Download Increased Expression of Cytoskeletal, Linkage, and Extracellular

Increased Expression of Cytoskeletal, Linkage, and
Extracellular Proteins in Failing Human Myocardium
Annette Heling, René Zimmermann, Sawa Kostin, Yoshi Maeno, Stefan Hein, Bruno Devaux,
Erwin Bauer, Wolf-Peter Klövekorn, Martin Schlepper, Wolfgang Schaper, Jutta Schaper
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Abstract—Experimental studies have shown that in hypertrophy and heart failure, accumulation of microtubules occurs that
impedes sarcomere motion and contributes to decreased ventricular compliance. We tested the hypothesis that these
changes are present in the failing human heart and that an entire complex of structural components, including
cytoskeletal, linkage, and extracellular proteins, are involved in causing functional deterioration. In explanted human
hearts failing because of dilated cardiomyopathy (ejection fraction ⱕ20%), expression of ␣- and ␤-tubulin, desmin,
vinculin, fibronectin, and vimentin was determined by Northern and Western blot analysis and compared with normal
myocardium from explants not used for transplantation. The mRNA for ␣- and ␤-tubulin was increased to 2.4-fold
(P⬍0.01) and 1.25-fold (NS), respectively; for desmin, 1.2-fold (P⬍0.05); for fibronectin, 5-fold (P⬍0.001); and for
vimentin, 1.7-fold (P⬍0.05). Protein levels for ␣-tubulin increased 2.6-fold (P⬍0.02); for ␤-tubulin, 1.2-fold
(P⬍0.005); for desmin, 2.1-fold (P⬍0.001); for vinculin, 1.2-fold (P⬍0.005); for fibronectin, 2.9-fold (P⬍0.001); and
for vimentin, 1.5-fold (P⬍0.005). Confocal microscopy showed augmentation and disorganization of all proteins
studied. In combination with the loss of myofilaments and sarcomeric skeleton previously reported, these changes
suggest cardiomyocyte remodeling. Increased fibronectin and elevated interstitial cellularity (vimentin labeling) indicate
progressive fibrosis. The present results suggest a causative role of cytoskeletal abnormalities and myofilament loss for
intrinsic contractile and diastolic dysfunction in failing hearts. (Circ Res. 2000;86:846-853.)
Key Words: heart failure 䡲 cardiomyopathy 䡲 cytoskeleton 䡲 fibrosis 䡲 structure
E
nd-stage human chronic heart failure is the common final
manifestation of a group of different diseases (such as
ischemic heart disease, hypertension, valve defects, dilated
cardiomyopathy, and others), and it is usually accompanied
by myocardial hypertrophy.1 Only a few animal models exist
to study this pathophysiologic situation, and these usually are
not able to mimic the slowly developing human situation.
Therefore, most studies concerning the mechanism of reduced function are carried out in tissue samples from human
hearts obtained at the time of cardiac transplantation, either
from ischemic hearts or from those with dilated cardiomyopathy (DCM). In this study, we present data obtained in failing
human hearts with DCM. In the late stages of this disease,
numerous degenerative structural alterations of the cardiomyocytes, as well as areas of focal fibrosis, are scattered
throughout the ventricular walls.2– 4
Decompensated hypertrophy occurs when increased cardiac
mass fails to normalize wall stress,5 and it has been postulated
that in the transition of compensated hypertrophy to a decompensated state, the cardiomyocyte cytoskeleton plays a key role
in causing contractile and diastolic dysfunction.6 – 8
Alterations of the cytoskeleton have been described in many
excellent studies, by Tsutsui et al6,7 and Tagawa et al,8 –11 in
hypertrophied and failing right ventricles of feline6,7,9 –11 and
canine8 myocardium. It was shown that the increase in tubulin
occurs at the mRNA as well as at the protein level and that an
increased viscosity and stiffness of the cardiomyocytes is the
consequence of increased amounts and abnormal polymerization
of microtubules.11 Evidence was presented that increased microtubular density imposes an intracellular load on the cardiac
myocyte that will result in impediment of sarcomere motion and
contribute to decreased compliance of the hypertrophied myocardium. In another study, microtubule stabilization and the
elevated expression of microtubule-associated protein-4 have
been associated with contractile dysfunction.12 It was furthermore shown that accumulation of ␤-tubulin and contractile
dysfunction may be related to the mechanical forces imposed on
the myocardium during the onset and progression of pressure
overload.13
The view that increased microtubular stabilization may be an
important factor in causing cellular stiffness and contractile
dysfunction in pressure-overload hypertrophy is shared by Wang
et al.14 On the other hand, Collins et al,15 investigating cytoskeletal gene expression in a similar experimental model, found the
opposite. The authors concluded that neither the level of
Received October 12, 1999; accepted February 25, 2000.
