This Article Abstract 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 Martin, J. L. Articles by Dillmann, W. H. Search for Related Content PubMed PubMed Citation Articles by Martin, J. L. Articles by Dillmann, W. H.

(Circulation. 1997;96:4343-4348.)
© 1997 American Heart Association, Inc.

Articles

Small Heat Shock Proteins and Protection Against Ischemic Injury in Cardiac Myocytes

Jody L. Martin, PhD; Ruben Mestril, PhD; Randa Hilal-Dandan, PhD; Laurence L. Brunton, PhD; ; Wolfgang H. Dillmann, MD

From the Department of Medicine, Divisions of Endocrinology and Metabolism (J.L.M., R.M., W.H.D.) and Cardiology (L.L.B.) and Department of Pharmacology (R.H.-D., L.L.B.), University of California, San Diego.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and B-crystallin protect against ischemic injury.

Methods and Results Our aim was to examine whether the overexpression of hsp27 and B-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or B-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45±7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, B-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29±6.6%) and neonatal (32±5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29±9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34±4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes.

Conclusions The increased expression of hsp27 and B-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of B-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Key Words: ischemia • adenovirus • myocytes • proteins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Recent studies have demonstrated that increased expression of the inducible heat shock protein 70 (hsp70i) protects against ischemic cardiac damage. The enhanced survival of myogenic cell lines stably transfected with hsp70i from ischemic injury demonstrates this protective effect.¹ More recently, it was reported that neonatal cardiomyocytes infected with hsp70i-expressing adenovirus were protected against simulated ischemia.² In addition, after global ischemia, the hearts of transgenic mice overexpressing hsp70i showed a decrease in infarct size and improved contractile recovery on comparison with transgene-negative littermates.^{3 4} Similarly, regional ischemia under in vivo conditions results in decreased infarct size in hsp70i transgenic mice.⁵ Several groups have also shown that whole animal preheat stress is associated with improved recovery from a subsequent ischemic event.^{6 7 8} Heat shock induces many proteins other than the inducible hsp70 that also may be involved in this protective effect.

Two candidate proteins for mediating a protective effect are the small heat shock proteins B-crystallin and hsp27 (hsp25 in rodents). hsp27 and B-crystallin share considerable sequence and structural similarity, associate in vivo, and are induced by oxidative stress.^{9 10 11 12} Both small heat shock proteins function as molecular chaperones in protein biosynthesis to facilitate protein folding and translocation.¹³ Protective effects of the small heat shock proteins have been described against several noxious stresses like hyperthermia, hypertonic stress, and various cytotoxic agents, including cytoskeletal disruptors, but their effect on ischemia-mediated injury in cardiac myocytes has not been explored. Examples of the protective effect against heat stress by small heat shock proteins include the demonstration that the amount of hsp27 expressed by transfection in rodent and Chinese hamster cell lines directly correlates with survival from hyperthermia^{14 15 16} and that ectopic expression of B-crystallin renders NIH 3T3 cells and glioma cells thermoresistant.^{17 18 19} Therefore, both hsp27 and B-crystallin can mediate stress tolerance, helping cells to endure a variety of stresses. Both small heat shock proteins are also associated with cytoskeletal structures, and stabilization of these elements may contribute to the increased stress tolerance. For example, hsp27 acts as an inhibitor of actin filament turnover in smooth muscle cells and seems to stabilize the actin filaments.^{20 21} Stable overexpression of hsp27 in Chinese hamster lung cells confers resistance to F-actin fragmentation induced by H₂O₂ and menadione.²² B-Crystallin stabilizes cytoskeletal structures in glioma cells.¹⁹ In cardiomyocytes, B-crystallin also associates with the intermediate filaments, especially desmin, an association that is strengthened under ischemic conditions.^{23 24}

