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and A. A. Knowlton Li Lin, S. C. Kim, Yin Wang, S. Gupta, B. Davis, S. I. Simon, G. Torre-Amione

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HSP60 in heart failure: abnormal distribution and role in cardiacmyocyte apoptosis

Li Lin,1,3 S. C. Kim,1,4 Yin Wang,1,5 S. Gupta,1 B. Davis,6 S. I. Simon,6 G. Torre-Amione,7

and A. A. Knowlton1,2

1Molecular and Cellular Cardiology, Cardiovascular Division, University of California, Davis, and 2Sacramento VeteransAffairs Medical Center, Sacramento, California; 3Department of Physiology, Second Military Medical University, Shanghai,People’s Republic of China; 4Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn,Germany; 5Ningxia Medical College, Yinchuan, People’s Republic of China; 6Department of Biomedical Engineering,University of California, Davis, California; and 7Methodist Hospital, Houston, Texas

Submitted 26 June 2007; accepted in final form 3 August 2007

Lin L, Kim SC, Wang Y, Gupta S, Davis B, Simon SI, Torre-Amione G, Knowlton AA. HSP60 in heart failure: abnormal distri-bution and role in cardiac myocyte apoptosis. Am J Physiol Heart CircPhysiol 293: H2238–H2247, 2007. First published August 3, 2007;doi:10.1152/ajpheart.00740.2007.—Heat shock protein (HSP) 60 is amitochondrial and cytosolic protein. Previously, we reported thatHSP60 doubled in end-stage heart failure, even though levels of theprotective HSP72 were unchanged. Furthermore, we observed thatacute injury in adult cardiac myocytes resulted in movement ofHSP60 to the plasma membrane. We hypothesized that the inflam-matory state of heart failure would cause translocation of HSP60 tothe plasma membrane and that this would provide a pathway forcardiac injury. Two models were used to test this hypothesis: 1) a ratmodel of heart failure and 2) human explanted failing hearts. Wefound that HSP60 localized to the plasma membrane and was alsofound in the plasma early in heart failure. Plasma membrane HSP60localized to lipid rafts and was detectable on the cell surface with theuse of both flow cytometry and confocal microscopy. Localization ofHSP60 to the cell surface correlated with increased apoptosis. In heartfailure, HSP60 is in the plasma membrane fraction, on the cell surface,and in the plasma. Membrane HSP60 correlated with increasedapoptosis. Release of HSP60 may activate the innate immune system,promoting a proinflammatory state, including an increase in TNF-�.Thus abnormal trafficking of HSP60 to the cell surface may be anearly trigger for myocyte loss and the progression of heart failure.

heat shock proteins; protein trafficking; plasma membrane; cytokines

HSP60 IS AN IMPORTANT MEMBER of the heat shock protein family,thought to be primarily a mitochondrial protein, although it isencoded by the nuclear genome. Previously, our laboratory (8)reported that HSP60 is doubled in end-stage heart failure.HSP60 binds Bax and Bak in the cytosol of the myocyte, andreduction in HSP60 precipitates apoptosis (6). Simulated is-chemia in cardiac myocytes resulted in translocation of HSP60to the plasma membrane before reoxygenation (5). Redistribu-tion of HSP60 to the plasma membrane was associated withmovement of Bax to the mitochondria and apoptosis.

Observations of HSP60 translocation in isolated myocytesmotivated studies on the redistribution of HSP60 in the failingheart, where apoptosis is thought to be a mechanism of myo-cyte death. To address this question, we developed a rat modelof heart failure with ligation of the left anterior descendingartery. Cardiac echo was used to follow changes in function

and chamber size. Studies were conducted long after coronaryligation and thus reflected the progressive changes of thefailing ventricle, rather than ischemia. Upregulation of HSP60correlated with increased expression of proinflammatory cyto-kines, brain natriuretic peptide (BNP), and atrial natriureticpeptide (ANP) 9 and 12 wk postligation. In addition, weexamined distribution of HSP60 in explanted human heartswith dilated (DCM) and ischemic cardiomyopathy (ICM). Wereport that HSP60 is present in the cell membrane as well as theplasma early on in the development of heart failure and thatthis abnormal distribution of HSP60 persists in advanced heartfailure. This has implications for the progressive nature ofheart failure, because antibodies to HSP60 are present in theplasma of many individuals and HSP60 is thought to be aligand for Toll-like receptor 4 (TLR-4), part of the innateimmune system (13). In fact, membrane HSP60 correlatedwith the presence of apoptosis. Thus the presence of HSP60in the plasma membrane is not a passive event; rather, itstranslocation is a significant response that exacerbates thedisease state.

