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Mechanistic Insights Into Nitrite-Induced Cardioprotection Using an Integrated Metabolomic/Proteomic Approach

Originally publishedhttps://doi.org/10.1161/CIRCRESAHA.108.187005Circulation Research. 2009;104:796–804

Abstract

Nitrite has recently emerged as an important bioactive molecule, capable of conferring cardioprotection and a variety of other benefits in the cardiovascular system and elsewhere. The mechanisms by which it accomplishes these functions remain largely unclear. To characterize the dose response and corresponding cardiac sequelae of transient systemic elevations of nitrite, we assessed the time course of oxidation/nitros(yl)ation, as well as the metabolomic, proteomic, and associated functional changes in rat hearts following acute exposure to nitrite in vivo. Transient systemic nitrite elevations resulted in: (1) rapid formation of nitroso and nitrosyl species; (2) moderate short-term changes in cardiac redox status; (3) a pronounced increase in selective manifestations of long-term oxidative stress as evidenced by cardiac ascorbate oxidation, persisting long after changes in nitrite-related metabolites had normalized; (4) lasting reductions in glutathione oxidation (GSSG/GSH) and remarkably concordant nitrite-induced cardioprotection, which both followed a complex dose–response profile; and (5) significant nitrite-induced protein modifications (including phosphorylation) revealed by mass spectrometry-based proteomic studies. Altered proteins included those involved in metabolism (eg, aldehyde dehydrogenase 2, ubiquinone biosynthesis protein CoQ9, lactate dehydrogenase B), redox regulation (eg, protein disulfide isomerase A3), contractile function (eg, filamin-C), and serine/threonine kinase signaling (eg, protein kinase A R1α, protein phosphatase 2A A R1-α). Thus, brief elevations in plasma nitrite trigger a concerted cardioprotective response characterized by persistent changes in cardiac metabolism, redox stress, and alterations in myocardial signaling. These findings help elucidate possible mechanisms of nitrite-induced cardioprotection and have implications for nitrite dosing in therapeutic regimens.

Although traditionally considered an inert byproduct of nitric oxide (NO) metabolism, the nitrite anion (NO2) is now recognized as an important bioactive molecule.1 It represents a source of NO, nitrosation and nitrosylation (nitros[yl]ation), yielding profound biological effects, especially in the context of hypoxia and ischemia.2,3 Nitrite has been shown to elicit NO-dependent vasodilatation,4,5 angiogenesis,6 and cardioprotection.7–11 Although the mechanisms of nitrite-induced cardioprotection remain elusive, proposals include alterations to mitochondrial respiration by direct modulation of the electron transport chain.7 Nitrite bioconversion to NO has varyingly been attributed to heme-dependent reductase activity,7,12,13 to reduction by other enzymes, including aldehyde dehydrogenase (ALDH)2 and xanthine oxidase,14 and to chemistries favored by low oxygen tension or pH.15 Cardioprotection by nitrite is manifest in isolated heart preparations,16 and low doses of nitrite prevent ischemia/reperfusion (I/R) injury in myocardial infarction.17 Thus, although great potential exists for nitrite-based therapeutics, a number of questions remain. (1) What is the dose–response relationship for nitrite-mediated cardioprotection? (2) What are the immediate (first-hour) and longer-term (24-hour) cardiac sequelae of elevations in plasma nitrite? (3) By what mechanisms does nitrite-induced cardioprotection occur? Using a metabolomic/proteomic approach, we demonstrate here that, following a brief systemic nitrite exposure in vivo, cardiac tissue experiences a rapid, dose-dependent wave of S-, N-, and heme nitros(yl)ation, followed by longer-term redox status alterations and cardiac proteomic changes, all of which may contribute to cardioprotection.

Materials and Methods

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

Nitrite Administration

Male Wistar rats (250 to 350 g) were given a single intraperitoneal injection of sodium nitrite dissolved in phosphate-buffered saline at an array of doses (0 [vehicle control], 0.1, 1.0, and 10 mg/kg body weight) with staggered administrations to allow tissue harvest at an identical chronobiological window.

Isolated Perfused Heart Preparation and Metabolomic and Redox Measurements

Blood and tissue harvest; quantitative analyses of nitroso/nitrosyl species, and NO oxidation products; measurements of ascorbate/dehydroascorbate and reduced/oxidized glutathione; and assessment of cardiac function of Langendorff-perfused hearts were performed as detailed in the online data supplement.

Isolation and Analysis of Cardiac Mitochondria and Cytoplasm

Preparation of mitochondria and cytosol and standard molecular biology/proteomic techniques used for differential display and protein identification, including 2D-PAGE analysis, Western blotting, in-gel digestion, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), and peptide mass fingerprinting, are described in detail in the online data supplement.

Phosphopeptide Enrichment and Liquid Chromatography–Mass Spectrometry

Following a 2-stage phosphopeptide enrichment, samples were analyzed by nanoflow high-performance liquid chromatography–coupled tandem MS as described in the online data supplement. Potential phosphopeptides were verified and assignments refined by manual interpretation of the original spectra.

