Actin is oxidized during myocardial ischemia

Free Radical Biology & Medicine, Vol. 30, No. 10, pp. 1171–1176, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter

PII S0891-5849(01)00514-7

Fast Track Paper ACTIN IS OXIDIZED DURING MYOCARDIAL ISCHEMIA SAUL R. POWELL, ELLEN M. GURZENDA,

and

SAYED EMAL WAHEZI

Maternal/Fetal Medicine Research Lab., Department of Obstetrics and Gynecology, Winthrop University Hospital, Mineola, NY, USA (Received 18 October 2000; Accepted 23 February 2001)

Abstract—Exposure of isolated rat hearts to 30 min global ischemia followed by 60 min reperfusion resulted in a significant 80% increase (p ⬍ .05) in actin content of carbonyl groups, which was associated with significant depression (p ⬍ .05) of postischemic contractile function. This result supports the hypothesis that one mechanism of postischemic contractile dysfunction may be oxidation of contractile proteins. © 2001 Elsevier Science Inc. Keywords—Actin, Protein oxidation, Protein carbonyls, Myocardial ischemia, Free radicals

INTRODUCTION

have demonstrated that myocardial proteins are oxidized during ischemia. Until now these proteins have not been identified, primarily because they are so numerous [13]. In this study we test the hypothesis that contractile proteins are susceptible to oxidative attack during ischemia and reperfusion. Using immunoblot techniques we demonstrate that actin is subject to oxidative modification during in vitro protocols that mimic myocardial stunning.

Ischemia/reperfusion results in cardiac injury ranging from short-term reversible contractile dysfunction to cellular necrosis and infarct with irreversible loss of function. Intermediate is myocardial “stunning,” prolonged reversible contractile dysfunction [1]. The mechanism of stunning-associated contractile dysfunction has been the subject of much research and debate. For example, one hypothesis is that Troponin (Tn), the regulatory element of the myofilament, is damaged during ischemia [2]. Other myofilament and structural cytoskeletal proteins, such as actin, myosin, myosin light chain, tropomyosin, ␣-actinin, and spectrin have been shown to be lost from ischemic tissue or subject to proteolytic cleavage [3– 8]. Little is known about the nature of the modification to the myofibrillar proteins that makes them susceptible to loss or proteolysis during ischemia. Numerous studies in a variety of models have established that oxidative modifications of proteins render them more susceptible to proteolytic attack [9,10]. Modifications include: oxidation of protein sulfhydryls; •OHmediated, site-specific oxidation of lysine, arginine, and histidine residues to carbonyl groups [11]; and tyrosine nitration by peroxynitrite [12]. We [13] and others [14]

MATERIALS AND METHODS

Animals and perfused heart preparation All studies were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 85-23, revised 1985) and were approved by the Institutional Animal Care and Use Committee. Male Sprague Dawley rats (275–300 g, Hilltop Farms, Scottsdale, PA, USA), were heparinized (500 units, ip); anesthetized (sodium pentobarbital, 70 mg/kg, ip); hearts removed rapidly and orthogradely perfused [15] at a constant pressure of 95 cm H2O as previously described [16]. The perfusate contained (mmol/l): NaCl 118, KCl 6.1, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, HEPES 1.0, and glucose 11.1; and was checked for ascorbate reactivity to assess for contaminating transition metals and treated with Chelex-100 (Bio-Rad Laboratories, Hercules, CA, USA) if necessary [17]. Left ventricular function was determined as the rate ⫻ pressure product and is calculated as the heart rate mul-

Address correspondence to: Saul R. Powell, Ph.D., Maternal/Fetal Medicine Research Lab., Dept. of Obstetrics/Gynecology, Winthrop University Hospital, 222 Station Plaza North, Suite 623, Mineola, NY 11501, USA; Tel: (516) 663-9371; Fax: (516) 663-8208; E-Mail: [email protected]. 1171

