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B-crystallin and HSPB2 in protecting the myocardium from ischemia-reperfusion-induced damage in a KO mouse model
1San Diego State University Heart Institute and The Department of Biology, San Diego State University, San Diego, California 92182; and 2National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730
Submitted 24 July 2003 ; accepted in final form 21 October 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B-crystallin (BC) and HSPB2, two sHSPs that are expressed in high levels in the heart, are in close proximity to one another; as a result, both genes were disrupted in a recently generated knockout (KO) mouse line. The BC/HSPB2 KO mouse line is currently the only model that features disruption of sHSPs normally expressed in the heart. Accordingly, we examined the cardiac morphology, function, and response to I/R-induced stress in BC-HSPB2 KO mice. Initial gross, light microscopic and echocardiographic characterization showed that the morphological and functional properties of hearts from adult KO mice were indistinguishable from age-matched wild-type (WT) mice. Electron microscopy showed that, compared with WT mouse hearts, KO mouse heart sarcomeres were relatively normal. Isolated perfused KO mouse hearts displayed normal contractility; however, when compared with WT, after I/R, KO mouse hearts exhibited a twofold reduction in contractile recovery, as well as increased necrosis and apoptosis. Additionally, when compared with WT, KO mouse hearts exhibited 43% less reduced glutathione, which is known to protect from I/R-induced damage. Thus, whereas neither BC nor HSPB2 is essential for myocardial development and function under nonstressful conditions, one or both are required for maximal functional recovery and protection from I/R-induced necrosis and apoptosis.
small heat shock protein; necrosis; apoptosis; glutathione; knockout mouse
The mammalian sHSP family includes B-crystallin (BC), HSP27,1 HSPB2, HSPB3, HSP20, HSP22, cvHSP, and A-crystallin, all of which are expressed in the heart, except A-crystallin, which is expressed primarily in the lens (7, 29, 45, 46). Among the sHSPs expressed in the heart, BC is present in the highest quantities, where it comprises up to 3% of total myocardial protein (26, 31, 32). Accordingly, numerous studies have focused on possible roles for BC in the cardiac stress response. Like other sHSPs, BC is a chaperone and it interacts with various proteins that can foster cell survival, such as cytoskeletal components (6, 13, 50, 52). For example, in cardiac myocytes BC is localized to the Z disks of sarcomeres, and the amount of Z disk-associated BC increases during ischemia, as does the level of BC phosphorylation (16–18). In addition to the localization of BC to Z disks, BC phosphorylation is also believed to provide protection from stress-induced myocyte death (35).
The misexpression or mislocalization of BC is associated with several human diseases, such as the autosomal-dominant desmin-related cardiomyopathy (36, 39, 49). Furthermore, overexpression of BC in cultured cardiomyocytes and in transgenic mouse hearts reduces I/R-induced cell death (8, 35, 41). These findings indicate that mutated BC can be deleterious, whereas overexpression of native BC enhances protection. Also several other studies have shown expression of BC in the heart to differ as a function of age, implying roles for it in myogenic differentiation and cardiac development (5, 20, 31).
To demonstrate whether sHSPs are required for proper cardiac development and protection from stress, mouse models featuring the targeted disruption of sHSP genes are necessary. Currently, only two sHSP knock-out (KO) mouse lines are available, one features disruption of the A-crystallin gene (9) and the other features BC-gene disruption (10). Because A-crystallin is not expressed in the heart (45), the BC KO is the only available mouse model relevant for studies of the effects of sHSP gene disruption on cardiac development and function. Subsequent to the generation of the BC KO mouse, the gene that encodes the sHSP and HSPB2 was found to reside just 863 bp upstream of the BC-gene oriented in the opposite direction (22, 47). Because of the targeting strategy employed and the close proximity of the BC and HSPB2 genes, both genes were disrupted in the BC KO (10). Even though it cannot be discerned whether phenotypic differences between BC/HSPB2 KO and WT mice are due to the absence of BC and/or the absence of HSPB2, the BC/HSPB2 KO mouse line is a valuable model with which to establish the necessities of these two sHSPs in cardiac development, structure, and function. Accordingly, we assessed the cardiac phenotype in BC/HSPB2 KO mice with the goal of evaluating whether the absence of these two sHSPs affects cardiac development, morphology, and/or function under nonstressed and I/R stress conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animal procedures were carried out in accordance with the Institutional Animal Care and Use Committee at San Diego State University, and they conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Approximately 250 adult mice were used in this study. The KO mice used in this study were originally generated by using a targeting vector that replaced exons one, two, and most of three, as well as the putative transcriptional regulatory sequences of the BC gene and portions of the linked HSPB2 gene (10). Electroporation of J1 embryonic stem cells (strain: 129S4/SvJae) selection and screening of targeted cells and blastocyst microinjection were performed as previously described (9). Founders were bred into a 129S6/SvEvTac background, accordingly, the KO mice used in this study, which were bred as previously reported, are in a mixed background (129S6/SvEvTac x 129S4/SvJae). Control WT mice used in this study were 129S6/SvEvTac, also as previously reported (10).
