Published online before print October 23, 2003, doi: 10.1161/01.RES.0000102401.77712.ED
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© 2003 American Heart Association, Inc.
Integrative Physiology |
B-Crystallin Modulates Protein Aggregation of Abnormal Desmin
From the Division of Molecular Cardiovascular Biology (X.W., R.K., J.R.), Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; Cardiovascular Research Institute and Division of Basic Biomedical Sciences (X.W., W.H., J.G., F.L.), University of South Dakota School of Medicine, Sioux Falls, SD.
Correspondence to Xuejun Wang, MD, PhD, Cardiovascular Research Institute/Division of Basic Medical Sciences, University of South Dakota School of Medicine, 1400 W. 22nd St, Sioux Falls, SD 57105. E-mail xwang{at}usd.edu
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Abstract |
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B-crystallin (CryAB) is the most abundant small heat shock protein in the heart. Upregulation of CryAB in desmin-related myopathy and its downregulation in end-stage congestive heart failure have both been reported. We previously demonstrated via cardiac-specific transgenesis that modest increases in normal CryAB are not detrimental to the heart, whereas expression of the R120G mutation of CryAB caused a desminopathy. It is generally believed that CryAB plays an important role in protecting the intermediate filaments, but the underlying mechanism is unclear. We hypothesized that CryAB protects the desmin filaments via preventing abnormal desmin protein from aggregating adversely. To test this hypothesis in vivo, mice expressing a desmin mutation that causes a desmin-related cardiomyopathy (D7) were bred into the R120G-CryAB transgenic (TG) background to examine the accumulation and aberrant aggregation of desmin protein. Despite lower mRNA expression of D7-des than in the D7-des TG hearts, the double-TG myocardium exhibited significantly higher desmin protein levels and dramatically more aberrant desmin aggregates than the D7-des TG hearts. The double-TG mice displayed a significantly stronger cardiac hypertrophic response, with the mice dying of congestive heart failure before 7 weeks. To explore the ability of wild-type (WT) CryAB to protect against mutant desmin, a desmin mutant was expressed in both the conventional and WT-CryAB stably transfected HEK cells. Significantly less aberrant desmin aggregation was observed in the WT-CryAB–overexpressing cells than in the HEK cells. The results suggest that CryAB modulates abnormal desmin aggregation and can serve a cardioprotective role.
Key Words: small heat shock protein • protein aggregation • protein degradation • molecular chaperone • transgenic mice
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Introduction |
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Alpha B-crystallin (CryAB) is the most abundant small heat shock protein in the heart.1–3 Significant upregulation of CryAB in the heart has been associated with familial hypertrophic cardiomyopathy and desmin-related cardiomyopathy (DRM), and its downregulation has been reported in end-stage congestive heart failure.4–6 Although some controversy over the role of CryAB in cardiac preconditioning remains,7 overexpression of CryAB can protect cardiomyocytes from ischemia and reperfusion injury in primary cell culture and in transgenic mice.8–10 Within limits, it is now clear that upregulation of wild-type (WT) CryAB alone in the heart is not detrimental,11 and whereas some of the mechanisms by which CryAB protects ischemic cardiac myocytes have been defined,12,13 the mechanism underlying the protection of desmin filaments by CryAB is unclear.
Genetic linkage analyses associated a missense mutation (R120G) of CryAB with familial DRM.14,15 The mutation is inherited in an autosomal-dominant manner14 and in vitro experiments confirmed that R120G-CryAB is dominant negative, with the mutant protein compromising CryAB polymer structure and chaperone function when mixed with WT-CryAB.16,17 Using cardiac-specific transgenesis, we showed that R120G-CryAB causes both aberrant desmin and CryAB aggregation as well as cardiomyopathy in a dominant-negative manner.11 Data accumulated so far indicate that CryAB may play an important role in the formation and/or maintenance of desmin filament networks, but it is unknown if CryAB is able to ameliorate DRM pathology.
