Cardiovascular Research 2006 70(2):231-239; doi:10.1016/j.cardiores.2005.10.021
Copyright © 2006, European Society of Cardiology
Nitric oxide is a preconditioning mimetic and cardioprotectant and is the basis of many available infarct-sparing strategies
Michael V. Cohen*,
Xi-Ming Yang and
James M. Downey
Departments of Physiology and Medicine, MSB 3050, University of South Alabama, College of Medicine, Mobile, AL 36688, United States
* Corresponding author. Tel.: +1 251 460 6812; fax: +1 251 460 6464. Email address: mcohen{at}usouthal.edu
Received 25 August 2005; revised 10 October 2005; accepted 11 October 2005
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Abstract
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Ischemic preconditioning is a powerful infarct-sparing intervention.
Intensive investigations have revealed many of the signaling
steps used to elicit this protection. One of the steps involves
activation of nitric oxide synthase (NOS) by phosphorylation,
with the production of NO and subsequent activation of guanylyl
cyclase, production of cGMP, activation of protein kinase G,
opening of mitochondrial K
ATP channels, and generation of reactive
oxygen species. The latter act as second messengers to activate
critical kinase cascades that trigger entrance into the preconditioned
state. Thus, NO exposure before ischemia can act as a powerful
preconditioning mimetic. Elevating NO just prior to or at reperfusion
can still be an effective cardioprotective strategy. Activation
of NOS or production of NO can be done pharmacologically with
exogenous agents to trigger this cascade. Many of these strategies
are already available and safe.
KEYWORDS Carbon monoxide; Cardioprotection; Natriuretic peptide; Nitric oxide; Phosphodiesterase inhibitor; Preconditioning; Statin
Ischemic preconditioning (IPC) was first introduced by Murry
et al.
[1] in 1986. There has not been a more potent cardioprotective
intervention described before or since. In Murry's dogs 4 brief
5-min coronary occlusions in the 40-min period preceding a continuous
40-min occlusion decreased infarct size by 75% from that seen
in dogs with only the prolonged coronary occlusion. This remarkable
protection has been documented in all species of experimental
animals studied to date
[2] as well as man
[3]. Because of the
impressive clinical potential of such an intervention, it has
been subjected to much scrutiny during the past two decades.
Because myocardial ischemia is not a clinically useful trigger
of this phenomenon, the mechanism and signaling of preconditioning
have been extensively studied in an attempt to identify a more
appropriate and useful stimulus that could safely and reliably
be applied in man to produce prophylactic protection against
ischemic events. The result of these studies is that much is
now known concerning IPC's mechanism. One might ask if nitric
oxide (NO) could be one of these agents.
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1. History of NO in preconditioning
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Although Bolli and his colleagues
[4] convincingly demonstrated
that endogenous NO was critical to the triggering of the second
window of preconditioning (late preconditioning), its participation
in the signaling of classical (early) preconditioning has until
recently been quite controversial. Woolfson et al.
[5] were
the first to test for the involvement of NO in ischemic preconditioning.
Isolated rabbit hearts were treated with
N
-nitro-
L-arginine
methyl ester (
L-NAME), a NO synthase (NOS) inhibitor, but no
effect against ischemic preconditioning was seen. Interestingly,
however, they noted that
L-NAME reduced infarct size in non-preconditioned
hearts, a phenomenon which has not been reported since. On the
other hand, Lochner et al.
[6] presented evidence that NO plays
a role in IPC in rat hearts. Ferdinandy et al.
[7] also saw
loss of protection in a pacing model of preconditioning when
NOS inhibitors were administered. Regrettably our own work contributed
substantially to the confusion surrounding a possible role of
NO in IPC. In an early study we investigated the contribution
of endogenous nitric oxide to the protection of ischemic preconditioning
[8]. In an isolated rabbit heart preparation we examined the
effect of
L-NAME added to the perfusate. Ischemic preconditioning
with 5 min of global ischemia and 10 min of reperfusion before
the 30-min index ischemia resulted in dramatic salvage of myocardium
(
Fig. 1). When
L-NAME (100 µM) was infused for 50 min
beginning 5 min before the 5-min preconditioning ischemia/10-min
reperfusion and ending at the end of 30 min of regional ischemia,
protection was not affected. The result was the same with 200-µM
L-NAME
[9]. The NO donor
S-nitroso-
N-acetylpenicillamine (SNAP)
given prior to ischemia mimicked IPC and protected the hearts
[8]. We concluded that exogenously administered NO could trigger
the preconditioned state but that endogenous production of NO
was not involved in IPC. For several years we relied on this
study to discount NO's participation in IPC's mechanism. However,
our very recent observations have permitted us to challenge
our original conclusion (see below).
