Nucleolin/C23 mediates the antiapoptotic effect of heat shock protein 70 during oxidative stress Bimei Jiang1, Bin Zhang1, Pengfei Liang2, Juan Song1, Hongbing Deng1, Zizhi Tu1, Gonghua Deng1 and Xianzhong Xiao1 Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan, China Department of Burns and Plastic Surgery, Xiangya Hospital, Central South University, Changsha, Hunan, China Keywords apoptosis; cardiomyocytes; heat shock protein 70; hydrogen peroxide; nucleolin ⁄ C23 Correspondence Xianzhong Xiao, Department of Pathophysiology, Xiangya School of Medicine, Central South University, Changsha, Hunan 410008, China Fax: +86 731 82355019 Tel: +86 731 2355019 E-mail: xianzhongxiao@xysm.net (Received 22 August 2009, revised 26 October 2009, accepted 23 November 2009) doi:10.1111/j.1742-4658.2009.07510.x Although heat shock protein 70 (Hsp70) has been shown to markedly inhibit H2O2-induced apoptosis in C2C12 cells, and nucleolin ⁄ C23 has also been implicated in apoptosis, the relationship of these two molecules is still largely unknown The aim of the current study was to investigate the potential involvement of nucleolin ⁄ C23 in the antiapoptotic mechanism of Hsp70 We found that primary cultures of neonatal rat cardiomyocytes underwent apoptosis upon H2O2 treatment, and in these cells nucleolin ⁄ C23 protein was highly unstable and had a half-life of less than h However, transfection with Hsp70 greatly stabilized nucleolin ⁄ C23 and also protected the cells from H2O2-induced apoptosis When nucleolin ⁄ C23 was knocked down with an antisense oligomer, H2O2-induced apoptosis became more severe, even in Hsp70-overexpressed cells, demonstrating an essential role of nucleolin ⁄ C23 in the antiapoptotic effects of Hsp70 Similar results were obtained by both nuclear morphology observation and caspase-3 activity assay Therefore, these data provide evidence that nucleolin ⁄ C23 is an essential downstream effecter of Hsp70 in the protection of cardiomyocytes against oxidative stress-induced apoptosis Introduction Characterized by cellular and nuclear shrinkage, cytoplasmic blebbing, chromatin condensation and DNA fragmentation [1,2], apoptosis can cause irreversible loss of terminally differentiated cardiac myocytes and, therefore, contributes significantly to the pathogenesis of many cardiovascular diseases Apoptosis has been identified in cardiac myocytes from patients suffering from myocardial infarction, diabetic cardiomyopathy, and end-stage congestive heart failure Apoptosis is a highly regulated programme of cell death and can be mediated by death receptors in the plasma membrane, as well as in the mitochondria and the endoplasmic reticulum [3] Studies on apoptotic pathways in cardiomyocytes have revealed several molecules as key regulators [4] Heat shock protein 70 (Hsp70) is a major stress-inducible heat shock protein that has been shown to protect cells from apoptosis induced by heat shock, tumour necrosis factor, growth factor withdrawal, oxidative stress and radiation [5,6] Hsp70 is also a major self-preservation protein in the heart; its overexpression enhances myocardial tolerance to ischaemia ⁄ reperfusion injury in both transgenic animals [7] and cell cultures [8] Although substantial progress has been made in understanding the control and mechanisms of apoptosis, how Hsp70 protects cardiomyocytes against apoptosis induced by a variety of stresses remains to be investigated Abbreviations DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hsp70, heat shock protein 70; PBS, phosphate-buffered saline; PI, pyridine iodination; SEM, standard error of the mean 642 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al Nucleolin ⁄ C23 is a 110 kDa multifunctional phosphoprotein It is abundantly expressed in both normal and cancerous cells [9], and ubiquitously distributed in the nucleolus, nucleus and cytoplasm of the cell, as well as at the cell surface [10] Nucleolin ⁄ C23 regulates many aspects of DNA and RNA metabolism, such as RNA polymerase I-mediated transcription, the folding, maturation, ribosome assembly and nucleocytoplasmic transport of newly synthesized pre-rRNAs [11] Because of its widespread distribution and broad range of involvement, as well as the cross-talking among molecules of different cellular processes, dissection of nucleolin ⁄ C23’s specific functions is