We previously generated a transgenic (TG) mouse line with cardiac-specific expression of a 7-amino-acid deletion (R173-E179) mutation of desmin (D7-des), a human DRM-linked desmin mutation.15,18 In this model, the desmin filament networks were disrupted by formation of characteristic aberrant desmin aggregates, but lifespan was unaffected.18 Further analyses showed that both CryAB mRNA and protein were upregulated in the hearts. Because overexpression of WT-CryAB alone at the levels observed in the mice is not detrimental to the heart, we hypothesized that upregulation of CryAB in the DRM heart was a compensatory response to either disruption of the desmin network or accumulation of mutant desmin protein.
In this study, to understand the interplay between the mutant desmin and CryAB at the whole organ level, we crossbred the D7-des mice (line 641) into a mouse line (line 708), which carries a single copy of the dominant-negative R120G-CryAB transgene. Line 708 displays very mild pathology at an early age and does not show increased mortality up to 18 months.11 The double-TG mice all died of congestive heart failure before 7 weeks of age. The cardiac desmin protein levels and aberrant desmin aggregates were dramatically increased relative to the single transgenics, and the pathology at the cellular level was much more severe. Further in vitro experiments showed that mutant desmin proteins formed significantly less aberrant aggregates in a WT-CryAB–overexpressing human embryonic kidney (HEK) cell line, compared with the untransformed HEK cells. The data suggest that CryAB can function in preventing misfolded desmin protein from aggregating and ameliorate the resulting pathology.
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Materials and Methods |
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Transgenic Mouse Lines
The D7-des and R120G-CryAB TG mouse lines have been described.11,18 Controlled by the
-myosin heavy chain (-MHC) promoter, the expression of both transgenes are cardiac specific. The line 641 males and the line 708 females were used for crossbreeding. Line 641 carries approximately 20 copies of D7-des and line 708 carries 1 copy of the R120G-CryAB transgene (Figure 1A). The first generation of offspring from the crossbreeding was utilized for all related analyses. The mice were genotyped using PCR and genomic Southern blots as previously described.11,18 The crossbreeding results in four genotypes of offspring: non-TG for either transgene (-/-, NTG), R120G-CryAB TG positive only (+/-, R120G TG), D7-des TG positive only (-/+, D7-des TG), and both R120G-CryAB and D7-des TG positive (+/+, double-TG). Normal Mendelian ratios of the various genotypes were obtained at birth. All TG mice used for this study are heterozygous for the transgene. The double-TG mice (+/+) are double heterozygotes. Institutional guidelines were followed in the care and use of the mice.
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RNA Isolation and Dot Blotting
Total RNA was isolated from the ventricle (including the left, right ventricles, and the ventricular septum) of 3 individual mice of each genotype group using Tri-Reagent (Molecular Research Center, Inc). For RNA dot blotting, 4 µg of total RNA was loaded. The blots were hybridized with radioactively labeled transcript-specific oligonucleotide probes and detected with the Storm 860 (Molecular Dynamics) as described.11
Semiquantitative Western Blots
Total protein extract was prepared from 4 mouse hearts for each genotype. Frozen ventricular tissue was homogenized in 1% SDS PBS (pH 7.4), mixed with 2x SDS-PAGE sampling buffer (1:1) and boiled for 5 minutes. Protein concentration was estimated using Bio-Rad Protein Assay reagent (Bio-Rad). To each lane, 15 µg of total protein was loaded and separated by SDS-PAGE. The protein was then transferred to a PVDF membrane and probed with a primary antibody cocktail including mouse monoclonal anti-actin, desmin (Sigma), and rabbit polyclonal anti-CryAB (StressGen) antibodies. Horse radish peroxidase (HRP)–conjugated anti-mouse and anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology, Inc) were used. The secondary antibody binding was detected with the ECL+Plus detection reagents (Amersham Pharmacia Biotech) and quantified using the Storm 860 (Molecular Dynamics) and ImageQuant software.
