PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Heart Fail. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2743030
NIHMSID: NIHMS108973

Cardiac Restricted Over-Expression of Membrane Type-1 Matrix Metalloproteinase in Mice: Effects on Myocardial Remodeling with Aging

Abstract

Background

The direct consequences of a persistently increased myocardial expression of the unique matrix metalloproteinase (MMP), membrane type-1 (MT1-MMP) on myocardial remodeling remained unexplored.

Methods/Results

Cardiac restricted MT1-MMPexp was constructed in mice using the full length human MT1-MMP gene ligated to the myosin heavy chain promoter, which yielded approximately a 200% increase in MT1-MMP when compared to age/strain matched wild type mice (WT). LV function and geometry was assessed by echocardiography in 3 month (“young”) WT (n=32) and MT1-MMPexp (n=20) mice, and compared to 14 month (“middle age”) WT (n=58) and MT1-MMPexp (n=35) mice. LV end-diastolic volume was similar between the WT and MT1-MMPexp young groups as was LV ejection fraction. In the middle age WT mice, LV end-diastolic volume and ejection fraction was similar to young WT mice. However, in the MT1-MMPexp middle age mice, LV end-diastolic volume was approximately 43% higher and LV ejection fraction 40% lower (both p<0.05). Moreover, in the middle age MT1-MMPexp mice, myocardial fibrillar collagen increased by nearly 2-fold and was associated with an approximate 3-fold increase in the processing of the pro-fibrotic molecule, latency-associated transforming growth factor binding protein. In a second study, 14 day survival following myocardial infarction was significantly lower in middle aged MT1-MMPexp mice.

Conclusions

Persistently increased myocardial MT1-MMP expression, in and of itself, caused LV remodeling, myocardial fibrosis, dysfunction and reduced survival following myocardial injury. These findings suggest that MT1-MMP plays a mechanistic role in adverse remodeling within the myocardium.

Keywords: matrix, myocardial remodeling, ventricular function, aging

INTRODUCTION

Left ventricular (LV) remodeling is generically defined as changes in myocardial architecture and structural composition, which in turn will affect overall LV geometry and function. While the LV remodeling process evokes changes within both the cellular and extracellular compartment, recent studies have demonstrated that changes in extracellular structure and composition occur with LV remodeling.16 Specifically, the induction and activation of a family of matrix proteases, termed the matrix metalloproteinases (MMPs), have been demonstrated to occur in patients and animals and are related to the degree of LV remodeling.16 Moreover, using transgenic and pharmacological approaches, a cause-effect relationship has been demonstrated between the induction of MMPs and the LV remodeling process.2,79 However, there are a large number of MMP types which are expressed within the myocardium, and a unique functionality may exist for each of these MMP types with respect the LV remodeling process. One of the more unique MMP types, which has been identified within the human myocardium, is the membrane type MMPs (MT-MMPs) of which the MT1-MMP subtype has been the most studied.3,1015 A significant increase in the myocardial levels of MT1-MMP has been identified in patients with LV failure, and the relative magnitude of this increase was greater than that of any other MMP sub-class.3 In animal models, MT1-MMP myocardial levels are increased early and appear coincident with adverse LV remodeling.1,9 However, a direct causative relationship between persistently increased myocardial levels of MT1-MMP and the LV remodeling process has not been established. The central hypothesis of this study was that a persistent and selective increase of MT1-MMP within the myocardium would result in LV structural remodeling, dysfunction, and an inability to respond to a pathological stimulus such as myocardial infarction.

METHODS

Overview and Rationale

A murine construct of cardiac restricted over-expression of MT1-MMP was developed and then utilized to examine LV function and geometry as a function of age. Determinants of LV matrix remodeling in this construct was examined by measuring indices of transforming growth factor-beta (TGF) signaling,1621 MMP and tissue inhibitor of MMP (TIMP) levels through combined immunochemical/biochemical approaches. In order to determine whether persistent induction of MT1-MMP altered the response to a pathological stimulus, survival following a myocardial infarction (MI) was examined.

MT1-MMP Over-expression and Aging

A myocardial restricted overexpression construct of MT1-MMP (MT1-MMPexp; alpha myosin heavy chain promoter (MHC)-linked to full length human MT1-MMP) was established in mice from the FVB background strain. The MT1-MMP full length human gene sequence (GenBank 793762 Accession #90925; 2369bp) was cloned into the alpha-MHC construct (courtesy of Jeff Robbins, Univ of Cinn, Clone 26, Genebank u71441). The incorporation of the MHC-MT1-MMP construct was confirmed by using a PCR protocol from tail clip DNA. Three independent lines of MT1-MMPexp mice were developed and following backcrossing and stable breeding patterns, approximately 50% from each litter were MT1-MMPexp positive. The MT1-MMPexp negative mice were used as reference, wild-type (WT) sibling controls. The MT1-MMPexp mice displayed no obvious phenotypic abnormalities. MT1-MMPexp and WT mice were maintained until 3 months (“young”;3.0±0.1 month) or 12–18 months (“middle aged”; average age: 14±1 month) of age and then randomized to undergo LV functional assessment and myocardial sampling or to undergo surgically induced MI. This latter age category was chosen since past studies have identified that after approximately 12 months old, mice begin to display age dependent changes in wound healing and myocardial remodeling.22,23 Terminal procedures were performed under isoflurane anesthesia (4%), in which the LV was removed and then processed for histochemistry or biochemical analysis. All animals were treated and cared for in accordance with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (National Research Council, Washington, 1996) and under an approved MUSC IACUC protocol (ARC# 2389).

