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Originally published In Press as doi:10.1074/jbc.M404616200 on June 7, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34882-34889, August 13, 2004
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Bcl-2 Is a Key Factor for Cardiac Fibroblast Resistance to Programmed Cell Death*

Maritza Mayorga{ddagger}§, Núria Bahi{ddagger}, Manel Ballester||, Joan X. Comella{ddagger}**, and Daniel Sanchis, Supported by a postdoctoral grant from the Generalitat de Catalunya and by the Fondo de Investigaciones Sanitarias (01/3023){ddagger}**{ddagger}{ddagger}§§

From the {ddagger}Group of Cell Signaling & Apoptosis, Departament de Ciències Mèdiques Bàsiques and ||Departament de Medicina, Facultat de Medicina, Universitat de Lleida, 25008 Lleida, Spain and the {ddagger}{ddagger}Laboratori de Recerca, Hospital Universitari Arnau de Vilanova, 25198 Lleida, Spain

Received for publication, April 26, 2004 , and in revised form, May 28, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiac fibroblasts play an essential role in the physiology of the heart. These produce extracellular matrix proteins and synthesize angiogenic and cardioprotective factors. Although fibroblasts of cardiac origin are known to be resistant to apoptosis and to remain metabolically active in situations compromising cell survival, the underlying mechanisms are unknown. Here, we report that cardiac fibroblasts were more resistant than dermal or pulmonary fibroblasts to mitochondria-dependent cell death. Cytochrome c release was blocked in cardiac fibroblasts but not in dermal fibroblasts treated with staurosporine, etoposide, serum deprivation, or simulated ischemia, precluding caspase-3 activation and DNA fragmentation. Resistance to apoptosis of cardiac fibroblasts correlated with the expression of the anti-apoptotic protein Bcl-2, whereas skin and lung fibroblasts did not express detectable levels of this protein. Bcl-xL, Bax, and Bak were expressed at similar levels in cardiac, dermal, and lung fibroblasts. In addition, the death of cardiac fibroblasts during hypoxia was not associated with the cleavage of Bid but rather with Bcl-2 disappearance, suggesting the requirement of the mitochondrial apoptotic machinery to execute death receptor-induced programmed cell death. Knockdown of bcl-2 expression by siRNA in cardiac fibroblasts increased their apoptotic response to staurosporine, serum, and glucose deprivation and to simulated ischemia. Moreover, dermal fibroblasts overexpressing Bcl-2 achieved a similar level of resistance to these stimuli as cardiac fibroblasts. Thus, our data demonstrate that Bcl-2 is an important effector of heart fibroblast resistance to apoptosis and highlight a probable mechanism for promoting survival advantage in fibroblasts of cardiac origin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Programmed cell death (PCD)1 is involved in heart morphogenesis during embryonic development (1). In the early postnatal period, cardiac myocytes exit the cell cycle and differentiate (2, 3). This implies that in the adult heart newly formed cells cannot replace dead cardiomyocytes although a limited capacity of proliferation has been described (4). The requirement of maintaining heart function in stress situations has selected mechanisms of resistance against stress-induced cell death in this organ. Indeed, an incomplete apoptotic phenotype of cardiomyocytes occurs in heart disease (5, 6). The molecular mechanisms involved in resistance against apoptosis in the heart are now being elucidated. It has been reported that cardiomyocytes are relatively resistant to Fas-induced cell death (7–9). On the other hand, cardiomyocyte mitochondrial dysfunction, comprising mitochondrial potential dissipation, permeability transition, and cytochrome c release, occurs in response to several stress stimuli such as postischemic reperfusion (10), H2O2-induced oxidative stress (11), or hypertrophy (12). However, the exact role that mitochondria play in cardiac apoptosis is still a matter of debate (13). In this regard, we have recently reported that cardiac myocytes are resistant to apoptosome-driven apoptosis by a mechanism involving the silencing of Apaf-1 expression (14).

Despite the relative resistance of cardiac myocytes to caspase-dependent death, it is well established that apoptosis accounts for cardiac cell death during ischemia/reoxygenation (15, 16), hypertension (17), and in maladaptive hypertrophy (18), although necrosis and autophagic cell death also play a relevant role (19). Interestingly, the space left by dead myocytes is filled by granulation tissue composed of several cell types, including macrophages, endothelial cells and fibroblasts (20), and newly synthesized extracellular matrix (ECM) (21, 22), which is produced mainly by cardiac fibroblasts (23). This observation implies that cardiac fibroblasts are able to endure and secrete the ECM that constitutes the fibrotic scar under situations that threaten survival of other cell types. Consistent with the enhanced survival potential of cardiac fibroblasts, it has been reported that these cells have reduced apoptosis and sustained proliferation during hypoxia (24), alcohol exposure (25), and oxidative stress (26, 27). However, the molecular mechanisms underlying the resistance of cardiac fibroblasts to stimuli inducing cell death are presently unknown.

