Cardiovascular Research

Cardiovascular Research 2000 48(1):101-110; doi:10.1016/S0008-6363(00)00154-1
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Cellular FLIP is expressed in cardiomyocytes and down-regulated in TUNEL-positive grafted cardiac tissues

Toshio Imanishi^a,*, Charles E. Murry^b, Hans Reinecke^b, Takuzo Hano^a, Ichiro Nishio^a, W.Conrad Liles^c, Leonard Hofsta^f, Koanhoi Kim^b, Kevin D. O'Brien^d, Stephen M. Schwartz^b and David K.M. Han^e

^aDivision of Cardiology, Department of Medicine, Wakayama Medical College, 811-1, Kimiidera, Wakayama City, Wakayama 641-8510, Japan
^bDepartment of Pathology, University of Washington, Seattle, WA, USA
^cDepartment of Medicine (Infectious Diseases), University of Washington, Seattle, WA, USA
^dDepartment of Medicine (Cardiology), University of Washington, Seattle, WA, USA
^eDepartment of Molecular Biotechnology, University of Washington, Seattle, WA, USA
^fDepartment of Cardiology, University of Maastricht, Masstricht, The Netherlands

^* Corresponding author. Tel.: +81-73-441-0621; fax: +81-73-446-0631 imanishi{at}wakayama.hosp.go.jp

Received 6 January 2000; accepted 23 May 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: c-FLIP is a natural homologue of caspase 8, and may antagonize activation of death pathways mediated by FADD. c-FLIP is highly expressed in the heart, and a recent report suggests that c-FLIP may protect against certain types of myocyte death. The present study was designed to define the expression patterns of c-FLIP in the heart. Methods: The expression pattern of c-FLIP in end-stage human hearts, and rat cardiomyocyte grafting models was analyzed by in situ hybridization, immunohistochemistry and TUNEL assay. In addition, to determine whether Fas-dependent pathway is active in cardiomyocytes in vitro, we examined whether activated monocytes can kill neonatal cardiomyocytes in a co-culture system. Results: c-FLIP mRNA and protein were abundantly expressed in normal cardiomyocytes from failing human heart. In animal models, c-FLIP protein was absent in TUNEL-positive grafted cardiomyocytes. Double staining demonstrated that c-FLIP-positive cells rarely had fragmented DNA, while TUNEL-positive cells rarely contained c-FLIP. Finally, activated monocytes induced death of neonatal rat cardiomyocytes via the Fas/FasL system. Conclusions: Loss of c-FLIP expression correlates with cardiomyocyte cell death. We hypothesize that diminished c-FLIP expression may predispose cardiomyocytes to apoptotic death.

KEYWORDS Apoptosis; Cardiomyopathy; Heart failure; Histo(patho)logy; Myocytes

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Recently, apoptosis has been proposed as a contributing cause of cardiac myocyte loss in ischemic–perfusion injury [1], myocardial infarction [2,3] and end-stage heart failure [4,5]. Ongoing myocyte apoptosis may lead to a progressive deterioration in myocardial function, culminating in end-stage heart failure [6]. Since genetic and biochemical studies have now identified specific death pathways mediated by death-dealing proteases (caspases), recognition of the factors responsible for the initiation or prevention of apoptosis may provide new insights for developing strategies to regulate the survival or death of cells.

The widely expressed protein Fas is a member of the tumor necrosis factor receptor family which can trigger apoptosis [7]. Death receptor-induced apoptosis is an important event in tissue homeostasis as exemplified by the accumulation of T cells in Fas-deficient mice [7,8]. However, Fas surface expression is not limited to the immune system [7]. The Fas/Fas ligand pathway has been implicated in cellular in a variety of tissues, including vascular smooth muscle and endothelium [9,10]. Recent studies have shown that cells have not only caspases but also specific proteins that block activation of caspases by death receptors. Interestingly, unlike the Bcl-2 family members which are potent inhibitors of apoptosis induced by growth factor withdrawal or -irradiation [11], these inhibitors appear to primarily block apoptosis induced by death receptors [12].

