winooski, VT, USA) and recorded at 1-min intervals. 4.9 Statistical analysis Data are presented as means ± standard error of the mean (SEM). Data for AngII-treated cardiomyocytes are mean values of at least three different experiments. Student’s t-test for paired observations or one-way analysis of variance, followed by a Dunnett's test was used to compare drug treatments and the control. P < 0.05 was considered statistically significant.
Conflict of interest: none declared.
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Funding This work was supported by the national Natural Science Foundation of China grant No. 31071044 to ZB Yu.
Figure legends
Fig. 1 Blood pressure, cardiomyocyte size and apoptotic rate of cardiomyocytes in transverse abdominal aortic constriction (TAC) and the sham-operated (CON) rats. (A) Mean blood pressure in carotid artery. (B) The length and width of cardiomyocytes. (C) Representative images of TUNEL-positive nuclei (green) in the myocardial sections with or without AngII treatment. Desmin in red indicates the cardiomyocyte. DAPI stains the nuclei in blue. Scale bar: 50 μm. (D) Apoptotic rates of cardiomyocytes in the TAC and CON groups with or without AngII or δV1-1 treatments. Values are mean ± SEM. The n = 6 rats in each group. * P < 0.05 or **P < 0.01 vs. CON, ##P < 0.01 vs. TAC.
Fig. 2 Expression and location of PKCδ in the left ventricular myocardium with or without AngII treatment. (A) Representive Western Blots of PKCδ and phosphorylated PKCδ (p-PKCδ) in the myocardium. β-actin is used as an internal control. (B) Quantitative analysis of PKCδ expression. (C) Quantitative analysis of p-PKCδ expression. (D) Representive Western Blots of Bcl-2. Mitochondrial lysates were immunoprecipitated with PKCδ antibody and then blotted for Bcl-2. The equal loading mitochondrial lysates were detected by Western Blot
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against PKCδ. Three independent samples were tested in each group. (E) Ratios of Bcl-2 to PKCδ indicate the interaction between Bcl-2 and PKCδ. (F) Representative confocal images scanned at nucleus level. PKCδ was stained in red fluorescence, mitochondria in green, and nuclei in blue. Yellow indicates co-localization of PKCδ and mitochondria. Scale bar: 20 μm. (G) Magnified views of the square box regions in E. (H) Quantitative analysis of Pearson's Correlation coefficient. Values are mean ± SEM. The n=6 hearts for each group in B and C and n = 15 cardiomyocytes from 3 hearts for each group in G. *P < 0.05 or **P < 0.01 vs. CON. ##P < 0.01 vs. TAC. &P < 0.05 vs. TAC with AngII treatment.
Fig. 3
Expression of Bcl-2 and phosphorylated Bcl-2 in the left ventricular
myocardium. (A) Representive Western Blots of Bcl-2 and phosphorylated Bcl-2 in the myocardium. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is used as an internal control. (B) Ratios of Bcl-2 to GAPDH. (C) Quantitative analysis of phosphorylated serine-70 Bcl-2 expression. (D) Quantitative analysis of phosphorylated serine-87 Bcl-2 expression. Values are mean ± SEM, n = 6 hearts for each group. **P < 0.01 vs. CON. ##P < 0.01 vs. TAC.
Fig. 4
Expression of Bcl-2 and phosphorylated Bcl-2 in mitochondria from the left
ventricular myocardium. (A) Representive Western Blots of Bcl-2 and phosphorylated Bcl-2 in mitochondria. ANT (adenine nucleotide transporter) is used as an internal control. (B) Ratios of Bcl-2 to ANT. (C) Quantitative analysis of phosphorylated serine-70 Bcl-2 expression. (D) Quantitative analysis
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of phosphorylated serine-87 Bcl-2 expression. Values are mean ± SEM. The n = 6 mitochondrial samples from 6 hearts for each group. **P < 0.01 vs. CON. ##P < 0.01 vs. TAC. &
P < 0.05 or &&P < 0.01 vs. TAC with AngII treatment.
Fig. 5
The amount of Bak and Bax in mitochondria and the affinity of Bcl-2 to Bak or
Bax in the myocardium. (A) Representive Western Blots of Bak and Bax in mitochondria. (B) The ratios of Bak to ANT represent the amount of Bak in mitochondria. (C) The ratios of Bax to ANT in mitochondria. Values are mean ± SEM. The n = 6 mitochondrial samples from 6 hearts for each group. (D) Representive Western Blots of Bak and Bax. Mitochondrial lysates were immunoprecipitated with Bcl-2 antibody and then blotted for Bak or Bax. The equal loading mitochondrial lysates were detected by Western Blot against Bcl-2. Three independent samples were tested in each group. (E) Ratios of Bak to Bcl-2 indicate the interaction between Bak and Bcl-2. (F) Ratios of Bax to Bcl-2 indicate the interaction between Bax and Bcl-2. *P < 0.05 or **P < 0.01 vs. CON. ##P < 0.01 vs. TAC. &&P < 0.01 vs. TAC with AngII treatment.
Fig. 6
Cytochrome c location in cardiomyocytes and caspase-3 activity in the left
ventricular myocardium. (A) Representative laser confocal images. Cytochrome c (Cyto c) was stained in red fluorescence, mitochondria in green, and nuclei in blue. Yellow indicates co-localization of Cyto c and mitochondria. Scale bar: 20 μm. (B) Magnified views of the square box regions in
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A. (C) Statistical analysis of Pearson's Correlation coefficient. (D) Caspase-3 activity in the homogenate of left ventricular myocardium. Values are mean ± SEM. The n = 15 cardiomyocytes from 3 hearts for each group in C and n = 6 hearts for each group in D. **P < 0.01 vs. CON. ##P < 0.01 vs. TAC.
