This Article Abstract Full Text (PDF) All Versions of this Article: 92/2/203    most recent 01.RES.0000052989.83995.A5v1 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 Morrison, L. E. Articles by Glembotski, C. C. Search for Related Content PubMed PubMed Citation Articles by Morrison, L. E. Articles by Glembotski, C. C. Related Collections Apoptosis Cell signalling/signal transduction

(Circulation Research. 2003;92:203.)
© 2003 American Heart Association, Inc.

Cellular Biology

Mimicking Phosphorylation of B-Crystallin on Serine-59 Is Necessary and Sufficient to Provide Maximal Protection of Cardiac Myocytes From Apoptosis

Lisa E. Morrison^*, Holly E. Hoover^*, Donna J. Thuerauf, Christopher C. Glembotski

From the San Diego State University Heart Institute and the Department of Biology, San Diego State University, San Diego, Calif.

Correspondence to Christopher C. Glembotski, Department of Biology, San Diego State University, San Diego, CA 92182. E-mail cglembotski{at}sunstroke.sdsu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
B-Crystallin (BC), a small heat shock protein expressed in high levels in the heart, is phosphorylated on Ser-19, 45, and 59 after stress. However, it is not known whether BC phosphorylation directly affects cell survival. In the present study, constructs were prepared that encode forms of BC harboring Ser to Ala (blocks phosphorylation) or Ser to Glu (mimics phosphorylation) mutations at positions 19, 45, and 59. The effects of each form on apoptosis of cultured cardiac myocytes after hyperosmotic or hypoxic stress were assessed. Compared with controls, cells that expressed BC with Ser to Ala substitutions at all three positions, BC(AAA), exhibited more stress-induced apoptosis. Cells expressing either BC(AAE) or (EEE) exhibited 3-fold less apoptosis than cells expressing BC(AAA), indicating that phosphorylation of Ser-59 confers protection. BC is known to bind to procaspase-3 and to decrease caspase-3 activation. Compared with cells expressing BC(AAA), the activation of caspase-3 was decreased by 3-fold in cells expressing BC(AAE). These results demonstrate that mimicking the phosphorylation of BC on Ser-59 is necessary and sufficient to confer caspase-3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress. These findings provide direct evidence that BC(S59P) contributes to the cardioprotection observed after physiologically relevant stresses, such as transient hypoxia. Identifying the targets of BC(S59P) will reveal important details about the mechanism underlying the cytoprotective effects of this small heat shock protein.

Key Words: cardiac myocytes • B-crystallin • apoptosis • phosphorylation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stress kinases, such as p38 mitogen-activated protein kinase (MAPK), are activated in the heart in response to potentially harmful stress, such as ischemia.^1–3 In cardiac myocytes, when activated by the MAP kinase kinase, MKK6, p38 stimulates the MAPK-activated protein kinase-2 (MAPKAPK-2), and the cells exhibit reduced apoptosis in response to stress.^4,5 Moreover, the inhibition of p38 increases cardiac myocyte apoptosis and myocardial tissue damage in response to stress.^5,6 Accordingly, the p38/MAPKAPK-2 pathway serves a key role in protecting cells against stress; however, the downstream events responsible for these cytoprotective effects are uncharacterized.

The small heat shock proteins hsp25 and hsp27 and B-crystallin are targets of p38-activated MAPKAPK-2.^5,7–10 Although hsp25 and hsp27 exhibit a broad tissue expression pattern, BC is expressed in a more restricted manner, being enriched in lens, neurons, skeletal muscle, and cardiac myocytes.^7,11–13 In the heart, BC is thought to account for up to 3% of the total protein.¹⁴ Overexpression of BC in cultured cardiac myocytes protects them from ischemia-induced cell death and stabilizes microtubules.¹⁵ In addition, overexpression of BC in transgenic mice protects the heart from ischemic damage.¹⁶

The mechanism by which BC protects cells from stress-induced damage is unclear; however, it likely involves phosphorylation. In response to various types of cellular stress, BC is phosphorylated on Ser-19, 45, and 59.⁸ Although the signaling pathways leading to Ser-19 phosphorylation are unknown, the extracellular signal-regulated protein kinase (ERK) MAPK pathway appears to be responsible for phosphorylation of Ser-45, and the p38 MAPK pathway is responsible for phosphorylation of Ser-59.^5,10,17 In the heart, oxidative stress can lead to the activation of p38 and MAPKAPK-2^18,19 and in cardiac myocytes, stress increases p38-mediated MAPKAPK-2, the latter of which phosphorylates BC on Ser-59.⁵ Moreover, blocking p38-mediated MAPKAPK-2 activation inhibits BC phosphorylation on Ser-59 and enhances apoptotic myocyte death.⁵ These results are consistent with a role for phosphorylation of BC on Ser-59 in mediating the cytoprotective actions of this small heat shock protein (hsp); however, there is no direct evidence supporting this hypothesis. Consequently, we performed the present study to assess the role of phosphorylation of BC on stress-induced apoptosis in a cardiac myocyte model system. Phosphorylation of BC at Ser-19, 45, and 59 was either blocked by expressing Ser to Ala mutations or was mimicked by expressing Ser to Glu mutants in cardiac myocytes. The effects of expressing these forms of BC on stress-induced apoptosis were then assessed. The stresses chosen for this study were sorbitol, a hyperosmotic stress known to strongly activate p38 and MAPKAPK-2 in cardiac myocytes, and hypoxia followed by reoxygenation, a stress that activates p38 in cardiac myocytes and mimics the well-studied, clinically relevant stress of ischemia/reperfusion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Neonatal Rat Ventricular Myocytes
Primary neonatal rat ventricular cardiac myocytes (NRVCMs) were prepared from 1- to 4-day-old Harlan Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, Ind) as previously described.⁵ Animals were cared for according to the Guide for the Care and Use of Laboratory Animals by the NIH.