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tiplied by the developed systolic pressure as described previously [16]. Hearts were excluded from the study if a developed systolic pressure of at least 70 mm Hg; a heart rate of at least 220 beats per minute; or a normal rhythm was not maintained during the equilibration period. Experimental protocol Hearts were equilibrated for 30 min; subjected to either 15 or 30 min normothermic no-flow global ischemia; and reperfused for 60 min. To account for changes that might occur during prolonged perfusion a set of hearts (n ⫽ 5) were perfused for a total of 120 min with no ischemia. At the end of the periods of equilibration, ischemia, or reperfusion, the atria were dissected off, and ventricles flash-frozen. Myofibrillar fraction (MF) isolation The MF was isolated as described by Wing et al. [18], with all procedures carried out at 4°C. Frozen hearts were pulverized and homogenized in Tris maleate, 10 mmol/l, pH 7.0, buffer containing (mmol/l): KCl, 10; MgCl2, 2; EGTA, 1; and the protease inhibitors: L-tosylamido-2-phenylethylchloromethyl ketone, 1; tosyl-Llysine chloromethylketone, 1; and N-ethylmaleimide, 5 (isolation buffer). The homogenate was centrifuged at 1500 ⫻ g for 10 min; the pellet resuspended in isolation buffer containing 1% Triton; and washed twice more with isolation buffer(-Triton). The final pellet was solubilized in Tris maleate, 10 mmol/l, pH 7.0, containing the protease inhibitors and 2% sodium dodecyl sulfate (SDS) (preloading buffer). Protein content was determined according to the Lowry [19] method. Protein separation and immunoblot techniques MF proteins (5 ␮g) were heat-denatured and separated on a 4 –20% Tris-HCl gel (Ready Gel, Bio-Rad Laboratories) using standard SDS-PAGE. The gel loading buffer contained the preloading buffer (see above), 62.5 mmol/l Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.002% bromophenol blue, and 0.9 mol/l 2-mercaptoethanol [20]. To visualize the proteins, the gels were stained for 5 min with Coomassie Blue 0.25% (in methanol/acetic acid/water, 50:10:40) and destained with methanol/acetic acid/water (50:10:40) overnight. For positive identification the proteins were transferred onto a PVDF membrane, blocked for 1 h with 1% bovine serum albumin in Tris (100 mmol/l) buffered saline (0.9%), pH 7.0, containing 0.1% Tween-20 (TBST) and probed with the appropriate 1° antibody in blocking

buffer. The 1° antibodies included monoclonal anti-␣sarcomeric actin (clone 5C5) (1:1000) and anti-Troponin T (TnT) (clone JLT-12) (1:200) obtained from Sigma Chemical Company (St. Louis, MO, USA). The membrane was washed three times with TBST; probed with 2° antibody (biotinylated anti-mouse IgG; Vectastain Elite ABC kit, Vector Labs, Burlingame, CA, USA) in blocking buffer; washed three more times with TBST; and developed with the avidin/biotin/horseradish peroxidase system (Vectastain Elite ABC kit) using 3,3⬘-tetramethylbenzidine (TMB) as a substrate. For immunoprecipitation experiments, MF was reacted with monoclonal anti-actin (clone AC-40 (mouse IgG2a subtype), Sigma) for 1 h at 4°C. Protein A beads (Sigma) (100 ␮l) were added to the antigen:antibody reaction and incubated at 4°C for 1 h, then centrifuged at 10,000 ⫻ g for 15 s, and washed with Tris maleate, 10 mmol/l, pH 7.0. The antigen was released from the antibody by heat denaturation at 85°C for 10 min in Laemmli buffer and centrifuged at 10,000 ⫻ g for 15 s. The released proteins were then separated using SDSPAGE and bands positively identified with monoclonal anti-␣-sarcomeric actin (clone 5C5) (1:1000) as described above.