References 1. Go AS, Mozaffarian D, Roger VL, et al. Executive Summary: Heart Disease and Stroke Statistics--2013 Update: A Report From the American Heart Association. Circulation 2013; 127:143-52. 2. Duquesnes N, Lezoualc'H F, Crozatier B. PKC-delta and PKC-epsilon: foes of the same family or strangers? J Mol Cell Cardiol 2011; 51:665-73. 3. Braun MU, LaRosee P, Schon S, Borst MM, Strasser RH. Differential regulation of cardiac protein kinase C isozyme expression after aortic banding in rat. Cardiovasc Res 2002; 56:52-63. 4. Kilts JD, Grocott HP, Kwatra MM. G alpha(q)-coupled receptors in human atrium function through protein kinase C epsilon and delta. J Mol Cell Cardiol 2005; 38:267-76. 5. Inagaki K, Chen L, Ikeno F, et al. Inhibition of delta-protein kinase C protects against reperfusion injury of the ischemic heart in vivo. Circulation 2003; 108:2304-7. 6. Xie MJ, Chang H, Wang YY, et al. Evidence that apoptotic signalling in hypertrophic cardiomyocytes is determined by mitochondrial pathways involving protein kinase Cdelta. Clin Exp Pharmacol Physiol 2010; 37:1120-8. 7. Schaub MC, Hefti MA, Harder BA, Eppenberger HM. Various hypertrophic stimuli induce distinct phenotypes in cardiomyocytes. J Mol Med 1997; 75:901-20. 8. van Kats JP, Danser AH, van Meegen JR, Sassen LM, Verdouw PD, Schalekamp MA. Angiotensin production by the heart: a quantitative study in pigs with the use of radiolabeled angiotensin infusions. Circulation 1998; 98:73-81. 9. Wollert KC, Drexler H. The renin-angiotensin system and experimental heart failure. Cardiovasc Res 1999; 43:838-49. 10. Westphal D, Dewson G, Czabotar PE, Kluck RM. Molecular biology of Bax and Bak activation and action. Biochim Biophys Acta 2011; 1813:521-31. 11. Ruvolo PP, Deng X, May WS. Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia 2001; 15:515-22. 12. Ito T, Deng X, Carr B, May WS. Bcl-2 phosphorylation required for anti-apoptosis function. J Biol Chem 1997; 272:11671-3. 13. Braun MU, LaRosee P, Simonis G, Borst MM, Strasser RH. Regulation of protein kinase C isozymes in volume overload cardiac hypertrophy. Mol Cell Biochem 2004; 262:135-43. 14. Roman BB, Geenen DL, Leitges M, Buttrick PM. PKC-beta is not necessary for cardiac hypertrophy. Am J Physiol Heart Circ Physiol 2001; 280:H2264-70. 15. Murriel CL, Churchill E, Inagaki K, Szweda LI, Mochly-Rosen D. Protein kinase Cdelta activation induces apoptosis in response to cardiac ischemia and reperfusion damage: a mechanism involving BAD and the mitochondria. J Biol Chem 2004; 279:47985-91.
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16. Hahn HS, Yussman MG, Toyokawa T, et al. Ischemic protection and myofibrillar cardiomyopathy: dose-dependent effects of in vivo deltaPKC inhibition. Circ Res 2002; 91:741-8. 17. Haldar S, Chintapalli J, Croce CM. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996; 56:1253-5. 18. Yamamoto K, Ichijo H, Korsmeyer SJ. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase
pathway normally activated at G(2)/M. Mol Cell Biol 1999; 19:8469-78.
19. De Chiara G, Marcocci ME, Torcia M, et al. Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences. J Biol Chem 2006; 281:21353-61. 20. Pattingre S, Bauvy C, Carpentier S, Levade T, Levine B, Codogno P. Role of JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy. J Biol Chem 2009; 284:2719-28. 21. Deng X, Xiao L, Lang W, Gao F, Ruvolo P, May WJ. Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem 2001; 276:23681-8. 22. Ku B, Liang C, Jung JU, Oh BH. Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. Cell Res 2011; 21:627-41. 23. Gustafsson AB, Gottlieb RA. Bcl-2 family members and apoptosis, taken to heart. Am J Physiol Cell Physiol 2007; 292:C45-51. 24. Ding J, Zhang Z, Roberts GJ, et al. Bcl-2 and Bax interact via the BH1-3 groove-BH3 motif interface and a novel interface involving the BH4 motif. J Biol Chem 2010; 285:28749-63. 25. Bassik MC, Scorrano L, Oakes SA, Pozzan T, Korsmeyer SJ. Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis. Embo J 2004; 23:1207-16. 26. Hulot JS, Fauconnier J, Ramanujam D, et al. Critical role for stromal interaction molecule 1 in cardiac hypertrophy. Circulation 2011; 124:796-805. 27.Chen L, Wright LR, Chen CH, Oliver SF, Wender PA, Mochly-Rosen D. Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7). Chem Biol 2001; 8:1123-9. 28. Wang YY, Jiao B, Guo WG, Che HL, Yu ZB. Excessive thyroxine enhances susceptibility to apoptosis and decreases contractility of cardiomyocytes. Mol Cell Endocrinol 2010; 320:67-75. 29. Yu ZB, Wei H, Jin JP. Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling. Am J Physiol Cell Physiol 2012; 303:C24-32. 30. Villa-Abrille MC, Cingolani E, Cingolani HE, Alvarez BV. Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening. Am J Physiol Heart Circ Physiol 2011; 300:H1237-51. 31. Yu ZB, Gao F, Feng HZ, Jin JP. Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading. Am J Physiol Cell Physiol 2007; 292:C1192-203. 32. Wang GW, Klein JB, Kang YJ. Metallothionein inhibits doxorubicin-induced mitochondrial cytochrome c release and caspase-3 activation in cardiomyocytes. J Pharmacol Exp Ther 2001; 298:461-8.
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Article type: Original Article
Multisite phosphorylation of Bcl-2 via PKCδ facilitates apoptosis of hypertrophic cardiomyocytes
Juan-Juan Sheng*, Yan Chen*, Hui Chang, Yun-Ying Wang, Bo Jiao, Zhi-Bin Yu
Department of Aerospace Physiology, Fourth Military Medical University, 169# Changlexi Road, Xi’an 710032, China
Running head: Mechanisms of apoptosis in hypertrophic cardiomyocytes
*These authors contributed equally to this study.
