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Am J Physiol Heart Circ Physiol 292: H3052-H3056, 2007. First published February 16, 2007; doi:10.1152/ajpheart.01355.2006
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HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway

Sanjiv Gupta and A. A. Knowlton

Molecular and Cellular Cardiology, Cardiovascular Division, University of California, Davis; and Sacramento Veterans Affairs Medical Center, Sacramento, California

Submitted 12 December 2006 ; accepted in final form 12 February 2007


   ABSTRACT
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METHODS
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 DISCUSSION
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The heat shock proteins (HSP) are a highly conserved family of proteins with critical functions in protein folding, protein trafficking, and cell signaling. These proteins also protect the cell against injury. HSP60 has been found in the extracellular space and has been identified in the plasma of some individuals. HSP60 is thought to be a "danger signal" to the immune system and is also highly immunogenic. Thus extracellular HSP60 is possibly toxic to the cell. The mechanism by which HSP60 is released into the extracellular space is unknown, as is whether it is released by cardiac myocytes. We investigated several different pathways controlling protein release including the classic, Golgi-mediated pathway. We found that HSP60 is released via exosomes, and that within the exosome, HSP60 is tightly attached to the exosome membrane.

heat shock protein; toll-like receptor; protein secretion

HEAT SHOCK PROTEINS (HSPs) are an essential family of protective proteins. These highly conserved proteins have important roles in protein folding, protein trafficking, and cell signaling. The HSPs target proteins for degradation via ubiquitination. They also protect the cell against injury, such as heat stress or hypoxia-reoxygenation. The HSPs are ubiquitous, and deletion of HSPs can be lethal.

HSP60 is a mitochondrial and cytosolic protein found in all cell types. It has also been detected in the extracellular space in a growing number of settings. HSP60 has been identified in the plasma of some individuals (15, 19), but the source of this extracellular (ex) HSP60 is unknown. A growing body of literature supports a role for HSP60 as a ligand of toll-like receptor (TLR) 4, and through this function, as a possible signal for "danger" to the immune system. HSP60 is highly immunogenic, and a number of individuals have antibodies to this protein, possibly as a result of antecedent bacterial infections. Thus exHSP60 can activate innate and adaptive immunity (1, 5, 18). Therefore, how HSP60 is released from the cardiac myocyte is of interest.

The most recognized mechanism for protein release from the cell is the classical pathway involving the endoplasmic reticulum and Golgi apparatus. In addition to this classical route, there are other nonclassical or alternative pathways, which have recently been reviewed in detail (17). These include 1) exosomes; 2) export via intracellular vesicles, most likely endosomes, as seen with high mobility group box 1 release; 3) direct transport across the membrane, as occurs with fibroblast growth factor-1; and 4) a flip-flop mechanism, as utilized by the hydrophilic acylated surface protein B protein in Leishmania (17).

We hypothesized that exosomes mediated the release of HSP60 from myocytes under basal as well as stressed conditions. To investigate this hypothesis, we explored the classical pathway, as well as alternative pathways for protein secretion. The mechanism of HSP60 release is reported here.


   METHODS
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 METHODS
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Isolation of adult cardiac myocytes. Adult cardiac myocytes were isolated from 3- to 4-mo-old male Sprague-Dawley rats weighing 250–300 g and cultured as previously described (22, 13). The animal protocol was approved by the University of California, Davis Animal Research Committee in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Hypoxia-reoxygenation. Hypoxia-reoxygenation was done using a hypoxia workstation (Forma), which produces near-zero oxygen. The medium was changed to DMEM base (no glucose, glutamine, or phenol red to prevent switching to glycolysis), and the cells were subjected to hypoxia for 2 h in an anaerobic workstation (model 1025, Forma Scientific, Marietta, OH; 4.8% CO2, 10.3% H2, and 84.9% N2) as previously described (8). After hypoxia, cells were returned to supplement Medium 199. This is a mild hypoxia treatment and does not cause necrosis, as evidenced by lack of lactate dehydrogenase release (9).

