- Email: [email protected]
reoxygenation-induced apoptosis in primary neonatal rat cardiomyocytes
Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Effect of hypoxia-inducible factor 1-alpha on hypoxia/reoxygenation-induced apoptosis in primary neonatal rat cardiomyocytes Xiaoou Wang a, Shuai Ma b, Guoxian Qi a,⇑ a b
Department of Cardiology, First Affiliated Hospital, China Medical University, Shenyang, China Department of Surgical Oncology, Department of General Surgery, First Affiliated Hospital, China Medical University, Shenyang, China
a r t i c l e
i n f o
Article history: Received 13 December 2011 Available online 29 December 2011 Keywords: HIF-1a Cardiomyocyte Hypoxia/reoxygenation Apoptosis
a b s t r a c t We studied the role of hypoxia-inducible factor 1-alpha (HIF-1a) in hypoxia/reoxygenation (H/R)-induced apoptosis in primary neonatal rat cardiomyocytes and its possible molecular mechanisms. Isolated neonatal and adult rat cardiac myocytes were cultured for 48 h and were submitted to 5 h of hypoxia followed by 2, 6, or 12 h of reoxygenation. Small interfering RNA was used to target the HIF-1a gene. Cardiac myocyte apoptosis induced by H/R was assessed by Annexin V-FITC apoptosis assay. HIF-1a, Bnip3 and caspase-3 levels were determined by real-time reverse transcription polymerase chain reaction and western blot for mRNA and protein, respectively. H/R resulted in severe injury in cultured rat cardiomyocytes and it upregulated HIF-1a and proapoptotic Bnip3 mRNA and protein expression. HIF-1a activity inhibited by siRNA significantly decreased (P < 0.01) the rate of apoptotic cardiomyocytes induced by 5 h of hypoxia followed by 6 h of reoxygenation compared with cardiomyocytes without siRNA treatment. Additionally, the expression of Bnip3 and caspase-3 was also markedly reduced. We conclude that HIF-1a is a key regulator of apoptosis of cardiomyocytes induced by H/R. H/R enhances primary neonatal rat cardiomyocyte apoptosis through the activation of HIF-1a and the mechanism might involve Bnip3 and caspase-3. HIF-1a may be a possible therapeutic target to limit myocardial injury after myocardial infarction. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Hypoxia/reoxygenation (H/R) injury is a complex process in cells, especially in cardiomyocytes. It has been demonstrated that hypoxia and reoxygenation lead to cell death, and it is believed that cell death in cardiomyocytes occurs via necrosis as well as apoptosis [1,2]. Necrosis is a destructive and uncontrolled process, whereas apoptosis is a highly regulated process of programmed cell suicide. Apoptosis is an important mechanism of cell death in cardiomyocyte H/R injury [1]. Hypoxia-inducible factor (HIF) transcription factors function as master regulators of gene expression in response to hypoxia [3]. HIF comprises a set of transcription factors that regulate the expression of approximately 200 genes, which can affect the cellular adaptive responses to hypoxia and/ or ischemia [4,5]. HIF is a heterodimer comprising a and b subunits, which are constitutively expressed. HIF-1a may function to regulate cell death or cell survival. HIF1a-dependent genes such as heme oxygenase-1, inducible nitric oxide synthase, cyclooxygnease-2 and vascular endothelial growth ⇑ Corresponding author. Address: Department of Cardiology, First Affiliated Hospital of China Medical University, Nanjing Street 155, Shenyang 110001, China. Fax: +86 24 8328 2226. E-mail addresses: [email protected] (X. Wang), shuaima0809@ hotmail.com (S. Ma), [email protected] (G. Qi). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.115
factor can regulate cell survival in ischemia/reperfusion (I/R) [6–9]. HIF-1a also activates the transcription of pro-death genes such as Bcl-2 family members Bcl-2/adenovirus E1B 19-kDa protein -interacting protein 3 (Bnip3) and BMF [10] and the death ligand FasL [11]. Bnip3 is a BH3-only protein that is primarily localized in the mitochondria [12–14]. Overexpression of Bnip3 leads to the activation of Bax/Bak, opening of the mitochondrial permeability transition pore, and cell death [13,15,16]. Bnip3 is a significant contributor to I/R injury by inducing mitochondrial dysfunction [13,17]. Therefore, the role of HIF-1a in apoptosis remains controversial and appears to be cell type and context specific. This study aimed to determine the function of HIF-1a in H/R -induced injury in primary neonatal rat myocytes and the underlying molecular mechanisms. The method of small interfering RNA (siRNA) was used to silence the expression of HIF-1a and observe the effect of changes in HIF-1a on apoptosis.
2. Materials and methods 2.1. Reagents Neonatal Sprague–Dawley rats were purchased from the Experimental Animal Center of China Medical University. Small interfering RNA was obtained from GenePharma Co., Ltd. Antibodies raised
1228
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
against the following proteins were used in this investigation: HIF1a, Bnip3 (Santa Cruz Biotechnology, Inc.), and cleaved caspase-3 (Cell Signaling). Lipofectamine 2000 was purchased from Invitrogen. Our experiments were carried out according to the EU Directive 2010/63/EU for animal experiments.
2.2. Cell culture Primary cultures of neonatal rat cardiac myocytes were prepared as previously described with some modifications [18]. Briefly, hearts were removed from 1- to 2-day-old Sprague-Dawley rats anesthetized by ether under aseptic conditions and placed in Ca2+- and Mg2+-free phosphate buffered saline (PBS). The hearts were washed with PBS, and the atria and aorta were discarded. The ventricles were minced with scissors into 1 mm3 fragments, and they were enzymatically digested four times for 10–15 min each with 7.5 mL of PBS containing 0.2% collagenase (Sigma type II). The liberated cells were collected by centrifugation at 300g and incubated in a 25 cm2 flask (Falcon) for 60 min at 37 °C in a humidified incubator with 5% CO2 air. The nonadherent cardiac myocytes were harvested and seeded into 6-well plates (8 105 cell/well) or 24-well plates (2 105 cells/well) and incubated. The cardiomyocytes were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone). The compound 5-bromo-29-deoxyuridine (BrdU; 100 mmol/L) was added during the first 48 h to inhibit proliferation of nonmyocytes [19]. Using this method, we routinely obtained contractile myocyte-rich cultures with 90–95% myocytes, as assessed by immunofluorescence staining with a monoclonal antibody against b-myosin heavy chain. The cardiomyocytes were then incubated in DMEM containing 10% FBS without BrdU, and all assays were performed after 48 h of incubation.
2.3. Protocol for H/R injury H/R to cardiomyocytes was undertaken as described previously [20]. Before hypoxic exposure, cell medium was replaced by modified Tyrode’s solution (in mmol/L: NaCl 136.9, KCl 2.68, Na2HPO412 H2O 8.1, KH2PO4 1.47, CaCl2 0.9, MgCl26 H2O 0.49; pH 7.2). The cardiomyocytes were transferred to and kept in an anaerobic incubator flushed with 5% CO2 and 95% N2 for 5 h. They were then reoxygenated for 2, 6 or 12 h with DMEM containing 10% FBS. For the control normoxic treatment, cells were maintained at 37 °C in a modular culture incubator with 5% CO2 in air. Cells were collected for the experiments as described below.
