Am J Physiol Heart Circ Physiol 295: H1394 –H1402, 2008.
First published August 8, 2008; doi:10.1152/ajpheart.00346.2008.
Cardiac-restricted overexpression of extracellular matrix metalloproteinase
inducer causes myocardial remodeling and dysfunction in aging mice
Juozas A. Zavadzkas, Rebecca A. Plyler, Shenikqua Bouges, Christine N. Koval, William T. Rivers,
Christy U. Beck, Eileen I. Chang, Robert E. Stroud, Rupak Mukherjee, and Francis G. Spinale
Medical University of South Carolina and the Ralph H. Johnson Veterans Affairs Medical Center, Charleston,
South Carolina
Submitted 2 April 2008; accepted in final form 4 August 2008
ventricular function
There are a large number of MMP types that are expressed
within the myocardium and include the soluble MMP types,
such as MMP-2 and MMP-9, as well as the membrane-bound
types such as membrane type-1 (MT1)-MMP (28, 38, 48).
Ubiquitous signaling molecules such as bioactive peptides,
cytokines, and reactive oxygen species have all been clearly
demonstrated to induce this portfolio of MMPs in vitro (7, 34,
36, 38). Although the unique functionality for each of these
MMP types with respect to the LV remodeling process is
beginning to be realized, upstream signaling pathways, which
may selectively induce MMPs, remain poorly understood. A
transmembrane protein, termed the extracellular MMP inducer
(EMMPRIN), was initially described to cause increased MMP
transcription in vitro (15, 46). An association between
EMMPRIN and MMP induction in tissue remodeling processes
such as tumor metastasis has been reported (18, 21, 46).
Moreover, increased levels of EMMPRIN have been identified
within the myocardium of patients with severe LV remodeling
(39). However, whether the increased myocardial induction of
EMMPRIN, in and of itself, can cause MMP induction and
adverse LV remodeling remains to be established. Accordingly, the present study tested the central hypothesis that
persistently elevated EMMPRIN levels within the myocardial
compartment of mice, to levels that have been observed previously within the failing human myocardium (39), would
adversely affect LV geometry and structure. The present study
measured LV function and geometry, relative myocardial
MMP content and activity, and collagen content in young and
old mice following the selective and persistent myocardial
overexpression of EMMPRIN.
METHODS
(LV) remodeling, defined as changes in the
structure and geometry of the LV myocardial wall and chamber, invariably occurs with cardiovascular disease states such
as myocardial infarction, hypertension, and cardiomyopathy,
as well as a function of age (1, 5, 8, 13, 33). An increased
expression of a family of matrix proteases, termed the matrix
metalloproteinases (MMPs), occurs in patients and animals
with the development and progression LV remodeling (6, 11,
12, 14, 16, 18, 22, 26, 39, 40, 42, 47). Moreover, with the use
of transgenic and pharmacological approaches, a cause-effect
relationship has been demonstrated between the induction of
MMPs and adverse LV remodeling (6, 12, 16, 19, 22, 26, 40).
EMMPRIN overexpression. A myocardial restricted overexpression
construct of EMMPRIN [EMMPRINexp; ␣-myosin heavy chain promoter (MHC) linked to full-length human EMMPRIN] was established in mice from the C57 background strain. The EMMPRIN
full-length human gene sequence (Genebank accession no. NM
001728; 1.6 kb) (15, 42) was cloned into the ␣-MHC construct (Clone
26, Genebank u71441; courtesy of Jeff Robbins, University of Cincinnati) (42). The incorporation of the MHC-EMMPRIN construct
was confirmed by using a PCR protocol from tail-clip DNA. Briefly,
digested DNA (2 ␮g) was subjected to PCR (37 cycles; Bio-Rad
iCycler; 170-8720XTU; Hercules, CA) using a forward primer (5⬘GGCCAGAAAACGGAGTTCAA-3⬘; Sigma-Genosys) and a reverse
primer (5⬘-GCGCTTCTCGTAGATGAAGA-3⬘; Sigma-Genosys)
specific to the EMMPRIN transgene. Southern blot analysis was
Address for reprint requests and other correspondence: F. G. Spinale,
Cardiothoracic Surgery, Rm. 625, 770 MUSC Complex, 114 Doughty St.,
Charleston, SC 29425 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
LEFT VENTRICULAR
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Zavadzkas JA, Plyler RA, Bouges S, Koval CN, Rivers WT, Beck
CU, Chang EI, Stroud RE, Mukherjee R, Spinale FG. Cardiacrestricted overexpression of extracellular matrix metalloproteinase
inducer causes myocardial remodeling and dysfunction in aging mice.