Results

Acute Systemic Exposure to Nitrite Leads to Rapid, Transient Elevations in Tissue Nitrite and Nitros(yl)ation Levels and a Selective, Long-term Perturbation of Cardiac Redox Tone

We have demonstrated previously that systemically administered nitrite rapidly and dose dependently equilibrates across multiple organ systems, including the heart, brain, liver, kidney, and lung.18 Elevated tissue nitrite levels can then directly nitros(yl)ate proteins in an NO-independent manner by a cooperative action of tissue hemes and thiols.18 Using an experimental design tailored to investigate the effects of an acute systemic nitrite exposure on the heart (Figure I in the online data supplement), we now extended these findings by characterizing the dynamics of changing redox, metabolomic, proteomic, and functional parameters of the heart in response to various doses of nitrite, or vehicle (PBS), over an extended period after administration.

To follow cardiac sequelae after a systemic burst in nitrite levels, hearts from animals administered a bolus of nitrite (1 mg/kg) were analyzed over a 48-hour time course after application. At each time point (0, 2, 5, 10, and 30 minutes and 1, 3, 12, 24, 36, and 48 hours), cardiac nitrite levels were measured, together with other NO-related metabolites, including S-nitrosothiols, N-nitrosamines (S- and N-nitroso products), and heme-nitrosyl species. Additionally, as a measure of cardiac redox tone, the ascorbate oxidation status (the ratio of dehydroascorbate to ascorbic acid) was determined. Analysis of nitrite recovered from perfused heart tissue revealed a rapid increase in tissue nitrite levels within the first 5 minutes after administration (Figure 1). Cardiac nitrite levels peaked between 10 and 30 minutes (increasing from high-nanomolar baseline concentrations to low-micromolar peak levels), then after ≈1 hour, dropped sharply to baseline levels or below for the remainder of 48 hours. The initial, short-lived cardiac nitrite elevations were accompanied by rapid rises in S- and N-nitroso and heme-nitrosyl levels (1 to 3 orders-of-magnitude from baseline values), with S-nitroso species undergoing the largest relative changes (Figure 1). Similar results were observed in other compartments, including brain, liver, kidney, lung, plasma, and erythrocytes (not shown).

Figure 1. Major, long-term perturbation in cardiac redox tone following brief elevations in nitrite. Hearts from animals administered a bolus dose (1 mg/kg) of nitrite by intraperitoneal injection were analyzed for up to 2 days after application (0, 2, 5, 10, and 30 minutes and 1, 3, 12, 24, 36, and 48 hours). Cardiac nitrite levels were determined by ion chromatography, S-nitrosothiol (RS-NO), N-nitrosamine (RN-NO), and heme nitrosylation (heme-NO) levels, by gas-phase chemiluminescence, and ascorbate oxidation status (ie, dehydroascorbate [DHA]/ascorbic acid [AA]), by spectrophotometry. Absolute values were normalized as percentage change from baseline (indicated by the cyan plane). Left axis shows nitrite and nitros(yl)ation; right axis, ascorbate oxidation status. Asterisk indicates the brief elevation in ascorbate oxidation accompanying the spike in nitrite levels; arrow, the 24-hour value during subsequent protracted elevation in ascorbate oxidation (mean values of 3 animals/time point; error bars omitted for clarity). Errors were less than ±15%.

These transient spikes in cardiac nitrite and nitros(yl)ation were associated with a small increase in ascorbate oxidation (Figure 1, asterisk), which returned to near baseline within 1 hour, consistent with direct chemical interaction between ascorbate and nitrite.19 However, whereas nitrite and nitros(yl)ation levels in subsequent hours remained close to baseline, cardiac ascorbate oxidation continued to rise, peaking at 36 hours and remaining substantially elevated even 48 hours after nitrite administration. Total ascorbate levels changed little, remaining slightly elevated (15% to 21%) between 12 and 48 hours; no redox changes were observed in time-matched controls (not shown). Because this progressive and protracted elevation in ascorbate oxidation occurred long after return of nitrite to baseline, it is unlikely to have been caused through direct oxidation by nitrite but rather through a cascade of events elicited by the initial nitrite elevation.

Dose–Response Relationship of Nitrite-Induced Perturbations of Cardiac Redox Tone Is Complex

We sought to characterize in further detail these persisting, nitrite-triggered redox perturbations by examining their dose–response relationship. Our array of nitrite doses (0.1, 1.0, and 10 mg/kg body weight) was chosen to produce physiologically/pharmacologically relevant increases in plasma concentrations (from nanomolar baseline levels to transient low-micromolar treatment levels), and concomitant tissue elevations of nitrite, as previously shown.18 Cardiac ascorbate and glutathione oxidation status was analyzed 24 hours after nitrite administration (when cardiac nitrite and nitros[yl]ation had long returned to baseline but cardiac ascorbate oxidation was markedly enhanced; see Figure 1, arrow). Interestingly, nitrite-induced ascorbate was elevated independent of the dose applied (Figure 2A, left). Glutathione oxidation was also perturbed following nitrite administration (Figure 2A, right); however, in contrast to ascorbate, it showed a complex (trimodal) dose–response relationship, decreasing in hearts from the lowest (0.1 mg/kg) and highest (10 mg/kg) nitrite doses, with essentially no change in hearts from the intermediate (1.0 mg/kg) dose. Like ascorbate, total tissue glutathione concentration increased slightly (14±4%). The observed perturbations in cardiac redox status were specific to distinct cellular redox couples and not associated with global oxidative stress, because total protein carbonyl levels were unchanged 24 hours after administration at all nitrite doses (not shown).