Immunoblot analysis of protein carbonyls Oxidation of actin was assessed by determining the presence of carbonyl groups using a standard kit (Oxyblot, Intergen Co., Purchase, NY, USA) following derivatization with 2,4-dinitrophenylhydrazine (DNPH) in the presence of trifluoroacetic acid (denaturant). The derivatized proteins (3 ␮g) were separated on a 4 –20% TRIS-HCl gel; transferred onto a PVDF membrane; probed using the 1° and 2° antibodies supplied in the kit and developed using chemiluminescence as previously described [13] or the avidin/biotin/horseradish peroxidase system as described above. After transference, gels were routinely stained with Coomassie Blue to assess transference among lanes. The developed membranes were digitized and analyzed using computer-assisted densitometry (SigmaScan, Jandel Scientific, Chicago, IL, USA). Intensities were adjusted for differences in protein content based on Coomassie Blue staining of complementary gels, if necessary. All experiments were designed so that each gel contained at least one sample from each experimental group. For identification of a carbonyl-containing band as actin, MF was reacted with monoclonal anti-actin (clone AC-40) for 1 h at 4°C, then reacted with DNPH, separated using SDS-PAGE, and probed for protein carbonyls as described. To ensure equal loading of protein, samples were analyzed for protein content prior to gel

Oxidation of actin

Fig. 1. Cardiac function following 15 or 30 min normothermic global ischemia. Isolated hearts were equilibrated for 30 min, subjected to 15 or 30 min normothermic global ischemia, followed by 60 min of reperfusion. A separate group of hearts was perfused for 120 min with no ischemia. Values are expressed as the mean (⫾ SEM) of four or five hearts. *p ⬍ .05 (RMANOVA) when compared with other treatment groups at corresponding time point.

loading and volumes adjusted to account for addition of the anti-actin. Immunoblot analysis of nitrotyrosine Immunoblot analysis of protein bound nitrotyrosine was performed as described by Viera et al. [21]. MF proteins (10 ␮g) were separated and transferred, probed with monoclonal anti-nitrotyrosine (clone 1A6 [1:500] from Upstate Biotechnology, Waltham, MA, USA), developed and analyzed as described above. Statistical analysis Differences between multiple groups were determined using one-way Analysis of variance (ANOVA) (post hoc test, Tukey test) or Repeated Measures ANOVA (RMANOVA) where the within-group variable was time. In all cases results were considered to be significant at the p ⬍ .05 level. RESULTS

Postischemic left ventricular function The effect of 15 and 30 min global ischemia on left ventricular function is illustrated in Fig. 1. By 60 min of reperfusion, the heart rate ⫻ pressure product was approximately 40% (30 min) or 67% (15 min) of the

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Fig. 2. Composite figure illustrating positive actin identification using (A) Immunoblotting and (B) Immunoprecipitation. The MF was isolated and separated as described in Materials and Methods. Panel A: lane 1, a representative gel after Coomassie Blue staining (gray scale); lane 2, identification of actin by probing with specific anti-actin monoclonal antibody; lane 3, identification of TnT by probing with specific anti-TnT antibody. Panel B represents immunostaining of immunoprecipitated actin (described in Materials and Methods): lane 1, no sample; lanes 2 to 4 represent serial dilutions of anti-actin antibody used for immunoprecipitation.

preischemic values in contrast to the nonischemic hearts perfused for 120 min, which still maintained approximately 72% of their original values. Overall, there were no significant differences in function between the last 50 min of perfusion of the nonischemic hearts and reperfusion of the 15 min ischemic hearts, whereas function was significantly less (p ⬍ .05, RMANOVA) in the hearts ischemic for 30 min.