Corresponding author: Dr. Zhi-Bin Yu Department of Aerospace Physiology, Fourth Military Medical University, 169# Changlexi Road, Xi’an 710032, China. Tel: 86-29-84774807, Fax: 86-29-84774387. E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1440-1681.12295 This article is protected by copyright. All rights reserved.
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Multisite phosphorylation of Bcl-2 via PKCδ facilitates apoptosis of hypertrophic cardiomyocytes
The activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to mitochondria, and subsequently triggers the apoptotic signaling pathway. The mechanisms remain elusive. The aim of this study was to investigate mitochondrial translocation of PKCδ might phosphorylate multiple sites of Bcl-2 which induced an imbalance between Bcl-2 and Bak or Bax, and then enhanced apoptotic susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII). Chronic pressure overload was induced by transverse aortic constriction model (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. AngII-treated hearts exhibited a higher rate of TUNEL positive cells, while PKCδ inhibition prevented the increase in AngII-induced apoptosis. The expression of PKCδ increased, but the expression of Bcl-2 decreased in the hypertrophied myocardium compared with the synchronous control. AngII-treatment activated PKCδ and induced translocation of PKCδ to mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl-2 at serine-87 and serine-70 sites. Bcl-2 with multisite phosphorylation released from mitochondria, meanwhile reduced its affinity to Bak and Bax. The imbalance between Bcl-2 and Bak/Bax induced the release of mitochondrial cytochrome c, and then activated the caspase-3 apoptotic pathway during AngII stimulation in hypertrophied cardiomyocytes. PKCδ inhibition reduced these effects induced by AngII. These results suggest that PKCδ can counteract the anti-apoptosis effect of Bcl-2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl-2 in
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hypertrophied cardiomyocytes.
Keywords: cardiomyocyte; apoptosis; PKCδ; Bcl-2; multisite phosphorylation
1. Introduction Patients with heart failure are characterized by deteriorated cardiac function, impaired labor ability, and unacceptably high mortality; thus heart failure has made a serious health problem which threats the public society.1 Hypertension is a major pathogenic factor which leads to heart failure. The chronic pressure overload induces compensatory cardiac hypertrophy, and then transit to decompensated heart failure. Apoptosis of cardiomyocytes plays a critical role in this transition. But the mechanisms underlying the highly apoptotic rate of hypertrophic cardiomyocytes remain elusive. Protein kinase C (PKC) is a pivotal point promoting cardiac hypertrophy signal pathway.2 There are expressions of PKCα, PKCβI, PKCβII, PKCε, and PKCδ in the rat myocardium.3 PKCα, PKCβI, PKCβII, or PKCε are independently involved in cardiac hypertrophy under different stimulations.2 PKCε predominantly mediates cardiac hypertrophy under chronic pressure overload of the heart. PKCδ, as a stress protein, is activated at the early stage of pressure overload and the phase of heart failure.2 Kwatra and colleagues reported that PKCε activation via the G-protein coupled receptor (GPCR) mediated cardiomyocyte hypertrophy; but the activated PKCδ under the GPCR
over-activation
condition
translocated
to
mitochondria
and
sarcolemma.4
Mochly-Rosen and colleagues found that the activated PKCδ translocated to mitochondria and promoted apoptosis of cardiomyocytes in response to cardiac ischemia and reperfusion damage.5 Our study showed that a high dose of angiotensin II induced not only much This article is protected by copyright. All rights reserved.
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hypertrophy, but also a high apoptotic rate through mitochondrial translocation of PKCδ in the hypertrophic cardiomyocytes.6 However, precise mechanisms underlying PKCδ-induced apoptosis are still being debated. Pressure overload activates a wide variety of cardiovascular bioactive substances in cardiac tissue, including angiotensin II (AngII), endothelin-1, and several peptide growth factors.7 Locally produced AngII, perhaps more than circulating AngII, is a potent stimulator of cardiac hypertrophy.8 Local AngII concentration is further elevated at the end stage of compensated hypertrophy.9 High dose of AngII activates mitochondrial translocation of PKCδ.6 The mitochondrial translocation of activated PKCδ might phosphorylate Bcl-2 located on the outer membrane of mitochondria. Prosurvival Bcl-2 can inhibit the activity of proapoptotic Bak via the interaction between Bcl-2 and Bak on the outer membrane of mitochondria. The balance between Bcl-2 and Bak modulates mitochondrial permeability transition pores.10 Bcl-2 contains at least three phosphorylation sites.11 The phosphorylation of the serine-70 site is required for potent survival phenotype of Bcl-2. In contrast, the phosphorylation of the serine-87 site may somehow reduce survival function even if serine-70 is intact. Translocation of PKCα to mitochondrial outer membranes has been shown to promote Bcl-2 phosphorylation at the serine-70 site in REH leukemic cells.12 These studies give a strong hint that mitochondrial translocation of PKCδ may phosphorylate serine-87 site of Bcl-2 and then reduce the inhibitory effects of Bcl-2 on Bak. Finally the hypertrophic cardiomyocytes will be susceptibility to apoptotic stimulations. In order to test our research hypothesis, a hypertension-induced myocardial hypertrophy rat model with transverse aortic constriction (TAC) has been established. Furthermore, we
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investigated the relationship between the mitochondrial translocation of PKCδ and multisite phosphorylation of Bcl-2, and then observed the balance between Bcl-2 and Bax on mitochondria in hypertrophic cardiomyocytes.