Am J Physiol Heart Circ Physiol 295: H1394 –H1402, 2008. First
published August 8, 2008; doi:10.1152/ajpheart.00346.2008.—The
matrix metalloproteinases (MMPs) play a pivotal role in adverse left
ventricular (LV) myocardial remodeling. The transmembrane protein
extracellular MMP inducer (EMMPRIN) causes increased MMP expression in vitro, and elevated levels occur in patients with LV failure.
However, the direct consequences of a prolonged increase in the
myocardial expression of EMMPRIN in vivo remained unexplored.
Cardiac-restricted EMMPRIN expression (EMMPRINexp) was constructed in mice using the full-length human EMMPRIN gene ligated
to the myosin heavy chain promoter, which yielded approximately a
twofold increase in EMMPRIN compared with that of the age/strainmatched wild-type (WT) mice; EMMPRINexp (n ⫽ 27) and WT (n ⫽
33) mice were examined at 3.2 ⫾ 0.1 or at 13.3 ⫾ 0.5 mo of age (n ⫽
43 and 26, respectively). LV end-diastolic volume (EDV) was similar
in young EMMPRINexp and WT mice (54 ⫾ 2 vs. 57 ⫾ 3 ␮l), but LV
ejection fraction (EF) was reduced (51 ⫾ 1 vs. 57 ⫾ 1%; P ⬍ 0.05).
In old EMMPRINexp mice, LV EDV was increased compared with
WT mice values (76 ⫾ 3 vs. 58 ⫾ 3 ␮l; P ⬍ 0.05) and LV EF was
significantly reduced (45 ⫾ 1 vs. 57 ⫾ 2%; P ⬍ 0.05). In
EMMPRINexp old mice, myocardial MMP-2 and membrane type-1
MMP levels were increased by ⬎50% from WT values (P ⬍ 0.05)
and were accompanied by a twofold higher collagen content (P ⬍
0.05). Persistent myocardial EMMPRINexp in aging mice caused
increased levels of both soluble and membrane type MMPs, fibrosis,
and was associated with adverse LV remodeling. These findings
suggest that EMMPRIN is an upstream signaling pathway that can
play a mechanistic role in adverse remodeling within the myocardium.
CARDIAC EMMPRIN AND REMODELING
AJP-Heart Circ Physiol • VOL
peptide will only yield a detectable UV emission when proteolytically
processed at a specific amino acid sequence, and the specificity of this
approach has been demonstrated previously (10). The LV myocardial
extracts were incubated (37°C; 2 h) in the presence and absence of the
MMP substrate, and excitation/emission was recorded (328/393;
FluoStar Galaxy, BMG Labtech). To convert the fluorescent readings
from this in situ assay to relative MMP activity, a recombinant active
MMP construct (MMP-2/-9 SE-244/237; Biomol) was utilized. Increasing concentrations of the known MMP-2/-9 catalytic domain
(0.15–2.5 ng/ml) were incubated with the MMP substrate to obtain a
linear relation between MMP activity and fluorescence. The linear
portion of the fluorescence emission-concentration curve (y ⫽
26461x; r2 ⫽ 0.99; P ⬍ 0.0001) was used to calibrate subsequent
fluorescent emission values obtained from the LV myocardial extracts
and determine a final MMP activity value (nanograms of MMP
catalytic activity per time).
Collagen biochemistry. Total myocardial collagen was determined
by quantifying hydroxyproline content in acid-hydrolyzed samples
(40). This biochemical assay was optimized for murine myocardial
samples. Briefly, 25 mg of LV myocardial free wall was hydrolyzed
overnight in hydrochloric acid. The hydrolyzed samples were reacted
with Ehrlich’s reagent, measured spectrophotometrically (550 nm),
converted to hydroxyproline values using a linearized set of hydroxyproline standards, and expressed as nanograms hydroxyproline
per milligrams of myocardial wet weight.
