This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 105/24/2899    most recent 01.CIR.0000019403.35847.23v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kirchhoff, S.R. Articles by Knowlton, A.A. Search for Related Content PubMed PubMed Citation Articles by Kirchhoff, S.R. Articles by Knowlton, A.A. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Nucleotide Protein UniGene Related Collections Other myocardial biology Apoptosis Cell biology/structural biology

(Circulation. 2002;105:2899.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Cytosolic Heat Shock Protein 60, Apoptosis, and Myocardial Injury

S.R. Kirchhoff, BA; S. Gupta, PhD; A.A. Knowlton, MD

From the Division of Cardiology Research, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Tex.

Correspondence to A.A. Knowlton, MD, Cardiovascular Medicine, TB172, University of California, Davis, One Shields Ave, Davis, CA 95616. E-mail annek{at}bcm.tmc.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Heat shock proteins (HSPs) are well known for their ability to "protect" the structure and function of native macromolecules, particularly as they traffic across membranes. Considering the role of key mitochondrial proteins in apoptosis and the known antiapoptotic effects of HSP27 and HSP72, we postulated that HSP60, primarily a mitochondrial protein, also exerts an antiapoptotic effect.

Methods and Results— To test this hypothesis, we used an antisense phosphorothioate oligonucleotide to effect a 50% reduction in the levels of HSP60 in cardiac myocytes, a cell type that has abundant mitochondria. The induced decrease in HSP60 precipitated apoptosis, as manifested by the release of cytochrome c, activation of caspase 3, and induction of DNA fragmentation. Antisense treatment was associated with an increase in bax and a decrease in bcl-2 secondary to increased synthesis of bax and degradation of bcl-2. A control oligonucleotide had no effect on these measurements. We further demonstrated that cytosolic HSP60 forms a macromolecular complex with bax and bak in vitro suggesting that complex formation with HSP60 may block the ability of bax and bak to effect apoptosis in vivo. Lastly, we show that as cytosolic (nonmitochondrial) HSP60 decreases, a small unbound fraction of bax appears and that the amount of bax associated with the mitochondria and cell membranes increases.

Conclusions— These results support a key antiapoptotic role for cytosolic HSP60. To our knowledge, this is the first report suggesting that interactions of HSP60 with bax and/or bak regulate apoptosis.

Key Words: apoptosis • cells • myocytes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The heat shock proteins (HSPs) are a family of endogenous, protective proteins. Two of these HSPs, 27 and 72 (inducible 70), have been shown to have antiapoptotic effects.^1–3 HSP60 is a primarily mitochondrial HSP that is expressed at high levels in the normal cell. Although much is known about the function of HSP60 in folding macromolecules, only limited studies have addressed its possible role in apoptosis.⁴ In mammalian cells, 75% to 80% of HSP60 is in the mitochondria, whereas 15% to 20% of HSP60 is extramitochondrial.⁵ We postulated that as key proteins and events in the apoptotic cascade involve the mitochondria, HSP60 may have an antiapoptotic role in the cell.

To address this question, we used antisense phosphorothioate oligonucleotides (AS) to reduce HSP60 by 50% in cardiac myocytes. We report here that this decrease in HSP60 precipitated apoptosis as manifested by the release of cytochrome c, activation of caspase 3, and DNA fragmentation. Bax increased, whereas bcl-2 decreased. As the proapoptotic bak is thought to be relatively abundant in the heart, it was also examined and found to undergo changes similar to bax. A control oligonucleotide had no adverse effects on the cells. We demonstrate that HSP60 interacts with bax and bak, and that bax can be depleted from the cytoplasm by precipitation with anti-HSP60 antibodies. As the HSP60 in the cytoplasmic fraction is decreased by AS treatment, a small fraction of unbound bax can be identified in the cytoplasm. The amount of bax associated with a mitochondrial/membrane fraction increases as HSP60 decreases. Bax alone is sufficient to induce cytochrome c release and apoptosis.⁶ Thus, this interaction between HSP60 and bax may be critical in preventing apoptosis in the normal cell. To our knowledge, this is the first report of the interaction of HSP60 with bax and bak, and the first report that changes in HSP60 can precipitate apoptosis. We propose that HSP60 has a pivotal antiapoptotic role.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Isolation of Cardiac Myocytes
Adult cardiac myocytes were isolated from male Sprague Dawley rats (Harlan, Indianapolis, Ind) and cultured as previously described.⁷ The cells were incubated at 37°C for 2 hours to allow adherence, and then treated with either AS to HSP60 or a scrambled sequence (SCR) as a control for the effects of phosphorothioate oligonucleotide; both were used at a concentration of 5.5 µmol/L (25 µg/mL). Cells without any treatment were used as a second control.