In an attempt to highlight the possible existence of mechanisms conferring survival advantage specifically to fibroblasts of cardiac origin, we decided to explore them with several inducers of apoptosis and to compare their response and the expression of several apoptotic regulators with those of primary dermal and pulmonary fibroblasts. Indeed, fibroblasts of cardiac origin were more resistant than dermal- and lung-derived fibroblasts to staurosporine (STS), etoposide, serum deprivation, and simulated ischemia. We further analyzed the intracellular mechanisms involved. Our data pointed to the blockade of cytochrome c translocation as the main mechanism of cardiac fibroblast resistance to apoptosis. Furthermore, cardiac fibroblasts expressed an easily detectable level of the anti-apoptotic protein Bcl-2, which is repressed in many cell types after development (28), including dermal (29) and lung (30) fibroblasts. Here, we provide evidence supporting the notion that the maintenance of Bcl-2 expression in cardiac fibroblasts confers their resistance to mitochondria-dependent apoptosis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and Treatments—We obtained neonatal cardiac fibroblasts from the heart of 2–4-day-old Sprague Dawley rats. After digestion of the hearts with type 2 collagenase (Worthington, Lakewood, NJ), cells were pelleted, seeded in 10-cm FALCON polystyrene dishes (BD Biosciences), and incubated for 45 min in Dulbecco's modified Eagle's medium (Sigma-Aldrich) with 10% fetal calf serum (Invitrogen) and antibiotics. Medium was removed to eliminate cardiomyocytes that did not attach to the non-coated plates, and these were replaced with fresh medium. Cardiac fibroblasts were allowed to grow until confluence was reached, then trypsinized, and passaged twice before use. Skin and lung fibroblasts were obtained by collagenase digestion of dorsal skin patches and lungs from the same pups used to obtain the heart fibroblasts. Dermal and lung fibroblasts were cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and used after two or three passages. At the time of performing the experiments, all cultures were between 70 and 90% confluent. Etoposide (Sigma) was used at 100 µM over 24 h. STS was added to the culture medium at the concentrations and times described in the figures. We added the pan-caspase inhibitor z-VAD-fmk (Enzyme System Products, Livermore, CA) at 100 µM when indicated. For serum and glucose deprivation, cells were rinsed twice in sterile PBS and cultured in Dulbecco's modified Eagle's medium without glucose and pyruvate (cat. no. D-5070 from Sigma). Simulated ischemia was performed by culturing cells in deprivation medium inside a hypoxic chamber (Billups-Rothenberg) in a mixture of 5% CO2 and 95% N2 following the manufacturer's instructions to attain a 0.1% oxygen concentration, and incubated at 37 °C for the time periods indicated in the figures.

Quantification of Cell Death—Detection of cell death was performed by the trypan blue exclusion assay at the end of treatment. Data are expressed as percentage of cell death in treated dishes versus equally seeded control dishes at the initiation of the treatments. Apoptosis was quantified as percentage of cells showing condensed or fragmented nuclear morphology versus total nuclei after nuclear staining with bis-benzimide Hoechst H33258 [GenBank] dye (Sigma). Cell death and apoptosis for each experimental condition were measured in duplicate, and error bars represent the S.E. of three independent experiments. DNA fragmentation was assessed by conventional agarose gel electrophoresis as previously reported (14).

Preparation of Cytosolic Extracts—Cytosolic fractions were obtained at the end of the treatments by mixing pelleted cells in a buffer without detergent containing 220 mM D-mannitol and 70 mM sucrose (Sigma). Cells were incubated on ice for 25 min and subjected to a serial centrifugation protocol as previously reported (31). Purity of the cytosolic extracts were checked by Western blot detection of lactate dehydrogenase (LDH) as a specific marker of the cytosolic fraction, and cytochrome oxidase subunit IV (COXIV) as a marker for mitochondrial membrane contamination. Images are representative results of three independent experiments.

Protein Extraction, Western Blotting, and Immunofluorescence—At the end of the treatments, cells were scraped from the culture dishes, pelleted, and washed with ice-cold PBS. After lysis in 95 °C prewarmed 125 mM Tris, 2% SDS (pH 6.8), cell lysates were centrifuged, and the supernatant was used as whole protein cell lysate. Protein concentration was then measured in total and cytosolic extracts by the Lowry assay. SDS-PAGE electrophoresis was performed, and protein was electrotransferred to Immobilon-P filters (Millipore, Bedford, MA) and reacted with relevant primary antibodies. Immunoblots were exposed to appropriate peroxidase-conjugated secondary antibodies and developed with the ECL System (Biological Industries, Israel) or the SuperSignal Substrate (Pierce/Cultek). For immunofluorescence detection of cytochrome c and activated caspase-3, cells were grown in 4-well plates (Nunc, Roskilde, Denmark) and were fixed with 4% paraformaldehyde (PFA), rinsed twice with PBS, and processed as described (14). Finally, cell nuclei were stained with Hoechst for 10 min at room temperature. Cells were rinsed twice with PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA).

Antibodies—Primary antibodies used in this work were: rabbit polyclonals anti-rat Bcl-xL (cat. no. 610211, BD Transduction Laboratories, Palo Alto, CA), anti-caspase-3 (cat. no. AB1899, Chemicon, Temecula, CA) diluted 1:3,000, anti-human-processed caspase-3 (cat. no. 9661, Cell Signaling, Beverly, MA) diluted 1:2,000, anti-human Bak (cat. no. 12-01-16348, Biocarta, Hamburg, Germany) diluted 1:5,000, anti-human Bax (cat. no. 2772, Cell Signaling) diluted 1:5,000, anti-Bim (cat. no. AAP-330, Stressgen Biotech, Victoria, BC, Canada) diluted 1:4,000, anti-human Smac/DIABLO (cat no. PSC-2409-C100, ProSci Inc.) diluted 1:3,000; goat polyclonal anti-rabbit lactate dehydrogenase (code 100–1173, Rockland, Gilbertsville, PA) diluted 1:5,000; rat monoclonals anti-mouse Apaf-1 (cat. no. ALX-804-349) diluted 1:4,000, anti-mouse Bid (cat. no. MAB860, R&D Systems, Minneapolis, MN) diluted 1:2,000; mouse monoclonals anti-mouse Bcl-2 clone 10C4 (NeoMarkers, Fremont, CA) diluted 1:2,000, anti-COX subunit IV (cat. no. A-21348, Molecular Probes, Eugene, OR) at 1:5,000, anti-rat cytochrome c (cat. no. 556432, BD PharMingen) diluted 1:500, anti-pigeon cytochrome c (cat. no. 556433, BD PharMingen) at 1:5,000, and anti-human-XIAP (cat. no. AAM-050, Stressgen) diluted 1:1,000. Horseradish peroxidase-conjugated secondary antibodies were donkey anti-rabbit (cat. no. NA9340V, Amersham Biosciences) at 1:10,000, rabbit anti-rat (cat. no. 61-9420, Zymed Laboratories, San Francisco, CA) at 1:5,000, and goat anti-mouse (cat. no. A9917, Sigma-Aldrich) diluted 1:10,000. For immunofluorescence, secondary antibodies were Rhodamine Red-conjugated donkey anti-mouse (code no. 715-295-150, Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 488-conjugated goat anti-rabbit (cat. no. A-11008, Molecular Probes), both at 1:500.

