Nitroxyl gets to the heart of the matter
Louisiana State University Health Sciences Center, Department of Molecular and Cellular Physiology, 1501 Kings Highway,
Shreveport, LA 71130
heart’s ability to pump blood efficiently
enough to meet the body’s metabolic
demands. Despite substantial advances
in our understanding of the underlying
pathophysiology (1) and the therapeutic
management of acute and chronic HF
(2) in recent years, the outlook of pa-
tients with these conditions remains
poor. Not only are mortality and mor-
bidity discouragingly high, but also the
patients’ quality of life remains impaired
because of a substantial symptom bur-
den. In the United States alone, HF is
responsible for almost 1 million hospital
admissions (more than for all forms of
cancer combined) and ?50,000 deaths
each year, with estimated annual costs
exceeding $20 billion (3, 4). Despite im-
proved patient information, beneficial
changes in lifestyle and better treatment
options, HF remains to be a major pub-
lic health problem in industrialized na-
tions and the leading cause of hospital-
ization in people older than 65 years. At
a time when other cardiovascular dis-
eases are on the decline, HF is rising
and likely to further escalate over the
coming decades because of an aging
population and increased survival from
the underlying causes such as coronary
heart disease and hypertension. A broad
spectrum of different drugs and various
guidelines for the treatment of HF exist
(5, 6). In the past, HF was mainly
viewed as a problem of diminished car-
diac output. Maximization of the latter
with positive inotropic (contractility en-
hancing) agents led to therapies that
initially improved functional capacity,
but increased mortality. Today, the ther-
apeutic focus is on reducing elevated
filling pressures that lead to the symp-
toms of congestion (7). Although most
recommendations agree on the major
drug classes for the first-line and ad-
junct therapy of HF, there is consider-
able controversy about the role of posi-
tive inotropic agents (8). Despite a
documented negative impact on survival,
these agents are still widely used, often
combined with vasodilators, to limit se-
vere episodes of HF or as a bridge to
transplantation. The rationale for com-
bining vasodilatation with positive ino-
tropic intervention lies in the possibility
to ‘‘unload’’ the heart, i.e., to reduce its
preload and afterload by venous and
due to unfavorable effects on cardiac
eart failure (HF) is a progres-
sively disabling and ultimately
fatal disease, which is charac-
terized by a decline in the
arterial dilatation, allowing to stimulate
cardiac output without increasing oxy-
gen consumption. Although conceptually
ideal compounds, currently used inodila-
tors (compounds with positive inotropic
and vasodilatior properties) tend to in-
crease myocardial oxygen demand at
higher doses, precipitating ischemia in
patients with coronary artery disease.
Clearly, there is room for improvement
in HF management, in particular with
regard to quality of life and survival.
In this issue of PNAS, Paolocci et al.
(9) describe the beneficial cardiovascular
effects of nitroxyl (HNO)†in the failing
heart. A well characterized canine
model of chronic heart failure was used
in which cardiac dysfunction is produced
by rapid ventricular pacing over a period
of weeks. The authors used sophisti-
cated hemodynamic analyses suited to
discriminate direct cardiac effects from
indirect effects secondary to changes in
preload and afterload to demonstrate
that nitroxyl increases myocardial con-
tractility and enhances relaxation (posi-
tive lusitropic effect) in failing hearts.
These effects were accompanied by ar-
terial and venous dilation. Paolocci’s
finding that the cardiotonic action of
HNO was unaffected by ?-receptor
blockade and additive to that of dobut-
amine is therapeutically significant not
only because the action of dopaminergic
agonists and phosphodiesterase inhibi-
tors are often attenuated in HF, but
also in view of the recent advent of
?-blockers and their negative inotropic
effects in certain clinical settings. In
contrast to nitric oxide (NO)-generating
compounds, HNO production was not
associated with increased plasma levels
of the second messenger, cGMP. In-
stead, enhanced concentrations of calci-
tonin gene-related peptide (CGRP)
were detected during HNO, but not NO
administration, suggesting that the
former may exert its favorable action, at
least in part, via this endogenous neu-
ropeptide. Although the same group
had previously observed positive inotro-
pic effects of HNO in healthy hearts
(10), the study outcome with this com-
pound in the setting of HF was not
obvious. Numerous experimental and
clinical studies in the past have demon-
strated that the same pharmacological
principle capable of increasing contrac-
tility in the normal heart can produce
negative inotropy in the failing heart
oxygen consumption and energetics.
