Published online before print April 22, 2004, doi: 10.1161/01.RES.0000129179.66631.00
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© 2004 American Heart Association, Inc.
Cellular Biology |
Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits ß-Agonist-Induced Cardiac Apoptosis
From the Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio.
Correspondence to Dr Evangelia G. Kranias, Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0575. E-mail Litsa.Kranias{at}uc.edu
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Abstract |
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Activation of the sympathetic nervous system is a common compensatory feature in heart failure, but sustained ß-adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction. In mouse cardiomyocytes, we recently reported that prolonged exposure to ß-agonists is associated with transient increases in expression and phosphorylation of a small heat-shock protein, Hsp20. To determine the functional significance of Hsp20, we overexpressed this protein and its constitutively phosphorylated (S16D) or nonphosphorylated (S16A) mutant in adult rat cardiomyocytes. Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-PKA pathway, as indicated by decreases in the number of pyknotic nuclei, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, and DNA laddering, which were associated with inhibition of caspase-3 activity. These protective effects were further increased by the constitutively phosphorylated Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties. Immunostaining studies and immunoprecipitations with Hsp20 or actin antibodies demonstrated that Hsp20 translocated to cytoskeleton and associated with actin on isoproterenol stimulation. These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against ß-agonist-induced apoptosis. Thus, Hsp20 may represent a novel therapeutic target in the treatment of heart failure.
Key Words: apoptosis • ß-agonist • small heat-shock protein • cardiomyocyte • cytoskeleton
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Introduction |
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Activation of the sympathetic nervous system is a common feature in heart failure patients.1 However, the initial benefits of increased circulating catecholamines become maladaptive over the long-term, and chronic stimulation of the ß-adrenergic neurohormonal axis contributes to the progression of heart failure and mortality in animal models and human patients.2,3 One of the underlying mechanisms in the detrimental effects of prolonged ß-agonist stimulation involves cardiomyocyte apoptosis or cell death.2–4 Recent studies have identified a variety of signaling players in the ß-adrenergic pathway-induced cardiomyocyte apoptosis.4–6 However, few studies have investigated the potential players that confer cardiomyocyte protection from apoptosis triggered by this pathway.
Several small heat-shock proteins (Hsp), such as 
B-crystallins and Hsp25, have been shown to enhance the survival of cells subjected to different forms of stress.7,8 In mammals, at least 10 different small heat-shock proteins have been described.9 These are Hsp25 (Hsp27/28, HspB1), myotonic dystrophy protein kinase-binding protein (MKBP, HspB2), HspB3, A-crystallin (HspB4), B-crystallin (HspB5), Hsp20 (P20, HspB6), cardiovascular heat-shock protein (cvHsp, Hsp7), Hsp22 (HspB8), Hsp9, and Hsp10. Hsp20 has initially been isolated from skeletal muscle in mixed complexes with B-crystallin and Hsp25, which implies that all 3 proteins might be involved in similar processes, at least in skeletal muscle, and probably also in heart.10 It has also been reported that Hsp20 is expressed in cardiac muscle, stomach, intestine, bladder, and blood,10,11 and it is the only small heat-shock protein that contains a cGMP/cAMP-dependent protein kinase (PKG/PKA) consensus phosphorylation site (RRAS).10 Stable overexpression of Hsp20 in Chinese hamster ovary (CHO) cells resulted in enhanced survival, after a heat shock, similar to B-crystallin.12 Furthermore, heat pretreatment of swine carotid artery was associated with increased Hsp20 levels and its phosphorylation at Ser16.13 The significance of upregulation of Hsp20 is unclear, but it could represent a cytoprotective response to stress. Actually, Hsp20 has been suggested to be an actin-binding protein, involved in cyclic nucleotide-mediated vasodilation and relaxation of rat smooth muscle, or in histamine-induced and phorbol ester-induced contraction of bovine carotid artery smooth muscle.14–16 In cardiomyocytes, treatment with a 13mer phospho-peptide analog of Hsp20 resulted in increased rates of myocyte contraction and relaxation, indicating that phosphorylated Hsp20 may have a regulatory role in cardiomyocyte physiology.17
Importantly, we recently found that prolonged (30 minutes) ß-agonist stimulation of cardiomyocytes induced expression and phosphorylation of Hsp20,18 suggesting that Hsp20 may play an important role in ß-adrenergic mediated cardiac responses. In the present study, we used recombinant adenovirus mediated ex vivo gene transfer to investigate the functional role of Hsp20 and its phosphorylation in cardiac survival/apoptosis after stimulation by the cAMP-PKA pathway, as well as its possible mechanisms. Our findings demonstrate that Hsp20 translocates to and interacts with actin cytoskeleton in response to isoproterenol stimulation and prevents ß-agonist-induced apoptosis in adult rat cardiomyocytes.
