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CLINICAL STUDY: HEART FAILURE

Apoptotic pathway activation from mitochondria and death receptors without caspase-3 cleavage in failing human myocardium

Fragile balance of myocyte survival?

Robert J. Scheubel, MD^*,*, Babett Bartling, MS^*, Andreas Simm, PhD^*, Rolf-Edgar Silber, MD^*, Kostas Drogaris, MS, Dorothea Darmer, PhD and Juergen Holtz, MD

^* Clinic for Cardiothoracic Surgery, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany
Institute of Pathophysiology, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany

Manuscript received July 12, 2001; revised manuscript received October 4, 2001, accepted November 1, 2001.

^* Reprint requests and correspondence: Dr. Robert J. Scheubel, Klinik für Herz- und Thoraxchirurgie, Martin-Luther-Universität Halle-Wittenberg, Ernst-Grube-Str. 40, D-06097 Halle/Saale, Germany.
robert.scheubel{at}medizin.uni-halle.de

	Abstract

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OBJECTIVES: Activation of the caspase cascade through the mitochondrial and/or death receptor pathway was investigated in the failing human myocardium, in which the mode and extent of the cascade activation are unknown.

BACKGROUND: In terminal heart failure, a loss of cardiomyocytes by overload-induced apoptosis is an attractive mechanism, explaining the progressive character of the disease. However, its relevance is unclear, because the specificity of probes for apoptotic deoxyribonucleic acid damage is under debate.

METHODS: Left ventricular specimens from 36 explanted failing and 21 nonfailing donor hearts were used for messenger ribonucleic acid detection by semiquantitative reverse-transcription polymerase chain reaction. From these groups, immunoblot analysis was performed in samples from nine failing and six nonfailing donor hearts.

RESULTS: In terminally failing hearts, there was a significant accumulation of cytochrome c in the cytosol, which was associated with activation of caspase-9 and downregulation of its inhibitor, caspase-9S. Similarly, the death receptor-induced pathway revealed activation of caspase-8, combined with downregulation of its inhibitors, flice-like inhibitory protein-L (FLIP_L) and FLIP_S. The unspecific caspase inhibitors, XIAP, hIAP-1 and hIAP-2, were also downregulated. However, the terminal effector caspase-3 was not activated, and its substrate gelsolin, acting in its uncleaved form as a feedback inhibitor of caspase-3, was not cleaved.

CONCLUSIONS: In the terminally failing human myocardium, the caspase cascade is partially activated in the presence of a consistent phenotype shift toward enhanced susceptibility to apoptosis. Although the system is still under a fragile control, the partial initiation of the apoptotic program may be of functional relevance also for the surviving cardiomyocytes.

Abbreviations and Acronyms   TUNEL   AIF   apoptosis-inducing factor   ANP   atrial natriuretic peptide   ATP   adenosine triphosphate   CAD   coronary artery disease   DCM   dilated cardiomyopathy   DNA   deoxyribonucleic acid   FLIP   flice-like inhibitory protein   hIAP   human inhibitor of apoptosis protein   HRP   horseradish peroxidase   IAP   inhibitor of apoptosis protein   IgG   immunoglobulin G   mc   monoclonal   mRNA   messenger ribonucleic acid   RNA   ribonucleic acid   RT-PCR   reverse-transcription polymerase chain reaction   pc   polyclonal   TNF   tumor necrosis factor   TUNEL   terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling   VAD   ventricular assist device   XIAP   X-linked inhibitor of apoptosis protein

Proteolytic activation of the cascade of caspases is considered as the central element of the apoptotic machinery. The cascade is started by autoactivation of initiator caspases, triggered either by release of apoptotic signals from the mitochondria or by ligand binding to death domain receptors (6). The mitochondrially activated initiator caspase-9, can be inhibited by its short isoform, caspase-9S (7), and the death receptor-activated initiator caspase-8, is inhibited by the flice-like inhibitory proteins (FLIP) FLIP_L and FLIP_S (8). Another group of inhibitory proteins, the family of human inhibitor of apoptosis proteins (hIAP), hIAP-1, hIAP-2 and X-linked inhibitor of apoptosis protein (XIAP), are potent suppressors of apoptosis by preventing the activation of initiator, as well as effector, caspases and by direct inhibition of activated (cleaved) caspases (9).