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Fig. 1 Infarct size as a percentage of risk zone volume for in vitro (left) and in vivo (right) rabbit heart models. Open circles represent individual data points and closed circles represent means with SEM. In the in vitro model ischemic preconditioning (PC) resulted in significant reduction in infarction, but N-nitro-L-arginine methyl ester (L-NAME) was unable to blunt this effect. In contrast, in the in vivo model PC elicited with 1 cycle of 5-min coronary occlusion/10-min reperfusion continued to salvage ischemic myocardium, but now this salutary effect was completely abolished by L-NAME. However, if preconditioning was accomplished with 3 cycles of ischemia/reperfusion instead of the customary one cycle, L-NAME could no longer abort protection.
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2. Reactive oxygen species are involved in IPC in the in situ heart
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Around that same time it was noted that reactive oxygen species
(ROS) were involved in the triggering of IPC. Forbes et al.
[10] reported that protection from diazoxide, a selective opener
of mitochondrial ATP-sensitive potassium channels (mK
ATP), could
be blocked with a ROS scavenger. Similarly Yao et al.
[11] reported
a ROS scavenger could also block protection from acetylcholine
in chick myocytes exposed to simulated ischemia. That led us
to formulate a hypothesis that occupancy of surface receptors
during the preconditioning ischemia led to opening of mK
ATP channels which caused the mitochondria to release ROS. The ROS
would then act as second messengers to activate protein kinase
C (PKC) and its protective pathways. That hypothesis was supported
by the study of Pain et al.
[12]. Because it can be shown that
all events leading to ROS formation must occur prior to the
onset of the ischemic insult, we have termed this the trigger
pathway.
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3. The cardiomyocyte ROS model
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To map out the signaling steps in the trigger pathway we resorted
to an isolated rabbit cardiomyocyte preparation. To quantitate
cell ROS production we used reduced MitoTracker red, a non-fluorescent
probe, which, when oxidized by ROS, becomes fluorescent and
binds to thiol groups in mitochondria. When cardiomyocytes were
exposed to ACh, bradykinin, or opioids, the G protein-coupled
receptor (GPCR) agonists known to trigger preconditioning, ROS
production was increased. We were able to show that in general
these agents act to cause a metalloproteinase-dependent activation
of the epidermal growth factor (EGF) receptor which, through
a Src-kinase-dependent mechanism, activated phosphatidylinositol
3-kinase (PI3-K) and Akt (
Fig. 2). Thus the increased ROS production
induced by these agents could be blocked by inhibiting metalloproteinase
[13], blocking the EGF receptor
[13], inhibiting Src kinase
[14], inhibiting PI3-K
[14,15] and Akt
[16], or closing mK
ATP channels
[14,17], respectively. But then we noted that
L-NAME
would also block ROS production
[16,17], implying NOS was indeed
part of the trigger pathway. Given our earlier results in the
isolated heart, these data in cardiomyocytes were confusing.
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Fig. 2 Current concept of the signaling that makes up the trigger pathway of preconditioning. Abbreviations: ACh=acetylcholine; Brady=bradykinin; cGMP=cyclic GMP; EGFR=epidermal growth factor receptor; eNOS=enothelial nitric oxide synthase; GC=guanylyl cyclase; HB-EGF=heparin-binding EGF-like growth factor; MMP=membrane metalloproteinase; NO=nitric oxide; PDK=phosphoinositide-dependent kinase; PI3K=phosphatidylinositol 3-kinase; PIP2, PIP3=phosphatidylinositol bisphosphate, trisphosphate; PKC=protein kinase C; PKG=protein kinase G; Pro=pro-HB-EGF; PTEN=phosphatase and tensin homologue on chromosome ten (the phosphatase that removes phosphate in the 3 position).
