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Proteasome mediates removal of proteins oxidized during myocardial ischemia
Free Radical Biology & Medicine 40 (2006) 156 – 164 www.elsevier.com/locate/freeradbiomed
Original Contribution
Proteasome mediates removal of proteins oxidized during myocardial ischemia Andras Divald a, Saul R. Powell a,b,* b
a Department of Medicine, Institute for Medical Research, North Shore – Long Island Jewish Health System, New Hyde Park, NY 11042, USA Department of Medicine, Long Island Jewish Medical Center Campus of the Albert Einstein College of Medicine, New Hyde Park, NY 11042, USA
Received 28 July 2005; revised 6 September 2005; accepted 22 September 2005 Available online 17 October 2005
Abstract Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 Amol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystinpretreated ischemic hearts was significantly ( P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly ( P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss ( 6.5%) of 3methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome. D 2005 Elsevier Inc. All rights reserved. Keywords: Proteasome; Actin; Myocardial ischemia; Protein oxidation; 3-Methylhistidine; Free radical
Introduction During and following an ischemic insult tissues undergo increased oxidative stress with enhanced production of oxidant species [1,2]. As a downstream effect proteins can undergo oxidative modifications, such as introduction of carbonyl groups and hydroxyl groups leading to increased protein hydrophobicity, oxidation of sulfhydryls and tyrosine groups which may lead to protein crosslinking (reviewed in [3]), and various other modifications. Oxidation of proteins during myocardial ischemia was first shown by Park et al. [4] who demonstrated increased protein carbonyls and mixed disulfides
Abbreviations: His, histidine; KH, Krebs-Henseleit; Lac, lactacystin; 3-MH, 3-methylhistidine; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid. * Corresponding author. Long Island Jewish Medical Center, Room B387, 270-05 76th Avenue, New Hyde Park, NY 11042, USA. Fax: +1(718) 470 1732. E-mail address: [email protected] (S.R. Powell). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.09.022
after perfusion of isolated hearts. Subsequently, we [5] confirmed this finding demonstrating that oxidation of soluble proteins appears to be nonspecific, and that zinc, possibly through an antioxidative mechanism, decreases protein oxidation. We have identified sarcomeric and cytoskeletal actins as major proteins oxidized [6,7] and more recently have shown that mitochondrial proteins are also oxidized [8]. The consequences of oxidation of these proteins are largely unknown, though it has been suggested that oxidative modification renders proteins more susceptible to proteolytic digestion [9]. During myocardial ischemia numerous myofibrillar and cytoskeleton proteins, such as the troponins, myosin, actin, and others [10 –15], have been shown to be selectively proteolysed and lost. Of these, actin has been shown to be rapidly lost from human myocardial explants under hypoxic conditions [13,14]. There has been much speculation as to the enzyme(s) catalyzing proteolysis of these proteins. Based largely on inhibitor studies, a significant role has been suggested for the Ca2+-dependent proteases, most notably,
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calpain [16,17]. To date, there have been no studies examining the role of the proteasome in removal of these oxidatively modified proteins despite numerous studies in a variety of nonischemic systems that demonstrate this function [18 – 20]. In the current study, we examine the hypothesis that proteasome plays a significant role in removal of proteins oxidized during myocardial ischemia. The initial rationale for this hypothesis is based on our recent study [21] that demonstrated an association between postischemic proteasome activity and levels of oxidized proteins, but did not show that proteasome actually degrades these proteins. We show that preischemic inhibition of the proteasome with the inhibitor, lactacystin (Lac), results in greater accumulation of oxidized proteins in the postischemic isolated rat heart preparation. Moreover, using loss of 3-methylhistidine (3-MH) as a marker for actin degradation, we show that lactacystin pretreatment can effectively block ischemia-mediated degradation of this contractile protein, directly implicating the proteasome in its removal.
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analyzed using the AcqKnowledge Ver 3.8.1 Data Analysis Package (Biopac Systems Inc.). Hemodynamic data are expressed as the heart rate systolic pressure product. Hearts were excluded from the study if they developed persistent arrhythmias, had systolic pressure lower than 70 mm Hg, or had a heart rate below 220 bpm during the equilibration period. Experimental protocol Hearts were equilibrated for 20 min and then subjected to 15 or 30 min normothermic global ischemia followed by up to 60 min of aerobic reperfusion. To inhibit proteasome, some hearts were perfused through a separate column with Lac, 2 Amol/L in KH, for 10 min, followed by a 3 min washout during the preischemic equilibration period. Following reperfusion the ventricles were snap-frozen in liquid N2. To minimize proteasome degradation and sample variability, specimens were stored at 70-C for no more than 6 weeks, and repetitive freeze-thawing was avoided [21].
Materials and methods Assay of proteasome activity Animals Male Sprague Dawley rats (250 – 300 g) were obtained from Taconic Farms (Germantown, NY) and allowed a 3-day inhouse acclimatization period and ad libitum access to food (Ralston Purina Co., St. Louis, MO) and water prior to experimental use. All protocols were approved by the Institutional Animal Care and Utilization Committee and were in compliance with the NIH Guide for the Care and Use of Laboratory Animals (revised 1996). Chemicals and reagents Chemicals and reagents were obtained from reputable sources. Lactacystin and suc-LLVY-AMC were obtained from Biomol Research Labs (Plymouth Meeting, PA).
Proteasome activity was determined in cell lysates using the method of Grune et al. [24] as previously described [21]. Samples were incubated T Lac, 20 Amol/L, to differentiate between proteasome and non-proteasome-mediated activity and T ATP, 28 – 56 Amol/L, to differentiate between 20S and 26S activities of the proteasome. ATP-independent 20S and ATP-dependent 26S proteasome activities were calculated from fluorescence readings as follows. 20S proteasome activity is calculated as the difference between total proteolytic activity and that remaining in the presence of Lac. 26S proteasome activity is calculated as the difference between total ATP-stimulated proteolytic activity and that remaining in the presence of Lac, minus 20S proteasome activity. Immunoblot analysis of protein carbonyls
Isolated heart preparation Male Sprague-Dawley rats were treated with 500 U heparin sodium (ip) and anesthetized with 100 mg/kg pentobarbital sodium (ip). The hearts were rapidly removed, placed in ice-cold heparinized saline, and perfused using retrograde aortic perfusion in the Langendorff mode [22] as previously described [23]. The perfusate was a modified Krebs-Henseleit (KH) buffer consisting of (mmol/L) NaCl 118, KCl 4.9, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11, CaCl2 2.5, and oxygenated with 95% O2/5% CO2, at 37-C, and a constant pressure equivalent to 90 cm H2O. Systolic pressure was monitored by means of a fluid-filled latex balloon (0.1 ml) connected to a pressure transducer that was inserted into the left ventricle and expanded to exert a physiologic end-diastolic pressure of 5 mm Hg. Hemodynamic data were captured using the Biopac MP100 Acquisition Module (Biopac Systems Inc., Goleta, CA) and
Protein oxidation was detected using the Oxyblot kit (Chemicon International, Temecula, CA) as previously described [5,6]. Basically, proteins were reacted with dinitrophenylhydrazine to tag carbonyl groups and then separated under reducing conditions using standard sodium dodecyl sulfate (SDS) polylyacrylamide gel electrophoresis (PAGE) [25]. Protein carbonyls were then determined using an immunoblot technique and antibody specific for dinitrophenylhydrazine as previously described [6]. Band size and intensities were evaluated using computer-assisted densitometry (SigmaScan Image Analysis software ver 1.20.09, Jandel Scientific (SSI), Richmond, CA). Degradation of actin Degradation of actin was determined by analysis of 3-MH based on the chromatography method of Wassner et al. [26].
