The concept of preconditioning
for protection against myocardial infarction has been
extended to protection against other forms of ischemic
injury, such as stunning and arrhythmia. Preconditioning
is a paradoxical phenomenon that involves a brief period
of ischemia, followed by a period of reperfusion, then
a long ischemia, which results in a smaller infarct,
despite the fact that the total ischemic burden is longer.
This phenomenon is of interest because it is paradoxical,
biologically important, and has practical value. By
understanding the mechanism of ischemic preconditioning,
it may be used in a beneficial manner in patients with
coronary artery disease, particularly those at high
risk of a future ischemic event, in settings such as
high-risk coronary angioplasty or in cardiac surgery.
Further, it might be possible to treat patients with
multiple risk factors, in a manner similar to statins
being used for patients without a primary cholesterol
disorder, with the expectation of improved functional
recovery and a decrease in arrhythmias and tissue injury,
if preconditioning was well understood and an inexpensive,
well-tolerated drug developed. |
|
Ion Channels
in Ischemic Preconditioning |
ATP-sensitive potassium
channels have been implicated in the mechanism of preconditioning
by the mutually exclusive phenomena that genuine preconditioning
can be blocked by full protection with KATP channel
openers and that maximal cardioprotection by these channel
openers cannot be supplemented by genuine preconditioning
and by the fact that both are blocked by KATP channel
blockers.
The ATP-sensitive potassium channel first discovered
in Japan and extensively studied, mitochondrial KATP,
not surface KATP channels, are thought to be primarily
involved in preconditioning. This is based on work by
many investigators that show the mitochondrial KATP
channel agonist, diazoxide, is cardioprotective. The
mitochondrial KATP channel blocker, 5-hydroxydecanoate
(5-HD), which appears to be selective for the mitochondria
in heart cells, blocks preconditioning. The surface
agonist P-1075 does not cardioprotect and the surface
inhibitor HMR1098 does not interfere with preconditioning.
The ability of protein kinases, specifically protein
kinase C (PKC), and the ability of adenosine as a trigger
to regulate the mitochondrial KATP channels were explored.
In isolated ventricular myocytes prior exposure to phorbol
mirastyl acetate (PMA), an activator of PKC, abbreviated
the latency and increase the amplitude of the response
to diazoxide. Adenosine also abbreviated the period
of latency and increased the amplitude of the diazoxide
response. These effects were blocked by the A1 receptor
antagonist sulfa phenol theophylline. The increase in
flavoprotein fluorescence could be blocked by 5-HD,
even after adenosine administration, and the effects
of adenosine were antagonized by polymixin B, a PKC
inhibitor. Adenosine was shown in other work to be cardioprotective,
and the cardioprotection was abolished by 5-HD (the
mitochondrial blocker) but not affected by HMR-1098
(a surface blocker).
The revised mechanism of preconditioning, therefore,
involves the same upstream factors, but the importance
has shifted to the mitochondrial KATP channel. PKC can
regulate the surface KATP channels, which may have some
role in vivo where the heart is repetitively undergoing
excitation, contraction and coupling. However, since
the work by Marban and colleagues has been mostly in
isolated ventricular myocytes that are electrically
quiescent and biased to the possibility of mitochondrial
cells playing the dominant role, the contribution of
the surface KATP channels in vivo cannot be excluded.
Yet, it is clear that cardioprotection can be obtained
simply by stimulating mitochondrial KATP channels in
isolated myocytes. |
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|
Marban and colleagues have hypothesized that the opening
of mitochondrial KATP channels may suppress apoptosis
and thereby decreases the total amount of cell death
during myocardial infarction. It is known that mitochondrial
KATP channels protect against cell death during ischemia
and reperfusion, that mitochondria feature prominently
in apoptosis, and increasing evidence suggests that
apoptosis contributes to myocardial infarction.
