(Circulation. 1997;96:3466-3476.)
© 1997 American Heart Association, Inc.
Articles |
Abundance and Location of the Small Heat Shock Proteins HSP25 and 
B-Crystallin in Rat and Human Heart
From the Max Delbrück Center for Molecular Medicine, Berlin, Germany (G.L., R.V., M.W., J.S., R.B.); the Clinic of Internal Medicine, Charité, Humboldt University Berlin, Germany (U.O., I.S.); the Department of Pathology, Philips University Marburg, Germany (H.-J.G.); and the Division of Cancer Research, Department of Pathology, University of Zürich (Switzerland) Medical School (R.K.).
Correspondence to Dr Gudrun Lutsch, Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str 10, D-13122 Berlin, Germany. E-mail lutsch{at}mdc-berlin.de
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Abstract |
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Background In the heart, there are high constitutive levels of the two related small heat shock proteins, HSP25 and
B-crystallin. To gain insight into their functional role, we have
analyzed abundance and location of both proteins in rat and
human hearts at different stages of development and in diseased
state.
Methods and Results Immunoblotting
analysis of rat ventricular tissue at fetal,
neonatal, and adult stages reveals the level of HSP25 to decline
strongly during development, whereas the level of B-crystallin
remains nearly constant. In parallel, the portion of
phosphorylated isoforms of HSP25 decreases as shown by
two-dimensional polyacrylamide gel electrophoresis. HSP25 is
detected in cardiomyocytes and endothelial
and vascular smooth muscle cells, whereas B-crystallin is detected
in cardiomyocytes only by
immunofluorescence and immunoelectron microscopy.
Both proteins colocalize in the I-band and M-line region of myofibrils
in cardiomyocytes. In diseased and transplanted adult human
hearts, HSP25 and B-crystallin levels are considerably elevated
compared with fetal hearts. In failing adult human hearts,
phosphorylated isoforms of HSP25 predominate, and
cardiomyocytes with a partial dislocation of HSP25 and
B-crystallin are observed.
Conclusions Differential accumulation and location of HSP25
and B-crystallin in heart tissue during development imply distinct
functions of both proteins, which seem to be involved in organization
of cytoskeletal structures. As judged by level,
phosphorylation state, and location of both small heat
shock proteins, diseased adult human hearts share features with fetal
hearts.
Key Words: ventricles • immunohistochemistry • heart failure • proteins
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Introduction |
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Stress or HSPs are known to exert housekeeping functions in normal cell metabolism as well as protective functions under stress conditions in virtually all cell types studied. In the myocardium, elevated levels of HSPs have been described to be associated with cardiac protection against injury caused by ischemia/reperfusion or other stressful treatments such as hypoxia, ATP depletion, glucose deprivation, and hypotonicity.1 2 Thus, stress proteins are thought to represent a natural defense system that enables myocardial cells to survive stressful situations. Evidence for a cardioprotective function of HSP70 has been obtained recently in transfection experiments using a myogenic cell line derived from murine embryonic heart.3 4 Confirmation came from investigations with transgenic mice hearts overexpressing HSP70 that exhibited an elevated resistance to ischemic/reperfusion injury.5 6
Little is known about the cardiac function of HSP25 and
B-crystallin, which occur at relatively high levels in the
myocardium.7 8 9 10 11 12 13 Both proteins are structurally
and functionally related.14 15 They share sequence
homology and the tendency to form oligomeric particles ranging in size
from 200- to 800-kD molecular mass or even more.16 17 18 Both
HSP25 and B-crystallin are found in unphosphorylated
and phosphorylated isoforms.
Phosphorylation of HSP25 occurs in response to a number
of mitogens and stress factors (see Reference 1515 and references cited
therein), including oxidative stress,19 20 whereas factors
influencing phosphorylation of B-crystallin are
largely unknown.21 22 Phosphorylation of
HSP25 is catalyzed by HSP25 kinase (MAPKAP kinase-2),23 24 25
whereas phosphorylation of B-crystallin is most
likely catalyzed by a cAMP-dependent protein kinase.21 A
further common property of both proteins is their chaperoning activity
as demonstrated in in vitro assays.26 27 At the cellular
level, HSP2528 and B-crystallin29 30 have
been shown to be important determinants of acquired stress
tolerance.