2. Results 2.1 AngII induces a higher apoptotic rate in hypertrophic cardiomyocytes As compared with the synchronous control group (CON), the mean blood pressure of carotid artery was significantly higher in 20 weeks of TAC group (Fig. 1A); the length and width of cardiomyocytes were increased (Fig. 1B); and the number of TUNEL-positive myonuclei of left ventricular myocardium was higher in 20 weeks of TAC group (Fig. 1C and 1D). Apoptotic rate of cardiomyocytes increased significantly in both TAC and CON groups after AngII treatment, the increment of apoptotic rate was bigger in the TAC group. The AngII-induced cardiomyocyte apoptosis was blocked by PKCδ inhibitor in the TAC and CON groups (Fig. 1D). Therefore, hypertrophic cardiomyocytes were susceptibility to AngII-induced apoptosis. 2.2 The increased and activated PKCδ translocates to mitochondria in hypertrophic cardiomyocytes PKCδ and p-PKCδ expression was higher in the TAC group. AngII alone or AngII plus PKCδ inhibitor treatment did not affect PKCδ and p-PKCδ expression (Fig. 2A, 2B, and 2C). Mitochondrial lysates were immunoprecipitated with PKCδ antibody and then blotted for Bcl-2 (Fig. 2D). Mitochondrial Bcl-2 was found to slightly interact with PKCδ in both CON and TAC hearts. Moreover, the amount of PKCδ co-precipitating with the mitochondrial
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Bcl-2 was strikingly increased in AngII-treated TAC hearts (Fig. 2E). PKCδ showed a striate distribution on sarcolemma by an immunofluorescent cytochemistry staining and MitoTracker green stain showed a crystal-like pattern of mitochondria in cardiomyocytes from TAC and CON rats (Fig. 2F and 2G). Pearson’s correlation coefficients which determined the amount of PKCδ and mitochondria colocalization were less than +0.7 in CON and TAC cardiomyocytes (Fig. 2H). AngII induced a subsarcolemma mitochondrial translocation of PKCδ in the CON cardiomyocytes, but an intermyofibrillar mitochondrial translocation of PKCδ in the hypertrophic cardiomyocytes. Pearson’s correlation coefficient was more than +0.7 only in the AngII-treated TAC group. In contrast, PKCδ inhibitor blocked these translocations of PKCδ (Fig. 2F and 2G). 2.3 Expression and phosphorylation of Bcl-2 in the left ventricular myocardium and mitochondria Total Bcl-2 expression was decreased (Fig. 3A and 3B), but Bcl-2 phosphorylated at serine-70 was increased in the left ventricular myocardium of TAC group compared with the CON group. AngII induced a further increase in serine-70 phosphorylation of Bcl-2 in both TAC and CON groups, but PKCδ inhibitor blocked these increase (Fig. 3C). Phosphorylation of Bcl-2 at serine-87 was significantly enhanced in the TAC group (Fig. 3D). AngII promoted more serine-87 phosphorylation of Bcl-2 in both TAC and CON groups, but PKCδ inhibitor reduced AngII-induced serine-87 phosphorylation of Bcl-2 (Fig. 3D). Bcl-2 located at mitochondria was significantly decreased in the TAC group. AngII induced a release of Bcl-2 from mitochondria in the TAC group, but PKCδ inhibitor blocked
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the release of Bcl-2 (Fig. 4A and 4B). Serine-70 phosphorylation of Bcl-2 was decreased in the TAC group with or without AngII treatment (Fig. 4C). However, serine-87 phosphorylation of Bcl-2 was significantly increased, but PKCδ inhibitor reduced the increase in AngII-treated TAC group (Fig. 4D). 2.4 The amount of Bak and Bax in the mitochondria The amount of Bak in the mitochondria was unchanged in the TAC and CON groups with or without AngII treatment (Fig. 5A and 5B). As compared with the CON group, the amount of Bax in the mitochondria was significantly increased, and AngII-treatment induced a further increase in the TAC group. PKCδ inhibitor resisted the increase in the mitochondrial Bax (Fig. 5C). Mitochondrial lysates were immunoprecipitated with Bcl-2 antibody, and then blotted for Bak or Bax (Fig. 5D). Mitochondrial Bcl-2 had a higher affinity to Bak than to Bax in the CON group (Fig. 5E and 5F). The affinity of mitochondrial Bcl-2 to Bak and Bax was reduced in the TAC group with or without AngII-treatment, however, the reduction in TAC with AngII-treatment was more than that in TAC without AngII-treatment (Fig. 5E and 5F). 2.5 Cytochrome c location and caspase-3 activity in the left ventricular myocardium with or without AngII treatment Cytochrome c showed a co-localization with the mitochondria in the TAC and CON groups (Pearson’s correlation coefficients > +0.7). AngII induced a loss of cytochrome c co-localization with the mitochondria (Pearson’s correlation coefficients < +0.7). PKCδ inhibitor resisted the release of cytochrome c from mitochondria (Pearson’s correlation coefficients returned to +0.7) (Fig. 6A, 6B and 6C).
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Caspase-3 activity was set as 100% in the control group. There were no differences between the CON and TAC groups in caspase-3 activity (Fig. 6D). AngII treatment significantly increased caspase-3 activity in the CON and TAC groups, but the increment of caspase-3 activity in the TAC group was more than that in the CON group. PKCδ inhibitor treatment made caspase-3 activity to return to a similar level of the control group (Fig. 6D).