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Myofibrillar proteins were isolated by pulverizing frozen ventricular tissue under liquid N2 and homogenizing 1 part tissue in 4 parts buffer containing (mmol/L): Hepes 10, NaCl 137, KCl 4.6, KH2PO4 1.1, MgSO4 0.6, EDTA 1, digitonin 0.01%, pH 7.4, plus 1 protease inhibitors (Protease Cocktail Set 1, EMD-Calbiochem, San Diego, CA). The homogenates were centrifuged at 10,000g for 20 min at 4-C, and the resulting pellets were treated with 1% Triton TX100, followed by dissolution of the sediment in Tris buffer, 10 mmol/L, containing 2% SDS. Myofibrillar proteins (2 mg) were precipitated with deoxycholate, 0.03%, and TCA, 25% (final concentrations), dried by washes with acetone and ether, followed by hydrolysis in 6 N HCl:TFA (2:1) at 120-C for 16 h. Aliquots (25 Al) of dried hydrolysates in HCl, 0.01 N, were buffered with sodium borate, 0.2 mol/L, pH 10, followed by derivatization with fluorescamine, 0.67 mg/ml, by heating at 80-C for 50 min. The derivatized amino acids were separated using high-pressure liquid chromatography (Series 410 LC pump, Perkin-Elmer, Boston MA) on a ˚´ ,4 Am, 4.6 250 mm; Waters, Novapak C18 column (60 A Milford, MA), and detected using fluorometric spectroscopy (Model 1700, Bio-Rad Labs., Hercules, CA) at excitation, 365 nm, and emission, 455 nm. Chromatography data were captured and analyzed using the PeakSimple Chromatography System ver 3.2.1 (SRI Instruments, Inc., Las Vegas, NV). Both 3-MH and unmodified histidine (His) peaks were identified using internal standards (10 and 100 Amol/L). Comparisons were made between integrated areas of 3-MH and His peaks to estimate the changes in the methylated amino acid. 3-MH content was expressed as the percentage of total myofibrillar His in each sample after a factor accounting for the ratio of sensitivities for fluorometric responses to His/3-MH was applied. This factor was determined from peak areas obtained using equimolar calibrating solutions of his and 3-MH, respectively, and was found to be 0.487 T 0.027 (N = 4).
actin (clone AC-40; Sigma-Aldrich, St. Louis, MO) which recognizes a C-terminus epitope common to all actin isoforms. To minimize interference by the heavy- and light-chain IgGs, the actin immuno-complex was captured using the TrueBlot reagent kit (eBioscience, San Diego, CA) according to the literature protocol. Briefly, the precleared homogenates were rotated with 6 Ag of antibody for 1 h and then with antimouse-IgG-coupled TrueBlot beads for an additional hour. The beads were washed exhaustively with TBS buffer followed by the addition of reducing Laemmli sample buffer containing 2% h-mercaptoethanol and boiled for 10 min. Immunoprecipitated samples were then separated using standard SDS-PAGE and then electroblotted to a PVDF membrane. As a positive control, a-sarcomeric actin (SigmaAldrich) was also immunoprecipitated. The membranes were then probed with the same antibody specific for actin (clone AC-40) followed by multiple washings of the membrane and subsequent incubation with peroxidase-conjugated secondary antibody (anti-mouse TrueBlot IgG) and visualized with enhanced chemiluminescence autoradiography. Immunoprecipitated actin was also probed for ubiquitin in separate membranes or after stripping of the actin-probed membrane and visualized as described.
Immunoblot analysis of ubiquitinated proteins
Inhibition of myocardial proteasome
Ubiquitinated proteins were assessed as previously described [21] following protein separation using standard SDSPAGE and electroblotting to a PVDF membrane. The membrane was probed with a polyclonal antibody (1:1000) specific for ubiquitin (EMD-Calbiochem, San Diego, CA) and developed using enhanced chemiluminescence autoradiography (Perkin-Elmer Life Sciences, Boston, MA) as previously described [21].
Preischemic inhibition of myocardial proteasome was effected by treatment of hearts with the irreversible inhibitor, Lac [27,28]. Preliminary studies examined several concentrations to determine the best inhibition profile (data not shown). For reasons discussed in the next section, preischemic inhibition of approximately 40 –50% that would persist for a minimum of 90 min was felt to be most desirable for these experiments. The concentration of Lac observed to produce this degree of inhibition was 2 Amol/L infused into the heart over a period of 10 min which resulted in a 51% decrease in 20S proteasome and a 42% decrease in 26S proteasome activities (Fig. 1) still present after 90 min.
Immunoprecipitation of actins Ventricular tissue was homogenized in buffer containing (mmol/L): Hepes 10, NaCl 137, KCl 4.6, KH2PO4 1.1, MgSO4 0.6, EDTA 1, digitonin 0.01%, pH 7.4, plus 1 protease inhibitors (Protease Cocktail Set 1, EMD-Calbiochem) and centrifuged at 10,000g, at 4-C, for 30 min. An aliquot containing 300 Ag of supernatant protein was immunoprecipitated with a monoclonal antibody specific for
Statistical analysis All results are expressed as mean T SE. Statistical significance of differences between two populations with equal variance was with a Student’s t test. Analysis of differences between multiple sample populations with equal variances was with one-way ANOVA followed by Tukeys test for post hoc analysis. Statistical differences of P < 0.05 were considered to be significant. All statistics were performed using the SigmaStat statistical analysis package (Jandel Scientific (SSI)). Results
Preischemic proteasome inhibition increases accumulation of protein carbonyls This series of experiments examined the effect of proteasome inhibition on hemodynamic recovery and protein
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Fig. 1. Inhibition of myocardial proteasome by lactacystin. Isolated hearts were perfused with buffer containing Lac 2 Amol/L for 10 min. Hearts were then perfused with buffer alone for an additional 90 min and then harvested and analyzed for proteasome activities. 20S proteasome activity is calculated as the difference between total proteolytic activity and that remaining in the presence of Lac. 26S proteasome activity is calculated as the difference between total ATP-stimulated proteolytic activity and that remaining in the presence of Lac, minus 20S proteasome activity. The values represent the mean T SE of four hearts. *P < 0.05 (Student t test) when compared with control hearts.
carbonylation after ischemia and reperfusion. Exposure to 15 min ischemia has minimal effect on recovery of function and inducing this degree of preischemic proteasome inhibition does not change this (data not shown). In this series of experiments, 30 min ischemia had no significant effects on recovery even though function was still depressed 31% (Fig. 2) after 60 min reperfusion. In hearts not subjected to ischemia, a 10-min pulse treatment with Lac, 2 Amol/L, resulted in a 17% decrease in hemodynamic function after 90 min of perfusion that was not significantly different from control hearts (data not shown). However, in hearts made ischemic, prior treatment with Lac did result in additional depression ( 40%) which was now significantly ( P < 0.05) less than preischemic hearts. Representative immunoblots depicting the effect of proteasome inhibition on bands of carbonylated proteins in hearts subjected to 15 or 30 min
Fig. 2. The effect of proteasome inhibition on postischemic recovery of hearts. Isolated hearts were subjected to 30 min normothermic global ischemia followed by 60 min of reperfusion. Lactacystin-treated hearts were perfused with buffer containing 2 Amol/L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. The values represent the mean T SE of four to seven observations. P < 0.05 (ANOVA, Tukeys) when compared with preischemic baseline values.