One class of death receptor inhibitors, identified first in viruses, is the caspase 8 (FLICE)-like inhibitor proteins (v-FLIPs) [12]. v-FLIPs have homology to the death effector domains (DEDs) of caspase 8 and 10, respectively [12,13]. DEDs are the domains which caspases employ to aggregate to one another and to FADD. Similar to DED constructs of caspase 8, the vFLIPs act as dominant negative inhibitors of FADD-mediated death. v-FLIPs apparently act by competing for binding of the prodomains of caspase 8 or 10 and thus blocking Fas-mediated apoptosis. Recently, a cellular FLIP homologue was independently identified in mammalian cells by several laboratories [14–18], including ours [19]. This molecule, called MRIT in our report, exists in several splice forms including an apparent full length homologue of caspase 8 within active proteolytic domains. Although several names have been proposed for this putative caspase inhibitor, the name c-FLIP has generally been accepted by analogy to v-FLIP and in recognition of the earlier publication.

The Fas and TNFR1 death receptors both signal death through caspase 8. Furthermore, clinical studies report elevations of plasma Fas/FasL and TNF/TNFR1 in patients with both chronic heart failure and myocarditis [20–23]. Therefore, c-FLIP is of special interest in the heart because of its possible role in preventing apoptosis via the Fas pathway. To gain better insight into the possible protective role of c-FLIP in cardiomyocytes, we examined c-FLIP expression in cardiomyocyte grafting models. In addition, to explore the role of c-FLIP in both end-stage heart and animal models, we tested whether c-FLIP protein expression may co-localize with apoptotic cells by simultaneous immunohistochemistry for c-FLIP protein and TUNEL assay. Finally, to determine whether the Fas death pathway is active in cardiomyocytes in vitro, we examined whether activated monocytes, which release biologically active soluble FasL as we have shown previously [24], can kill neonatal cardiomyocytes by using a co-culture system.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Isolation and activation of monocytes
Venous blood was collected from normal human volunteers using 0.2% dipotassium EDTA (K₂EDTA) as anticoagulant. Platelets were initially depleted by three washes in ice-cold PBS with 0.3 mM Na₂EDTA. Peripheral blood monocytes were isolated by magnetic cell sorting using CD14 immunobeads according to the manufacturer's instructions (MACS/VarioMACS^®, Myltenyi Biotec, Sunnyvale, CA, USA). Prior to incubation with magnetic CD14 immunobeads, the peripheral blood mononuclear cell fraction was separated from granulocytes and erythrocytes by centrifugation in Histopaque-1077 (Sigma, St. Louis, MO, USA). The monocytes were maintained in RMPI 1640 plus either 0.1% human serum albumin (fraction V, Sigma, Pro) or 10% heat-inactivated fetal calf serum (BioWhittaker, Walkersville, MD, USA) as designated, supplemented with 10 mM HEPES, 0.2 mM L-glutamine, 25 U/ml penicillin and 25 mg/ml streptomycin, at a concentration of 5x10³ cells/ml in 48-well polystyrene cell culture cluster plates (Costar, Cambridge, MA, USA) [24]. Cultured monocytes were activated by incubation with 100 mg/ml human IgG immune complexes (IC). Human IgG immune complexes (IC) were prepared by precipitation of the complex formed upon addition of goat anti-human IgG serum to purified human IgG [24].

2.2 Induction of neonatal cardiomyocyte apoptosis by co-cultured activated monocytes
To estimate cell death in cell-to-cell contact experiments, a chromium release assay was used [25]. The amount of ⁵¹Cr released into culture supernatant by neonatal cardiomyocytes exposed to activated monocytes or endothelial cells reflects the magnitude of cell death. Neonatal cardiomyocytes were plated in 24-well plates at 40–50% confluence and preincubated with ⁵¹Cr for 4 h. After two washes with PBS, control (unactivated) monocytes, IC-activated monocytes were added to the wells. Chromium release was measured after incubation for 24 h. Neonatal cardiomyocytes were also preincubated with blocking IgG1 to Fas (ZB4, 1 mg/ml) to investigate the involvement of the Fas/FasL pathway in monocyte-induced killing. An effector-to-target ratio of 100:1 for monocytes was used in these experiments.