Extraction of RNA and RT-PCR of Bcl-2—Total RNA was purified with the Tri Reagent method (Molecular Research Center, Inc., Cincinnati, OH) from 3 to 4 x 106 control cardiac and dermal fibroblasts, following the manufacturer's instructions. Equal amounts of total RNA were retrotranscribed using ThermoScript reverse transcriptase (Invitrogen) and random hexamers (Roche Applied Science). The presence of Bcl-2 transcripts was checked by PCR using the rat Bcl-2-specific primers Bcl2RnFWD 5'-TGCACCTGACGCCCTTCAC-3' and Bcl2RnREV 5'-ACACAGCCAGGAGAAATCAAACAG-3', which amplify a fragment of 293 bp. The annealing temperature was 58 °C, and elongation time was 30 s per cycle, in a GeneAmp PCR System 2700 thermocycler (Applied Biosystems, Foster City, CA). PCR products from cycles 25, 30, and 35 from retrotranscribed samples and control samples, where the RT reaction was omitted, were migrated in 2% agarose gels and visualized by ethidium bromide staining. Amplification of ribosomal gene L27 was carried out as the control for cDNA input in the amplification reaction (32).

Bcl-2 and Bax Transfection—Human Bcl-2 gene was cloned in pcDNA3 (31). Dermal fibroblasts were transfected by electroporation (Bio-Rad Gene Pulser II) at 240 V and 500 microfarads in 0.5 ml of cold PBS with 1 µg of pcDNA3-Bcl-2, or empty vector, plus 0.3 µg of pcDNA3-enhanced yellow fluorescent protein (EYFP) per 105 cells. Cardiac fibroblasts were electroporated at 330 V with 1 µg of pcI-His-Bax (generously supplied by Jean-Claude Martinou, University of Geneva) or the empty vector, plus 0.3 µg of pcDNA3-EYFP. In all the vectors used, expression was driven by the cytomegalovirus promoter. Transfected cultures were treated 48 h later with STS at a final concentration of 0.1 µM or serum deprivation, for 24 h. Nuclei were visualized by bis-benzimide staining in 4% PFA-fixed cells, mounted with Vectashield (Vector Laboratories) and visualized with an Olympus IX70 vertical epifluorescence phase-contrast microscope. Apoptosis was counted as the percentage of transfected (green) cells showing chromatin condensation or fragmentation. Data shown are the mean of three independent experiments in duplicate.

Bcl-2 Gene Silencing—The following primers were annealed by standard protocols and cloned into pSUPER.retro.puro (Oligoengine Inc., Seattle, WA) previously digested by HindIII and BglII, in order to obtain the rat-specific Bcl-2 small interfering RNA (siRNA) construct (pSRPrBcl-2i): 5'-gatccccCGAGTGGGATAACTGGAGATttcaagagaATCTCCAGTATCCCACTCttttt-3' and 5'-agctaaaaaCGAGTGGGATACTGGAGATtctcttgaaATCTCCAGTATCCCACTCGggg-3'; or the scrambled construct (pSRPrBcl-2scr): 5'-gatccccGAGTATGAATAGCGAAGGCttcaagagaGCCTTCGCTATTCATACTCttttt-3', and 5'-agctaaaaaGAGTATGAATAGCGAAGGCtctcttgaaGCCTTCGCTATTCATACTCggg-3'.

In order to test the efficiency of the siRNA construct in repressing Bcl-2 expression, both constructs were transfected by electroporation into Rat1 rat embryonic fibroblasts (generous gift of Dr. Martín-Zanca). Stable transfected pools of Rat1 cells were obtained by puromycin selection and amplified in presence of the antibiotic. SDS-protein extracts of Bcl-2i-Rat1 and Bcl-2scr-Rat1 cells were tested for the expression of Bcl-2 and Bcl-xL by Western blot. Then, 10 µg of pSRPrBcl-2i or pSRPrBcl-2scr were co-transfected with 3 µg of pCDNA-EYFP by electroporation in 106 cardiac (330 V) or 106 dermal fibroblasts (240 V) at 500 microfarads in a volume of 0.5 ml of ice-cold PBS. Cells were then seeded in six 60-mm culture dishes and cultured in standard medium for 96 h before the initiation of the treatments, as described in the figure legend. Blind counting was adopted to minimize observer bias. Apoptosis was quantified in the cultures by counting green (transfected) cells with fragmented nuclei, visualized by Hoechst staining. Between 300 and 500 green cells were counted for each condition. Data were expressed as percent of green cells with fragmented nuclei versus total transfected cells in the culture plate. Experiments were repeated three times with independently isolated, amplified, and transfected primary fibroblasts.