From the Department of Experimental Cardiology (A.H., S.K., Y.M., M.S., W.S., J.S.), Max Planck Institute, and Department of Cardiac Surgery (R.Z., S.H.,
E.B., W.-P.K.), Kerckhoff-Clinic, Bad Nauheim, Germany, and Centre Hospitalier (B.D.), Université de Rouen, France.
Correspondence to Jutta Schaper, MD, Department of Experimental Cardiology, Max-Planck-Institute, Benekestrasse D-61231, Bad Nauheim, Germany.
E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
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Heling et al
TABLE 1.
Cytoskeleton in Heart Failure
847
Clinical Characteristics of the Patient Population
No. of
Patients
Sex
Age, Years
LVEDP, mm Hg
EF, %
PAP Mean, mm Hg
LVEDD, mm
DCM (n⫽19)
17 male/2 female
50.8⫾8.3
29.1⫾9.1
16⫾3.9
30.1⫾8.5
69.9⫾5.6
Control (n⫽4)
4 male
48⫾10
8–12
65–70
15–18
35–56
All patients with DCM were treated with digitalis, angiotensin-converting enzyme inhibitors, ␤-blockers, and diuretics at individual
dosages. Depending on the individual situation of the patients, treatment with antiarrhythmic drugs was added.
EF indicates ejection fraction; PAP, pulmonary arterial pressure; and LVEDD, left ventricular end-diastolic diameter.
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␤-tubulin nor its polymerization state affected the contractile
performance of the overloaded left ventricle. The role of tubulin
accumulation and polymerization was also questioned by Bailey
et al,16 who failed to find an effect of colchicine on contraction
dynamics in isolated feline myocytes from hypertrophied hearts,
and by de Tombe,17 who studied the effects of colchicine and
taxol on rat trabecula contractility.
In 1991, our group described accumulation and disorganization of microtubules and desmin filaments in human tissue with
chronic heart failure.3 In continuation of this work, we tested the
hypothesis and present here quantitative protein and mRNA data
that show that an entire system of structural proteins, including
the cytoskeleton and proteins linking the intracellular milieu to
the extracellular matrix such as vinculin and fibronectin, are
involved in causing the functional and structural deterioration
characteristic of heart failure.
Quantification was done by densitometry either by using the gelanalyzing program Quantity One (Protein Imageware System) or by
scanning the immunoblots on a STORM 860 imager (Amersham
Pharmacia Biotech) using ImageQuant software. All data were expressed as mean⫾SEM. Statistical analysis by unpaired t test was
considered significant when P⬍0.05.
Standardization
To exclude the possibility that the tubulin measured originated from
an increased number of fibroblasts in fibrotic tissue, myocardium
was first evaluated by light microscopy, and samples showing
absence of fibrosis were selected. However, the contribution of an
Materials and Methods
Nineteen patients with end-stage heart failure (ejection fraction
ⱕ20%) due to DCM without any evidence of myocardial infarction
undergoing heart transplantation were studied. Hearts from donors
with normal left ventricular function not used for transplantation
served as controls. The study was approved by the institutional
ethical committee. All clinical data are presented in Table 1.
Tissue Sampling
At the time of transplantation, tissue was removed from the left
ventricular free wall excluding papillary muscles. Up to 3 samples
were used for either Western or Northern blot and 5 to 8 samples for
confocal microscopy. Samples for Western blot were also examined
by microscopy. Samples were immediately frozen in liquid nitrogen
and stored at – 80°C until further use.
Western Blot
All antibodies used for Western blot and immunofluorescence are
listed in Table 2.
SDS-PAGE (separating gels 12%) and immunoblotting were carried
out following routine protocols. At least 2 different gels were prepared
from each heart. For each lane, 30 ␮g of protein was loaded. Tissue
samples from 3 donors served as control.