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Fig. 3. Proteasome inhibition increases accumulation of oxidized proteins following myocardial ischemia. Control (C) hearts were perfused with buffer for 90 min. Ischemic/reperfused (I) hearts were subjected to 15 (top panel) or 30 min (bottom panel) normothermic global ischemia followed by 60 min of reperfusion. Lactacystin-treated (L) hearts were perfused with buffer containing 2 Amol/L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. Lactacystin plus ischemia/reperfusion (IL) hearts were pretreated with the proteasome inhibitor and then subjected to the ischemia reperfusion protocol. At the end of the treatment protocols, all hearts were harvested and analyzed for protein carbonyls. The membrane is representative of five to seven separate determinations. A densitometric analysis is presented in Fig. 4.
ischemia and 60 min reperfusion are illustrated in Fig. 3. In general, Lac alone (Fig. 3, lane L (top and bottom panels)) appeared to have marginal effects on the degree of protein carbonylation. Likewise, exposure to 15 min ischemia (Fig. 3, lane I, top panel) had no effect, even after treatment with Lac (Fig. 3, lane IL, top panel). Consistent with previous studies [6,21], 30 min ischemia appears to increase protein carbonylation across a wide range of molecular masses (Fig. 3, lane I, bottom panel), an effect which was enhanced in the presence of Lac-induced proteasome inhibition (Fig. 3, lane IL, bottom panel). This effect was quantitated using computer-assisted densitometry which is presented in Fig. 4. We observed that bands of proteins of molecular masses of 95 kDa (top panel), 50 kDa (middle panel), and 40 kDa (bottom panel) were reasonably consistent and were chosen for analysis. A trend is immediately apparent with + Lac alone, or 15 min ischemia T Lac having no significant effects on carbonyl content of these bands (Fig. 4). Treatment with 30 min ischemia generally results in approximately 2-fold increases in detectable carbonyls in all three bands (Fig. 4). However, proteasome inhibition followed by 30 min ischemia resulted in significantly ( P < 0.05) greater accumulations of protein carbonyls in all three bands (90 kDa, 6-fold; 50 kDa, 3.3-fold; 40 kDa, 3.6-fold) suggesting either enhanced formation or decreased degradation of protein carbonyls (Fig. 4).
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represented 12% of unmodified His in agreement with other studies that concluded that of the eight His in this protein, only one is methylated [29,30]. Lactacystin alone had no effect on the 3-MH:His fraction after 90 min of perfusion (Fig. 5). However, 30 min ischemia results in a significant ( P < 0.05) 15% decrease in the 3-MH:His fraction after 60 min of reperfusion, suggesting proteolysis of actin (Fig. 5). Pretreatment of these hearts with Lac prevented much of this decrease with only a 6.5% loss after 60 min of reperfusion, suggesting that proteasome mediates part of this decrease. Actin is not ubiquitinated during myocardial ischemia/reperfusion This experiment examined whether loss of actin was ubiquitin dependent and mediated by the 26S proteasome, or was degraded directly by the 20S proteasome. Ubiquitination of proteins isolated from hearts exposed to ischemia/ reperfusion is depicted in Fig. 6A. As predicted, hearts treated with Lac had higher levels of ubiquitinated proteins, with Lac-pretreated ischemic/reperfused hearts having the highest content (Fig. 6A – Lane ‘‘IL’’). For the purposes of these experiments, actin was immunoprecipitated from control (C), ischemic/reperfused (I), and Lac-pretreated ischemic/reperfused (IL) hearts, as well as an additional control sample containing purified actin (A) (Fig. 6B). When this membrane was stripped of the anti-actin antibody and then probed with an antibody specific for ubiquitin, no bands representing ubiquitinated homologues were visible in any of the lanes (Fig. 6C). The exact same results were obtained if the immunoprecipitated proteins were probed for ubiquitin or actin on separate membranes without the need for stripping Fig. 4. Densitometric analysis of protein oxidation data. Hearts were treated as described in Fig. 3. Illustrated above is the densitometric analysis of bands of proteins containing carbonyls at molecular masses of 95 kDa (top panel), 50 kDa (middle panel), and 40 kDa (bottom panel) (see arrows—Fig. 3). Density was calculated as the size of the band average density of the band. All values are presented as percentage of the control hearts (95 kDa, 7714 T 915; 50 kDa, 40,709 T 8236; 21,170 T 4334 arbitrary units) and represent the mean T SE of 5 to 7 separate determinations. aP < 0.05 (ANOVA, Tukeys) when compared with control hearts. bP < 0.05 (ANOVA, Tukeys) when compared with +Lac hearts. c P < 0.05 (ANOVA, Tukeys) when compared with 30 min ischemia hearts.
Inhibition of the proteasome prevents loss of 3-methylhistidine As an indicator of actin degradation loss of 3-MH following ischemia and reperfusion was examined. Initial experiments calculated 3-MH content standardized to initial protein content which yielded 102 T 38 Umol/mg protein in control hearts. The high variability reflects varying yields for protein precipitation and derivatization among sets of experiments. For this reason, 3-MH was standardized to His content. In control rat heart myofibrillar proteins, 3.05 T 0.18% of His was in the form of 3-MH, representing about 25% of the value obtained by the analysis of a hydrolysate of pure actin. In this pure actin preparation, 3-MH
Fig. 5. Proteasome inhibition prevents loss of 3-methylhistidine after myocardial ischemia. Control isolated hearts were perfused with buffer for 90 min. Lactacystin-treated hearts were perfused with buffer containing 2 Amol/ L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. Ischemia/reperfusion hearts were subjected to 30 min normothermic global ischemia followed by 60 min of reperfusion. Ischemia/ reperfusion + Lac hearts were pretreated with the proteasome inhibitor and then subjected to the ischemia reperfusion protocol. At the end of the treatment protocols, all hearts were harvested and analyzed for 3-MH and His content. The values are expressed as the ratio (%) of 3-MH:His and represent the mean T SE of four to seven hearts. *P < 0.05 (ANOVA, Tukeys) when compared with control or (+) Lac alone groups.
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Fig. 6. Actin is not ubiquitinated during myocardial ischemia/reperfusion. Control (C) hearts were perfused with buffer for 90 min. Ischemic/reperfused (I) hearts were subjected to 30 min normothermic global ischemia followed by 60 min of reperfusion. Lactacystin-treated (L) hearts were perfused with buffer containing 2 Amol/L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. Lactacystin plus ischemia/reperfusion (IL) hearts were pretreated with the proteasome inhibitor and then subjected to the ischemia reperfusion protocol. Panel A depicts ubiquitinated proteins from hearts treated according to the above protocols. Panel B depicts actin immunoprecipitated from samples ‘‘C’’, ‘‘I’’, and ‘‘IL’’. Lane A is a control containing immunoprecipitated purified actin. Panel C depicts the membrane in Panel B after stripping and probing with an antibody specific for ubiquitin.