LV Geometry and Function

Transthoracic echocardiography was performed in order to measure LV geometry and function.9 Two-dimensional M-mode echocardiographic recordings were obtained using a 40 MHz scanning head with a spatial resolution of 30 um (Vevo 660, VisualSonics, Toronto). Using long-axis views, LV end-diastolic volume, posterior wall thickness, ejection fraction and mass were computed. Following which, the mice were positioned on a feed-back temperature controlled operating table (Vestavia Scientific, Birmingham, AL) and maintained with 2% isoflurane anesthesia. A pre-calibrated 4 electrode-pressure sensor catheter (1.4 F, SPR-839, Millar Instruments, Houston, TX) was positioned in the LV via the right carotid artery. LV pressures and relative volumetric units were continuously recorded using a pressure-conductance unit (ARIA, MPCU-200, Millar) and integrated electrical stimulation (DAQ, PV Analysis Software, Millar). The placement of the LV conductance catheter, validation procedures, and algorithm have been described previously.9,24 With continuous recording of the LV pressure-volumetric signal, gentle digital pressure was placed on the abdomen in order to reduce venous return and then released. The isochronal LV pressure-volume points were used in order to compute an index of LV contractility defined as maximal LV elastance (Emax).24

Histomorphometry

LV sections (5 um) were stained with picro-sirius red for fibrillar collagen and the percent area of collagen within the LV computed.1,9 For the subsequent MI studies, LV sections were stained with hematoxylin and eosin for measurement of MI size using computer assisted planimetry (Sigma Scan, Media Cybernetics) where MI size was expressed as a percent of the total LV area. In order to compute the relative density of alpha smooth muscle actin (ASMA) positive cells within the interstitium, reflective of myofibroblasts,25 parallel LV sections were incubated with anti-ASMA (AB5694;1:200 dilution) overnight at 4degC, and specifically bound antisera visualized by a peroxidase reaction (Vector Labs Peroxidase Substrate Kit, SK4100). The LV sections were imaged at a final magnification of 20X and 10 random fields within the mid-myocardial region, devoid of any vascular compartment, were digitized and the number of ASMA positive cells computed.

MT1-MMP Immunohistochemistry

Frozen LV sections (7 um) were fixed in ice cold acetone for 5 minutes, washed, blocked with 10% goat serum (Sigma), and then in the primary MT-MMP antisera (AB815; 1:250 dilution) overnight at 4degC. The LV sections were then vigorously washed and incubated with a secondary antisera (AlexaFluor 488;1:250 dilution, Molecular Probes, WA), cover-slipped (Vectasheild Mounting Media) and imaged using confocal microscopy (Zeiss LSM 510; Plan-Apochromat 63X/1.4; 495/519 nm excitation/emission) as well as by difference interference contrast. In a second protocol, dual staining for both MT1-MMP and ASMA was performed in which the secondary antisera utilized for ASMA localization was at different excitation/emission wavelengths (650 nm/668 nm, AlexaFluor 647; 1:250 dilution).

Myocardial MMP/TIMP Levels

Substrate zymography was performed in order to assess the relative content of the gelatinases, MMP-2 and MMP-9.1,3,9 A positive control was utilized in all zymography measurements (2 ug, MMP-2/9 SE-244/237, BIOMOL). Immunoblotting was performed for MMP-13, the predominant rodent interstitial collagenase as well as for TIMP-1, -2 and -4.1,3 For the immunoblotting studies (10 ug protein), anti-sera (1:2500 dilution) corresponding to MMP-13 (3533,BioVision); MT1-MMP (AB221,Millipore), TIMP-1(AB8122), TIMP-2 (AB801) or TIMP-4 (AB8221). For the MT1-MMP immunoblotting and activity assays, LV myocardium was homogenized in ice-cold 250-mmol/L sucrose–20-mmol/L MOPS buffer. The homogenate was centrifuged (100,000g, 1 hour), and the membrane fraction resuspended in buffer. Positive controls for MMP-13 (CC068 Millipore), MT1-MMP (CC1043), TIMP-1 (CC1062), TIMP-2(CC1064), and TIMP-4 (CC1066) were included in every assay.

MT1-MMP Activity Assay

LV myocardial extracts (50 ug) were incubated with a specific MT1-MMP fluorogenic substrate (MMP-14 Substrate I, Cat. No. 444258; Calbiochem) which has been validated previously.10 The LV myocardial extracts were incubated (37°C, 2 hrs) in the presence and absence of the MT1-MMP substrate, and excitation/emission recorded (328/400, FluoStar Galaxy, BMG Labtech Inc, NC). In order to convert the fluorescent readings from this in-situ assay to relative MT1-MMP activity, a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, Cat. No. 475935; Calbiochem 7.8–125.0 ng/mL) was utilized in a parallel set of reactions.