TABLE 2. Antibodies Used for Western Blotting (WB) and
Immunofluorescence (IF)
Antigen
Clone
Host
Dilution:WB/IF
Company
Desmin
DE-U-10
Mouse
1:100/20
Sigma
Vinculin
hVIN-1
Mouse
1:100/40
Sigma
␣-Tubulin
DM 1A
Mouse
1:100/100
Sigma
␤-Tubulin
2-28-33
Mouse
1:100/100
Sigma
Fibronectin
Polyclonal
Rabbit
1:100/100
ICN
Vimentin
Vg
Mouse
1:100/10
Immunotech
Pan-actin
HHF35
Mouse
1:100
DAKO
Figure 1. Illustration of the localization of the proteins investigated. a, Isolated rat myocyte shows network-like longitudinally
oriented localization of ␤-tubulin. b, Schematic representation of
localization of desmin around the Z-disks of the sarcomeres. c,
Localization of vinculin (Vc) as a link between the intracellular
milieu and extracellular fibronectin via the integrins of the sarcolemma. Tubulin is present also in fibroblasts. Vimentin present in
fibroblasts and endothelial cells is not shown here.
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Figure 2. mRNA for ␣- and ␤-tubulin is significantly increased (A and C) as compared with control (con). Protein levels in failing hearts
are elevated for both isoforms in the Western blot (B and D). Blots were carried out for all 19 failing hearts and the controls, but only
representative lanes are shown in Figures 2, 3, 5, 6, and 7. E and F, Immunofluorescence for ␣-tubulin (green). E, In normal myocardium, the myocytes are regularly arranged and the tubulin fluorescence is present as a fine network in each cell. F, In failing myocardium, the tubulin fluorescence is more dense and irregular in numerous cells as compared with panel E.
increased number of fibroblasts cannot be entirely excluded. Immunoblotting for pan-actin was performed, and only samples showing a
similar density of the actin band at 42 kDa were used in this study.
The total protein applied to the gel was kept constant.
were superimposed for reconstruction in a 3-dimensional mode using
a Silicon Graphics Indy workstation and 3-dimensional multichannel
image processing software (Bitplane).
Northern Blot
All proteins studied, with the exception of vimentin, are
schematically represented in Figure 1. Vimentin is present in
fibroblasts and in endothelial cells.
Results
The following molecular probes were used for Northern blot analysis: human ␣- and ␤-tubulin and human vimentin (American Type
Culture Collection), human desmin (R. Zimmermann, unpublished),
rat fibronectin clone pRCabFN1 (K. Boheler, National Heart and
Lung Institute, London, UK), and a murine 18S ribosomal RNA
cDNA probe (a gift of I. Oberbäumer, Max-Planck-Institut, Martinsried, Germany).
Standard methods for Northern blots were used.18 Each gel was done
at least in duplicate to control for variability within and between gels.
For each well, 15 ␮g of total RNA was loaded. Tissue samples from 3
donors served as control.
Quantification of mRNA levels was performed using a PhosphorImager and ImageQuant software (Molecular Dynamics). For normalization, the data of each hybridization signal were divided by the
matching 18S signal. All data are presented as mean⫾SEM. Expression
was assessed by an unpaired t test. Statistical significance was accepted
as P⬍0.05.
Both the ␣- and ␤-tubulin isoforms showed an upregulation in the Northern blot, 2.4- and 1.25-fold respectively,
and 2.6- and 1.2-fold in Western blot (Figures 2A through
2D). The immunocytochemical localization of ␣- and
␤-tubulin demonstrated an increased density of microtubules in most of the cardiomyocytes as compared with
control tissue. Microtubules, normally visible as fine
filamentous structures, were disorganized and aggregated,
especially in the perinuclear area, ie, microtubular density
was increased (Figures 2E and 2F).
Immunolabeling and Confocal Microscopy
Desmin
From each left ventricle, 5 to 8 randomly chosen samples were
prepared as previously described.19 Omission of primary antibodies
served as negative controls. Preparations were viewed in a confocal
laser microscope (Leica) equipped with appropriate filter blocks. The
optical confocal sections taken through the depth of tissue samples at
0.5- to 1-␮m intervals were viewed and photographed individually or
Northern blot analysis exhibited a 1.2-fold increase of desmin
mRNA (Figure 3A), Western blot showed a 2.1-fold elevation (Figure 3B).
In the confocal microscope, a distinct desmin cross-striation
pattern is typical of normal myocytes (Figure 3C). In failing
␣- and ␤-Tubulin
Heling et al
Cytoskeleton in Heart Failure
849
Figure 4. A good correlation exists between LVEDP and
cytoskeletal protein content (tubulin and desmin).