Histology and Immunofluorescence Confocal Microscopy
For paraffin sectioning, hearts from littermates were perfusion fixed with 10% formalin. Trichrome staining was performed with the paraffin sections.18 Indirect immunofluorescence staining was performed on fixed myocardial cryosections as described previously.18 Hearts were excised, fixed by coronary perfusion with 3.7% paraformaldehyde, saturated with 30% sucrose solution, and embedded in Tissue-Tek O.C.T. (Sakura Finetek). Cryosections were air-dried, incubated with 0.1 mol/L glycine in PBS (pH 7.2) for 30 minutes, treated with 1% Triton X-100 for 60 minutes and with 0.1% pronase (Sigma) for 20 minutes, and blocked with 0.5% BSA/10% goat serum in PBS. The specimens were then incubated with mouse monoclonal antibodies to desmin (Sigma) and rabbit polyclonal antibodies to CryAB (Stressgen) overnight at 4°C and subsequently with TRITC conjugated anti-mouse IgG and FITC conjugated anti-rabbit IgG antibodies (Sigma) for 1 hour at room temperature for confocal microscopy.
Cell Culture Experiments
A clonal HEK cell line stably overexpressing WT-CryAB was created by transfecting the HEK cells with a mammalian expression vector harboring the WT-CryAB murine cDNA, which also contained a HA epitope tag at its 3' end. The mouse cDNAs for WT-desmin and a DRM-associated mutant (A360P/N393I) desmin were also inserted into the same mammalian vector and used for transient transfection experiments. Forty-eight hours after the transfection, the cells were collected for analysis. For immunolabeling, cells grown on cover glass were fixed first with 100% ethanol for 5 minutes and then with 4% paraformaldehyde for 20 minutes. For immunoblotting, cells grown on the Petri dishes were collected after being rinsed with cold PBS (pH 7.4) and collected by centrifugation (500g for 5 minutes). The cell pellets were treated with 1x protein sampling buffer and boiled for 5 minutes to extract total protein.
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Results |
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Expression of Endogenous and TG Desmin and CryAB
The normal function of CryAB depends on the formation of CryAB polymers. The incorporation of a mutant CryAB into the polymer could affect the normal functions of the associated WT-CryAB protein, and consistent with this hypothesis, R120G-CryAB is dominant negative, altering CryAB polymer structure and compromising its chaperone function in vitro.17 We previously demonstrated that cardiac TG expression of the mutant CryAB gives rise to abnormal desmin and CryAB aggregation, resulting in a cardiomyopathy.11 The pathology presentation of the R120G mice is transgene dosage dependent with the single-copy TG line (line 708) showing only mild disease at an early age, although the TG lines displayed the characteristic pathological changes: aberrant desmin organization and CryAB aggregation.
We wished to determine the interplay of the two mutants, D7 and R120G, in desmin and CryAB, respectively, on the course of the cardiomyopathies, and thus performed the double cross. As expected, the D7-des and R120G transgenes were transmitted from their respective parental lines 641 (D7: 20 copies) and 708 (R120G: 1 copy) to either single-heterozygous TG mice (ie, +/-, -/+) or double heterozygous TG mice (+/+) (Figure 1A). Normal litter sizes and Mendelian ratios of the four genotypes were obtained with no embryonic lethality observed in the single or double heterozygotes.
Endogenous desmin transcript levels were increased 2-fold in both D7-des TG and double-TG mice but not in the R120G TG mice (Figures 2B and 2C). The total level of desmin transcripts including both endogenous and transgenic were increased in both the D7-des TG and the double-TG groups. However, the level is significantly lower in the double-TG group than in the D7-des TG group. As endogenous desmin RNA levels remained constant, this indicates that the double TGs had relatively less D7-des mRNA, compared with the single-TG D7-des group. This is likely due to a downregulation of the -MHC promoter activity during the ensuing hypertrophic response. Similarly, the mRNA level of CryAB is also significantly lower in the double-TG mice than in the R120G-TG mice (Figures 1B and 1C).
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Expression of R120G-CryAB Alters Steady-State Desmin Levels and Enhances the Aberrant Aggregation of Mutant Desmin
CryAB protein has been found in the abnormal desmin aggregates in human DRM. CryAB protein levels are significantly increased in the D7-des DRM (Figure 2), reflecting the increase in the steady-state RNA levels (Figures 1B and 1C). Interestingly, cardiac CryAB mRNA and protein levels in the double-TG mice were not significantly different from those in the D7-des single[TG mice (Figures 1B, 1C, 2A, and 2
B). Despite fewer desmin transcripts (Figures 1B and 1C), the double-TG mice displayed significantly higher cardiac desmin protein levels relative to the D7-des TG mice (Figure 2), suggesting that in the presence of R120G-CryAB, desmin protein degradation is substantially compromised.