LTBP-1 Processing, TGF-R1 and Smad-2

One of the initial critical steps for matrix bound TGF to become a competent profibrotic signaling molecule, is through the proteolytic release from the latency TGF binding protein-1 (LTBP-1).16,21 In light of the fact that LTBP-1 is initially a high molecular weight protein, which is subsequently proteolytically processed to low molecular weights,16,21 LV extracts (20ug of total protein) were loaded onto 3–8% Tris Acetate gels (EA03785, Invitrogen, Carlsbad, CA). The LV extracts were rigidly maintained in a protease inhibitory cocktail (150 mM, EDTA: 1 mM, PMSF: 1 mM, aprotinin: 1 mg/ml, leupeptin: 1mg/ml, pepstatin). Immunoblotting was performed for LTBP-1 (SC28133; 1:200). In all of these studies, a positive control for LTBP-1 (30 ug, 3611-RF whole cell lysate, Cat# sc-2215, Santa Cruz Biotechnology, CA) was utilized. We then proceeded to determine whether and to what degree native LTBP-1 could be proteolytically processed by MT1-MMP. For these in-vitro studies, referent WT myocardial extracts (n=3; 30 ug) were incubated at 37degC for 2 hours, with increasing concentrations of the MT1-MMP catalytic domain (0.5–3 ug), and then subjected to LTBP-1 immunoblotting. Next, relative levels of the TGF-R1 were determined in LV extracts by immunoblotting (sc-398; 1:200). Finally, LV myocardial levels for a common intracellular convergence point of the TGF receptor transduction pathway, Smad-2.1719 For these studies, immunoblotting was first performed for total Smad-2, the membranes stripped and re-probed for phosphorylated Smad-2 (Cell Signaling, #3102/3104 respectively, 1:1000).

Myocardial Infarction

In these studies, old WT and old MT1-MMP mice underwent LV echocardiography, and following which, a thoracotomy was performed, the LV visualized, and the main left coronary artery ligated (8.0 Neurilon, Ethicon, K801).9 The intra-operative mortality (first 24 hours) was 15% and similar between groups. The mice were followed for 14 days post-MI at which time a second echocardiogram was performed and the LV harvested for histomorphometry and MT1-MMP measurements.

Data Analysis

LV function and geometry was compared between the referent control and aging groups using an analysis of variance (ANOVA) and pair-wise comparisons performed by a Bonferroni adjusted t-test. The zymographic/immunoreactive signals were analyzed using densitometric methods (Gel Pro Analyzer, Media Cybernetics) to obtain 2-dimensional integrated optical density (IOD) values. The IOD values were then computed as a percent of control values where the control values were set to 100% and comparisons performed by a separate t-test. For the MMP immunoassays, a Winsorized mean was utilized if extreme values existed in the data set. Between group differences in these values were compared using ANOVA followed by Bonferroni adjusted t-test. For the morphometric data (percent collagen, ASMA density), the data was first confirmed to conform to a Gaussian distribution, subjected to ANOVA and finally to Tukey’s test for mean separation. For the survival portion of the study, survival curves were constructed utilizing Kaplan-Meier probability estimates and 14 day post-MI survival compared utilizing a Chi-Square analysis. Values of p<0.05 were considered statistically significant. All statistical procedures were performed using the STATA statistical software package (Statacorp, College Station, TX). Results are presented as mean ± standard error of the mean (SEM). Final sample sizes for each protocol/experiment are indicated in the figure legend or table. The authors had full access to the data and take full responsibility for its integrity.

RESULTS

LV Function and Geometry (Table 1)

Table 1
Effects of Age on LV Geometry and Function with MT1-MMP overexpression

LV function measurements were performed under equivalent, ambient heart rates. LV systolic pressure was equivalent across the WT and MT1-MMPexp groups, as well as between young and middle aged mice. However, LV end-diastolic pressure and wall thickness were increased in both middle aged WT and middle aged MT1-MMPexp groups. LV end-diastolic volume and ejection fraction were similar between young WT and MT1-MMPexp groups, and was unchanged in the middle aged WT group. However, LV end-diastolic volume was increased and ejection fraction reduced in the middle aged MT1-MMPexp group. Emax was similar between young WT and MT1-MMPexp groups, was decreased in the middle aged WT group, and was unchanged in the middle aged MT1-MMPexp group. LV mass was increased in the middle aged WT group and was increased further in the middle aged MT1-MMPexp group.

Fibrillar Collagen Content and ASMA Density

Representative full LV sections from the young and middle aged WT and MT1-MMPexp groups under bright field to illustrate the significant changes in LV geometry, and following picro-sirius staining and polarized light imaging in order to demarcate the myocardial fibrillar collagen are shown in Figure 1. Relative LV fibrillar collagen was increased the young MT1-MMPexp group when compared to young WT values (0.95±0.14, 0.53±0.12 %, p<0.05 respectively). In the middle aged WT group, fibrillar collagen was increased compared to respective young WT values (0.82±0.08 %, p<0.05) and was increased by over 2-fold in the middle aged MT1-MMPexp group (2.26±0.59 %, p<0.05). The density of positive ASMA interstitial cells in the young WT and middle aged WT groups were similar (25±2 vs 23±1 cells/mm2), increased in the young MT1-MMPexp groups (65±5 mm2,p<0.05) and remained elevated in the middle aged MT1-MMPexp group (43±1 mm2, p<0.05).