3. Discussion The principal findings of this study are that the increased and activated PKCδ translocated to mitochondria and further phosphorylated serine-70 and serine-87 residues of Bcl-2 in hypertrophic cardiomyocytes. The multisite phosphorylation of Bcl-2 released from mitochondria, meanwhile reduced the affinity to Bak and Bax. Therefore, mitochondrial apoptosis pathway was easily activated by proapoptotic factors and AngII could induce a higher apoptotic rate in hypertrophic cardiomyocytes. 3.1 The activated PKCδ translocates to mitochondria in hypertrophic cardiomyocytes A lot of researches demonstrate that quite distinct hypertrophic stimuli converge on the level of PKC.2, 13 PKC consists of at least 12 isoforms which are expressed differentially in various species and organs. In adult rat heart the dominant PKC isoforms PKCα, PKCβI, PKCβII, PKCδ and PKCε have been identified on the protein level.3 Each isoform may have distinct subcellular locations, biological substrates, and differential responses to hypertrophic stimuli. An enhanced protein expression of PKCδ and PKCα was reported in both pressure-overload and volume-overload rat cardiac hypertrophy.3, 13 Roman and colleagues showed that PKCβ in PKCβ knockout mice was not necessary for the development of cardiac
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hypertrophy induced by different and independent hypertrophic stimuli such as phenylepinephrine infusion or aortic banding.14 Several studies found that PKCε content was not changed in cardiac hypertrophy;3, 13 but an increased expression of PKCε was found in aortic banding-induced cardiac hypertrophy in rats, guinea pigs and in severe human aortic stenosis.2 In contrast, Murriel and colleagues reported that PKCδ activation and its mitochondrial translocation had a critical proapoptotic role in cardiac responses following ischemia and reperfusion.15 PKCδ inhibition protects against myocardial ischemia and reperfusion injury.5,
16
Therefore, activation of PKCε caused cardioprotection whereas
activation of PKCδ increased damage induced by ischemia in vitro and in vivo. In the present study, abdominal aortic banding not only increased the protein amount of PKCδ and phosphorylated PKCδ, but also induced a mitochondrial translocation of the activated PKCδ in rat hearts. Kilts and colleagues found that stimulation of Gαq-coupled receptors in human atrium activates PKCε and PKCδ; while both translocate to the plasma membrane, PKCδ also redistributes to mitochondria.4 We also demonstrated that AngII stimulation induced mitochondrial translocation of PKCδ in cultured neonatal rat cardiomyocytes.6 However, the role of PKCδ mitochondrial translocation is unclear in hypertrophic cardiomyocytes. 3.2 The translocated PKCδ phosphorylates Bcl-2 at serine-70 and serine-87 residues The Bcl-2 family plays a critical role in mammalian programmed cell death pathways by suppressing or promoting apoptosis. Bcl-2 has a protective effect on cell apoptosis by forming heterodimers with Bax and/or Bak to neutralize the proapoptotic activity of Bax and/or Bak. The antiapoptotic function of Bcl-2 is dependent on its phosphorylation state rather than its expression level.11 Multiple kinases have been reported to phosphorylate Bcl-2.
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The activation of PKC induces a rapid serine-70 phosphorylation of Bcl-2 which suppresses apoptosis in growth factor-dependent cells.12 Further study demonstrates that mitochondrial localization of PKCα in association with Bcl-2 phosphorylation at serine-70 increase the human pre-B acute leukemia cell line REH cell resistance to drug-induced apoptosis.12 A lot of studies involved in agonists or inhibitors of PKC and the mutation of Bcl-2 indicate that serine-70 phosphorylation of Bcl-2 is required for the full and potent survival phenotype of Bcl-2.11 However, Bcl-2 has multiple phosphorylation sites including threonine-69, serine-70, and serine-87.11 Multisite phosphorylation of Bcl-2 results in inhibition of antiapoptotic function in prostate cancer cell lines.17 The study on S87A mutant Bcl-2 indicates that phosphorylation of the serine-87 site may somehow abrogate survival function even if serine-70 is intact.18 PKCα and ERK are able to phosphorylate Bcl-2 at serine-70 in vitro. Bcl-2 phosphorylation at serine-87 by activated p38MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.19 Three sites of Bcl-2 can be phosphorylated by c-Jun N-terminal protein kinase 1 (JNK1).20 Because PKCδ is also a classic stress-activated protein kinase like p38MAPK and JNK1,21 PKCδ located on mitochondria may phosphorylate serine-87 of Bcl-2 in cardiomyocytes. In the present study, Bcl-2 phosphorylation at serine-70 and serine-87 was higher in the hypertrophic myocardium. AngII activated PKCδ which induced a further increase in Bcl-2 phosphorylation at serine-70 and serine-87. Therefore, the amount of Bcl-2 serine-70 and serine-87 phosphorylation was positively related to PKCδ translocation to mitochondria.
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3.3 Serine-87 phosphorylation of Bcl-2 releases from mitochondria and promotes apoptosis in hypertrophic cardiomyocytes All BH3-only proteins in Bcl-2 family initiate apoptosis through the activation of Bax and Bak. The proapoptotic Bax and Bak are maintained in an inactive conformation through direct interactions with antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-XL, and Mcl-1.22 In response to an apoptotic stimulus, proapoptotic BH3-only proteins bind to and neutralize the antiapoptotic Bcl-2 proteins, thereby releasing Bax and Bak which oligomerize to form pores in mitochondrial outer membrane. This leads to permeabilization of mitochondrial outer membrane and release of proapoptotic proteins cytochrome c. Under normal conditions, Bax is localized to the cytosol, but, in response to death stimuli, Bax undergoes a conformational change that triggers its translocation to and insertion into mitochondrial outer membrane. However, Bak is always localized to the mitochondria as an integral membrane protein.23 Thus the balance between Bcl-2 and Bak/Bax is one of important factors to regulate permeability of mitochondrial outer membrane. In this study, the mitochondrial location of total Bcl-2 was considerably reduced in AngII-treated hypertrophic myocardium. The release of Bcl-2 from mitochondria induced a decrease in Bcl-2 with serine-70 phosphorylation and a relative increase in Bcl-2 with serine-87 phosphorylation in the hypertrophic myocardium. The affinity of multisite phosphorylated Bcl-2 to Bak and Bax was reduced in the hypertrophic myocardium. Therefore, except that proapoptotic BH3-only proteins bind to and neutralize the antiapoptotic Bcl-2 proteins, multisite phosphorylation of Bcl-2 also induce a Bcl-2 release from mitochondria. It may be a novel mechanism to induce an imbalance between Bcl-2 and Bak/Bax.