Exosome isolation from media. Exosomes were isolated according to the method of Savina et al. (20, 21). Briefly, 5 ml of media were collected on ice and centrifuged, first at 800 g for 10 min to remove any cells, and then at 12,000 g for 30 min to eliminate any cellular debris. Exosomes were then separated from the supernatant by centrifugation at 110,000 g for 2 h. The exosomal pellet was washed once in phophate-buffered saline (PBS) and then resuspended in 200 ul of PBS (exosomal fraction).

Acetylcholine esterase assay. The amount of released exosomes was quantified by measuring the activity of acetylcholine esterase, an enzyme that is specifically directed to these vesicles (21). Acetylcholine esterase activity was assayed following a previously described procedure by Savina et al. (20). Briefly, 40 µl of the exosome fraction was suspended in 110 µl of PBS. A portion (37.5 µl) of this PBS-diluted exosome fraction was then added to individual wells on a 96-well flat-bottomed microplate. Acetylthiocholine (1.25 mM) and 5,'5'-dithio-bis(2-nitrobenzoic acid) (0.1 mM) were then added to exosome fractions in a final volume of 300 µl, and the change in absorbance at 412 nm was monitored every 5 min. The data presented represent acetylcholine esterase enzymatic activity after 30 min of incubation.

Western blotting. Western blotting and analysis was performed as previously described (13, 16). After transfer, all membranes were stained with Ponceau S to verify quality of transfer and equal loading. Blots were blocked with Blotto (Bio-Rad) and developed with anti-HSP60 (1:15,000, StressGen). Anti-mouse IgG-horseradish peroxidase (Amersham/GE, Piscataway, NJ) was used at 1:1,000. Blots were also probed for caveolin-1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), HSC70 (1:1,000, Affinity BioReagents, Rochester, NY), and HSP90 (1:1,000, BD Transduction Laboratories, San Jose, CA). Anti-Na-K-ATPase (Abcam) was used at 1:5,000. Blots were developed with a chemiluminescent agent (Pierce, Rockford, IL) and exposed to X-ray film. Densitometric analysis was done as previously described (13, 16). The immunoprecipitation method has been well described (13).

Carbonate treatment. Carbonate treatment of the exosomes was done using the method of Fujiki et al. (7). Cells were treated with 1 M Na2CO3, pH 11.5, for 30 min at 37°C. Carbonate treatment opens and flattens membranes, releasing proteins that are not integrated into the membrane (2).

Proteinase K treatment. Carbonate-treated exosomes were resuspended in either 7 mM proteinase K, 10 mM EDTA, 5 mM N-ethylmaleimide (NEM), or 10 nM EDTA, 5 mM NEM, as a control. The membranes were incubated at 37°C for 10 min as described by Hardy et al. (10). The membranes were then centrifuged and resuspended in sample buffer.

Chemicals were obtained from Sigma, unless otherwise indicated. Concentrations of inhibitors were based on the literature (14, 20).

Statistical analysis. Results are expressed as means ± SE. Groups of data were analyzed by an ANOVA or an ANOVA on Ranks, where appropriate, followed by the Student-Newman-Keuls test or Dunn's test. A P < 0.05 was considered significant.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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To investigate the mechanism(s) underlying HSP60 secretion, both the classical and alternative secretory pathways were investigated. In pilot experiments, we found that unstressed cardiac myocytes released HSP60 but that the addition of a mild stress, 2 h of hypoxia, increased the amount of HSP60 released from myocytes without evidence of necrosis (no lactate dehydrogenase release). HSP60 secretion was studied in both unstressed myocytes and in myocytes treated with brief hypoxia.

