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Am J Physiol Heart Circ Physiol 289: H2193-H2202, 2005. First published July 1, 2005; doi:10.1152/ajpheart.00355.2005
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Clusterin: a protective mediator for ischemic cardiomyocytes?

P. A. J. Krijnen,1,5 S. A. G. M. Cillessen,1 R. Manoe,6 A. Muller,3,5 C. A. Visser,2,5 C. J. L. M. Meijer,1 R. J. P. Musters,3,5 C. E. Hack,4,5,6 L. A. Aarden,6 and H. W. M. Niessen1,5

Departments of 1Pathology, 2Cardiology, 3Physiology, and 4Clinical Chemistry, Vrije Universiteit Medical Center, Amsterdam, The Netherlands; 5Institute for Cardiovascular Research, Vrije Universiteit, Amsterdam, The Netherlands; and 6Sanquin Research, Central Laboratory for Blood Transfusion, Department of Immunopathology and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Submitted 11 April 2005 ; accepted in final form 29 June 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
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We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.

ischemia; acute myocardial infarction; membrane flip-flop

CLUSTERIN (also designated apolipoprotein J) is an extracellular, highly conserved and heavily glycosylated, disulfide-linked heterodimer with an apparent molecular mass of 75–80 kDa. Clusterin is constitutively expressed in almost all mammalian tissues by different cell types, including epithelial and specific neuronal cells. Furthermore, clusterin is found in all body fluids, including plasma, seminal plasma, milk, urine, and cerebrospinal fluid (1, 17).

Clusterin has been reported to play a role in a wide variety of processes and has been shown to interact with a wide range of molecules, including lipids (7, 8) and several components of the membrane attack complex (MAC) of complement (5, 18, 25, 32, 38, 43). In the blood, clusterin is associated with subclasses of high-density lipoproteins, which also contain apolipoprotein A1 and cholesteryl ester transfer activity (7). Furthermore, expression of clusterin is upregulated in acute myocardial infarction (37), atherosclerosis (15, 37), myocarditis (36), oxidative stress and heat shock (40), Alzheimer's disease (26), several cancers (19), and after injury in general (17).

Clusterin deposition was previously reported in the human heart after acute myocardial infarction (AMI) (39), where it is colocalized with complement factors C1q, C4, C3d, and C9 on cardiomyocytes within the infarcted area. In contrast, in an experimental myocardial infarction model in the rat, depositions of clusterin were only found in the peri-infarct zone cardiomyocytes that did not stain for MAC (35). Moreover, these myocytes appeared to synthesize clusterin as detected with in situ hybridization. Expression of the clusterin gene was also induced in ventricular myocytes in a mouse model of myocarditis (36).

Considering the conflicting data on its distribution in rat versus human myocardial infarction, we decided to examine in more detail the functional role of clusterin in the human heart. In addition, we investigated its effect on ischemic rat neonatal myoblast cells (H9c2 cells), as well as on ischemic isolated adult ventricular rat cardiomyocytes as in vitro models of AMI.


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Subjects. Recently deceased patients included in the study (Table 1) were transferred to the Department of Pathology for autopsy (Vrije Universiteit Medical Center). At autopsy the deceased subjects showed signs of a recently developed AMI, i.e., on histochemical examination they had decreased lactate dehydrogenase (LDH) staining (decoloration) of the affected myocardium. Different infarct durations were categorized into different phases, as is explained in the next paragraph. Clinical data with respect to the duration of AMI corresponded to the different morphological stages of AMI. Autopsies were performed as soon as possible, but at least within 24 h after death. The study was approved by the ethics committee of the Vrije Universiteit Medical Center, Amsterdam, The Netherlands. The investigation conforms to the principles of the Declaration of Helsinki. Use of leftover material after the pathological examination is part of the standard patient contract in our hospital.


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  		Table 1. Subjects characteristics

 
Cell culture and metabolic inhibition. Neonatal rat cardiomyoblasts [H9c2 (2–1) cells; American Type Culture Collection, Manassas, VA] were cultured in culture medium as described previously (31). For experiments, cells grown at confluency of 60–80% were used. Metabolic inhibition was achieved by incubating the cells in ischemic buffer containing (in mM) 0.9 CaCl2·H2O, 20 2-deoxy-D-glucose (Sigma, St. Louis, MO), 106 NaCl, 5 NaCN, 3.8 NaHCO3, 4.4 KCl, and 1 MgCl2·H2O in H2O, pH 6.6 under a 5% CO2 atmosphere at 37°C. Reperfusion was mimicked by incubating the cells subsequent to metabolic inhibition in normal culture medium under a 5% CO2 atmosphere at 37°C.