2.3 Tissue collection, preparation, and characterization
Human cardiac tissues were obtained from hearts removed from patients with end-stage ischemic heart disease (n = 7) and idiopathic dilated cardiomyopathy (n = 6), from nine men and four women, 26–63 years of age. Samples of myocardium were dissected from the hearts and placed in 10% neutral buffered formalin within 2 h of organ excision. After overnight fixation, the tissue samples were routinely processed and embedded in paraffin. Collection and use of these tissues was approved by the University of Washington Human Subjects Review Committee. The investigation conforms with the principles outlined in the Declaration of Helsinki [Cardiovascular Research, 1997;35:2–3].

2.4 Cardiac cryoinjury and cardiomyocyte grafting model
All experiments were approved by the University of Washington Animal Care Committee and were in accordance with federal guidelines. Syngeneic Fisher 344 rats (Simonsen Labs., Gilrey, CA, USA) were used for all grafting studies to prevent immune rejection. To identify the donor cardiomyocytes after grafting, the fetuses were tagged in utero by continuous administration of BrdU to their mothers for 1 week prior to delivery. Myocytes were isolated from 1–2-day-old pups by collagenase/pancreatin digestion as recently detailed [26]. The cells from four to five consecutive 10-min digestions were pooled and then preplated for 30 min to reduce non-myocyte contamination. Preparations averaged >90% cardiomyocytes, as evidenced by positive staining for sarcomeric isoforms of myosin heavy chain (MF-20 staining). Cells were used for grafting within 2 h of isolation.

The cardiac cryoinjury model has recently been described in detail [27]. In brief, adult male Fisher rats were anesthetized with ketamine–xylazine, intubated and mechanically ventilated with room air. The heart was exposed via left thoracotomy. Cryoinjury was performed by placing a 7-mm aluminium rod, precooled with liquid nitrogen, on the anterior surface of the heart for 15 s. This resulted in a disc-shaped zone of coagulation necrosis extending 1 cm laterally and approximately half way through the wall. Previous studies have shown that the events of inflammation and repair proceed virtually identically to myocardial infarction.

For grafting studies, 4x10⁶ cardiomyocytes were suspended in 70 µl of serum free medium and used immediately. Grafting was performed using a Hamilton syringe and a 30 gauge needle. The needle was inserted superficially into acutely necrotic muscle and the cell suspension injected. The chest then was closed and the rats allowed to survive for defined intervals from 1 day to 2 weeks. After sacrifice by pentobarbital overdose, the hearts were perfusion fixed with methyl Carnoy's fixative (methanol–glacial acetic acid–chloroform, 60:30:10, v/v/v) and routinely processed and embedded in paraffin for histology. The protocol conforms 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).

2.5 c-FLIP antibody preparation, characterization and localization
A peptide of the N-terminal death-effector of c-FLIP corresponding to amino acids 10–27 (sequence=EEALDTDEKEMLIFLCRD) was used to generate an affinity-purified rabbit polyclonal antibody, termed MAG1. The specificity of this antibody was confirmed as described previously [28]. As predicted, the antibody recognizes a 54 kDa c-FLIP in total cell lysates from cardiomyocytes. To demonstrate a localization of c-FLIP, Hela cells were transiently transfected with a c-FLIP expression vector that encodes full-length c-FLIP using lipofectamine (Gibco/BRL) as described previously [19]. In addition, the staining pattern of c-FLIP was determined with MAG1 antibody by using human vascular smooth muscle cells (VSMCs). Immunostaining was performed by incubation of cells on glass slides with the MAG1 antibody (titer=1:100) for 1 h at 37°C. c-FLIP immunoreactivity was detected with a biotinylated anti-rabbit IgG secondary antibody and streptavidin–fluoresceine. The cells were then stained with Hoechst 33342 at a final concentration of 4 µg/ml for 30 min at 37°C to identify cell nuclei.