The primary AS oligonucleotide used throughout this study corresponds to bases 109 to 123 (5'-TAAGGCTCGAGCATC-3'; TriLink).⁸ A second AS sequence was also used that corresponds to bases 27 to 41. Similar results were obtained with both AS sequences. The scrambled sequence (5'-GCTCGTGGTCAATAC-3') was used as a control for the effects of the phosphorothioate compound (SCR). All of the oligonucleotide sequences were screened in GenBank.

Further information about Methods is included in the online-only Data Supplement.

Statistics
Groups were compared by an ANOVA followed by the Student-Neumann-Keuls test. When appropriate, an ANOVA on ranks was performed followed by a Dunn test. P<0.05 was considered significant. All data are expressed as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
After 24 hours of treatment, the levels of HSP60 were significantly reduced in the AS group, but not in SCR or control (Figure 1A and 1C). There were no changes in the -sarcomeric actin levels on the same Western blot (Figure 1B). Examination of cytosolic and mitochondrial cell fractions by Western blotting showed that HSP60 was present in both fractions and that AS treatment decreased the amount of HSP60 in each fraction similarly (Figure 1D). Although the amount of HSP60 appears somewhat similar in the figure (approximately a 2-fold difference), far more total protein is present in the mitochondrial fraction, so as equal amounts of protein were loaded on the gel, far more HSP60 was present in the mitochondria.

View larger version (17K): [in this window] [in a new window]   Figure 1. A, Western blot demonstrating that levels of HSP60 were significantly decreased in AS-treated groups vs control and SCR. B, The same blot developed with -sarcomeric actin to show equal loading of samples. C, HSP60 levels after 24 hours of AS treatment. Shown are results of 4 experiments. Values are normalized to control. D, Western blot of cell fractions demonstrating HSP60 in both mitochondria (MIT) and cytosol (CYT) in control myocytes, and levels of HSP60 in both fractions decrease with AS treatment. C indicates controls; AS, antisense; and SCR, scramble. *P<0.05 vs all others.

With the decrease in HSP60, there was a decrease in cell viability by live/dead staining (Figure 2A), but no release of lactate dehydrogenase (LDH), a marker of necrosis (Figure 2B). As the membrane is relatively impermeable in apoptosis, this method may underestimate apoptotic cell death.^9,10 We performed a series of experiments to determine whether the reduction of HSP60 was causing apoptosis.

View larger version (21K): [in this window] [in a new window]   Figure 2. A, Graphic representation of live/dead assay showing cell viability. Units are displayed as number of live/total cells counted. n=6; *P<0.05 vs all others. B, LDH release from cells after AS, SCR, or no treatment (C) (n=8 to 10). All data collected at 24 hours.

A pivotal point in apoptosis is release of cytochrome c from the mitochondria. As shown in Figure 3A, cytochrome c was released with reduction of HSP60. Neither control cells nor SCR-treated cells had release of cytochrome c. To verify that the release of cytochrome c was due to apoptosis rather than disintegration of the mitochondria, citrate synthase activity was measured in the medium. There was no release of citrate synthase from the mitochondria in any of the groups (Figure 3C). Furthermore, citrate synthase activity was the same for the mitochondria from all groups. Likewise, ATP levels were the same in the three groups after 24 hours of treatment (control [C], 191.0±20.0; AS, 181.3±27.4; and SCR, 178.3±21.5 nmol/mg protein). To confirm activation of the caspase proteases, caspase 3 cleavage was assessed by Western blot and was only present in the AS-treated myocytes (Figure 3D). A terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL) assay showed that 28.7% of AS-treated cells were positive for DNA fragmentation (P<0.05 compared with SCR and control; for details, see the Data Supplement).

