Cardiomyocyte apoptosis is associated with increased wall stress in chronic failing left ventricle
L. Jianga,b,*,
Y. Huanga,
S. Hunyora and
C.G. dos Remediosb
a Department of Cardiology, Cardiac Technology Centre, Royal North Shore Hospital, St. Leonards, NSW, Australia
b Department of Anatomy and Histology, Institute for Biomedical Research, University of Sydney, NSW, Australia
* Corresponding author. Department of Physiology (F13), Institute for Biomedical Research, The University of Sydney, NSW 2006, Australia. Tel.: +61-2-93514526; fax: +61-2-93512058
E-mail address: lelej{at}physiol.usyd.edu.au
Received 22 July 2002;
revised 6 September 2002;
accepted 11 September 2002
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Abstract
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Aims We examined cardiomyocyte apoptosis in chronic heart failure (HF) and its possible link to elevated wall stress.
Methods and results Moderate HF was produced in sheep by sequential coronary microembolization. Six months later, the animals remained in a stable compensated haemodynamic state of HF. Apoptosis of cardiomyocytes in left ventricles was verified using Western blotting based on increased expression of: the apoptosis-associated death receptor Fas (1.5-fold); its ligand (FasL, 2.0-fold); and an upstream protease caspase-8 (2.7-fold) as well as its active cleavage peptide, p20 (5.6-fold). Previously we have reported the elevated expression of caspase-3 in the same animal model. The occurrence of apoptotic cardiomyocytes (0.3%) was quantified by TUNEL assays. Haemodynamic analysis indicated that ventricular dilatation, without wall thickening, caused a 2-fold increase in LV wall stress which, together with LV end-diastolic pressure, was linearly correlated with expression of Fas/FasL. Immunohistochemical studies localized FasL and caspase-8 to intercalated discs, suggesting that wall stress may play a role in initiating cardiomyocyte apoptosis.
Conclusion Apoptosis of cardiomyocytes in chronic HF is associated with increased wall stress, which may be responsible for the activation of a Fas/FasL and caspase-8 interaction in the region of intercalated discs.
Key Words: Apoptosis Fas/Fas ligand Caspase Chronic heart failure Wall stress Intercalated disc
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1. Introduction
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Loss of cardiomyocytes by apoptosis is associated with various cardiovascular diseases including acute myocardial infarction1 and end-stage heart failure (HF).2,3 In the latter, apoptotic cardiomyocytes are increased regardless of aetiology.2,4 Up-regulated caspase expression and activation were reported in apoptosis of isolated rat cardiomyocytes,5 in acute myocardial ischaemia and reperfusion injury in rabbit6 and rat,7 and in human cardiomyopathy and HF.8 More recently, caspase-8 activity was found increased in apoptotic cardiomyocytes in rat during reperfusion after ischaemia7 and in failing human heart.9 Caspase-3 activity was proved to be responsible for the cardiomyocyte apoptosis in HF rabbit obtained by ventricularpacing.10 Blocking of caspase-3-activated apoptosis improves contractility in failing myocardium in the same HF model.10 Cytokine death receptors such as Fas (APO-1/CD95), a member of the tumour necrosis factor receptor (TNFr) family, have been shown to induce apoptosis in cardiomyocytes in various heart diseases including myocardial infarction and HF.11,12 Recent reports indicated that Fas and FasL are constitutively expressed in the myocardium13 and are involved in cardiomyocyte apoptosis14despite earlier reports of low or undetectable expression in the heart. Stretch of myocytes has been linked to apoptosis in isolated papillary muscles and in solitary myocytes.15,16
HF in humans has a constellation of aetiologies, durations, severities, comorbidities and treatments. On the other hand, the relevance of single defect animal models to human HF has been questioned on the grounds that they fail to take account of such differences. Accordingly, we set out to study the occurrence and mechanism of apoptosis in a sheep model of chronic, stable, moderate HF. This model was produced by multiple sequential coronary microembolization and remained unmedicated.17 Histopathology18 revealed persistent infarct scars and interstitial fibrosis with adjacent tissue well perfused and free of ongoing ischaemia 6 months after the last embolization.
Using this model we reported elevated expression of caspase-3 and increased activity of DNase19 which prompted us to examine the upstream elements such as caspase-8, Fas and FasL and to localize changes within cardiomyocytes exposed to heightened wall stress.