Processing of tissue specimens. For immunohistochemistry we obtained heart tissue samples from patients who died after AMI. Myocardial tissue specimens were obtained from each subject from the infarcted site, as well as from the adjacent zone and from remote sites in the healthy part of the heart. These remote sites showed normal LDH-staining patterns and were studied as internal noninfarcted controls. Heart tissue samples from the left ventricle of patients who had died from other causes, not related to heart disease, were used as a control. None of these subjects had sepsis or malignancies. Before being prepared as cryosections, tissue specimens were stored at –196°C (liquid N2). Frozen sections were mounted on SuperFrost Plus glass slides (Menzel-Gläser, Braunschweig, Germany).

Antibodies. A MAb against human clusterin (MAb G7) was used for immunohistochemical analysis (gift of Dr. Brendan Murphy, Melbourne, Australia) and has been described previously (29). MAb against the {alpha}-chain of rat clusterin (Upstate Biotechnology, Waltham, MA) and MAb G7 were used for Western blot and fluorescence-activated cell sorting (FACS) analysis. MAb C3–15 against the complement factor C3d has been used previously for immunohistochemical studies (22). In FACS analysis, phycoerythrin (PE)-conjugated goat anti-mouse immunoglobulins (GAM-RPE; Dakopatts, Glostrup, Denmark) were used as a secondary antibody. In Western blot analysis, horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins (RAM-HRP, Dakopatts) were used as a secondary antibody. The MAbs were stored in PBS (1 mg/ml, pH 7.4). As controls, irrelevant MAbs (2 IgG1 and 1 IgG2a) were tested at concentrations similar to those used for the MAbs. Staining with these control MAbs always yielded negative results.

Immunohistochemistry. Frozen sections were stained as described previously (20, 21) as primary antibodies were used (anti-clusterin MAb, 1 to 750; MAb, C3–15 1 to 1500).

Microscopic criteria (6, 24) were used to estimate infarct duration in all myocardial tissue specimens. To allow a more reliable morphological judgment, corresponding paraffin slides were also made. We characterized jeopardized myocardium with macroscopic LDH-decolorization (but without microscopical changes as an early-phase infarction), with macroscopic LDH-decolorization with infiltration of polymorphonuclear neutrophils (leukocytes, PMNs) as a PMN-phase infarction, and with macroscopic LDH-decolorization with infiltration of lymphocytes and macrophages and with signs of fibrosis as a chronic-phase infarction. We categorized as reinfarctions those subjects with an explicit medical history of a second myocardial infarction within 2 wk after the first; one subject with chronic-phase morphology and one with early-phase morphology were classified as early-phase reinfarctions, whereas those subjects with chronic-phase morphology combined with PMN-phase morphology were classified as PMN-phase reinfarctions. In all cases, histologically assessed infarct duration corresponded with the clinical course.

Two investigators (P. A. J. Krijnen and H. W. M. Niessen) each independently judged and scored all slides for infarct age and anatomic localization of the proteins, as visualized by immunohistochemical staining. Scattered clusterin and complement single positive cardiomyocytes were counted at a high-powered field (HPF; x400 magnification) in 25 HPFs. The average number of positive cardiomyocytes per HPF was used in the calculations. To assess the anatomic relation between clusterin and complement depositions, serial slides were used. For the final scores, consensus was achieved by the two investigators.

Western blot analysis. H9c2 cardiomyoblasts were dissolved in SDS sample buffer containing {beta}-mercaptoethanol as a reducing agent, stirred, and heated for 10 min at 95°C. The samples were subjected to SDS-PAGE using 10% gels, transferred to nitrocellulose membranes, and immunoblotted by subsequent incubation with MAb against rat clusterin (1:1,000) and RAM-HRP (1:1,000). The blots were then visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, UK). For experiments with cycloheximide (CH) and actinomycin D (AMD; both from Sigma, St. Louis, MO), H9c2 cells were incubated with 0.21 µM CH and 10 µM AMD before (1 h) and during metabolic inhibition (2 h). Lysates were subsequently made in reducing Laemmli buffer and were analyzed through Western blot analysis.

