ARTICLE
INTRODUCTION
Myocardial apoptosis is a typical feature of human terminal cardiac
failure. However, the quantitative role of the apoptotic process for the
progression of failure and the stimuli inducing programmed cell death
of cardiomyocytes in the failing heart are unknown. Acute stretch of isolated
papillary muscle in vitro induces apoptosis [1], but distension
of the extent reported in these in vitro experiments is unlikely
to occur in vivo, even in terminal failure. Chronically overloaded
myocardium has phenotype changes with an apoptotic potential, such as
alterations in "death-domain" containing receptor systems [2], in apoptosis
modulating proteins of the Bcl-2 family [3] and in the cardiac angiotensin
system (see [4]), and these alterations are partially normalized by hemodynamic
unloading in heart failure patients with an implanted cardiac assist device
[5]. However, a direct role of these phenotype changes for the induction
of apoptosis could not yet been demonstrated. The proteolytic activation
of a cascade of caspases (a class of aspartate-specific proteases) is
considered as the effector machinery of programmed cell death. A critical
step in the activation of this terminal apoptotic cascade is the release
of proapoptotic factors from mitochondria (see below). Until recently,
disturbances in mitochondrial function have been seen mainly in the context
of a disturbed cellular energy production. Disturbed mitochondrial function
as a starting mechanism for the execution of cellular apoptosis is a new
point of view, which will be discussed here in the context of cardiac
failure.
Mitochondria in eukaryotic cells are assumed to stem from ancestral
endosymbiosis between nuclear cells and bacteria capable of exploiting
oxygen. As a consequence of that origin, mitochondria still own an autonomously
replicating and expressing genome of about 16.6 kilobases, the mitochondrial
DNA (mtDNA). This genome has only a restricted set of genes coding for
some proteins involved in oxidative phosphorylation (and for ribosomal
and transfer RNA). However, the majority of genes required for biosynthesis
and function of mitochondria are encoded in the nucleus. Therefore, damage
of mitochondrial DNA as a consequence of aging-associated mitochondrial
dysfunction (see below) can primarily affect a certain fraction of the
proteins of the mitochondrial respiratory chain, while nuclear encoded
respiratory chain proteins remain primarily unaffected. We will summarize
here the arguments indicating that this differential affection of respiratory
chain subunits can importantly contribute to the induction of programmed
cell death in aging and/or calcium-overloaded cardiomyocytes.
MITOCHONDRIA AND APOPTOSIS
The major regulator of the caspase cascade activation during the induction
of apoptosis is a protein complex or "apoptosome" at the outer mitochondrial
membrane [6]. This complex consists of cytosolic proteins of the Bcl-2
family, which are anchored in the outer mitochondrial membrane (such as
Bcl-2 or Bcl-xL), and of several proteins released from the
mitochondria. Surprisingly, one of such released mitochondrial proteins
was identified as the mature heme-containing form of cytochrome c [7].
A collapse of the membrane potential of the inner mitochondrial membrane
is associated with and involved in the mitochondrial release of apoptotic
proteins [8]. Anti-apoptotic proteins of the Bcl-2 family prevent this
collapse of the mitochondrial membrane potential, release of proapoptotic
mitochondrial signal proteins and activation of the caspase cascade. Therefore,
the regulation of this mitochondrial membrane potential is in the center
of apoptosis research.
MITOCHONDRIAL ALTERATIONS
RELATED TO AGING
The accumulating damage of cellular components, especially of the cellular
DNA, through reactive oxygen species (ROS) is considered as one of the
most important mechanisms contributing to the aging process. The mitochondrial
respiratory chain is a major source of ROS in the cell. Because of its
closed proximity to the respiratory chain, the mitochondrial genome is
especially exposed to oxidative free radicals and therefore frequently
damaged. This constellation in combination with an insufficiency of mitochondrial
DNA repair mechanisms results in a high mutation rate of coding sequences
of the mitochondrial genome. A hydroxyl-radical adduct of deoxyguanosine,
8-hydroxydeoxyguanosine (8-OH-dG) is considered as a marker of oxidative
DNA damage and is increased in human hearts with aging, as is the number
of deletions in mitochondrial DNA [9].
As mentioned above, the mitochondrial DNA is coding for 13 protein subunits
of the respiratory chain, while the other subunits are encoded in the
nucleus. The mitochondrially encoded proteins are contributing to all
complexes of the respiratory chain except for complex II, which is completely
encoded by nuclear DNA. Accumulation of damage to the mtDNA should consequently
lead to a defective synthesis of mt-genome dependent proteins of the respiratory
chain with subsequent impaired function of the complexes containing these
proteins, but no impairment in the function of complex II. There are three
expected consequences resulting from a defective respiratory chain: at
first, the mitochondrial energy output could be diminished due to impaired
proton pumping. Secondly, impaired pumping of protons through a defective
respiratory chain from the matrix to the intermembrane space causes an
increased generation of reactive oxygen species in the mitochondrial matrix.