It was our aim to determine whether increased expression of the small heat shock proteins hsp27 and B-crystallin in rat cardiac myocytes exerts a protective effect against ischemic injury. Recombinant adenovirus were used to overexpress the human hsp27 (huhsp27) and the rat B-crystallin in the sense and antisense orientation. This approach provides for high levels of transgene expression in the great majority of cardiac myocytes used in the experiments. We examined the effects of overexpression of these proteins on the extent of ischemic damage in both neonatal and adult rat cardiomyocytes. We also used the antisense orientation of the genes in the adenovirus as a means of specifically decreasing the levels of the corresponding endogenous small heat shock proteins. We demonstrated, to our knowledge for the first time, that the overexpression of hsp27 and B-crystallin protects against ischemic damage in cardiomyocytes and, further, that decreasing the level of the endogenous hsp25 renders these cells more susceptible to ischemic injury. We also documented that protection is seen with hsp70i overexpression in adult cardiomyocytes exposed to simulated ischemia.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Construction of Recombinant Adenovirus
We constructed several adenoviral vectors for this study, especially huhsp27 and B-crystallin, each in the sense and antisense orientations. In addition, an insertless adenovirus was constructed to be used as a control for effects of adenovirus infection. The 0.76-kb coding region of the human hsp27 cDNA (provided by Drs L. Weber and E. Hickey, University of Nevada [Reno])²⁵ and the 0.76-kb rat B-crystallin cDNA (from Drs J. Goldman and A. Iwaki, College of Physicians and Surgeons of Columbia University, New York, NY)²⁶ were used to make the constructs. The appropriate fragments were cloned between the enhancer/promoter of the cytomegalovirus immediate-early genes and the Simian virus 40 polyadenylation signal of the pACCMV. pLpA shuttle vector provided by Dr R. Gerard. Replication-deficient adenovirus was generated through homologous recombination of two plasmids (pJM17, a bacterial plasmid that contains the full-length adenoviral genome, and the shuttle vector) after cotransfection into E1 transformed human embryonic kidney 293 cells to produce E1-deleted adenovirus encoding the appropriate transgene.²⁷ The inducible rat hsp70 adenovirus was constructed in a similar manner as we previously described.² Viral stocks were generated by infecting confluent 293 cells, harvesting the cells, and concentrating the cells through CsCl ultracentrifugation. Viral stocks were then desalted through a Sepharose CL4B (Sigma Chemical Co) column into a Tris-buffered solution, plaque-titered, aliquoted, and stored at -70°C with 10% glycerol until use.

Isolation of Ventricular Cardiomyocytes
Neonatal cardiomyocytes were isolated through collagenase/pancreatin digestion and Percoll gradient separation from 1- to 2-day-old rats as previously described.²⁸ They were infected 1 day after isolation at a multiplicity of infection (MOI) of 10:1 in 2% heat-inactivated bovine serum containing DME. After 1 hour, the medium was changed to no-serum DME. On day 2 after infection, the cells were submitted to simulated ischemia, harvested for Western blot analysis, or immunostained.

Adult cardiomyocytes are isolated from 250- to 300-g Sprague-Dawley rats through collagenase perfusion as previously described.²⁹ They were infected 1 hour after plating onto laminin-coated dishes at an MOI of 10:1 in M199 supplemented with HEPES (7.7 mmol/L), taurine (5 mmol/L), creatinine (5 mmol/L), carnitine (2 mmol/L) and 1% BSA. The virus was washed away with a single media change. Then, 2 days later, they were processed in the same manner as the neonatal cells.

Simulated Ischemia
Simulated ischemia was achieved by placing cells in hypotonic, balanced salt solution consisting of 1.3 mmol/L CaCl₂, 5 mmol/L KCl, 0.3 mmol/L KH₂PO₄, 0.5 mmol/L MgCl₂, 0.4 mmol/L MgSO₄, 69 mmol/L NaCl, 4 mmol/L NaHCO₃, and 0.3 mmol/L Na₂HPO₄ without glucose or serum and made hypoxic for 6 hours (adult cells) or 12 hours (neonatal cells) at 37°C.¹ Hypoxia was attained with an airtight jar from which the oxygen was flushed with argon for 5 minutes to achieve <0.2% oxygen.¹ To maintain hypoxia, the oxygen-consuming GasPak System from BBL Microbiology Systems was used.¹ At the end of the experiment, the dishes were removed from the chamber with the medium, and cells were separately assayed for both enzyme and protein content.

Enzyme Quantitation
The cells were scraped into 2 mL of cold PBS and sonicated (Ultrasonic Homogenizer 4710; Cole-Parmer) for 30 seconds followed by a 10-minute centrifugation at 150g to remove cell debris. This supernatant is taken to represent total enzymatic activity remaining in the cells. The enzymes in the media and from the sonicated cells were quantified according to the Oliver and Rosalki method with the creatine phosphokinase (CK) determination kit (Sigma Diagnostics), and the Wroblewski and LaDue method was used in the lactate dehydrogenase (LDH) determination kit (Sigma Diagnostics) for LDH release. The amount of enzyme released in the nonstressed controls is subtracted as background from the value obtained from the ischemic cells. This value is then normalized to the amount released by cells infected with the control adenovirus (which contains no transgene). This is necessary to pool multiple primary cardiomyocyte experiments. While treated in precisely the same manner, there still may be significant variability in the extent of enzyme release between experiments (ie, 20% to 40% of the total amount of enzyme is released after 12 hours of simulated ischemia).