2.5. RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated and purified from cell pellets using the RNeasy mini kit as recommended by the manufacturer (Qiagen). Quantitative real-time RT-PCR was performed using an ABI Prism 7000 Real-Time PCR System (Applied Biosystems) and a SYBR PrimeScript RT-PCR Two-Step Kit (TaKaRa) according to the manufacturer’s protocols for the SYBER Green method. To ensure the fidelity of the mRNA extraction and reverse transcription, this value was normalized to the value of the internal standard glyceraldehyde phosphate dehydrogenase (GAPDH) for each analysis. 2.6. Western blot analysis Semiquantitative analysis of proteins in cell lysates was performed by western blotting [21]. Primary antibodies to HIF-1a, Bnip3, b-actin (Santa Cruz Biotechnology, Inc.) or cleaved caspase-3 (Cell Signaling) were diluted in 5% milk/TBST and incubated overnight at 4 °C (1:1000). IgG horseradish peroxidase-linked secondary antibodies was diluted 1:2000. Proteins were visualized using the enhanced chemiluminescence plus system (Pierce) according to the manufacturer’s instructions. 2.7. Annexin V-FITC apoptosis assay Apoptosis was assessed by the Annexin V-FITC Apoptosis Detection kit (KeyGEN) according to the manufacturer’s protocol. Briefly, cells were washed with ice-cold PBS, and then resuspended in 500 ll of binding buffer at 1 106 cells/ml. A total volume of 5 ll Annexin V-FITC stock solution was added to the cells, which were incubated in the dark for 15 min at room temperature. Immediately after mixing with 5 ll propidium iodide solution for another 5 min in the dark, the samples were analyzed by flow cytometry with use of a FACSort flow cytometer (Becton-Dickinson). Approximately 10,000 cells were counted in each of the samples, and data were analyzed using WinMDI software (v2.9, Bio-Soft Net). 3. Statistical analyses Data are presented as mean ± standard deviation (SD). Multiple comparisons between experimental groups were made using ANOVA followed by Dunnett’s post-hoc test, which was evaluated at the 0.05 level of significant significance. Statistical analyses were performed using Statistical Package for the Social Sciences software, version 15.0 (SPSS, Chicago, IL, USA).
2.4. HIF-1a siRNA transfection
4. Results
The cardiomyocytes were seeded in 6-well plates. The cells grew to 70–80% confluence after 2 days, and transfection with either a SMARTpool siRNA specific for HIF-1a (100 nmol/L) or a nontargeting siRNA as a negative control (100 nmol/L) using Lipofectamine 2000 (Invitrogen) was performed according to the manufacturer’s instructions. Cardiomyocytes were divided into three groups: the nontransfection, scramble and siRNA groups. The nontransfection group included cardiomyocytes without transfection with siRNA. The scramble group was cardiomyocytes transfected with nontargeting siRNA. The siRNA group was cardiomyocytes transfected with targeting siRNA. Each group of cardiomyocytes underwent normoxia, hypoxia for 5 or 5 h of hypoxia followed by 6 h of reoxygenation (5H/6R). Forty-eight hours after transfection, the cell experienced normoxia, hypoxia for 5 h or 5H/6R and were separately collected after reoxygenation for 6 h in preparation for the following experiments.
4.1. HIF-1a mRNA and protein levels are upregulated following cardiomyocyte apoptosis induced by H/R To investigate the relationship between HIF-1a and cardiomyocyte apoptosis, primary rat cardiomyocytes underwent normoxia, 5 h of hypoxia or 5 h of hypoxia followed by 2, 6 or 12 h of reoxygenation. We found that the expression level of HIF-1a and the amount of apoptotic cardiomyocytes were significantly increased (P < 0.01) after hypoxia compared with the normoxia group (Fig. 1). We also found that the rate of apoptotic cardiomyocytes was markedly upregulated after cardiomyocytes were treated by 5 h of hypoxia followed by 2 h of reoxygenation compared with that in the hypoxia group (P < 0.05, Fig. 1B), and it peaked at 6 h of reoxygenation (P < 0.01, Fig. 1B). Interestingly, exposure of cardiomyocytes to H/R caused a significant increase (P < 0.05) in the expression of HIF-1a mRNA and protein relative to the hypoxia
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
group and it peaked at reoxygenation at 6 h (Fig. 1C and D). These data provided evidence that HIF-1a might play a role in cardiomyocyte apoptosis induced by H/R. In the following experiments, we only used 5H/6R in cardiomyocytes. 4.2. Upregulation of Bnip3 expression is consistent with HIF-1a upregulation To determine Bnip3 expression levels, primary rat cardiomyocytes underwent normoxia, 5 h of hypoxia or 5 h of hypoxia followed by 2, 6 or 12 h of reoxygenation. The expression of Bnip3 mRNA and protein was markedly increased (P < 0.05,) after cardiomyocytes were treated by H/R compared with that in the hypoxia group, and it peaked with reoxygenation at 6 h (Fig. 2). The change
1229
in Bnip3 mRNA and protein levels was similar to that of HIF-1a in cardiomyocytes induced by H/R (Fig. 2C). Taken together, these results suggest that there is a relationship between HIF-1a and Bnip3 in cardiomyocyte apoptosis induced by H/R. In the following experiments, we only used 5H/6R in cardiomyocytes. 4.3. Inhibition of HIF-1a by siRNA decreases apoptotic cardiomyocytes induced by 5H/6R If HIF-1a contributes to cardiomyocyte apoptosis induced by 5H/6R, then it is possible that inhibition of HIF-1a can reduce cardiomyocyte apoptosis. To test this hypothesis, we used siRNA to silence the expression of HIF-1a. Transfection efficiencies were monitored by evaluating the expression of HIF-1a mRNA induced
Fig. 1. H/R results in cardiomyocyte apoptosis and upregulates HIF-1a expression. Cardiomyocytes were divided into the following groups: normoxia, exposed to 5 h of hypoxia or exposed to 5 h of hypoxia followed by 2, 6, or 12 h of reoxygenation (5H/2R, 5H/6R, or 5H/12R, respectively). Apoptosis of cardiomyocytes and HIF-1a expression were analyzed. (A) Apoptotic cell death was measured by staining with Annexin V-FITC/PI. (B) The percentage of apoptosis among the different experimental groups is shown in the upper panel. ⁄⁄P < 0.01, hypoxia versus normoxia; #P < 0.05, 5H/2R, 5H/12R versus hypoxia; ##P < 0.01, 5H/6R versus hypoxia. (C) Relative amount (fold induction relative to normoxic levels) of HIF-1a mRNA. ⁄⁄P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; #P < 0.05, 5H/12R versus hypoxia. (D) Top, western blot analysis of HIF-1a in cardiomyocytes in all the five groups. Bottom, a histogram shows the results of densitometric quantification of the HIF-1a/b-actin ratio. ⁄⁄ P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; #P < 0.05, 5H/12R versus hypoxia.
1230
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
Fig. 2. Bnip3 mRNA and protein levels are upregulated by H/R, consistent with levels of HIF-1a. (A) H/R significantly induced expression of the Bnip3 gene in cardiomyocytes. The relative amount (fold induction relative to normoxic levels) of Bnip3 mRNA is shown. ⁄⁄P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; # P < 0.05, 5H/12R versus hypoxia. (B) Top, western blot analysis indicates that H/R markedly increases Bnip3 expression in cardiomyocytes. Bottom, a histogram shows the results of densitometric quantification of the Bnip3/b-actin ratio. ⁄⁄P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; #P < 0.05, 5H/12R versus hypoxia. (C) HIF-1a and Bnip3 protein expression in cardiomyocytes. The highest expression of HIF-1a and Bnip3 protein was at 5H/6R.
by hypoxia before or after transfection in cardiomyocytes. The average transfection efficiency of 3 independent experiments (n = 3) was 39.52% ± 5.84%. Our data showed that siRNA targeting HIF-1a resulted in a marked reduction in the expression of HIF1a mRNA and protein after 5H/6R compared with that in the scramble and nontransfection groups (Fig. 3A and B, P < 0.01). The expression of HIF-1a mRNA and protein in the siRNA group after 5H/6R was higher than that with normoxia in the nontransfection group (P < 0.01). In the siRNA group, HIF-1a mRNA and
protein expression were not significantly different between hypoxia and 5H/6R conditions (Fig. 3A and B, P > 0.05). HIF-1a expression was inhibited by siRNA, and the number of apoptotic cardiomyocytes was significantly reduced compared with that in the scramble and nontransfection groups after 5H/6R (Fig. 3D, P < 0.01). However, the number of apoptotic cardiomyocytes in the siRNA group after 5H/6R was still higher than that with normoxia in the nontransfection group (Fig. 3D, P < 0.05). These data showed that inhibition of HIF-1a by siRNA partly rescued cardiomyocytes
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
1231
Fig. 3. Inhibition of HIF-1a by siRNA decreases cardiomyocyte apoptosis induced by 5H/6R. Cardiomyocytes were divided into three groups: nontransfection, scramble and siRNA groups. Cardiomyocytes of each group experienced normoxia, hypoxia for 5 h or 5H/6R. They were then subjected to Annexin V-FITC apoptosis assay, real-time RT-PCR and western blot analysis. (A) Relative amount (fold induction relative to normoxic levels) of HIF-1a mRNA. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. There was no significant difference between the siRNA group subjected to hypoxia for 5 h and 5H/6R. (B) Top, Western blot analysis of HIF-1a expression in cardiomyocytes. Bottom, a histogram shows results of densitometric quantification of the HIF-1a/b-actin ratio. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. (C) Apoptotic cells were evaluated by staining with Annexin V-FITC/PI. (D) The percentage of apoptosis among the groups is shown in the upper panel. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; # P < 0.05, siRNA 5H/6R versus nontransfection normoxia.