View larger version (29K): [in this window] [in a new window]   Figure 3. A, Western blot showing cytochrome c release from AS-treated myocytes. B, Western blot for -sarcomeric actin on the digitonin-permeabilized cells to demonstrate that equivalent numbers are present on plates. C, Graph showing citrate synthase activity in media vs cells (mitochondria); mU/µg total protein. n=10/group. D, Western blot for the two cleavage products that occur with activation of caspase 3 (11 and 17 kDa).

After 24 hours of AS treatment, Western blots of whole-cell lysate revealed a significant increase in bak and bax and a significant decrease in bcl-2 in the AS-treated cells compared with SCR and control (C) (Figure 4A and 4B). Studies were done to determine the mechanism of the changes in bax and bcl-2. By polymerase chain reaction no difference was observed in GAPDH, bcl-2, or bax mRNA among the three groups (Figure 5A). The primers screened for five different mRNAs, two of which, bcl-xl and bcl-xs, were present at very low levels and are not visible in the figure. Labeling of newly synthesized protein demonstrated a marked increase in bax, but no change in bcl-2, which was barely detectable after 8 hours of labeling (Figure 5B and 5C). To look at degradation, the myocytes were labeled for 12 hours with [S³⁵]methionine and then returned to their regular media for 8 hours. Bax showed no change, whereas bcl-2 decreased 40% in the AS-treated group (P<0.01 versus C/SCR, Figure 5D and 5E).

View larger version (18K): [in this window] [in a new window]   Figure 4. A, Western blots for bax, bak, and bcl-2, as labeled. Same samples are shown on all Western blots. B, Graphs summarize 5 experiments. Values are normalized to control. *P<0.05 vs all others.

View larger version (53K): [in this window] [in a new window]   Figure 5. A, Reverse transcriptase–polymerase chain reaction showing expression of GAPDH, caspase 3 (unlabeled band below GAPDH), bax, and bcl-2 at 24 hours of treatment for the three groups. B, New synthesis of bax. Cells were labeled with [S35]methionine from hour 20 to 24 of treatment. C, New synthesis of bcl-2 detected after labeling from hour 16 to 24. D, Degradation of bax. Cells were labeled from hour 4 to 16 of treatment, and bax was collected by affinity column. E, Degradation of bcl-2.

Normal cardiac myocytes were collected, sonicated, and immunoprecipitated with an anti-HSP60 antibody to determine whether HSP60 bound bcl-2, bax, or bak. As shown in Figure 6A, both bax and bak coimmunoprecipitated with HSP60, but not bcl-2 (not shown). To further confirm the cytosolic interaction of HSP60 and bax, exhaustive immunoprecipitation (IP) was performed on the cytosolic fraction from the myocytes. As shown in Figure 6B, repetitive IP with either anti-HSP60 (lane B1 to B3) or anti-bax (lane D1 to D3) completely depleted all HSP60 (upper panel) and bax (lower panel) in the cytosolic fraction (no mitochondria, no plasma membrane) of the myocytes. For comparison, lane A shows 40 µg of total cytosol and lanes C and E show 40 µg of protein post-IP with anti-HSP60 or anti-bax, respectively. No HSP60 or bax is detectable in lane B4 or C4. Thus, exhaustive IP with either anti-HSP60 or anti-bax completely depletes the cytosol of both HSP60 and bax. A nonspecific IgG immunoprecipitated neither HSP60 nor bax from control cells (lane F1 to F3, upper and lower panels), and there is no depletion of either HSP60 or bax (lane G, upper and lower panel, compared with lane A). Immunocytochemistry followed by exhaustive photon reassignment (EPR) was done to demonstrate that HSP60 and bax colocalize within the cell. As shown in Figure 7, both HSP60 and bax were widely distributed throughout the cell, but analysis of the processed (by EPR) data from the images shows that the majority of the protein labels colocalize (white in the figure) and little to no bax alone (green) or HSP60 alone (red) is seen. Likewise, subtracting bax signal from HSP60 signal removes most of the signal from the cell (HSP60 less bax). Thus, by immunocytochemistry the two proteins colocalize within the myocyte, and IP studies support the existence of a bax/HSP60 complex within the cytosol of the myocyte.