To determine the distribution of the increased desmin protein in the double-TG hearts, light and immunofluorescence confocal microscopy were used. In human DRM, aberrant desmin aggregates in cardiac myocytes stain dark green or blue, depending on whether the third dye utilized is green or blue in the trichrome staining.19 As expected, scattered blue aggregates were observed in the D7-des TG cardiomyocytes (Figure 3C). Strikingly, the blue aggregates almost filled the cardiomyocytes in the double-TG heart. Immunofluorescence microscopy also showed that desmin-positive aggregates filled the entire myocyte (Figure 3D). As previously reported,18 a few small desmin aggregates in the 1-month-old D7-des TG hearts and small CryAB-containing bodies in R120G-TG hearts could also be observed (Figure 4). No aberrant desmin aggregates were present in the R120G-TG heart at this stage (Figures 3B and 4
). Neither aberrant desmin aggregates nor CryAB aggregates were evident in the NTG hearts (Figures 3A and 4). These data demonstrate that abnormal aggregation of mutant desmin is enhanced in the presence of mutant CryAB protein.
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D7-des and R120G-CryAB Are Synergistic in Pathogenesis
Previously, we found that relatively low levels of R120G-CryAB resulted in significant pathology when compared with TG expression of D7-des. Similarly, the pathology in these mice was more severe than that observed in the desmin knockouts, leading us to hypothesize that the pathogenesis resulting from the mutated CryAB is caused by more than just a simple failure of protecting desmin networks.11,15 Crossbreeding the two TG mice provided an opportunity to further test the hypothesis. Strikingly, these mice developed much more profound disease phenotypes than either of the single-TG lines. This is manifested by the increased hypertrophic responses at both the molecular and organ levels (Figure 5, Table) and by dramatically shortened lifespan in the double-TG mice (Figure 6). At the molecular level, activation of fetal gene programs is common during the early stages of hypertrophy, with upregulation of atrial natriuretic factor (ANF) and ß-MHC and downregulation of -MHC in the ventricle.20,21 Changes in the transcripts of phospholamban (PLN) and the sarcoplasm reticulum Ca2+ pump (SERCA) can often serve as markers for changes in calcium handling and heart failure. The relative amounts of these transcripts, as well as those of cardiac -actin (c-actin) and skeletal -actin (s-actin) were determined (Figure 5). For the transcripts tested, no differences were detected between the NTG and the R120G-TG groups. ANF and ß-MHC were upregulated, and -MHC, PLN, and SERCA were significantly downregulated in the D7-des TG mice, confirming previous data.18 Compared with the D7-des TG mice, activation of the fetal genetic program was significantly more pronounced in the double-TG mice (Figure 5). In general, cardiac -actin can be downregulated but skeletal -actin is upregulated in cardiac hypertrophy.21 Interestingly, skeletal -actin transcript levels were significantly downregulated in the D7-des TG mice and downregulated even more in the double-TG mice (Figure 5). Consistent with the molecular responses, cardiac hypertrophy was also more severe in the double-TG mice: heart weight, ventricular weight, heart/body weight ratio, and ventricle/body weight ratio were all significantly greater in the double-TG mice than in the D7-des TG mice (Table).
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Single-TG mouse cohorts from either line 641 (D7-des) or line 708 (R120G-CryAB) live at least 18 months. In contrast to the normal lifespan in the face of modest pathology, the double-TG mice died within 7 weeks, with a majority dying from congestive heart failure between 4 and 5 weeks (Figure 6). Because, at these early times, only subtle pathology could be detected in either line carrying a single transgene, the massive pathology apparent in the double transgenics cannot be ascribed to a simple additive effect between the single transgenes. Rather, the striking morbidity and mortality in the double transgenics imply a direct or synergistic interaction, or critical loss of function between the mutant proteins. Subcutaneous edema, ascites, plural effusion, dilatation of the heart, and congestion of the liver and lungs were observed on autopsy or when the double-TG mice were euthanized at 4 to 7 weeks of age. Labored breathing, failure to grow, and a pronounced reluctance to move were also observed invariably a few days before the mice died.