Figure 1
Long axis LV sections stained with hematoxylin and eosin (left panels), and parallel sections stained with picro-sirius and viewed under polarized light (right panels) taken from young and middle aged WT and MT1-MMPexp mice. In addition, LV sections were ...

MT1-MMP Immunohistochemistry

LV sections were first examined for MT1-MMP relative content and distribution using confocal fluorescence microscopy (Figure 2). A clear and definitive signal for MT1-MMP could be observed along cardiac myocytes in the young WT LV sections. In the middle aged WT group, the intensity for MT1-MMP staining increased along the sarcolemmal-matrix interface. This type of distribution is consistent with the transmembrane characteristics of MT1-MMP.14,15 The greatest immunofluorescent signal was observed in both the young and middle aged MT1-MMPexp sections, with robust staining along the myocyte-matrix interface. LV sections were next subjected to dual immunoflourescence in which sections were stained for MT1-MMP as well as for ASMA. A positive signal for ASMA was observed within interstitial cells in all LV sections, consistent with the myofibroblast phenotype (Figure 2).25 Moreover, in the MT1-MMPexp sections, the increased MT1-MMP levels were spatially associated with these ASMA positive interstitial cells.

Figure 2
(A) Representative LV sections in which MT1-MMP was localized by immunofluorescence using confocal microscopy and by difference interference contrast. Increased relative abundance and staining intensity for MT1-MMP was observed along the myocyte-matrix ...

MMP/TIMP Levels and MT1-MMP Activity

Representative MT1-MMP immunoblots of LV myocardial membrane extracts are shown in Figure 3. MT1-MMP levels were increased in middle aged WT mice compared to young WT mice. MT1-MMP levels were increased by approximately 2-fold in both MT1-MMPexp groups. Representative MMP zymograms and MMP/TIMP immunoblots along with quantitative data are shown in Figure 4. Total MMP-2 levels were increased in the young and middle aged MT1-MMPexp groups, which was primarily due to a relative increase the active form of MMP-2. MMP-13 levels were increased in both the middle aged Wt and middle aged MT1-MMPexp middle aged groups. TIMP-1 levels were reduced in the middle aged MT1-MMPexp group, whereas TIMP-2 levels were increased in the young MT1-MMP group. TIMP-4 levels were increased in the middle aged WT group and in both the young and middle aged MT1-MMPexp groups. MT1-MMP proteolytic activity was assessed using a specific fluorogenic substrate and validated by increasing concentrations of a recombinant MT1-MMP construct (with a known catalytic activity (Figure 3). Myocardial MT1-MMP activity was increased by approximately 2-fold in both the young and middle aged MT1-MMPexp groups.

Figure 3
(A) LV myocardial membrane extracts from young wild type (WT; n=12), middle aged WT (n=10), young MT1-MMP overexpression (MT1-MMP; n=10), and middle aged MT1-MMP (n=10) mice were subjected to MT1-MMP immunoblotting. Total MT1-MMP myocardial levels, which ...
Figure 4
(A) LV myocardial extracts from young wild type (WT; n=12), middle aged WT (MA; n=10), young MT1-MMP overexpression (MT1-MMP; n=10), and middle aged MT1-MMP (MA; n=10) mice were subjected to gelatin zymography. Total MMP-2 levels (72 and 64kDa forms) ...

LTBP-1 Processing, TGF-R1 and Smad-2

Positive immunoreactive bands corresponding to the full length (180kDa) and proteolytically processed form (60 kDa) of LTBP-1 were observed in all LV myocardial extracts (Figure 5). Total myocardial levels of LTBP-1 were increased in the middle aged WT group and both MT1-MMP groups. The lower molecular weight form of LTBP-1 was increased to the greatest degree in the middle aged MT1-MMP group. Using increasing concentrations of a recombinant MT1-MMP catalytic domain, a relative reduction in the 180 kDa form of LTBP-1 and emergence of the 60 kDa form was observed in LV myocardial extracts (Figure 5). These results provided in-vivo and in-vitro evidence of MT1-MMP mediated proteolytic processing of LTBP-1. Accordingly, we examined whether a specific cleavage site for MT1-MMP exists within LTBP-1 in-silico, which in turn would yield the appropriate LTBP-1 fragment obtained from these immunoblotting experiments. The full length sequence for LTBP-1 (NCBI; AAI30290.1) was examined for MT1-MMP substrate binding/cleavage sites as described previously.26 From these initial mapping studies, a series of peptide mimics were next assessed for MT1-MMP proteolytic specificity. Three potential peptides were identified: A176: SGRSENIRTA, A42: SGRIGFLRTA, and B175A: SGAAMHMYTA. The peptides were aligned to each individual sequence of interest to identify potential binding/cleavage sites using the CLUSTAL W+ alignment algorithm with the BLOSSUM scoring matrix.27 Sequences demonstrating positive peptide alignment were analyzed for domain structure using the Simple Modular Architecture Research Tool to assess putative cleavage sites.28 Peptide A176 demonstrated a high concordance with LTBP-1 and significant alignment for MT1-MMP specific cleavage. Moreover, these in-silico mapping experiments predicted a LTBP-1 cleavage product of approximately 60 kDa which was obtained from the in-vivo/in-vitro experiments.

