(Circulation. 2000;101:2854.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Left Ventricular Hypertrophy in Ascending Aortic Stenosis Mice
Anoikis and the Progression to Early Failure
From the Harvard-Thorndike Laboratory and the Department of Medicine, Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass (B.D., X.Y., P.S.D., E.O.W., J.B., B.H.L.); the Department of Developmental Biology and Anatomy, University of South Carolina School of Medicine, Columbia (R.L.P., E.C.G., T.K.B., T.T.); and the Department of Neurosurgery, Baylor College of Medicine, Houston, Tex (V.V.D.).
Correspondence to Beverly H. Lorell, MD, Cardiovascular Division, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215. E-mail blorell{at}caregroup.harvard.edu
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Abstract |
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Background—To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice.
Methods and Results—Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7±0.7 versus 3.1±0.2 and 5.7±0.4 versus 2.7±0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0±0.8 and 25.2±1.8 versus 14.1±0.5 µm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for ß1-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides.
Conclusions—Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.
Key Words: myocytes • hypertrophy • heart failure • integrins • apoptosis
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Introduction |
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Although apoptotic myocytes have been observed in end-stage human failing hearts, the potential role of programmed cell death during the transition from adaptive hypertrophy to early failure is not known. We investigated a mouse model of ascending aortic stenosis (AS) with fidelity to those clinical features that characterize the transition from adaptive hypertrophy to very early failure, including an initial phase of compensation followed by the development of left ventricular (LV) systolic dysfunction. At the stage of very early failure, we observed that LV myocytes exhibit aberrant myocyte branching and ß1-integrin deposition in situ in association with a very low prevalence of myocyte apoptosis estimated by terminal dUTP nick end-labeling (TUNEL) and ligation of hairpin oligonucleotide probes. These observations raise the hypothesis that anoikis, the process of abnormal cell-cell and cell–extracellular matrix (ECM) contact that initiates the cell death program, occurs early and may contribute to the progression to very early heart failure.
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Methods |
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Animal Preparation and Hemodynamic Measurements
Ascending AS was performed in male FVB/n mice (body weight 12 to 15 g, Charles River Breeding Laboratories, Wilmington, Mass) as described by our laboratory.1 Age-matched animals underwent the same procedure without placement of the aortic ligature to serve as controls. In vivo LV pressure measurements were performed before euthanasia (n=12 to 16 per group) by methods previously described in AS rats2 3 4 5 and mice.1 AS animals and age-matched sham-operated controls were euthanized at 4 and 7 weeks after surgery (n=25 to 30 per group). This stage of 7-week AS, which was selected as the transition to failure, precedes the development of severe decompensation that develops by 13-week AS, which is characterized by severely increased mortality (>75%) and liver and lung congestion with pleural effusion.
2D Targeted M-Mode Echocardiography
After measurement of body weight, animals were lightly
anesthetized with intraperitoneal
ketamine 50 mg/kg and xylazine 2.5 mg/kg and studied in the
conscious condition on a warming pad. 2D guided M-mode
echocardiography in the mouse was performed with a
12-MHz transducer (Hewlett Packard) as described by our
laboratory.6 In addition to standard analysis of
LV wall thickness, cavity size, and endocardial fractional shortening,
we also used midwall fractional shortening as an index to estimate LV
systolic function, because endocardial edge chamber dynamics
may overestimate function.6 7 8 In addition, the
relationship between LV volume measured by
echocardiography and postmortem volume was
determined in mice (n=6) that underwent
echocardiography as described above immediately
followed by diastolic arrest of the heart via
aortocoronary potassium chloride perfusion in situ. The
paraformaldehyde-fixed LV was sectioned into ten 0.5-mm
sections from apex to base, the sections were stained with
hematoxylin-eosin, and the volumes of all sections were calculated by
use of an ellipsoid model and summed for determination of LV volume by
Simpson’s rule.9 These data demonstrate a strong
correlation between echocardiographic and postmortem
measurements of LV volume: volume=0.97xcalculated
echocardiographic volume-19.8 µL; r=0.94,
P=0.001.
