Redox Regulation of Mitochondrial ATP SynthaseNovelty and Significance
Implications for Cardiac Resynchronization Therapy
Rationale: Cardiac resynchronization therapy (CRT) is an effective clinical treatment for heart failure patients with conduction delay, impaired contraction, and energetics. Our recent studies have revealed that mitochondrial posttranslational modifications (PTM) may contribute to its benefits, motivating the present study of the oxidative regulation of mitochondrial ATP synthase.
Objectives: We tested whether CRT alteration of ATP synthase function is linked to cysteine (Cys) oxidative PTM (Ox-PTM) of specific ATP synthase subunits.
Methods and Results: Canine left ventricular myocardium was collected under conditions to preserve Ox-PTM from control, dyssynchronous heart failure (DHF), or hearts that had undergone CRT. In-gel ATPase activity showed that CRT increased ATPase activity by ≈2-fold (P<0.05) over DHF, approaching control levels, and this effect was recapitulated with a reducing agent. ATP synthase function and 3 Ox-PTM: disulfide bond, S-glutathionylation and S-nitrosation were assessed. ATP synthase from DHF hearts contained 2 novel disulfide bonds, between ATP synthase α subunits themselves and between α and γ subunits, both of which were decreased in CRT hearts (4.38±0.13- and 4.23±0.36-fold, respectively, P<0.01). S-glutathionylation of ATP synthase α subunit occurred in DHF hearts and was decreased by CRT (1.56±0.16-fold, P<0.04). In contrast, S-nitrosation of ATP synthase α subunit in DHF hearts was lower than in CRT hearts (1.53±0.19-fold, P<0.05). All modifications occurred at ATP synthase α subunit Cys294 and Cys to Ser mutation indicated that this residue is critical for ATP synthase function.
Conclusions: A selective Cys in ATP synthase α subunit is targeted by multiple Ox-PTM suggesting that this Cys residue may act as a redox sensor modulating ATP synthase function.
Cardiac resynchronization therapy (CRT) is a clinically effective treatment for dyssynchronous heart failure (DHF); however, the underlying molecular and cellular mechanisms remain poorly understood.1 An early clinical finding indicated that CRT improves chamber energetic efficiency,2 and our recent work has pinpointed that CRT directly affects ATP production by mitochondria, profoundly affecting the mitochondrial subproteome.3 Specifically, DHF and CRT alter the mitochondrial subproteome by modifying proteins involved in cellular redox control and oxidative phosphorylation (OxPhos) pathways, as manifested by changes in both protein quantity and posttranslational modifications (PTM).3
The mitochondrial electron transport chain is a primary source of reactive oxygen and nitrogen species (ROS/RNS), which can mediate oxidative-PTM (Ox-PTM), particularly targeting important mitochondrial components including the respiratory chain and matrix enzymes, as well as membrane phospholipids.4,5 Emerging evidence links Ox-PTM to dysfunction in ATP synthesis in the failing heart, highlighting potential therapeutic targets.6–8 On the other hand, Ox-PTM in the heart may serve as redox switches, sensing cellular redox state to regulate protein function.9–11 This is not unprecedented; for example, in plants, chloroplast F1FO-ATPase is subject to redox regulation, whereby ATP hydrolytic activity is regulated by the formation and reduction of a disulfide bond located in the γ subunit.12 Changes in redox state do not affect the rate of ATP binding to the catalytic site(s) or the torque for rotation, but long pauses caused by ADP inhibition are more frequent in the oxidized state.12 As well, introducing the redox modulated region of the plant ATPase into the yeast γ-subunit causes a defect in oxidative phosphorylation.13
In the present study, we report several ROS/RNS-related PTM that occur on ATP synthase in failing dyssynchronous hearts (DHF) and demonstrate that CRT can ameliorate these Ox-PTM, supportive of a protected phenotype and improved ATP production.