Statistics—Student's t test was used to compare the difference between the response of cardiac and non-cardiac fibroblasts for every experimental condition, as well as to compare the difference between control group versus treatment group in the same type of fibroblasts. A p value < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cardiac but Not Dermal or Pulmonary Fibroblasts Are Resistant to Stimuli Inducing Mitochondria-dependent Programmed Cell Death—Cell death was measured in primary cardiac, dermal and pulmonary fibroblasts exposed to stimuli known to promote mitochondria-driven PCD: the topoisomerase-II inhibitor etoposide, which induces DNA damage (33); the kinase inhibitor STS (33), and serum deprivation (34). Cell death induced by 24 h of exposure to 100 µM etoposide, 0.1 µM STS, and serum deprivation, was more than 2–3-fold higher in pulmonary and dermal fibroblasts than in cardiac fibroblasts (Fig. 1A). The dose dependence analysis of the effect of exposure to STS for 24 h on fibroblast viability indicated that cardiac fibroblasts were less affected than dermal fibroblasts in a wide range of STS concentrations known to readily kill a number of other cell types (Fig. 1B). Nuclear fragmentation detected by bis-benzimide staining was also 2–3-fold higher in non-cardiac fibroblasts (Fig. 1C). We carried out the same treatments in wild type and Bak/Bax double knockout mouse embryonic fibroblasts (MEF), in parallel experiments. Indeed, cell death was completely blunted in Bak/Bax double knockout MEFs (Fig. 1B and data not shown), confirming that STS, etoposide, and serum deprivation induced cell death mainly by activating mitochondria-dependent apoptosis in fibroblasts, as has been previously reported (33). These results indicate that fibroblasts of cardiac origin were more resistant than skin and lung fibroblasts to apoptotic cell death driven by the mitochondrial pathway.



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  		FIG. 1.
Cardiac fibroblasts are more resistant than lung and skin fibroblasts to cell death induced by the apoptotic mitochondrial pathway. A, fibroblast cell death after 24 h of culture in the presence of 100 µM etoposide, 0.1 µM STS or SD. Cell death was measured using the trypan blue exclusion assay and is expressed as percentage of cell death versus untreated cultures at time 0. B, dose-dependent effects of STS on cell viability after 24 h of exposure. Cell death in wild type and Bak/Bax double knockout MEFs treated with 1 µM STS are depicted to highlight the relevance of mitochondrial apoptotic pathway in this treatment. C, apoptosis elicited by the same treatments as in A was calculated as percentage of cells showing nuclear condensation or fragmentation. Values are means ± S.E. of three independent experiments in duplicate. Statistically significant differences between treatment groups and their controls (p < 0.05), except for Bak/Bax double knockout MEFs and serum-deprived cardiac fibroblasts. *, p < 0.05 versus skin and/or pulmonary fibroblasts.

 
Cardiac Fibroblasts Do Not Attain Caspase-3 Activation nor DNA Degradation after STS Treatment and Serum Deprivation—The resistance of cardiac fibroblasts to apoptosis was confirmed by Western blot detection of the activated fragment of caspase-3, p17. STS and serum deprivation did not activate caspase-3 significantly in cardiac fibroblasts (Fig. 2A), but induced the activation of virtually the complete pool of caspase-3 in skin fibroblasts (Fig. 2B). Conventional agarose gel electrophoresis of DNA extracts from cardiac and dermal fibroblasts treated with 1 µM STS or serum deprivation showed the absence of fragmented DNA in cardiac fibroblasts, whereas laddering was obvious in DNA extracts of dermal fibroblasts (Fig. 2, C and D). The role of caspases in DNA damage induced by STS and serum deprivation was highlighted by the fact that DNA degradation was inhibited in dermal fibroblasts pretreated with 100 µM pan-caspase inhibitor z-VAD-fmk (Fig. 2D, and data not shown). These results reveal that cardiac fibroblasts can block caspase-dependent apoptosis before entering the execution phase.



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  		FIG. 2.
Staurosporine and serum deprivation do not induce activation of executioner caspases nor DNA fragmentation in cardiac fibroblasts. A, activation of caspase-3 was analyzed by immunodetection of the cleaved activated fragment (17/19 kDa) in protein extracts from cardiac (Heart F) and dermal (Skin F) fibroblasts treated with STS or SD. B, Western blot of total caspase-3 in whole protein extracts of cardiac (Heart F) and dermal (Skin F) fibroblasts treated with 1 µM STS. The band at {cong} 32 kDa corresponds to full-length procaspase-3. Asterisk indicates a product of the cleavage of caspase-3 not corresponding with the length of the active fragment. C, DNA low molecular weight fragmentation resolved by 1% agarose-gel electrophoresis and visualized by ethidium bromide staining of DNA extracts from cardiac (Heart F) and dermal (Skin F) fibroblasts after treatment with 1 µM STS for the indicated time intervals. D, DNA fragmentation induced by 24 h of SD in the presence or absence of 100 µM pan-caspase inhibitor z-VAD-fmk (I). A representative experiment out of three is depicted.