METHODS

Heart Failure Model

A rat coronary ligation model of heart failure was developed usingthe approach of Tanonaka et al. (29), which results in changes in heatshock protein expression similar to those we have found in humanheart failure. The animal protocol was approved by the University ofCalifornia, Davis Animal Research Committee in accordance with theNIH Guide for the Care and Use of Laboratory Animals. Sixty-four 5-to 6-mo-old male Sprague-Dawley rats were anesthetized with acombination of ketamine, acepromazine, and xylazine. Followingintubation and placement on a respirator, a left lateral thoracotomywas performed in the fourth intercostal space, and a ligature (6.0nylon suture) was placed around the left anterior descending coronaryartery (LAD) 2 mm below its origin. Ischemia was verified by visualinspection, the chest was closed, and the rat was extubated andreturned to its cage. This large infarct, needed to generate heartfailure, had a 24-h mortality of 35.9%. Thereafter, there were onlyfour deaths in the remaining 41 rats, two at 6 wk and 2 at 11 wk, allfrom congestive heart failure (CHF). Fifteen of the rats underwentthoracotomy but no ligation (sham). Fourteen rats were studied at 8–9wk (9 wk group). Six rats were followed until 12 wk. Sham rats werefollowed for 9 or 12 wk after sham surgery.

Address for reprint requests and other correspondence: A. A. Knowlton,Molecular and Cellular Cardiology, Univ. of California, Davis, One ShieldsAve., Davis, CA 95616 (e-mail: aaknowlton@ucdavis.edu).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Heart Circ Physiol 293: H2238–H2247, 2007.First published August 3, 2007; doi:10.1152/ajpheart.00740.2007.

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Cardiac Echo

Rats were studied 9 and 12 wk after ligation to examine progres-sion of changes over time. The rats were anesthetized with 50 mg/kgketamine and 5 mg/kg xylazine, their chests were shaved, and anechocardiogram was done (Acuson, Sequoia model C512, 15-MHzprobe). Two-dimensional imaging was used to identify the short-axisposition. Three consecutive m-mode images were collected in theshort-axis view and saved for analysis of chamber size and fractionalshortening. In a small subset, while the rat was under anesthesia, acarotid artery catheter was passed into the left ventricle (LV) tomeasure LV end-diastolic pressure (LVEDP), confirming that the ratswere in heart failure with elevated LVEDP (19.0 � 0.6 vs. 1.7 � 0.3mmHg). Hearts were collected at 9 and 12 wk to track changes inHSP60. Heart-to-body weight ratio was recorded. Plasma sampleswere collected for assay and stored at �80°C until use.

Human Heart Samples

Human heart samples were collected at transplantation at BaylorCollege of Medicine as part of a program run by one of the authors (G.Torre-Amione). All explanted hearts are preserved by immediatelyfreezing them in liquid nitrogen. Failing heart samples came fromadults identified as having either DCM or ICM. This tissue bank offailing hearts has Institutional Review Board (IRB) approval at Baylor.Normal adult human heart samples were collected in a similar fashionfrom brain-dead donors by a private company (T-Cubed). Heart sampleswere provided to the authors without identifying information but with abrief medical history, sex, and age. Pre- and post-LV assist device(LVAD) samples were obtained at the time of procedure under anIRB-approved protocol at Baylor (G. Torre-Amione). All LVAD patientswent to transplant, and a post-LVAD sample was collected at the time oftransplant.

Separation of Plasma Membrane, Cytosol, and Mitochondria

Plasma membrane, cytosol, and mitochondria were prepared aspreviously described (2, 6, 26). Citrate synthase activity, an index ofmitochondrial integrity, was measured as previously reported (5, 24).Plasma membrane had no significant citrate synthase activity, and thedifference between mitochondrial and plasma membrane citrate syn-thase activity was �20-fold. Alkaline phosphodiesterase I activity wasmeasured as a marker of plasma membrane, and a 20-fold difference inactivity was detected between plasma membrane and the trivial activity inthe mitochondrial fraction (2). HSP60 in fractions was compared byWestern blotting with equal amounts of protein loaded per lane. PercentHSP60 per fraction was calculated based on relative amounts by Westernblotting corrected by total protein per fraction, using the followingequation: [(density of HSP60 band for fraction A/sum of density bandsfor all 3 fractions) � (total protein in fraction A)]/(sum of protein in all3 fractions).

Lipid Rafts

Lipid rafts were isolated using the approach of Oliferenko et al.(14) and Lafont et al. (9). The method is based on the insolubility ofthese structures in the nonionic detergent Triton X-100. Briefly, theheart tissue was homogenized in isolation medium containing 150mM NaCl, 5 mM EDTA, and 25 mM Tris �HCl, pH 7.4, supplementedwith a cocktail of protease inhibitors and 1% Triton X-100. Fourvolumes of OptiPrep were added to 2 volumes of homogenate.OptiPrep was diluted with the isolation medium to give 35, 30, 25, and20% (wt/vol) iodixanol. Equal volumes of sample and the fourgradients were added to a tube and centrifuged for 4 h at 160,000 g inthe SW41 Ti rotor. Lipid rafts were collected from the top interface.

Western Blotting and ELISA

Western blotting and analysis was performed as previously de-scribed (6, 12). Anti-HSP60 was obtained from StressGen (Vancou-

ver, Canada) and used at a concentration of 1:15,000. Caveolin-1(polyclonal; Santa Cruz Biotechnology) and flotillin antibodies(monoclonal; BD Transduction) were used at 1:1,000. Affinity-puri-fied secondary antibody-horseradish peroxidase (rabbit and mouse;Amersham) was used at 1:1,000. HSP60 was measured using ELISA(StressGen). Likewise, cytokines were measured using ELISA(Pierce, Rockford, IL).