Analysis of ß1-Integrin Protein
Levels
Additional hearts (n=6 per group) underwent
collagenase perfusion for isolated LV myocyte dissociation
to achieve a highly myocyte-enriched suspension (98% myocytes and
<2% fragments of endothelial and fibroblast cells) as
described by our laboratory.4 5 After
homogenization, proteins were quantified with a BCA
assay (Biocinchoninic Assay, Pierce Chemical). Proteins were separated
by SDS-PAGE on 12% gradient gels and transferred onto nitrocellulose
membranes by standard procedures. Membranes were blocked in 5%
milk/TBST (10 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, 0.05%
Tween-20) followed by incubation with a polyclonal rabbit IgG against
ß1-integrin (2 µg/mL). Bound antibody was
detected with donkey anti-rabbit IgG conjugated with horseradish
peroxidase (Amersham) and the chemiluminescent detection system
(Amersham). Observed band densities were quantified with an AlphaImage
system and densitometric software. The
ß1-integrin antibody was prepared with
affinity-purified ß1-integrin as an immunogen
as previously described.10
Confocal Microscopic Analyses
Hearts (n=6 to 7 per group) were removed and rinsed for 1 minute
in 0.1 mol/L PBS with 50 mmol/L KCl, pH 7.2, and subsequently
fixed overnight at 4°C in 4% paraformaldehyde
prepared in PBS. Vibratome sections (100 µm) from similar areas
of the LV free wall and the septum were stained in a 1:20 dilution of
rhodamine phalloidin (Molecular Probes) and imaged with a BioRad MRC
1000 confocal scanning laser microscope. A minimum of 5 optical
sections were collected from the free wall of the LV of each animal
with a Nikon x60 NA 1.4 lens. Myocyte widths (60 to 120 myocytes per
heart) were measured perpendicular to the long axis of the sarcomeres
from unbranched areas of the myocytes near an intercalated disk with
the length/profile function in the BioRAD MRC1000 COMOS program.
Myocyte length was obtained by measuring the distance between the
intercalated disks.
In addition, in situ TUNEL was analyzed by confocal microscopy. Vibratome sections (100 µm) from 3 different hearts from each group were cut from similar regions of the hearts and triple-labeled with (1) rhodamine phalloidin, which is specific for the f-actin in myocytes; (2) TOPRO-3, which is a DNA-specific stain used for localizing nuclei; and (3) a Boehringer Mannheim TUNEL kit with FITC as the reactive fluorophore, according to the manufacturer’s instructions. Positive controls for TUNEL staining consisted of treating sections with 2% DNase 1 for 1 hour at 37°C before proceeding with the TUNEL reaction staining protocol. Negative controls for the TUNEL reaction consisted of treating sections with the TUNEL reaction mixture without terminal transferase. Sections were subsequently examined with a BioRad MRC 1000 confocal microscope with an argon/krypton laser capable of imaging the 3 fluorophores in distinct channels. Sequential images were obtained with the x10 objective from 5 random fields per section in each of the 3 channels without adjustment of the microscope stage position. Images were subsequently overlaid in the BioRad Confocal Assistant program, and nuclei stained with both the TUNEL reaction and TOPRO-3 were considered positive for the apoptosis reaction. Colocalization of the TUNEL-positive nuclei with rhodamine phalloidin–stained myocytes was used to confirm that apoptosis was occurring in myocytes.
Vibratome sections (100 µm) collected adjacent to those used for the TUNEL staining were also stained with a primary antibody against heart ß1-integrin, which recognizes both the extracellular and intracellular domains, as previously described.10 11 12 Sections were incubated at 4°C overnight in the primary antibody, rinsed in PBS, and stained at 4°C overnight with Alexa 488 (Molecular Probes) conjugated to the secondary antibody. Sections were then rinsed in PBS and mounted in a 1:3 mixture of glycerin and PBS with 10 mg/mL DABCO (Sigma). In addition, isolated fixed LV myocytes from the 7-week AS mice and age-matched normal mice were stained with this polyclonal antibody by methods published by our laboratory.10 To determine the specificity of the ß1-integrin staining in the extracellular space in the tissue sections, sections were stained with a polyclonal antibody made against specific peptides of the cytoplasmic domain of the ß1-integrin (a gift from Dr Clayton Buck, Wistar Institute). Controls consisted of incubation in nonimmune serum rather than the primary antibody followed by incubation with conjugated antibody as described above.