Plasmids
BC(wt)
pcDNA3.1 HA-BC(wt) codes for wild-type rat BC cDNA with an N-terminal HA tag. This construct was derived from a full-length rat BC cDNA, which was provided by A. Iwaki (Neurological Institute, Kyushu University, Fukuoka, Japan),^12,20 and the N-terminal HA tag sequence (MYPYDVPDYA) was cloned into pcDNA3.1 using standard PCR protocols.

BC(SSE)
pcDNA3.1 HA-BC(SSE) codes for mutated BC with Ser-59 replaced with Glu to mimic phosphorylation at this site. It was derived using the QuikChange site-directed mutagenesis kit (Stratagene) and mutating pcDNA3.1 HA-BC(wt) from Ser-59 to Glu-59 using the following primers:

The mutated nucleotides are identified in bold and underlined.

BC(AAA)
pcDNA3.1 HA-BC(AAA) codes for mutated BC with Ser-19, -45, and -59 replaced with Ala to block phosphorylation at these sites. It was derived from pcDNA3.1 HA-BC(wt) by mutating Ser-19, Ser-45, and Ser-59 to Ala-19, Ala-45, and Ala-59 using the following primers:

BC(EEE)
pcDNA3.1 HA-BC(EEE) codes for mutated BC with Ser-19, 45, and 59 replaced with Glu to mimic phosphorylation at these sites. It was derived from pcDNA3.1 HA-BC(wt) by mutating Ser-19, Ser-45, and Ser-59 to Glu-19, Glu-45, and Glu-59 using the Glu-59 primer pair (see above) and the following primer pairs:

BC(AAE)
pcDNA3.1 HA-BC(AAE) codes for mutated BC with Ser-19 and 45 replaced with Ala and Ser-59 replaced with Glu to mimic phosphorylation at Ser-59 and to block phosphorylation at Ser-19 and 45. It was derived from pcDNA3.1 HA-BC(AAA) by mutating Ala-59 to Glu-59 using the Glu-59 primer pairs.

pCMV6
pCMV6 was used as an empty vector control for electroporation-mediated gene transfer TUNEL studies.

pCMVß-Galactosidase
pCMVß-gal was cotransfected with either HA-tagged BC constructs or control pCMV6 vector to allow for identification of cardiomyocytes that were transfected.

Recombinant Adenovirus
Adv-BC(wt), Adv-BC(AAA), Adv-BC(EEE), and Adv-BC(AAE)
The AdEasy system (Stratagene) was used for preparing recombinant adenoviral strains using previously described methods.^21,22 Viral titers were determined as previously described.²²

Transfection by Electroporation
Between 5 and 12x10⁶ cells were combined with the indicated amounts of plasmids in a total of 300 µL of minimal media. Cells were cotransfected with 30 µg of pCMVß-galactosidase and 100 µg of HA-tagged BC test constructs or the control vector pCMV6. (Preliminary experiments using various concentrations of the test constructs indicated that electroporation with 100 µg provided optimal expression levels of the relevant form of HA-tagged BC.) Cells were electroporated at 550 V, 25 µF, and 100 in a 0.2-cm gap electroporation cuvette (Bio-Rad) using a Gene Pulser II (Bio-Rad). After transfection, cells were plated for 16 hours in DMEM:F-12 supplemented with 10% FCS.

Transfection by AdV Infection
Cells were plated and maintained for 24 hours in DMEM/F-12 with 10% fetal bovine serum, as described in Cell Culture. Cultures were infected with Adv-GFP (control adenovirus), Adv-BC(wt), Adv-BC(AAA), Adv-BC(AAE), or Adv-BC(EEE) at a multiplicity of infection (MOI)=10 in DMEM/F-12 with 10% fetal bovine serum for 5 hours, as previously described.²² Cultures were then washed with DMEM/F-12 (1:1) and incubated in minimal medium until experimental maneuvers.

Western Analyses
Cultures composed of 10⁶ ventricular myocytes were lysed in Laemmli sample buffer, resolved on a 15% acrylamide gel, and transferred to a PVDF membrane in methanol transfer buffer. Membranes were probed with an HA-mouse monoclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution, 1:2000 rabbit BC, or 1:1000 rabbit BC S59P-specific antisera (Stressgen Biotechnologies Corp, Victoria, British Columbia, Canada). Membranes were washed and incubated with horseradish peroxidase–conjugated anti-IgG secondary antiserum (Santa Cruz Biotechnology). Visualization of immune complexes was carried out by enhanced chemiluminescence method using Western blotting detection reagents (NEN Life Science).

Apoptosis Assays
TUNEL
TUNEL analyses of cells plated on glass slides were carried out as previously described.⁴

Sorbitol Treatment
In experiments where cells were transfected by electroporation, cells were treated with 400 mmol/L sorbitol for 6 to 7 hours. In experiments involving adenovirus-mediated gene transfer, cells were treated with 400 mmol/L of sorbitol for 14 hours. (We found that in contrast to electroporation, after AdV infection, longer treatment times with sorbitol were required to induce apoptosis.)

Hypoxia/Reoxygenation Treatment
The cell culture hypoxia/reoxygenation (H/R) model has been previously described.²³ In experiments where cells were infected by AdV, the hypoxia time was 10 hours, and reoxygenation time was 24 hours.

DNA Laddering
Cultured NRVCMs were plated at a density of either 700 cells/mm² (sorbitol experiments) or 100 cells/mm² (H/R experiments). After adenovirus infection, cells were washed with DMEM/F12 (1:1) and then maintained in minimal medium until day 4. Cells were either incubated ±400 mmol/L sorbitol for 14 hours or submitted to hypoxia (10 hours)/reoxygenation (28 hours). Upon termination of the experiment, isolation of genomic DNA was performed as described previously.⁵

Caspase-3 Assay
Approximately 2x10⁶ cells were treated ±400 mmol/L sorbitol for 8 hours or hypoxia (12 hours)/reoxygenation (18 hours) and lysed in buffer containing 0.71% NP-40, 71 mmol/L Tris (pH 7.5), 0.71 mmol/L EDTA, and 212 mmol/L NaCl. Cellular debris was removed by centrifugation. Samples were combined with an equal volume of a reaction buffer (21 mmol/L HEPES, 105 mmol/L NaCl, 5.25 mmol/L DTT, and 50 µmol/L Ac-DEVD-AFC). The mixture was then incubated at 37°C for 1 hour and then loaded into a black 96-well titer plate. Background fluorescence resulting primarily from green fluorescent protein (GFP) contributed 18% of the total emission. Fluorescence was read on a Spectra Max GeminiXS fluorometer (Molecular Devices) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm using Softmax Pro version 3.1.2 software.