In Vitro Transcription and Translation

In vitro transcription and translation was done using a kit (Pro-mega) as previously described (7).

Immunocytochemistry

Immunocytochemistry was done as previously described (25). Themonoclonal antibody for HSP60 was obtained from StressGen andused at a 1:500 concentration.

Flow Cytometry

To detect HSP60 on the surface of cardiac myocytes, cardiacmyocytes were isolated from sham and CHF rats as previouslydescribed (27). Before calcium was reintroduced, the cells wereadjusted to 200,000–300,000 cells/ml, fixed with formalin for 15 minat 4°C, incubated with 10 �g/ml FITC-conjugated mouse anti-HSP60monoclonal antibody (BD Pharmingen) for 45 min at 37°C, washed,and analyzed using FACScan flow cytometry (Becton Dickinson, SanJose, CA). To detect intracellular HSP60, cells were permeabilizedwith 0.1 mg/ml lysophosphatidylcholine (Avanti Polar Lipids) inbuffered formalin for 15 min at 4°C before incubation with the HSP60antibody. In an alternative approach, cells were treated with pro-pidium iodide (PI) immediately after isolation, washed, fixed asdescribed above, and then incubated with anti-HSP60-FITC, as de-scribed above. After washing, flow cytometry was done to comparecells labeling with each or both of these fluorescent markers.

Flow cytometry was used to correlate the presence of HSP60 on themembrane with myocyte viability. Double-label flow cytometry wasdone to determine whether the presence of HSP60 on the plasmamembrane increased apoptosis. Caspase-3/7 activation was quantifiedusing a fluorescently labeled inhibitor of these caspases, which bindsonly to the activated form of the caspase (Vybrant FAM caspase-3 and-7 assay kit; Molecular Probes) (20). A similarly labeled inhibitor forcaspase-8 (Vybrant FAM caspase-8; Molecular Probes) was used toquantify caspase-8 activation. Flow cytometry allowed the identifica-tion of the percentage of cardiac myocytes with HSP60 on the plasmamembrane that had activation of caspase-3/7 and caspase-8 comparedwith cells that did not have HSP60 on the plasma membrane. For thecaspase studies, a biotin-labeled anti-HSP60 antibody (Labvision,Fremont, CA) was used. Avidin-Texas red was used to attach afluorescent tag.

Confocal Microscopy

Cardiac myocytes were seeded onto laminin-coated coverslips andincubated at 37°C for 2 h to allow adherence. To identify thelocalization of HSP60 on plasma membrane, cells were fixed with 4%paraformaldehyde for 10 min at room temperature, blocked in 2%BSA for 60 min at room temperature, incubated with 1 �g/ml mouseanti-HSP60 monoclonal antibody (StressGen) overnight at 4°C, anddeveloped with 4 �g/ml Texas red-conjugated goat anti-mouse anti-body (Abcam). Confocal microscopy at �600 magnification wascarried out using a Zeiss confocal laser scanning microscope.

Statistical Analysis

Results are means � SE. Data were analyzed using a one-wayANOVA followed by a Holm-Sidak test or an ANOVA on ranksfollowed by Dunn’s test or a Student-Newman-Keuls test. LVAD data

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were analyzed using a paired t-test. Antibody data were comparedusing a �2 test. A P � 0.05 was considered significant.

RESULTS

Heart Failure Model

Coronary ligation was chosen as a model of heart failureto model ischemic cardiomyopathy. By 9 wk post-coronaryligation, a fibrous scar had replaced the region of infarct andno ischemia was present. As shown in Fig. 1A, this model ofheart failure resulted in a significant increase in heart-to-body weight ratio at 9 and 12 wk. Cardiac echo was usedto measure LV dimensions in the short-axis view and tocalculate fractional shortening (Fig. 1B). LV diastolic di-mensions (LVDD) did not significantly increase until 12 wk(Fig. 1C), whereas fractional shortening was markedly de-creased at both 9 and 12 wk (Fig. 1D). Along with thesefunctional differences, significant changes in ANP, BNP,and TNF-� did not occur until 12 wk (Fig. 2, A–C). Thus,over the course of 12 wk, rats with coronary ligation showedprogressive CHF.

HSP60 in Heart Failure

Previously, our group (8) reported that HSP60 is doubled in theend-stage failing human heart. Total LV HSP60 levels increased�40–50% by 9–12 wk compared with controls (Fig. 2D) with amean value of 0.37 �g/mg soluble protein. Assay of plasmasamples showed that by 9 wk, levels of HSP60 were 12.3 � 2.1ng/ml plasma compared with 0 for controls (P � 0.001).

