Published online before print October 4, 2007, doi: 10.1161/CIRCRESAHA.107.155879
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© 2007 American Heart Association, Inc.
Cellular Biology |
Preconditioning Results in S-Nitrosylation of Proteins Involved in Regulation of Mitochondrial Energetics and Calcium Transport
From the Vascular Medicine Branch (J.S., E.M.) and Proteomics Core Facility (M.M., R.-F.S.), National Heart Lung and Blood Institute, NIH, Bethesda; and Department of Pathology (C.S.), Johns Hopkins University, Baltimore, Md.
Correspondence to Dr Elizabeth Murphy, Senior Scientist, NHLBI, NIH, Vascular Medicine Branch, Room 7N112, Building 10, Magnuson CC, 10 Center Dr, Bethesda, MD 20892. E-mail murphy1{at}mail.nih.gov
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Abstract |
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Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel
1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, -ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase 1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.
Key Words: preconditioning • S-nitrosylation • cardioprotection
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryReferences |
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Ischemic preconditioning (IPC) is a cellular adaptive phenomenon whereby brief episodes of myocardial ischemia and reperfusion (I/R) render the heart resistant to subsequent prolonged ischemic injury.1 Through activation of a complex cascade of signaling events, IPC has been shown to reduce arrhythmias, infarct size, and postischemic contractile dysfunction.2–5
Nitric oxide (NO) has been shown to be an important signal in cardioprotection.6–8 In acute IPC, NO has been shown to mediate protection at least in part by activation of guanylyl cyclase, resulting in the production of cyclic guanosine monophosphate (cGMP) and the activation of protein kinase G, which in turn leads to the opening of the mitochondrial KATP channel.6 Recently, it has also been shown that protein kinase G results in activation of an endogenous mitochondrial protein kinase C
that is involved in activation of the mitochondrial KATP channel.9,10 The opening of the mitochondrial KATP channel is reported to reduce mitochondrial Ca2+ loading11,12 and also to lead to generation of reactive oxygen species, which activate signaling cascades in a feed-forward manner to elicit cardioprotection.13,14
In addition to activating cGMP/protein kinase G–dependent signaling pathways, NO can directly modify sulfhydryl residues of proteins through S-nitrosylation, which has emerged as an important posttranslational protein modification.15–17 Furthermore, S-nitrosylation of critical protein thiols has been shown to protect them from further oxidative modification by reactive oxygen species.15,18,19 S-Nitrosylation has recently been suggested to be important in cardioprotection.20,21 We have recently shown that cardioprotection in female hearts involves inhibition of ICa-L by S-nitrosylation.20 A recent study has shown that S-nitrosothiols (SNOs) were detected in mitochondria isolated from IPC hearts, suggesting that protein S-nitrosylation may play an important role in IPC cardioprotection.21
The goal of this study was to examine the role of S-nitrosylation in cardioprotection and to identify S-nitrosylation–modified proteins and the functional effects of S-nitrosylation. We found that IPC results in S-nitrosylation of a number of proteins that have important roles in cardioprotection. We report the novel finding that cardioprotection results in S-nitrosylation and decreased activity of the mitochondrial F1-ATPase. A decrease in F1-ATPase activity would provide a mechanism for the reduced rate of decline in ATP observed in IPC hearts.1
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Materials and Methods |
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Animals
C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Me). All animals were adults, reproductively viable, and between 12 and 15 weeks of age at the time of experimentation. All animals were treated in accordance with NIH guidelines and the Guiding Principles for Research Involving Animals and Human Beings.
Treatment Protocol, Hemodynamic, and Infarct Size Measurements
Hearts were Langendorff perfused in the dark and randomly assigned to 1 of 6 groups (Figure 1, left). For details regarding the protocol, hemodynamic, and infarct size measurements, see the online data supplement at http://circres.ahajournals.org.
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), SNO assay (
) are shown. The results are shown at right (II): postischemic LVDP recovery, measured after 40 minutes of post-I/R (A); infarct size, measured after 2 hours of post-I/R (B); and SNO content of heart membrane fraction, measured before ischemia (C). *P<0.05 vs control in each group. Numbers are given in the figure.
Preparation of Crude Homogenate and Membrane Fractions From Mouse Hearts
All preparative procedures were performed in the dark to prevent light-induced cleavage of SNOs. The crude heart homogenate and the membrane fraction was prepared as described previously20 (see the online data supplement for details).