Taken together, these results suggest
that nitroxyl donors represent a novel
class of inodilator with potential for the
treatment of HF.
In most cases, not a single cause but
a combination of systolic dysfunction
(inability to contract and eject blood
normally) and?or diastolic dysfunction
(inability to relax and fill normally), en-
ergetic and vascular loading factors,
contributes to the manifestation of HF.
The type of cardiac dysfunction prevail-
ing and the accompanying hemodynamic
situation of the patient have an obvious
impact on the choice of pharmacological
treatment. The analysis of pressure-
volume loops obtained at different load-
ing conditions is among the best of all
current approaches to assess the con-
tractile behavior of the heart in vivo.
This approach, which also formed the
basis of the present studies by Paolocci
et al. (9), has a long-standing history in
experimental physiology, but its diagnos-
tic power in animal studies and clinical
investigations has only been realized in
the last two decades. In the past, effects
on myocardial contractility have been
difficult to evaluate because of the load
dependence of conventional measures of
ventricular function. Since a couple of
years ago, left ventricular end-systolic
pressure-volume relationships can be
assessed in the setting of a routine car-
diac catheterization procedure. Using a
conductance catheter with a microma-
nometer tip for continuous measure-
ment of intraventricular volume and
pressure in combination with an occlud-
ing device to rapidly vary venous inflow,
a largely load-independent measure of
cardiac contractility can be obtained
without altering the status of the heart
(11). In addition to the assessment of
systolic and diastolic function, additional
parameters allow estimation of the rela-
tive effects of vasodilation on cardiac
performance. In their studies, Paolocci
et al. (9) used a nitrate (nitroglycerin)
and a NONOate (DEA?NO) to gener-
ate NO and Angelis’ salt to generate
HNO. Although all three compounds
were used at equieffective doses as
See companion article on page 5537.
†Not to be confused with nitroxyl radicals, which are
spin traps for the detection of radical species by EPR
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no. 9 www.pnas.org?cgi?doi?10.1073?pnas.1031571100
judged by the degree of reduction in
systolic pressure, the cardiac effects of
NO were dramatically different from
those of HNO. This is most likely due
to differences in the chemical properties
of these two species, which dictates their
reactivity with endogenous biomolecules
and the signaling pathways affected.
NO is a ubiquitous endogenous mes-
senger and modulator of cell function,
which is produced from L-arginine by a
family of isoenzymes, the NO synthases
(NOS) (12). HF is associated with re-
duced expression of endothelial NOS
and increased vascular oxidative stress,
which translates into diminished NO
availability, endothelial dysfunction and
reduced vasodilator capacity (13). NO is
also the pharmacological principle of a
number of drugs collectively termed ni-
trovasodilators, which are used clinically
to control hypertensive crises, protect
patients from attacks of angina pectoris
and to unload the heart during acute
HF. Numerous other compounds, in-
cluding NONOates are available to ex-
perimentally generate NO (14). Not-
withstanding the principal difference
that nitroglycerin requires tissue metab-
olism to generate NO whereas DEA?NO
releases it spontaneously, the cardiac
effects of both compounds were similar
(9), indicating that their action was me-
diated by the same signaling mechanism.
Nitroxyl anion (NO?) is the one-elec-
tron reduction product of NO. Its chem-
istry is not very well understood and
complicated by the fact that it exists in
two electronic forms, a singlet and a
triplet state (15). A recent reevaluation
of its pKavalue revealed that at physio-
logical pH it exists largely in its proton-
ated form, HNO (16), which can readily
cross cell membranes. Whether nitroxyl
is formed in vivo is currently unclear.