Statistical Analyses
The results are presented as mean±SE or SD, as described in the figure legends. Unless indicated otherwise in the figure legends, statistical analyses included a one-way ANOVA followed by Neuman-Keuls post hoc analysis for multiple-group comparisons using SPSS software. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
B-Crystallin Is Required for Protection From Apoptosis
To determine whether overexpression of BC in primary cardiac myocytes could protect the cells from apoptosis after stress, cells were transfected with a construct encoding HA-tagged rat BC(wt) or with an empty vector (Figure 1A). After treatment with sorbitol, cultures were then assessed for apoptosis by TUNEL analysis. In cultures that were transfected with the empty vector control pCMV6, treatment with sorbitol caused an 10-fold increase in the number of TUNEL-positive cells (Figure 1B). However, compared with cells transfected with the empty vector, cells expressing HA-BC(wt) exhibited an 40% reduction in the number of TUNEL-positive cells after sorbitol treatment (Figure 1B). To assess whether BC displayed similar apparent cytoprotective effects in response to a different but physiologically relevant stress, a previously described cell culture model of hypoxia followed by reoxygenation (H/R)²³ was used. This is a well-characterized cell culture model that mimics the ischemia/reperfusion that occurs in individuals with advanced coronary artery disease.²⁴ Moreover, the ability of various forms of BC to protect cardiac myocytes from H/R-induced apoptosis has never been assessed. Compared with cells transfected with the empty vector, cells expressing HA-BC(wt) exhibited an 50% reduction in the number of TUNEL-positive cells after H/R (Figure 1C). These findings indicated that overexpression of BC(wt) can act in concert with endogenous BC to protect cells from apoptosis in response to two different stresses.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effects of BC overexpression on cardiac myocyte apoptosis. A, Western analysis. Cultured cardiac myocytes were transfected by electroporation with either an empty vector control, pCMV6, or with a vector encoding HA-BC(wt). Cultures were then extracted and submitted to SDS-PAGE followed by a Western blot using an HA antibody. B, TUNEL analysis of sorbitol-treated cells. Cultured cardiac myocytes were cotransfected with CMV-ß-gal and either pCMV6 or with a vector encoding HA-BC(wt). Cultures were maintained on glass slides for 3 days as described in Materials and Methods and then treated ±400 mmol/L sorbitol for 6 to 7 hours. The cells were then fixed and analyzed for ß-gal immunocytofluorescence and for TUNEL, as described previously and in Materials and Methods.4,23 ß-Gal–positive cells provided a means by which transfected cells could be identified; as previously observed, 5% of the cells visibly expressed ß-gal. Evaluation of the frequency of TUNEL staining of the ß-gal–positive cells provided an assessment of the portion of the transfected cells that underwent presumptive apoptosis. In each experiment, at least 100 ß-gal–positive cells from each of 3 cultures in each group were assessed for TUNEL staining. The values shown are the mean±SE of at least 3 experiments. *P<0.05 different from Con; P<0.05 different from all other values, as determined by ANOVA followed by Neuman-Keuls post hoc analysis of variance. C, TUNEL analysis of H/R-treated cells. Cells were plated as described in panel B and then treated ±H/R, as described in Materials and Methods. Cultures were fixed and assessed for DNA fragmentation by TUNEL analysis, also as described in Materials and Methods. The values shown are the mean±SE of at least 3 experiments. *P<0.05 different from Con; P<0.05 different from all other values.

Effects of Mutations That Mimic or Block BC Phosphorylation
Experiments were carried out to determine the effects of recombinant forms of BC that either mimic or block phosphorylation via Ser to Glu or Ser to Ala substitutions, respectively, at positions 19, 45, and 59 (Figure 2A). Western blot analyses confirmed that in cardiac myocytes expression levels of the HA-tagged transgenes were approximately equal (Figure 2B). (The reasons for the slightly decreased expression of BC(AAE) and BC(EEE) are unknown; however, preparation of multiple, independent constructs resulted in the same relative expression levels.) Compared with cells transfected with an empty vector (Con), cells expressing BC(wt) displayed a significant decrease in the number of apoptotic cells after sorbitol treatment (Figure 2C). In contrast, compared with cells expressing similar amounts of BC(wt), cells expressing BC(AAA) exhibited a significant increase in sorbitol-induced apoptosis (Figure 2C). In fact, BC(AAA)-expressing cells were more susceptible to cell death than control cells. These results were consistent with requirements for Ser-19, 45, and 59 in mediating the protective effects of BC. To test the impact of mimicking phosphorylation at Ser-59 only, cultures were transfected with BC(AAE). Cells expressing BC(AAE) exhibited 3.5-fold less apoptosis than those expressing BC(AAA) (Figure 2C). This dramatic enhancement in protection resulting from a single amino acid substitution [BC(AAA) versus BC(AAE)] indicates that phosphorylation of Ser-59 enhances the protective effects of BC. Further support for the importance of phosphorylation of Ser-59, compared with Ser-19 or 45, was the finding that BC(EEE) exhibited protective effects that were the same as BC(AAE) (Figure 2C), as did BC(SSE) (not shown). Cells expressing BC(AAE) or BC(EEE) displayed 33% less apoptosis than those expressing BC(wt), further supporting the importance of phosphorylation of Ser-59. Qualitatively similar results were obtained when cells were treated with H/R (Figure 2D), indicating that the various forms of BC exhibit the same cytoprotective properties in response to two very different stressors. To examine the effects of the various forms of BC using a different means of DNA transfer that would provide higher transfection efficiency, recombinant adenoviral (AdV) strains encoding HA-BC(wt) AAA, AAE, and EEE were prepared. (AdV-mediated DNA transfer results in nearly 100% transfection efficiency of cardiac myocytes²² compared with the 5% efficiency afforded by electroporation.) Western blot analyses demonstrated that the recombinant AdV strains encoded the expression of similar levels of each form of HA-BC without affecting the levels of endogenous BC (Figure 3A). TUNEL analyses after treatment with either sorbitol or H/R showed that using AdV-mediated gene transfer, the various forms of BC exhibited effects that were similar to those observed using electroporation (Figures 3B and 3C). These results indicated that in response to either sorbitol or H/R, the various strains of AdV-BC exhibited cytoprotective effectiveness with a rank order of