particularly challenging Recently, nucleolin ⁄ C23 has also been implicated in apoptosis All-trans retinoic acid-induced apoptosis leads to nucleolin downregulation and bcl-2 mRNA instability; overexpression of nucleolin ⁄ C23 in chronic lymphocytic leukaemia cells stabilizes bcl-2 mRNA [12,13] Our previous study showed that oxidative stress induces nucleolin ⁄ C23 cleavage and apoptosis [14], and that nucleolin downregulation induces apoptosis in C2C12 cells [15] These data suggest the roles of nucleolin ⁄ C23 in oxidative stress-induced apoptosis, but whether nucleolin ⁄ C23 participates in Hsp70-mediated protection against H2O2-induced apoptosis is not clear In the present investigation, we further studied the effects of nucleolin ⁄ C23 ablation on Hsp70-mediated protection from apoptosis and examined the expression and stability of nucleolin ⁄ C23 with Hsp70 overexpression in response to H2O2-induced apoptosis using primary culture cardiomyocytes A B C Results Overexpression of Hsp70 protects cardiomyocytes from H2O2-induced apoptosis First, to overexpress Hsp70 we transiently transfected cardiomyocytes with a plasmid carrying human Hsp70 cDNA, and examined the protein levels by western blot analysis with Hsp70 antiserum after 36 h The efficiency of transfection mediated by the Lipofectamine Plus reagent ranged between 30 and 50% (data not shown) As can be seen in Fig 1A, Hsp70 overexpression was confirmed in the cells transfected with Hsp70 cDNA, but not in the cells transfected with the vector alone To determine the effect of Hsp70 overexpression on H2O2-induced apoptosis, the above transfected cells were exposed to H2O2 (0.5 mmolỈL)1) for the indicated time periods and the percentages of apoptotic cells examined by morphological observation (Fig 1B) Fig Overexpression of Hsp70 renders cardiomyocytes resistant to H2O2 Cardiomyocytes were transiently transfected with fulllength Hsp70 plasmid pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) or pcDNA vector (pcDNA ⁄ C) (A) Immunoblot analysis of Hsp70 expression At 36 h post-transfection, cells were analysed by immunoblotting with antibodies against Hsp70 or GAPDH (as an internal control of protein loading) The results shown are representative of three independent experiments (B) Quantification of apoptotic cells by nuclear staining Untransfected or transfected cells were exposed to 0.5 mmolỈL)1 H2O2 for the indicated durations and stained with Hoechst 33258; apoptotic cells showing condensed chromatin fragments were scored and expressed as the percentage of the total cell number counted The results from three independent experiments are presented as mean ± SEM *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2 group (n = 3) (C) Caspase-3 activity assay Thirty-six hours after transient transfection, cells were treated with or without 0.5 mmolỈL)1 H2O2 At different time points, cells were harvested for protease activity of caspase-3 using the caspase colorimetric assay kit Data were obtained from four independent experiments *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2 group FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 643 Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al After 24 h of H2O2 exposure,  44% of untransfected control cells and 45% of cells transfected with the empty vector (pcDNA3.1) exhibited similar features of apoptotic cells, such as highly condensed nuclei and decreased cell size However, only 22% of the cells transfected with Hsp70 underwent apoptosis (Fig 1B) We also investigated the effects of Hsp70 on caspase-3 activation induced by oxidative stress Using an in vitro substrate-cleavage reaction, H2O2 treatment significantly induced the activation of caspase-3 in the untransfected control cells as well as cells transfected with the vector alone in a time-dependent manner, but Hsp70 overexpression significantly reduced caspase-3 activation in response to H2O2 treatment (Fig 1C) Increased nucleolin/C23 protein levels in Hsp70-transfected cells in response to H2O2-induced apoptosis We next examined the expression of nucleolin ⁄ C23 protein in these cells in response to H2O2 exposure Upon treatment with H2O2 for up to h, all cells showed a time-dependent decrease in the 110 kDa nucleolin ⁄ C23 fragment accompanied by the appearance and an increase in the 80 kDa fragment (Fig 