For both small HSPs, interactions with myofibrils and the cytoskeleton
have been described. HSP25 is involved in sustained contraction of
rabbit gastrointestinal smooth muscle induced by bombesin and protein
kinase C31 and colocalizes with actin in myofibrils of rat
cardiomyocytes32 and with microfilaments in
rat Sertoli cells.33 It inhibits actin polymerization in
vitro,34 35 and stabilization of the microfilament network
in rodent nonmuscle cells has been observed after overexpression of
HSP25 in vivo.36 B-crystallin, on the other hand,
associates with actin and desmin in heart tissue37 38 39 and
is thought to prevent aggregation of actin filaments at acidic
pH.38 39 In vitro, B-crystallin was shown to inhibit
the assembly of vimentin.40
In this study, we investigate the abundance and location of HSP25 and
B-crystallin in rat and human ventricular tissue at
different stages of development and in diseased state. By application
of immunoblotting,
immunofluorescence, and immunoelectron microscopy,
we have observed differential regulation of the abundance of both
proteins during ontogenesis. In cardiomyocytes, both
proteins colocalize in the I-band and M-line region of myofilaments,
suggesting their involvement in the assembly and function of
myofibrils.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Preparation of Protein Extracts of Rat Hearts
Experiments were performed with Wistar, WKY, and Sprague-Dawley rats held in accordance with institutional guidelines. Hearts of Wistar rats were prepared at different stages of development (fetal stages, embryonic days 15 and 20; postnatal stages, days 1, 3, 6, 12, 21, and 30; adult stage, 2 years); hearts of Sprague-Dawley and WKY rats were investigated at the ages of 2 and 8 months, respectively. The number of rats in each studied group ranged between 5 and 10. To obtain fetal hearts, female Wistar rats were killed by decapitation at the indicated days of gestation to remove fetuses from uteri. After the fetuses were decapitated, hearts were rapidly removed and immersed in ice-cold wash buffer (130 mmol/L NaCl, 30 mmol/L KCl, 10 mmol/L histidine, pH 7.4). Hearts of newborn and adult rats were prepared correspondingly. After removal of atria, isolated ventricles were blotted, immediately frozen in liquid nitrogen, and stored at -70°C. For protein extraction, two methods were used. In method 1, 20 to 80 mg of frozen ventricular tissue was placed into 16 volumes of ice-cold homogenization buffer I (250 mmol/L sucrose, 10 mmol/L histidine, pH 7.4) and homogenized 6 times for 10 seconds with 15-second intervals by use of a Brinkman Polytron PT 3000 homogenizer (Kinematica AG, Littau/Lucerne) set to 25 000 rpm. The homogenate (60 mg wet tissue per 1 mL) was filtered through polyamide gauze of 90-µm mesh (NeoLab), and samples of 0.1 mL were immediately frozen in liquid nitrogen and stored at -70°C. Protein determination was according to the method of Lowry et al,41 and the protein concentration of each sample was adjusted to 2 mg/mL. This protocol has been successfully used previously for immunochemical quantification of heart proteins.42 In method 2, 20 to 80 mg of frozen ventricular tissue was placed at room temperature in a glass-Teflon Potter homogenizer; 20 µL homogenization buffer II (2% SDS, 80 mmol/L Tris-HCl, pH 6.8, 10% glycerol) was added per 1 mg tissue and allowed to soak for half an hour. After homogenization, extracts were centrifuged for 5 minutes at 15 000g, and supernatants were stored at -20°C. For protein determination, the DC protein assay (Bio-Rad) was used.
Preparation of Protein Extracts of Human Ventricular
Tissue
Human fetal hearts were obtained after abortion at 16 to 22
weeks of pregnancy (hearts 1 through 4). No
pathohistological features were observed by
conventional light microscopy. Ventricular tissue from
adults was obtained from myocardial biopsies of heart transplant
recipients at different time points after transplantation (hearts 5
through 8). Because of limited availability, this material was used
only for SDS-PAGE followed by immunoblotting.
Ventricular tissue of explanted hearts of patients
suffering from end-stage heart failure was available in larger
quantities and hence was used for 2D-PAGE followed by
immunoblotting and
immunofluorescence microscopy (hearts 9 through
12). Both fetal specimens and biopsies from transplanted and explanted
hearts were immediately frozen in liquid nitrogen and stored at
-70°C. Extraction of proteins was performed as described for rat
hearts.
SDS-PAGE, 2D-PAGE, and Immunoblotting
For SDS-PAGE, homogenates were adjusted to 5%
ß-mercaptoethanol, 2% SDS, 80 mmol/L Tris-HCl, pH 6.8,
and 10% glycerol (final concentrations) and boiled for 3 minutes;
proteins (18 µg total protein) were separated on 7% to 15%
polyacrylamide gels as described by Laemmli.43 For
2D-PAGE, proteins were extracted by method 1, precipitated at -20°C
with 80% ethanol, and processed according to
O'Farrell.44 For Western blotting, separated proteins
were transferred onto nitrocellulose by semidry electroblotting at 0.8
mA/cm2, and membranes were processed for immunodetection
following the procedure described in the technical manual ProtoBlot for
Western Blot Alkaline Phosphatase System (Promega Corp). The following
antibodies were used: (1) polyclonal rabbit anti-HSP25 antibody
recognizing murine and rat HSP25,45 (2) polyclonal rabbit
anti-HSP25 antibody recognizing preferentially human
HSP25,46 (3) polyclonal rabbit anti-B-crystallin
antibody,29 and (4) monoclonal mouse anti-HSP70 antibody,
clone C92F3A-5, recognizing the inducible form (HSP70i) of the HSP70
family (StressGen). Proteins were visualized by alkaline
phosphatase–conjugated secondary antibodies (Sigma). Optical density
of immunoreactive bands was evaluated by integration over the whole
area of extinction by use of a 2202 Ultroscan laser densitometer (LKB).
