Circulation Research. 2001
Published online before print June 21, 2001,
doi: 10.1161/hh1301.092688
A more recent version of this article appeared on July 6, 2001
(Circulation Research. 2001;0:hh1301.092688.)
© 2001 American Heart Association, Inc.
Expression of R120G–
B-Crystallin Causes Aberrant Desmin and B-Crystallin Aggregation and Cardiomyopathy in Mice
Xuejun Wang,
Hanna Osinska,
Raisa Klevitsky,
A. Martin Gerdes,
Michelle Nieman,
John Lorenz,
Timothy Hewett
Jeffrey Robbins
From the Division of Molecular Cardiovascular Biology (X.W., H.O., R.K.,
T.H., J.R.), Children’s Hospital Research Foundation, Cincinnati, Ohio;
South Dakota Health Research Foundation–Cardiovascular Research
Institute (A.M.G.), Sioux Falls, SD; and Department of Molecular and Cellular
Physiology (M.N., J.L.), University of Cincinnati Medical Center, Cincinnati,
Ohio.
Correspondence to Jeffrey Robbins, Division of Molecular Cardiovascular Biology, Children’s Hospital Research Foundation, 3333 Burnet Ave, Cincinnati, OH 45229. E-mail jeff.robbins{at}chmcc.org
Abstract
Abstract—Upregulation
of 
B-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.
Key Words: transgenic • heart disease • mouse • cardiac • genetics
The small heat
shock–related protein B-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.
Results
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.
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Figure 1. TG DNA, mRNA, and protein levels. A, Genomic Southern blots show that transgene copies in WT-CryAB mice (upper band) are substantially higher than in R120G-CryAB animals. An EcoRI-SalI fragment (1.7 kb) derived from the -myosin heavy chain promoter region was used as a probe, and the endogenous gene (lower band) allowed internal lane controls for copy number determinations. B, Transcript levels of CryAB in different lines. All RNA samples were derived from 6-week-old hearts. Total CryAB mRNA levels showed good but not exact copy number dependency. Compared with the endogenous CryAB mRNA level in NTG hearts, CryAB mRNA levels in lines 13, 41, 11, 708, 25, and 134 were increased by 106-, 50-, 22-, 3.5-, 4.0-, and 9.0-fold, respectively. Histogram represents degree of overexpression relative to endogenous transcript; values obtained from 1 to 5 animals were averaged. C, Western blot analyses of CryAB in the insoluble and soluble fractions. Desmin, actin, and CryAB levels in the insoluble fraction were determined simultaneously by immunoblotting. GAPDH and CryAB levels in the soluble fraction were also determined simultaneously by immunoblotting. Actin and GAPDH were used as loading controls. D, Quantification of CryAB protein levels. Each of the R120G-CryAB TG lines (708, 25, and 134) had increased CryAB in the insoluble fraction relative to the comparable WT-CryAB TG lines. All data were normalized to the actin signal (for the insoluble fraction) or GAPDH signal (for the soluble fraction) in the same lane before being plotted. The experiment was repeated twice; error bars indicate SD. Compared with the endogenous CryAB protein level of NTG hearts, CryAB protein expression was increased in lines 13, 41, 11, 708, 25, and 134 by 10-, 9-, 8-, 5-, 5.2-, and 7-fold, respectively.
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Figure 2. Kaplan-Meier curve. Lines 11 (WT-CryAB) and 134 were analyzed. A majority of R120G-CryAB TG mice died of congestive heart failure between 25 and 28 weeks of age, and all died by 32 weeks. No increases in mortality rate relative to NTG controls were detected in WT-CryAB TG mice.
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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).
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Figure 3. Trichrome-stained myocardial sections. Shown are paraffin-embedded sections prepared from 10% buffered formalin perfusion–fixed hearts from 12-week-old mice. Lines 11 (WT-CryAB) and 708 and 134 (R120G-CryAB) were analyzed. Eosinophilic aggregates (arrows) are present in essentially every line 134 myocyte. Similar but substantially smaller aggregates were found in TG hearts of line 708, an R120G line with lower levels of transgene expression. No aggregates were observed in WT-CryAB TG hearts. Bar=20 µm.
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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).