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4. The trigger pathway
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As a result of these investigations many of the signaling steps
of IPC's trigger pathway have now been elucidated. As depicted
in
Fig. 2 we now believe that during preconditioning the ischemic
myocardial cell releases at least 3 receptor agonists that bind
to G
i or G
q protein-coupled receptors. They include bradykinin,
opioids, and adenosine. These agonists then initiate a complex
signaling cascade which ultimately leads to protection. To facilitate
the early investigations most of these steps were first established
with acetylcholine (ACh), an agonist of a well characterized
GPCR although ACh is not released by ischemic myocardial cells.
However, ACh's signaling exactly mimics that of opioids and,
with a small exception, bradykinin. Curiously adenosine bypasses
many of these early trigger steps and appears to activate a
downstream kinase cascade directly.
We believe that the trigger pathway proceeds as follows. There is considerable evidence that these agonists first cause transactivation of the EGF receptor [13,18,19]. After the released agonists bind to their respective receptors, the G protein's β
subunits are liberated and shuttle within the sarcolemma where, among other things, they activate a membrane metalloproteinase. The metalloproteinase cleaves heparin-binding epidermal growth factor-like growth factor (HB-EGF) from its inactive pro-form. HB-EGF then binds to the EGF receptor which dimerizes leading to auto-phosphorylation of critical tyrosine residues (termed transactivation) resulting in attraction of several proteins including Src tyrosine kinase and PI3-K which assemble into a signaling complex. PI3-K is activated and it phosphorylates membrane phosphatidylinositol bisphosphate in the 3 position. The 3-phosphorylated phospholipid then activates the phosphoinositide-dependent kinases (PDKs) which in turn activate Akt through phosphorylation [20].
Akt (or protein kinase B) in turn phosphorylates endothelial NOS (eNOS) [21] which catalyzes formation of the powerful small molecular messenger NO. NO stimulates soluble guanylyl cyclase to increase production of cGMP which in turn activates protein kinase G (PKG). Cytosolic PKG then phosphorylates some unknown target on the mitochondria's outer membrane which then through a PKC isoform in the mitochondria (likely 
) causes the ATP-sensitive K+ channel on the inner membrane to open [22]. As a result K+ enters the mitochondrion along its electrochemical gradient resulting in alkalinization of the matrix [23] and increased production of ROS. Although initially regarded to be deleterious elements promoting DNA and protein oxidation, membrane disruption, and organelle destruction, ROS are critical elements in this signaling pathway. Thus interference with ROS production after triggering of preconditioning with ischemia can abort cardioprotection while controlled production of ROS in lieu of another preconditioning stimulus can decrease infarction [24,25]. When released from mitochondria ROS act as second messengers and are thought to activate phospholipase C and PKC. That initiates the mediator pathway which leads to activation of PI3 kinase and extracellular signal-regulated kinase (ERK) at reperfusion [26,27] and inhibition of mitochondrial permeability transition pore formation [28]. The pore is thought to be the end-effector. The signaling within the mediator pathway is less well understood than that of the trigger pathway. However, it is now thought that the actual protection occurs in the reperfusion rather than ischemic phase, and repopulation of adenosine receptors [27] and activation of multiple kinases [26,27] are critical events.
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5. Adenosine uses a pathway independent of NO
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While both opioids and bradykinin trigger IPC through the NO-dependent
pathway described above, adenosine uses an entirely different
pathway. Adenosine-triggered protection cannot be blocked by
K
ATP channel blockers
[29], ROS scavengers
[29], or NO blockers
[30]. Adenosine appears to trigger IPC by coupling directly
to PKC. Although this observation was surprising since adenosine
presumably couples to the same G
i as bradykinin and opioids,
the different pathway followed by adenosine appears to afford
the organism increased security. The redundancy insures the
continuing ability to precondition the heart even if there is
blockade or interference with the trigger pathway at one of
the intermediate steps as EGFR, Akt or NO. It is presumed that
once PKC is activated by adenosine or some other preconditioning
trigger, the mediator pathway is the same independent of the
specific trigger.
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6. NO does participate in IPC in the in situ heart
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Obviously the cell data did not agree with our
[8] or Woolfson's
[5] previous observation that IPC's protection in an isolated
heart could not be blocked by
L-NAME. We now believe that those
observations were the result of use of the isolated heart model.
