Variation of NDRG2 and c-Myc expression in rat heart during the acute stage of ischemia/reperfusion injury
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- Sun, Z., Shen, L., Sun, X. et al. Histochem Cell Biol (2011) 135: 27. doi:10.1007/s00418-010-0776-9
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N-Myc downstream regulated gene 2 (NDRG2), a Myc-repressed gene, is highly expressed in heart tissue. NDRG2 increases in response to hypoxia-induced stress and is involved in hypoxia-induced radioresistance. However, little is known about the expression changes and possible roles of NDRG2 in the heart under hypoxia condition. Here, the authors show that NDRG2, mainly localized in cardiomyocyte cytoplasm, was significantly reduced in myocardial tissue after acute ischemia/reperfusion (I/R) injury. Meanwhile, c-Myc was up-regulated following acute I/R injury, and the expression of c-Myc was significantly inversely correlated with that of NDRG2. In addition, overexpression of c-Myc in primary cultured cardiomyocyte repressed NDRG2 expression. Furthermore, the increase of cardiomyocyte apoptosis was correlated with the decrease of NDRG2 protein during the acute phase of reperfusion. These data suggested for the first time that I/R injury-induced up-regulation of pro-apoptotic c-Myc expression may contribute to the down-regulation of anti-apoptotic NDRG2. This stress response might be involved in the novel mechanism of myocardial apoptosis induced by I/R injury in rat.
Acute myocardial infarction (AMI) results in significant morbidity and disability, and is one of the leading causes of death worldwide. Early reperfusion is clinically effective, and is the key strategy for the clinical management of AMI. Restoration of blood flow to ischemic tissue is essential in limiting the damage caused by AMI and salvaging organ function. However, reperfusion per se exerts detrimental effects including cell death and loss of myocardial and coronary function, a phenomenon which has been termed ischemia/reperfusion (I/R) injury (Verma et al. 2002). The pathophysiology of I/R injury has not yet been fully delineated, and has become a major focus of research due to its potential importance in clinical management.
The heart is particularly sensitive to hypoxia since it contains only limited reserves of high-energy phosphates. Both hypoxia and reoxygenation result in biochemical and functional changes (Lefer and Granger 2000). Alterations of oxygen supply also regulate the expression of specific proteins that are protective or deleterious (Matsui et al. 2007). These proteins might be the potential targets in the treatment of AMI and the preservation of residual myocardial function (Piacentini and Karliner 1999).
NDRG2 belongs to the NDRG family which comprises four members: NDRG1 ~ 4. The NDRG family is highly conserved among many species which is indicative of the importance of its biological functions (Zhou et al. 2001). Previous studies have shown that NDRG2 is highly expressed in human adult brain, liver, kidney, and, in particular, heart tissue (Hu et al. 2006). However, the biological functions of NDRG2 in these organs are still not clear. NDRG2 is also involved in cell growth (Takahashi et al. 2005), differentiation (Hu et al. 2006), neuro-degeneration (Mitchelmore et al. 2004), and acts as a cell stress-responding molecule which is sensitive to oxygen deprivation (Wang et al. 2008). A previous study demonstrated that the expression of NDRG2 was significantly up-regulated in tumor cell lines exposed to hypoxia and radiation, and protected cervical cancer Hela cells from radiation-induced apoptosis (Liu et al. 2010a).
It has been well established that cardiomyocyte apoptosis contributes significantly to the myocardial dysfunction in I/R, and apoptosis prevention reduces cardiomyocyte loss and preserves myocardial function (Green and Reed 1998). Although NDRG2 has been shown to contribute to hypoxia-induced radioresistance of Hela cells, no study has investigated the expression and role of NDRG2 in heart tissue subjected to I/R stress. Furthermore, NDRG2 is regulated by c-Myc, an oncogene related to cell apoptosis and differentiation (Zhang et al. 2006).
