The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
S-nitrosylation: integrator of cardiovascular
performance and oxygen delivery
Saptarsi M. Haldar1 and Jonathan S. Stamler1,2,3
1Department of Medicine and Cardiovascular Division, 2Harrington Discovery Institute, and 3Institute for Transformative Molecular Medicine,
Case Western Reserve University School of Medicine and University Hospitals Case Medical Center, Cleveland, Ohio, USA.
Delivery of oxygen to tissues is the primary function of the cardiovascular system. NO, a gasotransmitter that signals
predominantly through protein S-nitrosylation to form S-nitrosothiols (SNOs) in target proteins, operates coordi-
nately with oxygen in mammalian cellular systems. From this perspective, SNO-based signaling may have evolved
as a major transducer of the cellular oxygen-sensing machinery that underlies global cardiovascular function. Here
we review mechanisms that regulate S-nitrosylation in the context of its essential role in “systems-level” control
of oxygen sensing, delivery, and utilization in the cardiovascular system, and we highlight examples of aberrant
S-nitrosylation that may lead to altered oxygen homeostasis in cardiovascular diseases. Thus, through a bird’s-eye
view of S-nitrosylation in the cardiovascular system, we provide a conceptual framework that may be broadly appli-
cable to the functioning of other cellular systems and physiological processes and that illuminates new therapeutic
promise in cardiovascular medicine.
Oxygen and NO: co-evolution for common function
From the appearance of the simplest metazoans to the most com-
plex multicellular life forms, the ability to efficiently handle oxygen
has remained essential for survival and has therefore been subject
to intense evolutionary pressure. A single-cell organism must rap-
idly adapt its core homeostatic processes to fluctuations in oxy-
gen tension, functions retained in specialized cells of higher verte-
brates (1, 2). In addition to cell-autonomous pathways for oxygen
homeostasis, complex multicellular organisms have also developed
sophisticated mechanisms to efficiently coordinate oxygen delivery
and utilization across diverse organ systems (3–5). From this per-
spective, the human cardiovascular system in all its complexity has
evolved for the principal purpose of oxygen delivery.
Although oxygen itself can function as a signaling molecule (2,
6), its signaling repertoire is dependent largely on heme binding
and is therefore limited, as hemes do not generally convey cellular
signals. Thus, organisms have necessarily evolved parallel mecha-
nisms to precisely control oxygen flux and function. Utilization
of the ancient gasotransmitter NO, highly abundant in the pri-
mordial atmosphere and linked to anaerobic respiration, likely
co-evolved with oxygen to serve a common function — regulation
of aerobic respiration (i.e., oxygen delivery and utilization). While
NO, like O2, binds transition metal centers to elicit cellular signals,
the majority of its cellular influence is achieved through posttrans-
lational modification (PTM) of cysteine thiols, a process termed
S-nitrosylation (7). The universal presence of cysteine thiols in all
major classes of proteins greatly expands signaling possibilities,
and regulation of protein function via S-nitrosylation may be
viewed as the prototypical system for redox-based and gasotrans-
mitter-mediated signal transduction (8).
Recent reviews of S-nitrosylation have detailed the redox biochem-
istry of reactive nitrogen species (8–10) and cataloged the myriad
proteins and cellular processes known to be regulated by this mod-
ification across systems, including the cardiovascular system (11,
12). Nearly 1,000 S-nitrosylated proteins have been identified in the
heart alone (13, 14), and cross-talk with a plethora of other PTMs
has been described (15). Principles underlying reversibility, specific-
ity, and enzymatic control of S-nitrosylation have received particular
attention. Here, we take a thematic perspective that highlights the
essential role of protein S-nitrosylation in the systems-level control
of oxygen delivery and utilization, which is arguably the essential
function of the cardiovascular system. Using these physiological
insights, we highlight examples of how S-nitrosylation is dysregu-
lated in cardiovascular disease and how modulation of this signal-
ing mechanism holds therapeutic promise. Through this bird’s-eye
view of S-nitrosylation in the cardiovascular system, we provide a
conceptual framework that may be broadly applicable to cellular
systems, physiological processes, and diseases.
S-nitrosylation as a prototypical system of protein PTM
Systems governing PTM of proteins generally fall into two broad
categories, those with a ubiquitous sphere of influence (e.g., phos-
phorylation) and those with a more limited cellular purview (e.g.,
methylation). S-nitrosylation, like phosphorylation, is clearly
evolutionarily conserved and ubiquitous, affecting most, if not
all classes of proteins across all cellular compartments (8, 9, 16).