The 3 major agonists released by the ischemic heart are bradykinin,
opioids, and adenosine. In the isolated buffer-perfused heart
the absence of circulating kininogens would minimize any release
of bradykinin. And the absence of cardiac innervation would
attenuate opioid release. Thus virtually all of the triggering
would come from adenosine which, unfortunately, bypasses the
NO-dependent trigger pathway. Therefore, it would not be surprising
that NO is unimportant in the isolated heart. We hypothesized
that in the in situ heart where bradykinin and opioids would
be released by ischemic myocardium the NOS-cGMP-PKG cascade
would be important. Accordingly, we recently repeated our
L-NAME
experiments in the in vivo heart. IPC with 1 cycle of 5-min
ischemia/10-min reperfusion decreased infarction from 38.5±3.4%
in control hearts to 14.7±3.3% (
p<0.001).
L-NAME blocked
the protection of ischemic preconditioning (
Fig. 1). When the
number of preconditioning cycles was increased to 3,
L-NAME
could no longer block the protection. Presumably the additional
preconditioning cycles increased the amount of released adenosine
so that it alone was sufficient to protect even though the NO-dependent
pathways had been blocked. Although our initial observation
in isolated hearts was accurate, we mistakenly extrapolated
the conclusion to all models. Hopefully these new data will
set the record straight.
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7. NO is a cardioprotectant
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There has been an ongoing debate as to whether NO is a protectant
or a source of injury in the ischemic heart. NO can be a source
of deleterious peroxynitrite in the heart. Woolfson et al.
[5] proposed that
L-NAME protected by preventing peroxynitrite formation.
However, most others see NO as a protectant. Nossuli et al.
[31] documented infarct-sparing properties of exogenous peroxynitrite.
Several reports have documented the deleterious effect of NOS
blockers in ischemia-reperfusion injury. Williams et al.
[32] and Hoshida et al.
[33] observed that either
L-NAME or
L-nitro
arginine, both NOS inhibitors, given prior to coronary occlusion
in in situ rabbit preparations resulted in significantly larger
infarcts than in untreated animals. And
L-NAME can exacerbate
post-ischemic contractile function in isolated rat hearts
[34].
The strongest evidence that NOS and NO play important roles
in cardioprotection is derived from experiments in genetically
altered mice. Brunner et al.
[35] created transgenic mice overexpressing
human eNOS exclusively in cardiac myocytes. Left ventricular
pressure was reduced by a maximum of 33% and basal cardiac cGMP
was increased twofold, changes which were reversed by NOS blockade
with
L-NAME. Relative to baseline, recovery of left ventricular
developed pressure and d
P/d
t following ischemia were significantly
better in transgenic hearts (95–98%) than wild type hearts
(48–51%). Again
L-NAME abolished the difference. Jones
et al.
[36] studied strains of mice overexpressing either bovine
or human eNOS. In an in vivo preparation a coronary artery was
occluded for 30 min and infarct size was determined by triphenyltetrazolium
chloride (TTC) staining after 24 h of reperfusion. Myocardial
infarct size was reduced by 32–33% in the two transgenic
strains (
p<0.05 vs. non-transgenic mice).
Parallel experiments have been performed in eNOS knockout mice. Kanno et al. [37] studied a strain developed at the University of North Carolina (UNC). Isolated hearts with this genetic defect subjected to 30 min of global ischemia and 60 min of reperfusion had smaller, not larger, infarcts than wild type murine hearts (20.0% vs. 30.2% of risk zone, p<0.05). This seemingly anomalous result was further investigated. Despite the missing enzyme the eNOS knockout hearts released significant amounts of nitrite into the effluent during reperfusion, implying significant production of NO. And immunoblots showed that iNOS was markedly induced in the eNOS knockout hearts. It was the compensatory increase in iNOS that salvaged these hearts. This conclusion was further supported when Sharp et al. [38] evaluated responses of this UNC eNOS knockout strain of mouse as well as a second Harvard knockout strain in which there was no compensatory increase in iNOS. Whereas UNC mice exhibited a 52% reduction in myocardial infarction compared to wild type controls (p<0.05), the Harvard mice experienced an 84% increase in myocardial necrosis (p<0.05). Furthermore an iNOS inhibitor exacerbated the extent of myocardial damage in UNC mice, but had little effect in the Harvard mice. These data certainly support the contention that NOS and NO are critical components of the cardioprotective mechanism.