1232
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
from apoptosis induced by 5H/6R, and that siRNA inhibited HIF-1a expression during hypoxia as well as during reoxygenation. 4.4. Inhibition of HIF-1a by siRNA decreases the expression of caspase3 and Bnip3 The expression of capase-3 mRNA in the nontransfection group was markedly increased after 5H/6R compared with that after hypoxia (Fig. 4A, P < 0.01). Cleaved caspase-3, the activated form of caspase-3, was significantly increased after 5H/6R compared with that after hypoxia in the nontransfection group (Fig. 4B, P < 0.01). However, the effect of 5H/6R on caspase-3 was reduced when HIF-1a expression was knocked down by siRNA treatment compared with the scramble and nontransfection groups (Fig. 4A and B, P < 0.01). We also found that inhibition of HIF-1a by siRNA resulted in markedly decreased Bnip3 in cardiomyocytes treated with 5H/6R compared with that in the scramble and nontransfection groups (Fig. 4C and D, P < 0.01). Taken together, these results suggest that siRNA against HIF-1a protects cardiomyocytes from apoptosis induced by 5H/6R, and this is correlated with the activation of Bnip3.
5. Discussion The role of HIF-1a in the regulation of gene expression in response to hypoxia and as a nuclear factor has been studied extensively [4]. Hypoxia can induce HIF-1a protein accumulation and mediate a hypoxia-adaptational response. HIF-1a can stimulate the mitochondrial apoptotic pathway and cell death during hypoxia [22,23]. Some studies have also demonstrated that HIF-1a can protect cardiomyocytes from I/R-induced injury with preconditioning or postconditioning [24–26]. Recent reports have illustrated that HIF-1a triggers apoptosis following I/R through the induction of hypoxically regulated genes in the liver and gut [27,28]. However, the role of HIF-1a in H/R-induced apoptosis remains controversial. To address this problem, we first investigated the role of HIF-1a in response to apoptosis of cardiomyocytes induced by H/R. We cultured primary neonatal rat ventricular myocytes under different times of reoxygenation and examination of these cells revealed that mRNA and protein levels of HIF-1a were upregulated following apoptosis of cardiomyocytes (Fig. 1). These data suggest that an increase in HIF-1a stimulates apoptosis of cardiomyocytes. Therefore, we hypothesized that if an increase in
Fig. 4. Inhibition of HIF-1a by siRNA decreases the expression levels of caspase-3 and Bnip3. Cells were subjected to real time RT-PCR and western blot analysis. (A) Relative amount (fold induction relative to normoxic levels) of caspase-3 mRNA. &&P < 0.01, nontransfection 5H/6R versus hypoxia for 5 h; ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. There was no difference between the siRNA group subjected to hypoxia and 5H/6R. (B) Top, Western blot analysis of cleaved caspase-3 in cardiomyocytes. Bottom, a histogram shows results of densitometric quantification of the cleaved caspase-3/b-actin ratio. && P < 0.01, nontransfection 5H/6R versus hypoxia for 5 h. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. (C) Relative amount (fold induction relative to normoxic levels) of Bnip3 mRNA. ⁄⁄P < 0.01 nontransfection 5H/6R, scramble 5H/6R versus siRNA group 5H/6R. #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. (D) Top, Western blot analysis of Bnip3 in cardiomyocytes. Bottom, a histogram shows results of densitometric quantification of the Bnip3/b-actin ratio. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia.
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
HIF-1a stimulates apoptosis of cardiomyocytes, then a reduction in HIF-1a will suppress apoptosis of cardiomyocytes. In our study, siRNA against HIF-1a was transferred into cardiomyocytes. Our results demonstrated that knockdown of HIF-1a decreased apoptosis of cardiomyocytes (Fig. 3) and that HIF-1a is an important regulator of apoptosis in cardiomyocytes when induced by H/R. Caspase-3 is well known as one of the key executioners of apoptosis. Activation of caspases-3 requires proteolytic processing of its inactive zymogen into activated p17 and p19 subunits [29]. Cleaved caspase-3 is regarded as a primary activator of apoptosis. We found that the expression of caspase-3 mRNA and cleaved caspase-3 protein was significantly decreased in cardiomyocytes treated with H/R that received siRNA treatment against HIF-1a compared with that in cardiomyocytes without siRNA (Fig. 4A and B). Our data suggest that HIF-1a triggers apoptosis induced by H/R in cardiomyocytes via activation of caspase-3 protein expression in the executive phase of apoptosis. Recently, some reports have centered on the effects of HIF-1a on transactivation of the Bnip3 gene in certain cells such as cardiac myocytes [23,30,31]. The Bnip3 promoter has been identified to contain the binding sites for HIF-1a [32,33]. Bnip3 is a member of the ‘‘BH3-only’’ subfamily of proapoptotic Bcl-2 family proteins. The BH3 domain is essential for the cell death activity of these proteins, as well as for mediating heterodimerization with anti- or proapoptotic proteins, which regulate cell death [34]. Some studies have demonstrated that Bnip3 displays proapoptotic activity in cardiomyocytes [13,35]. To examine whether Bnip3 participates in HIF-1a-induced apoptosis in H/R in cardiomyocytes, we first examined the expression of Bnip3. We found that the expression of Bnip3 protein and mRNA was markedly upregulated after cardiomyocytes were subjected to H/R compared with that in the hypoxia group, and that the increase in expression of Bnip3 peaked at reoxygenation at 6 h, which is consistent with the expression of HIF-1a Fig. 2. We also found the HIF-1a siRNA treatment significantly reduced the expression of Bnip3 (Fig. 4C and D), which is consistent with a recent report that inhibition of HIF-1a expression markedly reduces Bnip3 expression [36]. We propose that reduction of HIF-1a has an anti-apoptotic effect on cardiomyocytes through down regulation of Bnip3 expression. However, many questions remain to be answered, including ‘‘how does HIF-1a activate its downstream protein Bnip3?’’ and ‘‘what is the mechanism of Bnip3 in HIF-1a-mediated apoptosis in cultured primary neonatal rat cardiomyocytes?’’. In conclusion, the present results suggest that apoptosis in primary neonatal rat cardiomyocytes by H/R involves activated HIF1a, Bnip3, and caspase-3, and that HIF-1a-mediates apoptosis probably via activation of genes encoding Bnip3. This new insight into HIF-1a function is important because HIF-1a may be an attractive therapeutic target to limit myocardial injury after myocardial infarction. References [1] F.Y. Ho, W.P. Tsang, S.K. Kong, T.T. Kwok, The critical role of caspases activation in hypoxia/reoxygenation induced apoptosis, Biochem. Biophys. Res. Commun. 345 (2006) 1131–1137. [2] R. Dworakowski, D. Dworakowska, I. Kocic, T. Wirth, M. Gruchala, M. Kaminski, J. Petrusewicz, S. Yla-Herttuala, A. Rynkiewicz, Cerivastatin and hypercholesterolemia reduce apoptosis of cardiomyocytes in guinea pig papillary muscle subjected to hypoxia/reoxygenation, Pharmacol. Rep. 58 (2006) 420–426. [3] N.M. Mazure, M.C. Brahimi-Horn, M.A. Berta, E. Benizri, R.L. Bilton, F. Dayan, A. Ginouves, E. Berra, J. Pouyssegur, HIF-1: master and commander of the hypoxic world. A pharmacological approach to its regulation by siRNAs, Biochem. Pharmacol. 68 (2004) 971–980. [4] P.T. Schumacker, Hypoxia-inducible factor-1 (HIF-1), Crit. Care Med. 33 (2005) S423–425. [5] G. Loor, P.T. Schumacker, Role of hypoxia-inducible factor in cell survival during myocardial ischemia-reperfusion, Cell Death Differ. 15 (2008) 686–690.