SNO Content and Biotin Switch S-Nitrosylation Detection
SNO in heart membrane fractions was detected by the 2,3-diaminonaphthalene (Sigma) fluorometric method.20,22 S-Nitrosylated proteins were detected using a modified biotin switch method,23 as described in our previous study20 (also see the online supplement). To identify additional S-nitrosylated proteins in IPC or GSNO-treated hearts, we tried several approaches. For samples from IPC hearts, after the biotin switch method the respective protein bands with the same migration as positive anti-biotin detection were excised from the EZ-blue–stained 4% to 20% SDS-PAGE gel and subjected to in-gel tryptic digestion. For the GSNO-treated samples, after the biotin switch method, the biotinylated proteins were purified on streptavidin–agarose beads (Sigma) for 1 hour at 25°C, and in-bead tryptic digestion was performed for the MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) peptide mass fingerprinting.
S-Nitrosylation Identification by Two-Dimensional DyLight Fluorescence Difference Gel Electrophoresis Proteomic Analysis
The newly developed DyLight maleimide sulfhydryl-reactive fluors (Pierce, Rockford, Ill) were used to replace the biotin-HPDP{N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide} in the biotin switch method at pH 7.0. The SNO-derived cysteine residues in each sample taken before sustained ischemia were individually labeled by DyLight 488 (for perfusion control), DyLight 549 (for IPC heart), and DyLight 649 (for GSNO-treated heart). After labeling, equal amounts of each sample were mixed together and subjected to 2D DyLight fluorescence difference gel electrophoresis (DyLight Fluor DIGE), ie, running on the same isoelectric focusing (pI 3 to 10; Amersham Biosciences, Piscataway, NJ) and 10% to 15% gradient SDS-PAGE (NextGen, Ann Arbor, Mich). Gels were scanned on a Typhoon 9400 variable mode imager (Amersham Biosciences) at a resolution of 100 µm. Each of the individual samples was visualized independently by selecting the individual excitation and emission wavelength with fluorescence scanning. All of the images scanned from the same gel were aligned by 2 internal fluorescence anchor spots in the gel and the image analysis was performed using single-stain analysis with intelligent noise correction algorithm processing by Progenesis Discovery software (Nonlinear Dynamics, Newcastle on Tyne, UK). DyLight-labeled protein will cause a positive/acidic shift (on isoelectric focusing) and thereby a left shift (on 10% to 15% SDS-PAGE) because each DyLight fluor molecule contains 3 to 4 negative charges. Meanwhile, the 
1-KDa mass from each DyLight fluor molecule will also cause a minor shift upwards. To sample the protein spots from a gel with a shifted DyLight pattern, the gel was poststained with SYPRO Ruby (Sigma) to ensure that a protein from a spot will provide sufficient sample for mass spectrometric (MS) identification. In addition, some spots with strong DyLight signal were directly picked from the gel for further clarification. The Ettan Spot Handling Workstation (Amersham Biosciences) performed automated extraction and in-gel trypsin digestion of selected protein spots according to the instruction. Peptides were analyzed using a MALDI-TOF mass spectrometer (4700 Proteomics Discovery System; Applied Biosystems, Foster City, Calif) for peptide mass fingerprinting and tandem mass spectrometry. Proteins were identified from the acquired spectra using the MASCOT database search function.
Enzyme Assays
All enzyme assays were conducted at 24°C in the dark in the presence of 0 to 1.0 mmol/L GSNO. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity in membrane fractions was assayed by the Pi-sensitive malachite green ATPase method.24 The reaction was started by adding Mg2+-ATP to a medium containing membrane fractions in the absence and presence of 1 µmol/L ionomycin (Ca2+ ionophore; Sigma). The reaction was stopped by trichloroacetic acid precipitation, and SERCA activity was measured in the presence of 1 mmol/L EGTA from the total ATPase activity. The mitochondria-enriched fractions were prepared by differential centrifugation from the trypsin-digested heart.25 The -ketoglutarate dehydrogenase (-KGDH) activity was measured by the addition of mitochondrial fractions to assay mixtures consisting of (in mmol/L) KH2PO4 25, EDTA 0.5, MgCl2 10, -ketoglutarate 5, CoASH 0.2, thiamine pyrophosphate 0.4, NAD+ 2, and 0.1% Triton X-100.26 The F1-ATPase in sonicated mitochondrial fractions was assayed by coupling ATP hydrolysis to NADH oxidation as a decrease in absorbance at 340 nm.25 The difference in reaction rates in the absence and presence of oligomycin (10 µg/mL; Sigma) represented F1-ATPase activity.