Nevertheless, it may be formed from
nitrosothiols (17), which are found to be
present in a variety of biological systems
(18). In experimental settings, nitroxyl
can be conveniently generated by using
Angelis’s salt (14). In fact, it was the
spontaneous decomposition of this inor-
ganic salt that led chemists to postulate
the existence of HNO at the turn of the
century (19). Angeli’s salt has been
shown to induce vasorelaxation and to
lower blood pressure (20, 21). Unlike
NO, which does not directly react with
sulfhydryl groups, HNO is a potent thiol
oxidant (22) and possesses a high affin-
ity for ferric heme proteins (23). The
physiological significance of these or-
thogonal properties of NO and HNO
are not entirely clear, but may offer an
explanation for the discrete effects of
these two redox congeners in the failing
heart. Although it has been suggested
that NO?and NO are redox-intercon-
vertable species (24), NO?may not be
readily oxidized to NO under all condi-
tions. Select nitroxyl donors, but not
Angeli’s salt, have been shown to un-
dergo facile oxidative conversion to re-
lax vascular tissue and inhibit platelet
aggregation in a manner indistinguish-
able from NO (25). The clear-cut di-
chotomy between the pharmacological
profile of Angeli’s salt and that of NO
donors observed by Paolocci et al. (9),
however, indicates that HNO to NO
conversion does not take place in every
tissue. This conclusion is further sup-
ported by their cGMP and CGRP mea-
surements, which suggest that the car-
diac effects of HNO and NO are
mediated by different signaling path-
ways. Besides the significance of these
findings for HF, Paolocci’s study also
offers a novel pharmacological avenue
for the modulation of CGRP levels.
CGRP is a 37-aa peptide that is syn-
thesized by alternative splicing of the
primary RNA transcript of the calcito-
nin gene in sensory neurons. Blood ves-
sels of all vascular beds are surrounded
by a dense network of CGRP-containing
nerve fibers, and most of the CGRP
circulating in plasma is thought to origi-
nate from perivascular nerves (26).
CGRP is the most potent vasodilator
known to date and thought to be in-
volved in the regulation of resting blood
pressure and regional blood flow (27),
particularly in the coronary circulation
(28). In addition, it is a cardiotonic
agent with positive inotropic and, in
normal subjects, positive chronotropic
(heart rate increasing) effects (26).
CGRP interacts with specific cellular
receptors that are coupled via G pro-
teins to adenylyl cyclase. The consecu-
tive increase in cAMP is considered the
principal mechanism responsible for
CGRP-mediated smooth muscle relax-
ation, although NO-dependent effects
(29) and opening of ATP-sensitive po-
tassium channels (30) have been de-
scribed as well. In addition to cAMP,
phospholipase C may be involved in the
stimulation of intracellular Ca2?concen-
trations in the heart (31). In patients
suffering from HF, CGRP has been
shown to reduce pulmonary and sys-
temic pressures and increase cardiac
performance without producing tachy-
cardia (32). This is consistent with the
absence of a change in heart rate with
HNO in Paolocci’s study and suggests a
possible inhibitory modulation of sym-
pathetic nervous activity (33) at the si-
noatrial and?or the arterial baroreceptor
reflex level, which clearly distinguishes
nitroxyl donors from other positive ino-
tropes, including levosimendan (34).
A number of conditions and stimuli
can cause the release of CGRP, includ-
ing ischemia, nicotine, capsaicin (35),
nitroglycerin, and a nitroxyl donor (but
not other NO generating compounds)
(36). An alternative way to increase
plasma CGRP is to slow down its enzy-
matic breakdown by inhibiting neutral
endopeptidase, but this is bound to af-
fect numerous other pathways. It ap-
pears fair to assume that the HNO-
induced CGRP increase in Paolocci’s
study (9) originated from perivascular
nerves. It would be interesting to see
whether the inotropic effects of HNO
are blunted in dogs depleted of endoge-
nous CGRP by prolonged infusion of
capsaicin (37). Further insight may also
be gained from studies in CGRP-defi-
cient mice (33). Such investigations
would not only confirm the cardiotonic
mechanism proposed for nitroxyl, but
may also shed new light on the role of
CGRP in HF. Both increased (38) and
reduced (39) plasma CGRP concen-
trations have been reported in HF.