2.6 Immunohistochemistry and TUNEL assay
The indirect avidin–biotin horseradish peroxidase and alkaline phosphatase visualization method for immunohistochemistry and TUNEL assay has been described previously [28,29]. The primary antibodies used for immunohistochemistry analyses include a polyclonal antibody against c-FLIP (MAG1, 1:100 dilution) and a monoclonal antibody recognizing all forms of sarcometric MHC in heart and adult skeletal muscle (MF20, Dako, Carpinteria, CA, USA, 1:1000). All antibodies were diluted in PBS containing 10% normal goat serum and 1% bovine serum albumin and incubated with the tissue sections for 1 h at room temperature. For negative controls, non-immune isotype matched IgG was substituted for the primary antibody. In addition, competitive inhibition of the MAG1 antibody with the immunizing peptide was performed by preincubation for 30 min to 1 h at 37°C.

2.7 Double staining
The first immunostaining using MAG1 antibody was developed with 3,3'-diaminobenzidine (Sigma), which produced a brown reaction product. The second sequence of staining was performed on the same sections for TUNEL, with an avidin–alkaline phosphatase–substrate system and Vector Red (Vector ABC kit, Vector Labs.) which produced a red product.

2.8 Preparation of riboprobes
Full-length c-FLIP cDNA (1.4 kb) was subcloned into pBluescript II (Stragene, La Jolla, CA, USA) as described previously [17]. To generate anti-sense and sense RNA probes for in situ hybridization, plasmids were linearized with XbaI and KpnI, respectively. The plasmids were then in vitro transcribed with ³⁵S-labeled -thio UTP (New England Nuclear-Dupont) following a modified method of Wilcox et al. [30]. To improve penetration into sections during hybridization, transcripts were shortened by alkaline hydrolysis to a calculated average length of 250 bases.

2.9 In situ hybridization
In situ hybridization was performed with ³⁵S-labeled riboprobes on 5-µm thick, deparaffinized sections of 4% paraformaldehyde-fixed tissue. Riboprobes were separated from unincorporated counts by passage over G-50 NICK columns (Pharmacia, Piscataway, NJ, USA). The peak radioactive fractions were treated with phenol–chloroform, and the aqueous phase was precipitated with ethanol and resuspended to 300 000 cpm/ml in TE buffer. A 1-µl volume of the riboprobe was mixed in 50 µl hybridization buffer, applied to each section, and hybridizations were performed at 55°C overnight. Washes included treatment with RNAase A (20 mg/ml, Sigma) for 30 min at 37°C and a stringent wash in 0.1xSSC at 55°C at 2 h. After dehydration in graded alcohols and air drying, slides were dipped in NTB2 emulsion (Eastman Kodak), exposed in the dark at 4°C for 14 days, and developed as described previously [30]. Hematoxylin was used as counterstain.

2.10 Western blotting
For detection of c-FLIP, whole cell lysates (30 µg/lane) and tissues including adult and fetal mouse heart were resolved by SDS–PAGE, transferred to PVDF membranes, and processed as previously described [31]. The protein concentration of the lysate was determined using the Pierce micro BCA reagents (Rockland, IL, USA). The MAG1 antibody was used at a dilution of 1:100 representing a final concentration of 1.4 µg/ml of peptide affinity purified IgG. The secondary antibody, an anti-rabbit IgG labeled with horseradish-peroxidase, was used at a dilution of 1:30000 (Bio-Rad, Hercules, CA, USA). Visualization of signal was by ECL (Amersham).

2.11 Statistical analysis
The cell death assay was repeated three times in separate experiments. Because data distribution in experimental groups was not normal, non-parametric statistical analysis was performed (Mann–Whitney rank sum test), using SPSS software. A P value less than 0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 c-FLIP expression and cell death in human heart tissue
We first tested cellular localization of c-FLIP protein by performing immunofluorescent staining. As shown in Fig. 1A, using the MAG1 antibody, we found that c-FLIP protein was both in cytoplasm and nucleus in human VSMCs. To confirm that c-FLIP protein can be found in both nuclei and cytoplasmic components, we constructed GFP–c-FLIP fusion protein and examined the localization of fusion protein upon transfection. As shown in Fig. 1C, transient transfection of GFP–c-FLIP fusion protein resulted in uniform distribution of c-FLIP in cytoplasm and nucleus of these cells. In contrast, immunofluorescent staining with anti-tubulin antibody revealed a clear cytosolic fibrillar distribution (Fig. 1D). c-FLIP distribution was examined in end-stage human hearts, including hearts from six patients with end-stage ischemic heart disease and five patients with dilated cardiomyopathy. We and others have previously reported abundant c-FLIP mRNA expressions in human heart tissues by Northern hybridization [14–19]. Consistent with these observations, c-FLIP mRNA was highly abundant in human cardiomyocytes from end-stage failing hearts (Fig. 2C), as evidenced by in situ hybridization of cells identified as cardiomyocytes by positive staining for sarcomeric isoforms of myosin heavy chain, MF20 (Fig. 2A). No specific signal was detected in control hybridizations performed with sense c-FLIP riboprobes (Fig. 2D). c-FLIP protein was also found in human cardiomyocytes with staining in the cytoplasm and, less commonly, the nucleus (Fig. 2B). The immunizing peptide completely abolished immunoreactivity (data not shown).

