S-nitrosylation: integrator of cardiovascular performance and oxygen delivery (original) (raw)
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 concentration. SNO-based signals regulate each of these determinants and therefore play an essential role in optimizing oxygen delivery. Furthermore, _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). Inasmuch as oxygen sensing and delivery are perturbed in all cardiovascular disease, it follows that dysregulated SNO signaling contributes to disease pathogenesis.
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; respiratory 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.
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 muscle (hypoxic vasodilation [HVD]) (41–43), and heart (augmenting 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 desensitization, 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 endocytotic 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 activation 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_-nitrosylation 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 transnitrosylase 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 conditions (35). In addition, GSNO has direct inotropic effects (50). GSNOR–/– mice also show constitutive increases in β2-AR abundance (28), as is seen in ischemia. Collectively, then, enhanced _S_-nitrosylation in GSNOR–/– mice underlies hypoxia-mimetic changes throughout much of the cardiovascular system (Figure 2). Similar 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 consumption 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 moderate hypoxic exercise (52, 53). In a manner that parallels myocardial β2-AR signaling, SNO-based signals inhibit β2-AR receptor desensitization 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 bioactive 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 mechanisms may support oxygen delivery across a broad range of exercise intensity and duration.
A parallel mechanism operates in skeletal muscle via hypoxia-dependent, stimulatory _S_-nitrosylation of the skeletal muscle ryanodine receptor (RYR1) (55–57), a key mediator of sarcoplasmic 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 normoxia, 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 colocalizes with RYR1 and produces H2O2 in proportion to ambient pO2, thus functioning as a physiological oxygen sensor (55). Accordingly, 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), regulatory 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 muscle. 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_-nitrosylation of RYR1, which can occur in settings of nitrosative stress, causes SR calcium leak and plays a maladaptive role in Duchenne muscular dystrophy (64), malignant hyperthermia (65), and exercise intolerance (66). Inasmuch as skeletal muscle dysfunction is commonly present in chronic heart failure (67, 68), perturbations in SNO-based signaling may underlie pathological 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 undergoes 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–coupled RYR2 denitrosylation involving GSNOR) (29). The importance 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, contractile 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 manner. 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 adaptive 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 function 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 magnitude. 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 established half a century ago by Guyton (78), who showed that HVD is inversely proportional to SaO2 and recapitulated by rbc containing desaturated but not saturated Hb (78). By contrast, HVD is independent of arterial pO2 (79–81). Guyton proposed that erythrocytes sequestered a vasoconstrictor in the lungs (78), and the critical importance of SaO2 (as distinguished from pO2) was overlooked at the time. Later, rbc were appreciated to liberate vasodilator 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 governed by changes in the quaternary conformation of Hb associated with changes in O2 concentration (3, 4, 10, 54, 81). The molecular 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 principal 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 progressively 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 microcirculation 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 permeability 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 (binding is favored in the T state) and alters the submembrane cytoskeletal 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 cardiovascular 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 autoregulation, 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 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. Accumulation 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 microcirculation. 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-ventilated 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 physiological 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-glutamyl 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 ventilatory 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 disrupted 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 transcriptional regulator of hypoxic adaptation (1), the mechanism of activation 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 transcriptional activity. Canonical HIF-1α targets include erythropoietin, VEGF, and GLUT1, which regulate erythropoiesis, angiogenesis, 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 classical 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 abundance has been found to decrease in rodent models of acute anemia, which could further augment SNO bioactivity and hypoxic adaptation (110). Notably, endogenous SNOs are critical for ischemic 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 suggest new therapeutic approaches.