Whether these changes are reflections
of a counterregulatory mechanism or
causally involved in disease progression
is not known. Physiologically, it would
make sense to increase CGRP early dur-
ing HF as it would complement other
compensatory systems aimed at improv-
ing cardiac efficiency. Prolonged stimu-
lation of CGRP release may lead to
gradual peptide depletion, offering an
explanation for the lower plasma levels
observed in this and other studies
CGRP-related immunoreactivity is
often found either together with NOS in
the same neuronal structures or in close
proximity to NOS-containing nerves.
Interestingly, NOS activity appears to be
involved in capsaicin-induced CGRP
release (40), and there is mounting evi-
dence to believe that NOS is capable of
producing NO?under certain conditions
(41, 42). Although admittedly specula-
tive, the mechanism of CGRP stimula-
tion by HNO may not just be a peculiar
pharmacological phenomenon, but in
fact represent an endogenous pathway
involved in the fine-tuning of CGRP
release. Whether all cardiovascular ef-
fects of HNO are due to CGRP remains
to be investigated. In addition to the use
of knockout animals this issue could be
addressed by administration of a CGRP
receptor antagonist. Should the response
to HNO be only partially blunted by
CGRP receptor blockade it might be of
interest to investigate whether inhibition
of Na??K?-ATPase (digitalis-like activ-
ity) or a Ca2?-sensitizing component are
involved in addition. Considering the
reactivity of HNO with thiols one possi-
ble site of action might reside at the
level of the ryanodine receptor, which
plays an important role in the regulation
April 29, 2003 ?
vol. 100 ?
no. 9 ?
of intracellular Ca2?transients (hence
contraction) by controlling Ca2?release
from the sarcoplasmic reticulum and
whose channel opening probability is
modulated by oxidation (43) and nitro-
sation (44) of critical thiols. Alterna-
tively, protein oxidation may shift the
association?dissociation equilibrium of
the regulatory protein FKBP12 with the
channel (45). Interestingly, the CGRP-
related peptide, adrenomedullin has
been shown to enhance cardiac contrac-
tility via cAMP-independent mecha-
nisms including Ca2?release from ryan-
odine-sensitive stores (46).
Clearly, there is much more to learn
about the biological chemistry of HNO?
NO?. Nevertheless, it looks as if there is
clear potential for therapeutic exploita-
tion of nitroxyl donors, and it appears
timely to consider intensifying research
efforts in this relatively new field. Not-
withstanding the fact that Angelis’ salt is
nothing more than an investigational
tool, the studies by Paolocci et al. (9)
are nothing less than a proof-of-princi-
ple for a potentially promising new class
of inodilator. Additional studies will be
required to address whether nitroxyl
donors are subject to tolerance develop-
ment, which often limits the effective-
ness of organic nitrates. Tolerance to
HNO might develop as a result of
CGRP depletion from peripheral nerves
or desensitization of signaling pathways
downstream of CGRP receptor activa-
tion, albeit there is no indication for this
to occur from infusion studies with
CGRP in man (47). To come up with a
drug candidate for commercial develop-
ment that was sufficiently stable, orally
available, and amenable to optimization
of its pharmacokinetic properties, struc-
tures are required that offer a variety of
possibilities for chemical derivatization.
Further aspects that demand investiga-
tion include the frequency of unwanted
side effects of nitroxyl donors such as
hypotension, headache, and gastrointes-
tinal symptoms, which limits the useful-
ness of other vasodilators, and the risk
of triggering ventricular arrhytmias.
Because of the overall hemodynamic
complexity of the different forms of HF
there is no single, straightforward ap-
proach for the therapeutic management
of all patients. Hence, we will continue
to require several individual agents with
distinct pharmacological profiles to cor-
rect specific hemodynamic abnormali-
ties. CGRP has shown potential for HF
management in clinical studies, but lacks
oral availability, is rapidly metabolized
and has thus to be given by continuous
infusion. With no selective CGRP-
mimetic on the horizon and a recently
renewed interest in inodilators (34), this
may be a unique chance for nitroxyl do-
nors. As with any new pharmacological
principle at this early discovery stage,
many obstacles have to be overcome
before a new lead compound can even-
tually enter the developmental phase.
Should nitroxyl donors pass these hur-
dles in the next couple of years, HF may
become the key indication for such com-
pounds in the future and HNO-based
inodilators a potentially useful addition
to the therapeutic arsenal available
for treatment of this life-threatening
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