In view of the open debate on the specificity of DNA damage indicators, we decided to analyze the expression and activation of caspases and their endogenous inhibitors in the terminally failing myocardium. The data demonstrate a fragile balance, without measurable activation, of effector caspase-3, on the one hand, but with depressed expression of caspase inhibitors and substantial activation of two initiator caspase pathways, on the other hand.

	Methods

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Myocardial specimens from cardiomyopathic and donor hearts.   Samples from 21 donor hearts not transplanted for clinical reasons were obtained in cardioplegic arrest and from 36 explanted hearts of patients undergoing transplantation due to dilated cardiomyopathy (DCM) (n = 24), coronary artery disease (CAD) (n = 9) or a mixed etiology (n = 3), with a mean ejection fraction of 25 ± 3%, were immediately snap-frozen in liquid nitrogen. Eighteen of the patients with failing hearts had beta-blocker therapy, in combination with angiotensin-converting enzyme inhibitor therapy in 14 of these patients, whereas 16 of the 18 patients without beta-blocker therapy received an angiotensin-converting enzyme inhibitor. Probes from all 57 hearts were used for reverse-transcription polymerase chain reaction (RT-PCR), whereas nine failing and six nonfailing donor hearts, as examples, were used for immunoblot analysis.

The local ethics committee approved the study of these human cardiac tissues, and the patients gave written informed consent.

Ribonucleic acid (RNA) extraction and semiquantitative RT-PCR.   Total RNA was isolated from ventricular specimens, and semiquantitative RT-PCR was performed in accordance with the protocol described previously (10,11). The primer sequences and specific characteristics of semiquantitative messenger ribonucleic acid (mRNA) analyses of the genes are available from Dr. Scheubel. The PCR products were quantified either relatively per 18S ribosomal RNA or absolutely per competitive RT-PCR (10), with internal standard complementary RNA molecules for hIAP-1 and XIAP.

Isolation of the cytosolic fraction and immunoblot analysis.   The human myocardium was minced at 4°C in buffer A (in mmol/l: 10 N-[2-hydroxyethyl] piperazine-N"-[4-butane sulfonic acid], 80 potassium chloride, 1 sodium-ethylenediamine-tetraacetic acid, 1 sodium-ethyleneglycoltetraacetic acid, 4 Dithiothreitol and 250 sucrose; 50 µg/ml saponin and 5 µl protease inhibitor [Sigma, Deisenhofen, Germany] cocktail per 100 mg tissue; pH 7.4), and after 30 min, it was homogenized with DSTROY-S pestles (Biozym, Oldendorf, Germany) and centrifuged for 10 min at 500 g. The supernatant was centrifuged at 10,000 g for 30 min, and the final supernatant was used as the cytosolic fraction. The protein concentration was determined by the bicinchoninic acid protein assay (Sigma). Fifty micrograms of protein of the cytosolic fraction were run on a 10% or 15% sodium dodecyl sulfate polyacrylamide gel. Protein extracts of cleaved caspase-3-positive cells (Jurkat and National Institute of Health 3T3) were purchased from Cell Signaling Technology (Frankfurt, Germany). Proteins were electroblotted onto nitrocellulose membrane (BioRad, München, Germany), blocked with 6% nonfat dry milk in Tris-buffered saline-Tween 20 (200 mmol/l Tris [hydroxymethyl] aminomethane, 300 mmol/l sodium chloride, 0.1% Tween 20; pH 7.5) and incubated with the respective anti-human primary antibody. Antibodies against caspase-3 (pc rabbit, Pharmingen, Heidelberg, Germany; polyclonal [pc] rabbit and goat, Santa Cruz Biotechnology, Heidelberg, Germany), cleaved caspase-3 (pc rabbit, Cell Signaling Technology), caspase-9/-9S (pc rabbit, Pharmingen), caspase-8 (pc rabbit, Pharmingen), FLIPS (monoclonal [mc] mouse, Santa Cruz Biotechnology), hIAP-1 (pc rabbit, R&D Systems), hIAP-2 (pc rabbit, Santa Cruz Biotechnology), XIAP (mc mouse, Becton Dickinson, Heidelberg, Germany), gelsolin (mc mouse, Sigma), fodrin (mc mouse, Chemicon, Hofheim, Germany), cytochrome c (mc mouse, Pharmingen) and manganese superoxide dismutase (provided by Dr. H. Noak, University Halle-Wittenberg) were applied. Blots were subsequently washed in TBST and incubated with specific peroxidase-coupled secondary antibodies (anti-goat immunoglobulin G [IgG]-horseradish peroxidase [HRP], Dianova, Hamburg, Germany; anti-rabbit and anti-mouse IgG-HRP, Amersham Pharmacia Biotech, Freiburg, Germany). Bound antibodies were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech) and finally quantified using a laser-densitometer with imaging system (Molecular Dynamics, Sunnyvale, California).

