Myocardial Ischemic Preconditioning Preserves Postischemic Function of the 26S Proteasome Through Diminished Oxidative Damage to 19S Regulatory Particle Subunits
Rationale: The ubiquitin proteasome system (UPS) becomes dysfunctional as a result of ischemia/reperfusion (I/R), which may lead to dysregulation of signaling pathways. Ischemic preconditioning (IPC) may prevent dysregulation by preventing UPS dysfunction through inhibition of oxidative damage.
Objective: Examine the hypothesis that early IPC preserves postischemic UPS function thus facilitating prosurvival signaling events.
Methods and Results: I/R decreased proteasome chymotryptic activity by 50% in isolated rat heart and an in vivo murine left anterior descending coronary artery occlusion model. Following IPC, proteasome activity was decreased 25% (P<0.05) in isolated heart and not different from baseline in the murine model. Enriched 26S proteasome was prepared and analyzed for protein carbonyl content. Increased (P<0.05) carbonylation in a 53-kDa band following I/R was diminished by IPC. Immunoprecipitation studies indicated that the 53-kDa carbonylation signal was of proteasomal origin. Two-dimensional gel electrophoresis resolved the 53-kDa band into spots analyzed by liquid chromatography/tandem mass spectrometry containing Rpt3/Rpt5 both of which could be immunoprecipitated conjugated to dinitrophenylhydrazine (DNPH). Higher amounts of DNPH-tagged Rpt5 were immunoprecipitated from the I/R samples and less from the IPC samples. I/R increased Bax levels by 63% (P<0.05) which was decreased by IPC. Lactacystin (lac) pretreatment of preconditioned hearts increased Bax by 140% (P<0.05) and also increased ubiquitinated proteins. Pretreatment of hearts with a proteasome inhibitor reversed the effects of IPC on postischemic Rpt5 carbonylation, cardiac function, morphology and morphometry, and ubiquitinated and signaling proteins.
Conclusions: These studies suggest that IPC protects function of the UPS by diminishing oxidative damage to 19S regulatory particle subunits allowing this complex to facilitate degradation of proapoptotic proteins.
Ischemic preconditioning (IPC) decreases the vulnerability of the myocardium to longer durations of ischemia as a result of preischemic exposure to short ischemic bursts as evidenced by improved postischemic hemodynamic function and reduced markers of injury.1 The mechanisms involved with IPC has been the subject of intense research and the earlier steps appear to involve signaling changes resulting in opening of the inward mitochondrial KATP channels2 and prevention of opening of the mitochondrial permeability transition pore.3 These early changes have been linked to decreased release of cytochrome c with diminished cellular apoptosis,3 as well as decreased production of oxidative species.4 We have shown that a downstream effect of preconditioning is diminished myocardial oxidative injury.5 One later effect of IPC is decreased levels of certain proapoptotic proteins, such as Bax.6 Earlier studies focused on diminished upregulation of these proteins to account for changes, although none examined the possibility that perhaps decreased levels may be attributable to improved degradation.
The ubiquitin proteasome system (UPS) degrades up to 70% of all intracellular proteins including many signaling proteins involved in cardiomyocyte cell death/survival, including several Bcl family members, such as Bax.7,8 It has been proposed that one way of regulating these reactive proteins is through degradation whereby a posttranslational modification, such as phosphorylation, acts as a signal for their ubiquitination and degradation by the 26S proteasome.9,10 The UPS becomes dysfunctional as a result of myocardial ischemia.11 We have proposed that one reason for the reported increases in some of these signaling proteins might be decreased degradation.12 The present study examines the hypothesis that early IPC preserves postischemic function of the UPS by diminishing oxidative damage to proteasome subunits, thus facilitating some of the signaling changes through improved degradation.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Experimental Models and Protocols
Isolated hearts were perfused with modified Krebs–Henseleit buffer as described previously13 and divided into 6 groups as described in Figure 1. Proteasome inhibitor-treated hearts were infused with buffer containing 2 μmol/L lactacystin (lac) or 0.5 μmol/L epoxomicin (epx) (Biomol Labs, Plymouth Meeting, Pa) for 10 minutes.
