Expression of R120G–αB-Crystallin Causes Aberrant Desmin and αB-Crystallin Aggregation and Cardiomyopathy in Mice
Abstract—Upregulation of αB-crystallin (CryAB), a small heat shock protein, is associated with a variety of diseases, including the desmin-related myopathies. CryAB, which binds to both desmin and cytoplasmic actin, may participate as a chaperone in intermediate filament formation and maintenance, but the physiological consequences of CryAB upregulation are unknown. A mutation in CryAB, R120G, has been linked to a familial desminopathy. However, it is unclear whether the mutation is directly causative. We created multiple transgenic mouse lines that overexpressed either murine wild-type CryAB or the R120G mutation in cardiomyocytes. Overexpression of wild-type CryAB was relatively benign, with no increases in mortality and no induction of desmin-related cardiomyopathy even in a line in which CryAB mRNA expression was increased ≈104-fold and the protein level increased by 11-fold. In contrast, lines expressing the R120G mutation were compromised, with a high-expressing line exhibiting 100% mortality by early adulthood. Modest expression levels resulted in a phenotype that was strikingly similar to that observed for the desmin-related cardiomyopathies. The desmin filaments in the cardiomyocytes were overtly affected, myofibril alignment was significantly impaired, and a hypertrophic response occurred at both the molecular and cellular levels. The data show that the R120G mutation causes a desminopathy, is dominant negative, and results in cardiac hypertrophy.
The small heat shock–related protein αB-crystallin (CryAB) was originally discovered and classified as a lens protein.1 CryAB is also found in nonlenticular tissues and is abundant in cardiac and skeletal muscle.2 3 CryAB binds both desmin and cytoplasmic actin and possesses molecular chaperone function in vitro.4 5 6 When a cell is subjected to stress, CryAB transits from the cytosol onto the cytoskeleton.7 Phosphorylation by mitogen-activated protein kinase, p38, and other kinases may regulate this translocation and presumably its chaperone function.8 9 The upregulation of the gene and subsequent accumulation of CryAB occurs in a number of cardiac disorders including familial hypertrophic cardiomyopathy and desminopathy,10 11 12 as well as degenerative neural pathologies such as Alexander and Alzheimer diseases.2 13 However, the pathophysiological significance, if any, of CryAB protein upregulation in muscle remains obscure.
A missense mutation (R120G) of CryAB has recently been linked to familial desmin-related myopathy (DRM), a disease that is characterized by intrasarcoplasmic accumulation of desmin.14 Restrictive, hypertrophic, and dilated cardiomyopathies have all been observed in the desminopathies and often result in death.12 15 Overexpression of R120G-CryAB in a muscle cell line caused formation of electron-dense aggregates containing CryAB in the center and desmin at the periphery.14 However, there is no direct in vivo evidence, outside of linkage analysis, proving that the missense mutation of CryAB causes DRM. Furthermore, if the mutation is directly causative, it remains to be explored how it leads to disease presentation.
To approach these issues, we generated multiple stable transgenic (TG) mouse lines that express different levels of either the wild-type (WT) or mutant CryAB proteins specifically in the heart. Whereas overexpression of WT CryAB protein was benign, expression of even very modest levels of R120G-CryAB protein led to aberrant desmin and CryAB aggregation, disruption of the desmin network, perturbation of myofibril alignment, and compromised muscle function.
Materials and Methods
An expanded Materials and Methods section is available online at http://www.circresaha.org.