ATP measurements. H9c2 cardiomyoblasts were subjected to increasing periods of metabolic inhibition (0, 20 min, 40 min, 1 h, 1.5 h, and 2 h) and subsequent reperfusion (2 h) as described in Cell culture and metabolic inhibition. The following steps were all performed at 4°C. The cells were collected and counted, and the cell concentrations were equalized accordingly. After centrifugation (400 g) the cells were incubated in 150 µl of 0.4 M perchloric acid (Sigma) for 30 min and subsequently centrifuged (2,000 g). The supernatants were collected, and the pH was restored to 7 by adding 10 µl of 5 M K2CO3 (Sigma). The samples were frozen (–80°C) until measurements were taken. ATP levels were measured in a bioluminescence assay, using a sensitive ATP determination kit (Biaffin, Kassel, Germany) and following the manufacturer's protocol. Samples were measured in a FluoroNunc MaxiSorp plate (Nalge Nunc International, Rochester, NY). Luminescence was measured by using a Tecan GENios Plus reader (Tecan Benelux, Mechelen, Belgium).

Purification of human serum clusterin. Human clusterin was purified from human serum by affinity chromatography on MAb G7 coupled to sepharose, essentially as described previously (29). After elution at pH 2.8, the eluate was dialyzed against PBS and stored at a concentration of 200 µg/ml at –20°C.

Flow cytometry. Annexin V (AV)-FITC (Bender Med Systems, Vienna, Austria) was used to assess flip-flop of the cell membrane. Propidium iodide (PI; Bender Med Systems) was used to assess membrane permeability and thus cell death. H9c2 cells were incubated in culture medium or ischemic buffer (see Cell culture and metabolic inhibition). To assess binding of human serum clusterin, the cells were subsequently incubated for 30 min at 37°C in serum-free medium containing 20 µg purified human serum clusterin/ml. The cells were then incubated with MAb G7 against human clusterin (1:100) or with the MAb against the {alpha}-chain of rat clusterin (1:100) for 30 min at 4°C and subsequently labeled with GAM-RPE (1:20) for 30 min at 4°C. Finally, the cells were stained with AV-FITC (1:40) for 30 min at 4°C and subsequently washed twice with PBS. PI (1:40) was added only a few minutes before measurement. The cells were then analyzed by flow cytrometry (FACStar; Becton Dickinson).

Live cell imaging of AV-FITC-labeled H9c2 cells. H9c2 cells were grown in Delta T culture dishes (Bioptechs, Butler, PA) and subjected to metabolic inhibition (2 h) as described in Cell culture and metabolic inhibition. The cells were then incubated with AV-FITC (1:40) in serum-free medium for 30 min at 37°C. The cells were subsequently washed twice with PBS (37°C) and then incubated with normal culture medium at 37°C. The cells were qualitatively analyzed by the use of a 3I Marianas digital imaging microscopy workstation (41). Pictures were taken immediately after several locations were stained, and these locations were memorized by the software. The culture dishes were then returned to a 5% CO2, completely dark atmosphere at 37°C. After 2 h and 4.5 h, pictures were taken at the memorized locations with a similar camera setup.

Morphologic analysis of effects of purified human serum clusterin on H9c2 cell viability. H9c2 cells, grown in 12-well plates, were subjected to culture medium or ischemic buffer for 2 h in the absence or presence of 20 µg purified human serum clusterin/ml. Pictures of the cells in culture were subsequently taken by using a Leica DM-R microscope (Leica, Wetzlar, Germany; x25 magnification). The cells were then counted, distinguishing between cells with normal morphology or rounded cells. The percentage of rounded cells of the total number of cells was then calculated.

Analysis of effects of purified human serum clusterin on viability of isolated adult rat cardiomyocytes. Animals were treated according to the national guidelines and with permission of the Animal Experimental Committee of the Vrije Universiteit.