Murine Left Anterior Descending Coronary Artery Occlusion Model
Mice were subjected to in vivo left anterior descending coronary artery (LAD) occlusion for 30 minutes as described previously.14 The IPC protocol consisted of 3 cycles of LAD occlusion for 3 minutes and 2 minutes reperfusion just before 30 minutes ischemia. In sham-treated hearts, the LAD was exposed and a 7-0 silk suture passed around but not closed. Infarct size was estimated based on triphenyltetrazolium chloride staining of viable tissue.
ATP- and non–ATP-dependent chymotryptic activities of the proteasome were determined as described previously.15,16
Electron Microscopy and Morphometry
Cardiac morphology and morphometry were assessed as described previously.17
Cytosolic proteins were separated using standard SDS-PAGE under reducing conditions on a 4% to 20% gel. Following transference, membranes were probed with various antibodies as listed in Online Data Supplement.
Preparation of Enriched 26S Proteasome Fraction and Two-Dimensional Gel Electrophoresis
The enriched 26S proteasome fraction was prepared and separated using 2D gel electrophoresis as described previously.18
In early experiments, enriched proteasome fractions were reacted with 2,4-dinitrophenylhydrazine (DNPH) using the Oxyblot kit (Millipore, Billerica, Mass) as described previously.13 In later experiments, a formulation change required us to abandon the kit and use freshly made reagents, as described previously.19
All targeted proteins were immunoprecipitated as described previously13 using the TrueBlot reagent kit (eBioscience, San Diego, Calif). For studies of protein carbonyls, proteins were first immunoprecipitated and then reacted with DNPH as described previously,17 or the enriched proteasome fraction was reacted with DNPH and then immunoprecipitated with anti-DNPH antibody (Sigma-Aldrich, St Louis, Mo).
Polymerase Chain Reaction
RT-PCR was performed using standard kits for RNA isolation (Qiagen Rneasy Mini Kit, Valencia, Calif), reverse transcription (Qiagen Omniscript kit), and polymerase chain reaction using the primers for Bax and GAPDH as the internal control.
Liquid Chromatography/Tandem Mass Spectrometry
Stained proteins from 1D and 2D gels underwent liquid chromatography/tandem mass spectrometry (LC/MS/MS) on an LTQ-MS instrument (Thermo Fisher Scientific, San Jose, Calif) as described previously.18
All values are expressed as the means±SEM of a minimum of 3 determinations. Differences between 2 independent samples were assessed using a 2-tailed Student t test. Differences between 3 or more groups were assessed using 1-way ANOVA, followed by Tukey procedure as the post hoc test. Differences were considered to be significant at P<0.05.
Preconditioning Preserves Postischemic Proteasome Function
Isolated hearts were subjected to the experimental protocols defined in Figure 1. After 30 minutes of ischemia and 60 minutes of reperfusion, the chymotryptic activity of the cardiac proteasome was significantly decreased with greatest effect on the ATP-dependent activity (−50%) (Figure 2), consistent with our previous study.16 IPC tended to protect all proteasome functions to some degree with greatest effects on the ATP-dependent activity which was now decreased by only 25%. The effects of IPC were not related to changes in levels of proteasome subunit protein (Online Figure V). Treatment with the proteasome inhibitor, lac, before IPC reduced proteasome activity by 78% (Figure 2) with similar decreases in lac-pretreated ischemic hearts. Treatment with the inhibitor epx reduced proteasome activity but to a lesser degree (Figure 2). To determine whether the protective effect of IPC can be observed in vivo, mice were subjected to 30 minutes LAD occlusion followed by reperfusion. After ischemia and 3 hours of reperfusion, cardiac proteasome activity was decreased by 49% and 40% (P<0.05), respectively (Figure 3A), which was completely prevented by IPC (Figure 3A). IPC decreased infarct zone size from 60% to less than 30% (P<0.05) (Figure 3B) after 20 hours of reperfusion.