Immunohistochemistry.   Samples were immediately fixed in buffered 4% formalin and paraffin-embedded. Sections of 8 µm were cut, dewaxed and rehydrated. Antigen retrieval was performed in 0.1 mol/l citric acid (pH 6.0) for 4 times 5 min at 100°C. Unspecific protein bindings were blocked with bovine serum (1:5, DAKO, Hamburg, Germany). For immunostaining of XIAP, a mc mouse antibody (as described in the previous text) was applied in a humidified chamber at 37°C for 2 h, followed by incubation with a fluorescein isothiocyanate-labeled anti-mouse IgG antibody (Sigma). Slides were embedded in Mowiol (embedding reagent) (Hoechst, Frankfurt, Germany). Omission of the primary antibody served as a negative control study. Bound antibodies were detected by fluorescence microscopy.

Cell culture of adult rat ventricular cardiomyocytes.   Ventricular cardiomyocytes of adult rats were prepared as described (12). Cultured cardiomyocytes were incubated with 10 µmol/l of epinephrine for 24 h and then washed with phosphate-buffered saline (pH 7.2), scraped off the dishes and incubated in buffer A (as previously described) for extraction of cytosolic proteins.

Data analysis.   Western blotting and RT-PCR analyses were evaluated by scanning the negatives of the gel images using a computer-based imaging system (AIDA evaluation software, Raytest, Straubenhardt, Germany). The optical density units of RT-PCR products and Western blots are given as the mean value ± SEM. The significance of comparison of mean values was determined by the unpaired Student t test, using a significance level of p < 0.05.

	Results

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Mitochondrially activated caspase pathway.   We quantified cytochrome c release from the mitochondria, expression of procaspase-9 and its anti-apoptotic isoform, caspase-9S and cleavage of procaspase-9 protein. Procaspase-9 mRNA expression in failing ventricles (1.41 ± 0.06) was not different from that in nonfailing ventricles (1.34 ± 0.06). However, mRNA of the inhibitory splice variant caspase-9S was downregulated by 50% (Fig. 1a). In immunoblot analysis, uncleaved procaspase-9 protein tended to be elevated, whereas the activated form of caspase-9 was three times higher in failing myocardium. Conversely, the inhibitory isoform caspase-9S protein could only be detected in donor hearts, but not in failing hearts (Fig. 1b and 1c).

