Ann Thorac Surg 2008;86:109-114. doi:10.1016/j.athoracsur.2008.03.057
© 2008 The Society of Thoracic Surgeons
Original Articles: Adult Cardiac
Programmed Cell Death in Idiopathic Dilated Cardiomyopathy is Mediated by Suppression of the Apoptosis Inhibitor Apollon
Seyedhossein Aharinejad, MD, PhDa,b,*,
Olena Andrukhova, PhDb,
Trevor Lucas, PhDb,
Andreas Zuckermann, MDa,
Georg Wieselthaler, MDa,
Ernst Wolner, MDa,
Michael Grimm, MDa
a Department of Cardiothoracic Surgery, Center of Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria
b Department for Cardiovascular Research, Center of Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria
Accepted for publication March 25, 2008.
* Address correspondence to Dr Aharinejad, Department of Cardiothoracic Surgery, Medical University of Vienna, Waehringer Guertel 18–20, Vienna, A-1090, Austria (Email: seyedhossein.aharinejad{at}meduniwien.ac.at).
Presented at the Forty-fourth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28–30, 2008.
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Abstract
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Background: Idiopathic dilated cardiomyopathy (DCM) is characterized by ventricular wall remodeling and an increased frequency of cardiac cell apoptosis. Apollon is a 528kD cell membrane-anchored protein that inhibits apoptosis by ubiquitinylation facilitating the degradation of Smac/Diablo and caspase-9. The present study tested the hypothesis that the Apollon/Smac system may mediate programmed cell death in DCM.
Methods: Apollon and caspase-9 protein expression was assessed in left ventricular biopsies of explanted failing hearts using Western blotting in 36 DCM patients undergoing cardiac transplantation and in 10 controls. Human cardiac cells were transfected with a plasmid containing the human Apollon complementary DNA or control vector and were subsequently stressed by hypoxia. Apollon, Smac/Diablo, and caspase-9 expression were then examined in cell lysates by real-time polymerase chain reaction and a transferase-mediated dUTP nick-end labeling assay was used to determine the apoptotic index.
Results: In DCM myocardial tissue, Apollon messenger (m)RNA and protein expression was down-regulated compared with control hearts (p < 0.001 and p < 0.005, respectively) concomitant with an increase in activated caspase-9 protein levels (p < 0.001). Cell stress resulted in increased apoptosis in cardiac cells in vitro and down-regulation of Apollon mRNA expression compared with control cells (p < 0.001). Transfection increased Apollon mRNA expression in cell lysates (p < 0.001) and completely prevented hypoxia-induced apoptosis associated with reduced expression of Smac/Diablo and activated caspase-9.
Conclusions: These results suggest that Apollon down-regulation plays a role in programmed cell death associated with DCM. Up-regulation of Apollon might therefore represent a novel therapeutic strategy in the treatment of DCM.
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Introduction
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Idiopathic dilated cardiomyopathy (DCM) is characterized by ventricular wall remodeling and reduced cardiac function leading to congestive heart failure. For patients with end-stage DCM, cardiac transplantation is currently the only accepted treatment [1]. The myocardium of DCM patients shows increased frequencies of cardiomyocyte apoptosis [2] and a reduction in the number of terminally differentiated cardiomyocytes impairs the ability of the myocardium to sustain contractile function [3].
Cellular degradation during apoptosis is primarily associated with activation of a cysteine-aspartic acid protease (caspase) family that can be inhibited by the inhibitor of apoptosis family proteins (IAPs), defined by the presence of one to three tandem baculoviral IAP repeats (BIRs) [4]. Overexpression of almost all known IAPs suppresses or delays apoptosis by binding to active caspases, restricting access to substrate proteins. Conversely, IAP protein activity is suppressed by proapoptotic factors such as Smac/Diablo [5].
Expression of the mouse antiapoptotic, baculoviral IAP repeat-containing protein 6 (Birc6), also known as Bruce and Apollon in humans, has been described in several organs including cardiac tissue [6, 7]. However, the antiapoptotic and physiologic role of this protein in heart tissue remains unknown. Apollon is a 528kD membrane-associated IAP that promotes cell survival. Apollon inhibits apoptosis by binding to caspase-3 and caspase-9 through its Bir domain and inhibits caspase activity through a C-terminal ubiquitination domain [6, 8, 9]. Decreasing Birc6 expression by RNA interference (RNAi) directly induces apoptosis [7, 10] and a recent report shows that gene-targeted knockout of Birc6 in mice results in defective placental development leading to embryonic and neonatal lethality [11]. Because Apollon is a chimeric E2/E3 ubiquitin ligase and ubiquitinates Smac/Diablo and active caspase-9 in vitro, Apollon is thought to enhance cell survival by antagonizing apoptosis induced by spontaneously released Smac/Diablo. Moreover, Smac/Diablo can induce apoptosis in Apollon deficient cells but not in Apollon expressing cells. Unlike other IAPs, Apollon can also promote degradation of the Smac/Diablo precursor and pro-caspase-9 [7].