Figure 5
(A) LV myocardial extracts from young wild type (WT; n=12), middle aged WT (MA;n=10), young MT1-MMP overexpression (MT1-MMP; n=10), and middle aged MT1-MMP (MA;n=10) mice were subjected to immunoblotting for latency-associated transforming growth factor ...

LV murine extracts were next subjected to immunoblotting for TGF-R1 and a common convergence point of the TGF-R1 receptor transduction pathway, Smad-2 .1719 Total TGF-R1 levels were increased in the middle aged MT1-MMP group (Figure 6). A common Smad, Smad-2 in the classical TGF signaling pathway was measured in which both total Smad-2 and the phosphorylated form of Smad-2 were quantified (Figure 6). Total phospho-Smad-2 levels were increased in the middle aged WT group and both MT1-MMP groups. Moreover, the ratio of phosphoryated/total Smad-2 was increased to the greatest extent in the middle aged MT1-MMP group.

Figure 6
(A) LV myocardial extracts from young wild type (WT; n=12), middle aged WT (n=10), young MT1-MMP overexpression (MT1-MMP; n=10), and middle aged MT1-MMP (n=10) mice were subjected to immunoblotting for transforming growth factor beta receptor-1 (TGF-R1) ...

Persistent MT1-MMP Myocardial Expression and MI

In a second study MI was induced and after the initial 24 hour recovery period, middle aged WT (n=30) and middle aged MT1-MMP mice (n=29) were followed for 14 days post-MI (Figure 7). Prior to MI induction, baseline LV end-diastolic volume (43±1 uL) and ejection fraction (65±1%) in the middle aged WT group were very similar to those obtained in the initial cohort of middle aged WT mice (Table 1). In the middle aged MT1-MMPexp group LV end-diastolic volume was increased (77±3 uL) and ejection fraction reduced (39±2%) in a similar pattern to the initial cohort of middle aged MT1-MMPexp mice (p<0.05; Table 1). The survival rate for middle aged WT mice was 57% and was significantly lower in the middle aged MT1-MMPexp mice (14%, Chi-Square: 12.5, p=0.001). Equivalent distribution of post-mortem findings were observed between the WT and MT1-MMPexp groups where approximately 10% of the deaths were due to myocardial rupture at the LV apical region, 70% were due to occult cardiac decompensation as evidenced by significant serous fluid accumulation within the thoracic space, and 20% revealed no significant transudate or serosanginuous fluid in the thoracic space and therefore the deaths were presumed to be of a arrhythmic origin. Representative LV full sections under bright field and under polarized light for both MI groups are shown in Figure 1. Computed MI size was equivalent between the middle aged WT and MT1-MMP groups (35±4, 38±7 %, respectively). At 14 days post-MI, fibrillar collagen was increased in the middle aged WT group within the MI and remote region when compared to respective young or middle aged WT values (10.2±1.26, 3.36±0.14 %, respectively, p<0.05). In the surviving middle aged MT1-MMPexp MI mice (n=4), fibrillar collagen was increased from referent control values as well as post-MI WT values within the MI and remote regions (19.5±1.93, 6.05±0.32 %, respectively, p<0.05). Total MT1-MMP levels were increased by 2-fold and the fully proteolytically active form of MT1-MMP (55 kDa) increased by nearly 10-fold in the middle aged-MT1-MMPexp group (Figure 7).

Figure 7
(A) In an additional set of studies, old WT mice (n=30) and middle aged MT1-MMP overexpression mice (MT1-MMPexp, n= 29) were subjected to coronary ligation to induce a myocardial infarction (MI) and followed for 14 days post-MI. Post-MI survival was significantly ...

DISCUSSION

Changes in the expression and activity of the large family of matrix metalloproteinases (MMPs) have been well documented in animal models and in clinical studies of LV remodeling.19 One class of MMPs with a diverse substrate portfolio as well as unique functional aspects is the membrane type MMPs (MT-MMPs) of which MT1-MMP can be considered prototypical. While past studies have associated changes in MT1-MMP levels with adverse LV remodeling,1,3,10,11 the functional and structural consequences of cardiac restricted over-expression of MT1-MMP (MT1-MMPexp) has not been explored. In the present study, persistent cardiac restricted MT1-MMPexp was induced in mice and the effects on LV structure and function were examined as a function of age. The unique findings from this set of investigations were 3-fold. First, in middle aged MT1-MMPexp mice, significant LV remodeling and systolic dysfunction occurred, which was accompanied by increased proteolytic MT1-MMP activity and collagen content. Second, persistent MT1-MMPexp, was associated with increased proteolytic processing of latency-associated TGF transforming growth factor (TGF) binding protein (ie;LTBP-1), increased TGF receptor-1 density, and increased phosphorylation state of a common transduction convergence point of TGF signaling, Smad-2. Third, myocardial infarction (MI) in middle aged MT1-MMPexp mice, resulted in worsened post-MI survival and exacerbated collagen accumulation. Taken together, the results from this study suggest that the increased myocardial MT1-MMP levels, equivalent to those levels observed previously in patients and animals with severe LV failure,1,3,11 directly contributes to adverse LV remodeling and dysfunction, a pro-fibrotic response, and poor adaptation to a pathological stimulus such as MI.