EMMPRIN immunohistochemistry. LV frozen sections were cut at
9 ␮m thickness and placed on glass slides. The sections were then
immersed in ice-cold acetone for 5 min and washed vigorously with
phosphate-buffered saline (PBS). The LV sections were then incubated in 10% normal goat serum for 90 min at room temperature,
washed in PBS, and then subsequently incubated in the rabbit antisera
directed specifically against the NH2 terminus of human EMMPRIN
(H-200; 1:50 dilution) overnight at 4°C. The LV sections were then
washed five times with PBS and incubated in 0.3% H2O2/PBS for 30
min. The LV sections were washed three times with PBS and
incubated in goat anti-rabbit sera (No. 6101; 1:250 dilution; Vector)
for 30 min at room temperature. The LV sections were then subjected
to the avidin-biotin reaction (ABC Kit; Vectastain Elite; Vector), and
specifically bound EMMPRIN antisera was visualized. The LV sections were then imaged on a Zeiss Axioskop 2 microscope (PlanNeofluar 63x/1, 25), and images were digitized (Zeiss AxioCam
MRc). Negative controls included the substitution of the primary
antisera with nonimmune rabbit serum.
Data analysis. Indexes of LV function and geometry were subjected to a two-way ANOVA in which the treatment effects were
EMMPRIN overexpression and age. Pairwise comparisons were performed using a Bonferroni adjusted t-test. Due to myocardial sampling limitations, subsets of mice from the four groups were utilized
in the biochemistry and immunohistochemistry studies. Mice were
randomized to the different assay protocols following the echocardiographic study. Final sample sizes for each assay are indicated. For the
EMMPRIN, MMP, and collagen biochemistry studies, all measurements were performed in duplicate. The zymographic/immunoreactive signals were analyzed using densitometric methods (Gel Pro
Analyzer; Media Cybernetics) to obtain two-dimensional integrated
optical density (IOD) values. The hydroxyproline values and the
EMMPRIN IOD values were subjected to ANOVA and Bonferroni
separation. The IOD values were also computed, where appropriate,
as a percentage of WT referent control values, where the control
values were set to 100%, and comparisons were performed by a
separate t-test. Between-group differences in these values were compared using a two-sample t-test. Results are presented as means ⫾ SE.
Values of P ⬍ 0.05 were considered statistically significant. All
statistical procedures were performed using the STATA statistical
software package (Statacorp, College Station, TX).
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performed to confirm the presence of the transgene. Three independent lines of EMMPRINexp mice were developed, and following
backcrossing and stable breeding patterns, ⬃50% of each litter was
EMMPRINexp positive. The EMMPRINexp-negative mice were used
as reference, wild-type (WT) sibling controls. The EMMPRINexp
mice displayed no obvious phenotypic abnormalities with respect to
growth and developmental characteristics. All animals were treated
and cared for in accordance with the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (NIH Pub. No.
85-23, Revised 1996). This study was approved by the Medical
University of South Carolina Institutional Animal Care and Use
Committee (AR1590).
Aging studies. EMMPRINexp and WT mice were maintained until
reaching ⬃3 (young) or 13 (old) mo of age, and then LV echocardiography was performed as described in Echocardiography. The latter
age category was chosen since past studies have identified that by
⬃12 mo old, mice begin to display age-dependent changes in wound
healing, immune response, and myocardial remodeling/metabolism
(9, 17, 20, 52).
Echocardiography. For all of the mice included in these studies,
transthoracic echocardiography was performed to measure LV geometry and function (19, 26). For these studies, the mice were anesthetized with isoflurane (2% in oxygen) and maintained at ambient body
temperature with a heating blanket. Heart rate was determined from a
surface electrocardiogram. Ambient heart rate was maintained between 400 and 500 beats/min, and the mice remained normothermic
throughout the procedure. Two-dimensional M-mode echocardiographic recordings were obtained using a 40-MHz scanning head with
a spatial resolution of 30 ␮m (Vevo 770; VisualSonics). With the use
of long-axis views, LV end-diastolic volume, ejection fraction, and
mass were computed (20). Following these studies, the isoflurane was
increased to 5%, and the LV was then harvested, quickly weighed,
flash frozen, and stored at ⫺70°C for subsequent biochemistry and
immunohistochemistry.