Histology
Hearts were rinsed free of blood, then fixed in 4% formalin (EMS, Ft. Washington, PA) and embedded in paraffin (University of Califiornia, San Diego, Core Facility, San Diego, CA). Five-micrometer sections were stained with either hematoxylin and eosin or Masson's trichrome (University of Califiornia, San Diego).
Echocardiography
Transthoracic two-dimensional guided M-mode echocardiography of ketamine-anesthetized mice was performed as described previously (44, 48) using a Sonos 5500, Agilent echocardiograph and an L15/6-MHz transducer. The parameters measured are shown in Table 2.
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Electron Microscopy
Hearts were excised and immediately placed into 0.15 M cacodylate buffer (Sigma, St. Louis, MO) containing 100 mM sucrose and then finely chopped into 
1-mm2 pieces. The pieces were incubated in 2% glutaraldehye-2% formaldehyde (EMS) in 0.15 M cacodylate buffer for 4 h, washed, and fixed again in 1% osmium (EMS) for 2 h. After being rinsed with dH2O, the tissue was dehydrated with acetone and infiltrated with epon (EMS) for polymerization. After sectioning was completed, grids were stained with uranyl acetate (Sigma) and lead citrate (Sigma) before being viewed on a Phillips IL410 transmission electron microscope.
Cardiac Gene Expression Analyses
RNA analyses. Frozen ventricles were pulvarized and sonicated in RNAzol B (Tel-Test; Friendswood, TX) as directed in the manufacturer's protocol. Two micrograms of RNA from each sample were blotted onto nitrocellulose by using a Bio-Rad DotBlot apparatus. After UV cross-linking, prehybridization was carried out in QuikHyb (Stratagene, Cedar Creek) for 1 h at 60°C. Oligonucleotide probes were labeled using T4 PNK (New England Biolabs) and [-32]ATP (Amersham; Arlington Heights, IL). Unincorporated label was removed via G-25 Microspin Columns (Amersham). Hybridization was carried out with >1 x 108 counts·min–1·µg of oligonucleotide probe–1 at 60°C for 1 h in QuikHyb, with 100 µl of sonicated salmon sperm DNA (10 mg/ml). The following oligo probes were used: ANF, 5'-AATGTGACCAAGCTGCGTGACACACCACAAGGGCTTAGGATCTTTTGCGATCTGCTCAAG-3'; BNP, 5'-CAGGTTGAGATATGTGTCACCTTGGAATTT-3'; 
MHC, 5'-GCTTTATTCTGCTTCCACCTAAAGGGCTGTTGCAAAGGCTCCAGGTCTGAGGGCTTC-3'; -skeletal actin, 5'-TGGAGCAAAACAGAATGGCTGGCTTTAATGCTTCAAGTTTTCCATTTCCTTTCCACAGGG-3'; and GAPDH, 5'-GGAACATGTAGACCATGAGTTGAGGTCATGAAG-3'.
Membranes were washed and exposed to a PhosphorImager screen. Dot intesities were quantified by ImageQuant and normalized to GAPDH levels.