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myocardium, desmin was disorganized and accumulated in
many cardiomyocytes (Figure 3D).
A good correlation was found between the amount of cytoskeletal proteins (desmin and tubulin) and the left ventricular
end-diastolic pressure (LVEDP) in controls and DCM patients
(Figure 4).
Vinculin
As compared with normal myocardium, vinculin was 1.2-fold
increased in patients with failing hearts in Western blot
(Figure 5A).
In the confocal microscope, vinculin showed a distinct localization at the intercalated disks and at the lateral sarcolemma in
normal myocardium. This localization was more intense in
failing hearts, corresponding to the results from Western blotting
(Figure 5B).
Fibronectin
The mRNA for fibronectin was 5-fold increased as compared
with controls (Figure 6A); the protein was increased 2.9-fold
(Figure 6B).
In immunolabeling, it was evident that fibronectin was present
in the extracellular space and in the basement membrane of
myocytes, smooth muscle cells, and endothelial cells (Figure
6C). The expression was more intense in the widened interstitial
space of failing hearts (Figure 6D). A close colocalization of
fibronectin and vinculin was shown by double labeling (Figures
6E and 6F)
Vimentin
Figure 3. Desmin is significantly increased on the mRNA and
protein levels as shown by Northern (A) and Western (B) blot
analysis (con indicates control). C and D, Confocal microscopy
for desmin. C, Normal human myocardium shows a regular
cross-striation at the Z-level and distinct intercalated disk labeling. D, In failing myocardium, the cross-striation pattern is disturbed and desmin is accumulated in many irregularly shaped
cells. Nuclei are red.
The mRNA for vimentin was upregulated 1.7-fold as compared with controls (Figure 7A), and the protein was increased 1.5-fold in patients with heart failure (Figure 7B).
Immunostaining of normal myocardium showed that vimentin was present in fibroblasts and endothelial cells (Figure 7C).
In failing hearts, fibroblasts were more numerous, corresponding
to the values from Western blot (Figure 7D).
Discussion
This study demonstrates for the first time significant changes
of cytoskeletal and membrane-associated proteins in human
hearts failing because of longstanding DCM. We show that
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Figure 5. A, Immunoblotting shows that vinculin is significantly
increased in failing myocardium (con indicates control). B and
C, Vinculin localization using confocal microscopy. B, In normal
myocardium, labeling at the intercalated disk, at the costameres, and in the T-tubular system is evident. C, In failing
myocardium, vinculin fluorescence is more abundant and disorganized. Red in panel C indicates lipofuscin granules.
tubulin and desmin are significantly increased, not only at the
protein level but also at the mRNA level. The same is true for
vinculin, fibronectin, and vimentin. In the confocal microscope, a distinct disorganization and accumulation of the
microtubules and desmin filaments were evident, which
supports the quantitative protein data. A good correlation was
found between protein content and LVEDP in controls and
patients with DCM. It is hypothesized, therefore, that not only
the loss of myofilaments as described previously2,3 but also
the increase in cytoskeletal and membrane-associated proteins may play a major role in the pathogenesis of contractile
and diastolic dysfunction in failing hearts.4
The role of the cytoskeleton as a stabilizing factor of cellular
structure and functional integrity is well established.20 Tubulin is
organized around the nucleus and in the longitudinal direction of
the cell, and it contributes to the stability of the contractile
apparatus in relation to the nucleus, mitochondria, and cellular
membrane.21 Tubulin turnover is continuous and high and is an
energy-consuming process. Therefore, it is especially interesting
that a ubiquitous accumulation of tubulin takes place in failing
hearts. It cannot be excluded at the present time that tubulin
synthesis plays a significant role in the transition from hypertrophy to the decompensated state in the human heart, as shown in
rats with developing hypertrophy due to aortic banding.22
The concept of microtubular involvement in contractile and
diastolic dysfunction in failing hearts, previously formulated by
Tsutsui et al6,7 and Tagawa et al11 in hearts with decompensated
hypertrophy, is supported and extended to the involvement of other
proteins by the data in human hearts presented here. Cellular
hypertrophy as present in experimental animals was also found in
our patients.23 One significant difference between both models,
however, is the fact that in the animal experiments the contractile
machinery was intact, indicating that the more chronic situation in
the human heart represents a unique pathophysiological entity. The
present data suggest that similarities develop in the final structural as
well as functional deterioration in failing hearts independently of the
cause of failure. It may be concluded that changes, not only of
tubulin, as reported by Tsutsui et al6,7 and Tagawa et al,11 but also
of desmin and other linkage proteins, in addition to the loss of
myofilaments and the occurrence of fibrosis, are the structural
correlate of the reduction of ejection fraction and the increase in
LVEDP as observed in our patients.