Figure 2. Complex dose response of nitrite-induced, long-term perturbations in cardiac redox tone and preconditioning. A, Ascorbate oxidation increases uniformly in response to nitrite, whereas glutathione oxidation is complex in its nitrite dose response. Hearts from animals administered a nitrite bolus in an array of doses (0 [control], 0.1, 1.0, 10 mg/kg) were analyzed after 24 hours: ascorbate oxidation status (DHA/AA) (left) and glutathione oxidation status (GSSG/GSH) (right) were determined by spectrophotometry (means±SEM; n=3). B, Dose-dependent cardioprotective or detrimental preconditioning by nitrite. Hearts, isolated from animals 24 hours after a bolus administration of nitrite in our array of doses, were perfused in Langendorff mode, subjected ex vivo to global ischemia (15 minutes) followed by reperfusion (30 minutes), and monitored for recovery of contractile function by measurements of diastolic pressure (EDP), rate-pressure product (RPP), and other hemodynamic parameters (mean values±SEM; n=4 to 5).

Only Low and High, but Not Intermediate, Doses of Nitrite Are Cardioprotective

To evaluate the impact of these nitrite-induced cardiac tissue alterations on the functional properties of the heart, we subjected isolated perfused hearts from nitrite-treated animals (24 hours after administration) to 0-flow ischemia (15 minutes) and assessed their recovery during reperfusion (30 minutes). Hearts from low (0.1 mg/kg) and high (10 mg/kg) doses of nitrite showed significant improvement in cardiac contractile recovery, as reflected in decreased end diastolic pressure (Figure 2B, left) and increased rate pressure product (Figure 2B, right) relative to controls. In contrast, hearts from an intermediate nitrite dose (1.0 mg/kg) did not differ from controls. Thus, cardiac preconditioning, as reflected by improved function after I/R, was afforded by prior nitrite exposure in a complex (trimodal) dose-dependent fashion that was similar in pattern to the glutathione oxidation status.

Nitrite Induces Alterations to Cardiac Mitochondria-Associated Proteins, PDIA3, COQ9, and ALDH2

To explore the mechanisms of nitrite-induced cardiac preconditioning, we characterized cardiac proteomic alterations using a global differential 2D-PAGE–based proteomic analysis. We analyzed hearts 24 hours after exposure to an array of nitrite doses, using a strategy of partitioning heart tissue into mitochondria and cytoplasm through differential centrifugation. Although largely uniform between treatment groups (as evidenced by the overall similarity of the stained gels; see Figure 3A), the cardiac mitochondrial proteome displayed several distinct protein changes in response to nitrite treatment: dose-dependent changes were consistently evident in 3 series of protein spots (series 1: Figure 3B, i, left, spots a through d; series 2: Figure 3B, ii, left, spots a through d; series 3: Figure 3B, iii, left, spots a through e; for additional clarity, see the movies in the online data supplement showing dose-dependent changes in each series and supplemental Figure II) within the pI 4.5 to 6.5 region of the gels, in trains suggestive of changes in pI-altering posttranslational modifications (PTMs). The patterns of spot changes were complex. For example, whereas the intensities of the 2 most acidic spots in series 1 (Figure 3B, i, left, spots a and b) followed roughly the same dose dependence as glutathione oxidation, the intensities of the 2 most basic spots in that same series (Figure 3B, i, left, spots c and d) followed the dose-dependence of ascorbate oxidation. Interestingly, the patterns of spot changes were distinct for each series. This suggests that there may be multiple mechanisms linking these proteomic changes to other nitrite-induced changes in the heart.

Figure 3. Nitrite-induced alterations to cardiac mitochondrial-associated proteins PDIA3, COQ9, and ALDH2 revealed by differential 2D-PAGE and MS analyses. Hearts from animals administered a nitrite bolus at 3 different doses were isolated 24 hours after administration, homogenized, subjected to differential centrifugation to isolate mitochondria and postmitochondrial cytoplasmic supernatant, followed by 2D-PAGE and silver staining. All samples were pooled from 3 animals per dose; gels are representative of a minimum of 3 replicates. A, Purified cardiac mitochondria, subjected to 2D-PAGE over the pI range 3 to 10 and molecular mass range 250 to 10 kDa, as indicated. B, Enlargements across treatment groups of 3 regions of 2D gels run with the same material as in A over the pI range 4 to 7 and molecular mass range 250 to 10 kDa (enlargements, left gels; full gels, supplemental Figure II). Members of spot series with nitrite-dependant alterations are circled in red. i, Series 1, train of 4 spots, labeled a through d, at approximately pI 6 and molecular mass of 60 kDa. ii, Series 2, train of 4 spots, labeled a through d, at approximately pI 5 and molecular mass 30 kDa. iii, Series 3, train of 5 spots, labeled a through e, at approximately pI 6.3 and molecular mass 57 kDa. A summary of changes observed in series 1 to 3 is found in supplemental Table II and in the supplemental movie files. The same spots from equivalent, preparative 2D-PAGE gels stained with Coomassie blue were subjected to in-gel digestion and MALDI-TOF MS (right graphs; spectra shown for 1 spot in each series over the approximate range m/z 700 to 3000). Peak lists were submitted to Mascot, for peptide mass fingerprint analyses against the rodent proteome. Spot series 1, 2, and 3 were determined to consist of isoforms of protein disulfide isomerase A3 (PDIA3; Mascot score, 221; expected value, 1.6×10−18), ubiquinone biosynthesis protein CoQ9 (CoQ9; Mascot score, 79; expected value, 8.6×10−5), and aldehyde dehydrogenase 2 (ALDH2; Mascot score, 205; expected value 6.2×10−17), respectively. Prominent peptide ions are labeled in the spectra with their observed m/z values and corresponding amino acid intervals (bold). T indicates trypsin autolysis peptide.