In Situ Ligation Assay
In additional hearts (n=2 per group), the TUNEL
assay13 as well as in situ ligation using hairpin
oligonucleotide probes was performed to identify
double-strand DNA breaks, which are highly specific for
apoptotic DNA fragmentation.14 15 16 In situ
ligation using hairpin oligonucleotide probes was
performed as described in detail by Didenko et al,15 with
some modifications. Briefly, paraformaldehyde-fixed LV
sections were deparaffinized with xylene and rehydrated in graded
alcohol concentrations. After a washing in water, sections were
incubated with 25 µg/mL proteinase K (Intergen) in PBS for 15
minutes. Sections were then rinsed with water. A mixture of 50
mmol/L Tris/HCl (pH 7.8), 10 mmol/L MgCl2,
10 mmol/L dithiothreitol, 1 mmol/L ATP, 15% polyethylene
glycol (8000 MW, Sigma) with hairpin oligonucleotide
probes at 35 µg/mL,16 and T4DNA ligase at 250 U/mL was
added. Sections were placed in a humidified box for 16 hours, then
washed in water 3 times for 20 minutes. Fluorescein-avidin
conjugate (Vector Laboratories) was added at 4 µg/mL in 50
mmol/L sodium chloride (pH 8.2) for 45 minutes. Sections were washed 3
times for 10 minutes in the same buffer and then rinsed in water before
the addition of the terminal deoxynucleotidyl
transferase mixture for TUNEL staining. For TUNEL staining to detect
free 3' DNA hydroxyl groups with terminal transferase, the published
procedure was used,13 modified to accommodate the use of
Texas Red as label rather than biotin. A mixture comprising 30
mmol/L Tris HCl (pH 7.2), 140 mmol/L sodium cacodylate, 1
mmol/L CoCl2, 0.1 mmol/L dithiothreitol,
8 µmol/L Texas Red dUTP (Molecular Probes), and 800 U/mL
terminal transferase was added for 1 hour at 37°C in a humidified
incubator. After 2 washings in water for 20 minutes, the sections were
counterstained with the DNA-binding dye DAPI (1 µg/mL), mounted in
Vectashield, and examined by fluorescence microscopy.
DNA Laddering
Genomic DNA was isolated from LV tissue (n=3 per group) with a
DNA isolation kit (Gentra). One microgram of extracted DNA was labeled
by enzymatic assay with 3'-terminal
deoxynucleotidyl transferase (TdT) as
described.17 The DNA was labeled in buffer containing
2 mmol/L CoCl2, 0.5 mmol/L DTT,
100 mmol/L potassium cacodylate, 166 nmol/L
[32P]dCTP (3000 Ci/mmol), 664 nmol/L dCTP, and
20 U TdT. The samples were incubated for 60 minutes at 37°C.
One-tenth of the total reaction volume was loaded on 1.8% agarose gel,
run at 100 V for 1.5 hours, dried, and exposed to a phosphor-sensitive
screen. As a standard positive control for tissue with massive in situ
apoptosis, thymus tissue undergoing involution was extracted
from mice 24 hours after subcutaneous injection of
dexamethasone (6 mg/kg).14 15
Statistical Analysis
All values are expressed as mean±SEM. Statistical
analysis of differences observed between the AS groups and the
control groups was done by ANOVA comparison or by ANOVA for repeated
measures where appropriate. An unpaired Student’s t test
was used for comparison among the groups at the same age after
operation. Statistical significance was accepted at the level of
P<0.05.
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Results |
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LV Hypertrophy and Hemodynamics
As shown in Table 1
, LV
weight was increased by 56% in 4-week AS mice and by 108% in 7-week
AS mice compared with age-matched controls. At these stages of
hypertrophy, there were no differences in body weight or RV
weight compared with controls. In the 4-week AS mice, absolute LV
systolic pressure was elevated and LV systolic
developed pressure per gram was similar compared with the age-matched
control group. In the 7-week AS mice, absolute LV systolic
pressure was lower than in the 4-week AS mice despite a greater
magnitude of hypertrophy. In the 7-week AS mice, LV
systolic developed pressure per gram was depressed compared
with the control group. In the absence of LV chamber dilatation (see
below), the ratio between systolic developed pressure and mass
is directly proportional to systolic wall stress and is a
measure of force development per unit of force-developing
myocardium.18 These data indicate the presence
of depressed systolic performance in 7-week AS mice. In
vivo LV end-diastolic pressure was higher in both AS groups
than in age-matched controls. Heart rate was similar between the
groups. For comparison, LV hemodynamics were also
measured in a small cohort of surviving 13-week AS mice with advanced
failure, in whom mortality is high (>75% mortality), that exhibited
lethargic cage behavior, tachypnea, foot-pad edema, and pleural
effusions. At end-stage failure, there is further depression of LV
systolic developed pressure and elevation of LV
end-diastolic pressure.