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Methods |
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Recombinant Adenoviral Constructs
Ad.Hsp20, Ad.S16A, and Ad.S16D encode the wild-type Hsp20, the nonphosphorylatable form of Hsp20, and the constitutively phosphorylated form of Hsp20, respectively (Figure 1). Ad.GFP was used as a control.
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Myocyte Isolation and Culture
Adult rat left ventricular myocytes were isolated from hearts of male Sprague-Dawley rats (
300 grams; Harlan Laboratory, Indianapolis, Ind), as previously described.19 Cardiomyocytes were plated on laminin-coated glass coverslips or dishes. After 1 to 2 hours, the dishes were washed and then infected with adenovirus at a multiplicity of infection of 500. The adenoviral-infected cells were maintained in medium for 16 hours before the addition of isoproterenol (ISO) (10 µmol/L, Sigma) or forskolin (FSK) (10 µmol/L, Sigma). All dishes were supplemented with ascorbic acid (0.1 mmol/L, Sigma) to prevent ISO oxidation. In some experiments, N-[2-(bromocinnamylamino) ethyl]-5-isoquinolinesulfonic acid (H-89, 20 µmol/L, Sigma) was added for 30 minutes before the addition of ISO.
Analysis of Apoptosis and Caspase-3 Activity
Cardiomyocytes were examined for the occurrence of apoptosis at 24 hour posttreatment of ISO by Hoechst staining, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay, and DNA laddering, as previously described.4,5 The activity of caspase-3 was determined with CaspACE Assay System (Promega), according to the manufacturer’s instructions.
Cell Lysis and Immunoblotting
Cultured cardiomyocytes were harvested and lysed for 20 minutes at 4°C in lysis buffer consisting of (mmol/L) 20 Tris-HCl (pH 7.5), 150 NaCl, 1% NP-40, 10% glycerol, 0.4 sodium orthovanadate, 10 sodium pyrophosphate, 10 sodium fluoride, 0.5 DTT, and 2 µL/mL protease inhibitor cocktail (Sigma). The supernatants obtained after centrifugation at 8000g for 10 minutes were then used for SDS-PAGE. For cell fractionation with detergent, cultured adult rat cardiomyocytes were washed once with medium and twice with ice-cold phosphate-buffered saline (PBS). Pelleted cells were resuspended in ice-cold lysis buffer containing (mmol/L) 10 Tris pH 7.5, 10 NaCl, 5 MgCl2, 1 phenylmethanesulfonyl fluoride, and 0.5% Triton X-100. This was centrifuged (5 minutes at 2000 rpm, 4°C) and the supernatant was used as the detergent-soluble fraction. The pellet was washed twice with the same buffer and then used as the detergent-insoluble fraction. Fractions were dissolved in SDS sample buffer (50 mmol/L Tris pH6.8, 2%SDS, 100 mmol/L DTT, and 10% glycerol) and proteins were separated by SDS-PAGE, followed by Western blot analysis, as previously described.5,17
Immunoprecipitation
Cultured cardiomyocytes from control, ISO-treated (1 hour), or H-89 pretreated (0.5 hour), followed by ISO-stimulation for 1 hour, were harvested and homogenized in PBS with 0.5% Tween-20. Antibodies specific for either actin or Hsp20 were linked to protein-A-Sepharose fast-flow immunoprecipitation beads. After preclearing of the homogenate, using beads alone, the homogenates were incubated overnight at 4°C with gentle agitation. Afterward, the beads were washed 3 times with PBS-Tween20. Proteins were eluted from the beads by incubating with SDS-sample buffer at 55°C for 30 minutes.