For quantitative determination of HSP contents, reference solutions of
recombinant murine and human HSP25 (StressGen), recombinant human HSP70
(StressGen), and bovine B-crystallin (kind gift of R. Chiesa, New
York, NY) were used, the protein content of which was determined by
quantitative amino acid analysis. Distinct amounts of proteins
were used to ascertain linearity between the amount of protein loaded
onto gels and the intensity of the obtained signals. Protein standards
usually were run in three different concentrations on the same blot.
Data of three independent experiments are presented. To relate
HSP content to tissue weight, ventricular protein content
was determined per gram of wet tissue weight. The
Table
shows that total
ventricular protein content raises about three times
between days 1 and 30 of postnatal development.
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Immunofluorescence Microscopy
For microscopic investigations, rat hearts were prepared as
described above. Samples of fetal and neonatal ventricles were fixed
with a solution containing 4% formaldehyde (freshly prepared from
paraformaldehyde), 0.5%
glutaraldehyde, 0.18 mol/L sucrose, and 0.1
mol/L phosphate buffer, pH 7.4, for 2 hours at room temperature.
Fixation of adult rat heart tissue by this procedure resulted in poor
HSP25 labeling. Therefore, all investigated samples were fixed in
parallel with a fixative in which glutaraldehyde was
omitted. Ventricular tissue of explanted human hearts was
fixed in 4% phosphate-buffered formaldehyde. For cryosectioning,
samples were infiltrated with 2.3 mol/L sucrose for several
hours at room temperature and frozen in liquid nitrogen.47
Semithin cryosections of 1-µm thickness were prepared with an
Ultracut E ultramicrotome equipped with a Cryocut FC4E cryoattachment
(Leica). Immunolabeling was done with polyclonal rabbit and goat
anti-HSP2545 46 and rabbit anti-B-crystallin
antibodies29 (Serotec). For identification of different
cardiac cell types, the following monoclonal mouse antibodies were
used: anti–-sarcomeric actin (clone 5C5, Sigma), anti–rat
endothelial cell (clone OX-43, Dianova), anti-human
CD31 (clone HC1/6, Serotec), and anti–smooth muscle actin antibodies
(clone asm-1, Boehringer). In double-labeling experiments with
anti–-sarcomeric actin antibodies, cryosections were postfixed with
methanol for 10 minutes at -20°C to improve immunolabeling. In
single- and double-labeling experiments, primary antibodies were
visualized by staining with DTAF- and/or Cy3-conjugated
species-specific secondary antibodies (Dianova). To suppress unspecific
labeling, cryosections were preincubated with a solution containing
20 mmol/L Tris-HCl, pH 8.4, 130 mmol/L NaCl,
0.05% Tween 20, and 1% BSA (1% BSA–Tris) for 30 minutes at room
temperature. Primary and secondary antibodies were diluted with the
same solution to a protein concentration of 10 to 50 µg/mL and
2 to 10 µg/mL, respectively. Incubation with primary
antibodies was performed overnight at room temperature; incubation with
secondary antibodies, for 1 hour at 37°C. Washing steps were carried
out with 1% BSA–Tris containing an additional 500 mmol/L
NaCl. Controls were performed with the IgG fraction of nonimmune serum
at the same protein concentration as used for the primary antibody and
with primary antibodies preincubated with the corresponding protein.
Tissue sections were evaluated with an Axioplan fluorescence
microscope (Carl Zeiss) with appropriate filter systems. Micrographs
were taken with a MC100 automatic camera (Carl Zeiss) with Kodak TMax
400 film.
Immunoelectron Microscopy
Fixation, sectioning of ventricular tissue, and
incubations were done as described above with the following
modifications: section thickness was adjusted to 50 to 70 nm, and
antibodies were detected with protein A–gold complexes of 10- and
15-nm diameter (obtained from J. Slot, University of Utrecht,
the Netherlands). Double-labeling experiments were carried out as
described by Griffiths48 with a fixation step after
labeling of the first antibody. After immunolabeling, cryosections were
embedded and stained by a mixture of 2% methylcellulose and 0.3%
uranyl acetate.47
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Levels of HSP25 and
B-Crystallin in Rat Hearts at Different
Stages of DevelopmentImmunochemical detection of HSP25,
B-crystallin, and, for
comparison, HSP70i in hearts of Wistar rats at different developmental
stages between embryonic day 15 and 2 years reveals that the amounts of
HSP25 and HSP70i are highest in embryonic and early postnatal hearts
and decrease with further heart development, whereas B-crystallin
levels remain virtually constant (Fig 1A). As Fig 2
shows, quantitative determination of
immunostained protein bands shows that the HSP25 content
(closed columns) declines from 3 to 4 µg/mg protein in
embryonic hearts to 
2 µg/mg protein in neonatal hearts at
day 1 and to 0.7 µg/mg protein at day 12. These values
correspond to 87 and 60 µg HSP25/g wet tissue weight at days 1 and
12, respectively, as determined from data shown in the Table
.