Overexpression of WT-CryAB Modulates Behavior of Mutant Desmin in Cultured Cells
To further test in vitro whether upregulation of WT-CryAB in DRM is compensatory to abnormal desmin organization and aggregation, we chose to compare the behavior of mutant desmin in conventional HEK cells and a HEK cell line that stably overexpressed WT-CryAB.
In cultured 293 HEK cells, transient transfection of WT-des led to formation of a desmin filament network that is apparently superimposed on the endogenous cytokeratin filament network (Figure 7A); transient transfection of the mutant desmin, however, gave rise to mainly aberrant desmin aggregates (Figure 7Ba). However, in the HEK cells that stably overexpress WT-CryAB, the mutant desmin protein formed significantly fewer aggregates and displayed a relatively high proportion of apparently normal desmin filaments (Figures 7B and 7C). With either conventional HEK cells or WT-CryAB–overexpressing HEK cells, over 90% of the WT-des–transfected cells displayed desmin as a fine filamentous network without discernible aggregates, 6% to 8% showed a mixture of desmin filaments and small desmin aggregates and less than 3% contained only desmin aggregates. Differences in the distribution of WT-des protein between the 2 types of HEK cells were not statistically significant (Figure 7C). However, the distribution of the mutant desmin among the following 3 categories (exclusively desmin aggregates [45%], exclusively desmin filaments [25%], or both aggregates and filaments [30%]) in WT-CryAB–overexpressing cells were significantly different from the mutant desmin distribution (74%, 10%, and 16%, respectively) in the conventional HEK cells (Figure 7C; P<0.0005, 
2 test).
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Discussion |
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The interaction between CryAB and intermediate filaments has been observed in both muscle and neural tissues under both physiological and pathological conditions.3,22 CryAB is phosphorylated and translocated from the cytosol to the cytoskeleton when cells undergo stress.23–25 In the normal heart, a majority of CryAB protein is in the soluble fraction with a small amount associated with the cytoskeletal components. CryAB relocates from the cytosol to desmin filaments at the z-line level during ischemia,2,26,27 and this association is believed to stabilize and protect the cytoskeleton from stress damage. This hypothesis has been supported by the establishment of a causative relationship between R120G-CryAB and familial DRM via both genetic linkage analyses and subsequent in vivo analyses of the mutation in TG mice.11,14 Unlike overexpression of the wild-type protein, very modest overexpression of R120G-CryAB in the heart leads to aberrant CryAB aggregation as well as abnormal desmin aggregation, the hallmark of DRM.11 The formation of desmin aggregates in the R120G-CryAB TG mouse hearts is unlikely due to a so-called "fatal attraction" of the mutant CryAB protein,28 because the predominant type of CryAB aggregate does not display any direct interaction with desmin-positive material.11 The formation of desmin aggregates may be caused indirectly by a loss of normal CryAB function due to the dominant-negative effects of the mutant CryAB. Consistently, abnormal electron dense material and desmin filament disorganization have been observed in the muscle cells of CryAB/HSPB2 double-knockout mice.29 Therefore, we hypothesize that when small amounts of abnormal desmin accumulate, the damaged protein interacts with CryAB, which acts as a chaperone, remains soluble, and is efficiently removed over time. When CryAB function is lost or compromised, abnormal desmin protein forms aberrant aggregates and disrupts the integrity of the desmin network, with subsequent pathological consequences. We previously demonstrated that expression of D7-des in the heart can also give rise to desmin aggregates. However, aberrant aggregate accumulation in the cardiac myocytes of the D7-des mice is relatively slow and, interestingly, both CryAB mRNA and protein levels are significantly upregulated, consistent with the hypothesis that CryAB may be important in preventing the mutant desmin protein from aggregating and/or enhancing degradation of the mutant protein. If true, expression of the mutant desmin in a background in which normal CryAB function is compromised should greatly enhance the accumulation and aberrant aggregation of desmin protein. This proved to be the case, and the D7-des and R120G-CryAB double-TG mouse hearts accumulate more desmin and formed larger and more abundant abnormal desmin aggregates than in either of the single-TG mouse hearts. Consequently, the double-TG mice developed more severe cardiac hypertrophy and died from congestive heart failure at a much younger age.