Immunofluorescence Staining
The cells growing on coverslips were fixed with methanol at –20°C for 10 minutes, dipped in cold acetone, and air-dried. Nonspecific reactions were blocked by a 30-minute preincubation in normal goat serum. Anti-actin (1/200, Sigma) and anti-Hsp20 (1/1600, RDI) were used for staining, whereas Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes) at a dilution of 1/600 in blocking buffer were used as secondary antibodies. The cells were imaged on a Zeiss Axiophot microscope interfaced with a SPOT camera (Diagnostic Instruments).
2-Dimensional Gel Electrophoresis and Liquid Chromatography-Tandem Mass Spectrometry
For 2-dimensional gel analysis, cardiomyocyte proteins were extracted using a solubilization solution of 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 20 mmol/L DTT, 20 mmol/L spermine, and 1 mmol/L PMSF. The extract was centrifuged, and the supernatant was processed for 2-dimensional gel electrophoresis. First dimensional separation was performed using the IPGphor isoelectric focusing system (Amersham Pharmacia Biotech) and the second dimension using SDS-PAGE. Protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for phosphorylation mapping analysis, as previously described.18
Statistical Analysis
All data are expressed as mean±SEM. Comparisons between control and treated cells were performed with a Student unpaired t test. Statistical significance of multiple treatments was determined by ANOVA and a post hoc Tukey test. P<0.05 was considered to be significant.
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Results |
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ISO Mediates Hsp20 Expression in Cardiomyocytes
We previously reported that ISO stimulation for 30 minutes in mouse cardiomyocytes was associated with a distinct phosphoprotein signal spot, with molecular mass of 17.4 kDa and pI of 5.5.18 This phosphoprotein was consequently identified and cloned. Sequence analysis indicated that this protein is a cardiac homologue of Hsp20, because it has a significant similarity (95%) with the rat skeletal Hsp20.18 Thus, an antibody to the cardiac Hsp20 was generated and used to examine the time course of Hsp20 expression in cultured adult rat cardiomyocytes in response to ISO. Quantitative immunoblotting indicated that the levels of Hsp20 significantly increased by 30 minutes, returned to basal levels by 2 hours, and further decreased below baseline by 12 hours after treatment (Figure 2A). There were no changes in levels of small heat-shock proteins, Hsp27, or
B-crystallin expression after ISO exposure of the rat cardiomyocytes (Figure 2B and 2C). These results indicate that Hsp20 appears to be transiently and distinctively upregulated by treatment of cardiomyocytes with ISO, in contrast to Hsp27 and B-crystallin.