 
Apoptotic Mitochondrial Pathway Is Blocked in Cardiac Fibroblasts before Cytochrome c Release—To provide insight into the molecular pathways involved in cardiac fibroblast relative resistance to mitochondria-driven caspase-dependent apoptosis, we analyzed the translocation of cytochrome c and Smac/DIABLO in control, STS-treated, and serum-deprived cells. Both treatments induced translocation of cytochrome c and Smac/DIABLO to the cytosolic fraction in dermal fibroblasts but not in cardiac fibroblasts (Fig. 3A). The release of pro-apoptotic factors from mitochondria (Fig. 3A) correlated with the activation of caspase-3 (Fig. 2A) and nuclear fragmentation (Fig. 3B). These results suggest that cardiac fibroblast resistance to mitochondria-driven apoptosis occurs upstream of cytochrome c release.



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  		FIG. 3.
Staurosporine- and serum deprivation-induced programmed cell death is blocked in cardiac fibroblasts before cytochrome c translocation. A, immunodetection of cytochrome c, Bax, and Smac/DIABLO in the cytosolic fraction of cardiac (Heart F) and skin fibroblasts (Skin F) treated for 3 and 6 h with 0.1 and 1 µM STS; or after 6 h of SD. COXIV is a marker of mitochondrial membrane contamination, and LDH is a cytosolic marker. B, immunofluorescence detection of cytochrome c (red) and nuclear morphology (blue) in control (C) heart (HF) and dermal (SF) fibroblasts and treated with 0.1 µM STS for 24 h. Arrowhead indicates cell with diffuse cytochrome c staining and fragmented nuclei. Images show representative results from three independent experiments.

 
Cardiac Fibroblasts Are More Resistant than Dermal Fibroblasts to Apoptosis Induced by Simulated Ischemia—Blood flow interruption leads to a complex pathological situation termed ischemia. In addition to a drop in oxygen tension, which inactivates the respiratory chain and, therefore, impairs the efficient production of energy, ischemia also leads to a local deficiency in trophic factors, precursors, and energy-providing metabolites such as glucose. We focused on comparing the response of cardiac and dermal fibroblasts to such an adverse situation because it has been described that cardiac fibroblasts have a certain capacity to overcome heart infarction, at least for a certain time period (35), and die, in part, by apoptosis (36). In our experimental model of serum and glucose deprivation plus hypoxia, dermal fibroblasts readily activated apoptosis (Fig. 4A). Activation of executioner caspases and nuclear fragmentation in dermal fibroblasts was correlated with the depletion of the cytochrome c pool (Fig. 4, B and C). Cardiac fibroblasts resisted up to 72 h of simulated ischemia (Fig. 4A), and caspase-3 activation was only detected from 72 h onwards (Fig. 4B). These data suggest that cardiac resistance to ischemia-induced apoptosis involves mechanisms acting upstream of cytochrome c release.



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  		FIG. 4.
Cardiac fibroblasts are resistant to PCD induced by simulated ischemia. A, apoptotic cells were counted as described under "Experimental Procedures" in cardiac (Heart F) and dermal (Skin F) fibroblast cultures exposed to serum, glucose, and oxygen deprivation for periods between 6 and 72 h in a hypoxic chamber. Values are mean ± S.E. of three independent experiments in duplicate. *, p < 0.05 versus controls; #, p < 0.05 versus cardiac fibroblasts at the same time point. B, cleaved (activated) caspase-3 was immunodetected in whole protein lysates from cardiac (Heart F) and dermal (Skin F) fibroblasts cultured in deprivation medium (SDGD) in normoxia or hypoxia. Representative Western blot is shown from three independent experiments. C, cytochrome c was detected by immunofluorescence (red), and nuclei were stained with bis-benzimide (blue) in cultures of cardiac (HF) and dermal (SF) fibroblasts exposed to simulated ischemia during 48 h. Images were captured at x400 and are representative of three independent experiments. Arrowhead points to a cell with cytochrome c redistribution and intact nucleus. Arrows point to cells with cytochrome c redistribution and fragmented chromatin. Asterisks denote cells depleted of cytochrome c showing fragmented nuclei.

 
Bcl-2 Is Expressed in Cardiac but Not in Dermal or Pulmonary Fibroblasts—Bcl-2 family proteins control the integrity of membranes in mitochondria, endoplasmic reticulum, and nucleus and are the major known regulators of apoptotic cytochrome c release (37). The inactivation of anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Bcl-xL, induces cytochrome c release by a mechanism involving pro-apoptotic Bcl-2 family proteins, namely Bax and Bak. Changes in the activity of anti-apoptotic Bcl-2 family proteins are orchestrated by another subset of pro-apoptotic Bcl-2 family members known as BH3-only proteins (38). Furthermore, by modulating both anti- and pro-apoptotic members of the Bcl-2 family, BH3-only protein Bid is responsible for the activation of cytochrome c release upon induction of the death receptor pathway (39). Therefore, we analyzed the expression of Bcl-2-related proteins in total protein extracts from cardiac, pulmonary, and dermal fibroblasts. Detection of Bcl-2-related proteins was undertaken with much attention to the reliability of the antibodies, because several commercially available antibodies against these proteins are not specific (40). Thus, we directly tested antibodies against Bax and Bak on extracts from double knockout MEFs (data not shown), and we chose antibodies against Bid, Bim, Bcl-xL, and Bcl-2 in contrast to the bibliography (40). BH3-only proteins Bid and Bim, pro-apoptotic Bcl-2 family members Bax and Bak, and anti-apoptotic Bcl-xL were expressed at similar levels in cardiac, pulmonary, and dermal fibroblasts (Fig. 5A). Pro-apoptotic proteins Smac, XIAP, and Apaf-1, which act downstream of the mitochondrial release of cytochrome c, were also expressed in all fibroblast cell types (Fig. 4A). Most interestingly, Bcl-2 was undetectable in protein lysates of pulmonary and dermal fibroblasts whereas cardiac fibroblasts expressed easily detectable amounts of this protein (Fig. 5A). Fibroblasts were passaged several times before being used. We discarded the possibility of in vitro-induced Bcl-2 re-expression in cardiac fibroblasts or Bcl-2 repression in dermal and pulmonary fibroblasts by analyzing Bcl-2 expression in extracts of freshly isolated fibroblasts (data not shown).