Continued loss of cardiomyocytes in patients with DCM heart failure remains a severe clinical problem, which must be addressed to advance therapeutical approaches, in particular with regard to donor organ shortage. Therefore, strategies that reduce the biologic signals responsible for myocyte loss and chamber remodeling should improve clinical outcome. Intervention aimed at prevention of apoptosis may be a step in this direction. In screening experiments with gene expression profiling and subsequent Western blotting, we identify here decreased Apollon and increased activated caspase-9 expression in DCM hearts and show that the Apollon-Smac/Diablo-caspase-9 system may mediate programmed cell death in DCM.
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Patients and Methods
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Patients
In this prospective study approved by the Ethics Committee of the Medical University of Vienna, a total of 36 DCM patients undergoing cardiac transplantation were included. Left ventricular patient biopsies from the explanted failing hearts of the patients who gave informed consent to be enrolled in the study were included in the data analysis. Patients with arterial hypertension, myocardial infarction, or valve diseases were excluded. Patients were coded and case histories and clinical test results documented. All biopsies were coded, snap frozen, and stored in liquid nitrogen until analysis. Table 1
summarizes the demographic and relevant hemodynamic parameters of the patient group. The control group included 10 donor biopsies where transplantation could not be performed due to quality reasons.
Complementary (c)DNA Array
Poly(A+)-RNA was isolated with the Oligotex-dT kit (Quiagen, Valencia, CA) and first-strand cDNA synthesis performed with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) on 2 µg poly(A+) ribonucleic acid (RNA). The RNA strand within the DNA-RNA duplex was degraded and the products were purified on a Sephadex G-50 spun-column (Pharmacia, Uppsala, Sweden). For reverse-strand priming, first-strand cDNA was used to generate [
-32P] deoxycytidine triphosphate (dCTP-) labeled second-strand cDNA for the cDNA arrays (GEArray Q Human Apoptosis Gene Array, SuperArray Bioscience, Frederick, MD). Pooled cDNA hybridization signals were quantified by the use of ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Isolation of Cardiac Cells
Myocardial tissue from human hearts (DCM patients) was treated with collagenase-2 (PAA, Pasching, Austria), passed through a 100-µm nylon sieve and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, nonessential amino acids, penicillin and streptomycin (PAA) at 37°C in 5% carbon dioxide until 90% confluent and were subsequently passaged with trypsin. Seven to ten percent of cells in culture were scored as cardiomyocytes by -actinin immunohistochemical staining and 90% to 93% of cells in culture were scored as fibroblasts.
Transfection of Cells
The Birc-6 expression vector pApollon (pcDNA3-myc-ApollonFull(wild)-zeo) was prepared by standard methods. Human cardiac cells from DCM patients were transfected with pApollon in the presence of Lipofectamine (Invitrogen, Carlsbad, CA) in serum-free DMEM. Cells transfected with the red fluorescent protein expression vector (pRFP) pDsRed2-C1 (Clontech Laboratories, Palo Alto, CA) alone served as controls. The transfection was performed in triplicate. Experiments were commenced 24 hours posttransfection.
Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated from homogenized heart biopsies and from cell culture pellets in TRIZOL (Invitrogen, Carlsbad, CA), reverse transcribed from an oligo dT-primer with maloney murine leukemia virus reverse transcriptase (Fermentas, Ontario, Canada), and PCR performed with FastStart DNA Master SYBR Green mix (Roche, Mannheim, Germany) on a Light Cycler instrument (Roche). The primer sequences were sense/antisense: Apollon: 5'-CCAAAGGTGGTGAGCTTCAT-3'/5'-TCTACCCAGCATGGAGGAAC-3'; SMAC/Diablo: 5' -GGAGCCAGAGCTGAGATGAC-3'/5'- CCAGCTTGGTTTCTGCTTTC-3'; and β2-microglobulin: 5'-GATGAGTATGCCTGCCGTGTG-3'/5'-CAATCCAAATGCGGCATCT-3'. The LCDA Version 3.1.102 (Roche) was used for PCR data analysis. The messenger (m)RNA expression levels of target genes in individual patient samples and cultures were normalized to the β2-microglobulin signal as a housekeeping gene and represent the means of assays performed in triplicate as described previously [12].