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Gene Transfer of Cardiac Hsp20 Inhibits Apoptosis Triggered by the cAMP-PKA Pathway
To examine whether Hsp20 is involved in ISO-induced apoptosis, we infected cardiomyocytes with a recombinant adenovirus encoding mouse cardiac Hsp20 (Ad.Hsp20). We observed 100% infection efficiency with either Ad.GFP or Ad.Hsp20 (Figure 3A). Importantly, there were no apparent morphological alterations or differences in the number of adherent cells and rod-shaped cells between the Ad.Hsp20-infected and empty vector Ad.GFP-infected groups (Figure 3A), although infection with Ad.Hsp20 resulted in 2-fold increases in Hsp20 protein levels in the cardiomyocytes (Figure 3B), similar to the observed increases in ISO-treated cardiomyocytes at 0.5 to 1 hour (Figure 2A). Because several reports showed that norepinephrine stimulates apoptosis in adult rat myocytes by activation of the cAMP-PKA pathway,3,4 we investigated whether overexpressed Hsp20 had any effects on ISO-induced apoptosis. For this purpose, adult rat cardiomyocytes were infected with either control Ad.GFP or Ad.Hsp20 for 16 hours and then subjected to ISO treatment for 24 hours, as previously described for ISO-induced apoptosis.4 ISO treatment of uninfected and Ad.GFP-infected cells caused the appearance of round and condensed nuclei (Figure 3C), which are hallmarks of cell apoptosis. A quantitative analysis revealed that 13%±1% of cells displayed pyknotic nuclei under basal conditions (Figure 3D), consistent with previous reports.4,5 However, on ISO treatment, the number of pyknotic nuclei increased by 2.7-fold (Figure 3d). Infection with Ad.GFP did not significantly alter the number of ISO-induced condensed nuclei (Figure 3D). In contrast, infection with Ad.Hsp20 significantly reduced the occurrence of ISO-induced apoptosis (by 31%±2%) (Figure 3D), compared with Ad.GFP infection.
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Consistent with the effects of the ß-adrenergic agonist ISO, exposure of cardiomyocytes to the adenylyl cyclase activator FSK increased the number of apoptotic cells by 2.6-fold, compared with control (Figure 3E). However, Ad.Hsp20-infected myocytes exhibited a significant decrease in the fraction of apoptotic cells, compared with Ad.GFP-infected myocytes. Pretreatment of myocytes with H-89, a widely used inhibitor of PKA, completely blocked the effect of ISO, and there were no significant differences between Ad.GFP-infected and Ad.Hsp20-infected groups (Figure 3F). These results demonstrate that overexpression of Hsp20 protects adult rat cardiomyocytes from apoptosis triggered by the cAMP-PKA pathway.
Overexpressed Hsp20 Protein Is Partly Phosphorylated at Ser16 in Adult Rat Cardiomyocytes on ISO Stimulation
It has been previously suggested that Hsp20 may be phosphorylated by PKA/PKG on Serine 16.11,16,20 Thus, the observed protective effects of Hsp20 on myocyte apoptosis could be mediated by either Hsp20 or its phosphorylated form. To determine the phosphorylation status of Hsp20 in Ad.Hsp20-infected myocytes before and after ISO treatment for 24 hours, we used 2-dimensional gel electrophoresis and LC-MS/MS analysis. There were no visible Hsp20 protein spots in the control or in Ad.GFP-infected myocytes (Figure 4A, 4B, 4F, and 4G) without or with ISO treatment. However, infection with Ad.Hsp20 resulted in the appearance of 1 Hsp20 protein spot (Figure 4C). After addition of ISO, there were 2 Hsp20 protein spots migrating with a similar molecular weight of 17.4 kDa but different pI of 5.7 and 5.5 (Figure 4H, X and Y). These 2 protein spots were excised and subjected to LC-MS/MS for phosphorylation mapping analysis. MS/MS spectrum results indicated that Ser16 in spot Y was phosphorylated (data not shown), whereas there was no typical P-peptide MS/MS spectrum that corresponded to spot X, consistent with our previous report.18 These results indicated that the overexpressed Hsp20 protein was partially phosphorylated at Ser16 in adult rat cardiomyocytes on ISO stimulation, and these findings were further confirmed by analysis of Ad.S16A-infected and Ad.S16D-infected myocytes. Ad.S16A and Ad.S16D encode the 2 Hsp20 mutants, which either block or mimic phosphorylation via mutation of Ser16 to Ala (S16A) or to Asp (S16D), respectively. The 2-dimensional gels indicated the presence of a single Hsp20 protein spot in the Ad.S16A-infected (Figure 4D and 4I) or Ad.S16D-infected (Figure 4E and 4J) myocytes, corresponding to the nonphosphorylated or phosphorylated forms, respectively, regardless of ISO treatment.