Protein analyses. Frozen ventricles were pulvarized and sonicated in homogenization buffer [50 mM Tris (pH 7.5), 250 mM Nacl, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml p-nitrophenyl phosphate leupeptin, 2 mM EDTA, 3 mM EGTA, 0.1 mM Na orthovanadate, 1 mM, 10 µg/ml aprotinin, and 0.1% Triton X-100]. Aliquots (75 µg of protein) were subjected to 12% SDS-PAGE followed by Western blot analysis using atrial natriuretic factor (ANF) antiserum (Peninsula IHC 9103), B-type natriuretic factor (BNP) antiserum (Peninsula IHC 9085), and anti-GAPDH (RDITRK5G4–6C5). Visualization of immune complexes was carried out by enhanced chemiluminescence (NEN Life Science), and immunoreactive bands were quantified by ImageQuant and normalized to GAPDH levels.
Global I/R. Age-matched (12–14 wk of age) WT and KO mice (n = 12 of each) were treated with heparin (500 U/kg, Sigma) and anesthetized with pentobarbital sodium (150 mg/kg, Sigma) administered intraperitoneally. Hearts were excised and placed into ice-cold modified Krebs-Henseleit buffer (KHB, Sigma). Aortas were cannulated and hearts were perfused in Langendorff mode by gravity flow. Hearts were then transferred to an isolated perfused heart apparatus and perfused at a constant pressure of 80 mmHg. A water-filled balloon connected to a pressure transducer (Gould Stathem P23 ID) was inserted into the left ventricle through the left atrium to monitor left ventricular developed pressure (LVDP, mmHg); data, collected using Powerlab, were processed with AD Instruments Chart 4 v4.12. Hearts were submersed in warm KHB (37°C) throughout the perfusion and paced at 400 Hz using Powerlab Stimulus Isolator (AD Instruments) at 0.5 mA; hearts were not paced during ischemia. Hearts were equilibrated for 30 min before being subjected to 25 min of no-flow ischemia, which was followed by reperfusion for 1.5–4 h, depending on the experiment. Functional recovery was expressed as a percentage of preischemic LVDP.
DNA fragmentation. Genomic DNA was isolated from whole hearts that were flash frozen in liquid nitrogen. Hearts were homogenized by hand with a glass homogenizer in digestion buffer [100 mM NaCl, 10 mM Tris (pH 8.0), 25 mM EDTA, and 0.5% SDS]. RNaseA was added to a final concentration of 3 mg/ml and incubated at 37°C for 30 min followed by the addition of 0.4 mg/ml Proteinase K and incubated at 55°C for 30 min. A 1/20 volume of 5 M NaCl was added to the samples, which were then extracted twice with phenol, once with chloroform-isoamyl alcohol (24:1), and the DNA was precipitated with isopropanol. The samples were centrifuged for 10 min at 4°C, and sedimented DNA was washed with 70% ethanol, air-dried, and dissolved in water. Ten micrograms of DNA per sample were fractionated on a 2% agarose gel.
Myoglobin. Approximately 800 µl of perfusate were collected at various times of perfusion and reperfusion. Two hundred microliters were analyzed at the indicated times (see figures) using the Wyntek Diagnostics Myoglobin/CK-MB apparatus courtesy of Dr. Brad Cunningham, Genzyme Diagnostics,
GSH and GSSG levels. GSH and GSSG levels were determined as previously described (1). Briefly, hearts were excised, homogenized in 5% sulfosalicylic acid, and centrifuged at 10,000 g for 10 min at 4°C. To determine total glutathione, 10 µl of supernatant were added to 700 µl of daily buffer (0.248 mg/ml NADPH, 143 mM NaH2PO4, 6.3 mM EDTA) and 100 µl of 6 mM 5,5-dithiobis-2-nitro-benzoic acid prepared in stock buffer (143 mM NaH2PO4, 6.3 mM EDTA) and 190 µl H2O, and the reaction was warmed for 15 min at 30°C followed by the addition of 10 µl GSSG reductase (266 U/ml, Sigma). Reaction progress was followed at 412 nm for 3 min. To determine GSSG, 2 µl of 2-vinylpyridine and 6 µl of triethanolamine were added to 100 µl of supernatant and allowed to stand for 60 min before proceeding as above for the assessment of total glutathione. GSH and GSSG standards were used for calibration.