Induction of Hypoxia In Vitro
Apoptosis was induced in human cardiac cell cultures in Anaerocult hypoxic chambers (Merck, Darmstadt, Germany) according to the manufacturer's protocol for 24 hours in triplicate. The percent oxygen in hypoxia experiments was approximately 0.5%. Duplicate platings were performed to examine apoptosis and mRNA.
Western Blotting Analysis
Tissue lysates (50 µg/lane) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to electrophoretic transfer onto a Nitrocellulose membrane (Bio-Rad, Hercules, CA). Gradient gels 4% to 20% (Bio-Rad) were used for Apollon protein separation. The blots were incubated with primary monoclonal anti-Apollon antibody (Bethyl, Montgomery, TX) or polyclonal activated caspase-9 antibody (Abcam, Cambridge, UK) prior to incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ). Protein loading was assessed by Ponceau staining and immunodetection was performed by chemiluminescence (Supersignal-West-Femto; Pierce, Rockford, IL). Bands were quantified by Easy Plus Win 32 software and specific protein signals normalized to loading controls (Herolab, Wiesloch, Germany). The protein expression levels of target genes in individual patient samples were corrected for protein loading control levels and were performed in triplicate.
Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assays
Cardiac cell cultures on chamber slides were air dried and TUNEL assays (In situ Cell Death Detection Kit) were performed according to the manufacturer's protocol (Roche Molecular Biochemicals, Basel, Switzerland) in triplicate. Slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) and embedded in AF1 antifadent (Citifluor, Leicester, UK). Digital images were obtained by fluorescence microscopy (Nikon, Melville, NY).
Statistical Analysis
Apollon and Smac/Diablo mRNA expression levels in biopsies of patients with DCM compared with control subjects were analyzed by nonparametric analysis of variance (Kruskal-Wallis test). Data in tables are expressed as mean ± standard deviation. All statistical analyses were performed using SAS version 9.1.3 (SAS Institute Inc, Cary, NC). The statistical significance level was set at p less than 0.05.
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Results
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Cardiac Apollon Expression is Selectively Downregulated in DCM
Gene profiling arrays indicated lower expression levels of Apollon in cardiac biopsies of patients with DCM compared with control heart tissue (Fig 1A). The expression of Apollon was then specifically examined in cardiac tissue homogenates of DCM patients and controls. Apollon mRNA and protein expression was significantly downregulated in cardiac biopsies of patients with DCM compared with control subjects on the mRNA level assessed by RT-PCR (Fig 1B; P < 0.001) and on the protein level assessed by quantitative Western blotting (Figure 1C, P < 0.005). These data strongly indicate that expression of the IAP Apollon is abnormally down-regulated in cases of idiopathic DCM.
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Fig 1. Reduced expression of Apollon in human dilated cardiomyopathy (DCM). (A) Representative gene array images show reduced hybridization to Apollon array sequences (arrowed) of labeled complementary DNA probes from DCM compared with normal (Control) heart biopsies. (B) Quantification of myocardial Apollon messenger (m)RNA expression by real-time polymerase chain reaction. Levels of Apollon mRNA are significantly downregulated in DCM compared with control heart biopsies (*p < 0.001 compared with control biopsies). (C) Representative Western blot images and quantification of myocardial Apollon protein expression corrected for protein loading control levels. Apollon protein is significantly and selectively down-regulated in DCM compared with control. Results are expressed as mean ± SD (*p < 0.005 compared with control biopsies.)
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In the same gene profiling arrays, higher expression levels of caspase-9 were observed in cardiac biopsies of patients with DCM compared with control heart tissue (Fig 2A). Protein expression levels of the caspase-9 active subunit p35 were significantly upregulated in cardiac biopsies of patients with DCM compared with control subjects examined by quantitative Western blotting (Fig 2B; C). Because the presence of the activated form of caspase-9 is a marker of apoptosis, our data demonstrate that levels of myocardial apoptosis are elevated in cases of idiopathic DCM.