View larger version (122K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Distribution of c-FLIP within cells. Human VSMCs were stained for both c-FLIP using MAG-1 antibody and nuclear morphology (Hoechst 33342) (A, B; respectively). To confirm the c-FLIP localization in cells, we constructed a GFP–c-FLIP fusion protein followed by examining the localization of fusion protein upon transfection. Note that transient GFP–c-FLIP fusion protein resulted in uniform distribution of c-FLIP in cytoplasm and nucleus (C). To confirm the cytoplasm, immunofluorescent staining with anti-tubulin antibody was examined (D).

 

View larger version (122K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Localization of c-FLIP protein and mRNA in normal regions of human hearts removed at the time of transplantation for ischemic cardiomyopathy. c-FLIP protein (B) and mRNA (C) are detected in cardiomyocytes identified by immunostaining of sarcomeric isoforms of myosin heavy chain using MF20 antibody (A). No detectable signal was present when sense probe was used to hybridize adjacent sections from corresponding human hearts (D). Bar in D=50 µm and applies to A–C.

 
As previously reported by others in end-stage heart failure [4,5], we observed TUNEL-positive cells in tissues from patients with end-stage heart failure. Most of these TUNEL-positive cells were interstitial cells, but rare TUNEL-positive cardiomyocytes were present. Double staining of end-stage heart samples with c-FLIP antibody and by TUNEL assay demonstrated that c-FLIP-positive cells were TUNEL-negative in both ischemic and dilated cardiomyopathy. Representative tissues are shown in Fig. 3, including two samples of ischemic cardiomyopathy and two of idiopathic cardiomyopathy.

View larger version (108K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Inverse correlation of c-FLIP expression and TUNEL positivity in end-stage human hearts. Sections were double stained using a combination of TUNEL assay and immunohistochemistry with c-FLIP antibody in end-stage human heart, including two hearts with ischemic cardiomyopathy (A–D) and two hearts with idiopathic dilated cardiomyopathy (E–H). Immunostaining for c-FLIP antibody yielded a brown color. The TUNEL stain was visualized by using an avidin–alkaline phosphatase substrate system, yielding red nuclei (fluorescent reaction product) as described [26]. Photomicrographs of slides examined by light microscopy (A,C,E,G) or fluorescence microscopy (B,D,F,H) are included. Note that c-FLIP-positive cells (brown) and TUNEL-positive cells (red) are distinct populations in all samples. Data are representative of eleven hearts, from six patients with ischemic cardiomyopathy and five patients with dilated cardiomyopathy. Bar in A=100 µm and applies to B–H.

 
3.2 c-FLIP expression and cell death following neonatal cardiomyocyte grafting
Neonatal cardiomyocytes have a high spontaneous rate of cell death after grafting into injured hearts [32,33], which represents a possible useful model for anti-apoptotic therapies. We therefore examined the relationship between cell death and c-FLIP expression in neonatal cardiomyocyte grafts. We examined the relationship between c-FLIP expression and cell death in neonatal cardiomyocytes transplanted into freeze-induced regions of rat heart. The identity of grafted cells in the myocardium were confirmed by staining for their BrdU tag in adjacent sections (Fig. 4A). By TUNEL assay, 52% of transplanted cells had fragmented DNA by 24 h. By double staining for TUNEL and c-FLIP, we found that TUNEL-positive myocytes were rarely c-FLIP positive (1.0%). In contrast, TUNEL-negative/c-FLIP-positive myocytes were observed frequently (10.0%) (Fig. 4B and C).