Desmin connects the sarcomeres in the transverse direction,
thereby inhibiting slippage during contraction. Desmin accumulation and disorganization were especially evident in cells lacking
contractile material. Given that desmin is localized around Z-lines
and that it connects neighboring sarcomeres, this disorganization
can be explained by the absence of a structural element with which
it can be associated. Using desmin knockout mice, Capetanaki et
al24 showed that desmin is essential for the maintenance of the
functional integrity of myofibrils, whole myofibers, and the general
cellular integrity, which was confirmed in mice with desmin null
mutations in which degeneration of cardiac muscle was observed.25
The findings of the present study are in agreement with those
reported in congenital myopathies and hereditary cardiac diseases.26–28 Therefore, the desmin augmentation found in human myocardium was interpreted as a compensatory mechanism to ensure
cellular integrity and sarcomere stability.
Heling et al
Cytoskeleton in Heart Failure
851
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Figure 6. Fibronectin is significantly
increased by Northern (A) and Western
(B) blot analysis (con indicates control).
C and D, Confocal images of fibronectin
(green) and actin (red) labeling. E and F,
Colocalization of fibronectin (red) and
vinculin (green). C, Normal myocardium
shows fibronectin as a fine line surrounding the myocytes and capillary
vessels. D, In failing myocardium, the
interstitial space is enlarged and
fibronectin is significantly increased,
leading to encapsulation of myocytes. E,
In this slightly tangential section, vinculin
is present at the intercalated disk, at the
lateral sarcolemma, and in the T system.
F, At high magnification of the boxed
area from panel E, the close spatial
localization of fibronectin and vinculin at
the T-tubular membrane and the sarcolemma is evident (arrows). Myocytes are
unstained (black).
Vinculin, a member of the family of membrane-associated
proteins, is situated at the costameres of the lateral sarcolemma
and at the intercalated disk19 and is one of the major components
of the linkage system that connects via the integrins the intracellular milieu with the extracellular matrix.29 –31 The ␤1integrins act as receptors for fibronectin. Their dynamic interaction with both the cytoskeleton and fibronectin is important for
structural changes during muscle cell differentiation (see Reference 32 for other relevant references).
We report here that vinculin was increased at the protein level.
In another study, we found that dystrophin, talin, and spectrin
were increased as well.21 This indicates that the entire group of
membrane-associated proteins is involved in the attempt to
stabilize the sarcolemma and its connections to the contractile
apparatus on the one hand and to the extracellular matrix on the
other hand. The stabilizing function of the cytoskeleton is of
importance when the contractile material of the cardiomyocytes
is reduced as a result of the progression from hypertrophy to
failure. In the early stages of the disease, therefore, this might
help the cardiomyocytes to maintain function and may be
regarded as a compensatory mechanism. Recent experimental
studies have shown that a dystrophin missense mutation in the
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Figure 7. Vimentin is significantly increased on the mRNA (A)
and protein (B) levels (con indicates control). C and D, Confocal
images of immunofluorescence for vimentin (red), with myocytes
counterstained with phalloidin (green). C, Normal myocardium
with few fibroblasts and capillaries. D, In failing myocardium, the
myocytes are separated by fibrotic material containing many
fibroblasts. Only few capillaries are visible. Red in myocytes
indicates lipofuscin granules.
mouse,33 muscle LIM protein (MLP) deficiency in mice,34 or
cleavage of dystrophin by enteroviral proteases35 result in the
clinical picture of DCM. On the basis of these data, the
hypothesis was put forward that DCM is a disease of cytoskeletal damage.36,37 This view, however, is in contradistinction to
the increased density of the major cytoskeletal components in
human hearts with DCM as reported here.