Hearts were excized from Wistar rats and were perfused retrogradely (Langendorff) at 37°C for 2 min with Tyrode buffer containing (in mM: 120 NaCl, 27 NaHCO3, 5.0 KCl, 2.0 NaH2PO4, 1.2 MgSO4, 10 glucose, 5 creatine, and 5 taurine; gassed with 95% O2-5% CO2) supplemented with 1 mM Ca2+, and followed by perfusion for 2 min with Tyrode buffer supplemented with 10 µM Ca2+. The heart was then perfused with recirculating Tyrode buffer supplemented with 10 µM Ca2+, 150 U/ml collagenase (class II, Worthington Biochem), and 0.5 mg/ml hyaluronidase (Sigma-Aldrich) at a flow rate of 10 ml/min for 20 min. Ventricular tissue fragments were incubated with HEPES buffer containing (in mM) 116 NaCl, 20 HEPES, 5.3 KCl, 1.13 NaH2PO4, 1.2 MgSO4, 10 glucose, 5 creatine, and 5 taurine, and 100 U/ml penicillin-streptomycin, 0.2% (wt/vol) fatty acid-free BSA, and 10 µmol/l Ca2+; pH 7.35), containing 150 U/ml collagenase and 0.5 mg/ml hyaluronidase during 8 min in a shaking water bath at 37°C. The obtained cell suspension was mixed (1:1) with HEPES buffer supplemented with 0.4 mM Ca2+, passed through a 200-µm mesh nylon gauze, and centrifuged for 1 min at 43 g. The cells were suspended in 35 ml of a HEPES-medium 199 mixture containing 0.4 mM Ca2+ and were allowed to sediment during 10 min at room temperature. This process was repeated with HEPES-M199 mixtures of 0.8 and 1.2 mM Ca2+, respectively. Finally, the cells were resuspended in M199 [supplemented with 5 mM creatine, 5 mM taurine, 100 U/ml penicillin-streptomycin, and 0.2% (wt/vol) fatty acid-free BSA], seeded onto laminin-coated Lab Tek II chamber slides (Nalge Nunc International, Rochester, NY), and allowed to adhere to the substratum for 1–2 h. Medium was then changed to supplemented M199 [i.e., M199 was supplemented with 5 mM creatine, 5 mM taurine, 100 U/ml penicillin-streptomycin, 0.2% (wt/vol) fatty acid-free BSA, 100 nM insulin, 0.1 nM L-triiodothyronine, and 2 mM carnitine].

Isolated ventricular rat cardiomyocytes were subjected to supplemented M199 or ischemic buffer for 30 min in the absence or presence of 20 µg purified human serum clusterin/ml, under a 5% CO2 atmosphere at 37°C. The cells were subsequently incubated with AV-FITC (1:40) in serum-free DMEM (containing 100 IU penicillin/ml, 100 µg streptomycin/ml, and 2 mM L-glutamin) for 30 min under a 5% CO2 atmosphere at 37°C. Afterward, the cells were washed twice with PBS (37°C) and then incubated with supplemented M199. PI was added immediately before analysis. The cells were analyzed by fluorescence microscopy using the three-dimensional live-imaging microscope.

Statistics. Statistics were performed with the SPSS statistical program (Windows version 9.0). To evaluate whether observed differences were significant, paired or nonpaired t-tests were used when appropriate. A P value (2-sided) of <0.05 was considered to represent a significant difference.


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Distribution of clusterin in human hearts after AMI. In agreement with a former study (39), we also found colocalization of clusterin and complement in the infarcted areas of the human heart in PMN-phase infarctions and PMN-phase reinfarctions (Fig. 1). In these large areas, complete colocalization of complement and clusterin was found. In addition, we also found cardiomyocytes within the infarcted area that stained positive for clusterin (Fig. 1C) but were negative for complement factor C3d (Fig. 1D). In contrast with the large areas that were positive for complement and clusterin, these clusterin-positive and complement-negative cardiomyocytes were found in all phases of infarction or reinfarction. Remarkably, the morphology of these cells was that of viable cells. Furthermore, these clusterin-positive and complement-negative cardiomyocytes were also found in the border zone and in the remote zone, although at lower numbers (data not shown). Notably, in the hearts of control subjects, almost no clusterin-positive cardiomyocytes were found (mean = 0.19 positive cardiomyocytes/HPF compared with a mean of 1.62 (chronic phase) or higher in the infarcted areas of AMI subjects).