To identify these protein spots, preparative amounts of purified mitochondria were subjected to 2D-PAGE analysis and Coomassie staining. A large-scale peptide mass fingerprint (PMF) survey of the protein spots across the gels was undertaken as a road map for protein identifications by in-gel digestion, MALDI-TOF MS, and PMF analyses (see supplemental Table I). When the spots from series 1, 2, and 3 were subjected to these analyses (Figure 3B, i through iii, right graphs, from representative spots within each series), PMF results revealed unambiguous matches to isoforms of protein disulfide isomerase (PDI)A3, ubiquinone biosynthesis protein CoQ9 (COQ9), and ALDH2, respectively.

Nitrite Induces Changes in the Nitration and Phosphorylation of Cardiac Myofilament, Energetic, and Signaling Proteins

In addition to differential 2D-PAGE analyses of mitochondria, we performed similar analyses on the postmitochondrial cytoplasm from hearts of nitrite-treated animals. These 2D gels (supplemental Figure III) revealed distinct nitrite-dependent alterations in spot intensity in a spot cluster corresponding to myosin light chain protein (MLC)1 isoforms (Figure 4A, spots a through g), and a train of spots corresponding to cardiac actin, as accentuated by the dye, Pro-Q Diamond, which stains protein phospho-isoforms and other acidic species (Figure 4B, spots a through e). Importantly, the MLC1 observations are consistent with previous reports suggesting changes in MLC1 phosphorylation occur in the context of cardioprotection.20,21 These observations motivated a more detailed characterization of stable nitrite-induced PTMs.

Figure 4. Alterations to other cardiac proteins, including myofilament, energetic, and signaling proteins, induced by brief nitrite exposure. Postmitochondrial cytoplasm, purified by differential centrifugation from the cardiac tissue of animals administered a nitrite bolus at different doses, was analyzed by 2D-PAGE over the pI range 4 to 7 and molecular mass range 250 to 10 kDa. A, Enlargements of the MLC1 region displaying nitrite-dependent changes in isoforms (spots a through g, circled in red). B, Equivalent gels, stained with the phosphoprotein-sensitive fluorescent dye Pro-Q Diamond; shown are enlargements of the actin region (actin, circled) displaying nitrite-dependent changes in actin migrational isoforms (spots labeled a through e). C, Protein nitration changes resulting from acute nitrite exposure. Equivalent amounts of cardiac tissue homogenates from nitrite-treated animals were analyzed by IEF (over the pI range 5 to 8), and then strips were placed side-by-side atop single second dimension gels and subjected to SDS-PAGE, followed by Western blotting using anti-nitrotyrosine anti-sera. Shown is a 2D Western blot over the molecular mass range 80 to 20 kDa. Nitrated protein spots a through h (a through e, changing with nitrite dose, circled in red): lactate dehydrogenase B (LDHB) (a); dehydrolipamide S-acetyl transferase (PDC-E2) (b); actin (c); unidentified cardiac proteins (d and e); F1 ATPase β subunit (f); GRP78 (g); MLC isoforms (h).

In the context of oxidative stress, the capacity of nitrite to produce nitrogen dioxide may be accompanied by posttranslational protein nitration. To assess whether brief nitrite exposure also leads to the nitration of cardiac proteins, we conducted 2D-PAGE and anti-nitrotyrosine Western analysis of whole heart homogenates 24 hours after nitrite treatment (Figure 4C). To control for blotting variability, isoelectric focusing strips from each nitrite-dose sample were placed side-by-side atop single 2D gels and subjected as one to SDS-PAGE and Western blotting. Whereas multiple proteins were found to be post-translationally modified by nitrite, nitration was not indiscriminate: the staining of some protein spots remained unchanged with increasing nitrite exposure, such as those corresponding to F1-ATPase β subunit and GRP78, both known targets of tyrosine nitration (spots f and g, Figure 4C). In contrast, qualitative changes in nitration were observed for lactate dehydrogenase B (LDHB) (spot a, Figure 4C), dehydrolipamide S-acetyl transferase (PDC-E2, a component of the pyruvate dehydrogenase complex) (spot b, Figure 4C), actin (spot c, Figure 4C), and other cardiac proteins (including spots d and e, Figure 4C). Whereas LDHB nitration appeared to increase with increasing nitrite dose, the nitration of other proteins showed a more complex behavior. Notably, several cardiac proteins were observed as nitrated under basal conditions (in controls). A summary of the nitrite-dependent changes in normalized relative intensity of each gel spot shown in Figures 3B and 4A through 4C is contained in supplemental Table II.

As the spot patterns we observed in several of our 2D gel analyses (eg, Figure 3B, series 1 through 3) resemble those resulting from phosphorylation, we investigated the cardiac phosphoproteome for alterations in response to nitrite administration. The MALDI-TOF mass spectra of peptides from PDIA3, COQ9, and ALDH2 acidic gel spots (as in Figure 3B, i through iii, spots a and b) contained ions corresponding to phosphopeptides from these proteins. For example, we detected an ion corresponding to the ALDH2 phosphopeptide, 431 to 438 (Figure 5A), bearing phosphorylation on Thr431, which has recently been reported as a functionally active phosphosite.22 Also evident in the MALDI-TOF mass spectra were several other potential phosphopeptides (data not shown) that correspond to known and novel phosphorylation sites, whose definitive assignments await confirmation and further characterization by other MS techniques.