HSP60 Distribution in Heart Failure

Previously, our group (5) has observed that HSP60 moves tothe plasma membrane of isolated adult cardiac myocytes fol-lowing hypoxia and reoxygenation. To determine whether thesame abnormal distribution of HSP60 occurs in the chronicinjury state of heart failure, LV samples were fractionated intoplasma membrane, cytosol, and mitochondrial fractions. Asshown in Fig. 3A, a significant amount of HSP60 (3.0 � 0.8%,P � 0.05) was detected in the plasma membrane fraction at 9wk, and this increased to 8.2 � 1.0% at 12 wk (P � 0.05 vs.9 wk and controls). Virtually no HSP60 was detected in controlplasma membrane fractions.

Fig. 1. A: heart-to- body weight ratio in sham vs. 9- and 12-wk congestive heart failure (CHF) rats. B: representative cardiac echo showing normal wall motionin sham and hypokinesis in CHF heart. Arrows point to septum in both echoes, with reduced wall motion in the CHF heart. C: left ventricular diastolic dimensions(LVDD; cm) in sham vs. 9- and 12-wk CHF rats. D: fractional shortening in sham vs. 9- and 12-wk CHF rats. In A–D, n 6–14/group. *P � 0.05 vs. sham.

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Because total HSP60 is increasing at the same time ascellular distribution is changing in CHF, we were interested inhow absolute levels of HSP60 changed in various fractions. Asshown in Fig. 3B, the overall pattern of change was similar, but

the increase in mitochondrial HSP60 at 9 wk was moremarked, as was the concomitant decrease in cytosolic HSP60.Although mitochondrial HSP60 increased, overall protein lev-els in the mitochondrial fraction were unchanged. Total mito-

Fig. 2. A: plasma atrial natriuretic peptide (ANP) con-tent. B: plasma brain natriuretic peptide (BNP) con-tent. C: plasma tumor necrosis factor (TNF)-�content. D: rat LV heat shock protein (HSP) 60level. Cytokine and HSP60 levels were determinedusing ELISA (n 5–19/group). *P � 0.05 vs.sham.

Fig. 3. A: cellular distribution of HSP60 in CHFvs. sham hearts (n 4–9/group). B: absolutelevels of HSP60 in cellular fractions. C: represen-tative fractions for sham and 2 CHF hearts (both9 wk). Two micrograms of total protein wereloaded in each lane: P or PM, plasma membrane;M or MIT, mitochondria; C or CYT, cytosol.Although the amount of plasma membraneHSP60 appears to be approximately one-third thatin mitochondria in failing heart samples, theplasma membrane only has 10–20% as muchprotein as the mitochondria. Therefore, totalHSP60 in the plasma membrane fraction is muchless than that in the mitochondria. D: nondenatur-ing Western blot showing cytosolic HSP60 run-ning at 60 and �90 kDa. Separation of the proteinis nonlinear. *P � 0.05 vs. sham. P � 0.05 vs.sham and 12 wk.

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chondrial protein at 9 wk was 1.27 � 0.05 mg/g tissue for theCHF group compared with 1.28 � 0.03 mg/g tissue for thecontrols. Thus there was a preferential increase in HSP60alone, rather than an increase in total mitochondrial protein.HSP60 is thought to be synthesized in the cytosol from anuclear transcript as a pre-HSP60 with a 26-amino acid mito-chondrial transport sequence (MTS) at the amino terminus(19). After transport to the mitochondria, the MTS is cleavedand some of the HSP60 returns to the cytosol (19). Thepresence of this 26-amino acid (molecular mass 3.1 kDa) MTSwas clearly distinguished from cleaved HSP60 by 10% SDS-PAGE (19). As shown in Fig. 3C, Western blot analysis ofmitochondrial HSP60 at 9 wk did not demonstrate the doubletthat would be expected if uncleaved HSP60 accumulated in themitochondria (Fig. 3C). Careful analysis of both total andmitochondrial HSP60 by Western blotting with a 12% SDS-PAGE showed no evidence of the preprotein form (P1), al-though after in vitro transcription and translation, this could beeasily detected (data not shown).

In the mitochondria, HSP60 with HSP10 forms a heptamer-ous barrel within which folding of imported proteins occurs.We next examined whether in the cytosol, the tertiary structure ofHSP60 was different. As shown in Fig. 3D, a nondenaturing gel

transferred to a membrane and developed for HSP60 demon-strated two bands: one at 60 kDa and one at 90 kDa. Thus HSP60in the cytosol is a monomer and also binds to one or more smallproteins, as has been reported in the literature (6, 18).

HSP60 Localization in End-Stage Human Hearts

Cell fractionation studies were done on samples of end-stageleft ventricles, removed at the time of transplant, to determinewhether the cellular distribution of HSP60 was altered, as inthe rat heart failure model. Seven DCM (5 male, 56.8 � 6.4 yrold) and six ICM hearts (6 male, 66.5 � 1.0 yr old) werestudied. As shown in Fig. 4A, both DCM and ICM hearts hadHSP60 in the plasma membrane fraction compared with nonein normal hearts (see Table 1). Based on the relative amountsand total protein in the mitochondria and plasma membrane,we estimated that 7.7% of HSP60 was associated with theplasma membrane in heart failure compared with 92.3% withthe mitochondria.