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2. Methods
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2.1. Chronic ovine heart failure
Global LV damage was induced in sheep by multiple, sequential and selective left coronary artery microembolization using 90µm polystyrene microspheres, as previously described.17 HF was defined as achievement of a left ventricular ejection fraction (LVEF) of <35%, which required 4±2 embolizations spaced 2 weeks apart. End-systolic and end-diastolic volumes were derived according to the modified Simpson's rule.20 LV pressure was measured using a 5F Mikro-Millar pressure transducer (Millar Instruments Inc., Houston, TX). LV end-diastolic meridional wall stress was also measured.21 The aetiology and histology of this model resembles human ischaemic cardiomyopathy.
Eight adult Merino-wether sheep were examined 6 months after establishment of HF. Five normal sheep served as controls. Protocols were approved by the Animal Care and Ethics Committee of the Royal North Shore Hospital (Sydney, Australia). The study complies with the declaration of Helsinki. Hearts were harvested and preserved in 10% phosphate buffered formalin or in liquid nitrogen.
Six months after HF was established the LV was dilated and there was evidence of interstitial and myocardial remodelling.18 Myocytes and myocyte nuclear density decreased across the LV free wall in non-infarcted zones. In contrast, LV replacement and interstitial fibrosis, myocyte diameter and nuclear length increased. Haemodynamics, contractile state, myocardial reserve, echocardiographic parameters including LVEF, and wall stress had been found to remain stable in a group of 21 sheep for 6 months. Neurohormonal axis parameters such as atrial natriuretic peptide (ANP), plasma aldosterone and renin activity and angiotensin II were activated at 3 months in these unmedicated HF animals. ANP remained twice the normal level at 6 months and LV collagen content more than doubled.
2.2. Western blotting
Protein extraction and western blotting (Fas, FasL and caspase-8) were performed as describedpreviously.19 Primary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and positive controls of human cell lysates were from Transduction Laboratories (Lexington, KY, USA).
2.3. Immunohistochemistry
Frozen sections (10µm) were treated in 0.1% Triton X-100 (5min), fixed in 4% paraformaldehyde, then incubated with 1% BSA and 20% horse serum in phosphate buffered saline for 30min at room temperature. Primary antibody was applied at a dilution determined by titration, followed by secondary biotinylated anti-rabbit IgG (Sapphire Bioscience, Alexandria, Australia) then was labelled with streptavidin-Cy3 (Sigma, Castle Hill, Australia). Cardiomyocytes were identified with anti-ß-myosin heavy chain antibody (Dr Joseph Hoh, University of Sydney), followed by secondary biotinylated anti-mouse IgG (Sapphire Bioscience) and streptavidin-FITC (Pierce, Rockford, CT, USA). Antibody specificity was demonstrated by: (1) omitting the primary antibody; and (2) preincubation of antibody with the epitope-peptide used to generate the corresponding antibody. Images were observed on a Leica TCS NT confocal microscope (Heidelberg, Germany) and processed using Photoshop 5.0 software (Adobe Systems, USA).
2.4. Caspase activity assay
A fluorescent AFC (7-amino-4-trigluoromethyl coumarin) substrate/inhibitor QuantiPak (Biomol, Plymouth Meeting, PA, USA) was used to detect caspase-3 and caspase-8 activities as described by the manufacturer. Assays were performed in 96-well black-walled microtiter plates. Each well contained 100µl assay buffer, substrate, inhibitor where appropriate, and the protein extract. Fluorescence intensity was determined using a Spectra MAX 250 microplate reader with MAX Pro v2.4 software (Molecular Devices, Sunnyvale, CA, USA). A linear correlation
was found between the change in fluorescence intensity due to the cleavage of the AFC fluorophore from the substrates by corresponding caspase activity.
2.5. TUNEL assays
Cardiomyocyte apoptosis was quantified22,23 by the TdT-mediated dUTP Nick-End Labelling (TUNEL) technique (Promega, Madison, WI, USA). Formalin-fixed sections (10µm) were treated with proteinase K (20µg/ml) and then with fluorecein-12-dUTP (50µm) in the presence of dATP (100µM) and TdT enzyme at 37°C. Positive controls were achieved by the treatment of DNase I (1µg/ml, Boehringer Mannheim, Australia). Nuclei were labelled with TOTO-3 (Sigma, St Louis, MO, USA).
2.6. Statistical analyses
Data are expressed as mean±SEM (n) unless otherwise indicated. Student's t-tests were performed using StatView (Abacus Concepts Inc., Berkeley, CA, USA). A probability
was consideredstatistically significant. Linear regression analyses were performed using SigmaStat (Jandel Scientific Software, San Rafael, CA, USA).