Afterward, the HSP25 level decreases more slowly and reaches about 0.2
µg/mg protein in adult animals, corresponding to 38 µg
HSP25/g wet tissue weight. In comparison, the HSP70i level is subject
to a more moderate development-dependent decrease, ranging from about
0.6 µg/mg protein in prenatal stages to about 0.2
µg/mg protein in adults (Fig 2, open columns) when related to
total ventricular protein, but remains virtually constant
at about 20 µg when related to gram wet tissue weight. By contrast,
the level of B-crystallin was determined to amount to 3 to 4
µg/mg protein in all stages studied (Fig 2, hatched columns).
Related to gram of wet tissue weight, however, the B-crystallin
level increases between days 1 and 30 from about 150 to 500
µg/g wet tissue weight, respectively. To exclude possible
strain specificities, we analyzed in parallel the cardiac HSP25
content in adult animals of two other rat strains using two different
methods of protein extraction (see "Methods"). Using both
methods, we found HSP25 levels of 0.20±0.02 µg/mg and
0.28±0.02 µg/mg (mean±SD) protein in WKY (8 months of age)
and Sprague-Dawley rat hearts (2 months of age), respectively, values
that are not significantly different from those of adult Wistar rats.
In conclusion, the immunochemical data suggest that the levels of the
investigated three stress proteins are differentially regulated during
rat heart development.
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Because it is known that HSP25 occurs in different
phosphorylation states, which may be of functional
importance, we analyzed the HSP25
phosphorylation status in developing hearts of Wistar
rats using 2D-PAGE and immunoblotting. Fig 1B shows
typical isoform patterns of rat ventricular tissues at
embryonic day 20 (a), at postnatal day 1 (b), and from adult rats (c).
Comparison of the isoform patterns demonstrates that in fetal hearts,
the portion of the diphosphorylated isoform (HSP25/3)
is highest compared with the unphosphorylated (HSP25/1)
and the monophosphorylated (HSP25/2) isoforms, whereas
in neonatal and adult hearts, the unphosphorylated and
the monophosphorylated isoforms dominate.
Location of HSP25 and B-Crystallin in Rat Hearts at Different
Stages of Development
Application of immunofluorescence microscopy
shows that in fetal and neonatal ventricular tissue, most
cardiac cells are strongly labeled with anti-HSP25 and
anti-B-crystallin antibodies. As Fig 3
shows, double-labeling of
ventricular tissue from neonatal rats at day 1 with
anti-HSP25 (Fig 3A) and anti–-sarcomeric actin antibodies (Fig 3B)
demonstrates that HSP25 labeling occurs in both cytoplasm and
myofibrils of cardiomyocytes. When double-labeling is
carried out with anti-HSP25 (Fig 3C) and anti-B-crystallin
antibodies (Fig 3D), nearly identical staining patterns are obtained
indicating colocalization of HSP25 and B-crystallin in
cardiomyocytes. Furthermore, it is obvious from Fig 3C and 3D that smooth muscle cells of blood vessels (thick arrows) and
endothelial cells (thin arrows) are not labeled by
these antibodies. This is confirmed by double-labeling experiments with
anti–B-crystallin (Fig 3E) or anti-HSP25 (not shown) and
anti–smooth muscle actin antibodies (Fig 3F), which demonstrate a lack
of B-crystallin staining in the area of the blood vessel. From these
data, it is concluded that HSP25 and B-crystallin colocalize in
cardiomyocytes of fetal and neonatal
ventricular tissue. In smooth muscle and
endothelial cells, both proteins do not seem to be
expressed at these growth stages. We cannot exclude, however, that they
are expressed below detectable levels or are present in a status
not detectable by the used antibodies.
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In accordance with the results of Western blotting,
ventricular tissue from 12-day-old and older rats reveals
much lower labeling intensity with anti-HSP25 antibodies compared with
samples from newborn animals, whereas labeling intensity with
B-crystallin antibodies remains nearly the same. As Fig 4
shows, on sections of
ventricular tissue from an adult rat, the labeling pattern
in cardiomyocytes has, however, changed from being mainly
cytoplasmic in fetal and neonatal hearts to mainly myofibrillar in
sections from older animals. A clear striated staining pattern is seen
in longitudinal sections of cardiomyocytes incubated with
anti-HSP25 (Fig 4A) and anti–B-crystallin antibodies (Fig 4C).