In the previous study, the authors have found that I/R injury, instead of ischemia alone, could lead to alteration in NDRG2 expression in rat heart. To make further investigation, in this study, the authors examined NDRG2 distribution and localization in rat cardiac tissue, and centered on variation of NDRG2 expression during the acute phase of reperfusion injury and tried to explore the possible regulatory mechanism. In addition, the authors analyzed the myocardial apoptosis in order to explore the possible relationship between NDRG2 and apoptosis induced by I/R in rat myocardium.
Materials and methods
Male Sprague–Dawley rats received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). Thirty rats weighing 260–290 g were randomly separated into five groups. One group of sham-operated rats was used as control. The remaining four groups undergone 30 min of ischemia followed by reperfusion for 3, 6, 12, or 24 h. To produce ischemia/reperfusion, the rats were anesthetized with pentobarbital sodium (Abbott, North Chicago, IL; 40 mg/kg, i.p.), and ventilated 60 times/min with a volume-cycled respirator. The left coronary artery was ligated using a previously described technique (Deloche et al. 1977). In brief, through a left thoracotomy incision, a ligature was placed around the left coronary artery, and 30 min later, the slipknot was released. After reperfusion for one of the aforementioned time periods, the rats were killed. Sham-operated animals were subjected to the same surgical procedures, except for coronary artery occlusion.
After treatment, injured myocardium was separated from the whole heart as completely as possible. The ischemic/reperfused cardiac tissues were rapidly frozen in liquid nitrogen and stored at −70°C for RT-PCR and western blotting analysis, or fixed in 4% paraformaldehyde for immunostaining.
Total RNA was extracted using TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA (2 μg) was reversely transcribed with reverse transcriptase (Promega, WI, USA). The first strand cDNA was used as the template for real-time quantitative PCR analysis. β-actin cDNA was used as an internal control to normalize variances. The primers used were as follows: NDRG2, 5′-GAGATATGCTCTTAACCACCCG-3′ (forward) and 5′-GCTGCCCAATCCATCCAA-3′ (reverse); β-actin, 5′-ATCATGTTTGAGACCTTCAACA-3′ (forward) and 5′-CATCTCTTGCTCGAAGTCCA-3′ (reverse). PCR was performed in a GeneAmp PCR system 2400 Thermal Cycler (Perkin–Elmer, Norwalk CT, USA). Conditions for PCR were 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C (30 cycles). The PCR products were separated on 1.5% agarose gels and bands were visualized using ultraviolet light.
Western blot analysis
Rat heart tissues or cardiomyocytes were lyzed in RIPA buffer [0.05 M Tris–HCl pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% Nonidet P-40 (NP-40), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, and 1 mg/ml leupeptin]. Protein concentrations were measured using the BCA protein assay. Proteins were separated by electrophoresis on SDS-PAGE and transferred onto a nitrocellulose membrane. After being blocked with 5% milk, the immunoblots were probed with anti-NDRG2 (1:500; Mouse; Abnova, Taiwan), anti-c-Myc (1:300; Rabbit; Santa Cruz Biotechnology, CA, USA), or anti-cleaved-caspase3 (1:500; Rabbit; Sigma, CA, USA) antibodies overnight at 4°C followed by incubation with the corresponding secondary antibodies. The blot was developed with a chemiluminescence substrate solution (Pierce) and exposed to X-ray film.
Immunohistochemistry and immunofluorescence
Heart tissues were fixed in 4% paraformaldehyde solution, dehydrated through graded solutions of ethanol, and embedded in paraffin. Serial sections (5 μm thick) were cut and mounted on glass slides (Fisher, Pittsburgh, PA). After dewaxing and microwave antigen retrieval, slides were incubated with 10% normal goat serum for 1 h and then overnight at 4°C with mouse monoclonal antibody to NDRG2 (1:100), rabbit polyclonal antibody to cleaved-caspase3 (1:300), or PBS as a control. Biotinylated anti-mouse IgG (Sigma) or anti-rabbit IgG (Sigma) was applied and detected with a streptavidin-peroxidase complex and 0.1% of 3,3′-diaminobenzidine (Sigma) in PBS with 0.05% H2O2 for 5 min at room temperature. In addition, slides were stained with hematoxylin and eosin for tissue morphology.