2) Although both the untransfected cells and the cells transfected with the vector alone started to show the 80 kDa fragment at time points as early as 2–4 h after H2O2 treatment, this smaller protein fragment appeared in the Hsp70-overexpressed cells at the h time point (Fig 2A,B) Overall, the change in nucleolin ⁄ C23 protein levels was least significant in cells transfected with Hsp70 cDNA (Fig 2B), suggesting a protective effect of Hsp70 on the degradation of nucleolin ⁄ C23 protein Prolonged half-life of nucleolin/C23 in Hsp70-transfected cells in response to H2O2 exposure To compare the stability of nucleolin ⁄ C23 protein in cells transfected with the vector alone and cells transfected with Hsp70 cDNA during H2O2 exposure, the cells were treated with H2O2 for h Protein synthesis was then inhibited by cycloheximide (5 lgỈmL)1) Western blot analysis revealed a time-dependent rapid decline in nucleolin ⁄ C23 110 kDa bands in H2O2-treated cells transfected with the vector alone; the half-life of the protein was estimated to be less than h (Fig 3, left panel) At the same time, the 80 kDa degraded product was detected in an increasing amount in H2O2-treated cells transfected with the vector alone shortly after cycloheximide treatment (2–8 h) By contrast, nucleolin ⁄ C23 in cells transfected with Hsp70 cDNA was more stable in response to H2O2 exposure, reaching its half-life at h after cycloheximide treatment (Fig 3, right panel) Overexpression of nucleolin/C23 protects cardiomyocytes from H2O2-induced apoptosis To determine the effect of nucleolin ⁄ C23 overexpression on H2O2-induced apoptosis in cardiomyocytes, we first transiently transfected cells with plasmids carrying human nucleolin ⁄ C23 cDNA or the empty vector Using western blot analysis we confirmed nucleolin ⁄ C23 overexpression only in cells transfected with nucleolin ⁄ C23 cDNA but not the empty vector (pcDNA ⁄ C) (Fig 4A) Then, transfected cells were exposed to H2O2 (0.5 mmolỈL)1) and apoptosis was assayed by both Hoechst nuclear staining (Fig 4B) H2O2 A Ctrl 0h 4h 8h 12 h Nucleolin/C23 GAPDH B PcDNA/C cells with H2O2 exposure Hsp70/C cells with H2O2 exposure 0h 0h 2h 4h 8h 2h 4h 8h Nucleolin/C23 GAPDH 644 Fig Changes in nucleolin ⁄ C23 protein levels in cells transfected with pcDNA vector (pcDNA ⁄ C) or Hsp70 cDNA (Hsp70 ⁄ C) during H2O2 exposure Cardiomyocytes were untransfected (A) or transiently transfected with pcDNA vector (pcDNA ⁄ C) or pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) for 36 h (B), and were treated with 0.5 mmolỈL)1 H2O2 for 0–8 h Cells were harvested and protein lysates were prepared for western blot analysis with monoclonal antibodies against nucleolin ⁄ C23 or GAPDH The results shown are the representatives of three independent experiments FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al Hsp70/C cells with H2O2 exposure h pcDNA/C cells with H2O2 exposure h CHX h 2h 4h 8h 0h 2h 4h 8h Nucleolin/C23 GAPDH Fig Nucleolin ⁄ C23 protein is stabilized in Hsp70-transfected cells during H2O2 exposure Cardiomyocytes were transiently transfected with pcDNA vector (pcDNA ⁄ C) or pcDNA ⁄ Hsp70 (Hsp70 ⁄ C) After treatment with 0.5 mmolỈL)1 H2O2 for h, cycloheximide (CHX; lgỈmL)1) was added At different time points (2–8 h), cells were harvested and protein lysates were prepared for western blot analysis with monoclonal antibodies against nucleolin ⁄ C23 or GAPDH The results shown are the representatives of three independent experiments and caspase-3 activity assays (Fig 4C) By counting apoptotic nuclei, at 24 h H2O2 exposure,  45% of cells transfected with the vector alone were apoptotic However, only 22% of cells transfected with nucleolin ⁄ C23 cDNA underwent apoptosis (Fig 4B) Consistently, H2O2 treatment significantly induced caspase-3 activation in cells transfected with the vector alone in a time-dependent manner, and nucleolin ⁄ C23 overexpression significantly reduced caspase-3 activation (Fig 4C) However, simultaneous Hsp70 overexpression afforded the cells no significant protection by nucleolin ⁄ C23 against oxidant stress (Fig 4D) In addition, H2O2 induced apoptosis in cardiomyocytes with a marked downregulation of Bcl-2 protein, and nucleolin overexpression could inhibit the downregulation of Bcl-2 protein (Fig 5A) Furthermore, we compared the stability of Bcl-2 mRNA in cells transfected