Immunostaining and Protein Analysis
Cell extracts were prepared by washing plates twice with ice-cold PBS and scraped into solution B containing 1% Triton x-100, 0.5% deoxycholate, and 5 µmol/L 2-mercaptoethanol, followed by vigorous vortexing and cooling on ice for 15 minutes, before 15 minutes of centrifugation at 12 000g.³² Protein concentration was determined according to the Bradford assay (BRL). Protein samples were fractionated on a 12% SDS–polyacrylamide gel and electrotransferred onto nitrocellulose using a semidry electrotransfer apparatus (BRL). The nitrocellulose membranes were reacted with polyclonal anti-rodent hsp25, monoclonal anti-human hsp27 (both from StressGen), or polyclonal anti-rodent B-crystallin (kindly provided by Drs K. Kegel, M. Head, and J. Goldman, Columbia University, New York, NY) at a 1:1000 dilution. Protein loading was normalized by using monoclonal antibodies to constitutive hsp70 (StressGen) and sarcomeric -actin (Sigma). Protein antibody complexes were visualized either with the Vectastain Elite kit (Vector) using diaminobenzidine plus nickel as a substrate or by using the Enhanced Chemiluminescence kit (Amersham). Immunoblots were scanned with a Scanjet 4C (Hewlett-Packard) and then digitized and analyzed with the NIH Image analysis program.

For immunostaining, cells were washed with PBS and fixed with cold methanol for 5 minutes. Cell dishes were hydrated with TBS containing 0.1% BSA (fraction V; Sigma), and the primary antibody was added at 1:100. The samples were then processed using the Vectastain ABC kit (Vector).

Statistical Analysis
Results are expressed as mean±SEM. Statistical significance was assessed with one-way ANOVA. Tukey-Kramer post-test comparisons were performed when appropriate.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Due to the shortcomings of transient transfection techniques resulting in a low percentage of cells receiving the transgene, we decided to use first-generation recombinant adenovirus to express the small heat shock protein transgenes. This system offers the advantage of efficient infection (>90%) and longer duration of transgene expression. After construction of the recombinant adenovirus as described above, we demonstrated efficient infection of both neonatal and adult cardiomyocytes (Fig 1). Adult cardiac myocytes (Fig 1B) maintained their rod-shaped structure and expressed an abundant amount of transgene in almost all myocytes. The same held true for neonatal cardiac myocytes, which exhibited a normal trapezoidal shape. There was no general stress response associated with adenoviral infection, as shown by the absence of the inducible hsp70 on Western analysis of extracts from cells 2 days after infection (data not shown).

View larger version (172K): [in this window] [in a new window]   Figure 1. Immunohistochemistry of cardiomyocytes infected with adenovirus encoding hsp27. Cells were fixed 2 days after infection and reacted with a monoclonal antibody that binds to the human hsp27 and developed with a Vectastain ABC kit. A, Noninfected adult cardiomyocytes cultured for 2 days after isolation. B, Hsp27 adenovirus–infected cardiomyocytes 2 days after infection showing virtually 100% transduction efficiency. C, Noninfected neonatal cardiomyocytes 3 days after isolation. The cells are plated in sera-containing media for 1 day and then cultured for 2 days in serum-free conditions. D, Neonatal cells infected with the hsp27 adenovirus 1 day after isolation and then fixed after 2 days in serum free media (scale bar, 50 µm).

Known amounts of recombinant human hsp27 proteins were run on denaturing polyacrylamide gels in parallel with extracts from control and huhsp27 adenovirus–infected cell and then electrotransferred. The level of transgene expression (Fig 2A, lanes 3 and 4 versus lane 5) was 100 ng/20 µg total protein (0.5%). Extracts from noninfected cells demonstrated the specificity of the antibody (Fig 2A, lane 2). Similar results were seen with the adult cardiomyocytes (data not shown). We also examined the effectiveness of the antisense huhsp27 infection by Western blot analysis. In neonatal cardiomyocytes, antisense infection reduces the endogenous hsp25 by 80% when normalized for loading with the heat shock cognate 70 protein (Fig 2B, lane 3 versus lane 1). In the adult cardiomyocytes, we were not able to detect a significant reduction using the antisense to huhsp27 (data not shown), probably due to a slower hsp25 protein turnover. The adenovirus encoding B-crystallin leads to effective transgene expression as demonstrated by Western blots (Fig 3A, lane 2 versus lane 1; Fig 3B, lane 1 versus lane 3). Adult cardiac myocytes express a higher level of endogenous B-crystallin than do neonatal myocytes (compare Fig 3A with 3B). These findings are in line with the in vivo results showing that B-crystallin levels are higher in adult rat hearts than in neonatal rat hearts.^{31 32} The level of endogenous B-crystallin does not seem to be significantly altered with the use of the antisense B-crystallin adenovirus in neonatal cardiomyocytes (Fig 3A, lane 4) or in adult myocytes (data not shown). This could be due to the slow turnover of B-crystallin protein. A very low B-crystallin turnover has been seen in the lens of the eye.