View larger version (50K): [in this window] [in a new window]   Figure 1 Comparison of left ventricular procaspase-9, activated caspase-9 and caspase-9S expression in patients with end-stage heart failure with nonfailing donor hearts. The messenger ribonucleic acid (mRNA) expression of caspase-9S was downregulated and the ratio of caspase-9S/procaspase-9 was decreased in patients with heart failure (a). Western blot analyses of procaspase-9, activated caspase-9 and caspase-9S protein in failing left ventricles, as compared with donor ventricles (b), illustrate increases of procaspase-9 by 25% and activated caspase-9 by 65% and an inverse relationship for caspase-9S, with no detection of caspase-9S in the failing myocardium (c). (*)p < 0.1; **p < 0.01; ***p < 0.001.

View larger version (34K): [in this window] [in a new window]   Figure 2 Immunoblot analysis of cytochrome c and manganese superoxide dismutase (MnSOD; as a marker of the mitochondrial matrix) in the cytosol (a) and total cell lysate (b) of left ventricular specimens from donor hearts and failing hearts. In the failing myocardium, cytosolic cytochrome c was 53% higher (c), whereas MnSOD was not different between the two groups in terms of the cytosolic fraction and total lysate. In cultured rat cardiomyocytes, cytochrome c release could only be detected after treatment with 10 µmol/l of norepinephrine for 24 h (d). *p < 0.05.

Receptor-activated caspase pathway.   This pathway includes the initiator procaspase-8 and the specific FLIP inhibitors. Compared with nonfailing ventricles, the myocardium of patients with heart failure was characterized by decreased mRNA expression of FLIP_L (–25%), as well as FLIP_S (–58%) (Fig. 3a), but no significant alteration of procaspase-8 mRNA expression (0.96 ± 0.05 in failing myocardium vs. 1.04 ± 0.9 in donor hearts). On immunoblot analysis, uncleaved procaspase-8 protein levels were similar in both groups, whereas activated caspase-8 tended to be higher (p = 0.065), and FLIP_S was significantly downregulated in the failing myocardium (Fig. 3). Similarly as for mitochondrially activated procaspase-9, the receptor-activated procaspase-8 was partially activated and its inhibitors were downregulated in the failing myocardium.

View larger version (33K): [in this window] [in a new window]   Figure 3 Analysis of left ventricular protein expression of procaspase-8, activated caspase-8, flice-like inhibitory protein (FLIP)S and FLIPL in the failing human myocardium, as compared with donor organs. The messenger ribonucleic acid (mRNA) expression of FLIPS and FLIPL is significantly downregulated in the failing myocardium (a). For activated caspase-8, there was an increase of 62% in the failing myocardium, as compared with donor hearts (b), whereas procaspase-8 was not different between the two groups. The FLIPS protein decreased significantly in the failing myocardium (b). (*)p < 0.1; *p < 0.05; ***p < 0.001.

View larger version (31K): [in this window] [in a new window]   Figure 4 Comparison of left ventricular expression of the inhibitor of apoptosis proteins, hIAP-1, hIAP-2 and XIAP, from patients with end-stage heart failure and donors. The messenger ribonucleic acid (mRNA) expression of hIAP-1, hIAP-2 and XIAP was downregulated in the left ventricles of patients with end-stage cardiomyopathy (a). Immunoblot analysis showed a decrease of hIAP-1 and XIAP in patients with terminal heart failure, as compared with donor organs (b). (*)p < 0.1; *p < 0.05; ***p < 0.001.

View larger version (51K): [in this window] [in a new window]   Figure 5 Immunoblot analysis of caspase-3 and its cleavage substrates gelsolin and fodrin in the left ventricles of patients with end-stage heart failure, as compared with donor hearts: 17- and 19-kDa–cleaved caspase-3 was exclusively detectable in cell lysates from cytochrome c–treated Jurkat and NIH 3T3 cell extracts. Neither donor hearts nor terminally failing hearts revealed caspase-3 activation when using an anti-active caspase-3 antibody (Cell Signaling Technology) (a). When performing an immunoblot with an antibody against the 32-kDa inactive and active caspase-3 proteins (Pharmingen), there was no cleavage product detectable, although procaspase-3 was detectable in a similar amount (b). For gelsolin, we found only the uncleaved form in both groups (c). The terminally failing hearts revealed significantly more cleaved (p < 0.001) and uncleaved (p < 0.05) fodrin, as compared with donor hearts (d). Equal protein loading of each patient has been shown by coomassie staining (e).