Statistical analyses. All values are expressed as means ± SE. Statistical significance was assessed with a two-tailed Student's t-test using SPSS software. Significance was defined by P < 0.05, and P values are indicated in the figure legends.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BC-HSPB2 KO mouse, the lens and several muscle types were morphologically characterized (10); however, a detailed analysis of cardiac morphology and function has not been carried out. Accordingly, we assessed gross and microscopic cardiac morphology and evaluated function, in vivo, using echocardiography, and in vitro, using an isolated-perfused mouse heart model.
Characterization of BC-HSPB2 KO mice. As part of our initial characterization of the KO mice, a longevity study was carried out. We found that through 10 mo of age there was no difference in the survival rates of WT and KO mice (Fig. 1). However, beginning at 11 mo of age, the KO mice exhibited significantly reduced survival. At 14 mo of age, 90% of the WT mice survived; however, all of the KO mice had died. On postmortem examination, the hearts of KO mice appeared no different from those of WT mice (not shown). Accordingly, the premature death of the BC-HSPB2 KO mice is most likely associated with malnourishment, as suggested in the initial report on these mice (10), and not cardiac functional deficit.
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Physical examination showed that the weights of the KO mouse hearts were the same as those from age- and gender-matched WT mice (Table 1). However, the liver weights and body weights of the KO mice were about 30% lower than WT. This combination of characteristics resulted in similar liver-to-body weight ratios but 38% greater heart-to-body weight ratios in the KO mice than in WT mice (Table 1). Although suggestive of cardiac hypertrophy, the higher heart-to-body weight ratio in the KO mice is actually the result of reduced body weight, which may be the result of malnutrition in the KO mice.
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To further assess possible affects of BC and HSPB2 gene disruption, the levels of expression of several cardiac genes that serve as markers of hypertrophy were analyzed. The mRNA levels for -skeletal actin and -myosin heavy chain were the same in the KO and WT mouse hearts; whereas the mRNA levels for ANF and BNP appeared lower in the KO than those of WT mouse hearts, this trend did not reach statistical significance (Fig. 2A). Moreover, Western analysis showed that the levels of ANF and BNP protein were the same in the WT and KO mouse hearts (Fig. 2B). Thus deletion of the BC and HSPB2 genes does not activate the expression of these hypertrophic marker genes.
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Morphology of BC-HSPB2 KO mouse hearts. To assess whether disruption of the BC and HSPB2 genes affected cardiac morphology, hearts from 9-mo-old KO and WT mice were processed for histology. Hematoxylin and eosin or trichrome staining revealed no tissue alterations or fibrosis in the KO mouse hearts (Fig. 3, A and B), consistent with previous observations (10).
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Because BC and HSPB2 both interact with myofilaments (6, 13, 47, 50, 52, 54), it was of interest to examine myofibril ultrastructure in the KO mouse hearts. Earlier analysis of tongue and soleus muscle from the KO mice demonstrated cell degeneration and vacuoles with irregular edges (10); however, these features were not apparent in the heart, as determined in the present study. Electron photomicrographs of cardiac tissue revealed that the myofibrils from WT and KO mouse hearts contained regular repeats of sarcomeres delineated by Z disks,- and parallel arrangements of myofibrils (Fig. 3, C and D). Thus deletion of the BC and HSPB2 genes had no apparent affect on myofibril structure.
Functional analysis of BC-HSPB2 KO mouse hearts. To assess the hemodynamic properties of BC-HSPB2 KO mouse hearts, two-dimensional guided M-mode echocardiography measurements were performed on 6-mo-old mice. All but one of the parameters measured were the same in KO and WT mouse hearts (Table 2). The one statistically different parameter was the left ventricular end-systolic dimension (LVESD), which was lower in the KO than in the WT mouse hearts (1.8 ± 0.02 vs. 2.3 ± 0.2 mm, respectively), suggesting enhanced left ventricular contractility in the KO mice. This possibility is supported by the slightly higher percent fractional shortening in the KO mouse hearts compared with WT, although this difference did not reach statistical significance. Thus deletion of the BC and HSPB2 genes had little affect on hemodynamic cardiac function as determined by echocardiography.