with nucleolin cDNA (pcDNA3.1-C23) or vector control (pcDNA3.1) in the presence or absence of H2O2 by incubating the cells with actinomycin D (5 lgỈmL)1) for up to h Bcl-2 mRNA in H2O2-treated vector control cells was found to be highly unstable In contrast, Bcl-2 in H2O2-treated nucleolin-overexpressed cells was significantly more stable (Fig 5B) Potentiation of apoptosis in Hsp70-transfected cells by nucleolin/C23 antisense oligomer Next, we used the gene knockdown approach with an antisense oligonucleotide to examine whether nucleolin ⁄ C23 is necessary in the antiapoptotic function of Hsp70 Fluorescein isothiocyanate (FITC)-labelled phosphorothioate oligodeoxynucleotides were successfully transfected into primary cultured cardiomyocytes with Lipofectamine 2000 and achieved a transfection efficacy of  80% (Fig 6A) Transfection of nucleolin ⁄ C23 antisense oligonucleotide selectively reduced the levels of endogenous nucleolin ⁄ C23 protein by more than 75% after 36 h, whereas transfection of the scrambled control oligonucleotide did not alter nucleolin ⁄ C23 protein levels (Fig 6B) Then, Hsp70 overexpressing cells were transfected with nonspecific control or nucleolin ⁄ C23 antisense oligomers and the percentages of apoptotic cells in response to H2O2 treatment were examined by three methods First, by nuclear staining the apoptotic rates were 9–25% in cells transfected with the control oligomers and much lower at 13 and 44% in cells transfected with the nucleolin ⁄ C23 antisense oligomer (Fig 6C) Second, the activity of caspase-3 was analysed as a marker of apoptotic cells using an in vitro substrate-cleaving reaction As shown in Fig 6D, H2O2-induced apoptosis was increased in cells transfected with nucleolin ⁄ C23 antisense oligonucleotide compared with those transfected with the scrambled control oligonucleotide, even in the presence of Hsp70 overexpression Finally, apoptosis was further assayed by flow cytometry with annexin V–FITC ⁄ pyridine iodination (PI) double staining, which identified apoptotic cells as annexin V positive, but PI negative This method obtained results consistent with those from the other two methods, showing that when nucleolin ⁄ C23 was knocked down with an antisense oligomer, H2O2induced apoptosis was enhanced, even with the Hsp70overexpressed cells (Fig 7A,B) Discussion Both our previous study [16] and the present study have provided evidence that oxidative stress induces apoptosis of cardiomyocytes, which can be characterized by nuclear condensation and cell shrinkage Interestingly, upon transfection with an Hsp70-containing plasmid, these same cells become much less sensitive to H2O2 exposure This observation was confirmed by both cell morphology and caspase-3 activity analyses FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 645 Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 Hsp70 has been shown nalling, effecter molecule stream events of caspase cathepsins, cytochrome c, Ctrl A pcDNA3.1 B Jiang et al to regulate apoptotic sigactivation, certain downactivation, the release of apoptosis-inducing factor pcDNA3.1-C23 Nucleolin/C23 GAPDH Percentage of apoptotic nuclei B 60 * 50 40 30 # 20 10 pcDNA3.1 + H2O2 pcDNA3.1-C23 + H2O2 Ctrl and even the death-associated lysosome permeabilization [17–21] Particularly, Hsp70 inhibits the release of cytochrome c and apoptosis-inducing factor from mitochondria and prevents apoptosis [22,23] Our previous study showed that Hsp70 overexpression inhibited H2O2-mediated release of Smac (the second mitochondria-derived activator of caspases), activation of caspase-3 and caspase-9, and apoptosis in C2C12 myogenic cells [24] Stankiewicz et al [25] found that Hsp70 overexpression stabilizes Mcl-1 protein in heat-shocked cells Hsp70 has also been suggested to play an important role in preconditioning, a phenomenon of protection of a heart from strong ischaemic insult by prior exposure to mild ischaemia or other mild stresses In the present study, we have shown that Hsp70 renders nucleolin ⁄ C23 enhanced stability; however, a decrease in nucleolin ⁄ C23 stability restrains such a protective effect of Hsp70 against the H2O2-induced apoptosis These data suggest that Hsp70 may inhibit apoptosis at multiple points and through a wide range of mechanisms Nucleolin is a major nucleolar phosphoprotein that belongs to