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Bcl-2 binds Bax through two interdependent interfaces to inhibit the proapoptotic oligomerization of Bax. The Bcl-2 homology 3 (BH3) region of Bax and the BH1-3 groove of Bcl-2 form the first BH3-BH1-3 groove interface. Other interaction sites form a second interface that includes helix 6 of Bax and the BH4 region of Bcl-2. Disrupted any interface can abolish Bcl-2-mediated inhibition of Bax oligomerization.24 Multisite phosphorylation of Bcl-2 may interfere in the second interface and thereby regulate the affinity of Bcl-2 to Bax. Bcl-2 phosphorylation at serine-87 seems to inhibit its binding to Bax.17 Korsmeyer and colleagues also demonstrate that phosphorylation of Bcl-2 inhibits its binding to proapoptotic family members.25 In the present study, the amount of Bak unchanged but Bax increased on mitochondria lysates. The imbalance between Bcl-2 and Bak/Bax may easily lead to permeabilization of mitochondrial outer membrane in the hypertrophic cardiomyocytes. Therefore, AngII induced a higher apoptotic rate in the hypertrophic cardiomyocytes.
4. Methods 4.1 Aortic constriction procedure Transverse abdominal aortic constriction (TAC) was performed as described previously.26 Briefly, male Sprague-Dawley rats weighing 150 ~ 200 g were subjected to the abdominal aortic banding procedure. All rats were anesthetized for surgery with sodium pentobarbital (40 mg/kg, i.p.). A midline laparotomy was performed, and a silver clip (0.6 mm ID) was positioned in the transverse aorta just below right renal artery. Sham-operated rats served as controls and were subjected to the same surgeries except for the creation of the aortic band. At the 20th week after surgery, blood pressure of left carotid artery was
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measured by a PE10-catheter connecting to a pressure transducer (SP844, MEMScAP, Norway). Data were recorded and analyzed by Chart 5 software (ADInstruments Inc., Sydney, Australia). All animal procedures were approved by the Animal Care and Use Committee at the Fourth Military Medical University. 4.2 TUNEL assay δV1-1 peptide [δ-PKC inhibitor, composed of amino acids 8 to 17 from δPKC plus a cysteine at the N terminus (C-SFNSYELGSL); AnaSpec, Inc.] was conjugated to TAT carrier peptide [amino acids 47 to 57 plus a cysteine at the N terminus (C-YGRKKRRQRRR); AnaSpec, Inc.] via a cysteine–cysteine S-S bond at their N termini, as previously described.27 There were three groups. The hearts in vehicle or AngII-treated group were balanced at the working heart mode for 30 min and then perfused cyclically with or without 10 nmol/L AngII (Sigma-Aldrich, St. Louis, MO, USA) for 60 min. The hearts in δV1-1 plus AngII-treated group were perfused with 500 nmol/L δV1-1, an inhibitor of PKCδ, at the balancing period for 10 min, and subsequently perfused cyclically with 10 nmol/L AngII plus 500 nmol/L δV1-1 for 60 min. After AngII treatment, all hearts were infused by 4% paraformaldehyde in PBS for 10 min at the Langendorff mode. The heart was then fixed overnight. After paraffin embedding, sections were fixed to glass slides. The 5-μm-thick sections were de-paraffinized by washing in xylene and a descending ethanol series. The sections were incubated with 20 μg/mL proteinase K for 20 min at room temperature. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using dUTP-FITC (FragEL™ DNA Fragmentation Detective Kit, Calbiochem, Darmstadt, Germany) was carried out according to the manufacturer’s protocols. To specifically identify This article is protected by copyright. All rights reserved.
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the TUNEL-positive nuclei of cardiomyocytes, sections were concomitantly stained with a rabbit anti-desmin antibody [1:50, Cell Signaling Technology, Inc. (CST), Danvers, MA, USA], followed by incubation with a tetramethyl-rhodamine-labeled (TRITC) secondary antibody (1:400; Molecular Probes, Eugene, OR, USA) for 90 min at room temperature. Tissue sections were also counterstained with 0.5 μg/mL 4’,6-diamidino-2-phenylindole (DAPI). Sections were examined at a 60×water objective using a laser-scanning confocal microscope (Olympus FV1000; Olympus Co., Ltd., Tokyo, Japan) equipped with the FV10-ASW system. Ten random fields per section were examined in a blinded fashion. 4.3 Preparation of the isolated cardiomyocytes and quantification of cardiomyocyte length and width Cardiomyocytes were isolated from rat hearts using a previously described technique.28 The heart was perfused in the Langendorff mode with non-circulating Ca2+-free Joklik solution (Sigma-Aldrich) containing 10 mmol/L HEPES and 10 mmol/L NaHCO3 at a constant flow of 8 mL/min for 5 min and then perfused with the circulating digestion Joklik solution containing 0.08% collagenase I (Sigma-Aldrich) plus 0.1% bovine serum albumin (BSA) for 30 min. Finally, the digestion Joklik solution was washed out with Joklik solution for 5 min. The ventricular myocardium was cut into small pieces and gently agitated. The cardiomyocytes were filtered through mesh screens, centrifuged for 3 min at 400×g, and resuspended in part in fresh Joklik solution with 1% BSA at room temperature. CaCl2 was added at 5 min interval to 1.25 mmol/L of Ca2+ concentration. The cardiomyocytes were transferred to a 0.3 mL chamber which was mounted on the stage of an inverted microscope (OlympusX71). The signal from a CCD camera was fed to a video-edge detector enabling This article is protected by copyright. All rights reserved.