1233
[6] B. Crowe, J.A. Poynter, M.C. Manukyan, Y. Wang, B.D. Brewster, J.L. Herrmann, A.M. Abarbanell, B.R. Weil, D.R. Meldrum, Pretreatment with intracoronary mimosine improves postischemic myocardial functional recovery, Surgery 150 (2011) 191–196. [7] A.R. Knudsen, A.S. Kannerup, H. Gronbaek, K.J. Andersen, P. Funch-Jensen, J. Frystyk, A. Flyvbjerg, F.V. Mortensen, Effects of ischemic pre- and postconditioning on HIF-1alpha, VEGF and TGF-beta expression after warm ischemia and reperfusion in the rat liver, Comp. Hepatol. 10 (2011) 3. [8] B.S. Rayner, T.T. Duong, S.J. Myers, P.K. Witting, Protective effect of a synthetic anti-oxidant on neuronal cell apoptosis resulting from experimental hypoxia re-oxygenation injury, J. Neurochem. 97 (2006) 211–221. [9] A.A. Samoilenko, The role of hypoxia-inducible factor family (HIF) proteins in the regulation of cells physiologic responses to hypoxia, Ukr. Biokhim. Zh. 82 (2010) 5–17. [10] K.A. Webster, R.M. Graham, N.H. Bishopric, BNip3 and signal-specific programmed death in the heart, J. Mol. Cell. Cardiol. 38 (2005) 35–45. [11] G. Yaniv, M. Shilkrut, R. Lotan, G. Berke, S. Larisch, O. Binah, Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes, Cardiovasc. Res. 54 (2002) 611–623. [12] G. Chen, R. Ray, D. Dubik, L. Shi, J. Cizeau, R.C. Bleackley, S. Saxena, R.D. Gietz, A.H. Greenberg, The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis, J. Exp. Med. 186 (1997) 1975–1983. [13] A. Hamacher-Brady, N.R. Brady, S.E. Logue, M.R. Sayen, M. Jinno, L.A. Kirshenbaum, R.A. Gottlieb, A.B. Gustafsson, Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy, Cell Death Differ. 14 (2007) 146–157. [14] Y. Lee, H.Y. Lee, R.A. Hanna, A.B. Gustafsson, Mitochondrial autophagy by Bnip3 involves Drp1-mediated mitochondrial fission and recruitment of Parkin in cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol. 301 (2011) H1924–1931. [15] K.M. Regula, K. Ens, L.A. Kirshenbaum, Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes, Circ. Res. 91 (2002) 226–231. [16] X. Feng, X. Liu, W. Zhang, W. Xiao, P53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death, EMBO J. 30 (2011) 3397–3415. [17] R.M. Graham, J.W. Thompson, J. Wei, N.H. Bishopric, K.A. Webster, Regulation of Bnip3 death pathways by calcium, phosphorylation, and hypoxiareoxygenation, Antioxid. Redox Signal 9 (2007) 1309–1315. [18] K. Goshima, Quabain-induced arrhythmias of single isolated myocardial cells and cell clusters cultured in vitro and their improvement by quinidine, J. Mol. Cell. Cardiol. 9 (1977) 7–23. [19] J. Sadoshima, L. Jahn, T. Takahashi, T.J. Kulik, S. Izumo, Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy, J. Biol. Chem. 267 (1992) 10551–10560. [20] W.T. Chang, Z.H. Shao, J.J. Yin, S. Mehendale, C.Z. Wang, Y. Qin, J. Li, W.J. Chen, C.T. Chien, L.B. Becker, T.L. Vanden Hoek, C.S. Yuan, Comparative effects of flavonoids on oxidant scavenging and ischemia-reperfusion injury in cardiomyocytes, Eur. J. Pharmacol. 566 (2007) 58–66. [21] J. Cheng, J. Du, Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulin-like growth factor-1 receptor, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1744–1751. [22] K.D. Mansfield, R.D. Guzy, Y. Pan, R.M. Young, T.P. Cash, P.T. Schumacker, M.C. Simon, Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation, Cell Metab. 1 (2005) 393–399. [23] Y.F. Zhou, X.W. Zheng, G.H. Zhang, Z.H. Zong, G.X. Qi, The effect of hypoxiainducible factor 1-alpha on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes, Cardiovasc. J. Afr. 21 (2010) 37–41. [24] Y. Guo, T. Yang, J. Lu, S. Li, L. Wan, D. Long, Q. Li, L. Feng, Y. Li, Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway, Life Sci. 88 (2011) 598–605. [25] Z. Cai, H. Zhong, M. Bosch-Marce, K. Fox-Talbot, L. Wang, C. Wei, M.A. Trush, G.L. Semenza, Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1 alpha, Cardiovasc. Res. 77 (2008) 463–470. [26] F. Du, L. Zhu, Z.M. Qian, X.M. Wu, W.H. Yung, Y. Ke, Hyperthermic preconditioning protects astrocytes from ischemia/reperfusion injury by upregulation of HIF-1 alpha expression and binding activity, Biochim. Biophys. Acta 1802 (2010) 1048–1053. [27] K.B. Kannan, I. Colorado, D. Reino, D. Palange, Q. Lu, X. Qin, B. Abungu, A. Watkins, F.J. Caputo, D.Z. Xu, G.L. Semenza, E.A. Deitch, R. Feinman, Hypoxiainducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury, Am. J. Physiol. Gastrointest. Liver Physiol. 300 (2011) G853–861. [28] R. Cursio, C. Miele, N. Filippa, E. Van Obberghen, J. Gugenheim, Liver HIF-1 alpha induction precedes apoptosis following normothermic ischemiareperfusion in rats, Transplant. Proc. 40 (2008) 2042–2045. [29] T. Hoshi, H. Sasano, K. Kato, N. Yabuki, S. Ohara, R. Konno, S. Asaki, T. Toyota, H. Tateno, H. Nagura, Immunohistochemistry of Caspase3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis, Anticancer Res. 18 (1998) 4347–4353. [30] R.M. Graham, D.P. Frazier, J.W. Thompson, S. Haliko, H. Li, B.J. Wasserlauf, M.G. Spiga, N.H. Bishopric, K.A. Webster, A unique pathway of cardiac myocyte death caused by hypoxia-acidosis, J. Exp. Biol. 207 (2004) 3189–3200.
1234
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
[31] A.E. Greijer, E. van der Wall, The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis, J. Clin. Pathol. 57 (2004) 1009–1014. [32] C. Kuiper, I.G. Molenaar, G.U. Dachs, M.J. Currie, P.H. Sykes, M.C. Vissers, Low ascorbate levels are associated with increased hypoxia-inducible factor-1 activity and an aggressive tumor phenotype in endometrial cancer, Cancer Res. 70 (2010) 5749–5758. [33] J.K. Chen, L.J. Hu, J. Wang, K.R. Lamborn, E.L. Kong, D.F. Deen, Hypoxia-induced BAX overexpression and radiation killing of hypoxic glioblastoma cells, Radiat. Res. 163 (2005) 644–653.
[34] K. Wang, X.M. Yin, D.T. Chao, C.L. Milliman, S.J. Korsmeyer, BID: a novel BH3 domain-only death agonist, Genes Dev. 10 (1996) 2859–2869. [35] D.A. Kubli, M.N. Quinsay, C. Huang, Y. Lee, A.B. Gustafsson, Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion, Am. J. Physiol. Heart Circ. Physiol. 295 (2008) H2025–2031. [36] R. Malhotra, D.W. Tyson, H.M. Rosevear, F.C. Brosius 3rd, Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells, BMC Cardiovasc. Disord. 8 (2008) 9.