EMMPRIN/MMP quantitation. The relative content of myocardial
EMMPRIN was determined by immunoblotting utilizing two distinct
approaches. First, an antisera that recognizes both human and mouse
EMMPRIN was utilized in quantitative immunoblotting as detailed
below. Second, an antisera that recognizes only human EMMPRIN
was utilized in parallel immunoblotting studies. In this manner, the
specificity and stability of expressing human EMMPRIN within the
myocardial compartment could be confirmed, as well as determining
the overall increase in total myocardial EMMPRIN achieved in this
mouse construct. For immunoblotting, myocardial extracts (10 ␮g)
were loaded onto 4 –12% Bis-Tris gels and subjected to electrophoretic separation. The separated proteins were then transferred to a
nitrocellulose membrane. Following a blocking and washing step, the
membranes were incubated for 30 min at 37°C in mouse antisera
directed against amino acids 1–190 of EMMPRIN (1:5,000 dilution;
B-5; Santa Cruz Biotechnology) or in rabbit antisera directed specifically against the NH2 terminus of human EMMPRIN (1:5,000 dilution; H-200; Santa Cruz). Initial immunoblotting studies demonstrated
that this dilution of the mouse antisera recognized both the human and
mouse form of EMMPRIN, whereas there was no cross-reactivity of
the rabbit antisera against WT mouse LV extracts. In addition, the
relative abundance of the gelatinases (MMP-2 and MMP-9) were
performed on LV myocardial extracts using quantitative zymography
as described previously (16, 19, 22, 26, 39, 40). Finally, immunoblotting was performed for MMP-13 (1:2,500; No. 3533; BioVision), the
predominant rodent interstitial collagenase, and MT1-MMP (1:2,500;
No. AB815; Chemicon). Recombinant standards (MT1, Calbiochem
No. 475937; EMMPRIN, Santa Cruz-2215; and MMP-13, Biomol No.
SE-246) were included on the immunoblots as positive controls.
Myocardial MMP activity. LV myocardial extracts (10 ␮g) were
incubated with a global MMP fluorogenic substrate (OmniMMP
Fluorogenic Substrate, P-126 Biomol) that is cleaved by all active
MMPs with equivalent kinetics (10, 29). This MMP fluorogenic
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RESULTS
Fig. 1. Representative left ventricular (LV) echocardiograms taken from the long-axis, parasternal approach in a young wild-type (WT) mouse, a young
extracellular matrix metalloproteinase (MMP) inducer
expression (EMMPRINexp) mouse, an old WT
mouse, and an old EMMPRINexp mouse. Significant
LV dilation was evident in the old EMMPRINexp
group, and the summary findings are presented in Fig.
2. Scale bars ⫽ 2 mm.
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LV function and geometry. LV function and geometry were
assessed by echocardiography in 3 mo (young; 3.2 ⫾ 0.1 mo)
WT (n ⫽ 33) and EMMPRINexp (n ⫽ 27) mice and compared
with WT (old; 13.3 ⫾ 0.5 mo; n ⫽ 26) and EMMRPINexp
(n ⫽ 43) mice. Representative long-axis, two-dimensional LV
echocardiograms are shown in Fig. 1. Summary results for all
of the echocardiographic measurements of LV volumes, function, and mass are shown in Fig. 2. LV end-diastolic volume
and mass were similar between the WT and EMMPRINexp
young groups, but LV ejection fraction was reduced in the
EMMPRINexp group. In the old WT mice, LV end-diastolic
volume and ejection fraction were similar to that of young WT
mice, but LV mass was increased. In the EMMPRINexp old
group, LV end-diastolic volume and mass were significantly
increased over respective WT old and young values. Moreover,
LV ejection fraction was significantly reduced in the old
EMMPRINexp mice when compared with respective old and
young values. Thus persistent myocardial EMMPRINexp
caused LV dilation, pump dysfunction, and hypertrophy in
aging mice.
EMMPRIN and MMP levels. The results from the EMMPRIN
immunoblotting studies are summarized in Fig. 3. Immunoreactive signals for EMMPRIN were identified in LV extracts
taken from both WT and EMMPRINexp mice. However, the
total EMMPRIN signal was increased by approximately twofold in both the young and old EMMPRINexp mice. Moreover,
the increased levels of myocardial EMMPRIN in the
EMMPRINexp mice were due to a specific induction of human
EMMPRIN. Representative zymograms for MMP-2 and quantitative data for young and old WT and EMMPRINexp mice
are shown in Fig. 4. Total MMP-2 levels were reduced in the
old WT mice but were substantially increased in the old
EMMPRINexp group. The size fractionation of the zymographic signal into the proMMP-2 (72 kDa) and active MMP-2
(64 kDa) form demonstrated increased levels of both in the
old EMMPRINexp group. In contrast with MMP-2 levels,
MMP-9 was reduced in the old WT mice and was significantly reduced in both the young and old EMMPRINexp
groups (Fig. 4). MMP-13 levels were slightly reduced in the
EMMPRINexp groups, but this did not reach statistical
significance (Fig. 5). However, MT1-MMP myocardial levels were increased in both the young and old EMMPRINexp
old groups (Fig. 5).