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on-line cardiomyocyte data acquisition using software (PTI, Lawrenceville, USA). The length and width of cardiomyocyte were quantified by comparison with a calibrated micrometer on the microscope stage.29 Other cardiomyocytes were resuspended in a warm Dulbecco’s modified Eagle’s medium (Invitrogen Corporation, Carlsbad, CA) containing (in mmol/L) 5 creatine, 2 L-carnitine, 5 taurine, 0.1 insulin, and 0.1% BSA. Cardiomyocytes were plated on culture chamber slides coated with 10 mg/mL laminin (Sigma-Aldrich) at approximately 3×104 cells per well and incubated in a CO2 incubator at 37℃. After settling on the slide, cardiomyocytes were treated with or without 10 nmol/L AngII for 60 min, or pre-incubated with 500 nmol/L δV1-1 for 30 min, and subsequently treated with 10 nmol/L AngII plus 500 nmol/L δV1-1 for 60 min. 4.4 Immunofluorescent cytochemistry and confocal analysis The cardiomyocytes were incubated with MitoTracker green (1:50,000; Invitrogen) for 30 min at 37°C. The treated cardiomyocytes were fixed in 4% paraformaldehyde for 30 min. Cardiomyocytes were permeabilized in 0.1% Triton X-100 in PBS for 30 min, blocked with 1% BSA in PBS for 60 min at room temperature, and then incubated with rabbit polyclonal anti-PKCδ antibody (1:100; Sigma-Aldrich) or anti-cytochrome c (1:50; CST) at 4°C overnight. The slides were rinsed twice in PBS and incubated with TRITC-labeled goat anti-rabbit secondary antibody (1:400; Molecular Probes) for 60 min. The slides were then washed in PBS, incubated in Hoechst 33258 (5 μg/mL; Sigma-Aldrich) for 30 min, and washed twice with PBS. Staining was observed using a laser-scanning confocal microscope equipped with the FV10-ASW system. The TRITC-, MitoTracker green- and Hoechst-labeled
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signals were visualized at 555 nm, 490 nm and 352 nm, respectively. Images were acquired at 60× water objective. The amount of two proteins colocalization was determined using Pearson’s correlation coefficient (r) as described previously.30 Images of cardiomyocytes were analyzed using the Image-Pro Plus software. The degree of colocalization is depicted as the r-values between 0 ~ +0.3 indicate little or no colocalization, +0.3 ~ +0.7 weak positive colocalization, and +0.7~ +1.0 strong positive colocalization.30 4.5 Isolation of mitochondria Mitochondria were isolated as described previously.6 The left ventricular myocardium was homogenized by a glass homogenizer in an ice-cold extraction buffer containing (in mmol/L) 20 Tris-HCl, 330 sucrose, 2.0 EDTA, and 0.2 phenylmethylsulfonyl fluorides (PMSF) at pH 7.4. After the centrifugation of these homogenates at 900×g for 15 min at 4ºC, the supernatants were again centrifuged at 10,000×g for 45 min at 4ºC to obtain the mitochondrial pellet. The supernatants were used as the cytosolic fraction. The mitochondrial pellet was washed and resuspended in a buffer (100 mmol/L KCl, 1 mmol/L EGTA, 20 mmol/L Tris-HCl, pH 7.4) to a concentration of 10 mg protein/mL. 4.6 Immunoprecipitation Immunoprecipitations were made using a Thermo Scientific Pierce Classic IP Kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacture protocol. Briefly, mitochondria pellet was lysed in an ice cold IP Lysis/Wash buffer. After an initial preclearing step of 60 min at 4°C, antigens were coupled to 10 μg of purified rabbit polyclonal anti-PKCδ or anti-Bcl-2 antibody (Sigma-Aldrich). Protein-antibody complexes were
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precipitated with a mix of 20 μL Pierce Protein A/G Agarose for 2 hour at 4°C. After 1 washing step with 200 μL of TBS, 3 washing steps with 200 μL of cold IP Lysis/Wash buffer and 1 with 200 μL of conditioning buffer, 50 μL of 2×reducing sample buffer was added to the Agarose spin column, and incubate at 80ºC for 5 minutes. The Agarose spin column was centrifuged to collect eluate. Ten μL of each sample was loaded and separated in a 12% SDS-PAGE. 4.7 Western blot analysis The myocardial protein extracts were resolved by SDS-PAGE using Laemmli gels as described previously.31 Ten percent gel was used for the examination of PKCδ and phosphorylated PKCδ (p-PKCδ); and 12% gel was used for the examination of Bcl-2, β-actin, ANT (adenine nucleotide transporter), and GAPDH (glyceraldehyde
3-phosphate
dehydrogenase). After electrophoresis, proteins were electrically transferred to PVDF membrane using a Bio-Rad semi-dry transfer apparatus. The blotted PVDF membranes were blocked with 1% BSA in Tris-buffered saline (TBS: 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5) and incubated with a rabbit polyclonal anti-PKCδ (1:1,000; Sigma-Aldrich), rabbit polyclonal anti-p Tyr311-PKCδ (1:1,000; CST), rabbit polyclonal anti-Bcl-2 (1:1,000; CST), rabbit polyclonal anti-p-Ser70-Bcl-2 (1:1,000; CST), rabbit polyclonal anti-p-Ser87-Bcl-2 [1:500; Santa Cruz biotechnology(SCB), Inc., CA, USA], rabbit polyclonal anti-ANT (1:200; SCB), mouse monoclonal anti-β-actin (1:4,000; Sigma-Aldrich), or mouse monoclonal anti-GAPDH (1:2,000; SCB) in TBS containing 0.1% BSA at 4°C overnight. The membranes were incubated with IRDye 700DX goat-anti mouse or IRDye 800CW goat-anti rabbit secondary antibodies (1:10,000) for 90 min at room temperature, and visualized using an
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Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). Quantification analysis of blots was performed with the NIH Image J software. 4.8 Caspase-3 activity assay Caspase-3 activity was measured as described previously.32 The left ventricular myocardium was homogenized in an ice-cold lysis buffer containing (in mmol/L) 20 MOPS, 100 NaCl, 1 EDTA, 10% sucrose, 10 dithiothreitol, and 0.1% CHAPS, and 1 PMSF at pH 7.5. The homogenate was centrifuged at 12,000×g for 5 min at 4°C and supernatant was collected. The protein concentration was determined by the Bradford method. For enzyme activity assay, a 96-well microplate was equilibrated to 37°C for 10 min. The cell lysates (1 mg protein/mL) and lysis buffer were added to each well and incubated for 10 min at 37°C before adding the substrate (160 μmol/L DEVD-pNA, Sigma-Aldrich). The absorbance at 405 nm was read using a microtiter reader (EL311s; Bio Tek Instruments, Inc., Winooski, VT, USA) and recorded at 1-min intervals. 4.9 Statistical analysis Data are presented as means ± standard error of the mean (SEM). Data for AngII-treated cardiomyocytes are mean values of at least three different experiments. Student’s t-test for paired observations or one-way analysis of variance, followed by a Dunnett's test was used to compare drug treatments and the control. P < 0.05 was considered statistically significant.