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Effect of hypoxia-inducible factor 1-alpha on hypoxia/reoxygenation-induced apoptosis in primary neonatal rat cardiomyocytes Xiaoou Wang a, Shuai Ma b, Guoxian Qi a,⇑ a b
Department of Cardiology, First Affiliated Hospital, China Medical University, Shenyang, China Department of Surgical Oncology, Department of General Surgery, First Affiliated Hospital, China Medical University, Shenyang, China
a r t i c l e
i n f o
Article history: Received 13 December 2011 Available online 29 December 2011 Keywords: HIF-1a Cardiomyocyte Hypoxia/reoxygenation Apoptosis
a b s t r a c t We studied the role of hypoxia-inducible factor 1-alpha (HIF-1a) in hypoxia/reoxygenation (H/R)-induced apoptosis in primary neonatal rat cardiomyocytes and its possible molecular mechanisms. Isolated neonatal and adult rat cardiac myocytes were cultured for 48 h and were submitted to 5 h of hypoxia followed by 2, 6, or 12 h of reoxygenation. Small interfering RNA was used to target the HIF-1a gene. Cardiac myocyte apoptosis induced by H/R was assessed by Annexin V-FITC apoptosis assay. HIF-1a, Bnip3 and caspase-3 levels were determined by real-time reverse transcription polymerase chain reaction and western blot for mRNA and protein, respectively. H/R resulted in severe injury in cultured rat cardiomyocytes and it upregulated HIF-1a and proapoptotic Bnip3 mRNA and protein expression. HIF-1a activity inhibited by siRNA significantly decreased (P < 0.01) the rate of apoptotic cardiomyocytes induced by 5 h of hypoxia followed by 6 h of reoxygenation compared with cardiomyocytes without siRNA treatment. Additionally, the expression of Bnip3 and caspase-3 was also markedly reduced. We conclude that HIF-1a is a key regulator of apoptosis of cardiomyocytes induced by H/R. H/R enhances primary neonatal rat cardiomyocyte apoptosis through the activation of HIF-1a and the mechanism might involve Bnip3 and caspase-3. HIF-1a may be a possible therapeutic target to limit myocardial injury after myocardial infarction. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Hypoxia/reoxygenation (H/R) injury is a complex process in cells, especially in cardiomyocytes. It has been demonstrated that hypoxia and reoxygenation lead to cell death, and it is believed that cell death in cardiomyocytes occurs via necrosis as well as apoptosis [1,2]. Necrosis is a destructive and uncontrolled process, whereas apoptosis is a highly regulated process of programmed cell suicide. Apoptosis is an important mechanism of cell death in cardiomyocyte H/R injury [1]. Hypoxia-inducible factor (HIF) transcription factors function as master regulators of gene expression in response to hypoxia [3]. HIF comprises a set of transcription factors that regulate the expression of approximately 200 genes, which can affect the cellular adaptive responses to hypoxia and/ or ischemia [4,5]. HIF is a heterodimer comprising a and b subunits, which are constitutively expressed. HIF-1a may function to regulate cell death or cell survival. HIF1a-dependent genes such as heme oxygenase-1, inducible nitric oxide synthase, cyclooxygnease-2 and vascular endothelial growth ⇑ Corresponding author. Address: Department of Cardiology, First Affiliated Hospital of China Medical University, Nanjing Street 155, Shenyang 110001, China. Fax: +86 24 8328 2226. E-mail addresses: [email protected] (X. Wang), shuaima0809@ hotmail.com (S. Ma), [email protected] (G. Qi). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.115
factor can regulate cell survival in ischemia/reperfusion (I/R) [6–9]. HIF-1a also activates the transcription of pro-death genes such as Bcl-2 family members Bcl-2/adenovirus E1B 19-kDa protein -interacting protein 3 (Bnip3) and BMF [10] and the death ligand FasL [11]. Bnip3 is a BH3-only protein that is primarily localized in the mitochondria [12–14]. Overexpression of Bnip3 leads to the activation of Bax/Bak, opening of the mitochondrial permeability transition pore, and cell death [13,15,16]. Bnip3 is a significant contributor to I/R injury by inducing mitochondrial dysfunction [13,17]. Therefore, the role of HIF-1a in apoptosis remains controversial and appears to be cell type and context specific. This study aimed to determine the function of HIF-1a in H/R -induced injury in primary neonatal rat myocytes and the underlying molecular mechanisms. The method of small interfering RNA (siRNA) was used to silence the expression of HIF-1a and observe the effect of changes in HIF-1a on apoptosis.
2. Materials and methods 2.1. Reagents Neonatal Sprague–Dawley rats were purchased from the Experimental Animal Center of China Medical University. Small interfering RNA was obtained from GenePharma Co., Ltd. Antibodies raised
1228
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
against the following proteins were used in this investigation: HIF1a, Bnip3 (Santa Cruz Biotechnology, Inc.), and cleaved caspase-3 (Cell Signaling). Lipofectamine 2000 was purchased from Invitrogen. Our experiments were carried out according to the EU Directive 2010/63/EU for animal experiments.
2.2. Cell culture Primary cultures of neonatal rat cardiac myocytes were prepared as previously described with some modifications [18]. Briefly, hearts were removed from 1- to 2-day-old Sprague-Dawley rats anesthetized by ether under aseptic conditions and placed in Ca2+- and Mg2+-free phosphate buffered saline (PBS). The hearts were washed with PBS, and the atria and aorta were discarded. The ventricles were minced with scissors into 1 mm3 fragments, and they were enzymatically digested four times for 10–15 min each with 7.5 mL of PBS containing 0.2% collagenase (Sigma type II). The liberated cells were collected by centrifugation at 300g and incubated in a 25 cm2 flask (Falcon) for 60 min at 37 °C in a humidified incubator with 5% CO2 air. The nonadherent cardiac myocytes were harvested and seeded into 6-well plates (8 105 cell/well) or 24-well plates (2 105 cells/well) and incubated. The cardiomyocytes were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) (Hyclone). The compound 5-bromo-29-deoxyuridine (BrdU; 100 mmol/L) was added during the first 48 h to inhibit proliferation of nonmyocytes [19]. Using this method, we routinely obtained contractile myocyte-rich cultures with 90–95% myocytes, as assessed by immunofluorescence staining with a monoclonal antibody against b-myosin heavy chain. The cardiomyocytes were then incubated in DMEM containing 10% FBS without BrdU, and all assays were performed after 48 h of incubation.
2.3. Protocol for H/R injury H/R to cardiomyocytes was undertaken as described previously [20]. Before hypoxic exposure, cell medium was replaced by modified Tyrode’s solution (in mmol/L: NaCl 136.9, KCl 2.68, Na2HPO412 H2O 8.1, KH2PO4 1.47, CaCl2 0.9, MgCl26 H2O 0.49; pH 7.2). The cardiomyocytes were transferred to and kept in an anaerobic incubator flushed with 5% CO2 and 95% N2 for 5 h. They were then reoxygenated for 2, 6 or 12 h with DMEM containing 10% FBS. For the control normoxic treatment, cells were maintained at 37 °C in a modular culture incubator with 5% CO2 in air. Cells were collected for the experiments as described below.
2.5. RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated and purified from cell pellets using the RNeasy mini kit as recommended by the manufacturer (Qiagen). Quantitative real-time RT-PCR was performed using an ABI Prism 7000 Real-Time PCR System (Applied Biosystems) and a SYBR PrimeScript RT-PCR Two-Step Kit (TaKaRa) according to the manufacturer’s protocols for the SYBER Green method. To ensure the fidelity of the mRNA extraction and reverse transcription, this value was normalized to the value of the internal standard glyceraldehyde phosphate dehydrogenase (GAPDH) for each analysis. 2.6. Western blot analysis Semiquantitative analysis of proteins in cell lysates was performed by western blotting [21]. Primary antibodies to HIF-1a, Bnip3, b-actin (Santa Cruz Biotechnology, Inc.) or cleaved caspase-3 (Cell Signaling) were diluted in 5% milk/TBST and incubated overnight at 4 °C (1:1000). IgG horseradish peroxidase-linked secondary antibodies was diluted 1:2000. Proteins were visualized using the enhanced chemiluminescence plus system (Pierce) according to the manufacturer’s instructions. 2.7. Annexin V-FITC apoptosis assay Apoptosis was assessed by the Annexin V-FITC Apoptosis Detection kit (KeyGEN) according to the manufacturer’s protocol. Briefly, cells were washed with ice-cold PBS, and then resuspended in 500 ll of binding buffer at 1 106 cells/ml. A total volume of 5 ll Annexin V-FITC stock solution was added to the cells, which were incubated in the dark for 15 min at room temperature. Immediately after mixing with 5 ll propidium iodide solution for another 5 min in the dark, the samples were analyzed by flow cytometry with use of a FACSort flow cytometer (Becton-Dickinson). Approximately 10,000 cells were counted in each of the samples, and data were analyzed using WinMDI software (v2.9, Bio-Soft Net). 3. Statistical analyses Data are presented as mean ± standard deviation (SD). Multiple comparisons between experimental groups were made using ANOVA followed by Dunnett’s post-hoc test, which was evaluated at the 0.05 level of significant significance. Statistical analyses were performed using Statistical Package for the Social Sciences software, version 15.0 (SPSS, Chicago, IL, USA).