Brefeldin A (BFA) inhibits the classical protein transport pathway. As shown in Fig. 1, A and C, 1-h pretreatment with 10 µg/ml BFA (14) had no effect on HSP60 release into the media under basal conditions. Hypoxia increased the release of HSP60 into the media, and this was not blocked by BFA treatment. In contrast, treatment with 15 nM dimethyl amiloride (DMA), an exosome inhibitor, as well as an inhibitor of both the H+/Na+ and Na+/Ca2+ exchanger, greatly reduced release of HSP60 (Fig. 1, B and C) (20). DMA also decreased the release of HSP60 following brief hypoxia (Fig. 1, B and C). Methyl-beta-cyclodextrin (5 mM, MBC), an inhibitor of lipid raft formation via depletion of membrane cholesterol, also reduced the release of HSP60 from the cardiac myocytes, as shown in Fig. 2, A and B (14). MBC, like DMA, reduced the release of HSP60 following hypoxia (Fig. 2, A and B). Thus BFA, an inhibitor of the classic pathway, had no significant effect on HSP60 release, whereas an exosome inhibitor (DMA) and a lipid raft inhibitor (MBC) both reduced release.


Figure 1
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  		Fig. 1. A: the results of Brefeldin A (BFA) pretreatment. *P < 0.05 vs. C, BFA. n = 6/group, 4 separate experiments. B: effect of dimethyl amiloride (DMA) pretreatment. *P < 0.05 vs. all others. +P < 0.05 vs. 2 h hypoxia (Hyp). n = 6/group, 4 separate experiments. C: representative Western showing release of heat shock protein (HSP)60 from BFA- and DMA-treated cardiac myocytes. Lanes 1 and 5, control cells; lane 2, BFA treated; lanes 3 and 7, hypoxia; lane 4, BFA treatment plus hypoxia; lane 6, DMA treatment; and lane 8, DMA treatment plus hypoxia. C, control; BFAHYP, BFA treatment plus hypoxia; DMAHYP, DMA treatment plus hypoxia.

 

Figure 2
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  		Fig. 2. A: graph summarizes the results of three separate experiments, n = 4/ group. B: Western shows the effect of methyl-beta-cyclodextrin (MBC) pretreatment on HSP60 release from cardiac myocytes. MBC/HYP, MBC treatment plus hypoxia. *P < 0.05 vs. all others; +P < 0.05 vs. C.

 
To investigate this further, exosomes were isolated from the media of cultured adult, rat cardiac myocytes (Fig. 3). As shown in Fig. 3A, HSP60 was present in the exosomes, and that the amount of HSP60 in the exosomes increased after brief hypoxia (2 h). Again, no lactate dehydrogenase release, a sign of necrosis and membrane disruption, was seen with this stress. To determine whether additional HSP60 was released independent of the exosomes, HSP60 expression in the exosomes was compared with the expression in the supernatant after the high-speed centrifugation to purify the exosomes. As shown in Fig. 3B, a trivial amount of HSP60 was seen in the media after removal of the exosomes.


Figure 3
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  		Fig. 3. A: Western of exosomes isolated from media following brief hypoxia (H) or hypoxia-reoxygenation (HR). HSP60 is present in both sets of exosomes. B: Western comparing media HSP60 in control (C) media after hypoxia (H) or hypoxia-reoxygenation (HR) versus media after exosome isolation. As demonstrated by Western, almost all HSP60 is removed by isolation of exosomes. Identical amounts of protein loaded in all lanes.

 
Both HSC70, the constituitively expressed HSP70, and HSP90 have been reported to be common components of exosomes (6). Four different isolations of exosomes were compared, as shown in Fig. 4. Western blots of the exosomes showed both HSC70 and HSP90 to be present, but there clearly was variation in the amount of these proteins. Lipid rafts are thought to be involved in exosome formation (4), and MBC blocked HSP60 release from the cell, implicating lipid rafts in HSP60 release. Analysis of caveolin-1 (probing the same blot used for the three heat shock proteins), a common component of lipid rafts, showed it also was present in the exosomes (Fig. 4). Thus caveolin levels remained relatively constant in four different preparations of exosomes, whereas variation was seen in levels of the HSPs, particularly HSC70 and HSP90.