Postischemic contractile recovery. Increased BC expression in transgenic mouse hearts has been shown to enhance functional recovery of isolated perfused hearts following global ischemia (41). Accordingly, we assessed the effects of BC and HSPB2 gene disruption on myocardial function following I/R of isolated perfused mouse hearts. Hearts were subjected to 30 min of equilibration, followed by 25 min of no-flow (global) ischemia and 90 min of reperfusion. As illustrated in Fig. 4, hearts from KO and WT mice exhibited similar LVDP (% maximum LVDP) values during equilibration and ischemia. However, on reperfusion, the KO mouse hearts exhibited lower contractile recovery than controls. This functional difference was evident within 5 min of reperfusion, when the LVDP of the KO mouse hearts was less than half that of WT mouse hearts (7.0 ± 1.8% vs. 18.2 ± 3.5%, respectively, P = 0.007). The functional deficit exhibited by KO mouse hearts continued throughout the 90-min reperfusion (25.65 ± 3.54% and 51.42 ± 5.09% for KO and WT, respectively, P = 0.0003).
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Analysis of I/R-induced necrosis and apoptosis. In the heart, I/R results in a combination of necrosis and apoptosis (23); accordingly, apoptosis and necrosis were measured by DNA fragmentation (laddering) and myoglobin release, respectively. Initial control experiments showed that after 25 min of ischemia, DNA laddering was not evident in isolated perfused WT mouse hearts until after 4 h of reperfusion (Fig. 5A). Whereas neither WT nor KO mouse hearts displayed DNA laddering after 5 h of perfusion only (Fig. 5B, lanes 1 and 2), 25 min of ischemia followed by 4 h of reperfusion resulted in more intense laddering in KO (Fig. 5B, lanes 5 and 6) than WT mouse hearts (Fig. 5B, lanes 3 and 4). Myoglobin release was evident only during the first 30 min of reperfusion and was about twpfold greater in KO than in WT mouse hearts (Fig. 5C). Based on myoglobin release, it was apparent that necrosis occurred very soon after reperfusion was initiated, reaching maximal levels after about 5 min and then returning to control values by 60 min. These results indicate that compared with WT, the KO mouse hearts exhibit greater apoptotic and necrotic tissue damage after I/R.
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Analysis of glutathione levels. Reactive oxygen species (ROS) are a major cause of I/R-induced myocardial damage. Reduced glutathione (GSH) serves an important role in neutralizing the damaging effects of ROS (27). Small HSPs contribute to reducing damage from ROS by raising the intracellular concentration of GSH compared with oxidized glutathione (GSSH) (34). Accordingly, we assessed GSH and GSSG levels in the hearts of BC-HSPB2 KO mice. When compared with WT, hearts from KO mice exhibited a 43% decrease in total glutathione (Fig. 6A). Additionally, when compared with WT, KO mouse hearts displayed 54% more oxidized glutathione (Fig. 6B). The GSH-to-GSSG ratio in hearts from BC-HSPB2 KO mice was 21.73 ± 0.818, which was decreased by 60% compared with WT, which was 56.79 ± 2.82 (Fig. 6C).2 These results indicate that the absence of BC and/or HSPB2 in the KO mouse hearts might lead to a reduced GSH-to-GSSG ratio, which is at least partly responsible for the increased damage following I/R.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BC and HSPB2 genes exhibited increased mortality compared with WT mice. It is presently unclear what causes the increased mortality in the KO mice; however, because they exhibit a slightly dystrophic phenotype (10), it is possible that they were less able to acquire food and water. This could also account for the lower body weights of the KO mice. Although we were able to study the KO mice up to about 8 mo of age, this increased mortality poses some limitation on the types of studies that can be undertaken on the BC-HSPB2 KO mice.