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Fig 2. Increased expression of caspase-9 active subunit p35 in human dilated cardiomyopathy (DCM). (A) Representative gene array images show increased hybridization to caspase-9 array sequences (arrowed) of labeled complementary DNA probes from DCM compared with normal (Control) heart biopsies. (B) Quantification of myocardial caspase-9 active subunit p35 protein expression. Results are expressed as mean ± SD. (*p < 0.001 compared with control biopsies). (C) Representative Western blot images of myocardial caspase-9 active subunit p35 protein expression corrected for protein loading control levels. Caspase-9 active subunit p35 protein is significantly up-regulated in DCM compared with control biopsies.
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Apollon Upregulation in Hypoxia-Stressed Cardiac Cells Promotes Abrogation of Smac/Diablo and Caspase-9
In human cardiac cell cultures transfected with pApollon, Apollon mRNA expression levels were significantly up-regulated compared with cardiac cells transfected with the pRFP control expression vector under normoxic and hypoxic conditions (Fig 3A;
p < 0.001). In response to hypoxia, Smac/Diablo mRNA expression levels significantly increased in human cardiac cells transfected with the control expression vector (Fig 3B; p < 0.001). Induction of Smac/Diablo expression was, however, completely abrogated in cultures transfected with pApollon (Fig 3B; p < 0.001). The slight reductions in hypoxic Smac/Diablo expression after Apollon transfection (Fig 3B, right panel) may indicate that pathways in addition to Apollon are involved in the control of Smac/Diablo expression during cardiac cell hypoxia.
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Fig 3. Quantification of Apollon and Smac/Diablo messenger (m)RNA in human cardiac muscle cultures under hypoxic and normoxic conditions. (A) Apollon mRNA expression levels were significantly up-regulated in cells transfected with pApollon compared with control transfections with red fluorescent protein (pRFP) vector (*p < 0.001). (B) In cells transfected with control pRFP vector, Smac/Diablo expression increased significantly under hypoxic conditions in human cultures ( p < 0.001), whereas Smac/Diablo mRNA expression levels were significantly down-regulated in cardiac cells transfected with pApollon in response to hypoxia (*p < 0.001). Data incorporate the standard deviations of 3 experiments.
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In response to hypoxia, protein expression levels of the caspase-9 active subunit p35 were also significantly increased in human cardiac cells transfected with the control expression vector (Figs 4A, B;
p < 0.001). Induction of caspase-9 active subunit p35 protein expression was significantly reduced in cultures transfected with pApollon (Fig 4A; p < 0.001).
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Fig 4. Quantification of caspase-9 active subunit p35 protein expression in human cardiac muscle cultures under hypoxic and normoxic conditions. Quantification (A) and representative Western blot images (B) of caspase-9 active subunit p35 protein expression corrected for protein loading control levels. Protein expression levels were significantly increased in response to hypoxia in human cardiac cells transfected with the control red fluorescent protein (pRFP) vector (p < 0.001). Induction of caspase-9 active subunit p35 protein expression was significantly reduced in cultures transfected with pApollon (*p < 0.001). Data incorporate the standard deviations of 3 experiments.
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In summary, these data clearly indicate that increased Apollon expression during hypoxia-induced cell stress can stimulate Smac/Diablo and caspase-9 abrogation in cardiac cells. Because hypoxia has been linked to the induction of apoptosis, we then investigated the role of Apollon expression on programmed cell death in the cardiac cell cultures.
Apollon Protects Cardiac Cells From Hypoxia-Induced Apoptosis
Following a hypoxic stimulus, human cardiac cell cultures underwent apoptosis as assessed by TUNEL staining (Fig 5A). Transfection of human cardiac cells with pApollon significantly decreased hypoxia-induced apoptosis compared with cells transfected with the pRFP control expression vector (Fig 5B; p < 0.001). These data indicate that Apollon upregulation in cardiac cells can prevent apoptosis mediated by hypoxia-induced cell stress associated with reduced expression of Smac/Diablo and activated caspase-9.
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Fig 5. Apollon inhibits hypoxia-induced apoptosis in cardiac muscle (CM) cells. (A) Representative transferase-mediated dUTP nick-end labeling assays showing induction of apoptosis in human cardiac cells exposed to hypoxia. (B) Quantification of the apoptotic index (AI) in culture expressed as the number of apoptotic events per 10–5 cells in human cardiac cells. Transfection with pApollon significantly protected against hypoxia-induced apoptosis (*P < 0.001) compared with cultures transfected with red fluorescent protein (pRFP) vector. Data incorporate the standard deviations of 3 experiments.