Fibronectin is the major extracellular matrix protein and is an
important component of the cellular basement membrane.38 It is
increased in hearts with DCM, resulting, together with an
increase of other extracellular proteins, in a significant degree of
fibrosis.4,39 Vimentin, the intermediate filament of fibroblasts
and endothelial cells, was studied because it represents an
indicator of the cellularity of the interstitium.40 It was significantly elevated because the number of fibroblasts was higher
than in control. Taken together, these findings indicate that the
synthesis of connective tissue components is continuous and will
result in progressive fibrosis despite pretransplantation treatment
of all patients with angiotensin-converting enzyme inhibitors. In
an experimental study, we have recently shown that fibrosis
development cannot be prevented indefinitely by this treatment,41 which would confirm the data presented here. Fibrosis
will contribute to an increased stiffness and loss of ventricular
compliance.42
The cytoskeleton also plays an important role in mechanotransduction across the cell surface, where integrins act as
mechanoreceptors.43 Transfer of force from integrins to the
cytoskeleton is thought to represent a proximal step in intracellular mechanical signaling that leads to global cytoskeletal
rearrangements.43 Furthermore, it has been shown that the
extracellular matrix controls cytoskeletal mechanics and structure, particularly by binding of fibronectin to integrins.44,45
Interestingly, increasing extracellular matrix contacts by binding
through an elevated number of integrins increases cytoskeletal
stiffness and apparent viscosity.44 Transferring these data to our
own findings, it may be assumed that the increased content of
fibronectin found in failing hearts is the inducer of changes of
cytoskeletal structure and function.
The question remains open whether the changes described
here are effects rather than causes of heart failure. The present
data, however, are more in support of the hypothesis that these
structural changes are contributory to the transition to heart
failure. This hypothesis will be tested in human hearts with
compensatory hypertrophy. Another question is whether the
cytoskeletal proteins either have a structural role only or participate in cellular signaling. As discussed recently, “chronic
increases in wall stress mediated by cytoskeletal pathways”
(page 558) as is the case in failing hearts with DCM, may play
a decisive role in cellular hypertrophy and cell survival.46 Recent
evidence concerning ␣- or ␦-sarcoglycan47 or LIM domain
protein34 – deficient mice strongly suggests that the cytoskeleton
might be involved in cellular signaling.
In conclusion, in myocytes from failing human hearts, the
major components of the cytoskeleton tubulin and desmin, as
well as the membrane-associated protein vinculin, are increased,
most probably as a mechanism compensatory for the loss of
contractile filaments, thereby contributing to an increased stiffness of individual cardiomyocytes. Furthermore, the linkage
protein vinculin and the extracellular matrix protein fibronectin
Heling et al
are augmented, which will, by interaction with the integrins,
further increase cellular stiffness. Fibronectin and vimentin
indicate a progressive development of fibrosis. The present data
support experimental reports showing the important role of
microtubule accumulation in failing hearts. They furthermore
show that changes of a whole complex of proteins, in addition to
the loss of contractile filaments and proteins of the sarcomeric
skeleton described previously,3 represent the structural basis for
the reduction of contractile function and compliance in failing
human hearts.21
Acknowledgment
We thank Gunther Schuster for computer assistance.
References
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1. Braunwald E. Pathophysiology of heart failure. In: Braunwald E, ed.
Heart Disease: A Textbook of Cardiovascular Medicine. Philadelphia, Pa:
WB Saunders Co Inc; 1980:453– 483.
2. Hein S, Scholz D, Fujitani N, Rennollet H, Brand T, Friedl A, Schaper J.
Altered expression of titin and contractile proteins in failing human myocardium. J Mol Cell Cardiol. 1994;26:1291–1306.
3. Schaper J, Froede R, Hein S, Buck A, Hashizume H, Speiser B, Friedl A,
Bleese N. Impairment of the myocardial ultrastructure and changes of the
cytoskeleton in dilated cardiomyopathy. Circulation. 1991;83:504–514.
4. Schaper J, Speiser B, Brand T. The cytoskeleton and extracellular matrix in
human hearts with dilated cardiomyopathy. In: Figulla HR, Kandolf R,
McManus B, eds. Idiopathic Dilated Cardiomyopathy. Berlin, Germany:
Springer-Verlag; 1993:75–80.
5. Grossman W, Jones D, McLaurin MP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975;56:56–64.
6. Tsutsui H, Ishihara K, Cooper G. Cytoskeletal role for contractile dysfunction
of hypertrophied myocardium. Science. 1993;260:682–687.