Myocardial MMP activity. Total myocardial MMP activity
was determined in crude LV extracts from all groups utilizing
a caged fluorogenic peptide where the fluorescence emission
was transformed to units of MMP catalytic activity (Fig. 6).
Fluorescence emission values obtained in all myocardial extracts fell within the linear portion of the emission-activity
curve generated using recombinant active MMP standards. In
the old EMMPRINexp group, LV myocardial MMP activity
was increased by approximately twofold compared with WT
values.
CARDIAC EMMPRIN AND REMODELING
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Fig. 2. Summary results from the LV echocardiograms for LV end-diastolic
volume, ejection fraction, and mass for young (n ⫽ 33) and old (n ⫽ 26) WT
and young (n ⫽ 27) and old (n ⫽ 43) EMMPRINexp mice. LV end-diastolic
volume was significantly increased in the old EMMPRINexp group when
compared with respective young or old WT values. LV ejection fraction was
lower in the young EMMPRINexp group and fell significantly in the old
EMMPRINexp group. LV mass increased in the old WT group and was
increased further in the old EMMPRINexp group. P ⬍ 0.05 *vs. young WT;
#vs. old WT; ⫹vs. young EMMPRINexp.
AJP-Heart Circ Physiol • VOL
Myocardial collagen content. LV hydroxyproline was similar between the young WT and EMMPRINexp groups (11.9 ⫾
0.7 vs. 14.3 ⫾ 1.2 ng/mg; n ⫽ 8 and 6, respectively) and also
similar in the old WT group (13.0 ⫾ 0.7 ng/mg; n ⫽ 5).
However, LV myocardial hydroxyproline increased significantly in the old EMMPRINexp group compared with either
young WT, old WT, or young EMMPRINexp values (26.0 ⫾
1.6 ng/mg; n ⫽ 10; P ⬍ 0.05).
EMMPRIN immunohistochemistry. As with the immunoblotting experiments, the antisera directed against the NH2
terminus of human EMMPRIN failed to produce a positive
signal by immunohistochemistry in WT sections. A clear
positive signal for EMMPRIN could be detected in the
EMMPRINexp mice, which demonstrated a distribution primarily along the sarcolemmal surface of myocytes. Representative images taken from WT and EMMPRINexp old LV
sections are shown in Fig. 7. At a higher magnification, a
distinct EMMPRIN staining pattern emerged around the sar-
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Fig. 3. EMMPRIN immunoblotting was performed in young (n ⫽ 8) and old
(n ⫽ 6) WT and young (n ⫽ 6) and old (n ⫽ 10) EMMPRINexp LV
myocardial extracts using 2 different antisera: a mouse EMMPRIN antibody
(Ab; EMMPRIN Ms/Human) that recognized both mouse and human
EMMPRIN (top) and a rabbit antibody that recognized only human
EMMPRIN (EMMPRIN Human; bottom). A significant increase in total LV
myocardial EMMPRIN was observed in the EMMPRINexp LV myocardial
extracts, which was primarily due to the specific overexpression of human
EMMPRIN. P ⬍ 0.05 *vs. young WT; #vs. old WT. ND, not determined.
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CARDIAC EMMPRIN AND REMODELING
colemmal-interstitial interface. Negative controls yielded an
absence of a positive immunoreactive signal. Thus, in this
EMMPRINexp construct, human EMMPRIN could be identified along the membrane surface of myocytes, consistent with
this transmembrane glycoprotein.
DISCUSSION
Fig. 4. Gelatin zymography was performed on LV myocardial extracts taken
from young (n ⫽ 14) and old (n ⫽ 12) WT and young (n ⫽ 12) and old (n ⫽
17) EMMPRINexp mice to identify the abundance and size fractionation for
MMP-2 and MMP-9. A significant decrease in the proform of MMP-2 (72
kDa) was observed in old WT mice but was increased in the old
EMMPRINexp mice (top). Moreover, an overall increase in the active form of
MMP-2 (64 kDa) was observed in the old EMMPRINexp mice (middle). An
overall reduction in MMP-9 abundance was observed in the old WT and young
EMMPRINexp mice and was further reduced in the old EMMPRINexp mice
(bottom). P ⬍ 0.05 *vs. young WT; #vs. old WT; ⫹vs. young EMMPRINexp.