To determine the full distribution of HSP60, cell fraction-ation was performed on additional samples to compare cytosol,mitochondria, and plasma membrane distribution of HSP60.As shown in Table 1, 1.7% of HSP60 was present in the plasma

Fig. 4. A: cellular distribution of HSP60 inhuman cardiomyopathy by Western blotanalysis. Hearts 1–3 have ischemic cardio-myopathy (ICM), and hearts 4–6 have di-lated cardiomyopathy (DCM). See Table 1for summary of data. Although the amountof plasma membrane HSP60 appears to beabout one-half that in mitochondria in failingheart samples, the plasma membrane onlyhas 10–20% as much protein as the mito-chondria. Therefore, total HSP60 in plasmamembrane fraction is much less than that inmitochondria. B: summary time course (left)of changes in cellular distribution in failingrat heart and comparison with end-stage hu-man heart (bars at right). C: immunocyto-chemistry of normal vs. failing human heartshowing distribution of HSP60 (brown).D: comparison of plasma membrane vs. mi-tochondrial HSP60 in human hearts pre- andpost-LV assist device (LVAD) implantation.Data labeled A– D represent 4 different pa-tients pre- and post-LVAD. Normal heartrepresents small biopsies from 5 normal do-nor hearts combined. Note HSP60 some-times runs as a doublet on gel, but this notconsistent. Doublet may represent posttrans-lational modification. *P � 0.05 vs. sham.P � 0.05 vs. sham and 12 wk.

Table 1. Distribution of cardiac HSP60 among mitochondria, plasma membrane, and cytosol in human and rat hearts

Human Rat

Normal LV DCM LV ICM LV CHF 12 wk LV Normal LV Isolated Adult Cardiac Myocytes

PM 0 1.7�0.2 2.4�0.2 8.2�1.0 0.5�0.3 0M 67.7�5.9 59.9�3.3 55.9�1.1 66.4�5.2 70.5�4.0 65.4�5.4C 32.3�5.9 38.5�3.5 41.7�1.2 25.4�3.9 29.0�4.1 34.7�5.4

Values indicate distribution of cardiac heat shock protein 60 (HSP60) among plasma membrane (PM), mitochondria (M), and cytosol (C) in human and rathearts as determined by cell fractionation followed by Western blotting (n 3–8/group, except for cardiac myocytes, where n 2). DCM, dilatedcardiomyopathy; ICM, ischemic cardiomyopathy; CHF, congestive heart failure; LV, left ventricle.

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membrane of DCM hearts and 2.4% in ICM hearts. Roughly40% of HSP60 was in the cytosol in these failing hearts. Forcomparison, cell fractionation was done on normal humanheart samples (all male, 54.0 � 2.1 yr old). None had a cardiachistory, although one was found to have atrial fibrillation at thetime of presentation. Normal human ventricle had no HSP60 inthe plasma membrane and 32.3% HSP60 in the cytosol. Figure4B shows a comparison of HSP60 distribution during devel-opment of heart failure in the rat compared with that inadvanced human failure. Both groups show similar plasmamembrane distribution of HSP60. As shown in Fig. 4C, HSP60expression was increased throughout the myocytes by immu-nocytochemistry, although the intensity varied across differentfields.

LVAD implantation represents an intervention in advancedheart failure that is associated with hemodynamic improve-ment, even though most of these patients go on to transplant.We analyzed paired pre- and post-LVAD samples and corre-lated this analysis with outcome: transplant, death, or clinicalimprovement. The seven LVAD patients (2 male, 56.3 � 7.8 yrold) had significant amounts of HSP60 in the plasma mem-brane (Fig. 4D). Comparing just mitochondrial and plasmamembrane distribution showed that at implantation, 12.1 �3.9% of HSP60 was in the plasma membrane, and at the timeof removal this value was 10.2 � 3.3% (Table 2, P NS).

Lipid Rafts

Lipid rafts are regions rich in signaling receptors andcytodomains and are hot zones of membrane activity, such asprotein transport. As shown in Fig. 5, HSP60 was found in lipidrafts prepared from explanted human hearts. Both caveolin-1 andflotillin, lipid raft markers, were found in association with HSP60(Fig. 5, A and B).

Is HSP60 Detectable on the Cell Surface?

If HSP60 is exposed on the cell surface, it can be recognizedby antibodies or released, which could activate the innateimmune system. Two different approaches were taken to ad-dress whether HSP60 was accessible to immunodetection by amonoclonal antibody. Isolated rat cardiac myocytes were pre-pared from failing and sham hearts at 9 wk. The myocytes wereimmediately fixed, developed with FITC-labeled anti-HSP60,and detected using flow cytometry. As shown in Fig. 6B,

myocytes from failing hearts expressed HSP60 on their sur-face. Control myocytes had only the baseline fluorescence seenwith a nonspecific antibody and with autofluorescence (Fig.6A). Mean events were 129.0 � 13.6 in the failing myocytescompared with controls at 58.4 � 13.6 (P � 0.05), and thesignal intensity was far greater in the failing myocytes asshown in Fig. 6, A and B. Signal from the normal myocytesincubated with anti-HSP60 antibody did not differ from con-trols treated with a nonspecific antibody ( Fig. 6A). Permeabi-lization with lysophosphatidylcholine exposed intracellularHSP60 to antibody as shown in Fig. 6, C and D. Positive eventsincreased in both groups, and there was no difference betweenthem after permeabilization, indicating that the 1.2-fold in-crease was due to detection of the upregulated plasma mem-brane HSP60.