View larger version (40K): [in this window] [in a new window]   Figure 6. A, Western blot demonstrating the coimmunoprecipitation of bax and bak with HSP60. C indicates myocyte lysate precipitated with protein G–agarose only as a control; +, IP with anti-HSP60 antibody followed by protein G–agarose. Arrows indicate bands for bax and bak. B, IP of cytosolic fraction (no mitochondria or plasma membranes) with anti-HSP60 and anti-bax. Upper panel shows immunoprecipitate developed with anti-HSP60 and lower with anti-bax. Lane A, 40 µg of total cytosol. B, Lanes 1 to 3, Successive IPs with anti-HSP60. Both HSP60 (upper panel) and bax (lower panel) immunoprecipitate. Lane C, 40 µg of protein post-IP. D, Lanes 1 to 3, Successive IPs with anti-bax. Both HSP60 (upper panel) and bax (lower panel) immunoprecipitate. Lane E, 40 µg of protein post-IP. F, Lanes 1 to 3 as in lane A, but with nonspecific IgG; lane F4 shows that neither HSP60 (upper panel) nor bax (lower panel) has been depleted from cytosol by nonspecific IgG.

View larger version (35K): [in this window] [in a new window]   Figure 7. EPR. HSP60 and bax images as labeled. C indicates EPR; CO-LOC, colocalization of signal for HSP60 and bax; HSP60 less bax, signal for bax was subtracted from HSP60 signal (virtually all signal was removed); white color, both signals present; red, HSP60 alone; and green, bax alone.

These experiments show an interaction between HSP60 and bax. Furthermore, they demonstrate that reduction in HSP60 results in apoptosis. The digitonin-permeabilized myocytes were used to delineate further the relation between HSP60 levels and bax distribution by dividing the cell into two fractions, cytosol and mitochondrial/membrane. The cytosol was immunoprecipitated with HSP60 after 24 hours of treatment with increasing amounts of AS. As shown in Figure 8A, bax coprecipitated with HSP60 from this cytosolic fraction. The supernatants from the IP and the remaining mitochondrial fraction (mitochondria, nuclei, membranes, etc) were examined by Western blot for bax. As shown in Figure 8A, as the level of AS increased, a small amount of bax becomes present in the cytosol. The mitochondrial fraction shows increasing amounts of bax present as the amount of AS is increased. Thus, as the AS concentration increases, HSP60 decreases (Figure 8B), and the amount of bax free of HSP60 increases (ie, the bax present in the supernatant after IP with anti-HSP60). Meanwhile, as HSP60 decreases and unbound bax increases, the amount of bax in the mitochondrial fraction increases.

View larger version (26K): [in this window] [in a new window]   Figure 8. Cells were divided into two fractions, cytoplasm and organelle/membrane by gentle digitonin permeabilization. A, Western blot for bax from representative experiment. Each series of samples is with increasing concentrations of AS (µg/mL as shown). Left side, Coimmunoprecipitation of bax with HSP60 from cytoplasmic fraction; center, supernatant from IP. A small but increasing amount of bax is present as HSP60 decreases with higher AS concentration. Right, Mitochondrial/membrane fraction. B, Western blot of intact cytoplasm fraction showing that as AS concentration increases, HSP60 decreases.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Within 24 hours of AS treatment, the level of HSP60 was significantly reduced in the cardiac myocytes. In contrast, control and SCR-treated cells showed no change in HSP60 levels. Along with this decrease in HSP60, there was an increase in cell death, but no LDH release, a sign of necrosis. Western blotting showed that the proapoptotic proteins bak and bax were both increased, whereas the antiapoptotic bcl-2 was decreased in AS-treated cells. These changes in bax, bak, and bcl-2 were accompanied by release of cytochrome c from the mitochondria. The citrate synthase assay confirmed that the mitochondria remained intact, as did the preserved ATP levels. The observed changes were accompanied by cleavage of caspase 3 and by DNA fragmentation within the nucleus. Thus, the reduction in HSP60 alone precipitated apoptosis. Decreasing the amount of HSP60 in the cytoplasm fraction resulted in "free" bax in the cytoplasm after IP with HSP60. Furthermore, the amount of bax associated with the mitochondrial/membrane fraction increased markedly as HSP60 decreased, suggesting that unbound bax immediately translocates to the mitochondria. The addition of bax to mitochondria has been shown to be sufficient to trigger cytochrome c release and subsequent caspase activation.^6,11 Our data support a model in which HSP60 complexes with bax preventing apoptosis. Reduction in HSP60, or dissociation from HSP60, can trigger apoptosis.