By contrast, other oxygen/redox-based modifications, including
hydroxylation and sulfenylation, have been identified to date with
specific classes of proteins and functions (2, 17). Here, we draw
parallels between S-nitrosylation and other important PTMs (e.g.,
phosphorylation, ubiquitinylation, acetylation) to provide a con-
ceptual framework for understanding the molecular machinery
that governs this fundamental biologic process (Figure 1).
In mammals, the principal sources of newly synthesized NO
are the three NOS isoforms (NOS1–3). Nitrate and nitrite may
also contribute to the NO reservoir (18, 19), particularly under
duress. The transfer of the NO moiety to cysteine thiols in target
proteins is carried out by peptide or protein nitrosylases, which
mediate either metal-to-Cys or Cys-to-Cys transfer. Metal-to-Cys
nitrosylases are proteins that transfer NO groups from transition
metals (e.g., Fe2+, Cu2+) to cysteine thiol. For example, mammalian
hemoglobin (Hb) undergoes auto-nitrosylation via intramolecu-
lar transfer of NO from heme iron (iron nitrosyl; HbFeNO) to a
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2013;123(1):101–110. doi:10.1172/JCI62854.
102 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
specific cysteine thiol in the β-globin chain (Cysβ93) (20, 21). Sim-
ilarly, transfer of metal-coordinated NO from cytochrome c (22)
or ceruloplasmin (23) is employed in the synthesis of S-nitrosog-
lutathione (GSNO). Cys-to-Cys nitrosylases (referred to herein as
transnitrosylases) are typically S-nitroso-proteins (SNO-proteins)
involved in Cys-to-Cys transfer of the NO group from donor to
acceptor protein (9, 24). The net nitrosylation (NO group trans-
fer) reaction is analogous to the group transfer reactions catalyzed
by ubiquitin ligases, palmitoyltransferases, or acetyltransferases
(Figure 1). Conversely, protein denitrosylation has been shown
to play a major role in decreasing cellular S-nitrosylation (25–29)
much the same way that protein phosphatases, deubiquitinases,
and deacetylases set cellular levels of their respective PTMs (9,
11). To date, two major enzymatic systems mediating protein
denitrosylation have been described (Figure 1): GSNO reductase
(GSNOR) (30, 31) and thioredoxin (Trx) (26, 32). The GSNOR
system (two GSNORs have been identified, but only one has been
studied in detail; ref. 25) denitrosylates GSNO, the major low-mo-
lecular-weight SNO in mammalian cells (33). Although GSNOR
acts directly only on GSNO, it governs protein S-nitrosylation by
influencing the cellular equilibrium that is maintained by trans-
nitrosylation reactions between SNO-proteins and GSNO (8, 24).
Studies of GSNOR-deficient organisms have demonstrated the
central role of GSNOR in SNO-based signal transduction and car-
diovascular function (refs. 27–29, 34, 35, and Figure 2). Members
of the TRX enzyme family (TRX1 and TRX2) mediate the deni-
trosylation of multiple SNO-protein substrates in the cytosol and
mitochondria (25, 36).
Studies over the past decade have established close parallels
between guiding principles for S-nitrosylation (SNO-based sig-
naling) and other PTM systems. Signals are propagated through
stimulus-coupled and spatiotemporally restricted interactions
within signaling complexes. The composition of SNO-based sig-
naling complexes includes NOSs (which provide the source of
NO), NO group donors including GSNO and other SNO-pro-
teins that can participate in transnitrosylation (propagation)
reactions, and denitrosylases (which curtail the signals) (Figure
1). By analogy to kinases, which align with substrates through
hydrophobic and ionic interactions, hydrophobic and charged
amino acids surrounding substrate cysteines (“SNO motifs”)
may provide for alignment with nitrosylases (8, 9, 37). Thus,
SNO-protein abundance reflects regulated equilibria between
S-nitrosylation and denitrosylation pathways, rather than rates
of NO production per se. The next section, which details the role
of SNO-based signaling in cardiovascular function and oxygen
homeostasis, illustrates the operation of these principles under
physiologic and pathophysiologic states.