Animal Model and Sample Preparation
Adult mongrel dogs underwent either a DHF or a CRT protocol, as previously described.3 Heart tissues were harvested under conditions to preserve Ox-PTM.14 Mitochondria were isolated by a differential centrifugation protocol as previously described3 and adapted to preserve Ox-PTM. Rat heart tissues were from Pel-Freez Biologicals (Rogers, AR), and mitochondria were isolated in the absence of N-ethylmaleimide (NEM). ATP synthase was isolated using ATP synthase immunocapture kit (Mitosciences) according to the manufacture's protocol. See Online Supplement available at http://circres.ahajournals.org.
HEK 293 cells used for exogenous expression of Cys mutants were cultured at 37°C in a 5% CO2 incubator in DMEM media (Mediatech) supplement with 10% FBS (HyClone).
The human ATP5A1 and ATP5C1 ORF and full-length cDNA were obtained from Open Biosystems. Cys to Ser mutants were created by QuickChange site-directed mutagenesis kit (Stratagene) with primers list in Online Table I. siRNAs for ATP5A1 and ATP5C1 gene were obtained from Applied Biosystems. Expression plasmids and siRNAs were introduced into HEK 293 cells by transient cotransfection with Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacture's protocol. See Online Supplement.
Induction of Ox-PTM in Isolated Mitochondria and ATP Synthase Complex With Oxidants
Isolated mitochondria or ATP synthase complex were resuspended in reaction buffer containing different reagents and induction performed as described in Online Figure I.
Gel Electrophoresis and Immunoblot
Clear Native PAGE (CNP) and Blue Native PAGE (BNP) were used to resolve intact mitochondrial protein complexes.15,16 Two-dimensional BNP/SDS-PAGE was performed to analyze complex subunit composition.16 One-dimensional SDS-PAGE was performed as described.3 For immunoblotting, proteins were transferred to PVDF (polyvinylidene fluoride) membranes before blotting. Densitometry was performed on scanned gels and immunoblots using Progenesis Workstation 2005 software (Nonlinear Dynamics). See Online Supplement for method and antibodies.
Biotin Switch Assay
S-nitrosation (SNO) was detected with a modified biotin switch assay as described.17 After streptavidin pulldown, the S-nitrosated ATP synthase α subunit was detected with anti–Complex V α subunit antibody (Invitrogen). See Online Supplement.
ATPase Activity Determination
ATPase activity was determined by in-gel (CNP) ATPase assay according to a previously described method.15 See Online Supplement.
Mitochondrial Respiration Measurement
Mitochondria were freshly isolated from left ventricular endocardium of a normal dog in the absence of NEM as described above. Mitochondrial respiration was measured as described.18 See Online Supplement.
Gel bands or spots were excised, digested with trypsin, and analyzed on an LTQ-Orbitrap LC-MS/MS (liquid chromatography-tandem mass spectrometry, Thermo Finnigan) with proteins identified by SEQUEST (Sage-N Research), according to our published methods.3,19 IPI database (IPI_RAT_v3.62) was used, protein name redundancy was removed, and isoforms were identified positively only if a peptide(s) was observed to correspond to an isoform unique amino acid sequence. See Online Supplement.
The 3-dimensional structure of bovine mitochondrial F1 ATP synthase from protein data bank (PDB entry No. 1E79) was modeled using PyMOL (Delano Scientific LLC).
All data are expressed as mean±SD. Comparisons between different groups were performed with the use of a 2-tailed, unpaired Student t test, with P<0.05 considered significant.
Reversal of Cys Oxidative Modification Contributes to the Beneficial Effect of CRT on ATP Synthase Activity
To test whether Cys oxidative modification correlates with mitochondrial dysfunction, we measured mitochondrial ATPase activity from adult mongrel dogs subjected to either DHF, CRT, or no tachypacing (sham), using CNP and subsequent in-gel ATPase activity assay (a method more sensitive than BNP15). Figure 1A shows ATP synthase activity in mitochondria from left ventricular endocardium obtained from DHF, CRT, and sham and isolated under Cys modification preserving conditions, unlike previous work on this model, in which the focus was on other nonoxidative PTM.3 Under these conditions, ATPase activity was significantly lower in DHF compared with control (Figure 1A, bottom panel) (26.13±7.71 versus 47.04±16.9, P<0.05). CRT significantly increased activity to control levels (53.99±13.53 versus 47.04±16.93, P=0.24). Importantly, incubation of mitochondria isolated from DHF hearts with 1 mmol/L dithiothreitol (DTT) restored ATPase activity to control levels (Figure 1A), suggesting that reversal of Cys oxidative modifications contributed to the beneficial effects of CRT on ATP synthase activity. Interestingly, the ATPase activity from sham mitochondria was also DTT sensitive, suggesting that oxidative modifications are present under baseline conditions. However, it is important to note that the degree of DTT sensitivity was much greater in DHF hearts and was significantly reduced in CRT hearts.