of antibodies (membranes not shown). This result suggests that actin is not ubiquitinated during myocardial ischemia and reperfusion of the magnitude or time frame used in these experiments. Discussion One of the major pathways for intracellular protein degradation in eukaryotic cells is a multicatalytic proteolytic complex, known as the proteasome (macroxyproteinase). The core catalytic complex of the proteasome contains 28 subunits arranged in a cylindrical structure composed of four rings, each containing seven subunits. The proteolytic center is located inside the cylinder and has ‘‘trypsin-like,’’ ‘‘chymotrypsinlike,’’ and ‘‘peptidylglutamyl-peptide hydrolase-like’’ activity. This complex is also known as the 20S proteasome [31]. Capping of each end of the 20S proteasome with a regulatory 19S complex, each containing at least 15 additional subunits, confers ATP and ubiquitin specificity [32,33]. This entire complex is known as the 26S proteasome which is responsible for turnover of important regulatory proteins, such as the cyclins and transcription factors, and plays an important role in antigen presentation [32 –34]. Proteins are targeted to the 26S proteasome following multiubiquitination by sequential addition of ubiquitins to the (-amino group of a protein lysine [32]. While somewhat controversial, recent evidence suggests that oxidatively modified proteins can be removed by the ‘‘core’’ 20S proteasome without requirement for ATP or ubiquitin [18 – 20,35]. Numerous studies have examined removal of proteins damaged during myocardial ischemia, yet none have examined the role of the proteasome. The current study examined the hypothesis that proteasome can mediate removal of proteins oxidized during myocardial ischemia. The salient observations were threefold: (1) inhibition of the proteasome with Lac increases postischemic accumulation of protein carbonyls; (2) 3-MH content is decreased in the postischemic heart, indicating
loss of myocardial actin, and this effect is partially blocked by proteasome inhibition; and (3) there appears to be no formation of ubiquitinated homologues of actin, suggesting proteolysis by the 20S proteasome. To demonstrate a role for the proteasome it was necessary to treat the hearts with the inhibitor, Lac. The selection of Lac was based on its irreversibility [27,28] which allowed for pulse treatment of the hearts, and for its high degree of specificity [36], which makes it preferable to MG132, which is a reversible inhibitor and is known to inhibit cysteine proteases, such as cathepsins and calpains, at concentrations not that different from that which inhibits the proteasome [36 – 38]. We [21] and others [39] have previously shown that myocardial ischemia of sufficient duration can result in significant inhibition of proteasome. So as not to inhibit proteasome completely a concentration and treatment duration that effected 40 to 50% inhibition of 20S and 26S proteasome prior to ischemia was utilized. Inhibition of this magnitude by this class of proteasome inhibitors is generally considered to be specific and not associated with significant inhibition of other proteases (reviewed in [36]). Inhibition of this degree marginally decreased hemodynamic recovery of postischemic hearts. While not of the same degree, this observation is in general agreement with what we have published previously [21], although a reversible inhibitor was used in this prior study without determination of degree of proteasome inhibition. In either case, improvement of function was clearly not observed as has been reported by other groups using the inhibitor, PS519 [40,41]. Formation of protein carbonyls was used as an indicator of oxidative modification of proteins during myocardial ischemia. While not the only oxidative modification to proteins that occurs during ischemia, it is generally considered to be representative, and we [5 – 8] and others [4,42,43] have consistently demonstrated increases. The increases in protein carbonyls in postischemic hearts that were observed in the
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current study are in general agreement with what we have published previously [6– 8,21]. Short duration ischemia did not result in significant accumulation of protein carbonyls and additional proteasome inhibition of the magnitude used in this study had no additional effect. Interpretation in combination with the observation that proteasome inhibition alone (no ischemia) does not result in significant accumulation of protein carbonyls suggests that excess proteasome is present and that the heart is not adversely effected by 40 to 50% proteasome inhibition unless subjected to increased oxidative stress, at least within the time frame of these experiments. On the other hand, longer ischemia resulted in accumulation of protein carbonyls, an effect which was significantly enhanced by the use of a proteasome inhibitor, suggesting that proteasome removes these modified proteins. An alternate explanation for this effect is that proteasome inhibition has been suggested to result in increased production of oxidative species and protein modifications [44]. The lack of effect of Lac on the nonischemic and short duration ischemic hearts suggests, but is not conclusive, that this is not the case. In order to refute this possibility, degradation of a specific protein was examined. We [6,7] have previously shown that sarcomeric and cytoskeletal actins are targets of oxidative injury in the postischemic heart, and that oxidized actin is one of the major components of the 50-kDa band (see Fig. 3) of carbonylcontaining proteins. Therefore, we quantitated actin proteolysis by measuring loss of 3-MH. 3-Methylation is a posttranslational modification of His that is generally considered to be peculiar to two myofibrillar proteins, actin and myosin [45,46]. Loss of this amino acid is an accepted indicator of proteolysis of muscle myofibrillar protein and would usually be representative of loss of both of these proteins [45]. However, in mammalian heart, myosin contains only methyllysine and not methylhistidines; thus, actin is the only source [46] and decreases are a quantitative indicator of its degradation only. We observed a 15% loss of 3-MH within 1 h of reperfusion, indicating a significant loss of this protein following myocardial ischemia, a result in general agreement with older studies [13,47] using different techniques. Actin is a long-lived protein, with a reported t 2 of 10.3 days [48]; thus, it is highly unlikely that normal turnover contributed to this loss. That proteasome mediates at least part of this degradation was indicated by the ability of Lac to prevent much of this decrease. It is no great surprise that Lac did not completely prevent actin degradation as the chosen dose does not completely inhibit proteasome. Nor should it be assumed that proteasome is the only proteolytic enzyme or complex involved in its degradation. While supportive of the overall hypothesis, these observations shed no light on which proteasome, the 20S or 26S, is actually mediating actin degradation. Since the 26S proteasome recognizes only ubiquitinated substrates [32], it should have been possible to isolate ubiquitinated homologues of actin, particularly in the presence of significant inhibition of 26S proteasome. Our results demonstrate significant inhibition of 26S proteasome coincident with increased accumulation of ubiquitinated proteins in agreement with previous observations
[21]. Yet, we failed to demonstrate the presence of any ubiquitinated homologues whether actin is immunoprecipitated from control, ischemic/reperfused, or Lac-pretreated ischemic/ reperfused hearts, suggesting that the 20S proteasome is mediating this effect. This conclusion is entirely consistent with studies of proteasome-mediated degradation of oxidized proteins in cellular systems as well as what is known about actin degradation. Studies by Davies and co-workers [18 – 20,35] in cell preparations have consistently shown that 20S proteasome mediates removal of oxidized proteins without requirement for ATP or ubiquitin. These investigators have theorized that certain oxidative modifications increase the hydrophobicity of a protein, either by introduction of hydrophobic groups or protein unfolding exposing hydrophobic patches, which targets the protein to the 20S proteasome (reviewed in [3]). While there is evidence that actin can be degraded via a ubiquitin-dependent pathway, this appears to be limited to increased catabolic states, such as starvation, posttraumatic injury, and sepsis, when proteins are catabolized as an alternate source of energy substrates [49 – 52]. In summary, these studies have shown that inhibition of the proteasome results in enhanced accumulation of oxidized proteins in the postischemic heart. Furthermore, actin degradation is increased in the postischemic heart, a process that is partially blocked by a proteasome inhibitor. Lastly, failure to isolate ubiquitinated homologues of actin provides strong evidence that the 20S proteasome mediates removal of this oxidized protein in a process independent of ubiquitin. In conclusion, these studies provide the first evidence that proteasome mediates removal of proteins oxidized during myocardial ischemia. Acknowledgment This study was supported by NIH R01 HL68936 (S.R.P.). References [1] Bolli, R.; Jeroudi, M. O.; Patel, B. S.; DuBose, C. M.; Lai, E. K.; Roberts, R.; McCay, P. B. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc. Natl. Acad. Sci. USA 86:4695 – 4699; 1989. [2] Powell, S. R.; Hall, D. Use of salicylate as a probe for OH formation in isolated ischemic rat hearts. Free Radic. Biol. Med. 9:133 – 141; 1990. [3] Grune, T.; Merker, K.; Sandig, G.; Davies, K. J. Selective degradation of oxidatively modified protein substrates by the proteasome. Biochem. Biophys. Res. Commun. 305:709 – 718; 2003. [4] Park, Y.; Kanekal, S.; Kehrer, J. P. Oxidative changes in hypoxic rat heart tissue. Am. J. Physiol. 260:H1395 – H1405; 1991. [5] Powell, S. R.; Gurzenda, E. M.; Wingertzahn, M. A.; Wapnir, R. A. Promotion of copper excretion from the isolated perfused rat heart attenuates post-ischemic cardiac oxidative injury. Am. J. Physiol. 277:H956 – H962; 1999. [6] Powell, S. R.; Gurzenda, E. M.; Wahezi, S. E. Actin is oxidized during myocardial ischemia. Free Radic. Biol. Med. 80:1171 – 1176; 2001. [7] Schwalb, H.; Olivson, A.; Li, J.; Houminer, E.; Wahezi, S. E.; Opie, L. H.; Maulik, D.; Borman, J. B.; Powell, S. R. Nicorandil decreases postischemic actin oxidation. Free Radic. Biol. Med. 31:607 – 614; 2001. [8] Khaliulin, I.; Schwalb, H.; Wang, P.; Houminer, E.; Grinberg, L.; Katzeff, H. L.; Borman, J. B.; Powell, S. R. Preconditioning improves