TG Mouse Lines
Both human genetic and in vitro biochemical data indicate that the missense mutation (R120G) of CryAB acts in a dominant-negative fashion.14 16 Therefore, we chose to use a TG approach in an attempt to show direct causality of the mutation in causing cardiovascular disease and to create an animal model suitable for longitudinal analyses of the pathogenic processes. To control for the possibility that alterations in the overall stoichiometries of either the CryAB transcript or protein pools might lead to a phenotype, WT murine CryAB was also used to generate TG lines in parallel with the R120G-CryAB construct (Figure 1⇓ online, available in the data supplement at http://www.circresaha.org). Three TG lines (lines 11, 13, and 41) were made using WT-CryAB cDNA, whereas three TG lines (lines 25, 134, and 708) expressed the R120G-CryAB transgene. Germline transmission was confirmed, and normal mendelian ratios were observed, indicating that no embryonic lethality occurred with either construct with those particular lines. Genomic Southern blotting (Figure 1A⇓) showed that the transgene copy numbers of the WT-CryAB lines 11, 13, and 41 were 22, 120, and 60, respectively. Those of R120G lines 708, 25, and 134 were 1, 1, and 3, respectively. CryAB transcript and protein levels determined (Figures 1B⇓ and 1C⇓). Consistent with the copy numbers, all of the WT-CryAB TG lines showed higher CryAB mRNA and protein levels as compared with the mutant lines (Figures 1B⇓ through 1D). Increases in soluble CryAB protein levels in the WT-CryAB TG lines accounted for essentially all of the increase in protein. In contrast, the R120G lines show significant increases in the insoluble, and presumably aggregated, fraction (Figure 1D⇓). We have now carried the WT-CryAB lines for ≈16 months. To date, there is no increase in mortality for the WT-CryAB TG mice relative to nontransgenic (NTG) littermates. However, line 134 R120G-CryAB TG mice, which express the transgene at lower levels than even those of line 11 (WT-CryAB), died at 5 to 7 months of age (Figure 2⇓). On dissection, the hearts were grossly enlarged and dilated. Atrial thrombosis and sometimes calcification were evident. Both pulmonary and hepatic congestion, pleural effusion, and/or ascites, as well as subcutaneous edema, were observed in the autopsies, a pathology consistent with death by congestive heart failure. Line 708, which expresses R120G, but at lower levels, also developed a similar phenotype but only after 12 to 16 months (data not shown). Line 25, which also contained a single copy of the transgene, has not been studied in detail, although the cardiomyocytes do show aggregates similar to those seen in lines 134 and 708. Line 11 (WT-CryAB) and lines 708 and 134 (R120G-CryAB) were chosen for detailed characterization, as line 11 is the WT-CryAB line of which the TG copy number and protein expression level are closest to the mutant line.
R120G-CryAB Causes Aberrant Desmin and CryAB Aggregation
The hallmark of DRM is the presence of aberrant desmin aggregates in myocytes of the affected muscle. These aggregates display a unique morphology at the ultrastructural level.15 We recently created and characterized a TG mouse model of DRM, in which the disease is caused by TG overexpression of a desmin cDNA that carries a mutation that causes human disease.17 Because CryAB associates with desmin, we wished to determine whether the R120G mutation resulted in a pathological outcome similar to DRM. If the R120G mutation is sufficient and causative for DRM, expression of the mutant protein in vivo should result in aggregate formation. We used light microscopy, immunofluorescence confocal microscopy, transmission electron microscopy, and immunoelectron microscopy to characterize potential aberrant desmin aggregation in 12-week-old TG hearts. Virtually every myocyte in the line 134 R120G CryAB TG hearts displayed eosinophilic aggregates in paraffin sections that were stained with Gomori’s modified trichrome (Figure 3⇓). Although not as readily apparent in the trichrome-stained sections, myocytes in the lower-expressing R120G lines showed a similar morphology in that aggregates, which stained intensely for CryAB, could be detected (Figure 2⇑ online). Line 11, the WT overexpressor, appeared normal (Figure 3⇓). The number and size of aggregates increased as the R120G animals aged but could not be detected in the WT-overexpressing lines at any age tested (data not shown).
The distribution and organization of both desmin and CryAB were investigated using immunofluorescence confocal microscopy. Immunostaining for desmin in cardiac myocytes showed that the desmin networks were well preserved in the WT-CryAB TG heart and the staining pattern was not different from that in NTG hearts (Figures 4a⇓ and 4b⇓). The normal desmin network was, however, disrupted in the R120G-CryAB TG hearts. The striated pattern was absent, especially in the central parts of the cell; aberrant aggregates of desmin were obvious (Figure 4c⇓). The immunolabeling in cardiac myocytes also demonstrated that CryAB distribution in WT-CryAB TG hearts was normal. The transverse striated pattern is apparent in both NTG and WT-CryAB cardiomyocytes (Figures 4d⇓ and 4e⇓), although the increased CryAB level in the WT-CryAB TG cells resulted in a more homogenous staining background with higher fluorescent intensity (Figure 4e⇓). Abnormal CryAB-positive aggregates were prominent in the R120G TG myocytes (Figure 4f⇓ and online Figure 2B⇑, available at http://www.circresaha.org) but were not present in either the WT-CryAB TG or the NTG cells. Double-immunostaining for desmin and CryAB was subsequently performed to decipher the relationship between the desmin and CryAB aggregates. Although some regions of the aberrant CryAB aggregates were desmin positive as evidenced by the yellow color (overlay), the desmin aggregates (red) were, for the most part, distributed outside of the CryAB (green) aggregates (Figure 4i⇓).