The ATPase activity of mitochondria from sham animals, obtained in the presence of alkylating or oxidizing reagents, is shown in Figure 1B. Blocking thiols on any free Cys residue with the alkylating reagent NEM during mitochondria isolation greatly improved ATPase activity at baseline and rendered it resistant to oxidant treatment. This enhanced basal activity is probably due to reversal of modest oxidation of the exposed free thiols by oxygen in the solution during the process of mitochondria isolation, despite our best efforts to preserve a reducing environment (see Methods section for details). However, importantly, the difference between the NEM-treated and untreated groups was large (≈2 fold), suggesting that the Cys oxidative modifications have a profound effect on ATPase activity. To confirm that the effect of oxidation on ATPase activity in DHF dogs occurs in vivo, independent of NEM treatment, we also measured the ATPase activity in mitochondria isolated from DHF, CRT, and sham dogs that were not treated with NEM. As shown in Online Figure I, in the absence of NEM, the DHF group still had significantly lower ATPase activity compared with controls (≈30%), whereas the activity was restored to control levels by CRT. This result is similar to our previous study3 in the same model, where CRT induces a 20% increase in ATPase activity compared with DHF. Taken together, these results demonstrate that the beneficial effect of CRT with respect to ATPase activity is independent of NEM. Because NEM and DTT may have broad effects on mitochondrial proteins other than ATP synthase, we next studied the effects of NEM and DTT on oxygen consumption rate in freshly isolated mitochondria from control dogs. As shown in Online Figure II, 20 mmol/L NEM treatment severely impaired respiration supported by either glutamate/malate or succinate. This suggests that NEM could inhibit multiple components of OxPhos (eg, substrate transport, the Krebs cycle, or electron transport), in addition to restoring ATP synthase activity in the in-gel assay. By contrast, treatment with 1 mmol/L DTT increased respiration substantially for both substrates. On a relative basis, state 2 increased more than state 3 respiration. As a result, the net effect of DTT on oxygen consumption rate (glutamate/malate) was a reduction from 5 to 2.3. This finding, although consistent with the enhanced ATP synthase activity observed in the in-gel assay, indicates that the reversal of mitochondrial oxidative modifications by DTT probably affects mitochondrial respiration at more than one site.
DHF Is Correlated With Disulfide Bond Formation in the ATP Synthase Complex
To assess the types of Cys modification present in the various experimental groups, we first assessed the presence of disulfide bonds in the ATP synthase by classic 2D-BNP/SDS-PAGE under nonreducing conditions and compared the results with those obtained under reducing conditions from DHF, CRT, and sham hearts. Figure 2A shows that mitochondria from DHF hearts have cross-linked products of ≈120 kDa and ≈100 kDa within the ATP synthase complex. These were reversible after DTT treatment (Figure 2A, bottom panel) and blocked by pretreatment with NEM (Figure 2C, left panel), indicating that cross-linking was a Cys-based modification, that is, disulfide bond formation. Two-dimensional nonreducing/reducing SDS-PAGE demonstrated that the disulfide bond complex was composed of ATP synthase alpha subunit (ATPα) and ATP synthase gamma subunit (ATPγ) (Online Figure III), confirmed by Western blot and verified by LC-MS/MS (Online Table II). These results indicate that the cross-links formed in DHF dog hearts are disulfide bonds. Figure 2B shows that the quantity of the ATPα-ATPα and ATPα-ATPγ disulfide bond complexes was significantly reduced (≈4 fold) after CRT treatment, compared with DHF animals. There were trace amounts of disulfide cross-linked complexes detected in sham dogs.
