MiniReview |
From the Department of Cardiology (A.H.), University of Heidelberg, Germany, and Cardiovascular Division (S.I.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.
Correspondence to Seigo Izumo, Cardiovascular Division, Beth Israel Deaconess Medical Center, 330 Brookline Ave (Room SL-201), Boston, MA 02215. E-mail sizumo{at}caregroup.harvard.edu
Key Words: apoptosis myocyte therapy signal transduction
| Introduction |
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| Targets for Intervention |
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Targeting the Proapoptotic Stimulus Acting on the
Myocyte
Current therapy directed at reducing myocardial ischemia,
hypertrophy due to increased afterload, and myocardial
remodeling already might include an antiapoptotic effect, as
these conditions are known to induce myocyte apoptosis, as
evidenced by experimental and pathologic studies.6 7 8 9 10 11 The
use of ß-blocking agents to treat hypertension, ischemic
heart disease, and chronic heart failure may suppress the
apoptotic effect of excess catecholamines. In
rabbit hearts, treatment with carvedilol reduced myocyte
apoptosis in response to
ischemia/reperfusion.12 Likewise,
angiotensin-converting enzyme inhibitors might
inhibit angiotensin IImediated myocyte
apoptosis,13 although studies in human and animal
heart disease are missing. Other strategies directed at reducing
myocyte stretch, oxidative stress during reperfusion, or improvement of
myocardial perfusion by growth factorinduced angiogenesis may all
reduce myocyte apoptosis by minimizing proapoptotic
stimulation of myocytes.14 15 16 17 In addition, inhibition of
death receptor stimulation by scavenging ligands with soluble Fas and
tumor necrosis factor (TNF) receptors or by synthetic receptor
antagonists constitutes another potential approach.
However, although Fas and TNF receptor 1 are expressed on cardiac
myocytes, the role of death receptor stimulation in myocyte
apoptosis and in clinical cardiac disease needs further
evaluation.6 12 18 19 20 21
Promotion of Antiapoptotic Signaling
Apoptosis is tightly regulated by several mechanisms to
prevent inadvertent cell loss. Among these mechanisms are
the antiapoptotic members of the Bcl-2 protein family (eg,
Bcl-2 or Bcl-xL), which inhibit the release of
proapoptotic factors from mitochondria, and apoptosis
repressor with a caspase recruitment domain, Fas-associated death
domain protein (FADD)like antiapoptotic molecule (FLAME-1),
and the decoy receptors (eg, TNF-related apoptosisinducing ligand
[TRAIL] receptor without an intracellular domain [TRID]),
which interfere with proapoptotic signaling through death
receptors.22 23 24 25 However, at present there is no
effective methodology other than gene overexpression available to
augment the efficacy of these regulatory mechanisms. In addition, it is
not known to which degree augmentation of these antiapoptotic
pathways could prevent myocyte apoptosis in clinical cardiac
disease, as the role of mitochondria-mediated versus death
receptormediated induction of apoptosis is not yet well
delineated.
Earlier studies in cultured cells of the neuronal and hematopoietic lineage indicated that growth factors provide a survival signal that renders cells resistant to apoptotic cell death. Recent observations support a role for growth factors also in the survival of cardiac myocytes. Among these factors are the insulin-like growth factor-1, cardiotrophin-1, and the neuregulins, which were shown to reduce myocyte apoptosis after ischemia, serum withdrawal, myocyte stretch, and treatment with the cardiotoxic chemotherapeutic drug doxorubicin.26 27 28 29 30 31 In addition, activation of the glycoprotein 130 subunit of the leukemia inhibitory factor/cardiotrophin-1 receptor confers protection against cardiac dilation induced by aortic banding in mice.32 In this study, inhibition of cardiac dilation correlated with a decrease in myocyte apoptosis. Intracellular signaling of the survival factors includes the ERK and phosphatidylinositol-3-kinase/Akt pathways,30 33 although the role of these pathways in myocyte protection still needs to be further substantiated. Initial attempts to treat chronic heart failure with recombinant human growth hormone, which increases serum concentrations of insulin-like growth factor-1, have so far created ambiguous results with regard to clinical outcome.34 35 Unfortunately, the effect of growth hormone treatment on myocyte apoptosis was not studied, and it is not known whether the treatment protocol affected apoptotic myocyte loss. Nevertheless, growth factor treatment deserves further studies, as different survival factors may differ in their protective potential. Also, cell surface receptors are more easily amenable to pharmacologic therapy.