SNO-based signaling regulates cardiovascular
performance and optimizes oxygen delivery
Systemic oxygen delivery is largely determined by microcirculatory
blood flow and, to a lesser extent, by blood O2 content, which is
a function of Hb O2 saturation (SaO2) and blood Hb concentra-
tion. SNO-based signals regulate each of these determinants and
therefore play an essential role in optimizing oxygen delivery. Fur-
thermore, S-nitrosylation allows for crosstalk between NO and
O2-sensing pathways to signal tissue oxygen levels and to effect
changes in O2 bioavailability (5). Here, we illustrate how the SNO-
based system exerts coordinated effects across multiple organs to
provide an integrated mechanism for sensing oxygen levels and
executing molecular responses to hypoxic cues (Figure 3). Inas-
much as oxygen sensing and delivery are perturbed in all cardio-
vascular disease, it follows that dysregulated SNO signaling con-
tributes to disease pathogenesis.
S-nitrosylation and myocardial performance during simulated hypoxia.
Signaling via the β2-adrenergic receptor (β2-AR) coordinates
hypoxic adaptation across multiple organs, including the lungs
(improved ventilation/perfusion matching) (38–41), skeletal mus-
cle (hypoxic vasodilation [HVD]) (41–43), and heart (augment-
ing contractility) (44). Through its influence on β2-AR signaling,
S-nitrosylation may regulate hypoxic responses. The G protein
receptor kinase 2 (GRK2), which mediates β2-AR desensitiza-
tion, undergoes agonist-coupled inhibitory S-nitrosylation in an
eNOS-dependent manner (28). Absent S-nitrosylation, cardiac
contractility (28) and peripheral vasodilation (45) decline during
maintained adrenergic stimulation (28). β-Arrestin2 (a scaffolding
protein that targets the β2-AR for internalization via endocytosis)
and dynamin (a core component of the clathrin-mediated endocy-
totic machinery) also undergo S-nitrosylation downstream of the
β2-AR, leading to enhanced receptor trafficking (34, 46). Although
the precise chain of molecular events is not fully understood, it is
known that GRK2, β-arrestin2, and dynamin are each complexed
with eNOS, and stimulation of the β2-AR leads to eNOS activa-
tion and subsequent S-nitrosylation of these proteins (34, 46,
47). Thus, coordinate S-nitrosylation events may serve to enable
β2-AR signaling by preventing desensitization and promoting
receptor recycling to facilitate oxygen transport (cardiac output)
and delivery (vasodilation). The abundance of SNO-GRK2 and
SNO-β-arrestin2 is diminished in eNOS–/– mice and enhanced in
GSNOR–/– mice (28, 34); eNOS and GSNOR thus promote S-ni-
trosylation and denitrosylation of these proteins, respectively,
through the intermediacy of GSNO. eNOS also binds dynamin
(46), but whether S-nitrosylation is mediated directly by a trans-
nitrosylase activity of NOS (48, 49) or via GSNO is not known.
GSNOR–/– mice further exhibit increases in cardiac output under
basal conditions, reflecting marked peripheral vasodilation (29) as
well perhaps as pronounced myocardial angiogenesis that results
from stimulatory S-nitrosylation of HIF-1α under normoxic con-
ditions (35). In addition, GSNO has direct inotropic effects (50).
GSNOR–/– mice also show constitutive increases in β2-AR abun-
dance (28), as is seen in ischemia. Collectively, then, enhanced S-ni-
trosylation in GSNOR–/– mice underlies hypoxia-mimetic changes
throughout much of the cardiovascular system (Figure 2). Sim-
Framework for PTMs and signaling. (A) Parallels between S-nitrosy-
lation and other PTMs (phosphorylation, ubiquitinylation, and acet-
ylation) highlight shared features of bona fide signaling systems.
S-nitrosylation is ubiquitous, reversible, and subject to enzymatic
control (by nitrosylases and denitrosylases), enabling spatiotempo-
ral and target specificity. The biochemistry of NO group transfer and
denitrosylation reactions is depicted. Note that auto–S-nitrosylation
is shown as an example of metal-to-Cys NO transfer (e.g., as occurs
from the Hb heme center to Cysβ93 to form SNO-Hb). However,
metal-to-Cys NO transfer between two different peptides or proteins
may also occur (e.g., between cytochrome C and glutathione to
form GSNO). (B) Denitrosylases. Two classes of denitrosylases are
shown, which comprise four enzymes in mammals, including two
GSNORs and two thioredoxins. GR, glutathione reductase; GSH,
reduced glutathione; GSNHOH, glutathione N-hydroxysulfenamide;
GSSG, oxidized glutathione; TrxR, Trx reductase.