AJP-Heart Circ Physiol • VOL
Changes in the expression and activity of the large family of
MMPs have been well documented in animal models and in
clinical studies of LV remodeling (1, 3–5, 8, 13, 16, 33, 38, 40,
42, 47). Moreover, transgenic models and pharmacological
MMP inhibition have demonstrated a cause-effect relation
between MMP activity and adverse LV remodeling (7, 13, 36,
47). However, the expression patterns and functionality of the
different classes and subtypes of MMPs are quite diverse and
are likely to be regulated by specific upstream signaling pathways (7, 14, 34, 36, 38, 47, 49). One such upstream signaling
pathway, which appears to be unique for MMPs, is the transmembrane protein, termed EMMPRIN (15, 18, 21, 46, 53).
Although past studies have reported associative changes
between EMMPRIN levels and adverse tissue remodeling
such as tumor metastasis (21, 23, 45, 46, 50, 51, 53), the
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Fig. 5. MMP-13 and membrane type (MT)-1-MMP levels were determined by
immunoblotting on LV myocardial extracts taken from young WT (n ⫽ 14)
and old (n ⫽ 12) WT and young (n ⫽ 12) and old (n ⫽ 17) EMMPRINexp
mice. There were no changes in relative MMP-13 levels between groups.
However, MT1-MMP myocardial levels were significantly increased in the
EMMPRINexp mice. P ⬍ 0.05 *vs. young WT; #vs. old WT.
CARDIAC EMMPRIN AND REMODELING
functional and structural consequences of the cardiac-restricted
EMMPRINexp had not been explored previously. In the
present study, cardiac-restricted EMMPRINexp was induced
in mice and the effects on LV structure and function were
examined in mice at ⬃3 and 13 mo of age. The relative
increase in total myocardial EMMPRIN achieved by
EMMPRINexp was similar to that previously measured in patients
with severe LV failure (39). Unique findings from this set of
investigations were threefold. First, in old EMMPRINexp mice,
significant LV remodeling and systolic dysfunction occurred
compared with those of age-matched WT controls. Second,
EMMPRINexp caused a specific induction of MMP-2 and
MT1-MMP within the LV myocardium, which was associated
with heightened myocardial MMP activity. Finally, LV myocardial collagen content was significantly increased in old
EMMPRINexp mice. Taken together, the results from this
study suggest that a persistent myocardial induction of
EMMPRIN, to levels that have been observed in patients with
severe LV failure and following myocardial infarction (31, 39),
can directly contribute to adverse LV remodeling.
EMMPRIN, which has also been known as basigen, CD147,
M6, and tumor cell-derived collagenase stimulatory factor, is a
glycoprotein that belongs to the immunoglobin superfamily
(15, 18, 46). EMMPRIN is a transmembrane protein that has
been shown to be heavily enriched on the plasma membrane of
cancer cells from a number of different cancer types (21, 23,
45, 50, 51, 53). For example, a robust increase in EMMPRIN has
been observed in samples taken from lung, bladder, and renal
carcinoma (46). Despite the nomenclature for EMMPRIN, this
transmembrane protein has been associated with a number of
diverse molecular events, which include tumor cell growth,
changes in cell viability such as anoikis, and tumor angiogenesis (21, 23, 45, 46, 50, 51, 53). For example, in a study by
Yang et al. (51), it was demonstrated that in breast cancer cells
expressing high levels of EMMPRIN, cell size was increased
and anoikis was decreased compared with those of cells expressing low levels of EMMPRIN. Thus there is in vitro
evidence to suggest that increased EMMPRIN will affect
biological processes independent of MMP induction such as
cell growth and viability. In the present study, EMMPRINexp
within the myocardial compartment for short periods of time
(weeks) did not appear to affect myocardial growth, since there
was no change in LV mass from WT values. However, with
chronic myocardial EMMPRINexp (months), LV mass significantly increased from age-matched WT values. This robust
LV hypertrophic response is likely due to several mechanisms.
First, the persistent myocardial EMMPRINexp and subsequent
MMP induction caused LV dilation and, by extension, increased LV wall stress. The increased LV wall stress, in turn,
would serve as a mechanical stimulus for myocardial hypertrophy. Finally, persistent myocardial EMMPRINexp may
have had a direct effect of EMMPRIN on myocardial cell
growth.