Spatial distribution of HSP60 was also imaged by confocalmicroscopy. As shown in Fig. 6E, HSP60 is clearly evident onthe surface of a fixed, but not permeabilized, failing myocytetreated with anti-HSP60 compared with a normal myocyte(Fig. 6F). Patterns varied, with some cells showing onlyHSP60 around the edge of the cell, whereas others had moreextensive labeling along the plasma membrane. Normal myo-cytes showed no significant labeling on the cell surface. Per-meabilization increased intracellular detection, since HSP60fluorescence was clearly present on sarcomeric bands in thecontrol myocytes (Fig. 6H). In failing myocytes, the pattern ofHSP60 expression varied after permeabilization. Some cellsshowed the typical sarcomeric pattern of expression, but othershad very abnormal patterns (Fig. 6G). These data clearly showsurface upregulation of HSP60 in myocytes from failing hearts.

Plasma Membrane HSP60 and Apoptosis

Cardiac myocytes were isolated from failing hearts at 9 wk.Cells were examined for uptake and binding of FAMcaspase-8, which is a cell-permeable compound that detectsactivated caspase-8, and this was compared with the presenceof HSP60 on the cell surface. As shown in Fig. 7, A and B,myocytes with HSP60 on the cell surface were far more likelyto have activation of caspase-8. For comparison, 20–25% ofnormal myocytes immediately after isolation are positive forcaspase-8 activation (Kim SC and Knowlton AA, unpublished

Fig. 5. A: Western blot showing HSP60 localizes in lipid rafts along withcaveolin-1. B: HSP60 colocalizes with flotillin in lipid rafts.

Table 2. Effect of LVAD implantation on HSP60 distributionin plasma membrane relative to mitochondria

DevicePre-LVAD

Membrane HSP60, %Post-LVAD

Membrane HSP60, % Outcome

TVAD 30.4 2.1 TransplantTVAD 7.1 6.2 TransplantNVAD 0.02 13.9 TransplantDVAD 21.8 28.4 Transplant,

poor prognosisDVAD 9.7 6.2 TransplantDVAD 6.6 7.2 TransplantDVAD 8.2 7.6 Transplant

Values are percentages of HSP60 in plasma membrane relative to that inmitochondria following left ventricular assist device (LVAD) implantation.DVAD, DeBakey LVAD; TVAD, Thoratec LVAD; NVAD, Norvasc LVAD.All patients were transplanted.

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observations). Figure 7B shows FAM caspase-8 distributedthroughout the cell, whereas HSP60 is localized in distinctclusters on the surface. Similarly, as shown in Fig. 7, C and D,HSP60 on the cell surface was associated with higher activa-tion of caspase-3/7, using myocytes from the same hearts.Figure 8D shows localization of FAM caspase-3/7 throughoutthe myocyte with surface localization of HSP60 evident on theedge of the myocyte. Thus surface HSP60 was associated withhigher activation of caspases and confirmed entry into theapoptotic cascade, supporting the hypothesis that cell surfaceHSP60 adversely effects myocyte viability.

To confirm that HSP60 on the cell surface did not correlatewith increased permeability, we studied cardiac myocytesfreshly isolated from failing hearts at 9 wk. Cells were notplated, to minimize changes from isolation and culture, butwere immediately incubated with PI. After washing, the cellswere fixed and incubated with anti-HSP60-FITC. Flow cytom-etry showed that there was no difference in PI uptake betweencells that had HSP60 on their surface and those that did not, assummarized in Fig. 7E. The high uptake of PI in both groupsreflects that the hearts were diseased, making cell isolationmore damaging to the myocytes, and the cells had not under-gone the initial plating step, which removes dead cells.

HSP60 is quite immunogenic, and antibodies to HSP60 arefound in the general population, possibly from prior bacterialinfections (there is high homology between bacterial and hu-man HSP60). Therefore, to determine whether heart failurewith both surface localization and release of HSP60 into theplasma is associated with increased antibodies to HSP60, ratplasma samples from sham controls and failing hearts werecompared for the presence of anti-HSP60 antibodies binding topurified recombinant human HSP60 (StressGen). This was

assessed using a special apparatus to load 20 different antibodysamples, and these were incubated with a nitrocellulose mem-brane containing a single band of HSP60 across the width ofthe membrane. Sixty percent of sham controls had antibodiesto HSP60 at a 1:2,500 plasma dilution along with 60% of 9-wkCHF rats and 100% of 12-wk CHF rats (P 0.045). At1:10,000 dilution, none of the control samples were positive foranti-HSP60 antibody, but 20% of 9-wk CHF rats and 55% of12-wk CHF rats were positive (Fig. 8A, P 0.004). Arepresentative slot blot is shown in Fig. 8B.