Ischemic Preconditioning Diminishes Oxidation of 19S Regulatory Particle Subunits
To determine whether proteasome subunits were subject to increased carbonylation during myocardial ischemia/reperfusion (I/R) and whether IPC could diminish this, a ≥300-fold enriched proteasome fraction was prepared (Online Figure II). Following separation, bands at 48-, 53-, and 114-kDa contained significant content of carbonyls (Figure 4A). The 53-kDa band was quantified and significantly increased (P<0.05) by 60% following I/R (Figure 4A). The band at 114-kDa was also increased but not significantly (data not shown). The intensity of the 53-kDa carbonyl signal was decreased to baseline by IPC (Figure 4A), which was prevented by lac-pretreatment. Immune probing of the stripped membrane showed that the 53-kDa band contained Rpt5 (Figure 4B) and the 114-kDa band contained Rpn2 (data not shown). The 48-kDa band was not recognized by any of the19S regulatory particle subunit antibodies tested. Because LC/MS/MS analysis of the 53-kDa band revealed the presence of multiple proteasome-related proteins, including 11 different 19S regulatory particle subunits (Online Table I), additional steps were taken to determine the identity of the oxidized subunit(s).
Intact 19S regulatory particles (including attached 20S proteasome) were immunoprecipitated with a monoclonal antibody for Rpt6 with immunoprecipitation of a parallel system with Bax antibody acting as the control. When equal volumes of supernatant from both systems were probed for protein carbonyl content, the 53-kDa band was present in the pellet fraction of the Rpt6-precipitated supernatant and visibly greater than the Bax-precipitated sample (Figure 5A, top), providing evidence that the signal was of proteasomal origin. The 48-kDa carbonyl band was not affected by immunoprecipitation and was shown by LC/MS/MS analysis to contain principally actin (Online Figure VIII) and other proteins not of proteasomal origin (data not shown). Decreased Rpt6 (Figure 5A, bottom) confirmed successful immunoprecipitation of 19S regulatory subunits. DNPH-tagged enriched proteasome fraction was subjected to 2D gel separation. Coomassie blue staining of the 2D gel revealed a single spot (Figure 5B, arrow 1) recognized by both the DNPH and Rpt5 antibodies in close proximity but distinct from an additional spot consistent with the 48-kDa (arrow 2) carbonyl spot. LC/MS/MS revealed that spot 1 contained the 19S regulatory particle subunits Rpt5 (PSMC3) and Rpt3 (PSMC4) (Figure 5C) as unique proteins (Online Table II), with 3 peptides detected for each. Other proteins were detected in this spot but were present in other spots not staining for DNPH and thus are background. Spot 2 contained principally actin (data not shown) previously shown to be a proteasome associated protein.20 To confirm that Rpt5 and/or Rpt3 are oxidized, an enriched proteasome fraction was reacted with DNPH and then immunoprecipitated with the anti-DNPH antibody with a Bax pull-down acting as the control. Figure 5D presents a 1D gel separation of the anti-DNPH precipitated proteins, which were subsequently probed for Rpt3 or Rpt5. In both cases, a large band at ≈48 to 50 kDa was present in the supernatant, which was recognized by either Rpt3 or Rpt5. A smaller band is clearly present in the immunoprecipitated pellet in both cases, but not in the Bax pellet lane, indicating that this band is DNPH-related and supporting the identification of the oxidized subunits. To confirm the effects of I/R and IPC, an anti-DNPH pull-down was performed on three sets of enriched proteasome tagged with DNPH and then probed with anti-Rpt5/Rpt3. A significant (P<0.05) 53% increase in DNPH-tagged Rpt5 was detected following I/R (Figure 5E). IPC resulted in an intermediate effect with this group not different from either the preischemic or I/R samples. No effect of I/R or IPC was apparent on levels of DNPH-tagged Rpt3 (data not shown). Identification of the subunit(s), as well as the effects of I/R and IPC, are further supported by 2D gel electrophoresis studies of the DNPH-tagged enriched proteasome fraction, which are described in detail in the Supplement (Online Figure VI).