Finally, an enhanced mitochondrial radical formation should cause an unstable
or decreased mitochondrial transmembrane potential ( ),
as will be discussed later.
THE MITOCHONDRIAL DEFENSE
AGAINST REACTIVE OXYGEN SPECIES
During normal respiratory conditions, 1-3% of the electron flux is continuously
converted to free radicals at several sites of the respiratory chain [10].
At high cellular oxygen concentration, a reduced availability of reduced
cofactors of the respiratory chain and a high transmembrane potential
of the inner mitochondrial membrane tend to enhance this mitochondrial
radical formation, which is substantially enhanced in presence of defects
or imbalances within the respiratory chain [10]. Mitochondria apparently
respond to this radical-induced oxidative stress with a defined antioxidant
defense cascade, consisting of four steps:
Step 1: Enzymatic radical scavenging, consisting of mitochondrial
superoxide-dismutase, the glutathione system and the mitochondrial catalase.
Step 2: If the enzymatic scavenging mechanisms are exhausted,
the oxidative stress is suggested to result in a so called "mild uncoupling".
This term means an increased proton conductance of the mitochondrial inner
membrane, not coupled to the ATP synthesis and resulting in a small dissipation
of the mitochondrial transmembrane potential [11]. Proteins of the recently
identified UCP family (for "uncoupling proteins") could be candidates
as mediators of mild uncoupling.
Step3: If under oxidative stress a certain 
decline is reached due to mild uncoupling, a reversible opening of the
so called "permeability transition" pore is suggested to follow. Since
this process increases the permeability of the inner mitochondrial membrane
for solutes up to 1,500 Dalton, the consequence of the reversible permeability
transition is a rapid and much stronger uncoupling than during step two,
resulting in an steep membrane potential decrease and in a stimulation
of the respiratory rate with increased removal of cellular oxygen. Apart
from activation by oxidative stress, this permeability transition is triggered
by calcium overload of the mitochondrial matrix, by ADP, by the conversion
of thiol groups to disulfide linkages and an elevated the mitochondrial
matrix pH. Using patch clamp techniques at the inner mitochondrial membrane,
a multiconductance ion channel, modified by Ca2+ and sensitive
to cyclosporin A, has been described as likely candidate for the permeability
transition pore [12]. The proteins possibly contributing to the pore formation
are not yet convincingly identified and the composition of the pore might
vary. A direct visualization of reversible permeability pore transition
in response to oxidative stress has been obtained recently in isolated,
single mitochondria from rat myocardium, directly illustrating the step
3 of the mitochondrial defense cascade in cardiac mitochondria [13].
Step 4: Ongoing oxidative stress in spite
of these described defense mechanisms is assumed to result in an "irreversible"
permeability transition, as has been shown in vitro in single cardiac
mitochondria [13]. This transition, if lasting long enough, results in
a complete breakdown of the mitochondrial membrane potential, a cessation
of ATP synthesis due to the uncoupling of the oxidative phosphorylation
and an hydrolysis of all available ATP. Furthermore, factors involved
in the induction of programmed cell death, such as the intermembrane protein
cytochrome c are liberated from the mitochondria. As mentioned above,
the cytochrome c released from the intermembrane space into the cellular
cytosol promotes apoptosis by acting in concert with ATP and APAF-1 to
activate procaspase 9. The cell death following cytochrome c release apparently
depends on the liberation of a sufficient amount of that protein, indicating
that at least a certain number of mitochondria per cell has to undergo
permeability transition before the cell is condemned to die. Thus, apoptotic
removal of a cell with a majority of oxidatively damaged mitochondria
can be considered as the ultimate step of the defense cascade prior to
collapse of the entire organ function, as it occurs in terminal congestive
heart failure. The final form of cell death following oxidative stress,
mitochondrial permeability transition and release of cytochrome c can
occur as necrosis or apoptosis, and the equilibrium between these two
forms of cell death probably depends on the availability of cellular ATP
levels required for progredience of the caspase activation.
Thus, steps 2-4 of the mitochondrial anti-oxidative defense affect the
regulation of the inner mitochondrial membrane potential, and this defense
is challenged, if the mitochondrial radical formation is enhanced due
to defects in the respiratory chain. In cells with a lowered activity
of respiratory chain complex I due to defects in the mitochondrial genome,
it has been shown that the mitochondrial inner membrane potential is lowered
[14]. We propose that such differences reflect a strong difference in
the susceptibility for programmed cell death. Similarly, we hypothesize
that the enhanced susceptibility of the chronically overloaded heart with
the cytosolic calcium overload of cardiomyocytes is partly due to a high
number of mitochondria with lowered membrane potential. Furthermore, we
propose that an important pathomechanism of the aging heart is a similar
shift towards lowered mitochondrial membrane potentials and towards enhanced
susceptibility for apoptosis by all kinds of proapoptotic stimuli.
CONCLUSION
Acknowledgement
H. Heinrich is generously supported by an educational grant from the
"Deutsche Herzstiftung".
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