EMMPRIN and MMP induction. Although the effects of
EMMPRIN on tissue remodeling and cell physiological process are likely to be diverse, one predominant feature of this
protein is the induction of MMPs (15, 18, 21, 23, 45, 46,
49 –51, 53). The MMPs constitute a large family of proteolytic
enzymes with subclasses that include the collagenases (i.e.,
MMP-1, MMP-13), the gelatinases (MMP-2, MMP-9), the
stromelysins (MMP-3), and the membrane-type MMPs, such
as MT1-MMP (16, 38, 39, 40, 47). It is becoming apparent that
there is a diverse enzymatic portfolio for these MMP types, as
well as different regulatory mechanisms. One critical control
point for MMP synthesis and release is transcriptional regulation, and the promoter region is unique for each MMP type (28,
49). Past in vitro studies have shown that increased EMMPRIN
exposure to either cancer or stromal fibroblasts results in a
specific rather than a global induction of MMPs (15, 16, 31, 45,
50). For example, increased levels of EMMPRIN from epithe-
Fig. 7. Representative LV sections taken from WT (n ⫽ 3) and EMMPRINexp old (n ⫽ 6) mice, which were stained using a selective human EMMPRIN
antisera. In WT and negative control sections (Neg Control), a complete absence of positive EMMPRIN staining was observed. However, in EMMPRINexp
sections, a specific immunoreactive signal was detected along the sarcolemmal surface of myocytes (arrows). At high power (top right), EMMPRIN
immunostaining could be appreciated to localize to the sarcolemmal-interstitial interface (arrows). Scale bar ⫽ 20 ␮m.
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Fig. 6. LV myocardial MMP activity was computed by incubating crude
myocardial extracts with a global MMP fluorogenic substrate and measuring
fluorescent emission. The mean values for LV myocardial MMP activity in the
young WT (n ⫽ 14) and old (n ⫽ 12) WT and young (n ⫽ 12) and old (n ⫽
17) EMMPRINexp mice have been plotted with respect to the standard curve.
MMP activity was significantly increased in the old EMMPRINexp myocardial
samples compared with WT and young EMMPRINexp values. P ⬍ 0.05 *vs.
young WT; #vs. old WT; ⫹vs. young EMMPRINexp.
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32, 51). Another research avenue is to identify upstream
stimuli that induce EMMPRIN, which in turn cause MMP
upregulation. In a study by Siwik et al. (35), it was demonstrated that the stimulation of the ␤-adrenergic receptor on
isolated rat ventricular myocytes caused an upregulation of
EMMPRIN, which in turn increased MMP production. Past
studies have reported previously that biologically active molecules such as cytokines, as well as oxidative stress, can
induce MMPs in cardiac fibroblasts and myocytes (7, 34, 36).
Thus, although it remains speculative, it is likely that increased
EMMPRIN levels can be induced by biological signaling
cascades that are operative in the context of LV remodeling
and failure. Indeed, increased EMMPRIN levels have been
reported in patients with cardiomyopathic disease or with
myocardial infarction, conditions in which several of these
signaling cascades would be operative (31, 39). The findings
from the present study provided new mechanistic evidence that
increased myocardial levels of EMMPRIN, in and of itself, can
cause LV remodeling and failure. Accordingly, identifying the
upstream signaling pathways causative for EMMPRIN induction with cardiovascular disease as well as the intracellular
signaling pathways by which EMMPRIN induces MMPs
would be important avenues of further research.