104 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
ilar SNO-based regulation of β2-AR signaling in particular, and
hypoxia-mimetic responses more generally, are likely operative in
other tissues, including the airways and alveoli, kidney, blood, and
skeletal muscle, as discussed below (Figure 3).
S-nitrosylation regulates striated muscle performance. Oxygen con-
sumption can increase 5- to 10-fold in exercising humans over the
course of minutes (51). Accordingly, skeletal muscle has evolved
efficient mechanisms to rapidly adapt to large shifts in oxygen
demand. Just as β2-AR activation increases cardiac contractility
during hypoxia (44), β2-AR–coupled increases in bioactive NO are
also critical for compensatory vasodilation during mild to moder-
ate hypoxic exercise (52, 53). In a manner that parallels myocardial
β2-AR signaling, SNO-based signals inhibit β2-AR receptor desen-
sitization in the periphery to facilitate adrenergic responses (45).
Specifically, as local O2 tension (pO2) begins to fall during exercise,
NO signals to increase blood flow by potentiating β2-AR–coupled
HVD in working muscle (53) via a mechanism that likely involves
SNO-GRK2 and inhibition of β2-AR desensitization (28, 45). As
exercise intensity and tissue hypoxia increase, the source of bioac-
tive NO becomes less dependent upon β-adrenergic mechanisms
(53) and shifts to rbc-based SNO delivery (3, 4, 10, 54) (see SNO
signaling and the respiratory cycle below). Together, these two mecha-
nisms may support oxygen delivery across a broad range of exercise
intensity and duration.
A parallel mechanism operates in skeletal muscle via hypox-
ia-dependent, stimulatory S-nitrosylation of the skeletal muscle
ryanodine receptor (RYR1) (55–57), a key mediator of sarcoplas-
mic reticulum (SR) calcium release and excitation-contraction
coupling (ref. 58 and Figure 2). S-nitrosylation of RYR1 occurs
only during hypoxia, increases the open probability (PO) of the
channel, and potentiates SR calcium release (57, 59–62). This
pO2-dependent SNO-RyR1 formation is mediated by NO derived
from neuronal NOS (nNOS) complexed with RYR1 (63). In nor-
moxia, by contrast, RYR1 undergoes stimulatory oxidation of
redox-sensitive cysteine thiols (55, 60). The source of oxidizing
equivalents is the SR-resident NADPH oxidase 4, which colocal-
izes with RYR1 and produces H2O2 in proportion to ambient pO2,
thus functioning as a physiological oxygen sensor (55). Accord-
ingly, both S-oxidation and S-nitrosylation stimulate RYR1, but
at different physiologic pO2, reflecting conditions from resting
to exercising muscle (56). Alternatively stated, when pO2 falls
into the hypoxic range (as occurs in exercising muscle), regula-
tory thiols in RYR1 become reduced and protein conformation
is allosterically altered in a manner that favors S-nitrosylation
(57). Conversely, S-nitrosylation is superseded by S-oxidation in
the normoxic conformation assumed by RYR1 in resting mus-
cle. Thus, coordinate S-nitrosylation and oxidation of Cys thiols
within RYR1, which are favored during hypoxia and normoxia,
respectively, allow redox control over the range of physiological
pO2. From a pathophysiologic perspective, excessive S-nitrosyla-
tion of RYR1, which can occur in settings of nitrosative stress,
causes SR calcium leak and plays a maladaptive role in Duch-
enne muscular dystrophy (64), malignant hyperthermia (65),
and exercise intolerance (66). Inasmuch as skeletal muscle dys-
function is commonly present in chronic heart failure (67, 68),
perturbations in SNO-based signaling may underlie pathological
S-nitrosylation regulates cardiomyocyte sig-
naling at critical oxygen-responsive nodal
points. The central roles are highlighted for
the denitrosylase GSNOR in physiologic con-
trol of β2-AR signaling, SR calcium release,
HIF-1α responses, and mitochondrial func-
tion. S-nitrosylation reactions that have been
proven by genetic criteria to occur through
the intermediacy of GSNO include those tar-
geting GRK2, RYR2, and HIF-1α. Ligand-de-
pendent S-nitrosylation of GRK2, β-arrestin2,
and dynamin is eNOS dependent. eNOS is
complexed with GRK2, β-arrestin2, and
dynamin, as depicted. Coordinate titration of
S-nitrosylation (envisioned via receptor-cou-
pled activity of transnitrosylases and den-
itrosylases) across multiple steps in these
pathways determine net signaling responses.