Apoptosis is a genetically-programmed process of active
form of cell suicide that is critical physiologically
in tissue homeostasis and differentiation. Apoptosis
may also be important pathologically in a variety of
human diseases, including ischemic heart disease. Accelerated
apoptosis may contribute to some of the vascular wall
injury associated with atherosclerosis and although
controversial, apoptosis has been argued to be involved
in heart failure.
The mitochondrial death pathway, known to be operative
in myocardial cells, and the death receptor pathway
are the two general pathways for apoptosis triggering.
Mitochondria, which comprise by volume 35% of myocardial
cells, lose cytochrome C, which is followed by activation
of caspases (proteases specific for the apoptotic cascade)
as a result of stress (oxidative, calcium overload,
UVA radiation, etc). Apoptosis then continues and the
cell autodigests and breaks up its own DNA. Marban and
colleagues conceive of the possibility that any of these
stressors could lead to the opening of peri-apoptotic
channels, the release of cytochrome C, activation of
downstream caspases, cleavage of death substrates, and
apoptosis.
In parallel, necrosis may occur and in real infarct
it is likely there is a mixture of apoptosis and necrosis.
The extent of apoptosis and the cells in the pathway
may be more fragile and susceptible to necrosis. Studies
conducted by Marban and colleagues established the basic
finding that apoptosis is occurring with hydrogen peroxide
and that apoptosis is inhibited by diazoxide and that
the protective effect is blocked by 5-HD. Studies have
shown that the upstream cytochrome C, which is usually
retained within the mitochondria, after exposure to
hydrogen exposure is evenly distributed through the
cytosol and the nuclei become shrunken and pyknotic.
5-HD at least partially blocks against the protective
effect of diazoxide. Caspase 3 is activated by peroxide,
but diazoxide markedly inhibits the activation of caspase
3 and 5-HD blocks the protective effect.
Marban and colleagues showed that mitochondrial depolarization
is one of the initial steps in the apoptotic cascade
by using mitochondrial membrane potential indicators.
Under exposure to oxidant stress nearly all the cells
their mitochondrial inner membrane potential. However,
diazoxide was very protective and significantly reduced
the number of cells that lost their membrane potential.
Diazoxide also exerts a very potent protective effect
on neurons—very interesting in terms of brain ischemia,
for which effective treatment is limited. Diazoxide
was shown to be very neuroprotective in cultured neonatal
rat cerebellar granule neurons. 5-HD blocked that neuroprotection.
Marban and colleagues have also found that pinacidil
and nicorandil are protective and that glibenclamide
blocks that protection. |
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|
During myocardial infarction,
cells die both by apoptosis and necrosis, pathways that
may interact. About 48 hours is required to obtain a
good estimate of myocardial infarction after coronary
occlusion in vivo. Although necrotic cell death should
be complete sooner than that, apoptosis is thought to
contribute to cells continuing to die during the 48-hour
period. Intense TUNEL positivity is found when looking
directly for apoptotic nuclei in the post-ischemic myocardium.
Broad-spectrum caspase inhibitors have been shown to
be protective against myocardial infarction. Transgenic
mice that overexpress a protective kinase, known to
decrease apoptosis, have markedly decreased infarct
sizes. Based on this evidence, Marban and colleagues
believe that at least during relatively brief myocardial
infarction cells die both by apoptosis and necrosis.
It is also known that drugs that recruit pharmacologic
cardioprotection inhibit apoptosis by oxidative stress,
a prominent feature of ischemia and reperfusion.
Thus, their working hypothesis is that preconditioning
is due to the inhibition of apoptosis induced by ischemia
and reperfusion. In virgin myocardium, more and more
cells die during the progression from long ischemia
to short ischemia. They conjecture that the relative
contribution of apoptosis is most prominent after brief
ischemia, and that necrosis is more prominent after
prolonged ischemia. Preconditioning and the protective
drugs are extending the viability of the myocardium
and may be doing this by suppressing the apoptotic component
of cell death. |
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