Treatment With a Proteasome Inhibitor Prevents the Beneficial Effect of Ischemic Preconditioning
IPC improved postischemic function over ischemia alone (79% versus 50%, respectively) (Figure 6). This was prevented by lac treatment before IPC at a concentration that had no further effects on recovery of function of nonpreconditioned ischemic hearts. Epoxomicin pretreatment before IPC partially prevented the beneficial effects (Figure 6). Perfusing nonischemic hearts with the inhibitors alone had no effect on function (Figure 6, bottom). Additional studies examined the effects of lac on morphology of ischemic and preconditioned hearts. Electron microscopy of ischemic heart revealed grossly swollen mitochondria with loss of cristae organization and cardiac muscle fibers which appear to be in contracture (Figure 7A). Mitochondrial cross sectional area was increased by 86% (P<0.05) and sarcomere length decreased by 33% (P<0.05) (Figure 7B), changes that, for the most part, were diminished by IPC. In general, pretreatment with lac diminished the beneficial effects of IPC as mitochondrial cross sectional area was increased by 49%, whereas the effects on sarcomere length were completely prevented (Figure 7). The effect of lac on ischemia (without IPC) was qualitatively not different from ischemia alone (data not shown).
Consequences of Proteasome Inhibition on Signaling Proteins
Levels of Bax and cytosolic ubiquitinated proteins were assessed after reperfusion to determine whether proteasome inhibition in the preconditioned heart has any effect. I/R caused a 63% increase (P<0.05) in Bax, which was prevented by IPC (Figure 8A). Lac pretreatment resulted in 131% and 140% increase in Bax in both nonpreconditioned and preconditioned ischemic hearts, respectively, yet no treatments had effects on Bax mRNA levels (Figure 8B). As a general indicator of the functionality of the proteasome and protein quality control, level of soluble ubiquitinated proteins was assessed. Although difficult to quantitate, a trend is apparent in which ubiquitinated proteins were increased following I/R, but decreased following IPC (Figure 8C). Pretreatment with lac resulted in a marginal increase in I/R hearts and completely prevented the protective effects of IPC.
The present study examined the hypothesis that IPC preserves postischemic function of the UPS by diminishing oxidative damage to proteasome subunits, thus facilitating some of the IPC-associated signaling changes through improved protein degradation. The salient findings of this study are: (1) IPC preserves postischemic function of the proteasome, in vitro and in vivo; (2) I/R oxidizes at least one 19S regulatory particle subunit, Rpt5, which is diminished by IPC; (3) inhibition of the proteasome before IPC prevents beneficial effects on Rpt5 oxidation, function, and morphology; and (4) the UPS appears to play a facilitative role in postischemic signaling associated with IPC, in part by degrading proapoptotic proteins.
Ischemic Preconditioning Preserves Postischemic Proteasome Function Through Diminished Oxidation of 19S Regulatory Subunits
This hypothesis was examined by subjecting hearts to a classical IPC protocol that produces consistent improvement in postischemic hemodynamic function.5 IPC preserved postischemic proteasome activity in an in vitro preparation and an in vivo murine model. The greatest protective effect was on the ATP-dependent function suggesting effects on the 19S regulatory particle which uses ATP to activate the 20S proteasome and unfold the protein substrate before presentation to the access channel.21 Previous studies22 have demonstrated postischemic 4-hydroxynonelation of several α-type 20S proteasome subunits and that preischemic treatment with an antioxidant can preserve postischemic proteasome activity.12 Oxidative damage to 19S regulatory particles has not previously been examined in the setting of myocardial ischemia. These studies demonstrate that at least one critical ATPase subunit is subject to significant oxidative carbonylation to a degree consistent with what we5,23 have previously reported for actin and other proteins. Identification of these subunits was supported by a combination of immunoprecipitation, 1D and 2D gel electrophoresis, and mass spectrometry. With regard to the 53-kDa protein(s), the anti-DNPH pull-down experiment suggests that both Rpt3 (S6b ATPase) and Rpt5 (S6a ATPase) account for the oxidation signal although only I/R and IPC-related changes in Rpt5 were observed. These 2 subunits share similar extensive homology and have been reported to form heterodimers.24 Carbonylation of Rpt3/Rpt5 is consistent with a previous study in SH-SY5Y cells showing unique sensitivity of these ATPase subunits to this type of oxidative modification.25 Although it is not clear why oxidation of these subunits should diminish proteasome activity, Rpt5 does play pivotal roles in docking of the “base” of the 19S regulatory particle to 20S proteasome α-rings26 and binding of the 19S particle “lid” to the base.21 Furthermore, RNA interference knockdown of Rpt3 has been reported to decrease proteasome activity.25 IPC-mediated diminished oxidative damage to Rpt5 is consistent with the improved ATP-dependent activity of the proteasome. Remarkably, oxidative damage to other 19S regulatory particle subunits was not observed, although considering that the 53-kDa band contains 11 different subunits, this possibility or the possibility of other oxidative modifications cannot be excluded. Carbonylation of 20S proteasome subunits was also not observed possibly attributable to sequestration of the catalytic sites within the “barrel,” which may explain its’ reported relative resistance to oxidative stress.15 Based on the results of the present study, it seems reasonable to suggest that diminished oxidation of proteasome units, particularly as part of the 19S regulatory particle, accounts for the protective effect in preconditioned hearts.