DISCUSSION

Coronary ligation in rats induced heart failure, which pro-gressed over the time of the study. At 9 wk, decreased frac-tional shortening and increased HSP60 were present, andHSP60 was already in the plasma. By 12 wk, BNP, ANP,TNF-�, and LVDD were all increased. Studies demonstratedthat not only was HSP60 localized in the plasma membranefraction but also, importantly, it was detectable on the exofacialsurface, where it is a potential antibody target or innate im-mune system ligand. Cell surface HSP60 was associated withapoptosis. A fundamental issue is HSP60 localization. WhenHSP60 is in the cytosol or mitochondria, it is antiapoptotic andprotective. When HSP60 is in the plasma membrane or extra-cellular, it becomes a danger to the cell. There is precedent forthis; cytochrome c is critical for redox when within the mito-chondria, but when in the cytosol it is a key participant in theapoptotic cascade.

HSP60 is primarily thought a mitochondrial protein, but aswe have shown, a significant amount of HSP60 is present in thecytosol in the normal heart, as has been observed in some other

Fig. 6. Localization of HSP60 to cell surface in heart failure (n 2–3/group). Fluorescence-activated cell sorting (FACScan) analysis is shown in A–D. Numbersare the mean number of events (�SE). A: normal cardiac myocytes treated with anti-HSP60-FITC after fixation but without permeabilization of cells. Red lineshows results from myocytes after labeling with a nonspecific antibody. B: cardiac myocytes from failing heart as in A. Labeling with nonspecific antibody isshown in red, as a control. P � 0.05 vs. control. C: effect of permeabilization with lysophosphatidylcholine on binding of anti-HSP60-FITC to normal cardiacmyocytes. D: same as in C, with cardiac myocytes from failing heart. Images in E–H are confocal images of failing and normal cardiac myocytes (magnification�600) with and without permeabilization. Some myocytes showed patches of HSP60, whereas others show HSP60 only visible on the edge of cell. E: confocalimage of nonpermeabilized failing cardiac myocyte. Patches of HSP60 are visible on the surface of the cell (arrows). F: confocal image of normal cardiacmyocyte. There was little to no labeling of this nonpermeabilized cell. G: confocal image of permeabilized failing cardiac myocyte. An abnormal labeling patternis shown compared with normal permeabilized myocyte in H. Labeling of mitochondrial HSP60 in the permeabilized cell shows striation pattern in normalmyocytes and in some of the failing myocytes. H: confocal image of permeabilized normal cardiac myocyte. Clear striations are visible.

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settings (6, 21, 23). Unexpectedly, at 9 wk, cytosolic HSP60dropped significantly and mitochondrial HSP60 was greatlyincreased without any increase in overall mitochondrial pro-tein. By 12 wk, the percentage and absolute amounts ofmitochondrial HSP60 were similar to those for the control, butHSP60 persisted in the plasma membrane fraction. Analysis ofend-stage human hearts, removed at the time of transplant,showed similar distribution of HSP60 to the failing rat hearts at12 wk. Total HSP60 in the end-stage human heart is doubledcompared with that in the failing rat hearts, which had a38–52% increase in total HSP60 (8). However, the failinghuman heart, sustained by medications and other therapies,persists in a state of heart failure much longer than theexperimental model.

Key questions are why is HPS60 in the plasma membranefraction, and what is the consequence? In the current study,surface localization of HSP60 was associated with apoptosis.There are a several reports that in mammalian cells, HSP60translocates to the plasma membrane (11). Localization ofHSP60 to the plasma membrane has been observed in severaldifferent types of cultured cells with a possible role in mem-brane transport or signaling (3, 22). However, more often,HSP60 has been found in association with the plasma mem-brane under conditions of stress or injury, as in the currentstudy. HSP60 localized to the cell surface in stressed aorticendothelial cells has been postulated as a possible mechanismof atherosclerosis mediated through an immune response (1,32). Spontaneous apoptosis and cell lysis have been associated

Fig. 7. A: summary of flow cytometry experiments show-ing that surface localization of HSP60 by flow correlateswith activation of caspase-8. In normal myocytes imme-diately after isolation, 20–25% are positive for caspase-8activation. For the failing heart cardiac myocytes, overall56.7 � 5.6% were positive for caspase-8 activation.B: confocal image of cardiac myocyte stained for acti-vated caspase-8 (green, FITC, showing diffuse stainingthroughout cell) and for HSP60 (red). Merged image isshown at right. C: summary results of 4 experimentsshowing increased activation of caspase-3/7 when HSP60is present on the cell surface. Overall, for the failing heartcardiac myocytes, 59.3 � 3.6% were positive for caspase-3/7 activation. D: confocal image of cardiac myocytestained for activated caspase-3/7 (green) and for HSP60localization to cell surface (red). Caspase-3/7 localizedthroughout the cell. HSP60 is shown as a fine red rim atthe edge of the cell. Merged image is shown at right. InA–D, n 6/group. E: uptake of propidium iodide (PI)after isolation. No correlation was present betweenHSP60 on the surface and PI uptake (n 10/group).