a large family of RNA-binding proteins [26] It plays a role in the pre-rRNA transcription Caspase-3 activity (fold increase) C4 * 3.5 2.5 # 1.5 0.5 pcDNA3.1 + H2O2 pcDNA3.1-C23 + H2O2 Ctrl * 3.5 2.5 # # # 1.5 0.5 70 DN A3 1 -H -H sp sp 70 pc DN A3 pc DN A 1- C2 + pc C2 1pc DN A3 pc DN A3 rl Ct Caspase-3 activity (fold increase) D 0.5 mmol·L–1 H2O2 646 Fig Overexpression of nucleolin ⁄ C23 protects cardiomyocytes from H2O2-induced apoptosis Cardiomyocytes were transiently transfected with full-length nucleolin plasmid (pcDNA3.1-C23) or pcDNA3.1 vector (A) Immunoblot analysis of nucleolin ⁄ C23 expression At 36 h post-transfection, cells were analysed by immunoblotting with antibodies against nucleolin ⁄ C23 or GAPDH (as an internal control of protein loading) The results shown are representatives of three independent experiments (B) Quantification of apoptotic cells by nuclear staining Untransfected or transfected cells were exposed to 0.5 mmolỈL)1 H2O2 for the indicated durations and stained with Hoechst 33258; apoptotic cells showing condensed chromatin fragments were scored and expressed as a percentage of the total cell number counted The results from three independent experiments are presented as means ± SEM *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2 group (n = 3) (C) Caspase-3 activity assay Thirty-six hours after transient transfection, cells were treated with or without 0.5 mmolỈL)1 H2O2 for 12 h and harvested for the determination of protease activity of caspase-3 using the caspase colorimetric assay kit Data were obtained from four independent experiments *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2 group (D) Caspase-3 activity assay Thirty-six hours after transfection with pcDNA3.1-C23 or pcDNA3.1-Hsp70 or transient cotransfection with pcDNA3.1-C23 and pcDNA3.1-Hsp70, cells were treated with or without 0.5 mmolỈL)1 H2O2 for 12 h and harvested for protease activity of caspase-3 Data were obtained from four independent experiments *P < 0.05 versus control group; #P < 0.05 versus pcDNA3.1 + H2O2 group FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al H2O2 A pcDNA3.1 pcDNA3.1 pcDNA3.1-C23 Bcl-2 GAPDH Bcl-2 mRNA/GAPDH mRNA (%) B 100 10 0.5 Time (h) Fig Effect of nucleolin ⁄ C23 on Bcl-2 protein expression and mRNA stability in the presence or absence of H2O2 (A) Western blot analyses showing the effect of nucleolin ⁄ C23 on Bcl-2 protein expression Total cellular proteins were isolated and separated by 12% SDS ⁄ PAGE and analysed by immunoblotting with anti-Bcl-2 Immunoblot analyses with GAPDH antiserum were used as the protein loading control (B) Real-time PCR showing the effect of nucleolin ⁄ C23 on Bcl-2 mRNA stability Cells transfected with pcDNA3.1-C23 or the empty vector were cultured in the absence or presence of H2O2 (0.5 mM) for h Actinomycin D (5 lgỈmL)1) was then added to the media and the cells were further incubated for various times (0–3 h) Total RNAs were prepared and subjected to real-time PCR analyses The amount of Bcl-2 mRNA was normalized by that of GAPDH All experiments were performed in triplicate and shown here by one representative *P < 0.05 versus pcDNA3.1-transfected control group and ribosome assembly that is implicated in the early stage of preribosomal ribonucleoprotein assembly and processing [27] Nucleolin has also been suggested to regulate cell proliferation and growth, cytokinesis, replication, embryogenesis and nucleogenesis [28] by forming large molecular complexes with other factors, such as casein kinase II, c-Myb, midkine, histone H1, nucleophosmin, p53 and protein phosphatase A number of studies, including our own, indicate that nucleolin ⁄ C23 may be one of the key components in the regulation of apoptosis [12–14,29–31] In the present study, we found that overexpression of nucleolin ⁄ C23 protects cardiomyocytes from H2O2-induced apoptosis, and that the mechanism is probably associ- ated with the increase in Bcl-2 mRNA stability (Fig 5) In addition, we have also established a strong relationship between nucleolin ⁄ C23 and the antiapoptotic effect of Hsp70 Nucleolin ⁄ C23 in Hsp70-transfected cells is significantly stabilized during H2O2 exposure, and simultaneous Hsp70 overexpression affords no additional protection against oxidant stress (Fig 4D) Therefore, Hsp70-transfected cells with a higher level of