Double-labeling with anti–-sarcomeric actin antibodies (Fig 4B)
shows that HSP25 colocalizes with actin and is therefore located in the
I band of myofibrils. In some myofibrils, a splitting of the stained I
bands into two parts separated by a dark zone is recognizable (insets
in Fig 4A and 4B). This region represents the Z line where thin
filaments of sarcomeres are anchored. Additionally, in strongly stained
cells, HSP25 labeling occurs also in the region of the M line being
located in the middle of the A band and characterized by the presence
of myosin filament bundling proteins. The same staining pattern is
observed when double-labeling experiments with anti–B-crystallin
and anti–-sarcomeric actin antibodies are performed, ie, both small
HSPs colocalize in the I-band and the M-line region of
cardiomyocytes. This colocalization is confirmed by
double-labeling experiments with anti-HSP25 (Fig 4C) and
anti–B-crystallin antibodies (Fig 4D). Again, splitting of the
stained I-band region and staining of the M-line region is recognizable
at higher magnification (insets in Fig 4C and 4D). Furthermore, it is
obvious that there are differences in staining patterns between
anti-HSP25 and anti–B-crystallin antibodies with respect to
nonmuscle cells. Blood capillaries located between
cardiomyocytes (thin arrows in Fig 4C) and smooth muscle
cells of larger vessels (thick arrow in Fig 4C) are labeled by the
anti-HSP25 antibody, whereas these areas are not labeled by the
anti–B-crystallin antibody (arrows in Fig 4D). This labeling
pattern is also observed in 30-day-old rats but not in earlier growth
stages analyzed.
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Immunoelectron microscopy was used as a second approach to localize
HSP25 and B-crystallin in rat ventricular tissue. In
fetal rat ventricular tissue (Fig 5A), a strong cytoplasmic labeling of
B-crystallin (10-nm gold particles) and HSP25 (15-nm gold particles)
is observed in cardiomyocytes by application of the protein
A–gold technique, while nuclei and mitochondria are not labeled. No
labeling is seen in endothelial cells and fibroblasts
at this growth stage. In ventricular tissue of adult rats,
double-labeling reveals that B-crystallin and HSP25 colocalize in
the I band of myofibrils in cardiomyocytes (Fig 5B). Here,
the I band is differentiated from the A band by its lower contrast and
the existence of the dark Z line. HSP25 but not B-crystallin is
furthermore observed in endothelial cells of
ventricular tissue from adult rats (not shown).
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Abundance and Location of HSP25 and B-Crystallin in Hearts of
Human Fetuses and Adults
To compare the data obtained for rat heart with those in humans,
abundance, isoform pattern, and location of HSP25 were analyzed
in hearts of fetuses and adults. Fetal hearts were obtained from
abortions; samples of adult hearts came from biopsies of heart
transplant recipients and from explanted hearts. As obvious from
immunoblots after SDS-PAGE (Fig 6A), in human fetal (hearts 1 through 4)
and transplanted adult hearts (hearts 5 through 8), pronounced HSP25
signals are observed, although the intensities differ to some extent.
Deviating from the situation in rats, the studied transplanted adult
human hearts apparently contained higher levels of HSP25 than three
(hearts 2 through 4) of the four fetal hearts analyzed.
Quantitative determination of HSP25 yielded values of 1.0, 0.9, 1.2,
and 0.9 µg/mg protein for the adult hearts, which are
significantly higher than the values determined for adult rat hearts.
In the same hearts, the levels of B-crystallin differ considerably
(strong labeling, hearts 1, 6, and 8; weak labeling, hearts 2 through 5
and 7), again indicating a differential regulation of abundance of both
proteins. In comparison, HSP70i was detected in all fetal hearts and in
only one adult hearts (heart 8). This finding parallels the decrease of
HSP70i level found in developing rat hearts (cf Fig 1A).
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In contrast to rat HSP25, human HSP25 is phosphorylated
at three sites,12 22 and consequently four isoforms
(unphosphorylated HSP25/1,
phosphorylated HSP25/2-4) are detected on 2D gels (Fig 6B). However, the occurrence of HSP25 isoforms varies: fetal heart 1
contains all four HSP25 isoforms (Fig 6B, a), fetal heart 2 contains
isoforms HSP25/1, HSP25/2, and HSP25/3 (Fig 6B, b), and fetal hearts 3
and 4 contain isoforms HSP25/1 and HSP25/2 (Fig 6B, c and d). In fetal
hearts 1 and 2, the phosphorylated isoforms
predominate, while in fetal hearts 3 and 4, the
unphosphorylated isoform predominates. For
analysis of HSP25 isoform patterns in adult human hearts, we
were restricted to the use of tissue samples of the right ventricles of
pathological hearts of patients with congestive heart failure that had
been explanted because of dilated cardiomyopathy
(hearts 9 and 10), ischemic heart disease (heart 11), and
congenital heart disease (heart 12) (Fig 6B, e through h,
respectively). In these hearts, phosphorylated isoforms
always predominate, although the extent of
phosphorylation varies. The HSP25 content of hearts 9
through 12 was determined to be 2.3, 0.6, 1.1, and 2.6 µg/mg
protein, respectively, which is the same range as in the investigated
transplanted hearts.