To validate these data, we also tested whether disulfide bonds could be induced in vitro with a generic oxidant treatment applied to isolated ATP synthase complex from rat heart mitochondria. As shown in Figure 2C, middle panel, CuCl2 treatment (100 μmol/L) resulted in extensive disulfide bond formation within the ATP synthase complex. As a control, NEM was used to block free Cys, and no cross-linked product was detected (n=3) (Figure 2C, left panel). With CuCl2 treatment, besides ATPα-ATPα and ATPα-ATPγ disulfide bonds, ATPα and the ATP synthase oligomycin sensitivity conferral protein (OSCP) disulfide bonds were also observed, as well as those between ATPγ-ATPγ and ATPγ-OSCP. As before, the identity of each subunit was confirmed by Western blot (Figure 2C, right panel) and by LC-MS/MS analysis (Online Table II). Thus, it is feasible that these additional disulfide bonds could occur with extreme oxidative stress.
DHF Increases S-Glutathionylation of the ATP Synthase Complex
We next tested whether ATPα was also the target of other Cys modifications, such as protein S-glutathionylation (S-Glu) and SNO. The extent of ATPα S-Glu was determined in isolated mitochondria from DHF, CRT, and sham dogs by Western blot, using an anti-GSH antibody (Figure 3A). Compared with sham animals, the extent of ATPα S-Glu was significantly increased in DHF, whereas the levels of S-Glu were partially normalized in CRT (Figure 3B). For validation, isolated ATP synthase complex from rat heart mitochondria was treated with GSSG and the resulting protein S-Glu was detected by Western blot. As shown in Figure 3C, the S-Glu of ATPα could be induced by GSSG in a dose-dependent manner.
CRT Increases SNO of the ATP Synthase Complex
SNO of ATPα was investigated by a modified biotin switch assay.17 As shown in Figure 4A, S-nitrosoglutathione (GSNO) treatment resulted in a significant increase of SNO of ATPα in isolated mitochondria when conditions for preserving Cys modifications were not used in DHF and CRT dogs. When mitochondria were isolated under Cys modification preserving conditions, untreated mitochondria from DHF had significantly less SNO compared with CRT or sham, as shown in Figure 4B. However, the extent of SNO induced by CRT was not as pronounced as other modifications. This suggests that SNO modification can only partly account for reversal of DHF-induced cross-linking with CRT, leaving the majority of Cys free in the CRT hearts. GSNO-induced, dose-dependent SNO of ATPα could also be demonstrated in the isolated rat heart mitochondrial ATP synthase complex (Figure 4C).
Cys294 of ATPα Is the Site for Disulfide Bond Formation, S-Glu, and SNO
We next performed mass spectrometry (MS) to identify the specific Cys residues modified by either disulfide bond formation or S-Glu. A differential labeling and MS strategy, outlined in Figure 5A, was used to identify the Cys residues involved in disulfide bonds. ATPγ contains 1 Cys, Cys103, and as expected, this Cys was differentially labeled by d5-N-ethylmaleimide in the presence of 100 μmol/L CuCl2 (Online Figure IV). However, in ATPα, both Cys244 and Cys294 were labeled with d5NEM (Online Figure IV), indicating that both could be oxidized by CuCl2 treatment. To determine which of the 2 Cys residues of ATPα were responsible for the disulfide bond, C-terminally FLAG tagged C244S and C294S mutants were prepared and expressed in HEK 293 cells by transient transfection. As shown in Figure 5B, with CuCl2 treatment, C294S but not C244S prevents the formation of the ATPα-ATPα disulfide bond, and the disulfide bond in C244S mutant was still reversible by DTT treatment. This result clearly demonstrates that Cys294 of ATPα is specifically involved in the disulfide bond formation.