Targeting Proapoptotic Signaling
Apoptosis mediated either by mitochondria or death
receptors involves proapoptotic signaling pathways or
apoptosis-specific regulatory proteins. Best known are the
proapoptotic members of the Bcl-2 protein family such as Bax,
Bad, and Bid, which induce a transition of mitochondria to a
proapoptotic state, and the adaptor protein FADD, which is
involved in the recruitment of upstream caspases in response to death
receptor stimulation.22 36 Although currently not
available, it may be possible to develop synthetic drugs that are
membrane permeable and interfere with the proapoptotic activity
of these proteins. In addition, pharmacologic inhibition of the
permeability transition pore may represent another target for
antiapoptotic treatment.37 38 Furthermore,
experimental studies in isolated cardiac myocytes and transgenic mice
have provided evidence for a role of "classical" intracellular
signaling pathways in promoting apoptosis. Stimulation of the
MKK3/p38
pathway caused increased myocyte death.39 40
Likewise, overexpression of Gs
or Gq
induced myocyte
apoptosis in transgenic mice and cultured cardiac myocytes,
respectively.41 42 However, the pleiotropic effect of
these signaling proteins in both cardiac myocytes and other cell
lineages may limit an approach that targets these signaling pathways
due to an increased risk of side effects.
Targeting the Downstream Execution Phase of Apoptosis
In most cellular models, apoptosis relies on the
activation of downstream executioner caspases (caspase-3, -6, or
-7) that cleave a plethora of cellular target
proteins.3 43 Inhibition of downstream caspases
constitutes a cellular regulatory mechanism exerted by IAPs (eg, IAP-1,
IAP-2, or XIAP).44 In addition, potent pharmacologic
agents have been developed that efficiently inhibit caspase activity
(eg, benzoxycarbonyl valine-alanine-aspartate-fluoromethylketone). We
currently do not know how to augment the endogenous
antiapoptotic activity mediated by the IAPs. On the other hand,
treatment with synthetic caspase inhibitors was shown to be
effective in a model of short-term myocardial ischemia and
reperfusion.45 In addition, some of these agents permeate
the cell membrane and might therefore permit systemic administration of
the inhibitor. However, caspase activation may not be
effective in inhibiting apoptosis mediated by
apoptosis-inducing factor (AIF).5
| Unresolved Questions |
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What Are the Potentials of an Antiapoptotic Treatment
Strategy?
The benefit that can be gained from antiapoptotic
treatment of cardiac disease is clearly related to the extent and
pathophysiologic significance of myocyte apoptosis in different
disease states. Obviously, as cardiac myocytes have no or at most a
very limited proliferative capacity,46 47 it would be
desirable to prevent myocyte apoptosis in any cardiac disease
with detectable myocyte apoptosis. Unfortunately, at
present the potentials of an antiapoptotic approach in
cardiac disease in terms of amount of myocardium that can
be salvaged and the impact on cardiac function and survival are largely
unknown. In heart failure, for example, pathophysiology is
multifactorial, and impaired excitation-contraction coupling,
alterations in intracellular filaments, interstitial
fibrosis, unfavorable chamber geometry, and neurohumoral deregulation
have all been implicated in the disease process.48 The
relative importance of these factors with regard to initiation and
progression of heart failure is still a matter of debate.
Interestingly, direct ultrastructural evidence (eg, by microscopy) for
myocyte loss in nonischemic heart failure has long been scant.