The effects of GSNOR (from observations
in GSNOR–/– mice) manifest as increases
in cardiac output under basal conditions, a
persistent state of systemic vasodilation,
and protection from ischemic insult, estab-
lishing a central role for GSNO in cardiovas-
cular hemodynamics and oxygen delivery.
Effects of GSNOR on mitochondrial targets
are inferred from studies using GSNO. HRE,
hypoxia response element.
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
crosstalk between these two tissues. Targeting key SNO-proteins
common to both tissues (e.g., HDAC2) (69–72) may represent a
new therapeutic approach.
Like skeletal muscle RYR1, RYR2 in cardiac muscle also under-
goes nNOS-dependent stimulatory S-nitrosylation (refs. 60, 72,
and Figure 2). However, unlike skeletal muscle (where NO can
directly modify RyR1), the transfer of an NO group from nNOS
to RyR2 requires GSNO (i.e., transnitrosylation) (62). In addition,
SNO-RyR2 is abundant during normoxia and stimulates channel
activity independently of oxygen concentration (29, 62). However,
pO2 may retain an influence on RYR2 S-nitrosylation in the heart
through the β2-AR (see above; specifically, through β2-AR–cou-
pled RYR2 denitrosylation involving GSNOR) (29). The impor-
tance of this regulatory pathway has been established by study of
GSNOR–/– mice, which exhibit depressed β-adrenergic inotropic
responses, impaired β-agonist–induced denitrosylation of RYR2,
and pathological calcium leak (29). Similarly, nNOS–/– hearts have
diminished SNO-RyR2, excessive diastolic SR calcium leak, con-
tractile dysfunction, and susceptibility to arrhythmias (73, 74). We
note that while the β2-AR system and RYR2 serve as important
examples of SNO-based regulation, S-nitrosylation likely controls
other aspects of cardiac homeostasis in an oxygen-dependent man-
ner. For example, emergent evidence suggests S-nitrosylation of
mitochondrial proteins may protect against myocardial ischemia
(75), potentially via prevention of pathologic protein oxidation
and inhibition of apoptosis. Likewise, inhibitory S-nitrosylation
of mitochondrial complex I, in certain contexts, may play an adap-
tive role in mechanoenergetic coupling (ref. 76 and Figure 2). As
disruption of the SNO/redox balance in myocytes is a hallmark
of human heart failure (77), restoration of this equilibrium may
provide a fruitful approach to restoring cardiac performance.
SNO signaling and the respiratory cycle. Oxygen delivery is a func-
tion of blood O2 content and blood flow. The ability to augment
blood O2 content is markedly constrained, varying linearly with
Hb concentration and SaO2. Conversely, modulation of regional
blood flow, which is proportional to vessel radius to the fourth
power, has a dynamic range encompassing several orders of mag-
nitude. Thus, volume and distribution of local blood flow are the
principal determinants of tissue oxygen delivery (10). Mammals
have a robust capacity to autoregulate systemic blood flow to
dynamically couple local oxygen demand with oxygen delivery — a
process termed HVD. The central role of rbc in HVD was estab-
lished half a century ago by Guyton (78), who showed that HVD is
inversely proportional to SaO2 and recapitulated by rbc contain-
ing desaturated but not saturated Hb (78). By contrast, HVD is
independent of arterial pO2 (79–81). Guyton proposed that ery-
throcytes sequestered a vasoconstrictor in the lungs (78), and the
critical importance of SaO2 (as distinguished from pO2) was over-
looked at the time. Later, rbc were appreciated to liberate vasodi-
lator SNOs during hypoxia. Specifically, circulating rbc transport
bioactive NO to the peripheral microcirculation and release it in
proportion to locally declining oxygen gradients, in a process gov-
erned by changes in the quaternary conformation of Hb associated
with changes in O2 concentration (3, 4, 10, 54, 81). The molecu-
lar basis for this effect involves a critical cysteine within the Hb
β-chain (Cysβ93) that exhibits dynamic S-nitrosylation coupled
to Hb allostery (3, 4, 20, 81). Oxygen binding to the heme-iron of
Hb promotes a transition from T state (in deoxygenated blood) to
R state (in oxygenated blood), during which heme-bound NO is
transferred to the thiol group of Cysβ93. This auto–S-nitrosylated
cysteine remains hydrophobically buried in the R configuration
and thus devoid of vasodilatory activity. With the transition from
R to T state as erythrocytes travel to increasingly hypoxic regions
of the systemic microcirculation, the NO group on Cysβ93 is
exposed to solvent and is released via transnitrosylative transfer to
glutathione or thiols of the rbc membrane protein AE-1 to form
GSNO and SNO AE-1 (10, 54, 82). In this manner, oxygen itself
serves as a principal allosteric regulator that couples physiological
release of O2 and bioactive NO. Inasmuch as blood flow is the prin-
cipal determinant of O2 delivery, this remarkable function of Hb
represents an elegant means of dynamically matching vasomotor
tone with local oxygen gradients (refs. 5, 80, and Figure 2). The
physiologic importance of SNO-Hb in human hypoxic adaptation
was recently demonstrated in an observational study of healthy
subjects undergoing progressive high-altitude acclimatization in
the Himalayas (83). Blood concentrations of SNO-Hb progres-
sively increased with ascent and were independently correlated
with exercise capacity at high altitude.