nucleolin ⁄ C23 protein become more resistant to oxidative stress and less susceptible to apoptotic death More importantly, the role of nucleolin ⁄ C23 in this scenario is indispensable, as the suppression of nucleolin ⁄ C23 expression by an antisense oligomer potentiates apoptosis, even in the Hsp70transfected cells These results also indicate that the nucleolin ⁄ C23 gene has a functional role in the growth control of cardiomyocytes, and its regulation may be closely associated with the susceptibility of the cell to the induction of apoptosis Caspases are important molecular mediators of apoptosis in the cell [32] Assays for caspase-3 activity have also confirmed that H2O2 exposure induces cardiomyocytes to undergo apoptosis and Hsp70 overexpression prevents H2O2-induced caspase activation; when nucleolin ⁄ C23 protein is suppressed by the antisense oligonucleotide, the protection of Hsp70 is lost Taken together, our results demonstrate an important relationship among the regulation of nucleolin ⁄ C23, the activation of caspase-3 and the induction of apoptosis under the setting of oxidative stress and Hsp70 overexpression More importantly, for the first time, we provide strong evidence that nucleolin ⁄ C23 is a downstream mediator of Hsp70’s antiapoptosis effects and it functions at the level of protein stability in cardiomyocytes It is our hope that such studies on the mechanisms of apoptosis in cardiomyocytes will provide a molecular basis for new therapeutic strategies targeting specific pathways to treat human heart disease Materials and methods Animals Neonatal Wistar rats (1-3 days) were purchased from the Animal Resource Center of Center South University The following procedures were approved by the Institutional Animal Care and Use Committee of the Center South University, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals All efforts were made to minimize the number of animals used and their suffering FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 647 Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al Cell culture and treatment Primary cultures of neonatal rat cardiomyocytes were cultivated as previously described [33,34] Briefly, hearts from A Hoechst 33258 staining GFP Hsp70/C cells B Ctrl Lip ScrODNs AsODNs neonatal Wistar rats (1–3 days after birth) were removed, minced and trypsinized at 37 °C with gentle stirring in D-Hanks buffer containing 0.1% trypsin (Gibco, Rockville, MD, USA) The cells were then centrifuged and resuspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 15% fetal bovine serum (Hyclone, Logan, UT, USA) After incubation at 37 °C for 120 min, the suspended cardiomyocytes were seeded at a density of · 105 cellsỈmL)1 All cells were cultured in DMEM containing 15% fetal bovine serum for 24 h before the initiation of experiments 5-Bromo-2¢-deoxyuridine (Sigma, St Louis, MO, USA) (0.1 mmolỈL)1) was added to the culture for 36 h to inhibit the proliferation of nonmyocytes H2O2 was first diluted in phosphate-buffered saline (PBS, pH 7.4) and further diluted in culture medium to a final concentration of 0.5 mmolỈL)1 for all treatments Cycloheximide (Sigma) was diluted in the medium to a final concentration of lgỈmL)1 Nucleolin/C23 Quantification of apoptotic cells Hsp70 At the predetermined time points after H2O2 exposure, cells were detached from tissue culture plates with trypsin ⁄ EDTA or cell scrapers, and collected together with the nonadherent cells After centrifugation at 500 g for min, the cells were fixed with 4% paraformaldehyde for 30 at room temperature and then washed once with PBS The fixed cells were then incubated with Hoechst 33258 (50 ngỈmL)1) for 30 at room temperature, and washed GAPDH 60 C Percentage of apoptotic nuclei Hsp70/C + NCL/C23-AsODNs Hsp70/C + NCL/C23-ScrODNs 50 * * 40 30 20 10 0 Caspase-3 activity (fold increase) D 12 24 Time (h) of H2O2 exposure Hsp70/C + NCL/C23-AsODNs Hsp70/C + NCL/C23-ScrODNs 2.5 * 36 * 1.5 0.5 0 648 12 Time (h) of H2O2 exposure 24 Fig Potentiation of apoptosis in Hsp70-overexpressed cells by nucleolin ⁄ C23 antisense oligomer (A) Transfection efficiency of phosphorothioate oligodeoxynucleotides in primary cultures of cardiomyocytes FITC-labelled phosphorothioate oligodeoxynucleotides were transfected into cells with Lipofectamine 2000 and cells were stained with Hoechst 33258 Right, the