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8. NO-promoting strategies protect both prior to ischemia and at reperfusion
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In light of this recognition many attempts to stimulate NO production
with exogenous pharmacologic agents have been made. Even before
the above evidence supporting the importance of NO was available,
investigators were administering
L-arginine, the required precursor
for production of NO, just before and/or during reperfusion
and observing smaller infarcts and improved recovery of post-ischemic
left ventricular function in dogs
[39], cats
[40], and rats
[34]. A variety of compounds known as NO donors are metabolized
in vivo to release NO. Many investigators have examined the
effect of SNAP. Kanno et al.
[37] demonstrated that SNAP raised
nitrite levels strikingly in the coronary effluent from buffer-perfused,
isolated wild type mouse hearts. In hearts undergoing 30 min
of global ischemia and 60 min of reperfusion SNAP infused into
the aortic cannula's side branch immediately after clamping
of the cannula decreased average infarct size from 30.2% in
untreated hearts to 16.5%. We
[8,9] have observed similar effects
in isolated rabbit hearts in which SNAP infused prior to the
index ischemia decreased infarction from 30.2±3.3% of
the risk zone in control hearts to 4.4±1.9%. Other NO
donors
[34,41–44], including sodium nitroprusside
[45] and nitroglycerin
[46,47], infused either before hypoxia or
ischemia or just before reperfusion similarly salvaged ischemic
myocardium and improved post-ischemic contractile function.
Conversely
L-NAME caused further deterioration. Nitroglycerin
can also trigger delayed preconditioning (discussed elsewhere
in this focused issue)
[47].
A new group of NO donors in which the NO moiety is attached to an aspirin backbone were synthesized to protect the gastric mucosa against the deleterious effect of aspirin. NCX 4016 (2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester) has multiple biologic actions including antithrombotic and platelet antiaggregatory effects and diminution of adherence of neutrophils to vascular endothelium. NCX 4016 resulted in rapid release of NO in rats [48] and man [49]. It also increased in a dose-dependent manner plasma cGMP levels and attenuated infarction in rats when administered orally for 5 days before 30 min of coronary artery occlusion [50]. NCX 4016 also diminished infarction in pig hearts in contrast to aspirin, although both drugs had similar inhibitory effects on platelet aggregation and thromboxane generation [51]. Interestingly in the latter model NCX 4016 had no effect on leukocyte adhesion to normal coronary arteries or those previously exposed to ischemia/reperfusion. This drug has been introduced for small clinical trials [52], but it has not yet been examined for an effect on ischemic myocardium.
Other strategies have also been used to increase circulating NO levels. Nitrite is typically thought of as the major oxidative metabolite of NO, an inert metabolic end-product with limited intrinsic biological activity. However, at low tissue pH and oxygen tension, conditions favored in ischemic myocardium, nitrite may be reduced to NO by disproportionation (acidic reduction) or by the enzymatic action of xanthine oxidoreductase. Solutions of sodium nitrite were infused 5 min before reperfusion into the left ventricular cavity of in situ mice in which coronary occlusion was maintained for 30 min followed by reperfusion for 24 h [53]. Infarct size was measured after staining with TTC. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitrosamines, and iron-nitrosylated heme proteins within 1 min of reperfusion. Nitrite administration decreased myocardial infarction by 67% compared with nitrate-treated controls. This protective effect was abolished in animals pretreated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO).
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9. PDE-5 inhibitors
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Kukreja and colleagues
[54–56] have championed the cardioprotective
properties of sildenafil (Viagra), probably the most widely
used drug for treatment of erectile dysfunction. It is a selective
inhibitor of phosphodiesterase-5 (PDE-5) that catalyzes the
breakdown of cGMP. Therefore, sildenafil enhances NO-driven
cGMP accumulation. When given to rabbits either intravenously
or orally it has a preconditioning-mimetic effect. Infarction
averaged 33.8±1.7% in untreated hearts, whereas infarct
size was significantly lower (10.8±0.9%) after 30 min
of sildenafil pre-treatment
[54]. Similar results have been
observed in rats
[57]. Following sildenafil injection in mice
eNOS mRNA increases with a peak at 45 min and inducible NOS
(iNOS) mRNA peaks at 2 h
[55]. Coronary occlusion 24 h later
was accompanied by a 75% decrease in infarction, and this salutary
effect was blocked by an iNOS inhibitor. A very recent investigation
in isolated adult mouse ventricular myocytes subjected to simulated
ischemia and reoxygenation demonstrated that sildenafil pre-treatment
decreased cell necrosis and apoptosis
[56]. Sildenafil-induced
protection against both necrosis and apoptosis was absent in
myocytes derived from iNOS knockout mice and attenuated in eNOS
knockout myocytes. Therefore sildenafil produces a direct protective
effect through the NO signaling pathway independent of hemodynamic
alterations. Although sildenafil itself does not increase NO
generation, it enhances sensitivity to NO by its effective inhibition
of cGMP degradation and thus augments PKG activation and the
following dependent events which lead to cardioprotection. Sildenafil
is currently used clinically for treatment of penile erectile
dysfunction and pulmonary hypertension. It has not yet been
evaluated in the treatment of ischemic heart disease, and this
may be a problem because of the induced hypotension caused by
concomitant use of this drug and nitrates.