For immunofluorescence histochemistry, nonspecific antibody-binding sites were first blocked with 1% bovine serum albumin (BSA; Sigma) in PBS, after which dewaxed slides were incubated with anti-NDRG2 alone or in combination with rabbit anti-cardiac troponin-I (cTnI; 1:50; Rabbit; Boster; China), or with PBS alone as a control. The combinations were visualized using a mixture of tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit (Sigma) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse (Sigma) secondary antibodies. Specimens were examined with a FV300 laser scanning microscope (N A = 0.70; Olympus, Tokyo, Japan) with the Olympus MicroSuite™ software (Magnificence ×200). Images were identically and minimally processed by importing them into Adobe Photoshop CS software (Adobe Systems, Inc, Mountain View, CA).
Neonatal cardiomyocytes culture
Primary cultures of neonatal rat cardiomyocytes were obtained from 1- to 2-day-old Sprague–Dawley rats, which were cultured and prepared as described previously (Ma et al. 2006). Simulated ischemia/reperfusion (SI/R) was performed by transferring cardiomyocytes into an ischemic buffer adapted from Esumi et al. (1991) containing (in mmol/l) NaCl 137, KCl 3.8, MgCl2 0.49, CaCl2·2H2O 0.9, HEPES 4 supplemented with deoxyglucose 10, sodium dithionate 0.75, KCl 12, and lactate 20, pH 6.5, for 2 h in a humidified cell culture incubator (21% oxygen, 5% CO2, 37°C). This buffer is designed to simulate the extracellular milieu of myocardial ischemia, with the approximate concentrations of potassium, hydrogen, and lactate ions occurring in vivo. After this simulated ischemia, the cells were cultured again in glucose-containing DMEM at 37°C in 95% air and 5% CO2 (reperfusion) for a serials of time period (2, 4, 8, 16, or 24 h).
DNA fragmentation was detected by fluorescence microscopy on paraffin-embedded left ventricle (LV) sections (4 μm) harvested at the end of surgery. Terminal transferase-mediated dUTP-biotin nick-end labeling (TUNEL) was performed with fluorescein-dUTP (In Situ Cell Death Detection Kit; Roche Diagnostics). Cell nuclei were stained with DAPI (Sigma). Slides were visualized on a LEICA-DMRA2 microscope.
Recording of ECG change during I/R and staining of myocardial infarct region after I/R in animal model
After the animal was anesthetized, four recording electrodes were placed subcutaneously into four limbs of rat with right orders. The heart rate and T wave changes were recorded by the electrocardiogram at each minute of the experiment.
At the end of the 24-h reperfusion period, mice were re-anesthetized and the ligature around the coronary artery was retied, and MI region was demonstrated by the Evans blue/2,3,5-triphenyltetrazoliumchloride (TTC) double staining method as described previously.
Determination of lactate dehydrogenase (LDH) release after SI/R in cellular model
The amount of lactate dehydrogenase (LDH) released into the culture medium from the injured cells after 6 h of simulated reperfusion was assayed using a Sigma assay kit. The optical density of the tetrazolium product was determined spectrophotometrically (Molecular Devices; Sunnyvale, CA) at a 490-nm wave-length. The percent LDH release was calculated as the ratio of the LDH released into the medium to the total LDH (release plus cellular content).
c-Myc were inserted into pWPT vector (Addgene, Cambridge, USA) by replacing GFP, to generate pWPT-c-Myc. Recombinant lentivirus particles were used to infect primary cultured cardiomyocytes. A pilot study with the control virus Lenti-pWPT-GFP demonstrated that 60–70% of the cells were infected, as detected by fluorescence microscope (Olympus, Tokyo, Japan; Supplemental Fig. 3).