To determine the Cys residues that undergo S-Glu, we subjected S-glutathionylated ATPα, obtained by treating isolated rat heart mitochondrial ATP synthase with GSSG, to trypsin digestion and analyzed by LC-MS/MS. Peptides with a mass difference of 305 Da, representing 1 glutathione moiety, were determined. Both Cys244 and Cys294 of ATPα were glutathionylated (Online Figure V). Interestingly, Cys294 has been previously shown to be S-nitrosated by GSNO treatment in isolated rat17 or mouse20 heart mitochondria. Taken together, these data show that Cys294 of ATPα is actively involved in various oxidative modifications including intermolecular disulfide bond formation, S-Glu, and SNO.
On the basis of the x-ray crystallographic determined protein structure of bovine mitochondrial F1 ATP synthase (PDB 1E7921), both Cys294 of ATPα and Cys103 of ATPγ are located on the surface of the ATP synthase, which makes them accessible for oxidant attack (Online Figure VI). To gain insight into the importance of these residues on the redox regulation of ATP synthase activity, Cys244 and Cys294 of ATPα, and Cys103 of ATPγ, were mutated to Ser and the mutant subunits were expressed in HEK cells. Exogenous expression of the constructs containing only the open reading frame of the corresponding subunit was induced, whereas RNAi was used to knock down the expression of endogenous ATPα and ATPγ subunits. As shown in Online Figure VII, A, siRNA knock-down decreased each of the endogenous subunits by 90%, with the majority of these being replaced with mutant ones. The mutants were able to form the intact F1FO-ATP synthase complex (Online Figure VII, A). Unexpectedly, however, measurements of the ATPase activity (normalized to the corresponding quantity of ATP synthase complex, based on Coomassie blue staining; Online Figure VII, B) showed that the expression of the wild-type subunit construct, in the presence of the siRNA, did not restore ATPase activity to levels found with the scrambled RNA transfection control. The reason for this is not clear, but because ATP synthase is a multisubunit protein complex, the function of this complex depends on the correct expression and assembly of each subunit. It is known that the 3′-untranslated region (3′-UTR) is involved in the regulation of expression of ATP synthase subunits.22 Therefore, we made additional expression constructs of the full-length cDNA for mutagenesis and transfection experiments (Figure 6A). Overexpression of subunit constructs containing the 3′-UTR partially restored the ATP synthase activity when the corresponding wild-type protein was present. As shown in Figure 6B, both C244S and C294S mutants have significantly lower ATP synthase activities when compared with the wild-type ATPα re-expression group (≈50% decrease), indicating that Cys244 and Cys294 are required for the functionality of ATP synthase complex.
Both wild-type and the C103S mutant of ATPγ also had significantly lower ATPase activities when compared with the transfection control, despite our efforts to optimize the knock down and transfection protocol (online Table III), indicating that there may be unknown mechanisms, eg, other PTM or chaperones missing that are required to regulate the γ subunit assembly into ATP synthase complex. For example, the newly identified chaperone, DAPIT (diabetes-associated protein in insulin-sensitive tissue), is required for the folding of ATP synthase.23 However, the C103S mutant had slightly more activity than its wild-type counterpart (≈10% increase). When the mutant cell lines were treated with 100 μmol/L CuCl2 or 1 mmol/L DTT, no significant differences in ATP synthase activity were detected for the mutants in response to either treatment when compared with its corresponding wild-type subunit (online Figure VIII), suggesting that the site-specific oxidations occurring in vivo are probably functionally important, although it currently remains unclear whether the rotation of γ subunit is needed for ATP hydrolysis.24
In our previous study using the CRT model,3 we showed that mitochondrial ATPase activity was increased with CRT as compared with DHF, and this correlated with reduced proteolysis and modest dephosphorylation. The largest changes in CRT, however, were upregulation of several key redox enzymes, leading to the current hypothesis that Ox-PTM might contribute to the beneficial effect of CRT. To maximize the detection of Ox-PTM, we performed the tissue collection and all downstream operations under conditions to preserve reversible Cys oxidative modifications. In the present study, we have shown for the first time that Cys oxidative modifications lead to mitochondrial ATP synthase dysfunction in DHF and that this effect is partially reversed by CRT. A number of Cys oxidative modifications can occur on the ATP synthase, including disulfide bond formation, S-Glu, and SNO, and they are specific to either DHF or CRT. With DHF, several subunits of the ATP synthase are cross-linked by intermolecular disulfide bonds. Specifically, disulfide bonds form between F1FO ATP synthase ATPα-ATPα and ATPα-ATPγ subunits, and S-Glu of ATPα was detected. These disulfide bonds were significantly decreased in CRT animals, as was the level of S-Glu. Although at present, we cannot quantify the extent of disulfide bonds versus S-Glu in the ATP synthase complex, it is clear that both can exist in the same sample, and both positively correlate with the loss of function of ATP synthase. In contrast, ATP synthase modification by SNO shows the opposite profile; it is decreased in DHF and then increased in CRT. In particular, we found that CRT can induce SNO of a modified cysteine after reversing the cysteine cross-linking and this might reverse the impaired function of the ATP synthase.