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  		FIG. 5.
Cardiac fibroblasts but not dermal or pulmonary fibroblasts express the anti-apoptotic protein Bcl-2. A, Western blot analysis of Bcl-2 family members and apoptotic regulators was performed in whole protein extracts from control cultures of fibroblasts from heart (H), lung (L), and skin (S). B, representative agarose gel image from three independent experiments of Bcl-2 mRNA semi-quantitative RT-PCR amplification from total RNA samples of cardiac and dermal fibroblasts at PCR cycles 25, 30, and 35, as well as from reactions at cycle 35 where the reverse transcription was omitted (35RT-). LMW, ladder of molecular mass. Ribosomal L27 transcript amplification was used as control of the initial cDNA input.

 
In an attempt to determine whether the inhibition of Bcl-2 expression in dermal fibroblasts occurred at the transcriptional or translational level, we carried out Bcl-2 mRNA semi-quantitative determinations. We found no detectable levels of Bcl-2 transcript in total RNA extracts from dermal fibroblasts whereas in cardiac fibroblasts, the Bcl-2 mRNA was amplified from 25 cycles onwards (Fig. 5B), suggesting that bcl-2 expression is regulated in primary fibroblasts at the transcriptional level. Specific expression of Bcl-2 in fibroblasts of cardiac origin suggests that it plays a role mediating the resistance of these cells against apoptosis.

Resistance of Cardiac Fibroblasts to Apoptosis Correlates with Bcl-2 Expression but Not with Expression or Post-translational Modification of Other Bcl-2-related Proteins—Bcl-2 expression was maintained in heart fibroblasts during treatment with 100 nM and 1 µM STS and serum deprivation and was not induced in dermal fibroblasts by either treatment, whereas anti-apoptotic Bcl-xL and pro-apoptotic Bak and Bax were expressed at similar levels independent of cell type and treatment (Fig. 6A). BH3-only proteins Bid, Bim, and Bad control PCD upstream of the above-mentioned Bcl-2 family members (38). They respond to apoptotic stimuli by increasing their expression and/or by post-translational modification, such as cleavage (Bid) or phosphorylation (Bim, Bad) (38). Enhanced expression and/or phosphorylation of BH3-only protein Bim isoforms, which are necessary for apoptosis induction in other cell types such as neurons (40), did not correlate with caspase-3 activation in fibroblasts (Fig. 6A). We did not test Bad expression/phosphorylation because, to our knowledge, there are no reliable and commercially available antibodies detecting endogenous levels of this protein in rat samples (Ref. 40 and data not shown). In addition, the resistance of cardiac fibroblasts to simulated ischemia-induced PCD lasted while Bcl-2 expression was maintained. Caspase-dependent apoptosis was significant in these cells from 72 h of hypoxia and did not correlate with Bid cleavage but rather with the disappearance of Bcl-2 (Fig. 6B). Altogether, these data demonstrate that, contrary to dermal and pulmonary fibroblasts, cardiac fibroblasts maintain Bcl-2 expression in normal conditions, and that activation of apoptosis correlates with a decrease in Bcl-2 expression rather than with transcriptional or post-translational modifications of other Bcl-2-related proteins.



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  		FIG. 6.
Activation of caspase-dependent apoptosis during hypoxia correlates with disappearance of Bcl-2 rather than with Bid cleavage and Bcl-xL expression in cardiac fibroblasts. A, immunodetection of Bcl-2 family proteins in protein extracts of cardiac (Heart F) and dermal (Skin F) fibroblasts treated for 6 and 24 h with 0.1 and 1.0 µM STS or 24 h of SD. A representative image from three independent experiments is depicted. B, cardiac and dermal fibroblasts were exposed to 24 h of SDGD or to serum and glucose deprivation plus hypoxia during periods from 6 to 72 h as reported under "Experimental Procedures." Bid, Bcl-2, Bcl-xL, and cleaved caspase-3 were immunodetected in total protein lysates. Images are representative of three independent experiments.

 
Expression of Bcl-2 Is Involved in the Resistance of Cardiac Fibroblasts to Apoptosis—We further aimed at providing functional evidence for the role of Bcl-2 expression in the resistance of primary cardiac fibroblasts to apoptosis. We obtained a construct allowing Bcl-2 gene silencing using siRNA. A 19-base sequence specific for rat bcl-2, (selected from several primers suggested by the siRNA designer, available at the web address: www.promega.com/siRNADesigner/default.htm), was synthesized followed by a short hairpin sequence and the same Bcl-2-specific sequence in reverse order. This primer and its reverse were annealed and cloned into the pSUPER.retro.puro vector allowing selection by puromycin. Primary fibroblasts are transfected at 20–30% efficiency by electroporation. Very few cells remained after a 10-day selection with puromycin and, in addition, did not proliferate. Thus, in order to test the efficacy of the Bcl-2 siRNA construct in repressing Bcl-2 expression, we decided to use the Rat1 rat embryonic fibroblast cell line, as described under "Experimental Procedures" (Fig. 7A, inset). Once the specificity of the siRNA construct in repressing Bcl-2 but not the Bcl-xL gene was verified, we performed transient transfection of cardiac and dermal fibroblasts. Bcl-2 gene silencing induced a higher apoptotic rate in cardiac cells treated with STS, serum, and glucose deprivation or simulated ischemia, whereas it had no effect on dermal fibroblasts, as expected (Fig. 7A). These data confirmed a major role of Bcl-2 in preventing drug-induced and hypoxia-induced apoptosis. Percentages of apoptosis were lower than in experiments done with non-transfected cells. This fact could be explained by a subestimation of apoptotic-transfected cells caused by the loss of green fluorescence preferentially in apoptotic cells.