Conflict of interest: none declared.
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Funding This work was supported by the national Natural Science Foundation of China grant No. 31071044 to ZB Yu.
Figure legends
Fig. 1 Blood pressure, cardiomyocyte size and apoptotic rate of cardiomyocytes in transverse abdominal aortic constriction (TAC) and the sham-operated (CON) rats. (A) Mean blood pressure in carotid artery. (B) The length and width of cardiomyocytes. (C) Representative images of TUNEL-positive nuclei (green) in the myocardial sections with or without AngII treatment. Desmin in red indicates the cardiomyocyte. DAPI stains the nuclei in blue. Scale bar: 50 μm. (D) Apoptotic rates of cardiomyocytes in the TAC and CON groups with or without AngII or δV1-1 treatments. Values are mean ± SEM. The n = 6 rats in each group. * P < 0.05 or **P < 0.01 vs. CON, ##P < 0.01 vs. TAC.
Fig. 2 Expression and location of PKCδ in the left ventricular myocardium with or without AngII treatment. (A) Representive Western Blots of PKCδ and phosphorylated PKCδ (p-PKCδ) in the myocardium. β-actin is used as an internal control. (B) Quantitative analysis of PKCδ expression. (C) Quantitative analysis of p-PKCδ expression. (D) Representive Western Blots of Bcl-2. Mitochondrial lysates were immunoprecipitated with PKCδ antibody and then blotted for Bcl-2. The equal loading mitochondrial lysates were detected by Western Blot
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against PKCδ. Three independent samples were tested in each group. (E) Ratios of Bcl-2 to PKCδ indicate the interaction between Bcl-2 and PKCδ. (F) Representative confocal images scanned at nucleus level. PKCδ was stained in red fluorescence, mitochondria in green, and nuclei in blue. Yellow indicates co-localization of PKCδ and mitochondria. Scale bar: 20 μm. (G) Magnified views of the square box regions in E. (H) Quantitative analysis of Pearson's Correlation coefficient. Values are mean ± SEM. The n=6 hearts for each group in B and C and n = 15 cardiomyocytes from 3 hearts for each group in G. *P < 0.05 or **P < 0.01 vs. CON. ##P < 0.01 vs. TAC. &P < 0.05 vs. TAC with AngII treatment.
Fig. 3
Expression of Bcl-2 and phosphorylated Bcl-2 in the left ventricular
myocardium. (A) Representive Western Blots of Bcl-2 and phosphorylated Bcl-2 in the myocardium. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is used as an internal control. (B) Ratios of Bcl-2 to GAPDH. (C) Quantitative analysis of phosphorylated serine-70 Bcl-2 expression. (D) Quantitative analysis of phosphorylated serine-87 Bcl-2 expression. Values are mean ± SEM, n = 6 hearts for each group. **P < 0.01 vs. CON. ##P < 0.01 vs. TAC.
Fig. 4
Expression of Bcl-2 and phosphorylated Bcl-2 in mitochondria from the left
ventricular myocardium. (A) Representive Western Blots of Bcl-2 and phosphorylated Bcl-2 in mitochondria. ANT (adenine nucleotide transporter) is used as an internal control. (B) Ratios of Bcl-2 to ANT. (C) Quantitative analysis of phosphorylated serine-70 Bcl-2 expression. (D) Quantitative analysis
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of phosphorylated serine-87 Bcl-2 expression. Values are mean ± SEM. The n = 6 mitochondrial samples from 6 hearts for each group. **P < 0.01 vs. CON. ##P < 0.01 vs. TAC. &
P < 0.05 or &&P < 0.01 vs. TAC with AngII treatment.
Fig. 5
The amount of Bak and Bax in mitochondria and the affinity of Bcl-2 to Bak or
Bax in the myocardium. (A) Representive Western Blots of Bak and Bax in mitochondria. (B) The ratios of Bak to ANT represent the amount of Bak in mitochondria. (C) The ratios of Bax to ANT in mitochondria. Values are mean ± SEM. The n = 6 mitochondrial samples from 6 hearts for each group. (D) Representive Western Blots of Bak and Bax. Mitochondrial lysates were immunoprecipitated with Bcl-2 antibody and then blotted for Bak or Bax. The equal loading mitochondrial lysates were detected by Western Blot against Bcl-2. Three independent samples were tested in each group. (E) Ratios of Bak to Bcl-2 indicate the interaction between Bak and Bcl-2. (F) Ratios of Bax to Bcl-2 indicate the interaction between Bax and Bcl-2. *P < 0.05 or **P < 0.01 vs. CON. ##P < 0.01 vs. TAC. &&P < 0.01 vs. TAC with AngII treatment.
Fig. 6
Cytochrome c location in cardiomyocytes and caspase-3 activity in the left
ventricular myocardium. (A) Representative laser confocal images. Cytochrome c (Cyto c) was stained in red fluorescence, mitochondria in green, and nuclei in blue. Yellow indicates co-localization of Cyto c and mitochondria. Scale bar: 20 μm. (B) Magnified views of the square box regions in
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A. (C) Statistical analysis of Pearson's Correlation coefficient. (D) Caspase-3 activity in the homogenate of left ventricular myocardium. Values are mean ± SEM. The n = 15 cardiomyocytes from 3 hearts for each group in C and n = 6 hearts for each group in D. **P < 0.01 vs. CON. ##P < 0.01 vs. TAC.
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