HSP60 is predominantly mitochondrial with 60% in the inner matrix, 20% in the inner membrane, and 15% to 20% extramitochondrial.⁵ We found that HSP60 complexes with both bax and bak as demonstrated by IP studies in untreated cardiac myocytes. Bax is in the cytosol in the normal cell as is bak, and both associate with the cytosolic fraction of HSP60, as shown by the current study. Immunocytochemistry followed by EPR demonstrated that HSP60 and bax colocalize. The lack of significant HSP60 signal without bax may result from incomplete permeabilization of the mitochondria by the fixation/permeabilization method that was used. Reduction in HSP60 releases bak and bax, and this may allow conformational changes, as well as the oligomerization, insertion into the outer membrane, and mitochondrial pore formation that are thought to occur with both proteins during apoptosis.^12–14 Both bak and bax induce the release of cytochrome c from the mitochondria. Binding to HSP60 in the normal cell may prevent bax and bak from oligomerizing and inserting into the mitochondrial membrane. In fact, our experiments show that with reduction in HSP60, bax redistributes from the cytosol to the mitochondria. During apoptosis, bax is found in the mitochondria along with HSP60; however, these two proteins are found in different compartments with bax localizing to the outer membrane and HSP60 associated with the inner membrane and inner matrix.^5,15,16 Thus, it is unlikely that the two proteins interact in the mitochondria. Our results suggest that HSP60 has a regulatory role for the activity of these proapoptotic proteins in the normal cell, and that HSP60 is a key antiapoptotic protein in the normal myocyte. This regulatory role in cell signaling is analogous to the role that HSP90 has in the cell.^17,18

Treatment with AS to HSP60 was associated with an increase in bax. The balance of antiapoptotic bcl-2 to proapoptotic bax is critical in mitochondrion-induced apoptosis.¹⁹ Our data show no change in mRNA for bax or bcl-2 with AS treatment. On the other hand, there was a significant increase in new synthesis of bax. In contrast, bcl-2 had increased degradation, but no change in synthesis.

HSP27 and HSP72 have been found to have antiapoptotic roles, but few studies have examined HSP60 in apoptosis.^1–3 Overexpression of HSP27 or HSP72 can inhibit apoptosis.^20,21 Experimental data suggest that HSP72 prevents activation of JNK and also that HSP72 may have antiapoptotic effects downstream of caspase 3 activation.^22,23 HSP60 has been found to form a complex with pro–caspase 3 and HSP10 in Jurkat cells, and appears to accelerate the activation of pro–caspase 3 during apoptosis.²⁴ In contrast, we observed that reduction of HSP60 resulted in activation of the apoptotic cascade. Our results are consistent with the observation that overexpression of HSP60 can prevent apoptosis.⁴

Antisense oligonucleotides can have nonspecific effects like any other experimental manipulation.²⁵ Careful sequence selection to avoid known problems and appropriate controls can avoid these problems.²⁶ We observed the same reduction of HSP60 and cell death via apoptosis with two different AS sequences. Neither the control phosphorothioate oligonucleotide used in this experiment nor any of the sequences used in previous work have affected HSP60 levels or caused cell death.²⁷ Thus, the observed changes are specific for the reduction in HSP60.

HSP60 is an abundant protein about which little is known with regard to its protective properties. A number of intriguing observations have been made about this protein. The heart of the hypoxia-tolerant turtle has very high levels of this protein compared with the hypoxia-intolerant human heart.²⁸ Increasing evidence supports the hypothesis that apoptosis is a mechanism of myocyte loss in heart disease.²⁹ HSP60 is primarily a mitochondrial protein, and the mitochondrion is the final common pathway to apoptotic destruction. In this report, we demonstrate that cytosolic HSP60 interacts with the proapoptotic bak and bax, but not with antiapoptotic bcl-2. Furthermore, we show that reduction in HSP60 is sufficient to precipitate apoptosis, and that cytosolic HSP60 has a key antiapoptotic role in the myocyte. Although opinion regarding the importance of mitochondria in apoptosis has waxed and waned, an argument can be made for the central importance of the mitochondria in the myocardium, and our data suggest that mitochondrial damage is a central event.