7. Tsutsui H, Tagawa H, Kent RL, McCollam PL, Ishihara K, Nagatsu M,
Cooper G. Role of microtubules in contractile dysfunction of hypertrophied
cardiocytes. Circulation. 1994;90:533–555.
8. Tagawa H, Koide M, Sato H, Zile MR, Carabello BA, Cooper G.
Cytoskeletal role in the transition from compensated to decompensated hypertrophy during adult canine left ventricular pressure overloading. Circ Res.
1998;82:751–761.
9. Tagawa H, Koide M, Sato I, Cooper G. Cytoskeletal role in the contractile
dysfunction of cardiocytes from hypertrophied and failing right ventricular
myocardium. Proc Assoc Am Physicians. 1996;108:218–229.
10. Tagawa H, Rozich JD, Tsutsui H, Narishige T, Kuppuswamy D, Sato H,
McDermott PJ, Koide M, Cooper G. Basis for increased microtubules in
pressure-hypertrophied cardiocytes. Circulation. 1996;93:1230–1243.
11. Tagawa H, Wang N, Narishige T, Ingber DE, Zile MR, Cooper G.
Cytoskeletal mechanics in pressure-overload cardiac hypertrophy. Circ Res.
1997;80:281–289.
12. Sato II, Nagai T, Kuppuswamy D, Narishige T, Koide M, Menick DR,
Cooper G. Microtubule stabilization in pressure overload cardiac hypertrophy. J Cell Biol. 1997;139:963–973.
13. Watson PA, Hannan R, Carl LL, Giger KE. Contractile activity and passive
strength regulate tubulin mRNA and protein content in cardiac myocytes.
Am J Physiol. 1996;271(2 pt 1):C684–C689.
14. Wang X, Li F, Campbell SE, Gerdes M. Chronic pressure overload hypertrophy and failure in guinea pigs, II: cytoskeletal remodeling. J Mol Cell
Cardiol. 1999;31:319–331.
15. Collins JF, Pawloski-Dahm C, Davies MG, Ball N, Dorn GW, Walsh RA.
The role of the cytoskeleton in left ventricular pressure overload hypertrophy
and failure. J Mol Cell Cardiol. 1996;28:1435–1443.
16. Bailey BA, Dipla K, Li S, Houser SR. Cellular basis of contractile
derangements of hypertrophied ventricular myocytes. J Mol Cell Cardiol.
1997;29:1823–1835.
17. de Tombe PP. Altered contractile function in heart failure. Cardiovasc Res.
1998;37:367–380.
18. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;
162:156–159.
19. Kostin S, Scholz D, Shimada T, Maeno Y, Mollnau H, Hein S, Schaper J.
The internal and external protein scaffold of the T-tubular system in cardiomyocytes. Cell Tissue Res. 1998;294:449–460.
Cytoskeleton in Heart Failure
853
20. Stromer MH. The cytoskeleton in skeletal, cardiac and smooth muscle cells.
Histol Histopathol. 1998;13:283–291.
21. Kostin S, Heling A, Hein S, Scholz D, Klövekorn W-P, Schaper J. The
protein composition of the normal and diseased cardiac myocyte. Heart Fail
Rev. 1998;2:245–260.
22. Watkins SC, Samuel JL, Marotte F, Bertier-Savalle B, Rappaport L. Microtubules and desmin filaments during onset of heart hypertrophy in the rat: a
double immunoelectron microscope study. Circ Res. 1987;60:327–336.
23. Scholz D, Diener W, Schaper J. Altered nucleus/cytoplasm relationship and
degenerative structural changes in human dilated cardiomyopathy. Cardioscience. 1994;5:127–138.
24. Capetanaki A, Milner DJ, Weitzer G. Desmin in muscle formation and
maintenance: knockouts and consequences. Cell Struct Funct. 1997;22:
103–116.
25. Milner DJ, Weitzer G, Tran D, Bradley A, Capetanaki Y. Disruption of
muscle architecture and myocardial degeneration in mice lacking desmin.
J Cell Biol. 1996;134:1255–1270.
26. Thornell LE, Johanson B, Erikson A, Lehto VP, Virtanen I. Intermediate
filament and associated proteins in the human heart: an immunofluorescence
study of normal and pathological hearts. Eur Heart J. 1984;5(suppl
F):231–241.
27. Thornell LE, Price MG. The cytoskeleton in muscle cells in relation to
function. Biochem Soc Transact. 1991;19:1116–1120.