As noted above, the hallmark of DRM is the appearance of electron-dense, granular aggregates in the cytoplasm of the affected cells.18 These structures were abundant in the R120G cardiomyocytes. Two populations of electron-dense aggregates, named type I and II, were present in the intermyofibrillar space (Figure 5⇓). Type I aggregates (Figure 5c⇓, asterisk) had a relatively low electron density and occupied a large portion of the central part of the cardiomyocyte. They were substantially larger and more regular in shape than type II. They had clear boundaries but were not enclosed in a membrane. In comparison with type II aggregates, they contained finer granules. Type II aggregates (Figure 5c⇓, arrow) were relatively small but more numerous. They were irregular in shape and surrounded by numerous fine filaments. Morphologically, type II aggregates resembled the desmin aggregates observed in TG animals that expressed the mutant desmin protein.17 Some of the type II aggregates appear to be associated with the nuclear envelope and others with the Z-band (Figure 5c⇓). No direct physical association between the two aggregate types was apparent (Figure 5c⇓), although occasionally we noted that type II aggregates were trapped in type I aggregates (data not shown). Immunoelectron microscopy analyses confirmed that type I aggregates were CryAB positive (Figure 6c⇓) with only a few scattered grains resulting from immunogold labeling with the desmin antibody (Figure 7b⇓). Type II aggregates were CryAB and desmin positive (Figures 6⇓ and 7⇓). A number of desmin-positive filaments outside of the type II aggregates were also observed, but no filamentous structure was associated with type I aggregates (Figure 7b⇓). Ultrastructural analyses also showed that the alignment of adjacent myofibrils at the Z-band was perturbed in the R120G-CryAB TG hearts (Figure 5c⇓). Generally in these animals, Z-band thickness was increased and its normal uniformity lost (Figures 5 through 7⇓⇓⇓). The pathologies were essentially identical between the left and right ventricles.
Expression of the CryAB Mutation Causes Cardiac Hypertrophy
Common outcomes of DRM are skeletal muscle atrophy and cardiac hypertrophy and, as expression of the mutant protein was restricted to the heart, we wished to determine whether the R120G mice recapitulated this aspect of the human disease. At the molecular level, activation of fetal genetic programs is common during the early stages of hypertrophy, with upregulation of atrial natriuretic factor and β-myosin and downregulation of α-myosin, phospholamban, and the sarcoplasm reticulum calcium pump. Transcript levels were measured at 1, 3, and 6 months of age as the R120G pathology developed (Figure 3⇑ online, available at http://www.circresaha.org). The relative amounts of these transcripts as well as those of desmin and total CryAB showed a pattern consistent with the morphological disarray apparent in the confocal and transmission electron microscopy analyses. That is, expression of R120G-CryAB but not overexpression of WT-CryAB triggered a hypertrophic response at the molecular level in the heart. By 3 months, hypertrophy was apparent at the gross level as well, with significant increases in the ventricular weight/tibial length ratios (Figure 4A⇑ online). Hypertrophy continued to develop and was even more pronounced at 6 months.
To determine whether the cardiomyocytes themselves were larger, the cell volume, profile area, and length, as well as the transverse sectional area (TSA), were measured at 3 and 6 months (Figure 4⇑ online). In both the left and right ventricles, cardiomyocyte size progressively increased (P<0.01) in the R120G-CryAB TG mice as compared with the NTG and WT-CryAB TG controls. At 3 months, the increase in cell size was due to increases in the TSA. By 6 months, both the cell length and TSA were larger, indicating that concentric hypertrophy at the cellular level occurs in both ventricles at 3 months and ventricular chamber dilatation occurs later on as the heart begins to fail. This is consistent with the clinical progression observed in many cardiovascular diseases, in which a compensatory hypertrophy is observed early, but later on decompensated heart failure presents. No abnormalities were observed in the cohort that overexpressed the WT protein.