View larger version (27K): [in this window] [in a new window]   Figure 2. Western blot analysis of neonatal cardiomyocytes. Protein samples (20 µg) were separated by 12% SDS-PAGE, electrotransferred to nitrocellulose, and subsequently reacted with human hsp27 antibody (A) or rodent hsp25 and hsp70 antibodies (B). A, Lanes 1 and 5, 30, and 120 ng recombinant human hsp27, respectively; lane 2, control virus-infected cells; lanes 3 and 4, cells infected with the hsp27 adenovirus. B, Lane 1, from noninfected cells; lane 2, cells infected with control virus; lane 3, from cells infected with hsp27 antisense adenovirus; and lane 4 from cells infected with the hsp27 adenovirus.

View larger version (36K): [in this window] [in a new window]   Figure 3. Western blot analysis of neonatal (A) or adult (B) cardiomyocytes. Samples were prepared as in previous figure and then blots were reacted with -actin and B-crystallin antibodies or rodent hsp25 and B-crystallin antibodies. A, Lane 1, noninfected cells; lane 2, B-crystallin adenovirus–infected cells; lane 3, control virus infected cells; lane 4, B-crystallin antisense adenovirus. B, Lane 1, noninfected cells; lane 2, control virus-infected cells; lane 3, B-crystallin adenovirus-infected cells.

After the effectiveness of infection and the time at which the small heat shock protein transgenes are maximally expressed were established, we submitted the cells to simulated ischemia or maintained them under normal conditions to assess the potential protective effects of hsp27 and B-crystallin. The results of experiments using adult and neonatal cardiac myocytes with altered levels of B-crystallin are shown in Fig 4. In neonatal and adult cardiac myocytes, a substantial decrease of ischemic injury was achieved by overexpression of B-crystallin. For example, the release of CK was decreased by 32±5.4% in neonatal cardiac myocytes; in adult cardiac myocytes, LDH release was decreased by 29±6.6% in B-crystallin–overexpressing cells. Infection of cells with control virus did not lead to significantly altered CK release. Expression of the B-crystallin antisense construct in neonatal or adult cardiac myocytes did not influence enzyme release. These results are in accord with the unchanged B-crystallin protein levels as shown in Fig 3.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of B-crystallin on enzyme release induced by ischemia. Cells were infected with control, B-crystallin, or B-crystallin antisense adenovirus 2 days before the stress (A, neonatal; B, adult). The data in this figure represent the pooling of at least three independent experiments, each performed in triplicate. **P<.01 vs control cells.

The results of experiments using adult and neonatal cardiac myocytes and altering the levels of hsp27 and hsp70i expression are shown in Fig 5. As determined by Western blot analysis, hsp25 levels are much lower in adult cardiac myocytes than in neonatal cardiac myocytes. In line with these results, we found that increasing hsp27 levels in adult cardiac myocytes resulted in decreased injury by simulated ischemia, as indicated by a 45±7.5% decrease in LDH release. In contrast, in neonatal cardiac myocytes, expression of the hsp27 transgene did not lead to decreased CK release. Expression of the antisense message to hsp27 leads to a significant decline of endogenous hsp25 protein and increased CK release. In contrast, in adult cardiac myocytes, the antisense hsp27 vector did not lower hsp27 protein levels as described above, and in line with these results, no increase in LDH release occurred. We also examined the effect of overexpression of hsp70i in adult cardiomyocytes. With this construct, we saw a 34±4.6% decrease in LDH release after ischemic injury, which is in accord with our previously published neonatal cardiomyocyte results (33±3%).²

View larger version (22K): [in this window] [in a new window]   Figure 5. Effects of hsp27 and hsp70i on enzyme release induced by simulated ischemia. Cells were infected with control, hsp27, hsp27 antisense, and hsp70i adenovirus 2 days before the stress (A, neonatal; B, adult). Data are a mean of at least three independent experiments, each performed in triplicate. *P<.05 vs control cells; **P<.01 vs control cells.