Location of Reactive Cys
In the present study, we have found an intermolecular disulfide bond between ATPα through Cys294 and an intermolecular disulfide bond between ATPα and ATPγ through Cys294 and Cys103, respectively. In addition, Cys294 of ATPα can also be modified by S-Glu and SNO. Based on the molecular model, Cys294 is located on the nucleotide binding domain surface, surrounded by several positively charged residues (Online Figure VI). Thus, it would probably be deprotonated at physiological pH, making it a good candidate for S-Glu and SNO.
Within an individual ATP synthase complex, the Cys294 of one of the ATPα subunits is located farther than 5 Å (the distance required for a disulfide bond to occur) from its neighboring ATPα or the Cys103 of ATPγ. Hence, it is unlikely that a disulfide bond between either ATPα-ATPα or ATPα-ATPγ occurs within a single complex. It is also unlikely that disulfide bonds occur between the ATPα from different ATP synthase complexes, because it is well documented that the subunits e and g are involved in the ATP synthase dimer interfaces resulting in the F1 subunits pointing away from each other.25,26
An alternate explanation is that these disulfide bonds could be formed before the individual subunits assemble into the ATP synthase complex, or when (or if) the protein/complex is aggregated. In all cases, the ATP synthase complex assembly will be compromised. In fact, DHF dog hearts have ≈20% less ATPα and ATP synthase beta subunit (ATPβ) content, and intact complex content than normal dogs, but this increases with CRT back to near control levels.3 This suggests that disulfide bond formation may represent a misfolded/aggregated form of the complex (but one with all subunits present).
The situation is different for both S-Glu and SNO, for which the physical spacing within the complex is not a factor. Rather, the main determinant would be whether the residues are accessible and if the Cys is buried or exposed, although for SNO there is no agreement on an amino acid consensus sequence.27,28 Importantly, Cys294 and Cys244 of ATPα are both found on the complex surface, and there is no amino acid sequence homology within 15 amino acid residues on either side of the modifiable Cys in the human or rat sequence.
Heart failure–induced oxidative stress causes redox imbalance, with low GSH levels and high GSSG levels, which could induce protein S-Glu.29 ATPα has already been reported to be the major mitochondrial protein that becomes glutathionylated under oxidative stress, at least in rat brain or in liver mitochondria that were isolated under a discontinuous Percoll gradient.30 Under these conditions, S-Glu of ATPα led to a substantial decrease of ATPase activity.30 Similarly, DHF dog hearts have a significantly higher level of S-Glu of ATPα compared with controls, and this increase correlated with the loss of function of the ATP synthase complex. S-Glu of Cys294 of ATPα, which adds a bulky negatively charged group to this residue, could potentially disrupt nucleotide binding to decrease ATPase activity.