We also found that under nonstressful conditions the BC-HSPB2 KO mice exhibited no discernible defects in cardiac development, morphology, or function. Although the observation that the BC-HSPB2 KO mouse hearts appeared normal was also made in the earlier report that described the generation of these mice (10), it is a surprising result, because BC is expressed in high levels in the embryonic heart and because both BC and HSPB2 are found in abundance in the adult heart (46), implying critical functions for these sHSPs. This apparent lack of phenotype could be because these sHSPs do not play critical roles in cardiac development, structure, and function. However, it is more likely that other sHSPs are sufficiently abundant so that under certain conditions they can compensate for the absence of BC and HSPB2. The high degree of structural similarity among the sHSPs expressed in the heart is consistent with such putative functional overlap (46). In fact, BC and HSPB2 are 32% identical at the amino acid level and 47% identical in the -crystallin domain. Moreover, whereas it is not known whether HSPB2 is phosphorylated during stress, several studies have demonstrated that, like BC, HSP27 is phosphorylated in response to stress, and that the phosphorylated forms are cytoprotective (2, 35), which supports potential functional redundancy between BC and HSP27. However, it should be noted that there is also evidence that the cytoprotective activities of BC and HSP27 do not correlate with phosphorylation. For example, in glioma cells, expression of BC where serines-19, -45, and -59 have been replaced by aspartic acid, which mimics phosphorylation at these sites, results in reduced chaperone-like activity (21). Also in isolated rabbit cardiac myocytes, ischemic preconditioning, which protects from cell death, did not result in increased phosphorylation of BC (3). This is similar to results that have been obtained with HSP27, showing that phosphorylation reduces chaperone-like activity (43), and that unphosphorylated forms of HSP27 protect against cell death in NIH 3T3 cells (33).
In contrast to the findings in unstressed hearts, when exposed to I/R BC-HSPB2 KO mouse hearts exhibited increased necrosis, apoptosis, and only half the contractile recovery of WT mouse hearts. These results are consistent with roles for BC and/or HSPB2 in protecting the myocardium from the functional deficits incurred on I/R stress. However, it is formally possible that the levels of some proteins, whose identities are not known, may be altered as a result of the gene distruption in this model and that these alterations might contribute to the observed phenotype, Although the precise mechanisms by which BC and/or HSPB2 contribute to this protection are not yet known, at least in part, they most likely involve the abilities of these sHSPs to serve as chaperones. For example, BC binds to and stabilizes the enzymatically inactive form of caspase-3, p24, thereby inhibiting caspase-3-dependent apoptosis (24). It has also been shown that HSPB2 colocalizes with mitochondria (37), indicating that it may protect from apoptosis by inhibiting cytochrome c efflux. Thus BC and HSPB2 may both inhibit the mitochondrial-dependent death pathways, albeit through different mechanisms.
Another mechanism by which BC and/or HSPB2 could protect the myocardium involves GSH. Overexpression of BC increases the level of GSH, which leads to protection from apoptosis induced by the ROS that are generated on oxidative stresses, such as reperfusion (12, 40). Thus it is possible that the absence of BC in the BC-HSPB2 KO mice leads to reduced levels of GSH. Consistent with this hypothesis are findings of a recent study showing that compared with hearts from WT mice, hearts from mice deficient in heat shock factor 1, a transcription factor known to be required for high expression of BC and other sHSPs, exhibit significantly lower levels of BC and GSH and a reduced GSH-to-GSSG ratio (53). Although GSH levels in the lenses of the BC-HSPB2 KO mice were found to be decreased by only about 10% compared with WT mice (25), in the present study we found that BC-HSPB2 KO mouse hearts possess about 45% lower levels of GSH and a decreased GSH-to-GSSG ratio compared with WT mouse hearts. Perhaps the absence of BC and/or HSPB2 has a greater impact on GSH levels in the metabolically more active cardiac tissue than in the lens. Nonetheless, the results from our study support the notion that reduced GSH leads to increased susceptibility to I/R-induced myocardial damage in the KO mice.
In addition to binding to proteins and organelles involved in apoptosis, BC and HSPB2 also bind to the cytoskeleton, including sarcomeric I bands, which reside adjacent to Z disks in cardiac and skeletal muscle (31, 54). This Z disk localization, which increases following I/R, has led to speculation that through their chaperone functions, BC and HSPB2 might stabilize sarcomeres during stress (4, 16, 17). Although the mechanism by which this localization is enhanced on I/R remains unclear, recent studies showing that BC can bind to the portion of titin that resides in the I band adjacent to the Z disk (16) suggest a possible means for targeting BC to this portion of the sarcomere.