R120G-CryAB Leads to Cardiac Dysfunction
Cardiac function in patients with DRM is often significantly compromised. As the disease progresses, the heart dilates, systolic function becomes compromised, and heart failure occurs. Considering the effects of R120G-CryAB expression on the cellular structure and organization, and the resultant hypertrophy, we wished to measure the impact of R120G-CryAB or WT-CryAB TG expression on cardiac function at 3 months of age. In the early stages of compensatory hypertrophy, contractile function is often maintained or even increased, whereas deficits in relaxation begin to occur.19 To divorce the system from endogenous β-adrenergic stimulation and determine whether any functional deficits presented at this stage, an isolated work-performing heart preparation20 was used (Figure 8A⇓). Contractile function, as measured using the left ventricular pressure waveform in the isolated work-performing heart, showed that +dP/dt was higher in the R120G-CryAB TG hearts than in the NTG and WT-CryAB TG controls (P<0.005). Relaxation, as measured by the first derivative of left ventricular pressure (−dP/dt), was significantly lower than in either control cohort (P<0.005).
By 6 months, the animals’ presentation was consistent with severe heart failure and, rather than the working heart, in vivo hemodynamics17 21 were used so that the neurohumoral axis, which helps to maintain cardiac function, could be taken into account. In vivo hemodynamics showed that baseline absolute values of both dP/dtmax and dP/dtmin were significantly decreased relative to the NTG and WT-CryAB TG controls. Even when stimulated via catecholamines, the hearts were unable to maintain normal contractility and an overt response to β-agonist stimulation via dobutamine infusion was substantially blunted (Figures 8B⇑ and 8C⇑). Determination of tau (Figure 8D⇑), the monoexponential time constant of relaxation, showed that the deficits in relaxation were relatively load independent. The data show that cardiac function is significantly compromised.
Cardiac and skeletal muscles contain the highest CryAB levels among the nonlenticular tissues. Upregulation of CryAB occurs in a number of cardiac disorders, including familial hypertrophic cardiomyopathy and DRM, but the functional consequences are unknown. Linkage of R120G-CryAB to familial DRM suggests that normal CryAB function, which presumably involves chaperone activity, is crucial for desmin filament formation and/or function. Interestingly, the mutant CryAB protein appears to be relatively resistant to degradation as compared with normal CryAB. As the TG mice age, total CryAB protein in the mutant CryAB hearts progressively increases, whereas CryAB protein levels stay relatively constant in the WT-CryAB TG heart despite much higher transcript levels (data not shown). The relationship between CryAB, desmin, and DRM pathogenesis remains obscure. The data presented in this study show that upregulation of normal CryAB is not, by itself, detrimental to the heart. However, in the intact animal, the R120G mutation results in both abnormal CryAB aggregation and aberrant desmin aggregation. The sequelae accurately recapitulate the progression of cardiovascular disease as the heart first attempts to compensate for the structural insult by hypertrophying but eventually transits into a decompensated, dilated state with heart failure as the final outcome.
No discernible phenotype presented in the WT-CryAB TG mice, supporting the conclusion that simple alterations in CryAB stoichiometry are not responsible for the phenotype. Oligomerization is required for CryAB to exert its molecular chaperone function,16 22 and this is consistent with the R120G-missense mutation of CryAB being dominant negative. The genetics of the R120G mutation indicate an autosomal dominant inherited pattern in the human disease,14 and in vitro analyses of R120G-CryAB polymers, or a mixture of R120G:WT-CryAB protein, result in altered morphology and compromised molecular chaperone function.16 22 The TG mice offer possible insights into the functional deficits that result. The type I and II aggregates in the R120G mice are intriguing. The two types of aggregates are distinguished both by their morphology and by their protein composition. Type I aggregates are CryAB positive and contain only traces of desmin, whereas type II aggregates are both desmin and CryAB positive and are similar to the characteristic desmin aggregates found in DRM hearts carrying desmin mutations. It is well established that WT CryAB binds to desmin and desmin filaments, especially when cells are stressed.4 23 24 We hypothesize that the relative paucity of desmin in the R120G-CryAB–loaded type I aggregates is due to the inability of the mutant CryAB to productively interact with desmin. The data also indicate that desmin aggregate formation in these mice is not due to a physical or biochemical interaction between desmin and the R120G mutant CryAB. Indeed, formation of the aberrant desmin aggregates may be caused by a loss of function of the mutant CryAB.