In the present study, mice with persistent MT1-MMPexp resulted in severe LV dilation, dysfunction, and hypertrophy as a function of age. In order to determine if intrinsic myocardial contractility was affected with MT1-MMPexp, load-independent indices of contractile function were assessed using pressure-conductance volumetry. These studies revealed that LV contractility was reduced as a function of age, but was not further impaired in the MT1-MMPexp mice. These observations would suggest that the reduced LV ejection performance in the middle aged MT1-MMPexp mice was most likely due to the significant alterations in chamber geometry as well as matrix remodeling. In addition, the present study demonstrated that the induction of a pathological stimulus (MI) in these middle aged MT1-MMP mice was associated with a poor compensatory response, as defined as reduced survival. These findings suggest that the persistent induction of MT1-MMP results in a more vulnerable myocardium when exposed to MI.

One of the more unexpected outcomes from these MT1-MMPexp studies was the changes in myocardial collagen content. Total myocardial collagen content was increased by nearly 2-fold when compared to respective wild-type values in the middle aged MT1-MMPexp mice. There are several possible factors for this shift in steady-state collagen content with MT1-MMPexp. First, increased MT1-MMP levels would heighten pericellular matrix proteolysis, change local cell-matrix interactions, and thereby affect steady state synthesis rates.13,15 Second, the relative increase in myofibroblasts with MT1-MMPexp would potentially result in increase net collagen synthesis. Third, the increased myocardial collagen content in the MT1-MMPexp mice may be the direct result of the diverse proteolytic profile of this membrane bound MMP.7,1215,29 For example, increased myocardial MT1-MMP induction was accompanied by heightened activation in the determinants of the pro-fibrotic signaling pathway: TGF. Full activation and release of TGF into the interstitium requires specific proteolysis of LTBP-1.21,29 In the present study, increased fibrillar collagen content occurred in the middle aged WT mice and was associated within increased LTBP-1 processing, TGF-R1 levels, and increased phosphorylation of a critical TGF intracellular signaling molecule, Smad-2. These associative observations suggest that the increased collagen accumulation with aging is likely due, in part to increased processing and activation of the TGF pathway. Through in-vivo, in-vitro, and in-silico approaches, the present study provided evidence for a mechanistic link between MT1-MMP proteolytic processing of LTBP-1. A recent in-vitro study in endothelial cells also demonstrated that MT1-MMP proteolytically processed LTBP-1.29 More importantly, phosphorylation of the intracellular signaling molecule Smad-2 occurred to the greatest degree in the aging MT1-MMPexp mice. Thus, while the present study provides only associative data, these unique findings suggest that the induction of MT1-MMP causes LTBP-1 processing and subsequently a profibrotic signaling cascade which culminates in increased myocardial collagen accumulation.

It has been demonstrated previously that once MT1-MMP undergoes translational processing and trafficking to the membrane, then a proteolytically competent enzyme exists.1215 In the present study, cardiac restricted MT1-MMPexp resulted in over a 2-fold increase in full length MT1-MMP within the myocardial membrane, which yielded a parallel increase in MT1-MMP activity. Thus, this study effectively increased MT1-MMP proteolytic activity within the myocardial compartment. MT1-MMP is an important pathway for proteolytically processing MMP-2 to the active form.7,13 Using a zymographic approach which provides a sensitive means to identify and size fractionate MMP-2, much higher levels of the active form of MMP-2 were observed in the MT1-MMPexp groups. The emergence of greater amounts of the 68kDa form would indicate that greater amounts of MMP-2 are being processed from the proform to the active form. These observations provide the first in-vivo evidence that selective induction of MT1-MMP within the myocardial compartment in and of itself causes increased levels of an active form of MMP-2. Increased activation of MMP-2 would further contribute to matrix instability and loss of cellular continuity in the MT1-MMPexp mice. MMP-13 levels, the predominant rodent collagenase, were increased in both the middle aged wild type and MT1-MMPexp mice. The increased MMP-13 levels in the aging myocardium would in turn, contribute to the instability and disruption of a normally functioning matrix. However, extrapolation of MMP protein levels to enzymatic activity must be done with caution and requires the consideration of a number of post-translational events including the relative levels of the endogenous MMP inhibitors- the TIMPs. In the present study, relative TIMP-1 levels fell in the middle aged MT1-MMPexp group, and relative TIMP-2 levels increased in the young MT1-MMPexp group. In the middle aged WT and the middle aged MT1-MMPexp groups, TIMP-4 levels were increased. Thus, TIMPs do not change in a uniform fashion as a function of age, and do not necessarily change in a uniform pattern with changes in relative MMP levels. This observation would suggest that TIMPs are differentially regulated within the myocardial compartment. Moreover, functional studies have identified unique roles for each of these TIMPs in the context of MMP processing, inhibition and matrix remodeling.7,30 Thus, a more comprehensive stoichiometric analysis of MMP and TIMP complexes in this transgenic system with aging would be necessary. In addition, TGF has been shown to upregulate TIMPs.20 Thus, increased TGF signaling would also contribute altered MMP/TIMP stoichiometry, which in turn would cause a shift in the balance of ECM turnover favoring ECM accumulation, and eventually fibrosis.