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Original Contribution
Proteasome mediates removal of proteins oxidized during myocardial ischemia Andras Divald a, Saul R. Powell a,b,* b
a Department of Medicine, Institute for Medical Research, North Shore – Long Island Jewish Health System, New Hyde Park, NY 11042, USA Department of Medicine, Long Island Jewish Medical Center Campus of the Albert Einstein College of Medicine, New Hyde Park, NY 11042, USA
Received 28 July 2005; revised 6 September 2005; accepted 22 September 2005 Available online 17 October 2005
Abstract Numerous proteins are known to be lost following myocardial ischemia/reperfusion yet little is known about the mediating proteinases. This study examines the hypothesis that proteasome plays a significant role in the removal of proteins oxidized during myocardial ischemia. Proteasome was inhibited by perfusing isolated rat hearts with buffer containing lactacystin, 2 Amol/L, for 10 min, which resulted in 51 and 42% decreases in 20S and 26S proteasome activities that persisted for a minimum of 90 min. Lactacystin pretreatment had minor effects on postischemic recovery of isolated hearts exposed to 30 min global ischemia and 60 min reperfusion. Protein carbonyl content of lactacystinpretreated ischemic hearts was significantly ( P < 0.05) increased. One band with approximate molecular mass of 50 kDa is known to contain oxidized actin. Actin degradation was quantitated by analysis of 3-methylhistidine which was significantly ( P < 0.05) decreased by 15% following 30 min ischemia and 60 min reperfusion. Pretreatment of ischemic hearts with lactacystin prevented much of the loss ( 6.5%) of 3methylhistidine. Probing immunoprecipitated actin with an antibody specific for ubiquitin revealed no bands containing ubiquitinated homologues of this protein. These observations support the conclusion that proteasome mediates removal of some of the proteins oxidized during myocardial ischemia/reperfusion, and that at least oxidized actin is removed by the 20S proteasome. D 2005 Elsevier Inc. All rights reserved. Keywords: Proteasome; Actin; Myocardial ischemia; Protein oxidation; 3-Methylhistidine; Free radical
Introduction During and following an ischemic insult tissues undergo increased oxidative stress with enhanced production of oxidant species [1,2]. As a downstream effect proteins can undergo oxidative modifications, such as introduction of carbonyl groups and hydroxyl groups leading to increased protein hydrophobicity, oxidation of sulfhydryls and tyrosine groups which may lead to protein crosslinking (reviewed in [3]), and various other modifications. Oxidation of proteins during myocardial ischemia was first shown by Park et al. [4] who demonstrated increased protein carbonyls and mixed disulfides
Abbreviations: His, histidine; KH, Krebs-Henseleit; Lac, lactacystin; 3-MH, 3-methylhistidine; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid. * Corresponding author. Long Island Jewish Medical Center, Room B387, 270-05 76th Avenue, New Hyde Park, NY 11042, USA. Fax: +1(718) 470 1732. E-mail address: [email protected] (S.R. Powell). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.09.022
after perfusion of isolated hearts. Subsequently, we [5] confirmed this finding demonstrating that oxidation of soluble proteins appears to be nonspecific, and that zinc, possibly through an antioxidative mechanism, decreases protein oxidation. We have identified sarcomeric and cytoskeletal actins as major proteins oxidized [6,7] and more recently have shown that mitochondrial proteins are also oxidized [8]. The consequences of oxidation of these proteins are largely unknown, though it has been suggested that oxidative modification renders proteins more susceptible to proteolytic digestion [9]. During myocardial ischemia numerous myofibrillar and cytoskeleton proteins, such as the troponins, myosin, actin, and others [10 –15], have been shown to be selectively proteolysed and lost. Of these, actin has been shown to be rapidly lost from human myocardial explants under hypoxic conditions [13,14]. There has been much speculation as to the enzyme(s) catalyzing proteolysis of these proteins. Based largely on inhibitor studies, a significant role has been suggested for the Ca2+-dependent proteases, most notably,
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calpain [16,17]. To date, there have been no studies examining the role of the proteasome in removal of these oxidatively modified proteins despite numerous studies in a variety of nonischemic systems that demonstrate this function [18 – 20]. In the current study, we examine the hypothesis that proteasome plays a significant role in removal of proteins oxidized during myocardial ischemia. The initial rationale for this hypothesis is based on our recent study [21] that demonstrated an association between postischemic proteasome activity and levels of oxidized proteins, but did not show that proteasome actually degrades these proteins. We show that preischemic inhibition of the proteasome with the inhibitor, lactacystin (Lac), results in greater accumulation of oxidized proteins in the postischemic isolated rat heart preparation. Moreover, using loss of 3-methylhistidine (3-MH) as a marker for actin degradation, we show that lactacystin pretreatment can effectively block ischemia-mediated degradation of this contractile protein, directly implicating the proteasome in its removal.
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analyzed using the AcqKnowledge Ver 3.8.1 Data Analysis Package (Biopac Systems Inc.). Hemodynamic data are expressed as the heart rate systolic pressure product. Hearts were excluded from the study if they developed persistent arrhythmias, had systolic pressure lower than 70 mm Hg, or had a heart rate below 220 bpm during the equilibration period. Experimental protocol Hearts were equilibrated for 20 min and then subjected to 15 or 30 min normothermic global ischemia followed by up to 60 min of aerobic reperfusion. To inhibit proteasome, some hearts were perfused through a separate column with Lac, 2 Amol/L in KH, for 10 min, followed by a 3 min washout during the preischemic equilibration period. Following reperfusion the ventricles were snap-frozen in liquid N2. To minimize proteasome degradation and sample variability, specimens were stored at 70-C for no more than 6 weeks, and repetitive freeze-thawing was avoided [21].
Materials and methods Assay of proteasome activity Animals Male Sprague Dawley rats (250 – 300 g) were obtained from Taconic Farms (Germantown, NY) and allowed a 3-day inhouse acclimatization period and ad libitum access to food (Ralston Purina Co., St. Louis, MO) and water prior to experimental use. All protocols were approved by the Institutional Animal Care and Utilization Committee and were in compliance with the NIH Guide for the Care and Use of Laboratory Animals (revised 1996). Chemicals and reagents Chemicals and reagents were obtained from reputable sources. Lactacystin and suc-LLVY-AMC were obtained from Biomol Research Labs (Plymouth Meeting, PA).
Proteasome activity was determined in cell lysates using the method of Grune et al. [24] as previously described [21]. Samples were incubated T Lac, 20 Amol/L, to differentiate between proteasome and non-proteasome-mediated activity and T ATP, 28 – 56 Amol/L, to differentiate between 20S and 26S activities of the proteasome. ATP-independent 20S and ATP-dependent 26S proteasome activities were calculated from fluorescence readings as follows. 20S proteasome activity is calculated as the difference between total proteolytic activity and that remaining in the presence of Lac. 26S proteasome activity is calculated as the difference between total ATP-stimulated proteolytic activity and that remaining in the presence of Lac, minus 20S proteasome activity. Immunoblot analysis of protein carbonyls
Isolated heart preparation Male Sprague-Dawley rats were treated with 500 U heparin sodium (ip) and anesthetized with 100 mg/kg pentobarbital sodium (ip). The hearts were rapidly removed, placed in ice-cold heparinized saline, and perfused using retrograde aortic perfusion in the Langendorff mode [22] as previously described [23]. The perfusate was a modified Krebs-Henseleit (KH) buffer consisting of (mmol/L) NaCl 118, KCl 4.9, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11, CaCl2 2.5, and oxygenated with 95% O2/5% CO2, at 37-C, and a constant pressure equivalent to 90 cm H2O. Systolic pressure was monitored by means of a fluid-filled latex balloon (0.1 ml) connected to a pressure transducer that was inserted into the left ventricle and expanded to exert a physiologic end-diastolic pressure of 5 mm Hg. Hemodynamic data were captured using the Biopac MP100 Acquisition Module (Biopac Systems Inc., Goleta, CA) and
Protein oxidation was detected using the Oxyblot kit (Chemicon International, Temecula, CA) as previously described [5,6]. Basically, proteins were reacted with dinitrophenylhydrazine to tag carbonyl groups and then separated under reducing conditions using standard sodium dodecyl sulfate (SDS) polylyacrylamide gel electrophoresis (PAGE) [25]. Protein carbonyls were then determined using an immunoblot technique and antibody specific for dinitrophenylhydrazine as previously described [6]. Band size and intensities were evaluated using computer-assisted densitometry (SigmaScan Image Analysis software ver 1.20.09, Jandel Scientific (SSI), Richmond, CA). Degradation of actin Degradation of actin was determined by analysis of 3-MH based on the chromatography method of Wassner et al. [26].