SNO signaling and pulmonary gas exchange. NO bioactivity exerts
control over ventilation-perfusion (V/Q) matching through a
dual mechanism: (a) a permissive action on the β2-AR (28, 34) (see
above), which may improve V/Q matching by enhancing alveolar
clearance of fluid (84) and (b) the process of hypoxic pulmonary
vasoconstriction (HPV), whereby the pulmonary arterial microcir-
culation preferentially perfuses well-ventilated alveolar units (refs.
5, 10, and Figure 3). Physiological trapping of NO by erythrocytes
involves capture or inactivation of NO by hemes of Hb and serves
as an important contributor to HPV (85–88). NO trapping during
hypoxia may be facilitated by regulation of rbc membrane NO per-
meability via conformation-dependent binding of Hb to the rbc
transmembrane protein AE-1 (54). In normoxia, the rbc plasma
membrane constitutes a significant barrier to NO entry mediated
by tight association between the submembrane cytoskeleton and
the cytoplasmic domain of AE-1. In hypoxia, Hb binds AE-1 (bind-
ing is favored in the T state) and alters the submembrane cytoskel-
etal scaffold in a manner that increases NO permeability, thereby
facilitating NO trapping (54, 89–91). As basal vasodilatory tone in
the pulmonary arterial circulation is set by a relatively high level of
local NO production (from eNOS), NO trapping during hypoxia
provides an important braking mechanism on vasodilation and,
consequently, regional pulmonary blood flow. In other words,
avid NO trapping by less-well-oxygenated erythrocytes perfusing
less-well-ventilated lung units, and attenuation of NO trapping by
well-oxygenated erythrocytes perfusing well-ventilated lung units,
can facilitate V/Q matching (5). pO2-regulated NO permeability
may also facilitate unloading of bioactive NO from SNO-Hb in the
transition from R state to T state in the systemic microcirculation
to mediate HVD and in the lungs to mitigate excessive pulmonary
vasoconstriction (refs. 10, 53, and Figure 3).
Defects in NO processing by rbc are associated with multiple car-
diovascular diseases, including sepsis (excess levels of SNOs in rbc)
and pulmonary arterial hypertension (PAH) (decreased rbc SNO
levels). In sepsis, uncontrolled production of SNOs (27, 82, 92),
known as nitrosative stress, is believed to contribute to multiorgan
failure with resultant disruption of NO-based vascular autoreg-
ulation, particularly V/Q matching in the lung and shunting in
tissues (27, 82, 93). SNO content is increased 20-fold in rbc from
humans with septic shock and acute respiratory distress syndrome
(27, 82), and vasoactivity of these rbc is dysregulated in a murine
lung bioassay (82, 93). The link between pO2 and SNO delivery
106 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
that underlies HVD is also overwhelmed in sepsis (94, 95), possibly
due to promiscuous transnitrosylation of exofacial rbc membrane
proteins that results in pO2-independent vasodilation. Accumula-
tion of rbc SNOs and loss of allosteric control of SNO release (93,
95) may help explain the severely dysregulated blood flow pattern,
which is greatly enhanced but chaotic, in the septic microcircula-
tion. This mechanism is supported by studies of GSNOR–/– mice,
which exhibit increased rbc SNO content, decreased basal vascular
tone (29), and excessive mortality during experimental models of
sepsis (27). Conversely, rbc in patients with PAH and hypoxemia
have reduced levels of SNO-Hb but preserved rbc NO trapping,
which reduces microcirculatory NO bioavailability. This defect
may promote excessive pulmonary vasoconstriction in well-ven-
tilated alveolar units and impair blood flow to hypoxic tissues in
the systemic circulation (96). As aberrant vascular autoregulation
in both the pulmonary and systemic circulation are hallmarks of
advanced heart failure (97), defects in SNO processing by rbc in
heart failure (98) likely also contribute to disease progression.