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Echocardiographic Assessment
In vivo 2D targeted M-mode echocardiograms were obtained in 4-week
and 7-week AS mice (Figure 1). As
reported in Table 2, LV posterior and
anterior wall thicknesses were increased in both 4-week and 7-week AS
mice relative to age-matched controls. In both AS groups, LV internal
diastolic dimension was slightly decreased and relative
wall thickness (an index of concentric hypertrophy) was
increased compared with age-matched control groups. In 4-week AS mice,
both endocardial fractional shortening and midwall fractional
shortening19,20 were preserved compared with age-matched
controls. In contrast, in 7-week AS mice, both endocardial fractional
shortening and midwall fractional shortening were depressed compared
with age-matched controls.
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Confocal Analysis of Morphology and
ß1-Integrin Localization
The confocal microscopic analysis of AS and control hearts
showed marked differences in wall thickness (Figure 2), as well as myocyte width (Figure 3 and Table 3). Both AS groups also showed slight
increases in myocyte length compared with controls. In the 7-week AS
animals, an increase in width of the intercalated disk and aberrant
branching of myocytes was observed in all hearts (Figure 3D, arrows).
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In the AS mice, the antibody staining for the
ß1-integrin showed an increased staining in the
ECM surrounding the myocytes as well as on the cell surface of the
myocytes (Figure 3
, B through D). As illustrated in Figure 3D, this staining pattern was prominent in all 7-week AS hearts
at the stage of early failure. These images represent a 10-µm
stacked image that provides the localization of antigens at the cell
surface in relation to the surrounding extracellular environment.
Staining at the Z line is also evident in these stacked confocal images
and is consistent with previous reports from our laboratory
describing the localization of the ß1-integrin
on the cell surface.10 Because this previous
analysis of ß1-integrin staining was
performed on isolated cells, isolated LV myocytes from 7-week AS mice
were also stained with the same antibody. As illustrated in Figure 3E, this analysis in isolated cells revealed a pattern
of localization of the antibody only at the Z bands, as previously
reported.10 No staining on the cell surface or the ECM was
evident in tissue sections when the preimmune antibody was used,
indicating that the pattern was not due to nonspecific binding in the
extracellular compartment (Figure 3A). In addition, further
staining with an antibody made against peptides from the cytoplasmic
domain of ß1-integrin (Figure 3F) showed
only intracellular staining. Taken together, these confocal images from
LV tissue showed aberrant staining of both the cell surface and
adjacent matrix in AS mice, whereas the isolated myocytes showed the
localization of ß1-integrin only on the cell
surface.
We next determined whether the abnormal pattern of
ß1-integrin staining in situ was related
predominantly to an increased expression of
ß1-integrin versus change in its localization
in AS hearts. ß1-Integrin protein levels were
measured by Western blotting in protein from LV myocytes from control
and AS hearts. As shown in Figure 4, LV
ß1-integrin protein levels were similar in
4-week and 7-week AS hearts compared with controls.
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Confocal Analysis of Apoptosis
In situ TUNEL in vibratome sections of LVs from each group was
performed (Figure 5). Five random
fields per vibratome section from 3 different hearts from each group
were obtained with the x10 objective in the confocal microscope. Each
field displayed 
3000 myocyte nuclei. Myocyte TUNEL loading was
extremely rare in the LVs of both age-matched control and 4-week AS
mice, with a range of 0 to 2 positive nuclei per field. In comparison,
TUNEL-positive myocyte nuclei were observed in all 7-week AS mice, with
a range of 0 to 17 nuclei per field. In corroboration, DNA laddering
was observed in 7-week AS mice and the positive control tissue
(involuting thymus after dexamethasone injection) but not
in 4-week AS mice or age-matched controls (data not shown).