Increased myocardial levels of MT1-MMP have been reported previously in the context of LV remodeling in humans and animals.1,3,11 Through the use of microdialysis in a large animal model, it has been demonstrated that increased myocardial MT1-MMP activity occurs very early following ischemia.10 The present study utilized a cardiac overexpression model of MT1-MMP, driven by a myosin heavy chain promoter, in order to induce myocardial MT1-MMP levels to those levels observed in these past studies. However, using the myocyte heavy chain promoter, the preponderance of expression will be restricted to the cardiac myocyte. LV myocardial fibroblasts robustly express MT1-MMP, and increased fibroblast levels of MT1-MMP have been reported in patients with end-stage LV failure.11 In the present study, interstitial density of ASMA positive cells, consistent with myofibroblasts,25 were increased with MT1-MMP induction and co-localized to the sarcolemmal sites of MT1-MMP expression. Whether increased density or phenotypic transformation of ASMA positive myocardial fibroblasts, was a consequence of MT1-MMP overexpression remains to be established. Moreover, whether MT1-MMP induction in fibroblasts as well as in cardiac myocytes may cause a more severe LV phenotype remains to be explored. The present study examined the consequences of MT1-MMP overexpression, but targeted downregulation of this MMP was not addressed. Thus, base upon past studies identifying increased MT1-MMP levels in the failing human myocardium and the results from the present study, more targeted and selective transgenic/pharmacological strategies to selectively interrupt MT1-MMP myocardial expression and activity in the context of LV remodeling would be warranted.

Acknowledgements

The authors wish to gratefully acknowledge Jeffery D. Molkentin University of Cinncinatti and the transgenic core facility of that institution for assistance in establishing the transgenic construct utilized in these studies.

Funding Sources

This study was supported by NIH grants HL059165, PO1 HL048788, HL078650, and a Merit Award from the Veterans’ Affairs Health Administration.

Footnotes

Disclosures

Francis G. Spinale: NIH and VA grant recipient

G. Patricia Escobar, Rupak Mukherjee, Juozas A. Zavadzkas, Stuart M. Saunders, Laura B. Jeffords, Allyson M. Leone, Christy Beck, Shenikqua Bouges, Robert E.