With the advent of terminal deoxynucleotidyl
transferase deoxy-UTP nick-end labeling (TUNEL) staining in heart
failure specimens, surprisingly high levels of apoptotic
myocyte loss have been reported. Extrapolation of apoptotic
rates based on TUNEL staining indicates that in some cases published
figures certainly overestimate the true incidence of apoptotic
myocyte loss. Additional research is necessary to estimate the maximal
treatment benefit that can be achieved in heart failure. In addition,
it remains to be determined whether inhibition of myocyte
apoptosis will prevent the initiation of clinical cardiac
disease or merely reduce disease progression.
Discrepancies may at least partly be due to current methodological shortcomings. For example, different protocols for tissue pretreatment before addition of the TUNEL reagents are in use by different laboratories. Direct comparison of different pretreatment protocols in myocardial tissue can result in >10-fold different TUNEL positive rates (our unpublished observation, 1999). In addition, despite initial hopes it has become increasingly evident that positive TUNEL staining is not specific for apoptotic cell loss, but may also reflect other physiological processes. In a recent study of ischemia and reperfusion, myocytes staining positive for TUNEL in light and electron microscopy actually did not show ultrastructural features typical for apoptotic cell death, but they showed alterations suggestive for necrotic cell deterioration.49 It is not known whether dying cells that exhibit necrotic morphology with positive staining for TUNEL will be amenable to therapeutic interventions targeted at apoptosis regulation. In addition, positive staining for TUNEL has been associated with DNA repair in myocardial specimens from patients with dilated cardiomyopathy, thus calling the specificity of TUNEL staining for apoptosis in cardiac myocytes further into question.50 Assessment of myocyte apoptosis is further complicated, as myocardium contains a large number of nonmyocyte cells that, although adding comparatively little to myocardial mass, contribute significantly (up to 50%) to the number of nuclei in myocardium. Indeed, cell typespecific analysis showed that nonmyocyte cell populations can significantly contribute to the total number of apoptotic cells in the ischemic myocardium.51 As has recently been discussed by Soonpaa and Field,46 attribution of a positively stained nucleus to a cell lineage may not always be reliable by light microscopic methodology, on which, so far, most observations have been based.
Will Myocyte Survival Translate Into Improved Myocyte
Function?
Another concern relates to the fact that prevention of myocyte
apoptosis might not necessarily be equivalent with full
functional recovery. In fact, stimulation of the death receptor Fas on
cardiac myocytes reduced the membrane potential and induced
afterdepolarizations, indicating a potentially increased propensity for
proarrhythmia of myocytes damaged by, but surviving stimulation
of the proapoptotic Fas receptor protein.52
Likewise, the immediate benefits of rescuing myocytes from TNF-induced
apoptotic cell death may be mitigated by the negative inotropic
effect of TNF that seems to be independent from its
proapoptotic activity.19 20 21 In addition, the
effect of inhibition of apoptosis might only be short-lived, as
only the mode of cell death (apoptotic versus
nonapoptotic) might be altered, whereas eventual cell demise
might not be prevented. Given these considerations, only little
evidence has so far been provided to support the notion that treatment
specifically targeted at apoptosis affects outcome in
myocardial disease. In a short-term model of myocardial
ischemia (30 minutes) and reperfusion (24 hours), Yaoita et
al45 showed that caspase inhibition led to a decrease in
infarct size from 67% to 52% of the myocardial area at risk.
Myocardial salvage was associated with an improvement in
ventricular contractility and left
ventricular end diastolic pressures. However,
it is not known whether this beneficial effect is persistent.
Definitely, further interventional studies are required that allow for
a better estimation of the therapeutic potentials of an
antiapoptotic approach in myocardial disease.
Is Antiapoptotic Treatment for Heart Disease Safe?