Figure 5. Phosphorylated and nonphosphorylated forms of ALDH2 peptide 431TIEEVVGR438 detected by MS analyses. Ions are labeled with their detected m/z values. The phosphorylated Thr431 residue is indicated on the sequences with a P inside a circle. A, MALDI-TOF mass spectrum of peptides from a 2D-PAGE spot assigned by PMF to ALDH2 from cardiac mitochondria of nitrite-treated animals (as in Figure 3B, iii, spot b), shown over the range m/z 900 to 990. Labeled are the unmodified ALDH2 peptide, 431 to 438, the phosphorylated species of the same peptide (the 80 u shift corresponds to phosphate addition), and another ALDH2 peptide, 150 to 157. B, ESI mass spectra recorded during LC-MS of peptides isolated, through in-solution digestion and phosphopeptide enrichment, directly from pooled heart homogenates of nitrite-treated animals. Shown are regions of the mass spectra containing the [M +2H]2+ molecular ions assigned to the unmodified (left; displaying the range m/z 451.0 to 453.0) and the phosphorylated (right; displaying the range m/z 491.0 to 493.0) species of the ALDH2 peptide, 431 to 438.

Recently, it has become appreciated that gel-based proteomics can lead to chemical artifacts resembling protein phosphorylation.23 To avoid potential artifactual chemical modifications associated with peptides extracted from gels, tryptic peptides were produced directly from pooled heart homogenates of control animals and pooled homogenates from nitrite-treated animals, by in-solution endoproteinase digestion. Peptides were then subjected to a 2-stage phosphopeptide enrichment procedure, involving calcium phosphate precipitation and titanium dioxide purification. Samples were then subjected to characterization by liquid chromatography (LC)-MS and tandem MS (MS/MS). LC-MS analysis of the samples from nitrite-treated animals detected the phosphorylated and nonphosphorylated forms of ALDH2 peptide 431 to 438 (Figure 5B) that were also found in the gel-derived samples. MS/MS spectra of this and other potential phosphopeptides from ALDH2, PDIA3, or COQ9 were not acquired during data-driven MS/MS peak selection because of their low relative abundances. However, LC-MS/MS analysis did reveal phosphopeptides that were abundantly present in the pooled nitrite-treated samples (Figure 6) but below detection in the control samples. Interpretation of MS/MS spectra led to identification of these phosphopeptides as belonging to filamin (FLN)C (Figure 6A) and to subunits of cAMP-dependent protein kinase A (PKA) (Figure 6B) and serine/threonine protein phosphatase 2A (PP2A) (Figure 6C). Because numerous diagnostic fragment ions were present in each spectrum, it was possible to assign unambiguously sites of phosphorylation to residues Ser2234 of FLNC, Ser83 of PKA regulatory subunit 1-α (PKA R1α, KAP0), and Ser9 of PP2A regulatory subunit A-α (PP2A A R1-α, 2AAA). These 3 proteins were not identified in our 2D-PAGE and PMF analyses, possibly because of the large size of FLNC, which likely renders it incompatible with our 2D-PAGE methodologies, and the potential comigration of PKA R1α and PP2A A R1-α with other, much higher–abundance proteins (their molecular masses and theoretical pI values place them within regions of our 2D gels with the highest spot densities). Although numerous additional phosphosites on other proteins were characterized by these LC-MS/MS analyses (such as Ser283 of tropomyosin α chain 1, TPM1, and Ser663 of sarcoplasmic/endoplasmic reticulum calcium ATPase 2, SERCA2, AT2A2; not shown), their potential variation with nitrite treatment was less clear. Further studies, such MS analyses incorporating stable-isotope labeled standards, will be necessary to quantitate potentially more subtle occupancy changes at these other sites and to determine their relevance to nitrite treatment.

Figure 6. Phosphospecies of FLNC, PKA, and PP2A subunits detected differentially in heart homogenates of nitrite-treated animals. Tandem mass spectra from 3 phosphopeptides present differentially in homogenates of nitrite-treated animals. A, FLNC phosphopeptide corresponding to amino acids 2232 to 2240, containing phosphorylated Ser2234 ([M +2H]2+m/z 509.2418). B, PKA regulatory subunit 1-α (KAP0) phosphopeptide, corresponding to amino acids 75 to 92, containing phosphorylated Ser83 ([M +2H]2+m/z 1028.9838). C, PP2A regulatory subunit A-α (2AAA) phosphopeptide, corresponding to amino acids 2 to 28, containing phosphorylated Ser9 ([M +4H]4+m/z 763.6262). Precursor ion spectra are shown as inset into product ion spectra (both over indicated m/z ranges). Precursor ions and prominent fragment ions are labeled with their observed m/z values; where applicable, their corresponding b/y ion designations, charge states, and phosphoric acid losses (−98) are labeled (bold). A summary of fragment ion data, including less abundant fragment ions detected, is indicated on the phosphopeptide sequence above each spectrum. In each case, the phosphorylation site (indicated with a P inside a circle) is identified unambiguously by numerous prominent diagnostic b and/or y ions.