28. Thornell LE, Carlsson L, Li Z, Mericskay M, Paulin D. Null mutation in the
desmin gene gives rise to a cardiomyopathy. J Mol Cell Cardiol. 1997;29:
2107–2124.
29. Geiger B. Vinculin, an intracellular protein localized at specialized sites
where microfilaments bundles terminate at cell membranes. Proc Natl Acad
Sci U S A. 1980;77:4127–4131.
30. Craig SW, Pardo JV. Gamma actin, spectrin, and intermediate filament
proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma
attachment sites. Cell Motil. 1983;3:449–462.
31. Pardo JV, Siliciano J, Craig SW. Vinculin is a component of an extensive
network of myofibril-sarcolemma attachment regions in cardiac muscle
fibers. J Cell Biol. 1983;97:1081–1088.
32. Enomoto MI, Boettiger D, Menko AS. ␣5-Integrin is a critical component of
adhesion plaques in myogenesis. Dev Biol. 1993;155:180–197.
33. Ortiz-Lopez R, Hua L, Su J, Goytia V, Towbin JA. Evidence for a dystrophin
missense mutation as a cause of X-linked dilated cardiomyopathy. Circulation. 1997;95:2434–2440.
34. Arber S, Hunter JJ, Ross J Jr, Hongo M, Sansig G, Borg J, Perriard J-C, Chien
KR, Caroni P. MLP-deficient mice exhibit a disruption of cardiac cytoarchitectural organization, dilated cardiomyopathy, and heart failure. Cell. 1987;
88:393–403.
35. Badorff C, Lee GH, Lamphear BL, Martone ME, Campbell KP, Rhoads RE,
Knowlton KU. Enteroviral protease 2A cleaves dystrophin: evidence of
cytoskeletal disruption in an acquired cardiomyopathy. Nat Med. 1999;5:
320–326.
36. Towbin JA. The role of cytoskeletal proteins in cardiomyopathies. Curr Opin
Cell Biol. 1998;10:131–139.
37. Towbin JA, Bowles KR, Bowles NE. Etiologies of cardiomyopathy and heart
failure. Nat Med. 1999;5:266–267.
38. Hynes RO. Fibronectins. Berlin, Germany: Springer; 1989.
39. Schaper J, Hein S, Scholz D, Mollnau H. Multifaceted morphological alterations are present in the failing human heart. J Mol Cell Cardiol. 1995;27:
857–861.
40. Traub P. Intermediate Filaments: A Review. Berlin, Germany: SpringerVerlag; 1985.
41. Zimmermann R, Kastens J, Linz W, Wiemer G, Schölkens BA, Schaper J.
Effect of long-term ACE inhibition on myocardial tissue in hypertensive
stroke-prone rats. J Mol Cell Cardiol. 1999;31:1447–1456.
42. Weber KT, Brilla CG, Janicki JS. Myocardial fibrosis: functional significance
and regulatory factors. Cardiovasc Res. 1993;27:341–348.
43. Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface
and through the cytoskeleton. Science. 1993;260:1124–1127.
44. Wang N, Ingber DE. Control of cytoskeletal mechanics by extracellular
matrix, cell shape, and mechanical tension. Biophys J. 1994;66:2181–2189.
45. Choquet D, Felsenfeld DP, Sheetz MP. Extracellular-matrix rigidity causes
strengthening of integrin-cytoskeleton linkages. Cell. 1997;88:39–48.
46. Chien KR. Stress pathways and heart failure. Cell. 1999;98:555–558.
47. Coral-Vazquez R, Cohn RD, Moore SA, Hill JA, Weiss RM, Davisson RL,
Straub V, Barresi R, Bansal D, Hrstka RF, Williamson R, Campbell K.
Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle:
a novel mechanism for cardiomyopathy and muscular dystrophy. Cell. 1999;
98:465–474.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Increased Expression of Cytoskeletal, Linkage, and Extracellular Proteins in Failing
Human Myocardium
Annette Heling, René Zimmermann, Sawa Kostin, Yoshi Maeno, Stefan Hein, Bruno Devaux,
Erwin Bauer, Wolf-Peter Klövekorn, Martin Schlepper, Wolfgang Schaper and Jutta Schaper
Circ Res. 2000;86:846-853
doi: 10.1161/01.RES.86.8.846
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