With immunofluorescence microscopy, the location of
HSP25 and B-crystallin was studied in human ventricular
tissue of an adult heart explanted because of dilated
cardiomyopathy. As Fig 7
shows, in this
pathophysiological situation, both proteins reveal
a similar staining pattern as described above for
ventricular tissue of adult rats. In human
cardiomyocytes, HSP25 (Fig 7A) and B-crystallin (Fig 7C)
colocalize with actin in the I band of myofibrils as shown by
double-labeling with anti–-sarcomeric actin antibodies (Fig 7B and 7D). In addition, splitting of the I band into two lines and staining
in the region of the M line are observed (insets in Fig 7A and 7C).
HSP25 is detected in vascular endothelial and smooth
muscle cells (Fig 7A), whereas B-crystallin is detected in
cardiomyocytes only (Fig 7C). In addition, regions with
altered location of HSP25 and B-crystallin are observed (Fig 7E and 7F). Here both proteins are located in the cytoplasm between
myofibrils, in the perinuclear region, and most prominently in
contracted cells, at the periphery of cardiomyocytes. This
indicates that profound changes in myofibril architecture have occurred
in some regions of the investigated diseased human heart.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Previous reports have revealed the importance of HSPs, especially HSP70, for heart function under adverse conditions,1 2 3 4 5 6 but little is known about the role of the cardiac small HSPs HSP25 and
B-crystallin. In this study, we have shown for the first time
differential regulation of abundance and location of HSP25 and
B-crystallin during heart development. Furthermore, we present
evidence for alterations in abundance, phosphorylation
state, and subcellular location of both proteins in diseased human
hearts.
In rat hearts, the HSP25 content is subject to a pronounced decrease to
6% in adult rats (0.2 to 0.3 µg HSP25/mg protein) compared with
the content of the earliest fetal stage analyzed (3 to 4 µg
HSP25/mg protein). This decline is less pronounced when the HSP25
content is related to gram of wet tissue weight. The values obtained
for adult rat hearts are somewhat lower than those reported earlier for
adult murine11 and rat hearts,49 most
probably because of the more efficient methods of protein extraction
used in our present study. In adult human failing and transplanted
hearts, however, we found 5- to 10-fold higher amounts of HSP25 than in
fetal human hearts. These data are in the same range as those found
earlier for an adult human heart by others.9 The amounts
determined for adult human hearts, however, do not necessarily reflect
basal levels of HSP25 because pathological processes and
pharmacological treatments may have caused increased abundance. In
contrast to HSP25, the content of B-crystallin remains nearly
constant at 3 to 4 µg/mg protein during rat heart development
when related to total ventricular protein content but
increases about 3-fold when related to gram of wet tissue weight. This
corresponds with data determined for adult murine, rat, bovine,
porcine, and human hearts9 11 49 and confirms the
existence of much higher levels of B-crystallin than of HSP25 in
adult rat heart tissue as found earlier in mice.11 In
accordance with our results, Bhat and Nagineni7 observed
similar levels of B-crystallin in fetal and adult rat heart tissue.
Kato et al,9 however, detected a dramatic increase in
cardiac B-crystallin level after birth. When comparing the methods
applied, one may speculate that formation of homologous and/or
heterologous high-molecular-weight complexes of B-crystallin may
have interfered with the assay applied by Kato et al.9 A
higher degree of complex formation in fetal and early postnatal stages
may have resulted in reduced immunodetectability by the ELISA test
because of limited accessibility of B-crystallin to the antibodies.
Complex formation is not of relevance, however, when Western blots of
SDS-treated samples are used as in our study and that of Bhat and
Nagineni.7 On the other hand, in adult human transplanted
and failing hearts, the levels of B-crystallin are considerably
higher than in fetal hearts, probably because of the same reasons
described above for HSP25. Taken together, the data suggest that HSP25
and B-crystallin levels are differentially regulated during
heart development. Furthermore, elevated levels of both proteins in
diseased hearts obviously resemble the situation in fetal hearts.
In our microscopic studies, similarities and differences in the
location of HSP25 and B-crystallin were observed in heart tissue.
Both proteins colocalize in cardiomyocytes, whereas in
endothelial and smooth muscle cells of adults, only
HSP25 and not B-crystallin is found. Although we cannot rule out
conclusively the existence of B-crystallin in the latter cell types,
the presented data suggest a specific, development-dependent
function of HSP25 in endothelial and smooth muscle
cells. This may be related to increasing blood pressure and its
regulation in dependence on rapidly changing humoral and neural
influences in adults. A differential development-dependent expression
of HSP25 and B-crystallin was also observed in skeletal muscle: In
slow and fast twitch fibers of rat hind-limb muscle, the levels of both
small stress proteins increase with birth and decrease to different
extents during postnatal growth.50 Furthermore,
alterations in HSP25 and B-crystallin levels have been described
after denervation and tenotomy of rat skeletal
muscles,50 51 indicating the involvement of neural and
nonneural factors in regulation of expression of both proteins in
vivo.