Isolation of Cardiomyocytes and Intracellular Ca2+ Measurements
Cardiomyocytes were isolated by the collagenase (Worthington Biochemical Corporation, Lakewood, NJ) perfusion method as described in our previous study.20 See the online data supplement for details.
Data Analysis
Results are expressed as means±SE. Statistical significance was determined by Student’s t test or ANOVA as required. Differences were regarded to be significant at P<0.05. Proteomic data searches were performed using the National Center for Biotechnology Information protein database.
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Results |
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IPC Reduces I/R Injury and Increases SNO Content
We were interested in examining the role of S-nitrosylation in cardioprotection. As shown in Figure 1A (right), IPC significantly increased the postischemic recovery of left ventricular developed pressure (LVDP). The LVDP at 40 minutes of reperfusion and 20 minutes of ischemia was 36.8±4.8% (n=8) of preischemic LVDP, whereas it was 62.1±6.7% (n=10) of preischemic LVDP, in IPC hearts. Consistent with the improved functional recovery, infarct size (Figure 1B) was also significantly less in IPC hearts (6.4±1.1%) compared with control hearts without IPC (28.2±5.6%). The SNO contents (Figure 1C) in membrane fractions of perfusion control heart was 3.4±0.6 pmol/mg protein, whereas there was almost a 3-fold increase of SNO content in IPC hearts (9.9±0.4 pmol/mg protein). After 20 minutes of ischemia and 40 minutes of reperfusion, the SNO content in IPC hearts was decreased to 5.6±0.4 pmol/mg protein, a value still significantly higher than perfusion control. Thus, IPC leads to an increased formation of SNO, consistent with a role for protein S-nitrosylation in IPC.
IPC Increases S-Nitrosylation Level of the L-type Ca2+ Channel 1 Subunit
We have previously reported that the L-type Ca2+ channel 1 subunit is among the predominant S-nitrosylated proteins in the membrane fraction of cardiac muscle and that S-nitrosylation of the L-type Ca2+ channel 1 subunit reduces Ca2+ entry via the channel, which reduces Ca2+ overload and contributes to cardioprotection in female hearts.20 Therefore we examined whether IPC results in any change in the level of S-nitrosylation of the L-type Ca2+ channel. Following the biotin switch method, we immunoprecipitated the membrane fraction with anti–L-type Ca2+ channel 1 antibody and then probed the immunoprecipitate with anti-biotin antibody. The anti-biotin signal of the L-type Ca2+ channel 1 (Figure 2A) was significantly higher in IPC hearts. After I/R, the level of S-nitrosylation of the L-type Ca2+ channel 1 was decreased but still higher than perfusion control.
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IPC Increases S-Nitrosylation Level of Other Proteins
To investigate other S-nitrosylated proteins in IPC hearts, in addition to the membrane fractions used in Figure 2, whole cell homogenates were also subjected to the biotin switch method. The EZ-blue–stained protein bands that comigrated with positive anti-biotin blots were excised and subjected to in-gel trypsin digestion and MS analysis. Several S-nitrosylated candidate proteins in IPC hearts were identified, including (Figure 3) the cardiac isoform SERCA (SERCA2a), glycogen phosphorylase, -KGDH, myomesin, and cardiac -myosin heavy chain.
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GSNO Preconditioning Increases LVDP Recovery and SNO Content
To determine whether an increase of NO/SNO elicits similar effects on S-nitrosylation and protection, we examined the effect of treatment with GSNO. Pharmacological preconditioning using GSNO has been reported in other studies.27,28 However, its molecular mechanism is still unclear. Perfusion of hearts with 0.1 mmol/L GSNO for 30 minutes did not significantly change hemodynamics (see the online data supplement). As shown in Figure 1, GSNO significantly improved postischemic recovery of LVDP (54.8±7.9% versus 36.8±4.8% in control) and also reduced infarct size (8.2±2.4% versus 28.2±5.6% in control). GSNO also increased the SNO content in the heart membrane fraction to a level comparable to IPC.