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Phosphorylation of Ser16 in Hsp20 Confers Enhanced Protection Against Apoptosis Activated by the cAMP-PKA Pathway
To define the functional significance of the nonphosphorylated and phosphorylated Hsp20 forms in cardioprotection from apoptosis activated by the cAMP-PKA pathway, we examined the effects of constitutively phosphorylated Hsp20 (S16D) or nonphosphorylatable Hsp20 (S16A), respectively, on ISO-induced apoptosis in adult rat cardiomyocytes. Western blot analysis confirmed similar expression levels (2.1-fold over Ad.GFP) of Hsp20 and its mutants in adult rat myocytes after 16 hours of infection with the recombinant adenoviruses (Figure 5A). ISO treatment increased nuclear fragmentation by 2.8-fold and Ad.GFP had no effect on the ISO-induced apoptosis (Figure 5B). However, myocytes infected with Ad.Hsp20 displayed a significant decrease in the number of apoptotic cells on ISO exposure (Figure 5B). This protective effect was further enhanced by Ad.S16D infection, which decreased the number of apoptotic cells by 42%±3%, compared with Ad.GFP infection. Importantly, the difference between Ad.S16D-infected and Ad.Hsp20-infected groups was significant (P<0.05). In contrast, Ad.S16A-infected cells exhibited no significant alteration in ISO-induced apoptosis, compared with the control (Ad.GFP). Treatment with FSK resulted in apoptosis similar to ISO (Figure 5C), and pretreatment of myocytes with H-89 indicated no difference in the number of apoptotic cells among the various groups (Figure 5D). These results suggest that phosphorylation of Ser16 enhances the protective effect of Hsp20 against cAMP-PKA activated apoptosis in the cardiomyocytes.
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TUNEL staining analysis also indicated that Ad.S16D-infected myocytes were less susceptible to ISO-induced cell death than the other groups (Figure 5E and 5F). The number of TUNEL-positive cells after ISO treatment was reduced by 28%±1% in the Ad.Hsp20-infected myocytes, compared with Ad.GFP-infected cells. This reduction was even greater (50%±1%) by Ad.S16D-infection, which appeared to confer full protection compared with cells without ISO treatment (P>0.05) (Figure 5F).
The enhanced cytoprotective effect of phosphorylation of Hsp20 at Ser16 was further analyzed using a DNA laddering assay (Figure 5G and 5H). Compared with cells infected with Ad.Hsp20, those infected with Ad.S16D displayed less DNA fragmentation on treatment with ISO, whereas those infected with Ad.S16A exhibited similar DNA fragmentation as observed in Ad.GFP-infected cells (Figure 5H).
Antiapoptosis of Hsp20 Is Mediated Through Decreases in the Activity of Caspase-3
Recent evidence indicates that many heat shock proteins are antiapoptotic and directly inhibit caspase activation.21 Thus, we investigated whether Hsp20 might confer protection against apoptosis by assessing the effects of our recombinant adenoviruses on caspase-3 activation (Figure 6). Overexpression of wild-type Hsp20 resulted in 10% inhibition of caspase-3 activation, and overexpression of Ser16-phosphorylated Hsp20 (S16D) resulted in a 25% reduction in caspase-3 activation, compared with cells infected with Ad.GFP. However, overexpression of nonphosphorylatable Hsp20 (S16A) had no effect and was associated with similar caspase-3 activation as the Ad.GFP control. These results suggest that the antiapoptotic effects of cardiac Hsp20 may involve inhibition of the processing and activation of caspase-3.