Discussion

Expanding on previous studies that have highlighted the ability of nitrite to confer cardioprotection,7–12 this study provides novel insights into the dose–response relationship, potential mechanisms, and consequences of nitrite-induced delayed cardioprotection. Our findings suggest that exposure to systemic nitrite elevations, such as that which occurs physiologically (eg, with exercise24), results in rapid uptake and metabolism of nitrite by the heart, with associated transient nitros(yl)ation of heart tissue. Despite the transience of elevated tissue nitrite and nitros(yl)ation levels, this exposure induces a protracted effect on cardiac redox status: it leads to an increase in ascorbate oxidation (DHA/AA) over 48 hours and a reduction of glutathione oxidation (GSSG/GSH) in a complex nitrite dose-dependent manner. The importance of these changes is underlined by the compelling correspondence between the patterns of GSSG/GSH changes and cardiac preconditioning, as manifested by improved recovery of contractile function following I/R. We speculate that these redox and cardioprotective changes reflect the influence of nitrite on the bioavailability, cross-talk, and signaling of NO and reactive oxygen species (ROS) (Figure 7). Both the levels and ratios of these species, and their interaction to form peroxynitrite (ONOO), appear a critical determinant of the preconditioning response.25,26

Figure 7. Schematic representation of the mechanisms underlying nitrite-induced late preconditioning. Nitrite, both directly and through NO release, induces the release of free radicals, including ROS. One mechanism of ROS increase includes NO-induced activation of cyclic GMP (cGMP), in turn activating a redox-sensitive protein kinase G (PKG), which opens mitochondrial KATP channels, enhancing local ROS production. NO, ROS, and peroxynitrite (OONO), when produced in an optimal balance resulting from effective nitrite concentrations and incipient conditions (eg, cellular redox status), lead to activation of cellular kinases (eg, PKA, PKCδ, and -ε). Nitrite-derived NO can also act via transient modification of electron transport chain components and S-nitrosation of proteins involved in regulation of mitochondrial energetics. Ultimately, these lead to cardioprotection mediated through the interplay of transcription factor signaling, proteomic, metabolic, and redox changes and alterations in contractile function.

Concurrent with these redox and functional changes, we further demonstrate that the heart undergoes significant proteomic alterations in response to brief nitrite elevations.

Affected proteins include: myofilament-related and energy-consuming proteins (eg, FLNC, actin, sarcomeric MLC), energy-producing proteins (eg, LDHB, PDC-E2), serine/threonine kinase signaling proteins (eg, PKA and PP2A) and proteins modifying mitochondrial function and redox stress (eg, PDIA3, COQ9, and ALDH2). Modifications of PDIA3, COQ9, and ALDH2 protein isoforms that, at least in part, are attributable to phosphorylation, have been identified here for the first time in the context of nitrite exposure. Moreover, LC-MS analysis of pooled nitrite-treated versus control animals demonstrated specific nitrite-induced protein phosphorylation of ALDH2, FLNC, PKA, and PP2A. Many of these proteins, and the pathways to which they belong, have recently been identified as critical mediators of cardioprotection arising from diverse preconditioning stimuli, underlining the robustness of conserved preconditioning pathways (Figure 7).20–22,27,28 For example, the phosphorylation of ALDH2 at Thr431 (Figure 5) is also regulated by PKCε and has been linked to cardioprotective increases in ALDH2 enzymatic activity.22,28

In accordance with other similar studies,26,27,29 novel and important, hypothesis-generating information has been gained from relatively unbiased proteomic investigations. In contrast to most cardioprotection proteomic analyses performed to date using isolated myocytes explanted 60 minutes after treatment, our studies were performed on whole heart preparations and uniquely studied the metabolome/proteome long after treatment (at 24 hours), yielding insights into potential mediators and pathways of late cardioprotection. Despite our extensive 2D-PAGE approach using cell compartment–specific proteomics, the protein changes we identified are by no means comprehensive. For technical reasons (eg, challenges of 2D-PAGE characterization of proteins with wide ranges in abundance and with limited solubility in 2D-PAGE buffers, etc), such proteomic approaches provide only a limited window into the biological pathways involved.20 Thus, many observations reported herein represent only the first stage of full structural and functional determination of nitrite-dependent protein alterations. Future studies are necessary to quantitate the nitrite dose response of the phosphosites we have described and to probe deeper into the proteome to investigate the quantitative changes, stoichiometry, and functional consequences of putative phosphorylation sites and their relationship to cardioprotection. Additionally, other novel and functionally active PTMs20,21,27,29,30 (eg, prolyl hydroxylation, nitration, oxidation, N-acetylglucosamination) should be investigated by methodologies tailored to their enrichment and characterization. As with other classes of proteins, PTM changes of myofilament proteins will require comprehensive characterization and functional assays to determine their contribution to cardioprotection.31,32 Additionally, although beyond the scope of this largely proteomic study, it will be necessary to test the hypothesis that the elaboration of NO and ROS are required for nitrite-induced delayed cardioprotection. This may be accomplished through administration of nitrite concurrently with antioxidants followed by a systematic assessment of its downstream effects, including its impact on recovery from I/R.26,33,34