All values in the text and figures are presented as mean ± SE of n independent experiments. All data were subjected to ANOVA followed by Bonferroni correction for post hoc t test. Probabilities values < 0.05 were considered to be statistically significant.
NDRG2 expression, distribution, and localization in normal rat heart
Expression and localization of NDRG2 in rat heart post I/R injury
The time-course of NDRG2 protein expression following coronary artery occlusion was assessed in tissue dissected from injured regions of the left ventricle in all groups. Following reperfusion, the expression of NDRG2 protein gradually decreased; the lowest level was found at 24 h, i.e., 12 h after NDRG2 mRNA had reached its lowest level (Fig. 2b, c).
Examination of NDRG2 and c-Myc protein level in heart post I/R stress
Apoptosis detection in rat heart post I/R stress
Examination of NDRG2, c-Myc, and cleaved-caspase-3 protein level in cultured cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R)
To further explore the relationship between c-Myc and NDRG2, overexpression of c-Myc using lentivirus containing c-Myc enhanced c-Myc expression while suppressed NDRG2 expression in primary cultured cardiomyocytes, which was detected by Western blot analysis (Fig. 6c, d). Therefore, this study provided evidences that rat NDRG2 expression was down-regulated by c-Myc.
In this study, NDRG2 is highly expressed in the heart (Boulkroun et al. 2002; Deng et al. 2003; Hu et al. 2006), and the authors demonstrate the cellular expression of NDRG2 is mainly located in cardiomyocytes. The expression of NDRG2 mRNA and NDRG2 protein reduced gradually over time following I/R stress. In addition, there existed a negatively relationship between the expression of NDRG2 and that of c-Myc in I/R-induced cardiomyocyte apoptosis. The observation here indicated that c-Myc might modulate the expression of NDRG2 protein in cardiomyocyte subjected to I/R, and this modulation may contribute to cardiomyocyte apoptosis.
The mechanisms of I/R-induced myocardial cell death, such as oxidative burst, endothelial cell dysfunction, and intracellular calcium overload, are always related to the changes of various proteins expression, which play critical roles in the pathophysiological process of I/R-induced myocardial injury. For example, toll like receptor 4-deficient mice exhibit reduced infarct size following myocardial ischemia (Oyama et al. 2004), while infarct size was increased in transgenic mice overexpressing caspase-3 subjected to myocardial I/R (Condorelli et al. 2001). Therefore, further investigation on the cellular and molecular mechanisms of I/R injury may provide therapeutic intervention in clinical care. Studies have demonstrated that promotion of anti-apoptotic protein or inhibition of pro-apoptotic proteins results in a reduced area of infarction in myocardium. For example, overexpression of the anti-apoptotic protein Bcl-2 reduces apoptosis and protects against myocardial I/R injury in transgenic mice. In addition, the proapototic Bax ablation also prevents myocardium from I/R injury in transgenic mice (Vila-Petroff et al. 2007).
NDRG2 belongs to the NDRG family which is involved in cellular differentiation and proliferation (Deng et al. 2003). Human NDRG2 was first cloned in the laboratory (Deng et al. 2001). On one hand, NDRG2 was considered as a candidate tumor suppressor gene due to its reduced expression in some cancer tissues and its induction of apoptosis in certain cancer cells (Lusis et al. 2005; Ma et al. 2008). On the other hand, NDRG2 has also been reported to have other biological functions: insulin could stimulate NDRG2 phosphorylation in the skeletal muscle cells (Burchfield et al. 2004). In addition, NDRG2 has also been identified as an early aldosterone-induced gene in rat kidney (Boulkroun et al. 2002). More recently, Liu et al. have reported that NDRG2 participates in oxLDL-induced macrophage activation and modulates ERK1/2-dependent PDGF and VEGF production, which has potential application in atherogenesis (Liu et al. 2010b). Though NDRG2 is highly expressed in myocardium, functions of NDRG2 in heart tissues or myocardial cells and the modulation of NDRG2 expression in heart was never studied before.