such that (AAE) displayed the greatest protection and AAA the least. Thus, either electroporation or AdV-mediated gene transfer resulted in similar profiles of cytoprotection for the various forms of BC, as assessed by TUNEL analysis.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of various forms of B-crystallin on sorbitol or H/R-induced apoptosis: TUNEL analysis. A, Diagram of the mutant forms of BC used in this study. B, Western blot analysis of BC expression. Cells were transfected by electroporation±empty vector (con) or with the BC constructs shown in panel A. Culture extracts were then analyzed by SDS-PAGE followed by Western blotting using an anti-BC antiserum, as previously described.5 C and D, TUNEL analysis. Cells were cotransfected with pCMV-ß-gal and BC constructs or empty vector, as described in panel B, plated on glass slides in serum-containing medium for 24 hours, and then maintained in serum-free medium for 48 hours. Cultures were then treated with sorbitol (panel C) or H/R (panel D) and then fixed and assessed for apoptosis by ß-gal immunocytofluorescence coupled with TUNEL analysis, as described in Figure 1 and in Materials and Methods. The data obtained with each construct were normalized to the treatment that resulted in the maximum number of TUNEL-positive cells, which was set to 100%; 17.6% or 16.1% of the cells transfected with BC(AAA) were TUNEL-positive after sorbitol or H/R treatment, respectively. Shown are the mean±SE of at least 3 experiments, each of which included at least 3 cultures for each treatment. P<0.05 different from Con; *P<0.05 different from wt.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of various recombinant strains of adenovirus B-crystallin on sorbitol or H/R-induced apoptosis: TUNEL analysis. A, Western blot analysis of transgene expression. Cells were infected with recombinant adenovirus strains designed to encode BC(wt), (AAA), (AAE), or (EEE), as described in Materials and Methods. Two days after infection, cultures were extracted and analyzed by SDS-PAGE followed by Western blotting using an anti-BC antiserum, as described in Materials and Methods and in Figure 2. B and C, TUNEL analysis. Cells infected with AdV, as described in panel A, were treated with sorbitol (panel B) or H/R (panel C) and then assessed for DNA fragmentation by TUNEL analysis, as described in Materials and Methods. The data were normalized to BC(AAA) (31% and 21.2% of the GFP-positive cells counted were TUNEL-positive after sorbitol or H/R treatment, respectively), which was set at 100%, as described in Figure 2. Shown are the mean±SE of at least 3 experiments, each of which included at least 2 cultures for each treatment. *P<0.05 different from wt; P<0.05 different from BC(AAA).

The cytoprotective effects of the various forms of BC were further analyzed using DNA fragmentation, or laddering, as a measure of the nucleosomal DNA cleavage, a hallmark of apoptosis.⁴ Compared with cells infected with AdV-Con, those infected with AdV-BC(wt) displayed less DNA fragmentation on treatment with sorbitol, indicating protection from apoptosis (Figure 4A). AdV-BC(AAA) exhibited no apparent protective effects and actually fostered somewhat more DNA fragmentation than infecting cells with AdV-Con. Infection of cells with either AdV-BC(AAE) or (EEE) resulted in levels of DNA fragmentation that were reduced significantly below levels observed in all other cultures. Similar results were obtained when these AdV strains were tested in cultures treated with H/R (Figure 5). Thus, in comparison to the TUNEL analyses, the DNA fragmentation analyses showed that in response to either sorbitol or H/R, the various strains of AdV-BC exhibited cytoprotective effectiveness against apoptosis with a rank order of

where (AAE) displayed the greatest protection and AAA the least. Thus, two different methods of BC transgene delivery and two different methods of assessing apoptosis supported the hypothesis that when phosphorylated on serine-59, BC protects cells from apoptosis in response to either hyperosmotic or hypoxic stress.

View larger version (38K): [in this window] [in a new window]   Figure 4. Effects of various recombinant strains of adenovirus B-crystallin on sorbitol-induced apoptosis: DNA ladder analysis. A, Cultured cardiac myocytes were infected with the recombinant AdV-BC strains shown, treated with sorbitol, the DNA was extracted, fractionated on an agarose gel, stained with ethidium bromide, and photographed. A 1-kb DNA ladder run in parallel was used for estimating fragment sizes (not shown); the fastest migrating band in each sample is the 200-bp band. B, The intensity of the 200-bp band (lowest) from each sample shown in panel A was assessed using Image Quant software (Molecular Dynamics). Each intensity was normalized to the maximum intensity, which was obtained with AdV-BC(AAA); shown are the mean intensities±SE from triplicate cultures. *P<0.05 different from wt; P<0.05 different from BC(AAA).

View larger version (38K): [in this window] [in a new window]   Figure 5. Effects of various recombinant strains of adenovirus B-crystallin on H/R-induced apoptosis: DNA ladder analysis. A, Cultured cardiac myocytes were infected with the recombinant AdV-BC strains shown, treated with H/R, and the DNA was extracted and fractionated as described in Figure 4A. B, The intensities of the 200-bp bands obtained from each sample shown in panel A were quantified and are plotted as described in Figure 4B. *P<0.05 different from wt; P<0.05 different from BC(AAA).