Figure 4
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  		Fig. 4. Westerns showing four separate exosome isolations (lanes 1–4). HSP90, HSC70 and lipid rafts (caveolin-1) are all common components of exosomes. HSP60 was present in all four exosome preparations. For unknown reasons, HSC70 migrated slightly faster in lane 2. The same blot is shown developed for each of the respective proteins.

 
To examine the localization of HSP60 within the exosome, exosomes were sonicated and the membrane and "cytosolic" fractions separated by centrifugation. As shown in Fig. 5, most of the HSP60 is associated with the membrane rather than the contents of the exosomes. In an alternate approach, the exosomes were treated with carbonate, disrupting the exosomes and flattening the membrane. As shown in Fig. 6, top, HSP60 remains with the membrane with this approach. To determine whether the HSP60 was embedded in the membrane or just tightly adherent to the surface, following carbonate treatment, the membranes were treated with 7 mM proteinase K for 30 min. at 37°C. As shown in Fig. 6, top, lane 2, proteinase K treatment removes HSP60 from the membrane. In contrast, Na-K-ATPase is present in the exosome membrane and is unaffected by proteinase K treatment (Fig. 6, bottom).


Figure 5
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  		Fig. 5. Exosomes were sonicated to create "cytosolic" and membrane fractions. A: content of exosomes. B: membrane fraction. Images are from a single blot, and samples in B are corresponding membrane fractions of exosome content fractions shown in A. Equal protein loaded in each lane.

 

Figure 6
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  		Fig. 6. Exosomes were treated with carbonate, which opens and flattens the membrane. Only proteins embedded in the membrane will remain. Subsequently, fractions were treated with proteinase K to digest proteins present on the surface of the membrane. Top, HSP60 is present in exosomal membrane after carbonate treatment plus the buffer for proteinase K (lane 1). Proteinase K plus carbonate treatment removes HSP60 (lane 2). Lane 3 shows carbonate treatment alone; lane 4 shows total exosome (i.e., no carbonate). Bottom, same blot developed for Na-K-ATPase. Na-K-ATPase, a transmembrane protein, is unaffected by treatment with proteinase K.

 
The function and significance of exosomes are still being defined. We were interested in whether exosomal HSP60 was ubiquitinated. HSP60 was immunoprecipitated from the exosomes and then analyzed by Western blotting. As shown in Fig. 7, left, exosomal HSP60 is ubiquitinated. When the same blot was probed with an antibody specific for polyubiquitination, no signal was seen. Total HSP60 for the same blot is shown in the Fig. 7, right. Whereas polyubiquitination is a signal for degradation, other ubiquitination modifications, such as monoubiquitination and ubiquitination Lys63 chains regulate cellular functions and have a role in establishing networks of protein interactions (9, 23).


Figure 7
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  		Fig. 7. Ubiquitination of HSP60 in exosomes. HSP60 was immunoprecipitated from exosomal membrane fraction (after carbonate treatment) and from the exosomal contents. Left: developed with an antibody binding to any ubiquitination (total ubiquitination). Lane 1, membrane-associated HSP60; lane 2, HSP60 immunoprecipitated from the exosome content. Right: HSP60 on the same blot. Antibody specific for polyubiquitination only did not bind to these samples, though it showed activity against other protein samples.

 
Exosomes can be quantified by measuring acetylcholine esterase activity. This enzyme is considered a marker of exosomes. As shown in Fig. 8A, DMA and MBC both inhibited exosome release from normal cardiac myocytes, whereas BFA had no effect. Two hours of hypoxia tripled the amount of exosomes released, and again DMA and MBC inhibited exosome release (Fig. 8B). In contrast, BFA had no effect on exosome release after hypoxia. Thus the effect of these three inhibitors on exosome release parallels their effect on HSP60 release.