We also examined the protective effect of B-crystallin expression against ischemic injury of various lengths in neonatal cardiomyocytes. The same degree of protection was observed at 4 hours (30±7.5% decrease in CK release) and 8 hours (36±9.2%), as shown in Fig 4 (32±5.4%) for 12 hours of simulated ischemia. It should be mentioned that there was no significant difference in enzyme release between cells infected with the control virus and noninfected cells and that all results are normalized to enzyme release by cells infected with the control insertless virus.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our results demonstrate that increased expression of the small heat shock proteins hsp27 and B-crystallin in adult cardiac myocytes provides for diminished injury from simulated ischemia. The ability of increased levels of B-crystallin to mediate increased protection against ischemic injury in neonatal and adult cardiac myocytes is of special interest. B-Crystallin was initially identified as a protein present in high concentrations in the lens of the eye, and its presence in the heart has only recently received attention. The precise mechanism by which the increased level of B-crystallin exerts a protective effect against ischemic injury is currently unclear, but protection may result from stabilization of specific cellular structures. B-Crystallin is associated with the desmin-based intermediate filaments, which play a role in anchoring I bands of the myofibril to the intercalated disc,²³ a region that seems to be an important target for ischemic damage in cardiac myocytes.³³ In contrast to the increased protection resulting from an enhanced B-crystallin expression mediated by infection with adenoviral vectors expressing the transgene in the sense orientation, placement of the B-crystallin gene in the antisense orientation did not alter ischemic injury. Assessment of the amount of B-crystallin in cells infected with adenovirus expressing the antisense transgene showed that no significant decrease occurred in B-crystallin protein levels. This is most likely due to the stability of the B-crystallin protein.³⁴

The protective effect provided by increased expression of hsp27 was limited to adult cardiac myocytes and therefore not as broadly based as that observed with B-crystallin. In neonatal cardiac myocytes, adenovirus-mediated hsp27 transgene expression did not result in a protective effect against ischemic injury; this is most likely due to the already high control level of hsp25 protein in neonatal cardiac myocytes, levels that markedly exceed that found in adult cardiac myocytes (unpublished data, 1996). In line with the lower level of hsp25 in adult cardiac myocytes (10% to 20% as much as in neonatal cardiomyocytes), increasing hsp27 levels through adenovirus-based transgene expression in adult cardiac myocytes resulted in increased protection against ischemic injury. A hsp27 dose-dependent protection also has been reported for cells exposed to a lethal heat shock.¹⁶ The precise mechanism by which hsp27 mediates this protective effect is currently unclear, but it has also been demonstrated that hsp27 associates with actin microfilaments and influences microfilament organization. Both B-crystallin and hsp27 therefore associate with the cytoskeleton and serve important functions in the maintenance of this important cellular structure. It is also possible the phosphorylation status of hsp27 markedly influences its function.^{21 22}

The importance of high hsp25 levels in neonatal cardiac myocyte for normal cell function and integrity is further emphasized by the results from hsp27 antisense expression in neonatal cardiac myocytes. Infection of neonatal cardiac myocytes with adenoviral vectors expressing antisense hsp27 results in a marked lowering of hsp25 levels, which leads to enhanced ischemic injury. In contrast to B-crystallin, the half-life of the hsp27 protein is shorter; within 2 days of hsp27 antisense expression, hsp25 protein levels are significantly lower in neonatal cardiac myocytes. In adult cardiac myocytes, hsp27 antisense expression did not lower hsp25 protein levels and ischemic injury was unchanged, again emphasizing the close correlation between hsp25/27 protein levels and the extent of ischemic injury. The consistent protective effect of overexpressing hsp70i was also demonstrated in adult cardiomyocytes.

In summary, our results show that increased expression of the small heat shock proteins B-crystallin and hsp27 in adult cardiac myocytes through an adenovirus vector–based approach leads to increased protection against injury mediated by simulated ischemia. It is therefore of interest to explore in future studies whether specific targets for this protective effect can be identified.

	Acknowledgments

 
This study was supported by an American Heart Association Grant-in-Aid and by US Public Health Service grants DK-07202 (Martin), K14-HL-03150-01 (Dr Mestril), R01-HL-49434-01 (Dr Dillmann), HL-41307 (Dr Brunton), and HL-07444 (Dr Hilal-Dandan) from the National Institutes of Health. We thank Gene Levin, PhD, for helpful advice and Roslyn Lara for her secretarial assistance.

	Footnotes

 
Reprint requests to Wolfgang H. Dillmann, MD, Department of Medicine, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0618.

Received June 11, 1997; revision received August 27, 1997; accepted September 15, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Mestril R, Chi SH, Sayen MR, O'Reilly K, Dillmann WH. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury. J Clin Invest.. 1994;93:759-767.
   
2. Mestril R, Giordano FJ, Conde AG, Dillmann WH. Adenovirus-mediated gene transfer of a heat shock protein 70 (hsp70i) protects against simulated ischemia. J Mol Cell Cardiol.. 1996;26:2351-2358.
   
3. Marber M, Mestril R, Chi SH, Sayen MR, Yellon DM, Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest.. 1995;95:1446-1456.
   
4. Plumier JCL, Ross B, Currie RW, Angelidis CE, Kazlaris H, Kollias G, Pagoulatous GN. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest.. 1995;95:1854-1860.
   