2.4. HIF-1a siRNA transfection
4. Results
The cardiomyocytes were seeded in 6-well plates. The cells grew to 70–80% confluence after 2 days, and transfection with either a SMARTpool siRNA specific for HIF-1a (100 nmol/L) or a nontargeting siRNA as a negative control (100 nmol/L) using Lipofectamine 2000 (Invitrogen) was performed according to the manufacturer’s instructions. Cardiomyocytes were divided into three groups: the nontransfection, scramble and siRNA groups. The nontransfection group included cardiomyocytes without transfection with siRNA. The scramble group was cardiomyocytes transfected with nontargeting siRNA. The siRNA group was cardiomyocytes transfected with targeting siRNA. Each group of cardiomyocytes underwent normoxia, hypoxia for 5 or 5 h of hypoxia followed by 6 h of reoxygenation (5H/6R). Forty-eight hours after transfection, the cell experienced normoxia, hypoxia for 5 h or 5H/6R and were separately collected after reoxygenation for 6 h in preparation for the following experiments.
4.1. HIF-1a mRNA and protein levels are upregulated following cardiomyocyte apoptosis induced by H/R To investigate the relationship between HIF-1a and cardiomyocyte apoptosis, primary rat cardiomyocytes underwent normoxia, 5 h of hypoxia or 5 h of hypoxia followed by 2, 6 or 12 h of reoxygenation. We found that the expression level of HIF-1a and the amount of apoptotic cardiomyocytes were significantly increased (P < 0.01) after hypoxia compared with the normoxia group (Fig. 1). We also found that the rate of apoptotic cardiomyocytes was markedly upregulated after cardiomyocytes were treated by 5 h of hypoxia followed by 2 h of reoxygenation compared with that in the hypoxia group (P < 0.05, Fig. 1B), and it peaked at 6 h of reoxygenation (P < 0.01, Fig. 1B). Interestingly, exposure of cardiomyocytes to H/R caused a significant increase (P < 0.05) in the expression of HIF-1a mRNA and protein relative to the hypoxia
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
group and it peaked at reoxygenation at 6 h (Fig. 1C and D). These data provided evidence that HIF-1a might play a role in cardiomyocyte apoptosis induced by H/R. In the following experiments, we only used 5H/6R in cardiomyocytes. 4.2. Upregulation of Bnip3 expression is consistent with HIF-1a upregulation To determine Bnip3 expression levels, primary rat cardiomyocytes underwent normoxia, 5 h of hypoxia or 5 h of hypoxia followed by 2, 6 or 12 h of reoxygenation. The expression of Bnip3 mRNA and protein was markedly increased (P < 0.05,) after cardiomyocytes were treated by H/R compared with that in the hypoxia group, and it peaked with reoxygenation at 6 h (Fig. 2). The change
1229
in Bnip3 mRNA and protein levels was similar to that of HIF-1a in cardiomyocytes induced by H/R (Fig. 2C). Taken together, these results suggest that there is a relationship between HIF-1a and Bnip3 in cardiomyocyte apoptosis induced by H/R. In the following experiments, we only used 5H/6R in cardiomyocytes. 4.3. Inhibition of HIF-1a by siRNA decreases apoptotic cardiomyocytes induced by 5H/6R If HIF-1a contributes to cardiomyocyte apoptosis induced by 5H/6R, then it is possible that inhibition of HIF-1a can reduce cardiomyocyte apoptosis. To test this hypothesis, we used siRNA to silence the expression of HIF-1a. Transfection efficiencies were monitored by evaluating the expression of HIF-1a mRNA induced
Fig. 1. H/R results in cardiomyocyte apoptosis and upregulates HIF-1a expression. Cardiomyocytes were divided into the following groups: normoxia, exposed to 5 h of hypoxia or exposed to 5 h of hypoxia followed by 2, 6, or 12 h of reoxygenation (5H/2R, 5H/6R, or 5H/12R, respectively). Apoptosis of cardiomyocytes and HIF-1a expression were analyzed. (A) Apoptotic cell death was measured by staining with Annexin V-FITC/PI. (B) The percentage of apoptosis among the different experimental groups is shown in the upper panel. ⁄⁄P < 0.01, hypoxia versus normoxia; #P < 0.05, 5H/2R, 5H/12R versus hypoxia; ##P < 0.01, 5H/6R versus hypoxia. (C) Relative amount (fold induction relative to normoxic levels) of HIF-1a mRNA. ⁄⁄P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; #P < 0.05, 5H/12R versus hypoxia. (D) Top, western blot analysis of HIF-1a in cardiomyocytes in all the five groups. Bottom, a histogram shows the results of densitometric quantification of the HIF-1a/b-actin ratio. ⁄⁄ P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; #P < 0.05, 5H/12R versus hypoxia.
1230
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
Fig. 2. Bnip3 mRNA and protein levels are upregulated by H/R, consistent with levels of HIF-1a. (A) H/R significantly induced expression of the Bnip3 gene in cardiomyocytes. The relative amount (fold induction relative to normoxic levels) of Bnip3 mRNA is shown. ⁄⁄P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; # P < 0.05, 5H/12R versus hypoxia. (B) Top, western blot analysis indicates that H/R markedly increases Bnip3 expression in cardiomyocytes. Bottom, a histogram shows the results of densitometric quantification of the Bnip3/b-actin ratio. ⁄⁄P < 0.01, hypoxia versus normoxia; ##P < 0.01, 5H/2R, 5H/6R versus hypoxia; #P < 0.05, 5H/12R versus hypoxia. (C) HIF-1a and Bnip3 protein expression in cardiomyocytes. The highest expression of HIF-1a and Bnip3 protein was at 5H/6R.
by hypoxia before or after transfection in cardiomyocytes. The average transfection efficiency of 3 independent experiments (n = 3) was 39.52% ± 5.84%. Our data showed that siRNA targeting HIF-1a resulted in a marked reduction in the expression of HIF1a mRNA and protein after 5H/6R compared with that in the scramble and nontransfection groups (Fig. 3A and B, P < 0.01). The expression of HIF-1a mRNA and protein in the siRNA group after 5H/6R was higher than that with normoxia in the nontransfection group (P < 0.01). In the siRNA group, HIF-1a mRNA and
protein expression were not significantly different between hypoxia and 5H/6R conditions (Fig. 3A and B, P > 0.05). HIF-1a expression was inhibited by siRNA, and the number of apoptotic cardiomyocytes was significantly reduced compared with that in the scramble and nontransfection groups after 5H/6R (Fig. 3D, P < 0.01). However, the number of apoptotic cardiomyocytes in the siRNA group after 5H/6R was still higher than that with normoxia in the nontransfection group (Fig. 3D, P < 0.05). These data showed that inhibition of HIF-1a by siRNA partly rescued cardiomyocytes
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
1231
Fig. 3. Inhibition of HIF-1a by siRNA decreases cardiomyocyte apoptosis induced by 5H/6R. Cardiomyocytes were divided into three groups: nontransfection, scramble and siRNA groups. Cardiomyocytes of each group experienced normoxia, hypoxia for 5 h or 5H/6R. They were then subjected to Annexin V-FITC apoptosis assay, real-time RT-PCR and western blot analysis. (A) Relative amount (fold induction relative to normoxic levels) of HIF-1a mRNA. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. There was no significant difference between the siRNA group subjected to hypoxia for 5 h and 5H/6R. (B) Top, Western blot analysis of HIF-1a expression in cardiomyocytes. Bottom, a histogram shows results of densitometric quantification of the HIF-1a/b-actin ratio. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. (C) Apoptotic cells were evaluated by staining with Annexin V-FITC/PI. (D) The percentage of apoptosis among the groups is shown in the upper panel. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; # P < 0.05, siRNA 5H/6R versus nontransfection normoxia.