Figure 8
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  		Fig. 8. Acetylcholine esterase activity was used to quantify exosomes present in the media after various treatments. Activity was normalized to total cellular protein per plate to correct for any variation in cell number. A: unstressed cardiac myocytes. Cells were treated with BFA, DMA, or MBC, as described. Both DMA and MBC inhibited exosome release. B: effect of a mild stress (2 h hypoxia). Both DMA and MBC inhibited exosome release. *P < 0.05 vs. C and BFA. BFA, DMA, and MBC indicate the three inhibitors, as described in text.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
HSP60 was released from adult cardiac myocytes in both the basal state and following mild stress through an exosomal-mediated process. To our knowledge, this is the first report of exosome release by cardiac cells. Lipid rafts were involved in this process, as inhibition of lipid raft formation reduced the release of HSP60. This is in contrast to observations on the release of HSP70 in exosomes, which was found to be independent of lipid rafts (14). This may reflect cell type differences: primary, adult cardiac myocytes versus L6 myotubes, 3T3 adipocytes and polymononuclear cells from the peripheral blood, or functional differences between the two proteins. The classical secretatory pathway did not play a significant role in HSP60 release from adult cardiac myocytes. HSP60 was found in exosomes along with HSC70 and HSP90, both of which are considered to be definitive components of exosomes (3, 12). In addition, exosomes are thought to contain lipid rafts, and caveolin-1 was found in associated with the exosomes. Others have not found HSP60 to be present in exosomes released by other cell types (11).

HSP60 in the exosome was closely bound to the membrane. Carbonate treatment did not remove HSP60; however, proteinase K treatment did remove HSP60 while leaving Na-K-ATPase intact. These results indicate that HSP60 is tightly bound to the surface of the membrane, rather than inserted into the membrane.

Exosomes have been found to contain ubiquitinated protein, and at least some of the exosomal HSP60 was ubiquitinated (2). Ubiquitination has been thought solely to target a protein for degradation. However, more recent work has shown that ubiquitination sites and modifications vary (2). Monoubiquitinated proteins have diverse functions unrelated to the proteosome, whereas polyubiquitinated proteins are usually cytosolic proteins targeted for degradation (2). Not all ubiquitination leads to protein degradation; rather, ubiquitination also is an important signaling modification (9, 23). Degradation-associated ubiquitination is characterized by the addition of multiple ubiquitin molecules, which is polyubiquitination. The results reported here demonstrate some monoubiquitination of the HSP60 in the exosomes but not polyubiquitination. Monoubiquitination is involved in signaling, and the ubiquitinated HSP60, which is associated with the exosome membrane, may have a signaling role. HSP60 has not previously been reported to be ubiquitinated.

A mild stress (brief hypoxia) tripled the release of exosomes from the cardiac myocytes, as measured by acetylcholine esterase activity. Both DMA and MBC reduced the release of exosomes, both from unstressed and stressed cells. BFA had no effect on exosome formation.

The current work shows for the first time that HSP60 is released from cardiac myocytes through exosomes, and that lipid rafts are involved, likely in formation of the exosomes. The release of exosomes by the highly differentiated cardiac myocyte has not been previously described. The function and fate of exosomes remains unknown. Potentially, these vesicles could have a role in intercellular signaling or be removed by the reticuloendothelial system. The HSP60 detected in the plasma of patients may have been in exosomes and released after freeze thawing of samples. Future in vivo studies will be needed to address the distribution and fate of exosomes. Further work will also need to examine the function of HSP60 ubiquitination and the role of exosomes in the cardiac myocyte.


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 ABSTRACT
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-077281 (to A. A. Knowlton) and HL-079071 (to A. A. Knowlton) and by a Merit Award from the Department of Veterans Affairs (to A. A. Knowlton).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Knowlton, Molecular & Cellular Cardiology, Division of Cardiovascular Medicine, Univ. of California, Davis, One Shields Ave., Davis, CA 95616 (e-mail: aaknowlton{at}ucdavis.edu)

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.


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