Identification of cardiac myofibrillar proteins Immunoblotting with a specific monoclonal antibody was used to identify actin. Fig. 2A (lane 1) shows a Coomassie blue stained SDS-PAGE gel demonstrating two distinct bands with molecular mass approximating 43 kDa, which is the expected mass of actin. Immunoblotting with specific monoclonal antibodies indicated that the higher molecular weight band was actin (Fig. 2A, lane 2) and the lower molecular band approximating 39 kDa was TnT (Fig. 2A, lane 3). To positively identify the 43 kDa band, the protein was complexed with an anti-actin antibody and precipitated with protein A conjugated to agarose beads. When released, immunoprecipitated actin was separated under denaturing conditions and probed with a specific monoclonal anti-actin, three bands were visible (Fig. 2B, lanes 2– 4 (antibody titration curve)). The band at 43 kDa is actin and the bands at approximately 55 and 25 kDa represent the heavy and light chains of IgG, respectively, which react with the 2°

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S. R. POWELL et al. Table 1. Densitometric Analysis of Actin Band Average intensity (Arbitrary units)

Treatment

28 ⫾ 3 40 ⫾ 6 50 ⫾ 6*

Preischemic Endischemic Postischemic

The relative content of carbonyls was quantified using densitometry. The effect of 30 min ischemia and 60 min reperfusion is shown in the above table. Each value is the mean (⫾ SEM) of samples derived from four individual hearts. *p ⬍ .05 (ANOVA) when compared with preischemic group.

Fig. 3. Composite figure illustrating (A) detection of protein carbonyls and nitrotyrosines during and following ischemia and (B) positive identification of oxidized band as actin. The MF was isolated and separated as described in Materials and Methods. Panel A: The effect of 30 min ischemia and 60 min reperfusion on actin content of protein carbonyls (top) and nitrotyrosines (bottom) (lane 1, preischemic; lane 2, 30 min ischemia; lane 3, 30 min ischemia and 60 min reperfusion). Panel B: Immunoshift of actin band (lane 1, protein carbonyls of MF isolated from heart subjected to 30 min ischemia and 60 min reperfusion; lane 2, protein carbonyls of MF [from same heart in lane 1] after treatment with anti-actin [described in Materials and Methods]; lanes 3 and 4, Coomassie blue staining [gray scale] of sister gel identical to lanes 1 and 2, respectively).

anti-mouse antibody. No actin band was visible when MF was omitted from the reaction (Fig. 2B, lane 1). Effect of ischemia and reperfusion on oxidation of contractile proteins Oxidation of contractile proteins was assessed by determining the introduction of carbonyl groups. As illustrated in Fig. 3A (top lanes 1–3), a broad band of immunoreactive protein was observed at 43 kDa. Based on molecular mass, this protein was suspected to be monomeric actin, which was confirmed by forming an immunocomplex. As shown in Fig. 3B, after treatment with anti-actin, the carbonyl-containing band at 43 kDa (lane 1) is not present (lane 2), but appears to be shifted up to approximately 68 kDa, which is the theoretical molecular mass of an actinIgG light chain or FAB fragment complex. We theorize this happens because after forming the antigen:antibody complex, the proteins were treated with DNPH under acid-reducing conditions, which may fragment the IgG molecule and somehow stabilize the complex. Analysis of Coomassie Blue stained sister gels re-

vealed a similar pattern with no overall difference in protein content between the 43 and 65 kDa bands (Fig. 3B, lanes 3 and 4). Actin normally appears to contain a significant quantity of carbonyls (Fig. 3A, top lane 1). Although we cannot rule out this background level as being artifactual, a preliminary experiment with purified actin (Sigma) demonstrated no increase in carbonyls during the isolation procedure (data not shown). After 30 min ischemia, actin contained 43% more (not significant) carbonyl groups (Fig. 3A, top lane 2; Table 1). After 30 min ischemia followed by 60 min reperfusion, content of actin carbonyl groups was significantly increased (p ⬍ .05, ANOVA) by 80% (Fig. 2A, top lane 3; Table 1). Analysis of Coomassie Blue stained complementary gels revealed no overall differences in protein content of this band amongst different samples, so the change is interpreted as an absolute increase in the number of carbonyls per actin. No changes in carbonyl content were observed after 15 min ischemia with or without 60 min reperfusion (data not shown). Further, there were no differences between carbonyl content of actin from the preischemic control hearts and the nonischemic hearts perfused for 120 min (data not shown). The effect of ischemia on nitration of actin tyrosines A monoclonal antibody specific for nitrotyrosine was used to determine if actin was vulnerable to attack by peroxynitrite. As shown in Fig. 3A (bottom lanes 1–3), the actin band had very little, if any, immunoreactivity even after 30 min ischemia and 60 min reperfusion. DISCUSSION