1232
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
from apoptosis induced by 5H/6R, and that siRNA inhibited HIF-1a expression during hypoxia as well as during reoxygenation. 4.4. Inhibition of HIF-1a by siRNA decreases the expression of caspase3 and Bnip3 The expression of capase-3 mRNA in the nontransfection group was markedly increased after 5H/6R compared with that after hypoxia (Fig. 4A, P < 0.01). Cleaved caspase-3, the activated form of caspase-3, was significantly increased after 5H/6R compared with that after hypoxia in the nontransfection group (Fig. 4B, P < 0.01). However, the effect of 5H/6R on caspase-3 was reduced when HIF-1a expression was knocked down by siRNA treatment compared with the scramble and nontransfection groups (Fig. 4A and B, P < 0.01). We also found that inhibition of HIF-1a by siRNA resulted in markedly decreased Bnip3 in cardiomyocytes treated with 5H/6R compared with that in the scramble and nontransfection groups (Fig. 4C and D, P < 0.01). Taken together, these results suggest that siRNA against HIF-1a protects cardiomyocytes from apoptosis induced by 5H/6R, and this is correlated with the activation of Bnip3.
5. Discussion The role of HIF-1a in the regulation of gene expression in response to hypoxia and as a nuclear factor has been studied extensively [4]. Hypoxia can induce HIF-1a protein accumulation and mediate a hypoxia-adaptational response. HIF-1a can stimulate the mitochondrial apoptotic pathway and cell death during hypoxia [22,23]. Some studies have also demonstrated that HIF-1a can protect cardiomyocytes from I/R-induced injury with preconditioning or postconditioning [24–26]. Recent reports have illustrated that HIF-1a triggers apoptosis following I/R through the induction of hypoxically regulated genes in the liver and gut [27,28]. However, the role of HIF-1a in H/R-induced apoptosis remains controversial. To address this problem, we first investigated the role of HIF-1a in response to apoptosis of cardiomyocytes induced by H/R. We cultured primary neonatal rat ventricular myocytes under different times of reoxygenation and examination of these cells revealed that mRNA and protein levels of HIF-1a were upregulated following apoptosis of cardiomyocytes (Fig. 1). These data suggest that an increase in HIF-1a stimulates apoptosis of cardiomyocytes. Therefore, we hypothesized that if an increase in
Fig. 4. Inhibition of HIF-1a by siRNA decreases the expression levels of caspase-3 and Bnip3. Cells were subjected to real time RT-PCR and western blot analysis. (A) Relative amount (fold induction relative to normoxic levels) of caspase-3 mRNA. &&P < 0.01, nontransfection 5H/6R versus hypoxia for 5 h; ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. There was no difference between the siRNA group subjected to hypoxia and 5H/6R. (B) Top, Western blot analysis of cleaved caspase-3 in cardiomyocytes. Bottom, a histogram shows results of densitometric quantification of the cleaved caspase-3/b-actin ratio. && P < 0.01, nontransfection 5H/6R versus hypoxia for 5 h. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. (C) Relative amount (fold induction relative to normoxic levels) of Bnip3 mRNA. ⁄⁄P < 0.01 nontransfection 5H/6R, scramble 5H/6R versus siRNA group 5H/6R. #P < 0.05, siRNA 5H/6R versus nontransfection normoxia. (D) Top, Western blot analysis of Bnip3 in cardiomyocytes. Bottom, a histogram shows results of densitometric quantification of the Bnip3/b-actin ratio. ⁄⁄P < 0.01, nontransfection 5H/6R, scramble 5H/6R versus siRNA 5H/6R; #P < 0.05, siRNA 5H/6R versus nontransfection normoxia.
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
HIF-1a stimulates apoptosis of cardiomyocytes, then a reduction in HIF-1a will suppress apoptosis of cardiomyocytes. In our study, siRNA against HIF-1a was transferred into cardiomyocytes. Our results demonstrated that knockdown of HIF-1a decreased apoptosis of cardiomyocytes (Fig. 3) and that HIF-1a is an important regulator of apoptosis in cardiomyocytes when induced by H/R. Caspase-3 is well known as one of the key executioners of apoptosis. Activation of caspases-3 requires proteolytic processing of its inactive zymogen into activated p17 and p19 subunits [29]. Cleaved caspase-3 is regarded as a primary activator of apoptosis. We found that the expression of caspase-3 mRNA and cleaved caspase-3 protein was significantly decreased in cardiomyocytes treated with H/R that received siRNA treatment against HIF-1a compared with that in cardiomyocytes without siRNA (Fig. 4A and B). Our data suggest that HIF-1a triggers apoptosis induced by H/R in cardiomyocytes via activation of caspase-3 protein expression in the executive phase of apoptosis. Recently, some reports have centered on the effects of HIF-1a on transactivation of the Bnip3 gene in certain cells such as cardiac myocytes [23,30,31]. The Bnip3 promoter has been identified to contain the binding sites for HIF-1a [32,33]. Bnip3 is a member of the ‘‘BH3-only’’ subfamily of proapoptotic Bcl-2 family proteins. The BH3 domain is essential for the cell death activity of these proteins, as well as for mediating heterodimerization with anti- or proapoptotic proteins, which regulate cell death [34]. Some studies have demonstrated that Bnip3 displays proapoptotic activity in cardiomyocytes [13,35]. To examine whether Bnip3 participates in HIF-1a-induced apoptosis in H/R in cardiomyocytes, we first examined the expression of Bnip3. We found that the expression of Bnip3 protein and mRNA was markedly upregulated after cardiomyocytes were subjected to H/R compared with that in the hypoxia group, and that the increase in expression of Bnip3 peaked at reoxygenation at 6 h, which is consistent with the expression of HIF-1a Fig. 2. We also found the HIF-1a siRNA treatment significantly reduced the expression of Bnip3 (Fig. 4C and D), which is consistent with a recent report that inhibition of HIF-1a expression markedly reduces Bnip3 expression [36]. We propose that reduction of HIF-1a has an anti-apoptotic effect on cardiomyocytes through down regulation of Bnip3 expression. However, many questions remain to be answered, including ‘‘how does HIF-1a activate its downstream protein Bnip3?’’ and ‘‘what is the mechanism of Bnip3 in HIF-1a-mediated apoptosis in cultured primary neonatal rat cardiomyocytes?’’. In conclusion, the present results suggest that apoptosis in primary neonatal rat cardiomyocytes by H/R involves activated HIF1a, Bnip3, and caspase-3, and that HIF-1a-mediates apoptosis probably via activation of genes encoding Bnip3. This new insight into HIF-1a function is important because HIF-1a may be an attractive therapeutic target to limit myocardial injury after myocardial infarction. References [1] F.Y. Ho, W.P. Tsang, S.K. Kong, T.T. Kwok, The critical role of caspases activation in hypoxia/reoxygenation induced apoptosis, Biochem. Biophys. Res. Commun. 345 (2006) 1131–1137. [2] R. Dworakowski, D. Dworakowska, I. Kocic, T. Wirth, M. Gruchala, M. Kaminski, J. Petrusewicz, S. Yla-Herttuala, A. Rynkiewicz, Cerivastatin and hypercholesterolemia reduce apoptosis of cardiomyocytes in guinea pig papillary muscle subjected to hypoxia/reoxygenation, Pharmacol. Rep. 58 (2006) 420–426. [3] N.M. Mazure, M.C. Brahimi-Horn, M.A. Berta, E. Benizri, R.L. Bilton, F. Dayan, A. Ginouves, E. Berra, J. Pouyssegur, HIF-1: master and commander of the hypoxic world. A pharmacological approach to its regulation by siRNAs, Biochem. Pharmacol. 68 (2004) 971–980. [4] P.T. Schumacker, Hypoxia-inducible factor-1 (HIF-1), Crit. Care Med. 33 (2005) S423–425. [5] G. Loor, P.T. Schumacker, Role of hypoxia-inducible factor in cell survival during myocardial ischemia-reperfusion, Cell Death Differ. 15 (2008) 686–690.