Conclusions

The present study confirms that nitrite is an effective cardioprotective agent that can facilitate persistent protection against myocardial ischemia and reperfusion. We propose for the first time that: (1) the protective influence of nitrite conforms to a complex dose-response relationship (only low and high doses are effective); (2) these doses induce a complex oxidant stress pattern with oxidized ascorbate and reduced glutathione corresponding to a functionally protected heart, and (3) the changes in redox status not only correlate functionally with protection but also correspond to a complex pattern of putatively protective protein alterations, including PTMs (phosphorylation and nitration), of proteins involved in serine/threonine kinase signaling, protection from oxidative stress, potential bioconversion of nitrite to NO, and the machinery of cellular metabolism. Although further studies will be needed to fully delineate the mechanisms of nitrite-induced cardioprotection, this study informs ongoing basic and translational studies by highlighting the importance of the dose–effect relationship for nitrite and the broad array of downstream targets possibly involved in its cardioprotective efficacy. Finally, seen in the context of the transient increases in circulating nitrite levels known to occur following physical exercise24 and ingestion of nitrite/nitrate-rich foods,3 our results may illuminate the role of these lifestyle-related factors in cardiovascular health and disease.

*Both authors contributed equally to this work.

Original received September 5, 2008; revision received February 4, 2009; accepted February 10, 2009.

Sources of Funding

This work was supported by NIH grants P41 RR10888 and S10 RR15942 and National Heart, Lung, and Blood Institute contract N01HV28178 (to C.E.C.); NIH grant S10 RR20946 (shared MS instrumentation grant to J. Zaia, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA); and NIH grants R01 HL69029 and R21 DA020644 and a Medical Research Council Strategic Appointment Award (to M.F.).

Disclosures

None.

Footnotes

Correspondence to Martin Feelisch, PhD, Professor of Experimental Medicine & Integrative Biology, Warwick Medical School, The University of Warwick, Gibbet Hill Rd, Coventry, CV4 7AL, United Kingdom. E-mail