The present study demonstrates increased carbonyl content of actin, the extent of which was related to the duration of ischemia, which is consistent with numerous reports of progressive production of oxidative species [22,23]. Actin was positively identified using a combination of immunoblotting and immunoprecipi-

Oxidation of actin

tation techniques. In the cell, this protein exists in two forms, globular or g-actin, and filamentous or f-actin. In the myofibril, f-actin is the predominant form. We suspect that the observed band represents depolymerized f-actin, resulting from oxidation [24] or from separation under denaturing conditions. Significant background oxidation of actin was observed in the nonischemic heart, which may represent the pool of senescent proteins estimated at any one time to be 10 –20% of all cellular proteins due to be degraded [9] or be indicative of some myofibril oxidation during isolation. Introduction of carbonyl groups into a protein is a multi-step process usually initiated by oxidation of the N-containing side chain of arginine, lysine, or histidine by •OH. Formation of •OH is catalyzed by a transition metal held as a co-ordination complex by the N in the amino acid side chain, thus is an example of site-specific oxidation (reviewed in [11]). Because there may be other means of introducing carbonyls into proteins that do not involve oxidation of amino acid residues, a potential limitation of this technique may be nonspecificity [11]. Peroxidation products of polyunsaturated fatty acids have been shown to react with protein sulfhydryl groups to form stable covalent thiolether adducts containing a carbonyl function [25]. However, this represents the end product of oxidative (•OH-mediated) processes, i.e., lipid peroxidation and sulfhydryl oxidation, and would not impact on interpretation of the results. It is thus reasonable to conclude that an increase in carbonyl groups indicates enhanced •OH-mediated oxidative injury to the protein. Exposure of proteins to peroxynitrite can result in the specific nitration of tyrosine residues [12]. However, little if any nitration of actin was observed, suggesting that peroxynitrite is not the major oxidizing species. It was observed that significant oxidation of actin correlated with the highest degree of postischemic contractile dysfunction. However, this does not necessarily constitute cause and effect, thus the relationship between actin oxidation and postischemic contractile dysfunction is unclear. Protein oxidation can cause formation of aggregates of proteins as a result of sulfhydryl crosslinking or reaction of one protein radical with an adjacent protein radical (e.g., two tyrosine radicals reacting to form a dityrosine) [9]. Previous studies have demonstrated that exposure of g-actin to a variety of oxidants can result in cross-linking and polymerization [26,27]. Cross-linking of actin and other contractile proteins could hinder the interactions of thick and thin filaments and interfere with excitation-contraction coupling and contractile function.

1175 SUMMARY

The major finding of this study was that actin is oxidized during ischemia and reperfusion through a process that involves oxidation of amino acid side chains with formation of a carbonyl group. These preliminary results support the hypothesis that oxidative injury to the myofilament may provide a plausible explanation for changes in contractile function. Acknowledgements — Funded in part by a grant from the Heart Council of Long Island, Inc. and by Winthrop University Hospital.

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ABBREVIATIONS

ANOVA—Analysis of variance DNPH—2,4-dinitrophenylhydrazine MF—Myofibrillar Fraction RMANOVA—Repeated measures analysis of variance TBST—Tris-buffered saline with Tween-20 TMB—3,3⬘-tetramethylbenzidine Tn—Troponin TnT—Troponin T