Therapeutic Benefit of CRT and Altered Cys Ox-PTM
Cys modifications are dependent on the redox status and antioxidant capacity of the myocyte. Our data indicate that DHF is associated with increased oxidative stress, whereas CRT improves antioxidant defenses, in particular, the thioredoxin/peroxiredoxin pathway.3 Enhanced ROS scavenging might also prevent NOS uncoupling and preserve physiological NO signaling mechanisms, including SNO.31 We and others have previously shown that ATPα could be S-nitrosated by in vitro GSNO treatment in a large-scale study mapping SNO sites in isolated rat heart mitochondria.17,32 Also, it has been established that SNO modification of this protein occurs after ischemic preconditioning (IPC) of hearts in a mouse model, and these authors suggested that SNO of this protein serves as a cardioprotective mechanism in failing heart.32,33 In the present study, we have found that SNO of ATPα was significantly decreased in DHF dog hearts, whereas it was recovered in the CRT dog hearts. This indicates that CRT can reverse Cys cross-links and induce SNO modification. However, the extent of cross-link reversal is more than can be accounted for by induction of SNO modification, whereas the overall ATPase activity is recovered in CRT dogs.
Source of Funding
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) grant P01HL77189-01 (to J.E.V.E., B.O'R., and D.A.K.), NHLBI Proteomics Contract N01-HV28180 (to J.E.V.E., B.O'R., and D.A.K.), Foundation Leducq (to D.A.K.), and the Peter Belfer Laboratory (to D.A.K.).
In June 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.48 days.
This manuscript was sent to Junichi Sadoshima, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Non-standard Abbreviations and Acronyms
- 2D BNP/SDS-PAGE
- 2-dimensional BNP/SDS-polyacrylamide gel electrophoresis
- ATP synthase alpha subunit
- ATP synthase beta subunit
- ATP synthase gamma subunit
- blue native polyacrylamide gel electrophoresis
- clear native polyacrylamide gel electrophoresis
- cardiac resynchronization therapy
- Direct Blue 71
- dyssynchronous heart failure
- liquid chromatography-tandem mass spectrometry
- mass spectrometry
- ATP synthase oligomycin sensitivity conferral protein
- oxidative posttranslational modifications
- posttranslational modifications
- polyvinylidene fluoride
- reactive oxygen/nitrogen species
- Received April 4, 2011.
- Revision received July 26, 2011.
- Accepted July 27, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Cardiac resynchronization therapy (CRT) has become an effective clinical intervention for dyssynchronous heart failure patients leading to improvement in heart function, clinical symptoms, and survival.
Mitochondrial ATP synthase function is attenuated in heart failure and is restored at least in part by CRT.
CRT affects the mitochondrial subproteome by specifically altering proteins that control the cellular redox state and oxidative phosphorylation (OxPhos) pathways, as manifested by changes in both protein quantity and posttranslational modifications (PTM) within the mitochondria.
What New Information Does This Article Contribute?
ATP synthase undergoes a number of cysteine-specific oxidative PTMs, disulfide bond formation, S-glutathionylation, and S-nitrosation in the failing heart.
CRT reverses disulfide bond formation and S-Glu but induces S-nitrosation.
A specific Cys residue (ATP synthase α subunit Cys294) competes for disulfide bond formation, glutathionylation, and nitrosation and thus may be a “redox switch” sensing redox status and altering ATP synthase function.
CRT is a clinically effective treatment for dyssynchronous heart failure; however, the molecular mechanisms underlying the beneficial effects of CRT remain largely unknown. We show that in failing hearts, ATPα undergoes specific oxidative modifications by 3 different Ox-PTM: intermolecular disulfide bonds, S-glutathionylation, and S-nitrosation These modifications can occur at Cys294, suggesting that in comparison with other cysteine residues present in ATP synthase, this amino acid has a high redox sensitivity. This implies that ATPα Cys294 acts as a redox switch that senses the redox potential of the local cellular environment, thereby regulating ATPase activity. With CRT, antioxidant protective systems are enhanced, and under these conditions, the disulfide bonds are reversed and S-glutathionylation decreases, resulting in greatly improved ATPase activity. Remarkably, CRT hearts appear to “sense” the improvement in the redox environment and respond by activating NO signaling and thereby inducing S-nitrosylation of Cys294, which may be potentially protective.