View larger version (100K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Inverse correlation of c-FLIP expression and TUNEL-positivity in neonatal cardiomyocyte grafting model. Photomicrographs showing cardiomyocytes 1 day following injection into cryoinjured rat hearts by immunohistochemical reaction against BrdU (top), and double staining for c-FLIP and TUNEL (center and bottom, respectively). Arrows indicate c-FLIP-positive and TUNEL-negative cardiomyocytes. Arrowheads indicate c-FLIP-negative and TUNEL-positive cardiomyocytes. Bar in A=50 µm and applies to B and C.

 
Given the strong expression of c-FLIP in myocardium in vivo and the diminished staining in grafted cardiomyocytes, we hypothesized that the cell isolation process might diminish c-FLIP expression. When c-FLIP expression was compared in adult and neonatal cardiomyocytes by Western blots immediately after isolation and after 1 day in culture, c-FLIP protein levels were markedly diminished by day 1 in culture (Fig. 5).

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Down-regulation of c-FLIP protein expression in cultured adult and neonatal cardiomyocytes. Western blot analysis of c-FLIP protein expression (left) in adult cardiomyocytes in vivo (lane 1) versus adult cardiomyocytes maintained in culture for 1 day. Western blot analysis of c-FLIP protein expression (right) in neonatal cardiomyocytes in vivo (lane 1) versus neonatal cardiomyocytes maintained in culture for 1 day (lane 2).

 
3.3 Activated monocytes kill neonatal cardiomyocytes
To examine whether the Fas-dependent pathway is active in isolated cardiomyocytes, we next tested whether activated monocytes can kill neonatal cardiomyocytes by using a co-culture system. A chromium release assay was used to determine the ability of monocytes, activated with immune complexes, to kill neonatal cardiomyocytes. As shown in Fig. 6, neonatal cardiomyocytes released significant Cr⁵¹ into the media when co-cultured with monocytes. This release of Cr⁵¹ was blocked by anti-Fas IgG1 (1 µg/ml) to a level comparable to the release caused by the addition of unstimulated monocytes to the neonatal cardiomyocytes. This suggests that neonatal cardiomyocyte death was mediated by a Fas/FasL-dependent pathway.

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Monocyte induced killing of cardiomyocytes. Monocytes were co-cultured with neonatal cardiomyocytes prelabeled with radiolabeled chromium. Monocytes activated by immune complexes caused increased release of radioactive chromium. Release of Cr51 was blocked by anti-Fas IgG1 (1 µg/ml, P<0.01) to a level comparable to release caused by addition of unstimulated monocytes to the neonatal cardiomyocytes. *, P<0.05 Mann–Whitney test for activated monocyte group vs. control.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Recently, Yaoita et al. reported in a rat model of myocardial infarction that treatment with the caspase inhibitor z-VAD-fmk led to a limitation in infarct size and an improvement of acute functional parameters [34]. Besides opening the door to the potential therapeutic use of caspase inhibitors, this finding focuses attention on the possible role of endogenous inhibitors of death pathways. Our data on c-FLIP expression in heart support and extend the observations of Rasper et al. [18], which showed that ‘Usurpin’ (c-FLIP) was decreased in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following rat ischemia–reperfusion injury [18]. We showed that c-FLIP RNA and protein were abundantly expressed in human cardiomyocytes. Double staining revealed that c-FLIP-positive cardiomyocytes were rarely apoptotic by TUNEL assay. Finally, activated monocytes, which release soluble FasL following stimulation as we have previously reported [24], could kill neonatal cardiomyocytes which could be inhibited by preincubation with a blocking anti-Fas IgG1, suggesting that neonatal cardiomyocyte death is mediated by a Fas-dependent pathway.

The principal caveat in both our study and that of Rasper et al. is that loss of c-FLIP could be a consequence of protein degradation or loss, unrelated to the mechanism of cell death. Against this possibility, Rasper et al. failed to show degradation of endogenous ‘Usurpin’ in Jurkat lymphocytes induced to die by ligation of Fas [18]. In addition, Scaffidi et al. showed that c-FLIP is recruited to the CD95 death-induced signaling complex (DISC), where it is partially proteolyzed but remains detectable by immunoprecipitation during Fas-induced death [35]. Thus, absence of c-FLIP could result from a transcriptional event or a protein turnover event that precedes sensitivity to specific stimuli.