Fig. 8. A: percentage of heart failure vs. sham control rats with plasmaanti-HSP60 antibody at 1:10,000 dilution. B: example of a slot blot. Onlysamples with a band lining up with HSP60 marker (shown at left) were scoredas positive (n 11–15/group).

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with expression of HSP60 and HSP70 on the cell surface inlymphocytes (17, 30). Previous work, as well as our own data,suggest that membrane expression of HSP60 can target cellsfor destruction and may mediate the induction of antibodiesagainst the cells themselves (4). In the current study, as heartfailure progressed there was an increase in antibodies toHSP60.

HSP60

HSP60 is primarily a mitochondrial protein, but in mamma-lian cells from 20 to 40% of cellular HSP60 has been reportedto be extramitochondrial (5, 23). Although HSP60 is thought toform a heptamerous barrel with HSP10 forming the barrelends, there are also data indicating that it exists at times in amonomeric form, as we saw in the cytosol, and it is able torefold proteins in this form (28). Other data suggest that HSP60is in equilibrium between the monomeric and heptamericforms (11).

HSP60 in synthesized in the cytosol with a mitochondriallocalization leader sequence (19). After translocation to themitochondria, a 26-amino acid peptide (3.1 kDa) containingthe mitochondrial localization sequence is cleaved from theamino terminus, and some HSP60 returns to the cytosol. Themechanism regulating distribution of HSP60 between the cy-tosol and mitochondria is unknown. We found an increase inmitochondrial HSP60 at 9 wk, but there was no evidence offailure to cleave HSP60. Little is known about the function ofHSP60 in the cytosol, although it is known to complex withBax, Bak, and Bcl-xL and is thought to have an antiapoptoticrole (6, 18).

HSP60: Paradoxically Injurious?

HSP60 as antigen. A number of pathological states havebeen reported to be associated with antibodies to HSP60. Inpatients with cardiomyopathy, 85% of those with DCM and42% with ICM had antibodies to HSP60 when a 1:200 dilutionof serum was examined (10). Portig et al. (16) found far fewerheart failure patients with antibodies to HSP60 (only 10% ofDCM and 1% of ICM) using a different approach. Nonetheless,the surface presentation of HSP60 on the myocyte combinedwith serum antibodies to this protein could target the myocytefor destruction via macrophage or neutrophil Fc recognition;this may be one mechanism fueling the downward spiral inheart failure. In the current study, we found an increase inantibodies to HSP60 as heart failure progressed with a clearincrease in antibody titer over time.

With the limited availability of donor hearts, the LVADprovides a bridge to transplant or recovery. We did not see areduction in membrane localized HSP60 with LVAD treat-ment, but the number of paired samples available was insuffi-cient to exclude that a reduction in HSP60 occurs with LVADtreatment.

HSP60 and innate immune system. HSP60 could also acti-vate the innate immune system. As we demonstrated, HSP60 isreleased into the plasma by the failing heart. Plasma HSP60could interact with monocytes or bind to anti-HSP60 antibodyand activate complement (15, 31, 33). This released proteincould also act in a paracrine or autocrine manner to activateTLR-4, one of the receptors for the innate immune system, forwhich HSP60 has been reported to be a ligand (13). Activation

of TLR-4 leads to the production of cytokines, includingTNF-�, which in itself is toxic to the myocyte. HSP60 ispresent in the plasma membrane and the plasma at 9 wk. Theincrease in plasma TNF-� was not seen until 12 wk, supportingthe suggestion that HSP60 helps drive the increase in TNF-�.Thus, by several different mechanisms, membrane and extra-cellular HSP60 can potentially target the cardiac myocyte fordestruction.

Further work is needed to elucidate the mechanisms control-ling cellular localization of HSP60 and the function of HSP60in the plasma membrane. The increase in HSP60 and itsredistribution likely have negative effects on the heart and maytag the myocyte for destruction, much in the way Fas operates.There are several key issues revealed in this study, includingwhat drives the increase in HSP60. The transient drop incytosolic HSP60 at 9 wk is puzzling; this is associated with anincrease in mitochondrial HSP60. By 12 wk, although totalHSP60 is elevated and is still present in the plasma membrane,there is a relatively normal distribution of HSP60 between themitochondrial and cytosolic fractions. As discussed, the pres-ence of HSP60 in the plasma membrane fraction on the cellsurface and in the plasma provides several possible pathwaysto myocyte destruction. Understanding the function and regu-lation of HSP60 may provide a valuable early prognosticmarker in heart failure and further our understanding of themechanisms of disease progression.

ACKNOWLEDGMENTS

We thank Paul Munch for surgical assistance.

GRANTS

This work was supported by HL077281 (to A. A. Knowlton), HL079071 (toA. A. Knowlton), the Department of Veterans Affairs (to A. A. Knowlton), andAI47294 (to S. I. Simon).

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