	Acknowledgments

 
This study was supported by a Veterans Affairs merit award (to Dr Knowlton) and National Heart, Lung, and Blood Institute Grant HL58515 (to Dr Knowlton). We thank Dr Jeanie B. McMillin for her suggestions on cytochrome c experiments and Drs Roger Rossen and Marco Marcelli for critical review of the manuscript.

	Footnotes

 
Further information is available in an online-only Data Supplement at http://circulationaha.org.

Guest editor for this article was Ivor J. Benjamin, MD, University of Texas Southwestern Medical School, Dallas, Tex.

Received January 28, 2002; revision received March 27, 2002; accepted March 28, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Li WX, Chen CH, Ling CC, et al. Apoptosis in heat-induced cell killing: the protective role of HSP-70 and the sensitization effect of the c-myc gene. Radiat Res. 1996; 145: 324–330.[Medline] [Order article via Infotrieve]
   
2. McMillan DR, Xiao X, Shao L, et al. Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis. J Biol Chem. 1998; 273: 7523–7528.[Abstract/Free Full Text]
   
3. Jolly C, Morimoto RI. Role of the heat shock response and molecular chaperones in oncogenesis and cell death. J Natl Cancer Inst. 2000; 92: 1564–1572.[Abstract/Free Full Text]
   
4. Lin KM, Lin B, Lian IY, et al. Combined and individual mitochondrial HSP60 and HSP10 expression in cardiac myocytes protects mitochondrial function and prevents apoptotic cell deaths induced by simulated ischemia-reoxygenation. Circulation. 2001; 103: 1787–1792.[Abstract/Free Full Text]
   
5. Soltys BJ, Gupta RS. Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (HSP60) in mammalian cells. Exp Cell Res. 1996; 222: 16–27.[CrossRef][Medline] [Order article via Infotrieve]
   
6. Jürgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA. 1998; 95: 4997–5002.[Abstract/Free Full Text]
   
7. Sun L, Chang J, Kirchhoff SR, et al. Activation of HSF and selective increase in heat shock proteins by acute dexamethasone treatment. Am J Physiol. 2000; 278: H1091–H1096.[Abstract/Free Full Text]
   
8. Venner TJ, Gupta RS. Nucleotide sequence of rat hsp60 (chaperonin, GroEl homolog) cDNA. Nucleic Acid Res. 1990; 18: 5309.[Free Full Text]
   
9. Yeh ETH. Life and death in the cardiovascular system. Circulation. 1997; 95: 782–786.[Free Full Text]
   
10. Bromme HJ, Holtz J. Apoptosis in the heart: when and why? Mol Cell Biochem. 1996; 163–164:261–275.
    
11. Bossy-Wetzel E, Newmeyer DD, Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 1998; 17: 37–49.[CrossRef][Medline] [Order article via Infotrieve]
    
12. Schlesinger PH, Gross A, Yin XM, et al. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci USA. 1997; 94: 11357–11362.[Abstract/Free Full Text]
    
13. Wei MC, Lindsten T, Mootha VK, et al. tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 2000; 14: 2060–2071.[Abstract/Free Full Text]
    
14. Nouraini S, Six E, Matsuyama S, et al. The putative pore forming domain of Bax regulates mitochondrial localization and interaction with Bcl-Xl. Mol Cell Biol. 2000; 20: 1604–1615.[Abstract/Free Full Text]
    
15. Eskes R, Desagher S, Antonsson B, et al. Bid induces the oligomerization and insertion of bax into the outer mitochondrial membrane. Mol Cell Biol. 2000; 20: 929–935.[Abstract/Free Full Text]
    
16. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med. 2000; 6: 513–519.[CrossRef][Medline] [Order article via Infotrieve]
    
17. Pratt WB. The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc Soc Exp Biol Med. 1998; 217: 420–434.[Abstract]
    
18. Knowlton AA, Sun L. Heat shock factor-1, steroid hormones and regulation of heat shock protein expression in the heart. Am J Physiol. 2001; 280: H455–H464.
    