image under blue fluorescence channel showing all cells with nuclear Hoechst staining; left, image under green fluorescence channel showing only cells positive for FITC-labelled oligomers (B) Knocking-down of nucleolin ⁄ C23 in cardiomyocytes The Hsp70-transfected cardiomyocytes (Hsp70 ⁄ C) were cotransfected with nucleolin ⁄ C23 scrambled (NCL ⁄ C23-ScrODNs) or antisense (NCL ⁄ C23-AsODNs) oligomers After 36 h, cells were harvested for western blot analysis with monoclonal antibody against nucleolin ⁄ C23 (NCL ⁄ C23) or GAPDH (C) Determination of the percentage of apoptotic cells by Hoechst staining Hsp70 ⁄ C cells were cotransfected with NCL ⁄ C23-ScrODNs or NCL ⁄ C23-AsODNs and then treated with H2O2 for 6–36 h The results from three independent experiments are shown as mean ± SEM *P < 0.05 versus NCL ⁄ C23-ScrODNs group (D) Determination of percentage of apoptotic cells by caspase-3 activity assay Hsp70 ⁄ C cells were transfected and treated as above, and assayed for caspase-3 activity Data were obtained from four independent experiments *P < 0.05 versus NCL ⁄ C23ScrODNs group FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al A Fig Flow cytometry analysis of apoptosis with annexin V–FITC ⁄ PI double staining Hsp70-transfected cardiomyocytes (Hsp70 ⁄ C) were cotransfected with the nucleolin ⁄ C23 scrambled (NCL ⁄ C23-ScrODNs) or antisense (NCL ⁄ C23-AsODNs) oligomers Twenty-four hours later, they were treated with 0.5 mmolỈL)1 H2O2 for or 12 h Cells were harvested and processed for annexin V–FITC and PI costaining and analysed by flow cytometry (A) Q3 cells were the control cells, Q4 cells were cells at the early apoptosis stage, Q2 cells were cells at the late apoptosis stage, and Q1 cells were necrotic cells The percentage of apoptotic cells was also calculated (B) The results are representative of three independent experiments Data are the mean ± SEM of triplicate samples *Significant difference (P < 0.05) versus the pcDNA3.1 control group; #significant difference (P < 0.05) versus the control group B again with PBS Cells were mounted on to glass slides and examined by fluorescence microscopy Apoptotic cells were identified by the condensation and fragmentation of their nuclei The percentage of apoptotic cells was calculated as the number of apoptotic cells divided by the total number of cells counted A minimum of 500 cells were counted for each slide Flow cytometry Both adherent and floating cells were collected after treatment, washed with ice-cold PBS and stained with FITC-conjugated annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and PI for 20 at room temperature in the dark The stained cells were then analysed by a flow cytometer FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 649 Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al (Beckman Coulter, Fullerton, CA, USA) FITC-conjugated annexin V binds to phosphatidylserine molecules only present at the surface of apoptotic cells where they were translocated from the internal side of the plasma membrane during apoptosis Cells were simultaneously stained with PI to discriminate membrane-permeable necrotic cells from FITC-labelled apoptotic cells Apoptotic cells were identified as those positive for annexin V–FITC staining but negative for PI staining, and the percentage of apoptotic cells in the total number of cells was calculated Western blot analyses Cells were washed with PBS and collected by centrifugation Cell pellets were resuspended with volumes of cold lysis buffer [50 mmolỈL)1 Tris ⁄ HCl (pH 7.5), 250 mmolỈL)1 NaCl, mmolỈL)1 EDTA, 50 mmolỈL)1 NaF, 0.5% Nonidet P-40] containing a protease inhibitor mixture (Roche Applied Science, Burgess Hill, UK) The cell lysate was incubated on ice for 30 and centrifuged at 10 000 g for 10 at °C The protein concentration of the supernatant was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA) Equal amounts of protein (5–10 lg) were loaded on to and separated by SDS ⁄ PAGE and transferred to a nitrocellulose membrane The blot was blocked with 2% albumin in 20 mmolỈL)1 Tris ⁄ HCl, pH 8.0, 150 mmolỈL)1 NaCl, 0.1% Tween 20 (TBST) overnight at °C and then incubated with the rabbit antiC23 (Sigma) or anti-(glyceraldehyde-3-phosphate dehydrogenase) (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for h After washing with TBST buffer three times for 