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  		FIG. 7.
The presence of Bcl-2 dictates the resistance of cardiac fibroblasts to apoptosis induced by STS, serum and glucose deprivation, and simulated ischemia. A, cardiac and dermal fibroblasts were co-transfected with EYFP and a vector allowing the inhibition of bcl-2 expression (siRNA Bcl-2) or a control vector, as described under "Experimental Procedures." Ninety-six hours later, cells were treated with 0.1 µM STS, deprived of serum and glucose (SDGD), or cultured under hypoxia without serum and glucose (Ischemia), for 24 h. Then, cells were PFA-fixed and stained with bis-benzimide. Apoptosis was counted as green (transfected) cells with nuclear fragmentation and expressed as percentage of total green cells. Values are means ± S.E. of three independent experiments. Inset shows Bcl-2 and Bcl-xL expression in stable transfected pools of the rat fibroblast cell line Rat1 expressing the Bcl2-siRNA vector (siRNA) or the control scrambled vector (scr), which were obtained as described above. *, p < 0.05 versus wild-type cells for each treatment. B, transient expression of Bcl-2 induces resistance to STS and SDGD-driven apoptosis in dermal fibroblasts. Fragmented nuclei were counted in Bcl-2 transiently transfected dermal fibroblasts as described under "Experimental Procedures." Treatments were initiated 48 h after transfection. STS was used at a final concentration of 0.1 µM for 24 h, and SDGD was maintained for 24 h. Data are expressed as condensed and fragmented nuclei per 100 total nuclei in transfected cells, i.e. cells expressing EYFP (mean ± S.E. of three independent experiments performed in duplicate; *, p < 0.05 versus wild-type cells).

 
Confirming the role of Bcl-2 in the resistance of heart fibroblasts to apoptosis, dermal fibroblasts transfected with a vector encoding for Bcl-2 (31) were more resistant to STS treatment and serum and glucose deprivation than cells transfected with empty vector (Fig. 7B). Actually, in Bcl-2-overexpressing dermal fibroblasts apoptosis dropped down to values similar to those observed in STS-treated cardiac fibroblasts, which express Bcl-2 (16.78% ± 3.01 in dermal fibroblasts expressing Bcl-2 versus 17.40% ± 2.12 in cardiac fibroblasts; n = 3). Furthermore, transient transfection of pro-apoptotic Bax in cardiac fibroblasts induced apoptosis despite Bcl-2 expression (50.08% ± 6.25 versus control, 5.58% ± 1.73, and STS, 17.40% ± 2.12; n = 3). Taken together, the above data suggest that apoptosis occurs through the mitochondrial pathway in primary fibroblasts and point to Bcl-2 as the main factor responsible for cardiac fibroblast resistance to programmed cell death.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cardiac injury resulting from myocardial infarction triggers the so-called cardiac remodeling, a complex phenomenon comprising changes of ventricular size, shape, and thickness (41). Remodeling palliates insufficient heart work, but finally leads to heart failure because of the loss of function of cardiomyocytes and a disproportionate accumulation of ECM, which increases tissue stiffness (42). During heart remodeling, cardiac fibroblasts proliferate and secrete ECM components that will fill up the space left by dead myocytes. The increased metabolism of cardiac fibroblasts, during stress situations promoting death of other cell types, underlies the development of resistance mechanisms against cell death.

The results presented here provide a likely explanation for the molecular mechanisms underlying the resistance of cardiac fibroblasts to PCD, and therefore at least in part, to cardiac fibroblast cell death. We report that primary cardiac fibroblasts are more resistant than fibroblasts of other origins, i.e. dermal and pulmonary fibroblasts, to cell toxicity triggered by stimuli inducing PCD. Treatment with STS, etoposide, serum deprivation, and simulated ischemia induced cell death at a much lower extent in fibroblasts of cardiac origin than in dermal fibroblasts. The high resistance of MEFs deficient for the Bcl-2-related pro-apoptotic proteins Bax and Bak, which are essential for mitochondrial dysfunction and PCD (33), confirmed the relevant role of the cytochrome c-dependent pathway in these experimental settings.

The main finding of the present work is that apoptotic cytochrome c release is blocked in isolated cardiac but not dermal fibroblasts, pointing to a mechanism of resistance to PCD that does not depend on cell type but on cell origin or level of differentiation. Inhibition of cytochrome c release correlated with constitutive expression of Bcl-2 but not of Bcl-xL and other Bcl-2 family members, which were equally expressed in all kinds of fibroblasts. We also show that Bid is cleaved in cardiac and dermal fibroblasts during simulated ischemia irrespective of executioner caspase activation. This suggests that mitochondria control the progression of death receptor-dependent PCD in fibroblasts and, by blocking this death pathway, cardiac fibroblasts are protected against a wide range of death stimuli. Furthermore, our data (showing resistance of cardiac fibroblasts up to 3 days from glucose and serum deprivation plus hypoxia) go beyond a previous report describing the lack of DNA degradation after 48 h of hypoxia alone (24). These findings are of pathophysiological relevance because cardiac fibroblasts play an important role in the healing of the ischemic lesion, where the drop in local oxygen tension is accompanied by a restriction in serum and glucose availability.