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3. Results
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3.1. Expression and distribution of FasL and Fas
FasL and Fas were detected in cardiomyocytes by western blotting. Constant protein samples were loaded in each lane. Laser-scanning densitometry was used to determine volume densities (AU) of the FasL band (Fig. 1(a)) detected by its corresponding antibody. FasL expression increased by 1.5-fold (293.3±59.2,
in controls and 448.7±64.6,
) in failing samples
. Mean volume densities (AU) of the Fas bands (Fig. 2(a)) showed a 2.0-fold increase (223.0±32.1,
for normals and 437.7±24.3,
for failing hearts;
).

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Fig. 1 Expression and distribution of FasL. (a) A representative Western blot stained for FasL. Lane 1 contains a positive control derived from human endothelial extract showing a reference band at 45kDa. Lanes 26 contain five control LV samples. Lanes 714 are samples of the eight failing LVs after 6 months of HF. (b) Confocal image illustrating FasL distribution in failing myocardium. Cardiomyocytes were identified using anti-ß-myosin heavy chain labelled with anti-mouse IgG-biotin-streptavidin-FITC (green). FasL was identified with anti-FasL labelled with anti-rabbit IgG-biotin-streptavidin-Cy3 (red). (c) Non-specific staining pattern observed after antibody waspreincubated with the epitope peptide used to generate the antibody. Arrows indicate intercalated discs.
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Fig. 2 Expression and distribution of Fas. (a) A western blot stained for Fas. Lane 1 is positive control for Fas derived from human Jurkat cell (reference band at 37kDa). Lanes 25 contain four control LV samples and lanes 612 are seven samples of failing LVs after 6 months HF. (b) Confocal image of Fas distribution in failing myocardium. Cardiomyocytes are stained green and Fas red. (c) Non-specific staining pattern after epitope peptide preincubation.
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The cellular localizationof FasL was examined by immunostaining of LV sections (Fig. 1(b)). Staining of FasL (red) was observed at the intercalated discs but was excluded from the nuclei (dark central structures). To show specificity of FasL labelling, the epitope peptide used to generate the antibody was preincubated with anti-FasL antibody and then applied to the tissue. This procedure eliminated positive staining of intercalated discs (Fig. 1(c)). Distribution of Fas in cardiomyocytes is demonstrated in Fig. 2(b). UnlikeFasL, Fas was not specifically localized near the sarcolemma but was distributed throughout the cytoplasm.Specificity of Fas labelling was also examined using epitope peptide preincubation, shown in Fig. 2(c).

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Fig. 3 Expression and distribution of caspases. (a) A western blot for caspase-8. Lane 1 contains a positive control derived from human Jurkat cell extract showing two reference bands at 55kDa for pro-caspase-8 and 20kDa for the p20 active peptide. Lanes 25 show four control samples and lanes 69 contain four samples of failing LVs after 6 months HF. (b) Confocal image demonstrating distribution of caspase-8 in normal myocardium. (c) Confocal image of caspase-3 distribution in normal myocardium. Cardiomyocytes are stained green and caspases red.
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Fig. 4 Activities of caspase-3 and caspase-8. Caspase-3 activity assay is shown in (a) (tissue homogenate from failing LV) and (b) (tissue homogenate from control LV). Top traces show the reaction system containing caspase-3 specific fluorescent peptide Ac-DEVD-AFC in the absence of tissue homogenate; middle traces are in the presence of LV tissue homogenates; while bottom traces are the same reaction system in the presence of a LV tissue homogenate containing caspase-3 inhibitor peptide Ac-DEVD-CHO. (c) Bar graph demonstrating both caspase-3 and caspase-8 activities. comparing failing samples with controls.
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Fig. 5 Detection of fragmented DNA of cardiomyocytes by TUNEL assay. Confocal images of LV sections stained for TUNEL assays are shown in (panels AF). All sections are triple-labelled for fragmented DNA (12-dUTP-fluorecein, green), cardiomyocytes (red) and nuclei (TOTO-3, blue). A, C and E display the red and green channels and B, D and F the red and blue channels of the confocal images. Dark spaces are the abundant capillaries containing erythrocytes (seen in A, C, and E, as yellow and in B, D, and F they are red) or interstitial tissues. Fragmented DNA in a section treated with DNase I as positive control is shown in A. A single apoptotic nucleus is seen in E (arrow, green) which is also aligned with the nucleus staining in F (arrow, blue). Nuclei stain blue but appear purple on a red background. Panel G shows the correlation between caspase-3 activity and percentage of apoptotic cardiomyocytes observed in each individual sheep LV.