15 each, the blot was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (diluted : 2000 in TBST buffer) at room temperature for h, and finally washed three times with TBST for 15 each Immunoreactivity was visualized using the enhanced chemiluminescence reaction (Amersham, Piscataway, NJ, USA) Caspase activity assay The activity of caspase-3 was measured by in vitro substrate-cleavage reactions using a commercial kit according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA) Briefly, cells cultured in 60 mm dishes were treated with 0.5 mmolỈL)1 H2O2 for the indicated time periods The cells (5 · 106) were lysed with 250 lL chilled cell lysis buffer on ice for 10 After centrifugation (10 000 g, min, °C), the protein concentration in the supernatant was determined using the BioRad protein assay Supernatants containing equal amounts of protein (corresponding to  · 105 cells) were used for caspase-3 colorimetric assays After protein samples were incubated with substrates at 37 °C for 1.5 h, the absorbance at 405 nm was measured using a microtitre plate reader 650 (Molecular Devices, Sunnyvale, CA, USA) Fold increases in caspase-3 activity over that before treatment were determined Lipofectamine-mediated gene transfection Transfection of cells was carried out following the manufacturer’s instructions (Lipofectamine 2000Ô, Invitrogen, Carlsbad, CA, USA) Briefly,  · 105 cells in mL appropriate complete growth medium per flask were grown at 37 °C in a CO2 incubator until the cells reached 70–80% confluence (24 h) After rinsing with serum-free and antibiotic-free medium, the cells were transfected with pcDNA3.1-Hsp70 (HspA1A was a gift from I Benjamin, University of Utah Health Sciences Center) (experimental group) or pcDNA3.1 (vector control), at lg DNA per 20 lL Lipofectamine, followed by incubation at 37 °C in a CO2 incubator for h The medium was then replaced with DMEM culture medium containing 20% fetal bovine serum Nucleolin/C23 antisense oligodeoxynucleotide The phosphorothioate oligodeoxynucleotides used in this study were manufactured by Bioasia Biotech (Shanghai, China) The sequence of nucleolin ⁄ C23 antisense oligodeoxynucleotide (NUL ⁄ C23-AsODNs), corresponding to the rat nucleolin ⁄ C23 translation initiation site, was 5¢-TG CGAGTTTCACCATGATGGC-3¢ A scrambled nucleolin ⁄ C23 oligodeoxynucleotide (NUL ⁄ C23-ScrODNs; 5¢-CTGA TGTCACGTCCATGTGAG-3¢) was used as the control The oligodeoxynucleotides were diluted in 10 mmolỈL)1 Tris (pH 7.4) and mmolỈL)1 EDTA and kept at )20 °C RT-PCR Total RNAs were prepared with the Rneasy kit (Qiagen, Cambridge, MA, USA) according to the manufacturer’s instructions and lg samples were reverse transcribed using oligo(dT) primers The levels of gene expression were quantified by real-time PCR, using a QuantiTect SYBR Green PCR Kit (Qiagen) and the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 40 sequential cycles, each including 95 °C for 10 s and 60 °C for 10 s Sequences of primers specific to each target cDNA were: nucleolin, forward, 5-CA ATCAGGCTGGAGTTGCAAG-3; and reverse, 5-TGGC CCAGTCCAAGGTAACTT-3 (amplicon size: 282 bp); GAPDH forward, 5-ACCACAGTCCATGCCATCAC-3; and reverse, 5-TCCACCACCCTGTTGCTGTA-3 (size: 440 bp) The specificity of PCR products was verified by melting curve analysis and electrophoresis on agarose gels The comparative threshold cycle method and an internal control (GAPDH) were used to normalize target gene expression FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 B Jiang et al Statistical analyses 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and hypoxia-induced apoptosis of cultured neonatal cardiomyocytes J Clin Invest 99, 2635–2643 FEBS Journal 277 (2010) 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS ... among the regulation of nucleolin ⁄ C23, the activation of caspase-3 and the induction of apoptosis under the setting of oxidative stress and Hsp70 overexpression More importantly, for the first... Xiao X (2009) ATP-binding domain of heat shock protein 70 is essential for its effects on the inhibition of the release of the second mitochondria-derived activator of caspase and apoptosis in C2C12... 642–652 ª 2009 The Authors Journal compilation ª 2009 FEBS 645 Nucleolin ⁄ C23 in antiapoptotic effect of Hsp70 Hsp70 has been shown nalling, effecter molecule stream events of caspase cathepsins,