S-Nitrosylated Protein Induced by GSNO Preconditioning
We also examined whether GSNO-mediated protection would result in S-nitrosylation of the L-type Ca2+ channel 1 subunit, which was S-nitrosylated in IPC hearts (Figure 2A). As shown in Figure 2B, the S-nitrosylation level of the L-type Ca2+ channel 1 subunit was also found to be significantly higher in GSNO-treated hearts compared with perfusion control. Thus, IPC and GSNO both significantly increase S-nitrosylation of the L-type Ca2+ channel.
To identify additional S-nitrosylated proteins occurring with GSNO treatment, after the biotin switch procedure to label the S-nitrosylated proteins, the samples were subjected to streptavidin–agarose chromatography. The MS analysis after in-bead tryptic digestion identified several S-nitrosylated proteins, including the mitochondrial F1-ATPase 1 subunit, SERCA2a, cardiac -myosin heavy chain, and myosin light chain 1. Interestingly, SERCA2a was also identified as an S-nitrosylated protein in IPC hearts (Figure 3).
Identification of S-Nitrosylated Proteins by DyLight Fluor DIGE Proteomic Analysis
We were interested in obtaining a direct comparison of S-nitrosylated proteins in IPC and GSNO-treated hearts. In addition, it would be useful to have a 2D method for S-nitrosylated proteins to provide better separation of proteins for MS identification. The biotin labeling of proteins in the biotin switch method is somewhat labile, and the biotin would be lost if gels were run under reducing conditions, as is common for most 2D methods. As described in Materials and Methods, we therefore developed a DyLight Fluor DIGE proteomic method that would allow labeling of S-nitrosylated proteins by DyLight–maleimide fluors and separation by a 2D fluorescence difference gel electrophoresis. Perfusion control samples were labeled with DyLight 488. As shown in Figure 4A, the very faint DyLight 488 signal suggests that a small level of endogenous S-nitrosylation was present under control conditions. IPC and GSNO significantly increased the protein S-nitrosylation, as shown by the increased fluorescence signal of DyLight 549 (labeling IPC sample) and DyLight 649 (labeling GSNO-treated sample). By overlaying the DyLight images to the Ruby images (Figure 4B), the Ruby protein spots with the same pattern as DyLight positive signal were chosen for MS identification, taking into account the shift attributable to DyLight modification of the proteins (arrowheads in Figure 4B). The S-nitrosylated proteins identified by DyLight Fluor DIGE proteomics are listed in Table.
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Functional Relevance of S-Nitrosylation to Cardioprotection
We identified several proteins that could be important in cardioprotection that were S-nitrosylated by either IPC or treatment with GSNO. We next examined whether S-nitrosylation altered the activities of these proteins. We examined the effect of GSNO treatment (1) on the activity of SERCA2a in the membrane fractions and (2) on the activity of -KGDH and F1-ATPase in the mitochondrial fractions. As shown in Figure 5A, GSNO concentration-dependently increased SERCA activity. GSNO also concentration-dependently increased the activity of -KGDH, either in a purified enzyme from porcine heart (Sigma, data not shown) or in our isolated mitochondrial fractions (Figure 5B). We next examined the effect of GSNO treatment on the activity of the mitochondrial F1-ATPase in submitochondrial particles prepared from sonicated mitochondrial fractions. As shown in Figure 5C, there was a decrease in the mitochondrial F1-ATPase activity with increasing concentrations of GSNO. This would be consistent with the decrease in ATP consumption observed previously in IPC hearts.1
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Effect of GSNO on Ca2+ Transient and Ca2+ Release by Caffeine in Cardiomyocytes
As shown in Figure 6A and B, GSNO (0.1 mmol/L) significantly decreased the Ca2+ transient in field-stimulated cardiomyocytes, consistent with reduced Ca2+ entry caused by inhibition of the L-type Ca2+ channel by S-nitrosylation and the resultant reduction of the Ca2+-induced Ca2+ release.20 Furthermore, the half-decay time of the Ca2+ transient (Figure 6C) was significantly shortened in GSNO-treated cardiomyocytes, consistent with increased SR Ca2+ uptake attributable to activation of SERCA2a by S-nitrosylation (Figure 5A). However, GSNO did not alter SR Ca2+, because Ca2+ release by caffeine in the presence of GSNO was comparable to the control (Figure 6B). These results show that GSNO could significantly decrease the cytosolic Ca2+ transient, which would reduce Ca2+ overload after I/R.