References

  • 1 Mazzone M, Carmeliet P. Drug discovery: a lifeline for suffocating tissues. Nature. 2008; 453: 1194–1195.CrossrefMedlineGoogle Scholar
  • 2 Zweier JL, Wang P, Samouilov A, Kuppusamy P. Enzyme-independent formation of nitric oxide in biological tissues. Nat Med. 1995; 1: 804–809.CrossrefMedlineGoogle Scholar
  • 3 Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008; 7: 156–167.CrossrefMedlineGoogle Scholar
  • 4 Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO III, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003; 9: 1498–1505.CrossrefMedlineGoogle Scholar
  • 5 Maher AR, Milsom AB, Gunaruwan P, Abozguia K, Ahmed I, Weaver RA, Thomas P, Ashrafian H, Born GV, James PE, Frenneaux MP. Hypoxic modulation of exogenous nitrite-induced vasodilation in humans. Circulation. 2008; 117: 670–677.LinkGoogle Scholar
  • 6 Kumar D, Branch BG, Pattillo CB, Hood J, Thoma S, Simpson S, Illum S, Arora N, Chidlow JH Jr, Langston W, Teng X, Lefer DJ, Patel RP, Kevil CG. Chronic sodium nitrite therapy augments ischemia-induced angiogenesis and arteriogenesis. Proc Natl Acad Sci U S A. 2008; 105: 7540–7545.CrossrefMedlineGoogle Scholar
  • 7 Shiva S, Sack MN, Greer JJ, Duranski M, Ringwood LA, Burwell L, Wang X, MacArthur PH, Shoja A, Raghavachari N, Calvert JW, Brookes PS, Lefer DJ, Gladwin MT. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J Exp Med. 2007; 204: 2089–2102.CrossrefMedlineGoogle Scholar
  • 8 Gonzalez FM, Shiva S, Vincent PS, Ringwood LA, Hsu LY, Hon YY, Aletras AH, Cannon RO III, Gladwin MT, Arai AE. Nitrite anion provides potent cytoprotective and antiapoptotic effects as adjunctive therapy to reperfusion for acute myocardial infarction. Circulation. 2008; 117: 2986–2994.LinkGoogle Scholar
  • 9 Bryan NS, Calvert JW, Gundewar S, Lefer DJ. Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice. Free Radic Biol Med. 2008; 45: 468–474.CrossrefMedlineGoogle Scholar
  • 10 Bryan NS, Calvert JW, Elrod JW, Gundewar S, Ji SY, Lefer DJ. Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2007; 104: 19144–19149.CrossrefMedlineGoogle Scholar
  • 11 Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, Patel RP, Yet SF, Wang X, Kevil CG, Gladwin MT, Lefer DJ. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest. 2005; 115: 1232–1240.CrossrefMedlineGoogle Scholar
  • 12 Shiva S, Huang Z, Grubina R, Sun J, Ringwood LA, MacArthur PH, Xu X, Murphy E, Darley-Usmar VM, Gladwin MT. Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration. Circ Res. 2007; 100: 654–661.LinkGoogle Scholar
  • 13 Gladwin MT, Kim-Shapiro DB. The functional nitrite reductase activity of the heme-globins. Blood. 2008.Google Scholar
  • 14 Li H, Cui H, Kundu TK, Alzawahra W, Zweier JL. Nitric oxide production from nitrite occurs primarily in tissues not in the blood: critical role of xanthine oxidase and aldehyde oxidase. J Biol Chem. 2008; 283: 17855–17863.CrossrefMedlineGoogle Scholar
  • 15 Benjamin N, O'Driscoll F, Dougall H, Duncan C, Smith L, Golden M, McKenzie H. Stomach NO synthesis. Nature. 1994; 368: 502.Google Scholar
  • 16 Webb A, Bond R, McLean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci U S A. 2004; 101: 13683–13688.CrossrefMedlineGoogle Scholar
  • 17 Gladwin MT, Schechter AN, Kim-Shapiro DB, Patel RP, Hogg N, Shiva S, Cannon RO III, Kelm M, Wink DA, Espey MG, Oldfield EH, Pluta RM, Freeman BA, Lancaster JR Jr, Feelisch M, Lundberg JO. The emerging biology of the nitrite anion. Nat Chem Biol. 2005; 1: 308–314.CrossrefMedlineGoogle Scholar
  • 18 Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Milsom AB, Rassaf T, Maloney RE, Bharti A, Rodriguez J, Feelisch M. Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat Chem Biol. 2005; 1: 290–297.CrossrefMedlineGoogle Scholar
  • 19 Dahn H, Loewe L, Bunton CA. Über die Oxydation von Ascorbinsäure durch salpetrige Säure. VI. Übersicht und Diskussion der Ergebnisse. Helv Chim Acta. 1960; 43: 320–333.CrossrefGoogle Scholar
  • 20 Arrell DK, Elliott ST, Kane LA, Guo Y, Ko YH, Pedersen PL, Robinson J, Murata M, Murphy AM, Marban E, Van Eyk JE. Proteomic analysis of pharmacological preconditioning: novel protein targets converge to mitochondrial metabolism pathways. Circ Res. 2006; 99: 706–714.LinkGoogle Scholar
  • 21 Arrell DK, Neverova I, Fraser H, Marban E, Van Eyk JE. Proteomic analysis of pharmacologically preconditioned cardiomyocytes reveals novel phosphorylation of myosin light chain 1. Circ Res. 2001; 89: 480–487.CrossrefMedlineGoogle Scholar
  • 22 Chen CH, Budas GR, Churchill EN, Disatnik MH, Hurley TD, Mochly-Rosen D. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Science. 2008; 321: 1493–1495.CrossrefMedlineGoogle Scholar
  • 23 Gharib M, Marcantonio M, Lehmann SG, Courcelles M, Meloche S, Verreault A, Thibault P. Artifactual sulfation of silver-stained proteins: Implications for the assignment of phosphorylation and sulfation sites. Mol Cell Proteomics. Available at: Epub ahead of print 2008; Oct 20.Google Scholar
  • 24 Rassaf T, Lauer T, Heiss C, Balzer J, Mangold S, Leyendecker T, Rottler J, Drexhage C, Meyer C, Kelm M. Nitric oxide synthase-derived plasma nitrite predicts exercise capacity. Br J Sports Med. 2007; 41: 669–673.CrossrefMedlineGoogle Scholar
  • 25 Ferdinandy P, Schulz R. Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning. Br J Pharmacol. 2003; 138: 532–543.CrossrefMedlineGoogle Scholar
  • 26 Bolli R. Preconditioning: a paradigm shift in the biology of myocardial ischemia. Am J Physiol Heart Circ Physiol. 2007; 292: H19–H27.CrossrefMedlineGoogle Scholar
  • 27 Mayr M, Liem D, Zhang J, Li X, Avliyakulov NK, Yang JI, Young G, Vondriska TM, Ladroue C, Madhu B, Griffiths JR, Gomes A, Xu Q, Ping P. Proteomic and metabolomic analysis of cardioprotection: Interplay between protein kinase C epsilon and delta in regulating glucose metabolism of murine hearts. J Mol Cell Cardiol. 2009; 46: 268–277.CrossrefMedlineGoogle Scholar
  • 28 Churchill EN, Disatnik MH, Mochly-Rosen D. Time-dependent and ethanol-induced cardiac protection from ischemia mediated by mitochondrial translocation of varepsilonPKC and activation of aldehyde dehydrogenase 2. J Mol Cell Cardiol. 2009; 46: 278–284.CrossrefMedlineGoogle Scholar
  • 29 Ngoh GA, Facundo HT, Hamid T, Dillmann W, Zachara NE, Jones SP. Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injury. Circ Res. 2009; 104: 41–49.LinkGoogle Scholar
  • 30 Ramirez-Correa GA, Jin W, Wang Z, Zhong X, Gao WD, Dias WB, Vecoli C, Hart GW, Murphy AM. O-linked GlcNAc modification of cardiac myofilament proteins: a novel regulator of myocardial contractile function. Circ Res. 2008; 103: 1354–1358.LinkGoogle Scholar
  • 31 Marston SB, Redwood CS. Modulation of thin filament activation by breakdown or isoform switching of thin filament proteins: physiological and pathological implications. Circ Res. 2003; 93: 1170–1178.LinkGoogle Scholar
  • 32 Kentish JC, McCloskey DT, Layland J, Palmer S, Leiden JM, Martin AF, Solaro RJ. Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle. Circ Res. 2001; 88: 1059–1065.CrossrefMedlineGoogle Scholar
  • 33 Tang XL, Takano H, Rizvi A, Turrens JF, Qiu Y, Wu WJ, Zhang Q, Bolli R. Oxidant species trigger late preconditioning against myocardial stunning in conscious rabbits. Am J Physiol Heart Circ Physiol. 2002; 282: H281–H291.CrossrefMedlineGoogle Scholar
  • 34 Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res. 1998; 83: 73–84.CrossrefMedlineGoogle Scholar

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