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Because TUNEL labeling may identify DNA fragmentation from
nonapoptotic processes, experiments were performed in
additional hearts from each group to determine whether myocyte TUNEL
labeling colocalized with in situ ligation of hairpin
oligonucleotide probes, which is considered to be
highly specific and sensitive for detection of apoptotic
double-strand DNA breaks.14 15 16 Consistent with
the quantitative TUNEL labeling analysis described above, TUNEL
labeling was absent or extremely rare in control and 4-week AS hearts
but was more common in 7-week AS hearts. In all instances, in situ
ligation was present and colocalized with TUNEL-positive myocyte
nuclei, as illustrated in Figure 6.
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Discussion |
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The present study demonstrates that the ascending AS mouse provides a model to study both initial compensated LV hypertrophy and very early systolic dysfunction. Our observations show that aberrant myocyte branching and abnormal ß1-integrin deposition appear in situ at this stage of early failure in association with a very low incidence of myocyte apoptosis as reported by positive TUNEL reaction, in situ ligation, and DNA laddering. These data raise the hypothesis that the dynamic changes in cell-cell and cell-ECM anchorage inherent in multicellular growth may be a signal that contributes to the development of apoptosis in chronic pressure overload hypertrophy.
Apoptosis and Early Heart Failure
Programmed cell death is critical for the regulation of
embryological development, immune cell growth, and controlled
dismantling of cells with defective transit through the cell
cycle.21 22 23 The activation of the highly regulated
caspase-protease cascade results in proteolytic damage to the nuclear
lamina, cytoskeleton, and internucleosomal cleavage of
DNA.24 25 The in situ TUNEL technique13 24 25
has been used to identify DNA fragmentation attributed to
apoptosis in studies of end-stage human and experimental heart
failure. The prevalence of TUNEL-positive cells has varied widely,
ranging from values as high as 35 to as low as 0.2 to 0.4 positive
nuclei per 100 myocytes.26 27 28 29
Using confocal microscopy, we observed that positive in situ TUNEL was
virtually absent in LV vibratome sections of both controls and 4-week
AS animals. Consistent with this finding in early adaptive
hypertrophy, Teiger et al17 reported a very
transient wave of TUNEL-positive myocytes immediately after aortic
banding in rats but observed no increase in apoptosis in early
hypertrophy 30 days after pressure overload. In contrast,
in the 7-week AS mice with early heart failure, we observed in situ
TUNEL of LV myocytes as well as DNA laddering by DNA gel
electrophoresis consistent with DNA internucleosomal cleavage.
Although the incidence of TUNEL-positive myocyte nuclei was very low,
the frequency of TUNEL-positive nuclei observed in 7-week AS mice was
similar to that described in a recent report of transgenic mice with
Gs
overexpression that were studied during the
transition to early failure.29 Regions of positive
labeling were not associated with infiltration of inflammatory
cells.
Limitations
A limitation of the TUNEL analysis is that nick
end-labeling of 3' DNA hydroxyl groups may not discriminate highly
regulated apoptotic DNA cleavage from nonspecific DNA
fragmentation due to necrosis, in vitro autolysis, RNA synthesis, or
DNA repair.14 15 16 30 31 Recent experiments by Didenko
et al14 15 16 that examined different forms of DNA damage
have demonstrated that in situ ligation of DNA with hairpin
oligonucleotides that are complementary to the ends of
double-strand DNA breaks is highly sensitive and specific in the
identification of apoptotic nuclei and discrimination between
other forms of nonspecific DNA fragmentation. Therefore, we performed
additional experiments that demonstrated that positive labeling for in
situ ligation colocalized with TUNEL-positive myocyte nuclei labeling
in all instances. Taken together, these data provide support that
myocyte apoptosis is not a ubiquitous outcome of pressure
overload and is extremely rare in early adaptive
hypertrophy. However, myocyte apoptosis is detected
at the stage of early failure. The onset of a very low frequency of
repetitive dropout of myocytes via apoptosis has the potential
to contribute to the loss of force-developing myocytes and promote the
gradual progression of systolic dysfunction. In pathological
hypertrophy, multiple signals may increase the risk of
apoptosis, including tissue hypoxia, free-radical
injury, local production of growth-stimulating peptides such as
angiotensin II, and perturbed calcium
regulation.32 33 However, anoikis has received little
attention as a potential signal of apoptosis in in vivo chronic
hypertrophy.