Stroud: None

References

1. Wilson EM, Moainie SL, Baskin JM, Lowry AS, Deschamps AM, Mukherjee R, Guy TS, St John-Sutton MG, Gorman JH, 3rd, Edmunds LH, Jr, Spinale FG. Region- and type-specific induction of matrix metalloproteinases in post-myocardial infarction remodeling. Circulation. 2003;107:2857–2863. [PubMed]
2. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nübe O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med. 1999;5:1135–1142. [PubMed]
3. Spinale FG, Coker ML, Heung LJ, Bond BR, Gunasinghe HR, Etoh T, Goldberg AT, Zellner JL, Crumbley AJ. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation. 2000;102:1944–1949. [PubMed]
4. Li YY, Feng Y, McTiernan CF, Pei W, Moravec CS, Wang P, Rosenblum W, Kormos RL, Feldman AM. Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices. Circulation. 2001;104:1147–1152. [PubMed]
5. Brower GL, Gardner JD, Forman MF, Murray DB, Voloshenyuk T, Levick SP, Janicki JS. The relationship between myocardial extracellular matrix remodeling and ventricular function. Eur J Cardiothorac Surg. 2006;30:604–610. [PubMed]
6. Asbun J, Villarreal FJ. The pathogenesis of myocardial fibrosis in the setting of diabetic cardiomyopathy. J Am Coll Cardiol. 2006;47:693–700. [PubMed]
7. Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev. 2007;87:1285–1342. [PubMed]
8. Bergman MR, Teerlink JR, Mahimkar R, Li L, Zhu BQ, Nguyen A, Dahi S, Karliner JS, Lovett DH. Cardiac matrix metalloproteinase-2 expression independently induces marked ventricular remodeling and systolic dysfunction. Am J Physiol Heart Circ Physiol. 2007;292:H1847–H1860. [PubMed]
9. Ikonomidis JS, Hendrick JW, Parkhurst AM, Herron AR, Escobar PG, Dowdy KB, Stroud RE, Hapke E, Zile MR, Spinale FG. Accelerated LV remodeling after myocardial infarction in TIMP-1-deficient mice: effects of exogenous MMP inhibition. Am J Physiol Heart Circ Physiol. 2005;288:H149–H158. [PubMed]
10. Deschamps AM, Yarbrough WM, Squires CE, Allen RA, McClister DM, Dowdy KB, McLean JE, Mingoia JT, Sample JA, Mukherjee R, Spinale FG. Trafficking of the membrane type-1 matrix metalloproteinase in ischemia and reperfusion: relation to interstitial membrane type-1 matrix metalloproteinase activity. Circulation. 2005;111:1166–1174. [PubMed]
11. Spruill LS, Lowry AS, Stroud RE, Squires CE, Mains IM, Flack EC, Beck C, Ikonomidis JS, Crumbley AJ, McDermott PJ, Spinale FG. Membrane-type-1 matrix metalloproteinase transcription and translation in myocardial fibroblasts from patients with normal left ventricular function and from patients with cardiomyopathy. Am J Physiol Cell Physiol. 2007;293:C1362–C1373. [PubMed]
12. Osenkowski P, Toth M, Fridman R. Processing, shedding, and endocytosis of membrane type 1-matrix metalloproteinase (MT1-MMP) J Cell Physiol. 2004;200:2–10. [PubMed]
13. Barbolina MV, Stack MS. Membrane type 1-matrix metalloproteinase: substrate diversity in pericellular proteolysis. Semin Cell Dev Biol. 2008;19:24–33. [PMC free article] [PubMed]
14. Wu YI, Munshi HG, Snipas SJ, Salvesen GS, Fridman R, Stack MS. Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking. Biochem J. 2007;407:171–177. [PubMed]
15. Baciu PC, Suleiman EA, Deryugina EI, Strongin AY. Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells. Exp Cell Res. 2003;291:167–175. [PubMed]
16. Rifkin DB. Latent transforming growth factor-beta (TGF-beta) binding proteins: orchestrators of TGF-beta availability. J Biol Chem. 2005;280:7409–7412. [PubMed]
17. Feng XH, Derynck R. Specificity and Versatility in TGF-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21:659–693. [PubMed]
18. Massague J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol. 2000;1:169–178. [PubMed]
19. Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang XF, Massague J. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 1992;71:1003–1014. [PubMed]
20. Kwak HJ, Park MJ, Cho H, Park CM, Moon SI, Lee HC, Park IC, Kim MS, Rhee CH, Hong SI. Transforming growth factor-beta1 induces tissue inhibitor of metalloproteinase-1 expression via activation of extracellular signal-regulated kinase and Sp1 in human fibrosarcoma cells. Mol Cancer Res. 2006;4:209–220. [PubMed]
21. Koli K, Saharinen J, Hyytiainen M, Penttinen C, Keski-Oja J. Latency, activation, and binding proteins of TGF-beta. Microsc Res Tech. 2001;52:354–362. [PubMed]
22. Danon D, Kowatch MA, Roth GS. Promotion of wound repair in old mice by local injection of macrophages. Proc Natl Acad Sci U S A. 1989 Mar;86(6):2018–2020. [PubMed]
23. Yang Y, Ma Y, Han W, Li J, Xiang Y, Liu F, Ma X, Zhang J, Fu Z, Su YD, Du XJ, Gao XM. Age-related differences in postinfarct left ventricular rupture and remodeling. Am J Physiol Heart Circ Physiol. 2008;294:H1815–H1822. [PubMed]
24. Nemoto S, DeFreitas G, Mann DL, Carabello BA. Effects of changes in left ventricular contractility on indexes of contractility in mice. Am J Physiol Heart Circ Physiol. 2002;283:H2504–H2510. [PubMed]
25. Eyden B. The myofibroblast: phenotypic characterization as a prerequisite to understanding its functions in translational medicine. J Cell Mol Med. 2008;12:22–37. [PubMed]
26. Kridel SJ, Sawai H, Ratnikov BI, Chen EI, Li W, Godzik A, Strongin AY, Smith JW. A unique substrate binding mode discriminates membrane type-1 matrix metalloproteinase from other matrix metalloproteinases. J Biol Chem. 2002;277:23788–23793. [PubMed]
27. CLUSTAL W+ alignment algorithm with the BLOSSUM scoring matrix (SeqWeb (v3.1.2) interface to the Genetic Computer Group. Wisconsin Package (v10.3) analysis software. San Diego, CA: Accelrys, Inc.;
28. Simple Modular Architecture Research Tool. Annes JP, Munger JS, Rifkin DB. Making sense of latent TGFbeta activation. J Cell Sci. 2003;116:217–224. SMART http://smart.embl.de/ [PubMed]
29. Tatti O, Vehviläinen P, Lehti K, Keski-Oja J. MT1-MMP releases latent TGF-beta1 from endothelial cell extracellular matrix via proteolytic processing of LTBP-1. Exp Cell Res. 2008;314:2501–2514. [PubMed]
30. Lovelock JD, Baker AH, Gao F, Dong JF, Bergeron AL, McPheat W, Sivasubramanian N, Mann DL. Heterogeneous effects of tissue inhibitors of matrix metalloproteinases on cardiac fibroblasts. Am J Physiol Heart Circ Physiol. 2005;288:H461–H468. [PubMed]