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3.2. Expression of caspase-8
The expression of caspase-8 protein was examined by western blotting using a polyclonal antibody against an epitope corresponding to amino acids 217350 within caspase-8 p20, the active subunit (Fig. 3(a)). This antibody recognizes both pro-caspase-8 and the p20 peptide. Constant protein loading (50µg) for each sample was applied to each lane. Two strong bands were detected of which one (MW 55kDa) corresponded to pro-caspase-8. Mean volume densities (AU) reveal a 2.4-fold increase in the failing samples (128.4±25.8,
) compared to the controls (302.8±67.2,
, failing), which was statistically significant
. The other band represents p20 which is barely visible in controls but clearly expressed in failing samples. Mean volume densities of the bands show a 5.6-fold increase in failing samples (149.4±26.6,
) compared to controls (26.9±4.2,
) that was significant
. The same antibody was used to observe the distribution of caspase-8 in LV by immunohistochemistry (Fig. 3(b)). Like FasL, staining of caspase-8 appears to be concentrated at intercalated discs. It is probable that this staining mainly detects pro-caspase-8. Caspase-3 (Fig. 3(c)) was observed in the perinuclear region, consistent with cellular distribution of the Golgi apparatus. However, a lack of epitope peptides for these antibodies precluded an evaluation of antibody specificity.
3.3. Enzymatic activity of caspase-3 and caspase-8 in chronic failing LV
Fig. 4 illustrates a caspase-3 activity assay. In the absence of tissue homogenates from a failing sample, fluorescence intensity of the substrate peptide for caspase-3 (Ac-DEVD-AFC) did not change during the 20min reaction period (Fig. 4(a)). In the presence of tissue homogenates of failing LV, the substrate is cleaved in a time-dependent manner. Caspase-3 activity is seen as an increase in fluorescence intensity starting at 10min (Fig. 4(a), middle trace). When the non-fluorescent competitive inhibitor (Ac-DEVD-CHO) was included in the reaction, the rate of fluorescence increase was substantially reduced indicating an inhibition of caspase-3 activity (Fig. 4(a), bottom trace). A caspase-3 activity assay for the control homogenates is shown in Fig. 4(b). Caspase-3 and caspase-8 activities in failing and control samples can be compared in Fig. 4(c).
3.4. Quantification of cardiomyocyte apoptosis by TUNEL assay
Confocal images of triple-labelled LV sections are shown in Fig. 5. A section treated with DNase I (1µg/ml) as positive control is shown in panel A (green) and total nuclei staining (blue) is shown in panel B on a background of myosin-stained cardiomyocytes (red) in all panels. A section of control LV after TUNEL staining (panel C) and total nuclei staining (panel D) shows no TUNEL-positive nuclei. Indeed, very low numbers of TUNEL-positive nuclei were observed in the controls compared to failing samples. Panels E and F display a section of failing LV with a solitary TUNEL-positive staining (E, green) aligned with the blue-stained nucleus of the same cardiomyocyte (F). On average, the number of nuclei per observation field (40x objective lens) was 80±13 (mean±SD,
) for control heart tissue sections and 72±22 (mean±SD,
) for failing hearts. At least five fields each of 132 sections from eight failing LVs and 76 sections of five control LVs were examined. We observed 0.3% TUNEL-positive nuclei in the failing LV samples, representing a 150-fold increase over 0.002% TUNEL-positive nuclei in controls
.
The correlation between caspase-3 activity and the percentage of apoptotic cardiomyocytes observed in each individual sheep sample is shown in Fig. 5(g). Clearly, control samples have low levels of apoptosis consistent with their low caspase-3 activity, whereas failing samples demonstrate a trend towards progressively higher levels of apoptosis corresponding to elevated levels of caspase-3 activity.
3.5. Correlation of FasL and Fas with wall stress and LV EDP
Functional and structural characterizations of the sheep used for this study are summarized in Table 1. A 20% reduction in LV wall thickness in the failing hearts was evident 6 months after the final embolization. LV dilatation also occurred, with doubling of LV volume, and a 137% increase in LVEDP. The rise in wall stress persisted over the study period. As shown in Fig. 6(a), FasL expression was linearly related to LV wall stress (FasL=202+4*LV wall stress,
,
,
), and LVEDP: (FasL=162+96*LVEDP,
,
,
). There was a similar correlation between Fas expression and LV wall stress (Fas=2123.9+9.85*LV wall stress,
,
,
), and LVEDP: (Fas=2147.5+209.6*LVEDP,
,
,
).