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Myofibrillar proteins were isolated by pulverizing frozen ventricular tissue under liquid N2 and homogenizing 1 part tissue in 4 parts buffer containing (mmol/L): Hepes 10, NaCl 137, KCl 4.6, KH2PO4 1.1, MgSO4 0.6, EDTA 1, digitonin 0.01%, pH 7.4, plus 1 protease inhibitors (Protease Cocktail Set 1, EMD-Calbiochem, San Diego, CA). The homogenates were centrifuged at 10,000g for 20 min at 4-C, and the resulting pellets were treated with 1% Triton TX100, followed by dissolution of the sediment in Tris buffer, 10 mmol/L, containing 2% SDS. Myofibrillar proteins (2 mg) were precipitated with deoxycholate, 0.03%, and TCA, 25% (final concentrations), dried by washes with acetone and ether, followed by hydrolysis in 6 N HCl:TFA (2:1) at 120-C for 16 h. Aliquots (25 Al) of dried hydrolysates in HCl, 0.01 N, were buffered with sodium borate, 0.2 mol/L, pH 10, followed by derivatization with fluorescamine, 0.67 mg/ml, by heating at 80-C for 50 min. The derivatized amino acids were separated using high-pressure liquid chromatography (Series 410 LC pump, Perkin-Elmer, Boston MA) on a ˚´ ,4 Am, 4.6 250 mm; Waters, Novapak C18 column (60 A Milford, MA), and detected using fluorometric spectroscopy (Model 1700, Bio-Rad Labs., Hercules, CA) at excitation, 365 nm, and emission, 455 nm. Chromatography data were captured and analyzed using the PeakSimple Chromatography System ver 3.2.1 (SRI Instruments, Inc., Las Vegas, NV). Both 3-MH and unmodified histidine (His) peaks were identified using internal standards (10 and 100 Amol/L). Comparisons were made between integrated areas of 3-MH and His peaks to estimate the changes in the methylated amino acid. 3-MH content was expressed as the percentage of total myofibrillar His in each sample after a factor accounting for the ratio of sensitivities for fluorometric responses to His/3-MH was applied. This factor was determined from peak areas obtained using equimolar calibrating solutions of his and 3-MH, respectively, and was found to be 0.487 T 0.027 (N = 4).
actin (clone AC-40; Sigma-Aldrich, St. Louis, MO) which recognizes a C-terminus epitope common to all actin isoforms. To minimize interference by the heavy- and light-chain IgGs, the actin immuno-complex was captured using the TrueBlot reagent kit (eBioscience, San Diego, CA) according to the literature protocol. Briefly, the precleared homogenates were rotated with 6 Ag of antibody for 1 h and then with antimouse-IgG-coupled TrueBlot beads for an additional hour. The beads were washed exhaustively with TBS buffer followed by the addition of reducing Laemmli sample buffer containing 2% h-mercaptoethanol and boiled for 10 min. Immunoprecipitated samples were then separated using standard SDS-PAGE and then electroblotted to a PVDF membrane. As a positive control, a-sarcomeric actin (SigmaAldrich) was also immunoprecipitated. The membranes were then probed with the same antibody specific for actin (clone AC-40) followed by multiple washings of the membrane and subsequent incubation with peroxidase-conjugated secondary antibody (anti-mouse TrueBlot IgG) and visualized with enhanced chemiluminescence autoradiography. Immunoprecipitated actin was also probed for ubiquitin in separate membranes or after stripping of the actin-probed membrane and visualized as described.
Immunoblot analysis of ubiquitinated proteins
Inhibition of myocardial proteasome
Ubiquitinated proteins were assessed as previously described [21] following protein separation using standard SDSPAGE and electroblotting to a PVDF membrane. The membrane was probed with a polyclonal antibody (1:1000) specific for ubiquitin (EMD-Calbiochem, San Diego, CA) and developed using enhanced chemiluminescence autoradiography (Perkin-Elmer Life Sciences, Boston, MA) as previously described [21].
Preischemic inhibition of myocardial proteasome was effected by treatment of hearts with the irreversible inhibitor, Lac [27,28]. Preliminary studies examined several concentrations to determine the best inhibition profile (data not shown). For reasons discussed in the next section, preischemic inhibition of approximately 40 –50% that would persist for a minimum of 90 min was felt to be most desirable for these experiments. The concentration of Lac observed to produce this degree of inhibition was 2 Amol/L infused into the heart over a period of 10 min which resulted in a 51% decrease in 20S proteasome and a 42% decrease in 26S proteasome activities (Fig. 1) still present after 90 min.
Immunoprecipitation of actins Ventricular tissue was homogenized in buffer containing (mmol/L): Hepes 10, NaCl 137, KCl 4.6, KH2PO4 1.1, MgSO4 0.6, EDTA 1, digitonin 0.01%, pH 7.4, plus 1 protease inhibitors (Protease Cocktail Set 1, EMD-Calbiochem) and centrifuged at 10,000g, at 4-C, for 30 min. An aliquot containing 300 Ag of supernatant protein was immunoprecipitated with a monoclonal antibody specific for
Statistical analysis All results are expressed as mean T SE. Statistical significance of differences between two populations with equal variance was with a Student’s t test. Analysis of differences between multiple sample populations with equal variances was with one-way ANOVA followed by Tukeys test for post hoc analysis. Statistical differences of P < 0.05 were considered to be significant. All statistics were performed using the SigmaStat statistical analysis package (Jandel Scientific (SSI)). Results
Preischemic proteasome inhibition increases accumulation of protein carbonyls This series of experiments examined the effect of proteasome inhibition on hemodynamic recovery and protein
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Fig. 1. Inhibition of myocardial proteasome by lactacystin. Isolated hearts were perfused with buffer containing Lac 2 Amol/L for 10 min. Hearts were then perfused with buffer alone for an additional 90 min and then harvested and analyzed for proteasome activities. 20S proteasome activity is calculated as the difference between total proteolytic activity and that remaining in the presence of Lac. 26S proteasome activity is calculated as the difference between total ATP-stimulated proteolytic activity and that remaining in the presence of Lac, minus 20S proteasome activity. The values represent the mean T SE of four hearts. *P < 0.05 (Student t test) when compared with control hearts.
carbonylation after ischemia and reperfusion. Exposure to 15 min ischemia has minimal effect on recovery of function and inducing this degree of preischemic proteasome inhibition does not change this (data not shown). In this series of experiments, 30 min ischemia had no significant effects on recovery even though function was still depressed 31% (Fig. 2) after 60 min reperfusion. In hearts not subjected to ischemia, a 10-min pulse treatment with Lac, 2 Amol/L, resulted in a 17% decrease in hemodynamic function after 90 min of perfusion that was not significantly different from control hearts (data not shown). However, in hearts made ischemic, prior treatment with Lac did result in additional depression ( 40%) which was now significantly ( P < 0.05) less than preischemic hearts. Representative immunoblots depicting the effect of proteasome inhibition on bands of carbonylated proteins in hearts subjected to 15 or 30 min
Fig. 2. The effect of proteasome inhibition on postischemic recovery of hearts. Isolated hearts were subjected to 30 min normothermic global ischemia followed by 60 min of reperfusion. Lactacystin-treated hearts were perfused with buffer containing 2 Amol/L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. The values represent the mean T SE of four to seven observations. P < 0.05 (ANOVA, Tukeys) when compared with preischemic baseline values.