Because BC is a chaperone, one possible mechanism by which it might confer protection against apoptosis is by binding to and altering the function of proteins involved in apoptosis. In support of this possibility is a recent study demonstrating that BC binds to and inhibits the proteolytic conversion of the inactive caspase-3 intermediate p24 to active caspase-3.²⁵ However, that study did not assess whether BC phosphorylation might be required for maximal inhibition of caspase-3 maturation. Accordingly, the effects of the various recombinant AdV strains on caspase-3 activation were assessed. In cells infected with AdV-Con, sorbitol or H/R increased caspase-3 activation (Figure 6A). Cultures were then infected with the test AdV-BC strains, and the effects of the various forms of BC on sorbitol or H/R-activated caspase-3 were assessed. Whereas overexpression of BC(wt) reduced caspase-3 activation by 15% to 30%, expression of BC(AAA) resulted in slightly more caspase-3 activation than BC(wt), although this increase did not reach statistical significance (Figure 6B). And, cells infected with AdV-BC(AAE) or (EEE) displayed 20% to 40% reductions in caspase-3 activation, compared with cells infected with AdV-BC(wt) (Figure 6B). In comparison to the apoptosis results, BC(AAE) displayed an 2-fold greater ability to inhibit caspase-3 activation compared with BC(AAA).

View larger version (29K): [in this window] [in a new window]   Figure 6. Effects of various recombinant strains of adenovirus B-crystallin on sorbitol- and H/R-mediated activation of caspase-3. A, Cells infected with AdV-Con were maintained in minimal medium±sorbitol (white bars) or in glucose-free media±H/R (black bars), as previously described.23 Cultures were then extracted and analyzed for caspase-3 activity, as described in Materials and Methods. Shown are the mean caspase-3 values±SE of at least 3 experiments, each of which included at least 3 cultures for each treatment. *P<0.05 different from Con using a 2-tailed independent Student’s t test. Note that the slightly elevated caspase-3 activation in the H/R control cells (left side black bar) is probably due to the glucose-free medium used as the base medium for the H/R maneuver.23 B, Cells were infected with the AdV strains shown and then cultured and treated with sorbitol or H/R followed by assessment of caspase-3 activation, as described in panel A and in Materials and Methods. Shown are the mean caspase-3 values±SE of at least 3 experiments, each of which included at least 3 cultures for each treatment. *P<0.05 different from wt; P<0.05 different from BC(AAA). C, Cultures infected and treated as in panel B were extracted and analyzed for total BC (top panels) and for BC Ser-59-P (bottom panels) by SDS-PAGE and Western blotting as described in Materials and Methods.

To determine whether the effects of the AdV-encoded forms of BC might be due to their abilities to alter either the expression level or phosphorylation status of endogenous BC, Western blot analyses were carried out. None of the AdV-encoded forms of BC affected the expression levels of endogenous BC (Figure 6C, Total BC). When these same blots were probed using an antiserum that detects BC S59P, the only AdV-encoded form of BC that was phosphorylated was HA-BC(wt), as expected (Figure 6C, Ser-59P wt). None of the AdV-encoded forms of BC had any effect on the level of endogenous BC phosphorylation on serine-59. These results indicate that the various forms of AdV-encoded BC affect sorbitol- or H/R-induced apoptosis by virtue of their abilities to inhibit (AAA) or mimic (AAE or EEE) BC S59P interaction with appropriate cellular targets.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In cardiac myocytes, p38 activation can result in protection from apoptosis and in MAPKAPK-2–mediated phosphorylation of BC on serine-59.⁵ However, it is not known whether such phosphorylation is required for BC-mediated cytoprotection. In the present study, BC(AAE), which mimics phosphorylation only on Ser-59, protected cardiac myocytes against apoptosis, whereas BC(AAA) conferred increased susceptibility to apoptosis. These results indicate that phosphorylation of BC on Ser-59 alone is necessary and sufficient for this hsp to confer maximal cytoprotection. The molecular mechanisms underlying the cytoprotection mediated by BC(S59P) or increased apoptosis conferred by BC(AAA) are unknown. The finding in the present study that BC(AAE) reduced caspase-3 activation suggests that the antiapoptotic effects of BC(S59P) may involve inhibition of the conversion of p24 caspase-3 to active caspase-3. Although it has been shown that BC can bind to p24 and decrease the rate of activated caspase-3 generation,²⁵ the role, if any, of BC serine-59 phosphorylation has not been established.

It is most likely that BC effects cytoprotection by interacting with many targets in addition to caspase-3. BC monomers, which are 25 kDa, associate into oligomers made of 32 subunits, reaching a mass of 645 kDa.²⁶ The mass of the oligomers decreases in response to cellular stresses that also increase the phosphorylation of BC,^27–29 suggesting that the phosphorylated BC monomers translocate to sites where they mediate cytoprotection. For example, cardiac ischemia causes BC translocation from a diffuse localization in the cytosol, to myofibrils,³⁰ where it binds to the I-band region of the Z-lines.³¹ This translocation takes place on a time frame consistent with a possible role for phosphorylation in mediating BC relocation.^31–33 The localization of BC to sarcomeres may allow myofibrils to maintain their correctly folded state and optimal function,³² similar to the way hsp27 stabilizes actin filaments in endothelial cells after stresses that activate p38.³⁴ Although these results imply that BC monomers would serve as effective chaperones, this concept is controversial. For example, in glioma cells, expression of BC (DDD), where serines-19, 45, and 59 have been replaced by aspartic acid, which mimics phosphorylation at these sites, resulted in oligomers that are smaller and display reduced chaperone-like activity.²⁸ However, a naturally occurring form of BC carrying an R120G mutation, which is known to cause desmin-related cardiomyopathy, forms oligomers that exceed 645 kDa; yet, this form is a poorer chaperone.³⁵ Thus, the relationships between BC oligomer size, phosphorylation status, and chaperone and cytoprotective activities remain unclear and will require additional study.