5. Hutter JJ, Mestril R, Tam EKW, Sievers RE, Dillmann WH, Wolfe CL. Overexpression of heat shock protein 72 in transgenic mice decreases infarct size in vivo. Circulation.. 1996;94:1408-1411.[Abstract/Free Full Text]
   
6. Donnelly TJ, Sievers RE, Vissern FLJ, Welch WJ, Wolfe CL. Heat shock protein induction in rat hearts: a role for improved myocardial salvage after ischemia and reperfusion? Circulation.. 1992;85:769-778.[Abstract/Free Full Text]
   
7. Currie RW, Tanguay RM, Klingma JG. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation.. 1993;87:963-971.[Abstract/Free Full Text]
   
8. Marber MS, Latchman DS, Walker JM, Yellon DM. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation.. 1993;88:1264-1274.[Abstract/Free Full Text]
   
9. Merck KB, Groenen PJTA, Voorter CEM, de Haard-Hoekman WA, Horwitz J, Bloemendal H, de Jong WW. Structural and functional similarities of bovine -crystallin and mouse small heat shock protein. J Biol Chem.. 1993;268:1046-1052.[Abstract/Free Full Text]
   
10. Zantema A, Verlaan-De Vries M, Maasdam, Bol S, Van der Eb A. Heat shock protein 27 and B crystallin can form a complex, which dissociates by heat shock. J Biol Chem.. 1992;267:12936-12941.[Abstract/Free Full Text]
    
11. Klemenz R, Frohli E, Steiger RH, Schafer R, Aoyama A. B-Crystallin is a small heat shock protein. Proc Natl Acad Sci U S A.. 1991;88:3652-3656.[Abstract/Free Full Text]
    
12. Head MW, Corbin E, Goldman JE. Coordinate and independent regulation of B crystallin and HSP27 in response to physiological stress. J Cell Physiol.. 1994;159:41-50.[Medline] [Order article via Infotrieve]
    
13. Jakob U, Gaestel M, Engel K, Buchner J. Small heat shock proteins are molecular chaperones. J Biol Chem.. 1993;268:1517-1520.[Abstract/Free Full Text]
    
14. Landry J, Chretien P, Lambert H, Hickey E, Weber LA. Heat shock resistance conferred by expression of the human hsp27 gene in rodent cells. J Cell Biol.. 1989;109:7-15.[Abstract/Free Full Text]
    
15. Chretien P, Landry J. Enhanced constitutive expression of the 27-kDa heat shock proteins in heat-resistant variants from chinese hamster cells. J Cell Physiol.. 1988;137:157-166.[Medline] [Order article via Infotrieve]
    
16. Lavoie JN, Gingras-Breton G, Tanguay RM, Landry J. Induction of Chinese hamster hsp27 gene expression in mouse cells confers resistance to heat shock. J Biol Chem.. 1993;268:3420-3429.[Abstract/Free Full Text]
    
17. Aoyama A, Frohli E, Schafer R, Klemenz R. B Crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection. Mol Cell Biol.. 1993;13:1824-1835.[Abstract/Free Full Text]
    
18. Blackburn R, Galoforo S, Berns CM, Ireland M, Cho JM, Corry PM, Lee YJ. Thermal response in murine L929 cells lacking B crystallin expression and B crystallin expressing L929 transfectants. Mol Cell Biochem.. 1996;55:51-60.
    
19. Iwaki A, Iwaki T, Tateishi J, Goldman JE. Sense and antisense modification of glial alpha B-crystallin production results in alterations of stress fiber formation and thermoresistance. J Cell Biol.. 1994;125:1385-1393.[Abstract/Free Full Text]
    
20. Miron T, Wilchek M, Geiger B. Characterization of an actin polymerization in vinculis-rich fraction of turkey gizzard smooth muscle. Eur J Biochem.. 1988;178:543-553.[Medline] [Order article via Infotrieve]
    
21. Lavoie JN, Hickey E, Weber L, Landry J. Modulation of actin microfilaments and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J Biol Chem.. 1993;268:24210-24214.[Abstract/Free Full Text]
    
22. Huot J, Houle F, Spitz DR, Landry J. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res.. 1996;56:273-279.[Abstract/Free Full Text]
    
23. Bennardini F, Wrzosek A, Chiesi M. B-Crystallin in cardiac tissue: association with actin and desmin filaments. Circ Res.. 1992;71:288-294.[Abstract/Free Full Text]
    
24. Barbato R, Menabo R, Dainese P, Carafoli E, Schiaffino S, Di Lisa F. Binding of cytosolic proteins to myofibrils in ischemic rat hearts. Circ Res.. 1996;78:821-828.[Abstract/Free Full Text]
    
25. Hickey E, Brandon SE, Sadis S, Smale G, Weber LA. Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia. Gene.. 1986;43:147-154.[Medline] [Order article via Infotrieve]
    