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Hsp20 Translocates to Actin Cytoskeleton on Isoproterenol Stimulation
To further elucidate the mechanism underlying the Hsp20 cytoprotective effects, we investigated a possible change in subcellular distribution of Hsp20 after treatment with ISO. In untreated cardiomyocytes, most of Hsp20 was found in the Triton X-100-soluble cellular fraction, although it was scarce in the insoluble fraction (Figure 7A). After 30 minutes or 1 hour of ISO treatment, Hsp20 was increased in the Triton X-100-insoluble fraction, suggesting its possible redistribution from cytosol to the cytoskeleton/nuclear compartment. This change was blocked by H-89 inhibition of PKA (Figure 7A). To further identify the cardiomyocyte compartment to which Hsp20 translocates, immunofluorescence staining studies were performed. Under nonstimulated conditions, Hsp20 was observed throughout the cytoplasm (Figure 7B), suggesting that cardiac Hsp20 is primarily a cytosolic protein, in agreement with Figure 7A. After ISO treatment, Hsp20 translocated to transverse bands, and no nuclear migration was observed (Figure 7E). Double staining with an antiactin antibody (Figure 7C and 7F) revealed colocalization of Hsp20 with actin (Figure 7G). Occasionally, in a small subset of the cultured adult rat cardiomyocytes, some weak cytoskeletal staining was visible under control conditions (Figure 7D). Therefore, we do not preclude the possibility that Hsp20 is partially colocalized with actin in the cytoplasm of cultured cardiomyocytes under basal conditions. However, ISO treatment induced Hsp20 redistribution to the cytoskeleton, and this was blocked by pretreated with H-89 (data not shown). These data suggest that the phosphorylation of Hsp20 by PKA correlated with its redistribution from the cytosol to the cytoskeleton in cultured adult rat cardiomyocytes.
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Furthermore, immunoprecipitations with an actin antibody indicated an association between Hsp20 and actin under basal conditions (Figure 7H). However, after ISO treatment, a significant increase in Hsp20-actin association was observed, which was prevented by H-89 pretreatment. A reciprocal immunoprecipitation study, using the Hsp20 antibody, also indicated increased actin-Hsp20 association on ISO treatment (Figure 7H). These findings suggest that Hsp20 phosphorylation enhances its interaction with actin and may stabilize the microfilaments, leading to protection against ß-agonist-induced cardiac apoptosis.
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Discussion |
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Apoptosis has been observed in various animal models of heart failure including ventricular pacing,22 pressure overload,23 and aged spontaneously hypertensive rats.24 In failing human heart,
80 to 250 cardiomyocytes per 105 cardiac nuclei commit apoptosis.25,26 In contrast, the baseline rate of apoptosis in healthy human hearts is only 1 to 10 myocytes per 105 cardiac nuclei. Recent studies using transgenic mice, which express a conditionally active caspase, demonstrated that very low levels of myocyte apoptosis, levels that are 4- to 10-fold lower than those observed in human heart failure, are sufficient to cause lethal, dilated cardiomyopathy.27 Thus, preventing the loss of the terminally differentiated cardiomyocytes becomes critical for the maintenance of normal cardiac function. However, both in vivo and in vitro studies have shown that enhanced ß-AR signaling promotes cardiac apoptosis,4,28–30 outweighing its short-term beneficial effects, which indicates that attenuation of the detrimental consequences of ß-AR signaling over the long-term is of crucial importance in heart failure.
Along these lines, small heat-shock proteins have been suggested to play a protective role in cardiomyocytes. In the present study, we showed that the upregulation of Hsp20 by ISO was transient and relatively specific in cultured adult rat cardiomyocytes. There were no alterations in the other small heat shock proteins Hsp27 and B-crystallin, consistent with previous reports in ISO-treated C6 rat glioma cells.31 Interestingly, these small heat-shock proteins, although they have considerable sequence homology with Hsp20, do not contain the RRAS consensus sequence for cAMP-PKA phosphorylation. Actually, Hsp20 is the only small heat shock protein that contains this sequence domain.10 In addition, we noted that Hsp20 was downregulated by ISO over the long-term. These findings suggest that Hsp20 is a putative candidate for the ß-adrenergic mediated effects in cardiomyocytes.