The finding in the present study that BC(AAA) leads to less protection from apoptosis than the other forms of BC tested is supported by several previous reports. In one study, it was shown that compared with BC(wt), BC(AAA) exhibited reduced chaperone-like activity when tested in an in vitro assay system.³⁶ In another report, when expressed in Escherichia coli, BC(AAA) displayed a nearly 10-fold reduction in the ability to protect cells from heat shock–induced death compared with BC(wt).²⁷ These findings support the notion that in the present study, BC(AAA) may confer enhanced apoptosis because of its reduced chaperone activity. Indeed, our findings that expression of BC(AAA) did not alter the phosphorylation of endogenous BC on S59 indicate that the ability of BC(AAA) to increase apoptosis is not because it acts in a dominant-negative manner on MAPKAPK-2. Instead, BC(AAA) may compete with BC(wt) for binding to downstream targets, such as p24 and procaspase-3; however, because BC(AAA) possesses reduced chaperone activity compared with BC, susceptibility to apoptosis increases.

In summary, this is the first study to report that mimicking Ser-59 phosphorylation of BC is necessary and sufficient to confer protection during hyperosmotic and hypoxic stress of cardiac myocytes. Future studies directed toward identifying targets of BC(S59P) will be required to better understand the molecular events underlying the potent cytoprotective roles of this small hsp in the stressed myocardium.

	Acknowledgments

 
This work was supported by grants from the NIH (HL-63975 and NS/HL-25037) to C.C.G. This work was supported by an award from the American Heart Association to H.E.H. The authors wish to thank Jason Wall for developing the caspase-3 assay. The authors wish to thank Dr Kanefusa Kato, Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Japan, for the S59P-specific BC antiserum.

	Footnotes

 
*Both authors contributed equally to this study.

Received August 15, 2002; revision received November 20, 2002; accepted December 9, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, Fuller SJ, Ben-Levy R, Ashworth A, Marshall CJ, Sugden PH. Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart: p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion. Circ Res. 1996; 79: 162–173.[Abstract/Free Full Text]

2. Maulik N, Watanabe M, Zu YL, Huang CK, Cordis GA, Schley JA, Das DK. Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett. 1996; 396: 233–237.[CrossRef][Medline] [Order article via Infotrieve]

3. Ping P, Murphy E. Role of p38 mitogen-activated protein kinases in preconditioning: a detrimental factor or protective kinase? Circ Res. 2000; 86: 921–922.[Free Full Text]

4. Zechner D, Craig R, Hanford DS, McDonough PM, Sabbadini RA, Glembotski CC. MKK6 activates myocardial cell NF-B and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner. J Biol Chem. 1998; 273: 8232–8239.[Abstract/Free Full Text]

5. Hoover HE, Thuerauf DJ, Martindale JJ, Glembotski CC. B-Crystallin gene induction and phosphorylation by MKK6-activated p38. J Biol Chem. 2000; 275: 23825–23833.[Abstract/Free Full Text]

6. Sato M, Cordis GA, Maulik N, Das DK. SAPKs regulation of ischemic preconditioning. Am J Physiol. 2000; 279: H901–H907.

7. Kato K, Shinohara H, Kurobe N, Inaguma Y, Shimizu K, Ohshima K. Tissue distribution and developmental profiles of immunoreactive B crystallin in the rat determined with a sensitive immunoassay system. Biochem Biophys Acta. 1991; 1074: 201–208.[Medline] [Order article via Infotrieve]

8. Ito H, Okamoto K, Nakayama H, Isobe T, Kato K. Phosphorylation of B-crystallin in response to various types of stress. J Biol Chem. 1997; 272: 29934–29941.[Abstract/Free Full Text]

9. Zu Y-L, Ai Y, Gilchrist A, Maulik N, Watras J, Sha’afi RI, Das DK, Huang C-K. High expression and activation of MAP kinase-activated protein kinase 2 in myocardium. J Mol Cell Cardiol. 1997; 29: 2159–2168.[CrossRef][Medline] [Order article via Infotrieve]

10. Kato K, Ito H, Kamei K, Inaguma Y, Iawamoto I, Saga S. Phosphorylation of B-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation. J Biol Chem. 1998; 273: 28346–28354.[Abstract/Free Full Text]

11. Bhat SP, Nagineni CN. B-Subunit of lens-specific protein -crystallin is present in other ocular and non-ocular tissues. Biochem Biophys Res Commun. 1989; 158: 319–325.[CrossRef][Medline] [Order article via Infotrieve]

12. Iwaki A, Iwaki T, Goldman JE, Liem RK. Multiple mRNAs of rat brain -crystallin B chain result from alternative transcriptional initiation. J Biol Chem. 1990; 265: 22197–22203.[Abstract/Free Full Text]

13. Iwaki T, Kume-Iwaki A, Goldman JE. Cellular distribution of B-crystallin in non-lenticular tissues. J Histochem Cytochem. 1990; 38: 31–39.[Abstract]

14. Lutsch G, Vetter R, Offhauss U, Wieske M, Grone HJ, Klemenz R, Schimke I, Stahl J, Benndorf R. Abundance and location of the small heat shock proteins HSP25 and B-crystallin in the rat and human heart. Circulation. 1997; 96: 3466–3476.[Abstract/Free Full Text]

15. Bluhm WF, Martin JL, Mestril R, Dillmann WH. Specific heat shock proteins protect microtubules during simulated ischemia in cardiac myocytes. Am J Physiol. 1998; 275: H2243–H2249.[Medline] [Order article via Infotrieve]

16. Ray PS, Martin JL, Swanson EA, Otani H, Dillmann WH, Das DK. Transgene overexpression of B crystalline confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J. 2001; 15: 393–402.[Abstract/Free Full Text]

17. Eaton P, Fuller W, Bell JR, Shattock MJ. B Crystallin translocation and phosphorylation: signal transduction pathways and preconditioning in the isolated rat heart. J Mol Cell Cardiol. 2001; 33: 1659–1671.[CrossRef][Medline] [Order article via Infotrieve]

18. Maulik N, Yoshida T, Zu YL, Sato M, Bannerjee A, Das DK. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am J Physiol. 1998; 275: H1857–H1864.[Medline] [Order article via Infotrieve]