View larger version (23K): [in this window] [in a new window]   Figure 6 Messenger ribonucleic acid (mRNA) expression of pro-atrial natriuretic peptide (ANP) as an overload indicator, several apoptosis inhibitors, apoptosis-inducing caspases and mitochondrially localized proteins in the left ventricles of terminally failing hearts treated with or without beta-blockers. Values are given in optical density units normalized to donor organ values. *p < 0.05. **p < 0.01. ***p < 0.001. AIF = apoptosis-inducing factor; FLIP = flice-like inhibitory protein.

	Discussion

Top Abstract Methods Results Discussion References

The process of proteolytic activation does not encompass the entire cascade equally: in contrast to the strong activation of initiator caspases-8 and -9, activation of the terminal effector caspase-3 and cleavage of the caspase-3 substrate gelsolin are below the limit of detection. Although the actin modulator gelsolin in its caspase-cleaved form promotes apoptosis (14), uncleaved gelsolin still acts as an inhibitor of caspase activation (15). Furthermore, other unidentified inhibitors and/or the residual function of the downregulated caspase inhibitors might have contributed to this incompleteness of the cascade activation. The cleavage of fodrin, as observed in the failing myocardium (Fig. 5d), is not a good indicator for caspase-3 activation, because fodrin can be cleaved independently from caspase-3 activation (16).

These findings, partially comparable to previous observations (17), instigate several questions: first, is there a functional relevance of cytochrome c release and initiator caspase activation in the failing myocardium? Second, what are the mechanisms of this partial activation of the cascade? Finally, what causes TUNEL-positive nuclear changes in only a small fraction of cardiomyocytes, although caspase-3 is not measurably activated in failing myocardial tissue extracts?

Functional relevance of mitochondrial caspase cascade activation.   We observed enhanced extramitochondrial cytochrome c, but no signs of enhanced cytochrome c resynthesis. In fact, mRNA expressions of cytochrome c and AIF, another releasable mitochondrial apoptogene, are downregulated in the failing myocardium, most pronounced in those hearts without beta-blockade before explantation (Fig. 6). In view of depressed cytochrome c expression and enhanced mitochondrial release, there must be a cytochrome c deficit in the oxidative phosphorylation in the respiratory chain of the mitochondria in the failing myocardium.

Lowered concentrations of adenosine triphosphate (ATP) and creatine phosphate have been reported in the failing human myocardium (18) and were ascribed to a primary attenuation in mitochondrial ATP synthesis (19). Our data, suggesting a mitochondrial cytochrome c deficit in the explanted hearts, yield an explanation for this postulated primary attenuation of ATP synthesis in the failing myocardium. Furthermore, this deficit must result in enhanced mitochondrial radical formation, as occurs upstream of any defect in the respiratory chain (20). Indeed, increased mitochondrial radical formation has been shown in the failing human myocardium (21).

The release of cytochrome c in the presence of low ATP availability should result in more necrotic forms of cell death (22). This is exactly what is observed in the failing myocardium: histochemical characterization of cardiomyocyte nuclei identified a more than fivefold higher occurrence of necrotic cardiomyocyte nuclei, as compared with apoptotic nuclei (3). Thus, we conclude that mitochondrial activation of the starting program of cell death in the failing myocardium is relevant for the enhanced mitochondrial radical formation, for the substantial incidence of necrotic processes under this condition and probably for the enhanced susceptibility of the failing myocardium to ischemic damage (23).

Mechanisms of partial caspase cascade activation.   The mechanisms for activation of initiator caspases in the failing myocardium include two aspects: the triggering event (at mitochondria and/or death receptors), on the one hand, and the phenotype alterations with downregulated endogenous caspase inhibitors, on the other hand.