Anoikis and Hypertrophy
In the present study, the combined morphological evidence of
increased myocyte branching, changes in the intercalated disk, changes
in myocyte size, and altered ß1-integrin
deposition were prominent at the stage of early failure in this model.
Previous studies in isolated myocytes have also suggested that changes
in myocyte cell shape appear long before overt failure.34
The data presented here suggest that chronic biomechanical
stress results in altered cell-cell and cell-ECM interactions as well
as an increase in cardiac myocyte size. Studies of apoptosis
using a variety of cell types have demonstrated that release of cells
from normal cell-cell and/or cell-ECM contacts are events that are
sufficient to trigger apoptosis, a process called
anoikis.35 36 37 Fibronectin and integrins, specifically
5ß1, have been shown
to be important components in triggering
apoptosis,36 37 and altered expression may be
present in cardiac hypertrophy.10 38 39
The release of myocytes from normal anchorage-dependent binding to
collagen and/or fibronectin via the
5ß1-integrin to an
anchorage-independent status could be an important signal in triggering
apoptosis during chronic hypertrophic growth.
Previous reports, as well as the staining pattern of isolated hypertrophied myocytes in this study, suggest that ß1-integrins are confined to the Z bands of the myocyte; however, these studies did not use confocal microscopy of tissue, so staining of the ECM would not have been evident. This study provides new observations that support the concept that abnormal myocyte-ECM anchorage develops during chronic hypertrophy. The data obtained from staining with antibodies against ß1-integrin indicated an increase in the presence of immunoreactive material both on the cell surface and in the ECM surrounding the myocytes. The staining in the ECM appeared to increase from 4- to 7-week AS compared with controls. Measurement of ß1-integrin protein levels by Western blots indicated that there was no significant change in the amount of ß1-integrin from isolated myocytes from AS and control hearts. However, these data do not account for ß1-integrin immunoreactive material in the ECM. Control experiments using preimmune antisera and antisera specific to only the cytoplasmic domain of ß1 showed no staining in the ECM. The characterization of specific integrins as well as the immunoreactive material that appears to be shed into the ECM is currently under way.
Shedding of the integrins into the ECM is consistent with cell growth.40 41 Because integrin attachment to the ECM represents fixed anchorage points,42 it is logical to assume that these points of attachment must repeatedly change as myocyte growth and changes in morphology occur. This would be expected to be more prominent in the later stages of hypertrophy (7-week AS), when the myocyte shows branching as well as increases in size. Thus, we propose that this shedding of the integrin-stainable material is a significant feature of hypertrophic growth; however, the precise mechanism by which this event happens is not clear. One likely mechanism is the shedding of ß1-integrin due to the presence of cell surface proteases, such as the matrix metalloproteases or the ADAMS proteins (A Disintegrin and Metalloprotease).40 41 43 44 Both classes of proteases are associated with integrins45 46 47 and could be important components in the interactions between the ECM and cell surface that contribute to the dynamic balance between cell growth and apoptosis.
In summary, these observations in AS mice support the hypothesis that myocyte apoptosis develops during the transition from hypertrophy to early failure in response to chronic biomechanical stress. Future experiments are necessary to investigate integrin–ADAMS protein regulation in hypertrophy and to conclusively demonstrate that changes in myocyte anchorage to the ECM contribute to apoptosis signaling in adult myocytes.
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Acknowledgments |
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This study was supported in part by NHLBI grant HL-38189 (Drs Lorell, Weinberg, Ding), a grant from the NASA National Space Biomedical Research Institute (Dr Lorell), and HL-37669 (Drs Borg and Price). We thank Dr Clayton Buck for his generous gift of antibody against the cytoplasmic domain of ß1-integrin. We appreciate the technical assistance of Kimber Converso and Lijie Ma in performance and analyses of the mouse cardiac echocardiography. We also appreciate the technical assistance of Hop Ngo in performance of the in situ ligation assays and thank Dr David S. Baskin for useful methodological discussion.
Received November 5, 1999; revision received December 14, 1999; accepted January 25, 2000.
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