SNOs regulate ventilation. During hypoxia, mammals increase total
lung ventilation by augmenting breathing rate (hypoxic ventilatory
drive) and tidal volume, both of which are regulated by SNO-based
signals. The central limb in this response is classically initiated by
hypoxia-sensing cells in the carotid body, which relay to nNOS-rich
neurons in the brainstem nucleus tractus solitarius (nTS). nNOS
activation in the nTS is critical for the hypoxic ventilatory response
and likely involves formation of low-molecular-weight SNOs (99,
100). Injection of low-molecular-weight SNOs, in particular GSNO
or S-nitroso-L-cysteine, into rodent nTS dramatically increases
minute ventilation in a manner that closely mimics the physiolog-
ical effects of hypoxia (101). Furthermore, nTS injection of a low-
mass fraction derived from deoxygenated blood, which contains
Hb-derived SNOs, reproduces the effects of GSNO, whereas a low-
mass fraction derived from oxygenated blood has no effect. Using
pharmacological and genetic approaches, it was also discovered
that enzymatic processing of GSNO to cysteinylGlySNO by γ-glu-
tamyl transpeptidase (γ-GT) was required for GSNO to augment
minute ventilation, and mice deficient in γ-GT were shown to have
a grossly abnormal ventilatory response to hypoxia (101). Together,
these data show that endogenous SNOs (likely those derived from
deoxygenated rbc) can act at the level of the nTS to mediate the ven-
tilatory response to hypoxia (101) and SNO-based signaling may
play a more pervasive role in controlling the drive to breathe, e.g., in
carotid body chemoreceptors. In addition to central effects, SNOs
can also augment ventilation via bronchodilation (102, 103) and
possibly via effects on contractile function of breathing muscles
(e.g., diaphragm, intercostals) (55, 56). These findings also suggest
a link between aberrancies in SNO signaling (104) and the dis-
rupted breathing pattern/mechanics (104, 105) that are frequently
observed in patients with heart failure.
SNOs and the cellular response to hypoxia versus anemia. Systemic
hypoxia (decreased pO2) and anemia (decreased rbc mass and blood
Hb concentration) both result in reduced oxygen delivery to tissues.
Although both stressors activate HIF-1α, a ubiquitous transcrip-
tional regulator of hypoxic adaptation (1), the mechanism of acti-
vation differs. In sustained hypoxia, arterial O2 delivery is reduced
due to low pO2 and SaO2. In this setting, inactivation of cellular
O2 sensors (in particular, O2-dependent prolyl hydroxylases) results
in the stabilization/accumulation of HIF-1α and enhanced tran-
scriptional activity. Canonical HIF-1α targets include erythropoi-
etin, VEGF, and GLUT1, which regulate erythropoiesis, angio-
genesis, and glucose utilization, respectively (1, 5). On the other
hand, anemia reduces blood O2 content through a reduction in Hb
concentration while preserving PaO2 and SaO2. Therefore, while
both hypoxia and anemia are associated with reduced O2 delivery,
the relative sparing of SaO2 during anemia fails to trigger classi-
cal O2-dependent HIF-1α signaling (5). Rather, HIF-1α activation
during anemia occurs because S-nitrosylation — generating SNO-
pVHL, SNO-PHD2, and SNO-HIF-1α — serves to activate HIF-1α
under normoxic conditions (106–109). In addition, GSNOR abun-
dance has been found to decrease in rodent models of acute ane-
mia, which could further augment SNO bioactivity and hypoxic
adaptation (110). Notably, endogenous SNOs are critical for ische-
mic cardioprotection in mouse models (35). Inasmuch as anemia
is a robust predictor of adverse outcomes in patients with ischemic
heart disease (111–113) and heart failure (114), these experimental
data strongly suggest that aberrant SNO-based signals can mediate
the detrimental effects of anemia in these clinical settings and sug-
gest new therapeutic approaches.