1233
[6] B. Crowe, J.A. Poynter, M.C. Manukyan, Y. Wang, B.D. Brewster, J.L. Herrmann, A.M. Abarbanell, B.R. Weil, D.R. Meldrum, Pretreatment with intracoronary mimosine improves postischemic myocardial functional recovery, Surgery 150 (2011) 191–196. [7] A.R. Knudsen, A.S. Kannerup, H. Gronbaek, K.J. Andersen, P. Funch-Jensen, J. Frystyk, A. Flyvbjerg, F.V. Mortensen, Effects of ischemic pre- and postconditioning on HIF-1alpha, VEGF and TGF-beta expression after warm ischemia and reperfusion in the rat liver, Comp. Hepatol. 10 (2011) 3. [8] B.S. Rayner, T.T. Duong, S.J. Myers, P.K. Witting, Protective effect of a synthetic anti-oxidant on neuronal cell apoptosis resulting from experimental hypoxia re-oxygenation injury, J. Neurochem. 97 (2006) 211–221. [9] A.A. Samoilenko, The role of hypoxia-inducible factor family (HIF) proteins in the regulation of cells physiologic responses to hypoxia, Ukr. Biokhim. Zh. 82 (2010) 5–17. [10] K.A. Webster, R.M. Graham, N.H. Bishopric, BNip3 and signal-specific programmed death in the heart, J. Mol. Cell. Cardiol. 38 (2005) 35–45. [11] G. Yaniv, M. Shilkrut, R. Lotan, G. Berke, S. Larisch, O. Binah, Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas (CD95/Apo-1) receptor: Fas activation and apoptosis in hypoxic myocytes, Cardiovasc. Res. 54 (2002) 611–623. [12] G. Chen, R. Ray, D. Dubik, L. Shi, J. Cizeau, R.C. Bleackley, S. Saxena, R.D. Gietz, A.H. Greenberg, The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis, J. Exp. Med. 186 (1997) 1975–1983. [13] A. Hamacher-Brady, N.R. Brady, S.E. Logue, M.R. Sayen, M. Jinno, L.A. Kirshenbaum, R.A. Gottlieb, A.B. Gustafsson, Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy, Cell Death Differ. 14 (2007) 146–157. [14] Y. Lee, H.Y. Lee, R.A. Hanna, A.B. Gustafsson, Mitochondrial autophagy by Bnip3 involves Drp1-mediated mitochondrial fission and recruitment of Parkin in cardiac myocytes, Am. J. Physiol. Heart Circ. Physiol. 301 (2011) H1924–1931. [15] K.M. Regula, K. Ens, L.A. Kirshenbaum, Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes, Circ. Res. 91 (2002) 226–231. [16] X. Feng, X. Liu, W. Zhang, W. Xiao, P53 directly suppresses BNIP3 expression to protect against hypoxia-induced cell death, EMBO J. 30 (2011) 3397–3415. [17] R.M. Graham, J.W. Thompson, J. Wei, N.H. Bishopric, K.A. Webster, Regulation of Bnip3 death pathways by calcium, phosphorylation, and hypoxiareoxygenation, Antioxid. Redox Signal 9 (2007) 1309–1315. [18] K. Goshima, Quabain-induced arrhythmias of single isolated myocardial cells and cell clusters cultured in vitro and their improvement by quinidine, J. Mol. Cell. Cardiol. 9 (1977) 7–23. [19] J. Sadoshima, L. Jahn, T. Takahashi, T.J. Kulik, S. Izumo, Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy, J. Biol. Chem. 267 (1992) 10551–10560. [20] W.T. Chang, Z.H. Shao, J.J. Yin, S. Mehendale, C.Z. Wang, Y. Qin, J. Li, W.J. Chen, C.T. Chien, L.B. Becker, T.L. Vanden Hoek, C.S. Yuan, Comparative effects of flavonoids on oxidant scavenging and ischemia-reperfusion injury in cardiomyocytes, Eur. J. Pharmacol. 566 (2007) 58–66. [21] J. Cheng, J. Du, Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulin-like growth factor-1 receptor, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 1744–1751. [22] K.D. Mansfield, R.D. Guzy, Y. Pan, R.M. Young, T.P. Cash, P.T. Schumacker, M.C. Simon, Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation, Cell Metab. 1 (2005) 393–399. [23] Y.F. Zhou, X.W. Zheng, G.H. Zhang, Z.H. Zong, G.X. Qi, The effect of hypoxiainducible factor 1-alpha on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes, Cardiovasc. J. Afr. 21 (2010) 37–41. [24] Y. Guo, T. Yang, J. Lu, S. Li, L. Wan, D. Long, Q. Li, L. Feng, Y. Li, Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway, Life Sci. 88 (2011) 598–605. [25] Z. Cai, H. Zhong, M. Bosch-Marce, K. Fox-Talbot, L. Wang, C. Wei, M.A. Trush, G.L. Semenza, Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partial deficiency of HIF-1 alpha, Cardiovasc. Res. 77 (2008) 463–470. [26] F. Du, L. Zhu, Z.M. Qian, X.M. Wu, W.H. Yung, Y. Ke, Hyperthermic preconditioning protects astrocytes from ischemia/reperfusion injury by upregulation of HIF-1 alpha expression and binding activity, Biochim. Biophys. Acta 1802 (2010) 1048–1053. [27] K.B. Kannan, I. Colorado, D. Reino, D. Palange, Q. Lu, X. Qin, B. Abungu, A. Watkins, F.J. Caputo, D.Z. Xu, G.L. Semenza, E.A. Deitch, R. Feinman, Hypoxiainducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury, Am. J. Physiol. Gastrointest. Liver Physiol. 300 (2011) G853–861. [28] R. Cursio, C. Miele, N. Filippa, E. Van Obberghen, J. Gugenheim, Liver HIF-1 alpha induction precedes apoptosis following normothermic ischemiareperfusion in rats, Transplant. Proc. 40 (2008) 2042–2045. [29] T. Hoshi, H. Sasano, K. Kato, N. Yabuki, S. Ohara, R. Konno, S. Asaki, T. Toyota, H. Tateno, H. Nagura, Immunohistochemistry of Caspase3/CPP32 in human stomach and its correlation with cell proliferation and apoptosis, Anticancer Res. 18 (1998) 4347–4353. [30] R.M. Graham, D.P. Frazier, J.W. Thompson, S. Haliko, H. Li, B.J. Wasserlauf, M.G. Spiga, N.H. Bishopric, K.A. Webster, A unique pathway of cardiac myocyte death caused by hypoxia-acidosis, J. Exp. Biol. 207 (2004) 3189–3200.
1234
X. Wang et al. / Biochemical and Biophysical Research Communications 417 (2012) 1227–1234
[31] A.E. Greijer, E. van der Wall, The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis, J. Clin. Pathol. 57 (2004) 1009–1014. [32] C. Kuiper, I.G. Molenaar, G.U. Dachs, M.J. Currie, P.H. Sykes, M.C. Vissers, Low ascorbate levels are associated with increased hypoxia-inducible factor-1 activity and an aggressive tumor phenotype in endometrial cancer, Cancer Res. 70 (2010) 5749–5758. [33] J.K. Chen, L.J. Hu, J. Wang, K.R. Lamborn, E.L. Kong, D.F. Deen, Hypoxia-induced BAX overexpression and radiation killing of hypoxic glioblastoma cells, Radiat. Res. 163 (2005) 644–653.
[34] K. Wang, X.M. Yin, D.T. Chao, C.L. Milliman, S.J. Korsmeyer, BID: a novel BH3 domain-only death agonist, Genes Dev. 10 (1996) 2859–2869. [35] D.A. Kubli, M.N. Quinsay, C. Huang, Y. Lee, A.B. Gustafsson, Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion, Am. J. Physiol. Heart Circ. Physiol. 295 (2008) H2025–2031. [36] R. Malhotra, D.W. Tyson, H.M. Rosevear, F.C. Brosius 3rd, Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells, BMC Cardiovasc. Disord. 8 (2008) 9.