As mentioned, HSP25 and B-crystallin colocalize in
cardiomyocytes at all growth stages analyzed. Their
subcellular location, however, varies from being primarily cytoplasmic
in poorly differentiated cardiomyocytes to mainly
myofibrillar at the level of the I-band and the M-line region in
well-differentiated cells. Concerning HSP25, we obtained similar
results in recent studies using isolated perfused rat hearts and
isolated cardiomyocytes.32 We could not
confirm, however, the location of B-crystallin at the Z line
described previously for adult
cardiomyocytes37 39 and skeletal muscle
fibers.51 Obviously, the use of cryosections as applied in
our studies allows better resolution of myofibril fine structure than
the earlier experiments performed with isolated
cardiomyocytes or muscle fibers. Partial dislocation of
HSP25 and B-crystallin from myofibrils to cytoplasmic, most
prominently to subsarcolemmal, regions was observed in some regions of
diseased human hearts, again reminiscent of the situation in fetal
cardiomyocytes.
The location of HSP25 and B-crystallin as described in our paper is
in accordance with several lines of experimentation that suggest the
involvement of both small HSPs in modulation of the cytoskeleton and in
muscle contraction. HSP25 isolated from yeast, turkey, and Ehrlich
ascites tumor cells inhibits polymerization of actin in
vitro,34 35 52 with unphosphorylated
monomeric HSP25 being the active component in this
process.35 Involvement of HSP25 in organization of actin
filaments was also demonstrated in vivo by transfection experiments,
because overexpression of human HSP25 in murine fibroblasts caused
increased stability of microfilaments against stress exerted by heat,
cytochalasin D, or reactive oxygen metabolites.20 36
Furthermore, in glomerular podocytes of kidneys and in
Sertoli cells of testes, HSP25 seems to be important both in
maintaining the normal structure and in pathophysiologic cytoskeletal
changes by regulating organization of actin.33 53 In
smooth muscle cells, HSP25 was shown to be involved in sustained muscle
contraction in response to bombesin or protein kinase C,31
which may be mediated by its interaction with actin. Similar to HSP25,
overexpression of B-crystallin in rat and human glioma cells results
in stabilization and antisense modification in disorganization of the
microfilament network.54 Association of B-crystallin
with actin was also observed by affinity chromatography
of rat heart extracts and binding studies with isolated
proteins.38 39 These methods also revealed association of
B-crystallin with cardiac desmin.39 The inhibition of
vimentin polymerization by B-crystallin in vitro40 is a
further indication for a possible involvement of B-crystallin in
modulation of intermediate filament structure.
As shown in this article, phosphorylation of HSP25
seems to play a role during heart development.
Phosphorylated isoforms of HSP25 predominate in fetal
rat hearts, whereas the portions of unphosphorylated
and monophosphorylated HSP25 increase with development.
In contrast, phosphorylated isoforms of HSP25
predominate in failing adult human hearts, similar to the situation in
fetal rat hearts. It should be pointed out, however, that a comparison
of rat and human HSP25 phosphorylation status may be
problematic because of possible species differences as well
as other influences such as stress conditions in the case of fetal
human hearts and drug treatment and pathological processes in the case
of adult human hearts. These influences could contribute to the obvious
variability between samples of different individuals shown in Fig 6A and 6B. In other experimental systems, phosphorylation
of HSP25 can be induced by a variety of stimuli including heat,
arsenite, calcium ionophores, tumor promoters, cytokines,
certain growth factors, and oxidative stress (Reference 1515 and
references cited therein19 20 ). Because most of these
treatments induce alterations of the microfilament
system,35 these data are compatible with a role of HSP25
phosphorylation in modulation of the actin
cytoskeleton. This is further supported by transfection experiments
with phosphorylation-deficient mutants of HSP25
providing arguments for a phosphorylation-dependent
stabilization of microfilaments against different kinds of stress in
vivo.20 36 Interestingly, there are recent findings that
phosphorylation of HSP25 is also induced in vascular
endothelial cells in vitro by fluid shear
stress,55 which is accompanied by extensive rearrangements
of the microfilament cytoskeleton. This finding is in accordance with
our data on HSP25 accumulation in endothelial cells of
the vasculature of adult rat and human myocardium in
vivo.