In the mitochondria, one possible mechanism for apoptogene release is the cytosolic calcium overload of the failing myocytes, which causes mitochondrial calcium overload, an established factor for cytochrome c release (24). Tumor necrosis factor (TNF) receptor-1 stimulation should be considered as a major factor for caspase-8 activation, because the TNF system is involved in the deterioration of cardiac function in severe heart failure (25). However, an activated myocardial TNF system also confers some anti-apoptotic protection (26,27), which is certainly of relevance for maintenance of the fragile anti-apoptotic balance in the failing myocardium, as demonstrated in our report.

Phenotype shifts in the failing myocardium are generally ascribed to three factors: enhanced mechanical load, exaggerated neuroendocrine activity and inflammatory activation. Enhanced mechanical load is probably not the decisive factor for these pro-apoptotic phenotype changes, because we could not obtain a re-normalization of the depressed expression of most caspase inhibitors in failing hearts under hemodynamic unloading by ventricular assist device (VAD) (11). Consistent with a role of neuroendocrine overactivation for depressed inhibitor expression, is our observation that this depression was less pronounced in patients treated with beta-blockers (Fig. 6). We assume that inflammatory activation is a major reason for the depressed expression of apoptosis inhibitors in heart failure, because inflammatory activation often remains elevated under VAD application (28), and caspase inhibitor expression remains depressed under VAD, despite hemodynamic unloading (11).

Apoptotic cardiomyocyte nuclei without caspase-3 activation?.   Although 0.05% of cardiomyocytes demonstrated DNA changes, as detectable by the TUNEL technique, we could not demonstrate cleavage of caspase-3 or its substrate gelsolin in extracts from these hearts, by using various antibodies that detected cleaved caspase-3 in apoptotic Jurkat and NIH 3T3 cell extracts (Fig. 5a and 5b). Probably, these biochemical analyses are not sensitive enough to detect cleavage processes in tissue extracts with only 0.05% apoptotic myocytes. Other reports on caspase-3 activation in the failing myocardium are conflicting, with minimal (29) or massive (17) caspase-3 activation, but without detectable cleavage of polyadenosine diphosphate-ribose-polymerase (17,29), another substrate of effector caspases. Furthermore, it has been shown that the TUNEL method can also label DNA repair in nonapoptotic myocytes (4), and that there are apoptotic processes that progress independent of caspase activity and that are mediated by release of AIF or other apoptogenes from mitochondria and their translocation to the nucleus (30,31). Presently, it is unclear whether such a caspase-independent apoptosis occurs in overloaded myocardium; in ischemic myocardium, apoptosis includes caspase-3 activation and is attenuated by caspase-inhibitors (32).

Conclusions.   Our data demonstrate that cytochrome c release from cardiomyocyte mitochondria contributes to partial activation of the apoptotic caspase cascade in the terminally failing myocardium of humans. Although endogenous caspase inhibitors are downregulated, the cascade is still kept in a fragile balance, because the effector caspase-3 is not yet activated, and the gelsolin switch is not yet cleaved into a pro-apoptotic mode. Activation of TNF signaling, as indicated by caspase-8 cleavage, may contribute to maintaining survival. Although only a tiny fraction of cardiomyocyte nuclei shows TUNEL-positive DNA alterations, the cytochrome c release in the failing myocardium is of functional relevance for impaired ATP production and enhanced radical formation, and probably also in cardiomyocytes that escape necrotic or apoptotic cell death.

	Acknowledgments

 
We appreciate the cooperation of the patients and the staff of the Clinic for Cardiothoracic Surgery in Halle/Saale, Germany. We thank R. Busath, B. Heinze, T. Wangemann and A. Graul for their technical assistance, as well as Dr. Heinroth-Hoffmann, Institute of Pharmacology, for administration of adult rat cardiomyocyte cultures.

	Footnotes

 
This work was supported by grants from the German Bundesministerium für Bildung und Forschung, BMBF (01ZZ9512.TV3, TV7).

	References

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