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  		Fig. 1. Localization of clusterin and complement factor C3d on cardiomyocytes within infarcted area of the human heart. Colocalization of clusterin (A) and complement factor C3d (B) in the human heart in a polymorphonuclear neutrophils (leukocytes; PMN)-phase infarction (magnification x25). Scattered cardiomyocytes positive for clusterin (C), but negative for complement factor C3d (D), within infarcted area of early-phase infarction (magnification x400). Detail of clusterin positive cardiomyocytes with predominantly membranous (E, arrows) or predominantly cytosolic (F, arrow) staining (magnification x630).

 
There was a marked difference in the pattern of clusterin staining between the clusterin-positive and complement-positive fibers and the clusterin-positive and complement-negative fibers. In the former, clusterin was predominantly located on the plasma membrane (Fig. 1E) and to a much lesser extent in the cytoplasm, whereas in the latter, clusterin staining was weaker and predominantly found in the cytoplasm of the cardiomyocytes (Fig. 1F). At this moment we do not know whether intracellular positivity of clusterin is derived from a local production by cardiomyocytes and/or whether it enters cardiomyocytes from the circulation in the human heart. Notably, local synthesis of clusterin within cardiomyocytes has been shown in the rat (35, 36).

In the infarcted area of early-phase and PMN-phase infarctions and of early-phase reinfarctions, the numbers of single clusterin-positive cardiomyocytes were significantly higher than those of single complement-positive cardiomyocytes (P = 0.000, P = 0.013, and P = 0.037, respectively; Fig. 2). Although we observed single clusterin-positive cardiomyocytes within the border zone and remote area, the numbers of single clusterin-positive versus single complement-positive cardiomyocytes in these areas did not significantly differ (data not shown), except for the border zones of early-phase reinfarctions (P = 0.007), although this group consisted of only three subjects.



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  		Fig. 2. Numbers of cardiomyocytes positive for complement or clusterin during different phases of infarction in the human heart. Bars represent number of complement-positive (white bars) or clusterin-positive (gray bars) cardiomyocytes per high-powered field (HPF) in different phases of infarction of infarction area. For each subject, average number of positive cardiomyocytes per HPF was determined from 25 HPFs scored. Error bars represent means ± SE; n, number of subjects.

 
The numbers of clusterin-positive cardiomyocytes within the infarcted area of all phases of infarction and reinfarction were significantly higher than in control subjects (early phase, P = 0.000; PMN phase, P = 0.000; chronic phase, P = 0.002; early-phase reinfarction, P = 0.009; and PMN-phase reinfarction, P = 0.014). Outside the infarcted area, only in the border zones of PMN-phase infarctions was the number of clusterin-positive cardiomyocytes significantly higher than in control subjects (P = 0.027).

Increased expression of clusterin in H9c2 cells after ischemia. To further establish putative local production of clusterin in ischemically challenged solitary cardiomyocytes, we applied Western blot analysis of reduced samples of H9c2 lysates. The {alpha}-chain of rat clusterin migrated to a height of ~40 kDa as was reported earlier (4), confirming that the blot detected clusterin. Figure 3A, lane 1, shows a low basal level of clusterin in H9c2 cells under control conditions. The expression of clusterin was increased during ischemia. As soon as 20 min after ischemia, clusterin levels were markedly increased. After 4 h of ischemia, the expression levels of clusterin were still elevated compared with baseline expression but was not further increased compared with 20 min of ischemia. Remarkably, the expression of clusterin decreased rapidly, i.e., as soon as 20 min after reperfusion with culture medium (Fig. 3B). The expression of clusterin was inhibited by translation inhibitor CH and also by transcription inhibitor AMD (Fig. 3C). Expression of clusterin was very low in the control (first lane) and in the control plus CH (second lane). AMD appeared to induce a slight increase in clusterin expression when added to control cells (third and fourth lanes). One hour of ischemia markedly increased clusterin levels (fifth lane), whereas both CH and AMD decreased the expression of clusterin to almost the control levels (sixth, seventh, and eighth lanes). Notably, although the metabolic inhibition resulted in ATP depletion, after 40 min of ischemia, ATP was still left to allow protein synthesis (data not shown).