19. Misao J, Hayakawa Y, Ohno M, et al. Expression of bcl-2 protein, an inhibitor of apoptosis, and bax, an accelerator of apoptosis, in ventricular myocytes of human hearts with myocardial infarction. Circulation. 1996; 94: 1506–1512.[Abstract/Free Full Text]
    
20. Mehlen P, Schulze-Osthoff K, Arrigo A-P. Small stress proteins as novel regulators of apoptosis. J Biol Chem. 1996; 271: 16510–16514.[Abstract/Free Full Text]
    
21. Wang Y, Knowlton AA, Christensen TG, et al. Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells. Kidney Int. 1999; 55: 2224–2235.[CrossRef][Medline] [Order article via Infotrieve]
    
22. Meriin AB, Yaglom JA, Gabai VL, et al. Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72. Mol Cell Biol. 1999; 19: 2547–2555.[Abstract/Free Full Text]
    
23. Jäättelä M, Wissing D, Kikolm K, et al. HSP70 exerts is anti-apoptotic function downstream of caspase-3-like proteases. EMBO J. 1998; 17: 6124–6134.[CrossRef][Medline] [Order article via Infotrieve]
    
24. Xanthoudakis S, Roy S, Rasper D, et al. HSP60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis. EMBO J. 1999; 18: 2049–2056.[CrossRef][Medline] [Order article via Infotrieve]
    
25. Burgess TL, Fisher EF, Ross SL, et al. The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense method. Proc Natl Acad Sci USA. 1995; 92: 4051–4055.[Abstract/Free Full Text]
    
26. Knowlton AA. Antisense, heat shock proteins, and the heart.In: Kukreja RC, Hess ML, eds. Heat Shock Proteins in Myocardial Protection. Georgetown, Tex: Landes Bioscience; 2000: 87–95.
    
27. Nakano M, Mann DL, Knowlton AA. Blocking the endogenous increase in HSP72 increases susceptibility to hypoxia and reoxygenation in isolated adult feline cardiocytes. Circulation. 1997; 95: 1523–1531.[Abstract/Free Full Text]
    
28. Chang J, Knowlton AA, Wasser J. Expression of heat shock proteins in turtle and mammalian hearts: relationship to anoxia tolerance. Am J Physiol. 2000; 278: R209–R214.
    
29. Olivetti G, Abbi R, Quaini J, et al. Apoptosis in the failing human heart. N Engl J Med. 1999; 336: 1131–1141.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				B. J.J.M. Brundel, L. Ke, A.-J. Dijkhuis, X. Qi, A. Shiroshita-Takeshita, S. Nattel, R. H. Henning, and H. H. Kampinga Heat shock proteins as molecular targets for intervention in atrial fibrillation Cardiovasc Res, June 1, 2008; 78(3): 422 - 428. [Abstract] [Full Text] [PDF]

				D. Chandra, G. Choy, and D. G. Tang Cytosolic Accumulation of HSP60 during Apoptosis with or without Apparent Mitochondrial Release: EVIDENCE THAT ITS PRO-APOPTOTIC OR PRO-SURVIVAL FUNCTIONS INVOLVE DIFFERENTIAL INTERACTIONS WITH CASPASE-3 J. Biol. Chem., October 26, 2007; 282(43): 31289 - 31301. [Abstract] [Full Text] [PDF]

				L. Lin, S. C. Kim, Y. Wang, S. Gupta, B. Davis, S. I. Simon, G. Torre-Amione, and A. A. Knowlton HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2238 - H2247. [Abstract] [Full Text] [PDF]

				C. Xu, Y. Lu, Z. Pan, W. Chu, X. Luo, H. Lin, J. Xiao, H. Shan, Z. Wang, and B. Yang The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes J. Cell Sci., September 1, 2007; 120(17): 3045 - 3052. [Abstract] [Full Text] [PDF]

				S. Gupta and A. A. Knowlton HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3052 - H3056. [Abstract] [Full Text] [PDF]

				X. Zhu, H. Zhao, A. R. Graveline, E. S. Buys, U. Schmidt, K. D. Bloch, A. Rosenzweig, and W. Chao MyD88 and NOS2 are essential for Toll-like receptor 4-mediated survival effect in cardiomyocytes Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1900 - H1909. [Abstract] [Full Text] [PDF]