26. Iwaki A, Iwaki T, Goldman JE, Liem RKH. Multiple mRNAs of rat brain -crystallin B chain alternative transcriptional initiation. J Biol Chem.. 1990;265:22197-22203.[Abstract/Free Full Text]
    
27. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology.. 1988;163:614-617.[Medline] [Order article via Infotrieve]
    
28. Iwaki K, Chi SH, Dillmann WH, Mestril R. Induction of hsp70 in cultured rat neonatal cardiomyocytes by hypoxia and metabolic stress. Circulation.. 1993;87:2023-2032.[Abstract/Free Full Text]
    
29. Hilal-Dandan R, Urasawa K, Brunton LL. Endothelin inhibits adenylate cyclase and stimulates hydrolysis in cardiac myocytes. J Biol Chem.. 1992;267:10620-10624.[Abstract/Free Full Text]
    
30. Welch WJ, Feramisco JR. Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol.. 1985;5:1229-1237.[Abstract/Free Full Text]
    
31. Kato K, Shinohara H, Kurobe N, Inaguama Y, Shimizu K, Ohshima K. Tissue distribution and developmental profiles of immunoreactive B-crystallin in the rat determined with a sensitive immunoassay system. Biochim Biophys Acta.. 1991;1074:201-208.[Medline] [Order article via Infotrieve]
    
32. Benjamin IJ, Shelton J, Garry DJ, Richardson JA. Temporospatial expression of the small HSP/B-crystallin in cardiac and skeletal muscle during mouse development. Dev Dyn.. 1997;208:75-84.[Medline] [Order article via Infotrieve]
    
33. Ganote C, Armstrong S. Ischaemia and the myocyte cytoskeleton: review and speculation. Cardiovasc Res.. 1993;27:1387-1403.[Free Full Text]
    
34. Maiti M, Kono M, Chakrabart B. Heat induced changes in the conformation of and B crystallins: unique thermal stability of crystallin. FEBS Lett.. 1988;236:109-114.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				A. Havasi, Z. Li, Z. Wang, J. L. Martin, V. Botla, K. Ruchalski, J. H. Schwartz, and S. C. Borkan Hsp27 Inhibits Bax Activation and Apoptosis via a Phosphatidylinositol 3-Kinase-dependent Mechanism J. Biol. Chem., May 2, 2008; 283(18): 12305 - 12313. [Abstract] [Full Text] [PDF]

				X.-Y. Lu, L. Chen, X.-L. Cai, and H.-T. Yang Overexpression of heat shock protein 27 protects against ischaemia/reperfusion-induced cardiac dysfunction via stabilization of troponin I and T Cardiovasc Res, April 25, 2008; (2008) cvn091v2. [Abstract] [Full Text] [PDF]

				I. Kardys, N. Rifai, O. Meilhac, J.-B. Michel, J. L. Martin-Ventura, J. E. Buring, P. Libby, and P. M Ridker Plasma Concentration of Heat Shock Protein 27 and Risk of Cardiovascular Disease: A Prospective, Nested Case-Control Study Clin. Chem., January 1, 2008; 54(1): 139 - 146. [Abstract] [Full Text] [PDF]

				G. Li, I. S. Ali, and R. W. Currie Insulin-induced myocardial protection in isolated ischemic rat hearts requires p38 MAPK phosphorylation of Hsp27 Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H74 - H87. [Abstract] [Full Text] [PDF]

				J.-F. Legare, A. Oxner, O. Heimrath, T. Myers, and R. W. Currie Heat shock treatment results in increased recruitment of labeled PMN following myocardial infarction Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3210 - H3215. [Abstract] [Full Text] [PDF]

				S. Turakhia, C. D. Venkatakrishnan, K. Dunsmore, H. Wong, P. Kuppusamy, J. L. Zweier, and G. Ilangovan Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27 Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3111 - H3121. [Abstract] [Full Text] [PDF]

				B. C. Blunt, A. T. Creek, D. C. Henderson, and P. A. Hofmann H2O2 activation of HSP25/27 protects desmin from calpain proteolysis in rat ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1518 - H1525. [Abstract] [Full Text] [PDF]

				J.-T. Hsu, Y.-C. Hsieh, W. H. Kan, J. G. Chen, M. A. Choudhry, M. G. Schwacha, K. I. Bland, and I. H. Chaudry Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2982 - H2987. [Abstract] [Full Text] [PDF]

				P. Rafiee, M. E. Theriot, V. M. Nelson, J. Heidemann, Y. Kanaa, S. A. Horowitz, A. Rogaczewski, C. P. Johnson, I. Ali, R. Shaker, et al. Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27 Am J Physiol Cell Physiol, November 1, 2006; 291(5): C931 - C945. [Abstract] [Full Text] [PDF]

				H. Wei, W. Campbell, and R. S. Vander Heide Heat shock-induced cardioprotection activates cytoskeletal-based cell survival pathways Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H638 - H647. [Abstract] [Full Text] [PDF]