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  		Fig. 3. Western blot analysis of clusterin expression in ischemically challenged H9c2 cells. A: H9c2 lysates, corrected for protein content, were separated on SDS-PAGE under reducing conditions and analyzed for clusterin by immunoblotting. I, metabolic inhibition. B: same experiment as in A, except that I cells were exposed to a fixed time of ischemia (1 h) and increasing time of reperfusion (R). C: effect of cycloheximide (CH) and/or actinomycin D (AMD) on clusterin expression by H9c2 cells. Control cells (C) and cells subjected to 2 h of I in absence or presence of 0.21 µM CH and/or 10 µM AMD.

 
Binding of clusterin on jeopardized H9c2 cells. Using flow cytometry, we analyzed whether clusterin could bind to H9c2 cardiomyoblast cells and assessed the conditions for such binding. FITC-labeled human recombinant AV (a marker for membrane flip-flop) and PI (a marker for cell death) were used to determine whether cells were viable (negative for both AV and PI), damaged (single AV positive), or dead (AV/PI positive or single PI positive). Anti-rat clusterin MAb or MAb G7 against human clusterin were used to detect binding of endogenous or purified human clusterin, respectively. With the use of Western blot analysis, MAb G7 was found not to cross-react with rat clusterin (data not shown). In control and ischemic conditions, endogenous rat clusterin was found to bind predominantly to H9c2 cells that were PI positive (Fig. 4, A and B, right). Only a minority of the clusterin-positive cells were single AV positive (<5%). Ischemia increased the number of PI-positive cells that bound clusterin (Fig. 5B, right). However, clusterin is also present in blood (7), and, therefore, we established the role of the exogenous clusterin by analyzing the binding of purified human serum clusterin to these H9c2 cells (Fig. 4, C and D). Also with exogenous clusterin, of the cells that were positive for human clusterin, one population was PI positive; another was PI negative (Fig. 6, A and B, left). The PI-negative cells that were positive for human clusterin were all positive for AV, which was also found for endogenous rat clusterin. The numbers of single AV, but clusterin-positive, cells were substantially higher when human clusterin was added than with endogenous clusterin. Because endogenous clusterin was found predominantly in and on H9c2 cells that were positive for PI, it seems, therefore, that endogenous clusterin is not secreted in high amounts to subsequently bind single AV-positive cells but rather resides within cells and is presumably only visualized in irreversible-damaged cells with leaking membranes.



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  		Fig. 4. Flow cytometric analysis of binding of endogenous rat clusterin to H9c2 cells. Control (A) or ischemically challenged (B, 2 h) H9c2 cells were stained with MAb against rat clusterin that was visualized with phycoerythrin (PE)-conjugated goat anti-mouse (GAM). Control (C) and ischemically challenged (D, 2 h) H9c2 cells were incubated with 20 µg purified human serum clusterin/ml and stained with MAb G7 against human clusterin, followed by PE-conjugated GAM. Cells were also stained with annexin V (AV)-FITC and propidium iodide (PI) to assess cell damage or cell death. Graphs were divided into four quarters to define positivity for different stainings. C and D, right: AV and clusterin staining of the gated clusterin-positive and PI-negative cells.

 


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  		Fig. 5. Live cell imaging analysis of membrane flip-flop in time, visualized by AV-FITC binding. H9c2 cells were ischemically challenged (2 h), stained with AV-FITC and analyzed by live cell imaging. A–D: differences in AV-FITC positivity between cells; focal patches (A, arrows), large aggregates (C and D, arrows I and II), and entirely positive (B) (magnification x16). E–H: loss of AV-FITC positivity, H9c2 cells 2 h after staining [E, differential interference contrast (DIC) + FITC channels; F, FITC channel], and same H9c2 cells 4.5 h after staining (G, DIC + FITC channels; H, FITC channel) (magnification x16). Arrows III and IV depict patches of AV-FITC positivity that have disappeared after 4.5 h. Arrows V depict patches of AV-FITC positivity that are still visible after 4.5 h.