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Fig. 6 Linear correlation between FasL/Fas expression and LV wall stress and LVEDP. The figure was constructed using the mean value of the Fas/FasL expression as determined by western blots for the sheep against their LV wall stress/end-diastolic pressure. The relationship between FasL expression and LV wall stress was: FasL=202+4*LV wall stress, , , ; FasL expression and LVEDP: FasL=162+96*LVEDP, , , ; the expression between Fas expression and LV wall stress was: Fas=2123.9+9.85*LV wall stress, , , ; whereas between Fas expression and LVEDP it was: Fas=2147.5+209.6*LVEDP, , , .
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4. Discussion
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4.1. Involvement of FasL, Fas and caspase-8 in the failing heart
Downstream caspases can be activated either by upstream caspases or by mitochondrial proteins. Upstream caspase-8 proenzyme is recruited when FasL binds to the Fas receptor resulting in the formation of a signalling complex comprising the receptor, the adaptor protein FADD/MORT1, and pro-caspase-8.24 Caspase-8 then activates the effector protease (caspase-3), thus triggering apoptosis.25 Alternatively, caspase-3 can be activated by caspase-9 via a mitochondrial pathway which involves the release of active caspase-9 from an apoptosome by the action of cytochrome c and dATP.26
The constitutive expression of Fas and FasL in the cardiomyocytes is consistent with an earlier publication.13 We also detected an elevated expression of Fas, FasL and increased caspase-8 activity in the failing heart. This strongly suggests that the Fas/FasL-initiated activation of caspase-8 signalling pathway may be responsible for cardiomyocyte apoptosis.
4.2. Distribution of Fas/FasL and caspase-3/caspase-8
FasL is mainly localized in the intercalated discs where electrical and molecular exchanges occur between adjacent cardiomyocytes and wheremechanical stress is transmitted. The specificity of this location is strongly supported by the peptide preincubation observation. Protein changes in gap junctions have been observed in pathologicalconditions such as myocardial infarction and hibernation.27,28 Desmin, known to be located at the intercalated discs, is cleaved in a dog model of HF.29 Interestingly, like FasL, caspase-8 also localizes at the intercalated discs. Given the interaction between Fas and FasL takes place on the membrane of adjacent cells30 and caspase-8 is the first recruitment of this interaction,31 this arrangement isconsistent with the initiation of apoptosis. The distribution of caspase-3 is also consistent with its role as a downstream caspase that targets proteins including lamin A and B in the nuclear lamina, poly (ADP-ribose) polymerase, and nuclear endonucleases.
The distribution of Fas is not limited to the sarcolemmal membrane but appears to be scattered throughout the cytoplasm. It is likely that more than one form of Fas is detected by the immunostaining because Fas can also exist assoluble forms (sFas) due to a splicing variant32 which may protect cells from apoptosis,33 sFas may compete for binding with FasL but does not form an active trimer, thereby inhibiting apoptosis.34 Alternatively, Fas receptor may be translocated from cytoplasm to cell membrane before it becomes functional, as is the case for other receptors such as Bax and cell surface adhesion receptors.35,36
While our data suggest colocalization of FasL and caspase-8, Fas was not seen at the intercalated discs. This is probably explained by the fact that the great majority of cardiomyocytes are not apoptotic and only become so when Fas is present. The ability to demonstrate colocalization of these elements in apoptotic cardiomyocytes is hampered by the rare and short-lived nature of this event.
4.3. Wall stress and apoptosis
Cardiomyocyte apoptosis can be induced by mechanical stretch.15,16 Our data showed that LV wall stress was significantly elevated when HF was established, with a sustained rise over the following 6 months. Furthermore, we also found a strong linear relationship between FasL levels and both LV wall stress and LVEDP which implicates a role of wall stress in the activation of the Fas/FasL signalling pathway.
In summary, our study shows that apoptosis remains activated during untreated, chronic, ischaemic HF of moderate severity and that a Fas/FasL-initiated caspase activation may be responsible for this. Our findings suggest that activation of this process may take place in the region of intercalated discs due to a sustained elevation of wall stress caused by chamber dilatation and cardiomyocyte stretch.
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Acknowledgments
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We acknowledge funding support from the National Heart Foundation of Australia and the Australian Government's Cooperative Research Centres Scheme (CRC for Cardiac Technology). We thank Professor J. Hoh of the Department of Physiology, University of Sydney, for his gift of the anti-ß-myosin heavy chain antibody.
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