Proteasome Inhibition Diminishes the Beneficial Postischemic Effects of Ischemic Preconditioning
If the UPS has any role in IPC, diminishing its protective effect on the proteasome should abolish the beneficial effects in the postischemic period. The lack of cardiac-specific genetic models of constitutive proteasome necessitated pretreating isolated hearts with a buffer containing a proteasome inhibitor before the preconditioning protocol. We13 have shown that perfusing hearts with 2 μmol/L lac for 10 minutes decreases proteasome activity by 40%, which in combination with ischemia, resulted in 73% and 78% loss of activity in nonpreconditioned and preconditioned hearts, respectively (Figure 4). The greater effect of lac on the preconditioned hearts can be explained by direct inhibition. Inhibitors have greater affinity for active proteasome, thus the greatest effect is in hearts having the highest activity.27 In addition, there is an indirect effect caused by the abolishment of the beneficial effects of IPC on proteasome subunit oxidation. This may be attributable to enhanced production of reactive oxygen species during early reperfusion as a result of blockade of IPC, thus producing additional inhibition additive to the direct effect. Overall, this suggests that the UPS regulates the signaling pathways involved in the early phases of IPC (discussed in next section). According to this interpretation, proteasome activity should be relatively equal in the lac-treated ischemic and preconditioned hearts, which was the observed effect. Proteasome inhibition also decreased the beneficial effects of IPC on function, without additional effects on nonpreconditioned hearts, an observation somewhat similar to a previous study.28 In the present study, the effect of lac was not attributable to a nonspecific activity, because the inhibitor, epx, had similar, albeit less pronounced, effects, possibly related to its high selectivity for the chymotryptic activity of the proteasome. This in turn can cause less inhibition of overall catalytic activity because of loss of cooperativity between catalytic subunits.29 The changes in postischemic function correlated with similar alterations in myocardial morphology and morphometry. I/R resulted in morphological changes consistent with necrosis which was diminished by IPC an effect prevented by proteasome inhibition. Considered in its entirety, the inhibitor studies link cardiac structure with hemodynamic function and provide cause and effect between proteasome activity and beneficial effects of IPC.
Proteasome Inhibition Alters a Representative Signaling Protein and Levels of Ubiquitinated Proteins
This phenomenon was illustrated by examining Bax, a proapoptotic member of the Bcl family of proteins that plays a major role in cardiomyocyte death signaling during I/R30 and IPC,6 and is degraded through a ubiquitin-dependent pathway.7 Bax levels are reported to increase after I/R and decrease after IPC,6 similar to that observed in the present study. The assumption that changes in Bax expression were attributable to gene transcription are not supported by the present results because no change in Bax mRNA was detectable, suggesting that perhaps increases are attributable to diminished UPS-mediated degradation. This possibility is supported by the proteasome inhibition studies which showed large increases in Bax yet no changes in mRNA, consistent with earlier in vitro studies.31 The inhibitor studies also provide cause and effect as Bax levels increase when proteasome is dysfunctional, yet when protected by IPC, return to baseline. When IPC is blocked by lac, proteasome shows enhanced dysfunction and Rpt5 oxidation and greatly increased Bax levels.