 


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  		Fig. 6. Effect of purified human serum clusterin on viability of ischemically challenged H9c2 cells and isolated adult rat cardiomyocytes. A: percentage of rounded H9c2 cells upon ischemic challenge as assessed as a measure for cell damage. Control and ischemically challenged H9c2 cells in presence or absence of 20 µg purified human serum clusterin/ml. Percentage of rounded cells was calculated by dividing number of rounded cells by total number of cells. Bars represent means percentage of rounded cells, and error bars represent means ± SE. B: percentage of viable isolated adult rat cardiomyocytes was assessed by using an AV-FITC and PI staining. Control and ischemically challenged cells in presence or absence of 20 µg purified human serum clusterin/ml were tested with fluorescence microscopy using three-dimensional live imaging microscopy. Bars represent percentages of unstained and thus viable cardiomyocytes, and error bars represent means ± SE.

 
Cells were also analyzed with MAb G7 against human clusterin in the absence of purified human serum clusterin or in the absence of the primary MAbs against rat or human clusterin. In both cases, no clusterin signal was recorded. Results of these control experiments proved that the clusterin signal was specific (data not shown).

Flip-flop of the membrane as reversible phenomenon. The results described above indicate that clusterin can bind to H9c2 cells that were positive for AV, i.e., flip-flopped H9c2 cells. The fate of these AV-positive H9c2 cells in time was analyzed by live cell imaging (Fig. 5). Within a population of ischemically challenged (2 h) H9c2 cells, there were marked differences in AV-FITC positivity between cells, varying from a few focal patches (Fig. 5A, arrows) to large aggregates (Fig. 5C, arrows I and II) to cells that were entirely AV-FITC positive (Fig. 5B). Cells with extensive AV positivity, as depicted in Fig. 5B, died relatively soon (within 2 h) after staining. For example, the cell shown in Fig. 5C died ~30 min after staining (Fig. 5D, arrows I and II depict the identical AV aggregates as seen in Fig. 5C). Most cells with AV positivity, similar to those in Fig. 5A, still had a normal morphology 4.5 h after staining, the time frame in which experiments were performed with clusterin, whereas other cells with similar AV positivity died within that time frame. Cells that maintained normal morphology and also appeared to lose their AV positivity over a period of 4.5 h after staining were also recorded (Fig. 5, E–H). The image of cells shown in Fig. 5E (see Fig. 5F for just the FITC channel) taken 2 h after staining shows a number of cells that were positive for AV. At 4.5 h after staining (Fig. 5, G and H), the same cells as depicted in Fig. 5, E and F, appeared to have lost AV positivity compared with 2 h after staining. Arrows III and IV in Fig. 5, E–H,show the disappearance of AV aggregates after 4.5 h. In addition, a lot of AV patches have disappeared after 4.5 h. Arrows V in Fig. 5, F and H, show aggregates that have not disappeared after 4.5 h, underlining that there still is an AV-FITC signal.

Effect of purified human clusterin on ischemic cardiomyocytes. The results described above indicated that part of ischemically challenged AV-positive cardiomyocytes appeared to be reversibly damaged. We assessed the effect of clusterin binding on the viability of ischemic cardiomyocytes (Fig. 6). We quantified morphologically viable and dead H9c2 cells exposed to ischemic challenge in the presence or absence of 20 µg clusterin/ml (Fig. 6A). Rounded cells were considered dead because all these cells were AV/PI-positive in flow cytometry analysis (not shown). Under control conditions, 4.08 ± 0.46% of the cells were rounded without clusterin, compared with 4.69 ± 0.59% in the presence of clusterin. A marked increase in the percentage of rounded cells (31.53 ± 4.61%) was seen after 2 h of ischemia in the absence of clusterin. Interestingly, the presence of purified human serum clusterin during ischemia significantly decreased the percentage of rounded cells to 12.75 ± 1.99% (P = 0.020).

In addition, we quantified isolated adult ventricular rat cardiomyocytes, exposed to ischemic challenge, in the presence or absence of 20 µg clusterin/ml with AV and PI staining (Fig. 6B). Under control conditions, 61.46 ± 1.72% of the cells were viable (i.e., negative for AV-FITC and/or PI), without clusterin compared with 58.41 ± 2.22% with clusterin. After 30 min of ischemia the percentage of viable cells was significantly decreased to 38.61 ± 3.07% (P < 0.001). Clusterin significantly increased the percentage of viable cells to 58.38 ± 2.75% (P < 0.001) under ischemia.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Several studies have suggested a cytoprotective role for clusterin via its inhibiting effect on complement (16, 25, 29, 38). A former study (39), in which clusterin was found to colocalize with complement on jeopardized human cardiomyocytes after AMI, suggests that this might also happen in the infarcted human heart. In the present study we show for the first time the existence of single cardiomyocytes that are positive for clusterin but negative for complement within the human heart after AMI, suggesting a role for clusterin irrespective of complement. Indeed, we found a protective effect of clusterin on ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes in the absence of complement. Both the human tissue study and the in vitro experiments therefore imply a function of clusterin independent of complement.