Concerning a possible function of HSP25 and B-crystallin in
cardiomyocytes, we hypothesize that both proteins may
facilitate the correct insertion of actin into myofilaments during
myofibrillogenesis. High levels of phosphorylated HSP25
as found in fetal rat hearts could be related to extensive formation of
new thin myofilaments at these early growth stages. Later in cardiac
development, HSP25 and B-crystallin may support the turnover of thin
myofilaments mediated by their postulated chaperoning
activities.26 27 The constant level of B-crystallin
during rat heart development, on the other hand, may be due to its
additional participation in organization of intermediate filaments as
concluded from previous literature data. The involvement of both HSP25
and B-crystallin in myofibril assembly and function is supported by
the finding that impaired function of human diseased hearts correlates
with depletion of both proteins from myofibrils.
In summary, the available data suggest that HSP25 and B-crystallin
are involved in organization of myofibrils and components of the
cytoskeleton. However, differential regulation of abundance and
location of both proteins during development of rat heart tissue imply
distinct functions: HSP25 seems to be involved in actin dynamics in
different cell types, while B-crystallin function seems to be
related to actin and intermediate filament organization in
cardiomyocytes. From these data, it is concluded that the
recently proven cardioprotective role of both small heat shock
proteins56 is exerted by maintaining the morphological and
functional integrity of components of the contractile
apparatus and the cytoskeleton. In diseased human hearts,
elevated levels of HSP25 and B-crystallin, elevated levels of
phosphorylated isoforms of HSP25, and the tendency to
cytoplasmic location of both proteins in cardiomyocytes are
similar to features in fetal hearts. Future studies will show to which
extent these alterations correlate with disorganization of myofibrils
and cytoskeletal elements.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported by grants Be 1464/2-1 to Dr Benndorf and Lu 499/3-1 to Dr Lutsch of the Deutsche Forschungsgemeinschaft and by grant 01229101 of the Bundesministerium für Forschung und Technologie to Dr Schimke. Bovine
B-crystallin was a generous gift
of Dr R. Chiesa, New York, NY. We thank U. Gerhard, C. Kemsies, E.
Kotitschke, and M. Schmidt for excellent technical assistance and G.
Grelle for quantitative amino acid analysis of HSP
solutions. Received December 31, 1996; revision received June 13, 1997; accepted June 26, 1997.
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References |
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 C. Schafer, P. Clapp, M. J. Welsh, R. Benndorf, and J. A. Williams HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells Am J Physiol Cell Physiol, December 1, 1999; 277(6): C1032 - C1043. [Abstract] [Full Text] [PDF]
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 T. Imura, S. Shimohama, M. Sato, H. Nishikawa, K. Madono, A. Akaike, and J. Kimura Differential Expression of Small Heat Shock Proteins in Reactive Astrocytes after Focal Ischemia: Possible Role of beta -Adrenergic Receptor J. Neurosci., November 15, 1999; 19(22): 9768 - 9779. [Abstract] [Full Text] [PDF]
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A. I. Ibitayo, J. Sladick, S. Tuteja, O. Louis-Jacques, H. Yamada, G. Groblewski, M. Welsh, and K. N. Bitar HSP27 in signal transduction and association with contractile proteins in smooth muscle cells Am J Physiol Gastrointest Liver Physiol, August 1, 1999; 277(2): G445 - G454. [Abstract] [Full Text] [PDF]
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I. J. Benjamin and D. R. McMillan Stress (Heat Shock) Proteins : Molecular Chaperones in Cardiovascular Biology and Disease Circ. Res., July 27, 1998; 83(2): 117 - 132. [Abstract] [Full Text] [PDF]
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C. Liu, R. R. Gilmont, R. Benndorf, and M. J. Welsh Identification and Characterization of a Novel Protein from Sertoli Cells, PASS1, That Associates with Mammalian Small Stress Protein hsp27 J. Biol. Chem., June 16, 2000; 275(25): 18724 - 18731. [Abstract] [Full Text] [PDF]
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H. E. Hoover, D. J. Thuerauf, J. J. Martindale, and C. C. Glembotski alpha B-crystallin Gene Induction and Phosphorylation by MKK6-activated p38. A POTENTIAL ROLE FOR alpha B-CRYSTALLIN AS A TARGET OF THE p38 BRANCH OF THE CARDIAC STRESS RESPONSE J. Biol. Chem., July 28, 2000; 275(31): 23825 - 23833. [Abstract] [Full Text] [PDF]
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R. Benndorf, X. Sun, R. R. Gilmont, K. J. Biederman, M. P. Molloy, C. W. Goodmurphy, H. Cheng, P. C. Andrews, and M. J. Welsh HSP22, a New Member of the Small Heat Shock Protein Superfamily, Interacts with Mimic of Phosphorylated HSP27 (3DHSP27) J. Biol. Chem., July 13, 2001; 276(29): 26753 - 26761. [Abstract] [Full Text] [PDF]
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K. N. Bitar HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction Am J Physiol Gastrointest Liver Physiol, May 1, 2002; 282(5): G894 - G903. [Abstract] [Full Text] [PDF]
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