19. Nakano A, Baines CP, Kim SO, Pelech SL, Downey JM, Cohen MV, Critz SD. Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK. Circ Res. 2000; 86: 144–151.[Abstract/Free Full Text]

20. Iwaki T, Iwaki A, Tateishi J, Goldman JE. Sense and antisense modification of glial B-crystallin production results in alterations of stress fiber formation and thermoresistance. J Cell Biol. 1994; 125: 1385–1393.[Abstract/Free Full Text]

21. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A. 1998; 95: 2509–2514.[Abstract/Free Full Text]

22. Craig RC, Larkin A, Mingo AM, Thuerauf DJ, Andrews C, McDonough PM, Glembotski CC. p38 MAPK and NF-B collaborate to induce interleukin-6 gene expression and release: evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system. J Biol Chem. 2000; 275: 23814–23824.[Abstract/Free Full Text]

23. Craig R, Wagner M, McCardle T, Craig AG, Glembotski CC. The cytoprotective effects of the glycoprotein 130 receptor-coupled cytokine, cardiotrophin-1, require activation of NF-B. J Biol Chem. 2001; 276: 37621–37629.[Abstract/Free Full Text]

24. Marber MS. Ischemic preconditioning in isolated cells. Circ Res. 2000; 86: 926–931.[Free Full Text]

25. Kamradt MC, Chen F, Cryns VL. The small heat shock protein B-crystallin negatively regulates cytochrome c- and caspase-8-dependent activation of caspase-3 by inhibiting its autoproteolytic maturation. J Biol Chem. 2001; 276: 16059–16063.[Abstract/Free Full Text]

26. Haley DA, Horwitz J, Stewart PL. The small heat-shock protein, B-crystallin, has a variable quaternary structure. J Mol Biol. 1998; 277: 27–35.[CrossRef][Medline] [Order article via Infotrieve]

27. Muchowski PJ, Wu GJ, Liang JJ, Adman ET, Clark JI. Site-directed mutations within the core "-crystallin" domain of the small heat-shock protein, human B-crystallin, decrease molecular chaperone functions. J Mol Biol. 1999; 289: 397–411.[CrossRef][Medline] [Order article via Infotrieve]

28. Ito H, Kamei K, Iwamoto K, Inaguma Y, Nohara D, Kato K. Phosphorylation-induced change of the oligomerization state of B-crystallin. J Biol Chem. 2001; 276: 5346–5352.[Abstract/Free Full Text]

29. Chiesi M, Longoni S, Limbruno MU. Cardiac -crystallin, III: involvement during heart ischemia. Mol Cell Biochem. 1990; 97: 129–136.[Medline] [Order article via Infotrieve]

30. 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]

31. Golenhofen N, Ness W, Koob R, Htun P, Schaper W, Drenckhahn D. Ischemia-induced phosphorylation and translocation of stress protein B-crystallin to Z lines of myocardium. Am J Physiol. 1998; 274: H1457–H1464.[Medline] [Order article via Infotrieve]

32. Golenhofen N, Htun P, Ness W, Koob R, Schaper W, Drenckhahn D. Binding of the stress protein B-crystallin to cardiac myofibrils correlates with the degree of myocardial damage during ischemia/reperfusion in vivo. J Mol Cell Cardiol. 1999; 31: 569–580.[CrossRef][Medline] [Order article via Infotrieve]

33. Golenhofen N, Arbeiter A, Koob R, Drenckhahn D. Ischemia-induced association of the stress protein B-crystallin with I-band portion of cardiac titin. J Mol Cell Cardiol. 2002; 34: 309–319.[CrossRef][Medline] [Order article via Infotrieve]

34. Huot J, Houle F, Marceau F, Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res. 1997; 80: 383–392.[Abstract/Free Full Text]

35. Bova MP, Yaron O, Huang Q, Ding L, Haley DA, Steward PL, Horwitz J. Mutation R120G in B-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function. Proc Natl Acad Sci U S A. 1999; 96: 6137–6142.[Abstract/Free Full Text]

36. Derham BK, van Boekel MA, Muchowski PJ, Clark JI, Horwitz J, Hepburne-Scott HW, de Jong WW, Crabbe MJ, Harding JJ. Chaperone function of mutant versions of A- and ßB-crystallin prepared to pinpoint chaperone binding sites. Eur J Biochem. 2001; 268: 713–721.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				A. C. Grey and K. L. Schey Age-Related Changes in the Spatial Distribution of Human Lens {alpha}-Crystallin Products by MALDI Imaging Mass Spectrometry Invest. Ophthalmol. Vis. Sci., September 1, 2009; 50(9): 4319 - 4329. [Abstract] [Full Text] [PDF]

				R. Whittaker, M. S. Glassy, N. Gude, M. A. Sussman, R. A. Gottlieb, and C. C. Glembotski Kinetics of the translocation and phosphorylation of {alpha}B-crystallin in mouse heart mitochondria during ex vivo ischemia Am J Physiol Heart Circ Physiol, May 1, 2009; 296(5): H1633 - H1642. [Abstract] [Full Text] [PDF]

				J. Radhakrishnan, I. M. Ayoub, and R. J. Gazmuri Activation of caspase-3 may not contribute to postresuscitation myocardial dysfunction Am J Physiol Heart Circ Physiol, April 1, 2009; 296(4): H1164 - H1174. [Abstract] [Full Text] [PDF]

				E. Chung and L. A. Leinwand Rescuing Cardiac Malfunction: The Roles of the Chaperone-Like Small Heat Shock Proteins Circ. Res., December 5, 2008; 103(12): 1351 - 1353. [Full Text] [PDF]

				K. A. Huey, R. R. Roy, H. Zhong, and C. Lullo Time-dependent changes in caspase-3 activity and heat shock protein 25 after spinal cord transection in adult rats Exp Physiol, March 1, 2008; 93(3): 415 - 425. [Abstract] [Full Text] [PDF]