In the infarcted heart two sources of clusterin exist: an exogenous source (namely, the blood) and an endogenous source (namely, clusterin expressed by cardiomyocytes themselves) (35, 36). We have also found this endogenous source of clusterin in H9c2 cells, which increased subsequent to ischemia. Notably, metabolic inhibition, a well-known method of inducing ischemia (11), results in ATP depletion. Dependent of the ischemic time frame, residual ATP does exist, which still favors protein synthesis. Next to this, it has been shown by others (42) that metabolic inhibition induces the synthesis of stress proteins like heat-shock proteins. At this moment, however, we do not know the contribution of the endogenous clusterin in the protection of ischemically challenged cardiomyocytes. We did find that the addition of exogenous clusterin to ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes resulted in a significant higher level of protection compared with cells deprived of exogenous clusterin. Using flow cytometry analysis, we found that clusterin bound predominantly to H9c2 cells that were positive for AV. Part of this AV-positive population was PI negative. Although AV is commonly regarded as an early marker for apoptosis, evidence has been published suggesting that the loss of membrane asymmetry resulting in AV positivity may to a certain level be reversible (9, 12, 30). Indeed, our live cell imaging experiments also show that in a population of ischemically challenged H9c2 cells, there were marked differences in the amount of AV positivity, varying from a few patches to large aggregates or even entire cell positivity. We also have now shown (see Fig. 5) that AV positivity, specifically when focal, is a reversible phenomenon. In concurrence with these findings, in experiments measuring the cytotoxic effects of type II secretory phospholipase A2 (sPLA2) on H9c2 cells, we found evidence for a reversible membrane flip-flop. sPLA2 among others bound to single AV-positive cells and thereby managed to induce extra cell death compared with cells deprived of sPLA2, implicating that at least part of the AV-positive cells apparently were not dead but were reversibly damaged (31). Therefore, this implies a mechanism in which clusterin binds to and possibly protects ischemically challenged cardiomyocytes. The mechanism whereby clusterin protects these cardiomyocytes is not yet clear. In noncardiomyocytes it was found that clusterin might protect reversibly damaged cells in a way resembling the effect of small heat-shock proteins (13, 23, 33, 34). Alternatively, it was suggested that clusterin binds to cellular debris and thereby protects cells from debris-inflicted damage (2, 3, 14). In renal proximal tubule cells, clusterin provided complement-independent protection against gentamicin-induced cytotoxicity but offered only marginal protection against ATP depletion-induced injury (10). In contrast, we found protection of clusterin independent of complement in ischemically challenged H9c2 cells in which ATP was strongly depleted (data not shown). Therefore, this suggests that the mechanism of the complement-independent cell protective effect of clusterin differs between renal proximal tubule cells and cardiomyocytes. The mechanism of protection of clusterin in cardiomyocytes requires further study.

Nevertheless, the cytoprotective effect of clusterin on cardiomyocytes could have important therapeutic implications. Upregulation and cytoprotection of clusterin has been suggested in a number of different cardiovascular-related pathophysiological conditions, such as atherosclerosis (15, 28, 37) and myocarditis (27, 36). An artificial enhancement of local clusterin levels and/or an increase of clusterin in the blood during cardiovascular disease might therefore limit the severity of damage and ultimately lead to a more favorable prognosis for patients by saving reversibly damaged cardiomyocytes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
H. W. M. Niessen is a recipient of the Dr. E. Dekker program of the Netherlands Heart Foundation (D99025)


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. J. Krijnen, VU Univ. Medical Center, Dept. of Pathology, De Boelelaan 1117, 1007 MB Amsterdam, The Netherlands (e-mail: paj.krijnen{at}vumc.nl)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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