Several recent findings have demonstrated that the Z disk serves as a major location of signal transduction machinery in cardiac myocytes (15), making this an excellent target for the cytoprotection-oriented chaperone function of BC, HSPB2, and other sHSPs. For example, the Z disk serves as the interface between the sarcomeres and the cytoskeleton, and the localization of the Z disk near the sarcolemma, along with the connection between the Z disk and the basement membrane via costameres, are ideal for transmitting signals from outside/in, and inside/out. Among signaling machinery localized to the Z disk is the recently described cardiac stretch sensor (28). Thus the localization of BC and HSPB2 to Z disks positions these abundant chaperones in an ideal environment to contribute to the maintenance of signals that foster myocardial cell survival. As such, BC and HSPB2 may play central roles in maintaining the structural integrity of various signaling complexes at the Z disk.
Although BC and HSPB2 share many structural features and the Z disk localization, there are numerous differences in the characteristics of these two sHSPs. For example, HSPB2 binds to myotonic dystrophy protein kinase (DMPK), whereas BC does not; by binding to DMPK, HSPB2 reduces the enzyme's susceptibility to thermodenaturation while increasing its kinase activity (47). The DMPK is the locus of mutations that cause myotonic dystrophy (11). Accordingly, it is thought that by stabilizing DMPK, HSPB2 contributes to maintaining proper skeletal muscle development, structure, and function (47). Thus it is possible that certain aspects of the BC-HSPB2 KO mouse phenotype (e.g., skeletal muscle atrophy) are due to reduced DMPK activity resulting from the absence of HSPB2. Moreover, although HSPB2 is able to form oligomers with HSPB3, it cannot bind to BC, HSP27, or HSP20 (46), which form oligomers with one another; these oligomers are thought to be involved in their chaperone functions, and whereas BC and HSP27 bind to actin filaments in C2C12 cells, HSPB2 does not exhibit such filamentous localization (46). It is interesting to note that based on developmental profiles of BC expression, several studies have suggested that it may be required for myogenic differentiation and cardiac morphogenesis (14, 19). Although HSPB2 does not appear to interact with actin filaments because it localizes to Z disks and its expression is tightly regulated during the myogenic program, some investigators believe it may also be critical for cardiac morphogenesis (46). However, the apparently normal hearts in the BC-HSPB2 KO mice indicate that neither of these sHSPs is required for cardiac morphogenesis.
In conclusion, the present study has characterized the cardiac phenotype of mice in which both the BC and HSPB2 genes have been disrupted. Because both genes are disrupted in the BC-HSPB2 KO mice, and because there are no mouse lines featuring disruption of either gene alone, or, for that matter, disruption of any other sHSP known to be expressed in the heart, it is impossible to conclude at this time whether either or both of these sHSPs is/are responsible for the observed cardiac effects. Nonetheless, the BC-HSPB2 KO mouse model has served as a valuable tool that has allowed us to determine that at least one of these two sHSPs is required for maximal protection against the damage and functional deficits incurred by I/R.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by The National Institutes of Health Grants HL-63975 and NS/HL-25037 (to C. C. Glembotski).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 In the mouse and rat, HSP25 is the homolog of human. For simplicity, in this paper, HSP27 is used to mean HSP27 in human and HSP25 in mouse or rat. 
2 A review of the literature indicates that the GSH-to-GSSG ratio seems to be somewhat different depending on the mouse strain and the tissues under study. For example, in the liver, heart, and brain of 1-mo-old CD-1 mice, the GSH-to-GSSG ratios are about 92, 35, and 50, respectively (30), whereas in 4-mo-old C57BL/6 mice these same tissues exhibited GSH-to-GSSG ratios of 61, 36, and 230, respectively (42). In 129/SvJ x BALB/c mouse hearts, the GSH-to-GSSG ratio is about 10 (53).
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B-crystallin to Z lines of myocardium. Am J Physiol Heart Circ Physiol 274: H1457–H1464, 1998.B crystallin confers simultaneous protection against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. FASEB J 15: 393–402, 2001.-Crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner. Eur J Biochem 242: 56–66, 1996.[ISI][Medline]
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P < 0.0001 different from WT mice as determined by two-tailed Student's t-test.