				O. Zolk, C. Schenke, and A. Sarikas The ubiquitin-proteasome system: Focus on the heart Cardiovasc Res, June 1, 2006; 70(3): 410 - 421. [Abstract] [Full Text] [PDF]

				J.-h. Xi, F. Bai, R. McGaha, and U. P. Andley Alpha-crystallin expression affects microtubule assembly and prevents their aggregation FASEB J, May 1, 2006; 20(7): 846 - 857. [Abstract] [Full Text] [PDF]

				H. Cao, N. Dronadula, F. Rizvi, Q. Li, K. Srivastava, W. T. Gerthoffer, and G. N. Rao Novel Role for STAT-5B in the Regulation of Hsp27-FGF-2 Axis Facilitating Thrombin-Induced Vascular Smooth Muscle Cell Growth and Motility Circ. Res., April 14, 2006; 98(7): 913 - 922. [Abstract] [Full Text] [PDF]

				T. De Celle, F. Vanrobaeys, P. Lijnen, W. M. Blankesteijn, S. Heeneman, J. Van Beeumen, B. Devreese, J. F. M Smits, and B. J. A Janssen Alterations in mouse cardiac proteome after in vivo myocardial infarction: permanent ischaemia versus ischaemia-reperfusion Exp Physiol, July 1, 2005; 90(4): 593 - 606. [Abstract] [Full Text] [PDF]

				C. Zhang, Z. Xu, X.-R. He, L. H. Michael, and C. Patterson CHIP, a cochaperone/ubiquitin ligase that regulates protein quality control, is required for maximal cardioprotection after myocardial infarction in mice Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2836 - H2842. [Abstract] [Full Text] [PDF]

				T. M. Casey, P. G. Arthur, and M. A. Bogoyevitch Proteomic Analysis Reveals Different Protein Changes during Endothelin-1- or Leukemic Inhibitory Factor-induced Hypertrophy of Cardiomyocytes in Vitro Mol. Cell. Proteomics, May 1, 2005; 4(5): 651 - 661. [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. M. Hollander, J. L. Martin, D. D. Belke, B. T. Scott, E. Swanson, V. Krishnamoorthy, and W. H. Dillmann Overexpression of Wild-Type Heat Shock Protein 27 and a Nonphosphorylatable Heat Shock Protein 27 Mutant Protects Against Ischemia/Reperfusion Injury in a Transgenic Mouse Model Circulation, December 7, 2004; 110(23): 3544 - 3552. [Abstract] [Full Text] [PDF]

				J. Inserte, D. Garcia-Dorado, M. Ruiz-Meana, L. Agullo, P. Pina, and J. Soler-Soler Ischemic preconditioning attenuates calpain-mediated degradation of structural proteins through a protein kinase A-dependent mechanism Cardiovasc Res, October 1, 2004; 64(1): 105 - 114. [Abstract] [Full Text] [PDF]

				C. Depre, S.-J. Kim, A. S. John, Y. Huang, O. E. Rimoldi, J. R. Pepper, G. D. Dreyfus, V. Gaussin, D. J. Pennell, D. E. Vatner, et al. Program of Cell Survival Underlying Human and Experimental Hibernating Myocardium Circ. Res., August 20, 2004; 95(4): 433 - 440. [Abstract] [Full Text] [PDF]

				G.-C. Fan, G. Chu, B. Mitton, Q. Song, Q. Yuan, and E. G. Kranias Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits {beta}-Agonist-Induced Cardiac Apoptosis Circ. Res., June 11, 2004; 94(11): 1474 - 1482. [Abstract] [Full Text] [PDF]

				H. Mao, Y. Wang, Z. Li, K. L. Ruchalski, X. Yu, J. H. Schwartz, and S. C. Borkan Hsp72 Interacts with Paxillin and Facilitates the Reassembly of Focal Adhesions during Recovery from ATP Depletion J. Biol. Chem., April 9, 2004; 279(15): 15472 - 15480. [Abstract] [Full Text] [PDF]

				B. Bullard, C. Ferguson, A. Minajeva, M. C. Leake, M. Gautel, D. Labeit, L. Ding, S. Labeit, J. Horwitz, K. R. Leonard, et al. Association of the Chaperone {alpha}B-crystallin with Titin in Heart Muscle J. Biol. Chem., February 27, 2004; 279(9): 7917 - 7924. [Abstract] [Full Text] [PDF]

X. Sun, J.-M. Fontaine, J. S. Rest, E. A. Shelden, M. J. Welsh, and R. Benndorf Interaction of Human HSP22 (HSPB8) with Other Small Heat Shock Proteins J. Biol. Chem., January 23, 2004; 279(4): 2394 - 2402.