Based on amino acid sequence homology and in vitro protein function assays, CryAB is classified as a member of the small heat shock protein family and is thought to be a molecular chaperone.30 However, its chaperone function has not been directly tested in intact animals before. The present data combined with our previous data11 strongly support a role for CryAB in molecular chaperone function, at least for desmin. It has been recently reported that overexpression of CryAB in cultured astrocytes dissolves aggregates formed by overexpression of the glial fibrilous protein, a neural intermediate filament protein.22 These data imply that the role of CryAB in vivo as a chaperone could be critical in certain cell types and this role may, at least partially, explain its antiapoptotic action in cardiomyocytes, through its interaction with desmin and other proteins.
An alternative interpretation of the result of the present study is that the R120G-CryAB protein directly interacts with the D7-des protein and mutually facilitates the proteins’ aggregation and accumulation. Because the aggregates formed in the double-TG hearts were both desmin and CryAB positive (Figure 4), this possibility cannot be completely eliminated. However, existing evidence argues against this alternative interpretation. WT-CryAB is able to interact D7-des protein because the desmin aggregates were also CryAB positive in the D7-des single-TG heart (Figure 4). Nevertheless, mutant CryAB is unable to productively directly interact with wild-type desmin protein.11 In the R120G-CryAB single-TG heart, we found previously two morphologically and biochemically different types of aberrant aggregates: one is predominantly CryAB positive, the other desmin positive. No physical interaction was evident between the two types of aggregates. No desmin-positive material was found to directly interact with the CryAB aggregates.11 The presence of D7-des protein did not increase the abundance of CryAB protein in the double-TG heart despite the presence of one copy of R120G-CryAB transgene (Figure 2).
Upregulation of desmin at both mRNA and protein levels has been reported in familial hypertrophic cardiomyopathy and pressure overloaded cardiac hypertrophy and failure.4,31,32 Therefore, the increase in desmin protein levels and increased aberrant desmin aggregation might be secondary to the more severe cardiac hypertrophy and failure in the double-TG mice. It is likely that the two mutant proteins can also act through different pathways and so synergistically result in more severe cardiac hypertrophy and congestive heart failure as seen in the double-TG mice. We think it is likely that R120G-CryAB causes cardiomyopathy via mechanisms other than or in addition to disruption of the desmin filament network. However, the increase in desmin in the double-TG heart was not paralleled by an increase in endogenous desmin expression, because the endogenous desmin mRNA levels in the double-TG and D7-des single-TG did not significantly differ (Figure 1). It is therefore unlikely that the increased desmin protein levels and abundance of desmin aggregates in the double-TG heart are due to an increase in the synthesis of endogenous desmin. The more likely possibility is degradation rates of the mutant desmin protein reduced.
In vitro experiments provide additional data that are consistent with CryAB and desmin functionally interacting. In cell culture-based experiments, we found that expression of a human DRM-linked mutant desmin leads to much less desmin protein aggregation in the WT-CryAB–overexpressing HEK cells than in regular HEK cells and that, in the presence of overexpression of WT-CryAB, more mutant desmin protein tends to form filamentous structures instead of aberrant aggregates. The data further suggest that CryAB is capable of reducing aberrant aggregation of abnormal desmin proteins.
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Acknowledgments |
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Acknowledgments
This work was supported by NIH grants HL56370, HL60546, HL52318, HL61638, and HL69779 (to J.R.), P20RR17662 and P20RR16479 (to X.W. and F.L.), and HL72166 (to X.W.) and by an American Heart Association Postdoctoral Fellowship and a Scientist Development Grant as well as a grant from University of South Dakota School of Medicine (to X.W.).
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Footnotes |
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Original received August 18, 2003; revision received October 8, 2003; accepted October 9, 2003.
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