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Discussion |
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S-Nitrosylated Proteins and Cardioprotection
The consistent relationship between the increase of protein S-nitrosylation and reduced infarct size suggests that S-nitrosylation might play a cardioprotective role. We found that cardioprotection was associated with increased S-nitrosylation of a number of proteins and we further showed that SNO altered enzymatic activities of these proteins. Of particular interest, we observed that GSNO treatment resulted in increased S-nitrosylation of the mitochondrial F1-ATPase, which resulted in decreased activity. It has been reported that
50% of the ATP generated during ischemia by glycolysis is consumed by reverse mode of the mitochondrial F1-ATPase.29 Therefore inhibition of the F1-ATPase during ischemia would conserve ATP. Indeed, inhibition of the F1-ATPase was an early hypothesis to explain the reduced rate of decline in ATP observed during ischemia in IPC hearts.30 Furthermore, the ATP consumed by the reverse mode of the F1-ATPase is used to maintain the mitochondrial 
and transport Ca2+ into the mitochondria. Thus inhibition of the F1-ATPase could be beneficial by conserving cytosolic ATP and by reducing Ca2+ uptake into the mitochondria. However, when the rate of the F1-ATPase was measured in submitochondrial particles, no differences in activity were observed between IPC and non-IPC hearts.30,31 Because S-nitrosylation is easily reversed, particularly if the samples are exposed to light and metal ions,32 it is likely that the IPC mediated S-nitrosylation of the F1-ATPase would be lost during isolation of the submitochondrial particles in those studies.
It has recently been reported that IPC prevents the I/R-induced loss of activity of -KGDH.26 Interestingly, we found that IPC resulted in increased S-nitrosylation of -KGDH. We further found that GSNO treatment increased activity of -KGDH, perhaps contributing to the IPC-mediated maintenance of -KGDH activity. It is tempting to speculate that the S-nitrosylation of -KGDH protects it from oxidative damage during I/R.
The original report describing IPC1 indicated that IPC reduced the accumulation of lactate during sustained ischemia. IPC has also been reported to reduce ischemic acidosis,33,34 and Weiss et al demonstrated that IPC results in the attenuation of glycogenolysis.34 It is therefore of interest that IPC increased S-nitrosylation of glycogen phosphorylase, the enzyme responsible for catabolism of glycogen. Furthermore, by DyLight Fluor DIGE proteomic analysis, several other important mitochondrial proteins, such as aconitase, creatine kinase, malate dehydrogenase, acyl-CoA dehydrogenase, complex I-75 KDa, heat shock protein 60, and electron transfer flavoprotein , were also found to be S-nitrosylated in IPC and GSNO-treated hearts (Table). Future studies will be directed at determining whether S-nitrosylation alters the activities of these metabolic enzymes.
In this study, we found that IPC resulted in an increase in S-nitrosylation of the L-type Ca2+ channel 1 subunit. This is consistent with our previous study of cardioprotection in hypercontractile female hearts, in which we observed increased S-nitrosylation that resulted in decreased Ca2+ entry via the L-type Ca2+ channel.20 The increase in S-nitrosylation of the L-type Ca2+ channel and reduced Ca2+ entry resulted in reduced I/R injury.18,20 Consistent with this hypothesis, we have previously reported that preconditioning reduces cytosolic Ca2+ levels during ischemia,33 and in this study, we found that GSNO treatment also led to reduction of the cytosolic Ca2+ transient (Figure 6). NO has been reported to modulate the activity of SERCA2a.35 Protein modification mediated by NO carriers could result from S-nitrosylation or from other secondary oxidative modifications such as S-glutathiolation.35–37 We found that both IPC and GSNO result in S-nitrosylation of SERCA2a, and we further showed that GSNO treatment causes an increase in SERCA2a activity (Figures 5A and 6
). An increase in SERCA2a activity during ischemia and early reperfusion would provide for improved Ca2+ uptake into the SR, which could reduce cytosolic Ca2+ and reduce diastolic Ca2+ during I/R, making SERCA2a a plausible target for cardioprotection.38 Indeed, adenoviral-mediated overexpression of SERCA2a has been reported to reduce infarct size and improve function following ischemia.39 In addition, the SR has been suggested to be a primary target of reperfusion protection.40 Ca2+-induced Ca2+ release is the well-known molecular mechanism of excitation–contraction coupling in cardiac muscle. The overall effect of S-nitrosylation on intracellular Ca2+ handling, ie, decreased Ca2+ entry by the inhibition of the L-type Ca2+ channel and increased SR Ca2+ uptake by the activation of SERCA2a, will lead to the attenuation of the rise in cytosolic Ca2+ during ischemia and Ca2+ overload during reperfusion.