We cannot conclude that the changes in Bax are the proximate cause of the diminished protective effect of IPC in the proteasome inhibitor–treated heart. Rather, loss of IPC-mediated protection is a culmination of interference with UPS-mediated regulation of many pathways, best illustrated by the changes in levels of ubiquitinated proteins which demonstrated a pattern similar to Bax. Consistent with what we11 have previously shown, I/R resulted in increased ubiquitinated proteins, whereas IPC diminished this, an effect blocked by treatment with a proteasome inhibitor. Many of these ubiquitinated proteins are signaling proteins marked for degradation; thus, it is reasonable to suggest that during I/R, production of oxidative species damages the proteasome leading to diminished proteasomal degradation resulting in progressively dysregulated signaling pathways dependent on the degree of proteasome dysfunction. This is essentially the feed-forward amplification loop that has been proposed to account for cell injury when proteasome is inhibited.8 By diminishing production of oxidative species,4 IPC may interfere with this loop by protecting proteasome during early reperfusion, allowing it to reregulate these pathways. Inhibiting the proteasome with lac prevents these pathways from being reregulated leading to progressively more dysregulation. Because some of these pathways may be responsible for production of oxidative species by mitochondria and other sites, a progressive increase in oxidative damage occurs as evidenced by the enhanced Rpt5 oxidation. This interpretation is consistent with prior studies showing that proteasome inhibitor–mediated cell injury may have an associated component of oxidative damage32 possibly related to accumulation of Bax.33 Additional evidence for the importance of the UPS in regulating myocardial signaling pathways is provided by a study suggesting that preischemic changes in PTEN account for loss of IPC protection in mice deficient in the immunoproteasome subunit LMP2 (β1i).34 Given the diverse pathways regulated by the UPS, it is unlikely that only changes in PTEN account for the loss of IPC protection in this model. Although generally supportive, this study34 did focus on immunoproteasome, not constitutive, proteasome; thus, how this study impacts the present study is not clear.
Summary and Conclusion
In summary, the present study has shown that one effect of IPC is preservation of proteasome function through diminished oxidative damage to proteasome subunits, such as Rpt5. Preservation of proteasome activity may facilitate degradation of proapoptotic proteins, such as Bax. Pharmacological blockade of this protection abolishes the cardioprotective effect of IPC leading to increased Rpt5 oxidation and increases in Bax protein. Taken together, these studies support the hypothesis that the UPS facilitates myocardial IPC and may represent a potential therapeutic target for cardiac preservation.
Sources of Funding
This work was supported by NIH grant HL68936 (to S.R.P.) and American Heart Association Grant 0835335N (to A.V.G.).
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Novelty and Significance
What Is Known?
During myocardial ischemia/reperfusion, the ATP-dependent activities of the ubiquitin proteasome system (UPS) can become dysfunctional leading to signaling changes that favor cell death.
Ischemic preconditioning decreases postischemic production of oxidative species and thereby oxidative damage to myocardial proteins.
Ischemic preconditioning converts cell death signals associated with ischemia/reperfusion to prosurvival signals and improves postischemic recovery.
What New Information Does This Article Contribute?
Ischemia/reperfusion promotes oxidation of the 19S regulatory particle subunit of the 26S proteasome complex.
Ischemic preconditioning partially prevents postischemic oxidation of 19S regulatory particle subunits and preserves the function of the ubiquitin proteasome system.
Functional UPS mediates some of the postischemic signaling events associated with ischemic preconditioning.
The UPS plays an important role in the regulation of cardiac signaling pathways. The proteasome has been shown to become dysfunctional following ischemia/reperfusion; however, the mechanism remains unclear, although some studies suggest that oxidative injury may be involved. Ischemic preconditioning decreases oxidative damage and prevents many of the signaling changes associated with ischemia/reperfusion. This study shows that one mechanism for dysfunctional UPS might be the oxidation of at least one subunit of the 19S regulatory particle responsible for proper docking to the 20S proteasome and ATP-dependent functions. Myocardial ischemic preconditioning diminished oxidation of this subunit and preserved the function of the proteasome while, at the same time, decreasing the levels of Bax, a prodeath signaling molecule. Together with other studies in this area, our results suggest that the UPS is both a recipient, as well as a critical downstream facilitator, of the effects of ischemic preconditioning by promoting degradation of multiple signaling proteins, many of which can favor cell death. Future studies might elucidate the role of damage to this subunit in proteasome dysfunction associated with other cardiac pathologies.
Original received October 22, 2009; resubmission received February 25, 2010; revised resubmission received March 26, 2010; accepted April 16, 2010.