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Figure 4. Immunofluorescence confocal analyses. a through c, Immunostaining for desmin in isolated left ventricular myocytes. Distribution of desmin in line 11 WT-CryAB TG (wt-tg) myocytes was identical to the NTG myocytes. Desmin distribution in line 134 R120G-CryAB TG myocytes (c) was altered dramatically. Typical striated pattern was absent, and irregular desmin-positive aggregates were present. d through f, CryAB immunostaining in left ventricular myocytes. Immunostaining of CryAB in NTG myocytes displayed weak transverse striations in a homogenous staining background, similar to the pattern observed in the line 11, WT-CryAB myocytes. Staining pattern in the R120G-TG myocytes differed, in that numerous CryAB-positive aggregates were prominent in the central part of the cells. Imaging gain used for collecting the NTG image was 3-fold that used for the WT-TG and R120G-TG images. g through i, Double immunostaining for desmin and CryAB in cryosections. Relationship between CryAB aggregates and desmin aggregates in R120G-CryAB TG myocytes was explored by double staining. As shown by the overlay image, some desmin aggregates overlapped portions of the CryAB aggregates, but the majority lay outside these structures. The area outside of both types of aggregates appears completely black because desmin and CryAB aggregates fluoresce so brightly that it was necessary to scale the imaging gain to very low levels to visualize aggregate morphology. Bar=20 µm.
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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.
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Figure 5. Electron microscopy. Sections were obtained from 3-month-old NTG (a), WT-CryAB TG (b), and R120G-CryAB TG (c) left ventricles. In both panels a and b, sarcomeres are organized normally with clearly distinguishable I- and A-bands, and M-lines as well as regularly aligned Z-bands. Mitochondria are typically arranged along myofibrils. The sarcoplasmic reticulum is closely associated with sarcomeres. No major differences between NTG and WT-CryAB TG samples are apparent, although there are subtle alterations in the space where mitochondria are localized (panel a vs panel b). In R120G-CryAB TG hearts (c), two populations of abnormal aggregates are present. Type I aggregates (*) are relatively large, regularly shaped, and of low electron density. Type II aggregates (arrows) are smaller and irregularly shaped, display relatively high electron density, and are associated with Z-bands. Z-band morphology, particularly near the aggregates, is perturbed. Bar=1.0 µm.
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Figure 6. Anti-CryAB immunogold labeling. CryAB distribution was analyzed at the ultrastructural level. Left ventricular sections were obtained from NTG (a), WT-CryAB TG (b), and R120G-CryAB TG (c and d) 12-week-old mice. CryAB distribution in the WT-CryAB TG and NTG hearts is similar, although there is some increased labeling in the intermyofibrillar space (a and b). Both the type I (*) and II (arrow) aggregates in R120G-CryAB TG hearts (c and d) are CryAB positive, but staining in type I aggregates was twice the intensity found in type II aggregates. Bar=0.5 µm.
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Figure 7. Antidesmin immunogold labeling. Distribution of desmin was analyzed at the ultrastructural level. Left ventricular sections were obtained from NTG (a) or R120G-CryAB TG (b) 12-week-old mice. Immunogold particles in the NTG heart were in the intermyofibrillar space, mainly at the Z-line level and intercalated disks. In R120G-CryAB TG hearts (b), most label was localized in type II aggregates (*), with type I aggregates being only slightly positive. No desmin labeling was evident in areas immediately surrounding type I aggregates, whereas a number of desmin-positive filaments surrounded the type II aggregates. Bar=0.5 µm.
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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 ß-myosin and downregulation
of -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 ß-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).
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Figure 8. Functional analyses. A, Isolated work-performing heart preparations were used to examine left ventricular function at 3 months. No differences were detected between NTG and WT-CryAB TG mice (n=6). B and C, Closed chest intact mouse model and dobutamine infusion protocols have been described in detail.21 D, Tau, the monoexponential time constant of relaxation, is relatively load-independent and was determined from primary data. Each experimental set (n=5) consisted of 23-week-old animals. Cardiovascular function was measured at baseline (no dobutamine) and under increasing ß-adrenergic stimulation. The three cohorts were all subjected to the same regimen, which included a series of 3-minute infusions of increasing concentrations of dobutamine and measurement of cardiovascular indices in the last 30 seconds of each period to obtain peak response. Pressure signals from both the COBE and Millar transducers were recorded using a MacLab 4/s data acquisition system (AD Instruments). The software directly determines arterial systolic and diastolic pressure, mean arterial pressure, heart rate, left ventricular systolic pressure, developed pressure, and both positive (dP/dtmax) and negative (dP/dtmin) dP/dt. Data were analyzed using a mixed, two-factor ANOVA with repeated measures on the second factor. At all points, there were no statistically significant differences between the NTG and WT-CryAB cohorts. When necessary, post hoc comparisons were performed by single degree-of-freedom contrasts. Compared with NTG and WT-CryAB animals, both dP/dtmax and dP/dtmin differed significantly at the indicated points. *P 0.05, #P0.005.
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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 ß-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.
Discussion
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.
Acknowledgments
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.).
Footnotes
Original received February 6, 2001; revision received May 8, 2001; accepted May 8, 2001.
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