In this study, it was found that there was a gradual and time-dependent decrease in the expression of NDRG2 mRNA and NDRG2 protein, although the lowest level of mRNA was reached before that of the protein. A possible explanation to this finding is that more time is required for protein synthesis or degradation than for the rapid process of mRNA transcription.
It has been shown that some protooncogenes, such as c-myc, c-fos, and c-jun, are implicated in the pathogenesis of gene-mediated myocardial remodeling and myocardial or cerebral I/R in experimental animal models. Wechsler et al. have reported that the post I/R stunning state is associated with early gene changes, such as the increase of c-Myc gene (Wechsler et al. 1994). In addition, lots of studies have shown that c-Myc promoted cell cycle progression and simultaneously sensitized cells to a wide variety of apoptosis triggering factors. For example, c-Myc was a critical determinant of apoptosis induced by TNF-α (Klefstrom et al. 1994). Meanwhile, c-Myc overexpression markedly accelerated apoptosis of murine myeloid 32D cells denied IL-3 (Askew et al. 1991). Furthermore, c-Myc was up-regulated in some neurons after focal cerebral ischemia and blocked c-Myc expression reduced the ischemic damage, suggesting that c-Myc may be involved in ischemic cellular events (Huang et al. 2001). In conclusion, the elevation of c-Myc induced by I/R may contribute to the apoptotic process of injured cells.
Although apoptosis plays an important role in the myocardial injury post I/R, the regulation mechanisms involved in cardiomyocyte apoptosis are not fully understood. It is reported that NDRG2 was up-regulated by hypoxia and radiation, and protected human cervical cancer Hela cells from radiation-induced apoptosis (Liu et al. 2010a). Meanwhile, overexpression of c-Myc, the NDRG2 suppressor, can also accelerate cell apoptosis triggered by oxygen deprivation (Brunelle et al. 2004). In the experimental model, reperfusion stress led to serious myocardial apoptosis, which showed a rapid increase during 24 h of I/R injury, and this change was positively correlated to that of c-Myc and negatively correlated to NDRG2 protein in rat heart post I/R. From the results of experiments, the authors led to the following hypothesis: I/R stress up-regulates pro-apoptotic c-Myc expression in cardiomyocyte, which may repress NDRG2 expression and aggravate myocardial apoptosis in the acute stage of reperfusion. NDRG2, suppressed by c-Myc, is possibly involved in the apoptosis mechanism of rat myocardium post I/R injury.
Recently, several studies have explored the possible relationship between NDRG2 and Myc in different species and tissues. In N-Myc deficient mutant mouse embryos, there is no significant elevation of NDRG2 expression (Okuda and Kondoh 1999). However, the ectopic expression of c-Myc represses human NDRG2 expression via interaction with the NDRG2 core promoter (Zhang et al. 2006). In addition, another group in the laboratory also found that rat NDRG2 and c-Myc expression were inversely correlated during the process of liver regeneration (Yang et al. 2010). From the experiment, there was also an inverse correlation between NDRG2 and c-Myc in rat cardiac tissue post I/R or in primary cardiomyocytes. Whether NDRG2 is repressed directly or indirectly by Myc on its core promoter region needs to be further investigated.
In conclusion, the authors demonstrate that NDRG2 is highly expressed in cardiomyocyte. It was also observed that NDRG2 was down-regulated following the induction of c-Myc in rat heart post I/R injury. c-Myc might regulate the expression of NDRG2 in cardiomyocyte subjected to I/R, and this regulation may contribute to rat cardiomyocyte apoptosis post I/R. In future studies, knock-down or overexpression of NDRG2 will be helpful to clarify the role of NDRG2 in heart following I/R injury. And elucidating the biological function of NDRG2 in heart post I/R injury may provide a novel area for therapeutic intervention in clinical coronary heart disease.
The research is supported by the National Natural Science Foundation of China (no. 30600314, 30801309, and 30830054), and National Key Basic Research and Development Program (no. 2010CB529705). The authors thank members of the laboratory for helpful discussions.