Interestingly, Thome et al. [12] found that c-FLIP protein levels correlated positively with resistance to Fas-mediated death observed in lymphocytes following activation by phytohemagglutinin. The same cells also fail to recruit caspase 8 (FLICE) after presentation of FasL. In contrast, they also found that c-FLIP does not protect from staurosporine or growth factor withdrawal. More recently, Algeciras-Schimnich et al. used the TAT membrane transport sequence to create a membrane-permeant chimera with c-FLIP. This chimera was able to protect Jurkat cells from Fas-mediated death [36]. These findings suggest that c-FLIP is a reasonable candidate for a regulator of cell death via the Fas–FasL pathway.

We have previously reported that monocytes/macrophages rapidly release soluble FasL following stimulation [24]. The role of soluble FasL, however, is still controversial. FasL is synthesized as a transmembrane, 37-kDa type II protein. This protein, however, can be cleaved by a metalloprotease to form a smaller, soluble molecule [37,38]. Older studies suggested that soluble FasL may be ineffective in inducing apoptosis or could actually down-regulate the proapoptotic activity of membrane-bound FasL [39,40]. More recent studies, however, indicate that biologically active soluble FasL is rapidly released in vitro by normal human monocytes and macrophages during phagocytosis and following activation with phytohemagglutinin, immune complexes, or superantigen [41]. This soluble FasL can function in vitro as a cytokine to induce apoptosis in susceptible cells. The current studies demonstrates that the death of neonatal cardiomyocytes can be induced by activated monocytes and this response is mediated by Fas–FasL system because this response can be blocked by anti-Fas IgG1. If we allowed neonatal cardiomyocytes to stay in vitro for 24 h, they lost or diminished c-FLIP protein levels. In addition, in our in vivo data only 1% of c-FLIP positive implanted cells showed cell death, whereas 57% of c-FLIP negative implanted cells were positive for TUNEL. So, c-FLIP may protect against Fas-mediated cardiomyocyte cell death.

Finally, these ex vivo manipulations of neonatal cardiomyocyte grafts may provide a useful model for therapeutic interventions because we can examine whether an overexpression of anti-apoptotic molecule (e.g. adenoviral c-FLIP) in neonatal cardiomyocytes decreases the apoptotic cells in grafted cardiac tissues or not.

It is important that these in vivo studies of antiapoptotic molecules, except for the antisense studies of BCl-XL [42], are phenomenological. The absence of c-FLIP in dying cells, indicated by simultaneous staining with TUNEL and an c-FLIP antibody, may be a consequence of protease generated during death rather than a cause. Although we have no direct way of answering this concern, in vitro studies with death receptor-activated death in lymphocytes show that c-FLIP is truncated to a form still recognized by the MAG-1 antibody or antibodies directed at the same peptide sequence [36]. A further caution is the use of TUNEL to identify dead cells. TUNEL data clearly depend on the method used. For example, this reported value of TUNEL-labeled cells obtained from atherosclerotic plaques ranged from less than 2% up to 60% [43]. Thus, TUNEL positivity probably only reflects occurrence of death rather than giving an absolute value [44]. To provide experiment-to-experiment consistency in the current study, however, TUNEL analyses were always performed including human tonsil and rat thymus as positive controls of human and rat samples, respectively.

In conclusion, although c-FLIP RNA and protein are abundantly expressed in cardiomyocytes, c-FLIP protein is down-regulated in TUNEL-positive grafted cardiomyocytes in animal models. More importantly, c-FLIP-positive cardiomyocytes rarely show evidence of apoptosis in failing hearts. We suggest that expression levels of c-FLIP may determine whether cardiac cells die or not. If so, c-FLIP is a reasonable candidate for a regulator of cardiac cell death and may be a novel target for end-stage heart and rejected heart transplant. Future investigations will be required to confirm this hypothesis.

Time for primary review 27 days.

	Acknowledgements

 
This work is supported by NIH grants HL03174 and DK02456.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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