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Fig. 3. Proteasome inhibition increases accumulation of oxidized proteins following myocardial ischemia. Control (C) hearts were perfused with buffer for 90 min. Ischemic/reperfused (I) hearts were subjected to 15 (top panel) or 30 min (bottom panel) normothermic global ischemia followed by 60 min of reperfusion. Lactacystin-treated (L) hearts were perfused with buffer containing 2 Amol/L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. Lactacystin plus ischemia/reperfusion (IL) hearts were pretreated with the proteasome inhibitor and then subjected to the ischemia reperfusion protocol. At the end of the treatment protocols, all hearts were harvested and analyzed for protein carbonyls. The membrane is representative of five to seven separate determinations. A densitometric analysis is presented in Fig. 4.
ischemia and 60 min reperfusion are illustrated in Fig. 3. In general, Lac alone (Fig. 3, lane L (top and bottom panels)) appeared to have marginal effects on the degree of protein carbonylation. Likewise, exposure to 15 min ischemia (Fig. 3, lane I, top panel) had no effect, even after treatment with Lac (Fig. 3, lane IL, top panel). Consistent with previous studies [6,21], 30 min ischemia appears to increase protein carbonylation across a wide range of molecular masses (Fig. 3, lane I, bottom panel), an effect which was enhanced in the presence of Lac-induced proteasome inhibition (Fig. 3, lane IL, bottom panel). This effect was quantitated using computer-assisted densitometry which is presented in Fig. 4. We observed that bands of proteins of molecular masses of 95 kDa (top panel), 50 kDa (middle panel), and 40 kDa (bottom panel) were reasonably consistent and were chosen for analysis. A trend is immediately apparent with + Lac alone, or 15 min ischemia T Lac having no significant effects on carbonyl content of these bands (Fig. 4). Treatment with 30 min ischemia generally results in approximately 2-fold increases in detectable carbonyls in all three bands (Fig. 4). However, proteasome inhibition followed by 30 min ischemia resulted in significantly ( P < 0.05) greater accumulations of protein carbonyls in all three bands (90 kDa, 6-fold; 50 kDa, 3.3-fold; 40 kDa, 3.6-fold) suggesting either enhanced formation or decreased degradation of protein carbonyls (Fig. 4).
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represented 12% of unmodified His in agreement with other studies that concluded that of the eight His in this protein, only one is methylated [29,30]. Lactacystin alone had no effect on the 3-MH:His fraction after 90 min of perfusion (Fig. 5). However, 30 min ischemia results in a significant ( P < 0.05) 15% decrease in the 3-MH:His fraction after 60 min of reperfusion, suggesting proteolysis of actin (Fig. 5). Pretreatment of these hearts with Lac prevented much of this decrease with only a 6.5% loss after 60 min of reperfusion, suggesting that proteasome mediates part of this decrease. Actin is not ubiquitinated during myocardial ischemia/reperfusion This experiment examined whether loss of actin was ubiquitin dependent and mediated by the 26S proteasome, or was degraded directly by the 20S proteasome. Ubiquitination of proteins isolated from hearts exposed to ischemia/ reperfusion is depicted in Fig. 6A. As predicted, hearts treated with Lac had higher levels of ubiquitinated proteins, with Lac-pretreated ischemic/reperfused hearts having the highest content (Fig. 6A – Lane ‘‘IL’’). For the purposes of these experiments, actin was immunoprecipitated from control (C), ischemic/reperfused (I), and Lac-pretreated ischemic/reperfused (IL) hearts, as well as an additional control sample containing purified actin (A) (Fig. 6B). When this membrane was stripped of the anti-actin antibody and then probed with an antibody specific for ubiquitin, no bands representing ubiquitinated homologues were visible in any of the lanes (Fig. 6C). The exact same results were obtained if the immunoprecipitated proteins were probed for ubiquitin or actin on separate membranes without the need for stripping Fig. 4. Densitometric analysis of protein oxidation data. Hearts were treated as described in Fig. 3. Illustrated above is the densitometric analysis of bands of proteins containing carbonyls at molecular masses of 95 kDa (top panel), 50 kDa (middle panel), and 40 kDa (bottom panel) (see arrows—Fig. 3). Density was calculated as the size of the band average density of the band. All values are presented as percentage of the control hearts (95 kDa, 7714 T 915; 50 kDa, 40,709 T 8236; 21,170 T 4334 arbitrary units) and represent the mean T SE of 5 to 7 separate determinations. aP < 0.05 (ANOVA, Tukeys) when compared with control hearts. bP < 0.05 (ANOVA, Tukeys) when compared with +Lac hearts. c P < 0.05 (ANOVA, Tukeys) when compared with 30 min ischemia hearts.
Inhibition of the proteasome prevents loss of 3-methylhistidine As an indicator of actin degradation loss of 3-MH following ischemia and reperfusion was examined. Initial experiments calculated 3-MH content standardized to initial protein content which yielded 102 T 38 Umol/mg protein in control hearts. The high variability reflects varying yields for protein precipitation and derivatization among sets of experiments. For this reason, 3-MH was standardized to His content. In control rat heart myofibrillar proteins, 3.05 T 0.18% of His was in the form of 3-MH, representing about 25% of the value obtained by the analysis of a hydrolysate of pure actin. In this pure actin preparation, 3-MH
Fig. 5. Proteasome inhibition prevents loss of 3-methylhistidine after myocardial ischemia. Control isolated hearts were perfused with buffer for 90 min. Lactacystin-treated hearts were perfused with buffer containing 2 Amol/ L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. Ischemia/reperfusion hearts were subjected to 30 min normothermic global ischemia followed by 60 min of reperfusion. Ischemia/ reperfusion + Lac hearts were pretreated with the proteasome inhibitor and then subjected to the ischemia reperfusion protocol. At the end of the treatment protocols, all hearts were harvested and analyzed for 3-MH and His content. The values are expressed as the ratio (%) of 3-MH:His and represent the mean T SE of four to seven hearts. *P < 0.05 (ANOVA, Tukeys) when compared with control or (+) Lac alone groups.
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Fig. 6. Actin is not ubiquitinated during myocardial ischemia/reperfusion. Control (C) hearts were perfused with buffer for 90 min. Ischemic/reperfused (I) hearts were subjected to 30 min normothermic global ischemia followed by 60 min of reperfusion. Lactacystin-treated (L) hearts were perfused with buffer containing 2 Amol/L of the proteasome inhibitor for 10 min followed by perfusion with buffer alone for 90 min. Lactacystin plus ischemia/reperfusion (IL) hearts were pretreated with the proteasome inhibitor and then subjected to the ischemia reperfusion protocol. Panel A depicts ubiquitinated proteins from hearts treated according to the above protocols. Panel B depicts actin immunoprecipitated from samples ‘‘C’’, ‘‘I’’, and ‘‘IL’’. Lane A is a control containing immunoprecipitated purified actin. Panel C depicts the membrane in Panel B after stripping and probing with an antibody specific for ubiquitin.