Therapeutic potential of modulating S-nitrosylation
Decreased levels and/or impaired bioavailability of SNO-mod-
ified proteins have been observed in a variety of disease states
characterized by tissue hypoxia (5), including congestive heart
failure (98). To the extent that NO donors (e.g., isosorbide dini-
trate, nitroglycerin) have beneficial effects in patients with heart
failure and ischemic heart disease (115–118), these therapies
may function, in part, via modulating S-nitrosylation of key
myocardial proteins (e.g., RYR2, HIF-1α, and β2-AR signaling
components including GRK2). More efficient methods of SNO
delivery to the myocardium (e.g., low-molecular-weight SNOs,
SNO-loaded rbc) may have clinical benefits in this setting (50).
In particular, rbc represent an attractive vehicle for SNO deliv-
ery: (a) rbc interface with endothelium to form the largest aggre-
gate intercellular interaction in the human body and therefore
can exert beneficial effects across multiple organs; (b) rbc deliver
both NO bioactivity and oxygen under the control of pO2-based
allostery and thereby facilitate autoregulatory control (3); and
(c) rbc provide unique access to the microcirculation that is dys-
functional in many cardiovascular diseases. The importance of
rbc-mediated SNO delivery is reflected in the clinical observa-
tion that anemia is strongly associated with adverse outcome
in patients with cardiovascular disease, even with modest lev-
els of blood loss (111–114). Paradoxically, liberal restoration of
plasma Hb concentration via standard clinical practices (i.e.,
transfusion of banked erythrocytes, administration of erythro-
SNO-based integration of oxygen utilization and homeostasis across
organ systems. SNO-based signals exert coordinated effects across
multiple organ systems to provide an integrated mechanism for sensing
oxygen levels and executing molecular responses to hypoxic cues. The
roles of SNOs in cardiac and skeletal muscle performance; respira-
tory cycle functions (vasodilator and vasoconstrictor function of Hb),
including HVD and alveolar ventilation and perfusion matching (rbc NO
permeability and trapping); the central ventilatory drive; and chronic
adaptation to hypoxemia and anemia (HIF-1α signaling) are depicted.
SNO-mediated activation of HIF-1α has been demonstrated in multiple
tissues, including the kidney (127). Specific details shown in the kidney
inset are derived, in part, from observations in the heart, brain, and
other tissues. EPO, erythropoietin.
108 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 1 January 2013
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A crucial role for GRK2 in regulation of endothelial
cell nitric oxide synthase function in portal hyper-
poietin) has failed to demonstrate improved tissue oxygenation
or clinical benefit in this setting, and multiple studies indicate
that this practice might actually be harmful (119–122). One con-
tributory explanation for this apparent paradox is that banked
blood is depleted of SNO and fails to deliver bioactive NO, and
can therefore exacerbate tissue hypoxia (123–125). Accordingly,
strategies aimed at restoring SNO levels in rbc (96, 126) may rep-
resent an attractive strategy to improve NO delivery, myocardial
performance, and tissue oxygenation.
O2 sensing and coupling mechanisms at the cellular, tissue, and
integrated system levels involve critical roles for S-nitrosyla-
tion–based signaling, a ubiquitous and evolutionarily conserved
mechanism for control of cellular function. In particular, SNOs
have been shown to regulate the activities of ion channels, recep-
tors, respiratory proteins, and enzymes that ultimately transduce
hypoxic signals into increased alveolar ventilation, matched alveo-
lar perfusion, augmentation of cardiac and skeletal muscle perfor-
mance, and enhanced microcirculatory blood flow. Dysregulation
of the cardiovascular system in heart failure can be understood
in these terms to represent global impairments of O2 delivery,
equated with reduced bioavailability of SNOs and their hypox-
ia-mimetic signaling function. Increased understanding of how
SNO-based signals execute control of myocardial, skeletal, vascu-
lar, and hematologic function will continue to pave the way for
new cardiovascular therapeutics.
This work was supported by the NIH (grants HL075443,
HL095463, and AI080633 to J.S. Stamler; grant HL086614 to S.M.
Haldar) and the Defense Advanced Research Projects Agency (grant
N66001-10-C-2015 to J.S. Stamler).
Address correspondence to: Jonathan S. Stamler, 2103 Cor-
nell Road, Room 5-542, Cleveland, Ohio 44106, USA. Phone:
216.368.5726; Fax: 216.368.2968; E-mail: jonathan.stamler@
case.edu. Or to: Saptarsi M. Haldar, 2103 Cornell Road, Room
4-525, Cleveland, Ohio 44106, USA. Phone: 216.368.3581; Fax:
216.368.0556; E-mail: firstname.lastname@example.org.
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