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10. HMG-CoA reductase inhibitors
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Statins, inhibitors of HMG-CoA reductase, were introduced to
lower serum cholesterol. However, their many pleiotropic actions
have been utilized to affect many biological processes, and
may even be more important than their effects on lipids
[58].
Statins upregulate NOS activity predominantly by posttranscriptional
mechanisms
[59,60] and increase NO production under baseline
conditions and after hypoxia
[61]. Statins increase eNOS expression
by increasing eNOS mRNA stability
[59]. Additionally statins
activate Akt, resulting in phosphorylation of eNOS and further
increases in NO production
[62]. Aortic rings from rats treated
18 h before sacrifice with simvastatin released twice as much
NO as aortic rings from untreated animals
[63].
L-NAME inhibited
the NO release.
Statins have been shown to be cardioprotective in a variety of animal models. In isolated rat hearts perfused with polymorphonuclear leukocytes subjected to 20 min of global ischemia and 45 min of reperfusion, post-ischemic left ventricular function was significantly better in hearts pretreated with simvastatin [63]. In in situ mice undergoing 30 min of coronary occlusion and 24 h of reperfusion, pretreatment with simvastatin significantly reduced infarction by more than 50% (p<0.01) [64]. This salvage by simvastatin was completely lost if the Harvard strain of eNOS knockout mice was substituted for wild type animals. Fluvastatin 20 min prior to left coronary artery ligation in rats was sufficient to preserve myocardial blood flow and decrease infarct size following 50 min of ischemia and 60 min of reperfusion [65]. L-NAME abolished fluvastatin's effect on infarction. Postulated mechanisms of this statin infarct-sparing effect include decreased polymorphonuclear leukocyte infiltration, decreased leukocyte rolling, and decreased expression of adhesion molecules [58]. Statins have been used clinically for many years for the treatment of hypercholesterolemia, and are currently being administered very early in the treatment of patients with acute coronary syndrome [66]. However, the effectiveness of these drugs as adjuncts to percutaneous coronary intervention has not yet been examined.
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11. Natriuretic peptides
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Natriuretic peptides (NP) are endogenous hormones released by
distended atria and ventricles. Both atrial NP (ANP) and brain
NP (BNP) bind to membrane NP receptors which are expressed on
cardiomyocytes. Receptor binding results in activation of particulate
guanylyl cyclase, distinct from the soluble guanylyl cyclase
in the NO-cGMP-PKG signaling cascade. Both BNP administered
before the index ischemia in rats
[67] and ANP infused a few
minutes before reperfusion in rabbits
[68] decreased infarct
size. Examination of signaling in isolated rabbit cardiomyocytes
confirms that ANP binds to a membrane NP receptor/particulate
guanylyl cyclase which results in activation of PKG (unpublished
observation). Unexpectedly, however, 1H-[1, 2, 4]oxadiazolo-[4,
3-a]quinoxalin-1-one (ODQ), a putative soluble guanylyl cyclase
inhibitor, blocked ANP's signaling and the natriuretic peptide's
infarct-sparing quality
[68]. Although not widely appreciated
there is experimental evidence that ANP stimulates eNOS and
NO production
[69–72]. So it appears that, at least in
part, the cardioprotection triggered by natriuretic peptides
involves increased production of NO. A clinical trial of the
use of ANP as an adjunct to percutaneous coronary intervention
in the treatment of acute myocardial infarction is ongoing
[73].