Originally, overexpression of Bcl-2 was identified as the main cause of lymphoblastic leukemia (43) and was found to provide a survival signal (44). Although the mechanisms by which Bcl-2 induces cell survival are still a matter of debate (37), they include blockade of cytochrome c translocation (45). This suggests that expression of Bcl-2 in fibroblasts of cardiac origin is involved in the blockade of cytochrome c release and in resistance against toxic stimuli. Maintenance of Bcl-2's constitutive expression in cardiac fibroblasts is interesting because Bcl-2 is down-regulated in most tissues after birth and remains restricted to lymphoid tissue, precursor hematopoietic cells, thymocytes, glandular epithelia, stem cells within the gastrointestinal system, and differentiated long lived cells such as neurons (46). In particular, bcl-2 is expressed in many cell types of the skin during embryonic life but is repressed after birth in all skin cells but the epithelium (29). Down-regulation of Bcl-2 expression in dermal fibroblasts must involve transcriptional regulation and/or alteration of the Bcl-2 mRNA stability because, as presented here, Bcl-2 transcript was almost undetectable in these cells. In addition, lung-derived fibroblasts also lacked Bcl-2 expression. It is again in accordance with results published by others, showing that pulmonary fibroblasts lose Bcl-2 expression during lung maturation (30). Finally, our data pointing to Bcl-2 as a relevant mediator of cardiac fibroblast resistance to hypoxia are consistent with previous reports where endogenous or induced overexpression of Bcl-2 has been involved in the resistance of other cell types, mainly tumoral cells, to hypoxia (47). Furthermore, overexpression of Bcl-2 in the fibroblast cell line REF52 has been shown to allow E1a-induced proliferation during anoxia by blocking apoptosis induced by this transcription factor (48).

Here, we also provide experimental evidence in support of a major role of Bcl-2 in the inhibition of cytochrome c release and enhanced survival of cardiac fibroblasts. First, inhibition of Bcl-2 expression increased apoptosis in cardiac but not in dermal fibroblasts during STS treatment, serum and glucose deprivation, or simulated ischemia. Second, forced expression of Bcl-2 in dermal fibroblasts conferred resistance to STS-induced and serum- and glucose-induced apoptosis at the same level as in wild-type cardiac fibroblasts. And third, overexpression of pro-apoptotic Bax in cardiac fibroblasts overrode the anti-apoptotic effect of Bcl-2, demonstrating that cardiac fibroblasts are able to complete apoptosis if Bcl-2-controlled steps are bypassed.

Although it is beyond the scope of the present work, it is tempting to speculate about the role of Bcl-2 as cell cycle controller in cardiac fibroblasts. Indeed, Bcl-2 prolongs G0 phase, thus delaying cell cycle progression by a mechanism involving increased expression of the Cdk-2 inhibitor p27kip1 and the retinoblastoma family protein p130 (49). This delaying effect of Bcl-2 in cell cycle entry is of relevance because premature entry into S phase under adverse conditions (such as hypoxia) has been associated with apoptosis.

In summary, we provide evidence for the existence of a constitutive anti-apoptotic mechanism acting in cardiac, but not in dermal or pulmonary fibroblasts, which involves the maintenance of Bcl-2 expression and blocks cytochrome c translocation, arresting drug-induced and ischemia-induced PCD before the execution phase. This anti-apoptotic mechanism could be involved in the resistance of cardiac fibroblasts to cell death during heart disease, allowing these cells to remain active when other cell types are dying. Altogether, our data unveil Bcl-2 as a key factor regulating cardiac fibroblast survival and, therefore, regulating the role of cardiac fibroblasts in scar formation and heart remodeling.


   FOOTNOTES
 
* The present work has been supported by grants from the Fundació Marató de TV3 (to J. X. C. and M. B.) and the Fondo de Investigaciones Sanitarias (01/3023 and PI020116, to D. S. and PI020051, to J. X. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by a Young Researcher Award from the Generalitat de Catalunya (to J. X. C.). Back

Supported by a grant from the Fondo de Investigaciones Sanitarias (PI020116, to D. S.). Back

** Both authors are co-senior authors. Back

§§ To whom correspondence should be addressed: Group of Cell Signaling & Apoptosis, Facultat de Medicina, Universitat de Lleida, Montserrat Roig, 2 25008 Lleida, Spain. Tel.: 34-973-70-24-14; Fax: 34-973-70-24-38; E-mail: daniel.sanchis{at}cmb.udl.es.

1 The abbreviations used are: PCD, programmed cell death; COXIV, cytochrome c oxidase subunit IV; ECM, extracellular matrix; EYFP, enhanced yellow fluorescent protein; MEF, mouse embryonic fibroblast; PFA, paraformaldehyde; siRNA, small interfering RNA; STS, staurosporine; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone; PBS, phosphate-buffered saline; SD, serum deprivation; RT, reverse transcriptase; SDGD, serum and glucose deprivation. Back


   ACKNOWLEDGMENTS
 
We thank Stanley J. Korsmeyer for providing the genetically modified mouse embryonic fibroblasts and Dionisio Martín-Zanca for providing the rat fibroblast cell line Rat1. We also thank Jean-Claude Martinou for the pcI-His-Bax construct.



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 DISCUSSION
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