Overexpression of Hsp20 in adult rat cardiomyocytes inhibited apoptosis, activated by the cAMP-PKA pathway. Furthermore, inclusion of H-89, before ISO-treatment, conferred similar protective effects. Although these findings may be challenged by the reported ß-AR blockade property of H-89,32 further studies using a PKA-pseudo-phosphorylated Hsp20 (S16D) mutant confirmed the protective role of Hsp20 in ISO-induced apoptosis. There are multiple mechanisms that may explain the cardioprotection of Hsp20 (Figure 8). The reduced caspase-3 activation, associated with Hsp20 or Ser16 phosphorylated Hsp20 (S16D), suggests that these antiapoptotic effects may involve inhibition of the conversion of procaspase-3 (p24) to active caspase-3. In addition to caspase-3, it is most likely that cardiac Hsp20 enhances cytoprotection by interacting with the actin cytoskeleton. In skeletal tissue, Hsp20 has been identified as an actin-binding protein and the association with actin is dependent on the phosphorylation state of Hsp20 at Ser16.14 Recent biochemical studies in rat heart tissue also showed that Hsp20 is associated with B-crystallin and localizes to distinct transverse bands in a similar pattern as B-crystallin and sarcomeric actin.17 However, confocal imaging of Hsp20 in smooth muscle tissues revealed that Hsp20 was present throughout the cytoplasm.33 Interestingly, our data demonstrated that Hsp20 in cultured adult rat cardiomyocytes undergoes dynamic intracellular redistribution from its cytoplasmic localization into the meshwork of filaments in response to ISO stimulation. A similar phenomenon with Hsp20 translocation to stress fibers has been observed in the rat cardiac myoblast cell line H9C2 after proteasomal inhibition.34 However, under heat stress, Hsp20 has been reported to migrate partially into the nucleus, with very few cells displaying a faint sarcomeric association of Hsp20.35 These results suggest that Hsp20 redistributes from the soluble to the insoluble nuclear/cytoskeletal fraction in response to the stress state of the cell. Therefore, it is possible that the relocalization of Hsp20 may be protective for the collapsed intermediate filament network or cytoskeletal protein damage, induced by stress stimulation. Indeed, Hsp20 colocalizes with actin after ISO stimulation, and this redistribution is blocked by PKA inhibition. Moreover, the Hsp20 association with actin appears to be dependent on its cAMP-phosphorylation state, consistent with previous reports in bovine carotid artery smooth muscle14,36 and in vitro studies using recombinant Hsp20.14 Taken together, these findings may explain the higher degree of cytoprotection by the constitutively phosphorylated (S16D) than wild-type Hsp20. Importantly, infection with the nonphosphorylatable Hsp20 (S16A) reduced protection from apoptosis, compared with the wild-type form, possibly caused by its reduced actin-stabilization activity, or reduced binding to downstream targets, such as p24 and procaspase-3.
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In summary, the present study demonstrates that phosphorylation of cardiac Hsp20 at Ser16 is associated with reduced cardiac apoptosis induced by ß-agonist stimulation. Thus, cardiac Hsp20 phosphorylation may confer cardioprotection against apoptosis, suggesting that increased expression of Ser16-phosphorylated Hsp20 in vivo may be a powerful therapeutic approach for improving the function of failing cardiomyocytes. However, future studies involving the generation of genetic models with altered cardiac expression of Hsp20 will be desired to better-understand the potent cytoprotective and physiological roles of Hsp20 in vivo.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-26057, HL-64018, and HL-52318 (E.G.K.). We thank Brent Baldwin at the Proteomics Core of University of Cincinnati Medical Center for performing 2-dimensional gel electrophoresis for this study.
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Footnotes |
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Original received November 19, 2003; resubmission received February 27, 2004; revised resubmission received April 6, 2004; accepted April 12, 2004.
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References |
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