Whereas apoptosis of cardiac myocytes in the adult
myocardium is considered to have a detrimental effect,
apoptosis of other cell lineages is
physiologic, such as the elimination of
autoreactive lymphocytes in the thymus or the deterioration and
shedding of epithelial cells in the intestine and the skin. In
addition, deficiency of the proapoptotic regulator Bax affects
male fertility, suggesting an important role for apoptosis in
spermatogenesis and male fertility.53 The potential
implications of inhibiting physiological
apoptosis is exemplified by the history of the
antiapoptotic regulatory protein Bcl-2
(B-cell lymphoma 2), which was
initially characterized as an oncogene in human B-cell lymphomas. Only
later it became obvious that Bcl-2 represents a mammalian
homologue of the antiapoptotic regulator ced-9 of
Caenorhabditis elegans and primarily inhibits cell death
instead of promoting cellular replication.54 55
Furthermore, animal studies and the analysis of Fas mutations
in the hereditary Canale-Smith syndrome (autoimmune lymphoproliferative
syndrome, type Ia) showed that inhibition of Fas signaling may be
associated with lymphadenopathy, neoplastic disease, and autoimmune
disease such as hemolytic anemia and
thrombocytopenia.56 57 58 59 Inhibition of apoptosis
may also bear a considerable risk of teratogenicity. Loss of function
of caspase-3, caspase-9, apaf-1, and Bcl-xL
causes embryonic lethality in mice associated with severe brain
malformation.60 61 62 63 64 Likewise, mice deficient in FADD and
caspase-8 die in utero because of impaired myocardial development and
cardiac dilation.65 66 These observations raise the
possibility that drugs directed at apoptotic mechanisms may be
associated with significant side effects limiting their clinical
use.
Given these considerations, is the prevention of myocyte apoptosis still a sound and viable goal? To answer this question, disease dynamics and timing need to be taken into account. On one hand, in chronic congestive heart failure, slowly progressive aortic stenosis and chronic valvular regurgitation, the apoptotic stimulus is present more or less continuously, probably requiring permanent antiapoptotic treatment to reduce myocyte loss. On the other hand, in acute cardiac events such as myocardial infarction, unstable angina, or reperfusion injury, apoptosis seems to occur in a limited time window. In the latter situation, short-term treatment targeted at common downstream events of apoptosis might be an effective approach and promalignancy and autoimmune side effects will be of minor concern, whereas salvage of a substantial number of cardiac myocytes is possible.45 In addition, interference with the apoptotic cascade may offer the unique opportunity to prevent cell death after the lethal stimulus has reached the cell, thus increasing the time window for effective treatment such as the restoration of myocardial blood flow after myocardial infarction. In this situation, inhibition of downstream caspases despite their widespread distribution might be an attractive molecular target. In contrast, chronic treatment will clearly entail the risks indicated by the physiological role of apoptosis in different tissues and by phenotype analysis of mice deficient in apoptosis-associated genes. To circumvent this problem, it would be of importance to define targets that are unique to the regulation of apoptosis in cardiac myocytes. Potential candidates will include upstream regulatory steps during the initiation of myocyte apoptosis. However, upstream regulation of myocyte apoptosis is not yet well characterized and may include signaling cascades that exert pleiotropic effects, once again entailing the risks of multiple side effects. Defining the target for chronic treatment of myocyte apoptosis will therefore become a more challenging approach.
| Conclusion |
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), thus reducing the treatment
benefit of a strategy merely aimed at myocyte survival. In addition,
safety and side effects of chronic antiapoptotic treatment
remain a major concern. The identification of apoptotic
regulatory pathways that are specific for cardiac myocytes or the
better characterization of the time course of myocyte apoptosis
in cardiac disease might reduce or even prevent the risks of
antiapoptotic treatment. Keeping the concerns associated with
chronic inhibition of apoptosis in mind, an
antiapoptotic approach for cardiac disease still figures among
the most attractive future therapeutic options for cardiac disease.
| Acknowledgments |
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Received September 13, 1999; accepted December 8, 1999.
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