EMMPRIN and myocardial remodeling. In the present
study, persistent EMMPRINexp caused LV dilation and subsequent dysfunction in aging mice. The LV dilation was likely
due to EMMPRIN-mediated MMP induction and subsequently increased MMP activity. Indeed, the present study
directly demonstrated that prolonged EMMPRINexp was
associated with increased myocardial MMP activity. This
increased MMP activity would in turn cause a loss of
myocardial matrix stability and myocyte adhesion, which
would favor myocyte remodeling and ultimately changes in LV
chamber geometry. One of the findings from the present study
was that EMMPRINexp was associated with increased total
collagen content. Increased myocardial collagen content occurred in the old EMMPRINexp mice, when the determinants
of collagen degradation were also increased, suggesting that
collagen synthesis rates were higher than overall degradation
rates. There are several likely contributory factors for this shift
in steady-state collagen content with EMMPRINexp. First, the
induction of MMP-2 and MT1-MMP by EMMPRIN would
heighten pericellular matrix proteolysis, change local cellmatrix interactions, and thereby affect steady-state synthesis
rates. For example, with chronic volume overload, it has been
reported that the initial increase in MMP activation and collagen degradation was followed and then followed by collagen
accumulation and the progression to LV failure (3, 4). Second,
the increased MT1-MMP levels induced by EMMPRIN may
cause the proteolysis and the activation of several profibrotic
signaling molecules (28, 38, 44). Specifically, MT1-MMP can
proteolytically process cytokines and chemokines, which in
turn would stimulate collagen synthetic pathways (44). Finally,
enhanced EMMPRIN signaling likely caused the heightened
activation of intracellular pathways, such as MAPKs, which in
turn would cause the formation of transcription factors favoring
collagen synthesis (2, 32). Thus, although it remains speculative, it
is likely that the increased myocardial collagen content that
occurred with prolonged myocardial EMMPRINexp was due to a
summation of these proposed mechanisms. The increased myocardial content that occurred in the EMMPRINexp mice would
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lial sarcoma cells caused an induction of MMP-2, but not
MMP-9, in peritumoral fibroblasts (23). In an in vivo study,
breast cancer clones transfected with EMMPRIN caused an
induction of MMP-2 in harvested tissue extracts (53). Moreover, in glioma cell or monocyte cultures, EMMPRIN induced
the expression of MT1-MMP (30, 31). In the present study,
myocardial EMMPRINexp caused an increase in the levels of
specific MMP types, notably MMP-2 and MT1-MMP. The
association between the upregulation of these specific MMP
types by EMMPRIN is of potential importance for several
reasons. First, through the use of a transgenic construct, it has
been demonstrated that MMP-2 likely contributes to adverse
LV remodeling (26) and is significantly upregulated within the
myocardium of patients and animals with LV failure (6, 12, 16,
19, 22, 38 – 40). Second, MT1-MMP is proteolytically diverse
and can likely process a number of matrix proteins and signaling molecules involved in LV remodeling (14, 37, 38, 44). The
increased levels of MT1-MMP observed in the present study
caused by EMMPRINexp are similar to those found in patients
with end-stage LV failure (39). Finally, an important pathway
for the conversion of proMMP-2 to active MMP-2 is mediated
by MT1-MMP (14, 37, 38, 41). In the present study, long-term
myocardial EMMPRINexp was associated with increased
MT1-MMP levels as well as higher levels of active MMP-2,
which in turn caused higher MMP activity within the myocardium. These past observations along with the present results
suggest that EMMPRIN induces a specific portfolio of MMPs
within the myocardium, which in turn may contribute to the
progression of LV remodeling and failure.
EMMPRIN signaling and activation. Although it is recognized that EMMPRIN is a transmembrane protein, and increased levels have been associated with MMP induction, the
precise cell-signaling pathway remains to be established. However, through the use of recombinant constructs of specific
EMMPRIN domains and coculture experiments, it is apparent
that the signaling pathway for this glycoprotein is complex (15,
21, 23, 45, 46, 50, 51, 53). One uniform finding is that the
soluble extracellular domain of EMMPRIN is capable of interacting with membrane-bound EMMPRIN on nearby stromal
cells, which in turn can cause the induction of MMPs (23, 45,
50, 51). For example, Tang et al. (45) reported that the
exposure of a soluble EMMPRIN construct induced MMP-2
expression in tumor-associated fibroblasts. Moreover, past
studies have suggested that membrane-bound EMMPRIN can
undergo proteolytic processing by MMPs to yield a soluble
form, which acts in a pericellular fashion (23, 45). These
findings suggest that EMMPRIN may act in a paracrine fashion, through interaction with membrane-bound EMMPRIN on
nearby cells, such as endothelial cells and fibroblasts. In the
present study, human EMMPRIN overexpression was driven
by the MHC, which would in effect limit the expression to the
cardiac myocyte. However, abundant native mouse EMMPRIN
was detected within the myocardium of both WT and transgenic mice. Thus it is likely that enhanced EMMPRIN signaling occurred in all myocardial cell types. However, this issue
remains speculative and warrants further study. The intracellular signaling pathways, which are activated following
EMMPRIN engagement, are an area of active investigation (2,
31, 32, 51). However, it does appear that the activation of
mitogen-activated protein kinases (MAPKs) is an essential
intracellular event for mediating the effects of EMMPRIN (2,
CARDIAC EMMPRIN AND REMODELING
GRANT
This study was supported by a merit award from the Veterans Affairs
Health Administration (to F. G. Spinale).
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30.
CARDIAC EMMPRIN AND REMODELING