Relationship Between SNO and Other PC-Signaling Mechanisms
It has been well established that NO is an essential component of IPC (see Figure 7).6,8 However, other signaling molecules have also been shown to be essential. How do we reconcile the observation that inhibition of any of these pathways blocks IPC? Two explanations are generally proposed. It has been suggested that IPC is a linear pathway, and therefore inhibition at any part of the pathway blocks IPC. This model would suggest that there is a single final effector. An alternative proposal is that IPC involves multiple signaling pathways and to achieve protection requires integration of these multiple pathways. In this latter case, inhibition of one arm of the protective signaling cascade may or may not block cardioprotection depending on the strength of the cardioprotective initiator (eg, 1 versus 4 cycles of IPC) and the length of the sustained ischemic period. These factors may account for some of the variability of the data in the literature. Recent data have suggested that inhibition of the mitochondrial permeability transition (MPT) pore is an important (if not final effector) component of cardioprotection. Many of the cardioprotective signals appear to converge on inhibition of MPT.5,9,10 MPT is regulated by reactive oxygen species and calcium, and there may be multiple mechanisms that can inhibit MPT. Our data suggest that NO has additional actions that can also reduce ischemic injury and reduce MPT opening. As discussed, S-nitrosylation and inhibition of the L-type Ca2+ channel would reduce Ca2+ loading of the myocytes, which would reduce Ca2+ available to activate the MPT. S-Nitrosylation of the SERCA2a, which has been previously reported to reduce infarct size and improve function following myocardial ischemia,39 would reduce cytosolic and mitochondrial Ca2+, which would reduce MPT. We also found a number of mitochondrial targets, such as the F1-ATPase, that were S-nitrosylated, and many of these targets might also result in inhibition of MPT. We found that the addition of GSNO resulted in inhibition of the F1-ATPase. Inhibition of the F1-ATPase during ischemia would reduce breakdown of glycolytic ATP and accelerate the fall in the mitochondrial membrane potential. The reduction in mitochondrial membrane potential would reduce reactive oxygen species generation and would reduce the driving force for Ca2+ entry into the mitochondria; these would both result in less activation of MPT. Our working hypothesis is that IPC results in activation of several interacting pathways that all seem to converge to block activation of the MPT, but the mechanisms may be different.
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Summary |
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IPC has been reported to increase the formation of NO.41,42 Our study suggests that the increase in NO occurring in IPC hearts results in protein modifications such as S-nitrosylation, which is also likely to be involved in cardioprotection. An increase in SNO content in the mitochondrial fraction has been reported recently in IPC rat hearts.21 Recent studies have shown that protein S-nitrosylation could affect cell death or survival by reversibly regulating mitochondrial respiration21 and redox status.43 Because S-nitrosylation is a reversible modification, it can modify cysteine residues and thereby protect them from irreversible oxidation during sustained ischemia.15,18,44 In addition, S-nitrosylation can alter enzymatic activity, and this altered activity may play a role in cardioprotection.15,44 IPC might provide an environment that favors SNO, for example, favorable ion content, pH, and redox equilibrium. The increase of SNO contents and S-nitrosylated proteins in IPC hearts suggests that protein S-nitrosylation, similar to phosphorylation, may play an important role in IPC. The data suggest that protein S-nitrosylation might elicit cardioprotective effects by regulating intracellular Ca2+ handling, mitochondrial energetics, and sarcomeric ultrastructure. Further investigation correlating protein S-nitrosylation modification and functional regulation will provide a better understanding of the molecular mechanisms of its cardioprotective effect and provide new therapeutic opportunities and targets for intervention in ischemic injury.
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Acknowledgments |
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We thank Dr Jason Williams of the Protein Microcharacterization Facility in the National Institute of Environmental Health Sciences, NIH, for help with MALDI-TOF peptide mass fingerprinting.
Sources of Funding
J.S. and E.M. were supported by the NIH Intramural Program. C.S. was supported in part by NIH grant HL-39752.
Disclosures
None.
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Footnotes |
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This manuscript was sent to Joseph Loscalzo, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received May 9, 2007; revision received August 29, 2007; accepted September 20, 2007.
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References |
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