of antibodies (membranes not shown). This result suggests that actin is not ubiquitinated during myocardial ischemia and reperfusion of the magnitude or time frame used in these experiments. Discussion One of the major pathways for intracellular protein degradation in eukaryotic cells is a multicatalytic proteolytic complex, known as the proteasome (macroxyproteinase). The core catalytic complex of the proteasome contains 28 subunits arranged in a cylindrical structure composed of four rings, each containing seven subunits. The proteolytic center is located inside the cylinder and has ‘‘trypsin-like,’’ ‘‘chymotrypsinlike,’’ and ‘‘peptidylglutamyl-peptide hydrolase-like’’ activity. This complex is also known as the 20S proteasome [31]. Capping of each end of the 20S proteasome with a regulatory 19S complex, each containing at least 15 additional subunits, confers ATP and ubiquitin specificity [32,33]. This entire complex is known as the 26S proteasome which is responsible for turnover of important regulatory proteins, such as the cyclins and transcription factors, and plays an important role in antigen presentation [32 –34]. Proteins are targeted to the 26S proteasome following multiubiquitination by sequential addition of ubiquitins to the (-amino group of a protein lysine [32]. While somewhat controversial, recent evidence suggests that oxidatively modified proteins can be removed by the ‘‘core’’ 20S proteasome without requirement for ATP or ubiquitin [18 – 20,35]. Numerous studies have examined removal of proteins damaged during myocardial ischemia, yet none have examined the role of the proteasome. The current study examined the hypothesis that proteasome can mediate removal of proteins oxidized during myocardial ischemia. The salient observations were threefold: (1) inhibition of the proteasome with Lac increases postischemic accumulation of protein carbonyls; (2) 3-MH content is decreased in the postischemic heart, indicating
loss of myocardial actin, and this effect is partially blocked by proteasome inhibition; and (3) there appears to be no formation of ubiquitinated homologues of actin, suggesting proteolysis by the 20S proteasome. To demonstrate a role for the proteasome it was necessary to treat the hearts with the inhibitor, Lac. The selection of Lac was based on its irreversibility [27,28] which allowed for pulse treatment of the hearts, and for its high degree of specificity [36], which makes it preferable to MG132, which is a reversible inhibitor and is known to inhibit cysteine proteases, such as cathepsins and calpains, at concentrations not that different from that which inhibits the proteasome [36 – 38]. We [21] and others [39] have previously shown that myocardial ischemia of sufficient duration can result in significant inhibition of proteasome. So as not to inhibit proteasome completely a concentration and treatment duration that effected 40 to 50% inhibition of 20S and 26S proteasome prior to ischemia was utilized. Inhibition of this magnitude by this class of proteasome inhibitors is generally considered to be specific and not associated with significant inhibition of other proteases (reviewed in [36]). Inhibition of this degree marginally decreased hemodynamic recovery of postischemic hearts. While not of the same degree, this observation is in general agreement with what we have published previously [21], although a reversible inhibitor was used in this prior study without determination of degree of proteasome inhibition. In either case, improvement of function was clearly not observed as has been reported by other groups using the inhibitor, PS519 [40,41]. Formation of protein carbonyls was used as an indicator of oxidative modification of proteins during myocardial ischemia. While not the only oxidative modification to proteins that occurs during ischemia, it is generally considered to be representative, and we [5 – 8] and others [4,42,43] have consistently demonstrated increases. The increases in protein carbonyls in postischemic hearts that were observed in the
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current study are in general agreement with what we have published previously [6– 8,21]. Short duration ischemia did not result in significant accumulation of protein carbonyls and additional proteasome inhibition of the magnitude used in this study had no additional effect. Interpretation in combination with the observation that proteasome inhibition alone (no ischemia) does not result in significant accumulation of protein carbonyls suggests that excess proteasome is present and that the heart is not adversely effected by 40 to 50% proteasome inhibition unless subjected to increased oxidative stress, at least within the time frame of these experiments. On the other hand, longer ischemia resulted in accumulation of protein carbonyls, an effect which was significantly enhanced by the use of a proteasome inhibitor, suggesting that proteasome removes these modified proteins. An alternate explanation for this effect is that proteasome inhibition has been suggested to result in increased production of oxidative species and protein modifications [44]. The lack of effect of Lac on the nonischemic and short duration ischemic hearts suggests, but is not conclusive, that this is not the case. In order to refute this possibility, degradation of a specific protein was examined. We [6,7] have previously shown that sarcomeric and cytoskeletal actins are targets of oxidative injury in the postischemic heart, and that oxidized actin is one of the major components of the 50-kDa band (see Fig. 3) of carbonylcontaining proteins. Therefore, we quantitated actin proteolysis by measuring loss of 3-MH. 3-Methylation is a posttranslational modification of His that is generally considered to be peculiar to two myofibrillar proteins, actin and myosin [45,46]. Loss of this amino acid is an accepted indicator of proteolysis of muscle myofibrillar protein and would usually be representative of loss of both of these proteins [45]. However, in mammalian heart, myosin contains only methyllysine and not methylhistidines; thus, actin is the only source [46] and decreases are a quantitative indicator of its degradation only. We observed a 15% loss of 3-MH within 1 h of reperfusion, indicating a significant loss of this protein following myocardial ischemia, a result in general agreement with older studies [13,47] using different techniques. Actin is a long-lived protein, with a reported t 2 of 10.3 days [48]; thus, it is highly unlikely that normal turnover contributed to this loss. That proteasome mediates at least part of this degradation was indicated by the ability of Lac to prevent much of this decrease. It is no great surprise that Lac did not completely prevent actin degradation as the chosen dose does not completely inhibit proteasome. Nor should it be assumed that proteasome is the only proteolytic enzyme or complex involved in its degradation. While supportive of the overall hypothesis, these observations shed no light on which proteasome, the 20S or 26S, is actually mediating actin degradation. Since the 26S proteasome recognizes only ubiquitinated substrates [32], it should have been possible to isolate ubiquitinated homologues of actin, particularly in the presence of significant inhibition of 26S proteasome. Our results demonstrate significant inhibition of 26S proteasome coincident with increased accumulation of ubiquitinated proteins in agreement with previous observations
[21]. Yet, we failed to demonstrate the presence of any ubiquitinated homologues whether actin is immunoprecipitated from control, ischemic/reperfused, or Lac-pretreated ischemic/ reperfused hearts, suggesting that the 20S proteasome is mediating this effect. This conclusion is entirely consistent with studies of proteasome-mediated degradation of oxidized proteins in cellular systems as well as what is known about actin degradation. Studies by Davies and co-workers [18 – 20,35] in cell preparations have consistently shown that 20S proteasome mediates removal of oxidized proteins without requirement for ATP or ubiquitin. These investigators have theorized that certain oxidative modifications increase the hydrophobicity of a protein, either by introduction of hydrophobic groups or protein unfolding exposing hydrophobic patches, which targets the protein to the 20S proteasome (reviewed in [3]). While there is evidence that actin can be degraded via a ubiquitin-dependent pathway, this appears to be limited to increased catabolic states, such as starvation, posttraumatic injury, and sepsis, when proteins are catabolized as an alternate source of energy substrates [49 – 52]. In summary, these studies have shown that inhibition of the proteasome results in enhanced accumulation of oxidized proteins in the postischemic heart. Furthermore, actin degradation is increased in the postischemic heart, a process that is partially blocked by a proteasome inhibitor. Lastly, failure to isolate ubiquitinated homologues of actin provides strong evidence that the 20S proteasome mediates removal of this oxidized protein in a process independent of ubiquitin. In conclusion, these studies provide the first evidence that proteasome mediates removal of proteins oxidized during myocardial ischemia. Acknowledgment This study was supported by NIH R01 HL68936 (S.R.P.). References [1] Bolli, R.; Jeroudi, M. O.; Patel, B. S.; DuBose, C. M.; Lai, E. K.; Roberts, R.; McCay, P. B. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc. Natl. Acad. Sci. USA 86:4695 – 4699; 1989. [2] Powell, S. R.; Hall, D. Use of salicylate as a probe for OH formation in isolated ischemic rat hearts. Free Radic. Biol. Med. 9:133 – 141; 1990. [3] Grune, T.; Merker, K.; Sandig, G.; Davies, K. J. Selective degradation of oxidatively modified protein substrates by the proteasome. Biochem. Biophys. Res. Commun. 305:709 – 718; 2003. [4] Park, Y.; Kanekal, S.; Kehrer, J. P. Oxidative changes in hypoxic rat heart tissue. Am. J. Physiol. 260:H1395 – H1405; 1991. [5] Powell, S. R.; Gurzenda, E. M.; Wingertzahn, M. A.; Wapnir, R. A. Promotion of copper excretion from the isolated perfused rat heart attenuates post-ischemic cardiac oxidative injury. Am. J. Physiol. 277:H956 – H962; 1999. [6] Powell, S. R.; Gurzenda, E. M.; Wahezi, S. E. Actin is oxidized during myocardial ischemia. Free Radic. Biol. Med. 80:1171 – 1176; 2001. [7] Schwalb, H.; Olivson, A.; Li, J.; Houminer, E.; Wahezi, S. E.; Opie, L. H.; Maulik, D.; Borman, J. B.; Powell, S. R. Nicorandil decreases postischemic actin oxidation. Free Radic. Biol. Med. 31:607 – 614; 2001. [8] Khaliulin, I.; Schwalb, H.; Wang, P.; Houminer, E.; Grinberg, L.; Katzeff, H. L.; Borman, J. B.; Powell, S. R. Preconditioning improves
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