				A. Dimberg, S. Rylova, L. C. Dieterich, A.-K. Olsson, P. Schiller, C. Wikner, S. Bohman, J. Botling, A. Lukinius, E. F. Wawrousek, et al. {alpha}B-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis Blood, February 15, 2008; 111(4): 2015 - 2023. [Abstract] [Full Text] [PDF]

				J.-K. Jin, R. Whittaker, M. S. Glassy, S. B. Barlow, R. A. Gottlieb, and C. C. Glembotski Localization of phosphorylated {alpha}B-crystallin to heart mitochondria during ischemia-reperfusion Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H337 - H344. [Abstract] [Full Text] [PDF]

				R. T. Clements, N. R. Sodha, J. Feng, S. Mieno, M. Boodhwani, B. Ramlawi, C. Bianchi, and F. W. Sellke Phosphorylation and translocation of heat shock protein 27 and alphaB-crystallin in human myocardium after cardioplegia and cardiopulmonary bypass. J. Thorac. Cardiovasc. Surg., December 1, 2007; 134(6): 1461 - 1470.e3. [Abstract] [Full Text] [PDF]

				I. J. Benjamin, Y. Guo, S. Srinivasan, S. Boudina, R. P. Taylor, N. S. Rajasekaran, R. Gottlieb, E. F. Wawrousek, E. D. Abel, and R. Bolli CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3201 - H3209. [Abstract] [Full Text] [PDF]

				T. Yamaguchi, H. Arai, N. Katayama, T. Ishikawa, K. Kikumoto, and Y. Atomi Age-Related Increase of Insoluble, Phosphorylated Small Heat Shock Proteins in Human Skeletal Muscle J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2007; 62(5): 481 - 489. [Abstract] [Full Text] [PDF]

				J. A. Wall, J. Wei, M. Ly, P. Belmont, J. J. Martindale, D. Tran, J. Sun, W. J. Chen, W. Yu, P. Oeller, et al. Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6 Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2462 - H2472. [Abstract] [Full Text] [PDF]

				D. J. Thuerauf, M. Marcinko, N. Gude, M. Rubio, M. A. Sussman, and C. C. Glembotski Activation of the Unfolded Protein Response in Infarcted Mouse Heart and Hypoxic Cultured Cardiac Myocytes Circ. Res., August 4, 2006; 99(3): 275 - 282. [Abstract] [Full Text] [PDF]

				J. den Engelsman, D. Gerrits, W. W. de Jong, J. Robbins, K. Kato, and W. C. Boelens Nuclear Import of {alpha}B-crystallin Is Phosphorylation-dependent and Hampered by Hyperphosphorylation of the Myopathy-related Mutant R120G J. Biol. Chem., November 4, 2005; 280(44): 37139 - 37148. [Abstract] [Full Text] [PDF]

				J. P. DeLany, Z. E. Floyd, S. Zvonic, A. Smith, A. Gravois, E. Reiners, X. Wu, G. Kilroy, M. Lefevre, and J. M. Gimble Proteomic Analysis of Primary Cultures of Human Adipose-derived Stem Cells: Modulation by Adipogenesis Mol. Cell. Proteomics, June 1, 2005; 4(6): 731 - 740. [Abstract] [Full Text] [PDF]

				R. Shashidharamurthy, H. A. Koteiche, J. Dong, and H. S. Mchaourab Mechanism of Chaperone Function in Small Heat Shock Proteins: DISSOCIATION OF THE HSP27 OLIGOMER IS REQUIRED FOR RECOGNITION AND BINDING OF DESTABILIZED T4 LYSOZYME J. Biol. Chem., February 18, 2005; 280(7): 5281 - 5289. [Abstract] [Full Text] [PDF]

				J. J. Martindale, J. A. Wall, D. M. Martinez-Longoria, P. Aryal, H. A. Rockman, Y. Guo, R. Bolli, and C. C. Glembotski Overexpression of Mitogen-activated Protein Kinase Kinase 6 in the Heart Improves Functional Recovery from Ischemia in Vitro and Protects against Myocardial Infarction in Vivo J. Biol. Chem., January 7, 2005; 280(1): 669 - 676. [Abstract] [Full Text] [PDF]

				K. Dahlin, E. M. Mager, L. Allen, Z. Tigue, L. Goodglick, M. Wadehra, and L. Dobbs Identification of Genes Differentially Expressed in Rat Alveolar Type I Cells Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 309 - 316. [Abstract] [Full Text] [PDF]

				Y. Chen, A.-P. Arrigo, and R. W. Currie Heat shock treatment suppresses angiotensin II-induced activation of NF-{kappa}B pathway and heart inflammation: a role for IKK depletion by heat shock? Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1104 - H1114. [Abstract] [Full Text] [PDF]

				L. E. Morrison, R. J. Whittaker, R. E. Klepper, E. F. Wawrousek, and C. C. Glembotski Roles for {alpha}B-crystallin and HSPB2 in protecting the myocardium from ischemia-reperfusion-induced damage in a KO mouse model Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H847 - H855. [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]

				X. Wang, R. Klevitsky, W. Huang, J. Glasford, F. Li, and J. Robbins {alpha}B-Crystallin Modulates Protein Aggregation of Abnormal Desmin Circ. Res., November 14, 2003; 93(10): 998 - 1005. [Abstract] [Full Text] [PDF]

				N. W. O'Brien, N. M. Gellings, M. Guo, S. B. Barlow, C. C. Glembotski, and R. A. Sabbadini Factor Associated With Neutral Sphingomyelinase Activation and Its Role in Cardiac Cell Death Circ. Res., April 4, 2003; 92(6): 589 - 591. [Abstract] [Full Text] [PDF]

				K. A. Webster Serine Phosphorylation and Suppression of Apoptosis by the Small Heat Shock Protein {alpha}B-Crystallin Circ. Res., February 7, 2003; 92(2): 130 - 132. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 92/2/203    most recent 01.RES.0000052989.83995.A5v1 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 Morrison, L. E. Articles by Glembotski, C. C. Search for Related Content PubMed PubMed Citation Articles by Morrison, L. E. Articles by Glembotski, C. C. Related Collections Apoptosis Cell signalling/signal transduction