				B.-J. Kim, S.-W. Ryu, and B.-J. Song JNK- and p38 Kinase-mediated Phosphorylation of Bax Leads to Its Activation and Mitochondrial Translocation and to Apoptosis of Human Hepatoma HepG2 Cells J. Biol. Chem., July 28, 2006; 281(30): 21256 - 21265. [Abstract] [Full Text] [PDF]

				A. Y. W. Chang, J. Y. H. Chan, J. L. J. Chou, F. C. H. Li, K.-Y. Dai, and S. H. H. Chan Heat shock protein 60 in rostral ventrolateral medulla reduces cardiovascular fatality during endotoxaemia in the rat J. Physiol., July 15, 2006; 574(2): 547 - 564. [Abstract] [Full Text] [PDF]

				L. Szalay, T. Shimizu, T. Suzuki, H.-P. Yu, M. A. Choudhry, M. G. Schwacha, L. W. Rue III, K. I. Bland, and I. H. Chaudry Estradiol improves cardiac and hepatic function after trauma-hemorrhage: role of enhanced heat shock protein expression Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2006; 290(3): R812 - R818. [Abstract] [Full Text] [PDF]

				G.-C. Fan, X. Ren, J. Qian, Q. Yuan, P. Nicolaou, Y. Wang, W. K. Jones, G. Chu, and E. G. Kranias Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury Circulation, April 12, 2005; 111(14): 1792 - 1799. [Abstract] [Full Text] [PDF]

				J. Trial, R. D. Rossen, J. Rubio, and A. A. Knowlton Inflammation and Ischemia: Macrophages Activated by Fibronectin Fragments Enhance the Survival of Injured Cardiac Myocytes Experimental Biology and Medicine, June 1, 2004; 229(6): 538 - 545. [Abstract] [Full Text] [PDF]

				N. C Chi and J. S Karliner Molecular determinants of responses to myocardial ischemia/reperfusion injury: focus on hypoxia-inducible and heat shock factors Cardiovasc Res, February 15, 2004; 61(3): 437 - 447. [Abstract] [Full Text] [PDF]

				Y.-x. Shan, T.-L. Yang, R. Mestril, and P. H. Wang Hsp10 and Hsp60 Suppress Ubiquitination of Insulin-like Growth Factor-1 Receptor and Augment Insulin-like Growth Factor-1 Receptor Signaling in Cardiac Muscle: IMPLICATIONS ON DECREASED MYOCARDIAL PROTECTION IN DIABETIC CARDIOMYOPATHY J. Biol. Chem., November 14, 2003; 278(46): 45492 - 45498. [Abstract] [Full Text] [PDF]

				L. He and J. J. Lemasters Heat Shock Suppresses the Permeability Transition in Rat Liver Mitochondria J. Biol. Chem., May 2, 2003; 278(19): 16755 - 16760. [Abstract] [Full Text] [PDF]

				P. Gromov, G. L. Skovgaard, H. Palsdottir, I. Gromova, M. Ostergaard, and J. E. Celis Protein Profiling of the Human Epidermis from the Elderly Reveals Up-regulation of a Signature of Interferon-{gamma}-induced Polypeptides That Includes Manganese-superoxide Dismutase and the p85{beta} Subunit of Phosphatidylinositol 3-Kinase Mol. Cell. Proteomics, February 1, 2003; 2(2): 70 - 84. [Abstract] [Full Text] [PDF]

				S. Gupta and A.A. Knowlton Cytosolic Heat Shock Protein 60, Hypoxia, and Apoptosis Circulation, November 19, 2002; 106(21): 2727 - 2733. [Abstract] [Full Text] [PDF]

				Q. Xu Role of Heat Shock Proteins in Atherosclerosis Arterioscler. Thromb. Vasc. Biol., October 1, 2002; 22(10): 1547 - 1559. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 105/24/2899    most recent 01.CIR.0000019403.35847.23v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kirchhoff, S.R. Articles by Knowlton, A.A. Search for Related Content PubMed PubMed Citation Articles by Kirchhoff, S.R. Articles by Knowlton, A.A. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Nucleotide Protein UniGene Related Collections Other myocardial biology