We observed significant differences in copy number and expression levels for the WT versus R120G transgenes (Figure 1⇑). The explanation probably lies in the embryonic lethality of embryos containing higher copy numbers of the R120G transgene. In fact, an R120G-CryAB founder with ≈30 copies of the transgene died at 8 weeks and so could not be bred. Another founder, who was mosaic for the transgene, produced TG pups who all died from congestive heart failure before 4 weeks. When analyzed, these mice had 40 copies of the R120G transgene, and the histology (at 3 weeks) showed tremendous aggregate accumulation in the cardiomyocytes (data not shown).
Previously we produced a mouse model of DRM by cardiac-specific expression of a transgene containing a desmin mutation that causes human disease.17 Those mice did not exhibit the severe morbidity and mortality that present in the R120G animals. Similarly, desmin-null mice also exhibit a less severe pattern of morbidity and mortality.25 26 27 Thus, both loss-of-function and dominant-negative alleles of desmin result in disease, but the pathology is markedly less severe than that resulting from the R120G-CryAB mutation. R120G TG mice develop cardiac hypertrophy that is concentric at an early stage (3 months) but leads to dilation and failure by 5 to 7 months. All mice from line 134, which have only three copies of the transgene, die during this period. Unfortunately, there is a lack of human data in terms of mutant/WT protein expression with which we can compare the mouse data. The earlier adulthood high mortality of line 134 relative to lines 708 and 25 is almost certainly due to the higher dosage. The higher level of mutant protein expression gives rise to a more pronounced phenotype at an earlier time. We have noted that line 708 TG mice also tend to die prematurely, but this is apparent only in the older adult population, and statistically significant data have not yet been accumulated, although there are alterations at the cardiomyocyte level (Figure 2⇑ online, available at http://www.circresaha.org). The desmin-null mice also die prematurely, but a substantial percentage of the null cohort survives for up to a year.25 26 The desmin mutation–induced DRM TG cohorts live for at least 18 months.17 These observations suggest that the cardiac dysfunction caused by R120G-CryAB must be due to more than just a loss of desmin function and imply either additional roles for CryAB that can impact on the general cytoskeletal architecture or other, as-yet-undefined targets for CryAB interaction(s).
In the young adults (3 months), systolic function is actually increased as measured using the isolated working heart preparation (Figure 8A⇑). Although this may appear to be somewhat surprising, at this early stage, the R120G TG mice show concentric hypertrophy (Figure 4⇑ online). The hypertrophic myocardium displayed an increased +dP/dt in response to changes in preload, and we have observed similar +/−dP/dt profiles in the early stage of concentric hypertrophy induced by pressure overload. Occurrence of hypertrophy at this stage might be due to functional deficit in diastole. Recently, we have evaluated in vivo left ventricular function on 3-month-old mice. Similarly, baseline +dP/dtmax of the R120G TG mice was unaffected (X. Wang and J. Robbins, unpublished observations, 2001). Histological changes at 3 months include primarily concentric hypertrophy with minimal indications of the more advanced changes that accompany failure. We observed the more severe histological changes and increased mortality at 5 to 7 months and beyond and believe the observed increases in +dP/dt at 3 months of age are consistent with the early stages of a compensatory hypertrophy. The primary performance effect of the mutation is decreased diastolic function, which most likely leads to initial compensation at 3 months and ultimate failure at 7 months and beyond.
In cardiomyocytes, desmin filaments link adjacent myofibrils to one another, to the cell membrane, and to the nuclear envelope.26 As other intermediate filaments do in other cells, the desmin filaments play an important role in maintaining the structural integrity of myocytes,25 and thus it is not surprising that alterations in a molecular chaperone, which functions in their transport, can have severe consequences. Previously, Vicart et al14 showed, via transfection of muscle cell cultures with the R120G mutant, that characteristic aggregates developed. Our data significantly extend these observations, showing that stable expression of a mutated chaperone in the heart can lead directly first to a compensated hypertrophy and eventually to heart failure. By combining a combination of biochemical and whole-animal approaches, it should be possible to define the role(s) of CryAB, both in normal cardiomyocyte function and in cardiovascular disease.
This work was supported by NIH Grants HL56370, HL41496, HL56620, HL52318, HL60546, and HL56620 (to J.R.) and HL62459 (to A.M.G.), and by the American Heart Association, Ohio Valley Affiliate (to X.W.).
Original received February 6, 2001; revision received May 8, 2001; accepted May 8, 2001.
- © 2001 American Heart Association, Inc.
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