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Comment
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Screening for genetic expression programs that are activated during the gradual loss of ventricular function associated with idiopathic DCM has been fruitful in the identification of novel pathways involved in myocardial biology. The genetic heterogeneity in DCM pathology has been revealed by the identification of mutations in the genes of some patients encoding for proteins that interact with the cytoskeleton [13]. It is increasingly being recognized that the process of apoptosis also plays a key role in myocardial cellular loss during DCM. The etiology of DCM is controversial and the role of hypoxia versus stretch and inflammation questionable by some investigators. In this paper, we examined the role of hypoxia-mediated apoptosis in vitro. Although this is likely different to the stimuli in vivo, which cause apoptosis during the progression of DCM, regions of local myocardial hypoxia in the failing DCM heart provide conditions for the induction of cellular apoptosis and preventing this process may be a promising therapeutic strategy for heart failure patients [14].
Here we identify transcriptional down-regulation of the IAP Apollon in failing human DCM heart tissue on the mRNA and protein levels compared with normal healthy heart tissue. In the same tissue samples, we also show an increase in the activated form of caspase-9 indicating apoptosis in human DCM heart tissue on the protein level compared with normal healthy heart tissue. This result is consistent with other studies showing up-regulation of caspase-9 in failing human hearts [15]. With apoptotic protease activating factor, activated caspase-9 is an integral component of the apoptosome, which controls downstream caspase activation leading to demise of the cell. The IAP expression may suppress apoptosis by binding to active caspase-9 and restricting access to substrate proteins, thus suppressing the cellular effects of proapoptotic signaling. In response to an apoptotic stimulus such as hypoxia, Smac/Diablo may be released from the mitochondria into the cytosol where interaction with IAPs takes place through N-terminal IAP-specific binding motifs, which suppress the role of Apollon in promoting cellular survival [9]. The balance between Apollon and Smac/Diablo expression may therefore dictate the cellular response to this pathway in a proapoptotic cellular environment such as hypoxia [5]. During the response of cardiac cells to hypoxia, we identify here the up-regulation of Smac/Diablo mRNA in vitro as a key transcriptional process during the apoptotic induction phase. The cellular effects of Smac/Diablo in sequestering Apollon activity during the apoptotic induction phase can be overcome by overexpression of Apollon through transfection, leading to dramatic decreases in the rate of apoptosis in cardiac cells.
In summary, we show that the Apollon system regulating Smac/Diablo and activated caspase-9 is active in idiopathic DCM. These data indicate that targeting the Apollon- Smac/Diablo pathway may represent a viable therapeutic strategy to modulate apoptosis in the myocardium of patients with idiopathic DCM.
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Discussion
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DR JOSEPH C. CLEVELAND, JR. (Denver, CO): If I could take the privilege of asking, I think that obviously with the inhibitor studies that is the next step for you, but where ultimately do you see this working? Do you think that this somehow will have some sort of a favorable signaling profile in the myocytes themselves, in the extracellular matrix, which is also known to be dysfunctional in this setting? Is it the myocytes, the matrix, both, neither?
DR AHARINEJAD: We assume, based on the electron microscopy studies—and I totally agree with you that the extracellular matrix is definitely a very significant part of heart failure, specifically in dilated cardiomyopathy. But there are signs, and we are just beginning to learn this, how apoptosis can also affect the cellular components of the extracellular matrix, including fibroblasts. So the answer is definitely just on the electron microscopy level, yes, but we would also expect some benefits at least towards the cellular components of the extracellular matrix, including fibroblasts.
DR JOHN V. CONTE, JR. (Baltimore, MD): Can you tell us how the in vivo study is being done in the animals, how you are actually delivering?
DR AHARINEJAD: We have two ways. For expression, we are using adenoviruses. In the large animals we go via the coronary sinus like we do in our patients, and for small animals, we perform intramyocardial injections, which is much more tricky. And for the compounds, which are being designed by our Swedish partner, it will just be delivered systemically IV, which is much easier.
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Acknowledgments
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The authors would like to thank Dr Mikihiko Naito, University of Tokyo, Japan for the kind gift of pcDNA3-myc-ApollonFull(wild)-zeo. This study was supported by the CARDIOWORKBENCH EU-grant # PL 018671 to Dr Aharinejad.
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References
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Abstract
Introduction