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12. Carbon monoxide
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Carbon monoxide is produced by the endogenous degradation of
heme by several heme oxygenase enzymes: the constitutively expressed
HO-2 and the inducible HO-1. Like NO, CO is a versatile signaling
molecule and, also like NO, it possesses vasorelaxing properties.
Certain transition metal carbonyls liberate CO under appropriate
conditions and function as CO-releasing molecules (CORM). Effects
of the water-soluble tricarbonylchloro(glycinato) ruthenium
II (CORM-3) have been examined in isolated, buffer-perfused
rat hearts undergoing global ischemia for 30 min and reperfusion
for 60 min
[74] and in in situ mouse hearts subjected to 30
min of coronary occlusion and 24 h of reperfusion
[75]. CORM-3
was administered either in the last minutes of ischemia and/or
the early minutes of reperfusion. This CO-releasing compound
diminished infarction in these models by 50–75%. Although
the mechanism of this salutary effect has not been fully elucidated,
closing of mitochondrial K
ATP channels aborts the infarct-sparing
property of CO
[74]. Inhaled CO in rats also diminished infarction
following a 30-min occlusion of the left anterior descending
coronary artery and activated Akt, eNOS and cGMP in myocardium
[76]. Wortmannin, a PI3-K inhibitor,
L-NAME, and methylene blue,
a soluble cGMP inhibitor, each attenuated the protection by
CO. This protective effect, therefore, was at least in part
dependent on activation of the NOS signaling cascade. Interestingly
NO may in part protect ischemic myocardium by triggering CO
signaling generated by the activation of heme oxygenase
[77].
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13. NO: a potential role in cardioprotection
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As detailed in the foregoing discussion NO supplied in multiple
forms can salvage ischemic myocardium and diminish infarction.
NO applied prior to ischemia clearly acts as a signal in a cascade
that triggers entrance into the preconditioned state. Much more
elusive is its specific role when administered at reperfusion.
Augmenting NO at reperfusion appears to be associated with protection
but its mechanism at that time remains speculative. It is acknowledged
that NO has multiple biologic actions, including effects on
inflammation, endothelial expression of adhesion molecules,
and anti-platelet and antithrombotic properties. These latter
effects may certainly enhance any cardioprotective quality of
NO
[34]. However, documentation of NO's infarct-sparing ability
in cell-free, isolated, buffer-perfused hearts diminishes the
importance of the above attributes, and the isolated cardiomyocyte
studies further support a role of NO on cardiomyocytes in the
absence of endothelial cells.
It is important to differentiate between strategies in which pharmacologic agents are administered before the index ischemia as opposed to those in which they are administered at the time of reperfusion. In a patient presenting to the emergency room after the onset of myocardial infarction, pre-ischemic therapy can no longer be initiated. In this case reliance must be placed on those strategies found to be effective when applied after the onset of ischemia. Agents like nitrates, nitrites, ANP and CORM have shown such efficacy in animal models, but unfortunately lack confirmation in clinical trials. Finally, most patients with acute myocardial infarction already receive nitrate therapy and it is possible that they may already be maximally protected.
Preconditioning with a treatment before ischemia could also have a clinical role. In iatrogenic ischemia as occurs with cardiac surgery the patient can be preconditioned, although other cardioprotective strategies such as cooling and cardioplegia have been the treatments of choice. Since it can never be predicted when a coronary thrombus might occur, it would be impossible to time the administration of a single dose of a preconditioning mimetic to have a clinically significant effect. But chronic treatment would be expected to have a prophylactic protective effect either by continuing stimulation of the pathway outlined in Fig. 2 or triggering delayed preconditioning or the second window of protection (described elsewhere in this focused issue). That of course requires that tachyphylaxis [78] be avoided. Statin and nitrate therapies would be expected to be candidates for this strategy. We have reviewed several papers above which indicate that NO can trigger the second window type of protection.
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14. Conclusion
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Cardioprotection either as a prophylactic measure or as a therapeutic
intervention during myocardial infarction is as yet an unrealized
dream. Although the concept is attractive, admittedly not all
questions have been answered by the ongoing intensive investigation.
Nonetheless there are sufficient safe and seemingly effective
approaches that can be used even now. A coalition between the
medical community and the pharmaceutical industry is necessary
to have this concept emerge from the research laboratory into
the clinical arena.
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Notes
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Time for primary review 25 days
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References
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Abstract
1. History of NO...