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Cardiovascular Research 2005 66(2):410-419; doi:10.1016/j.cardiores.2004.11.029
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Copyright © 2004, European Society of Cardiology

Age-dependent changes in myocardial matrix metalloproteinase/tissue inhibitor of metalloproteinase profiles and fibroblast function

Merry L. Lindseya,*, Danielle K. Goshorna, Christina E. Squiresa, G. Patricia Escobara, Jennifer W. Hendricka, Joseph T. Mingoiaa, Sarah E. Sweterlitscha and Francis G. Spinalea,b

aDivision of Cardiothoracic Surgery Research, Room 629, Strom Thurmond Research Building, 770 MUSC Complex, Medical University of South Carolina, 114 Doughty Street, P.O. Box 250778, Charleston, SC 29425, United States
bRalph A. Johnson Veterans Administration Medical Center, Charleston, SC 29425, United States

* Corresponding author. Tel.: +-1 843 876 5186; fax: +-1 843 876 5187. Email address: lindseml{at}musc.edu

Received 30 August 2004; revised 3 November 2004; accepted 24 November 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: To evaluate the effects of aging on left ventricular (LV) geometry, collagen levels, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) abundance, and myocardial fibroblast function.

Methods: Young (3-month-old; n=28), middle-aged (MA; 15-month-old; n=17), and old (23-month-old; n=16) CB6F1 mice of both sexes were used in this study. Echocardiographic parameters were measured; collagen, MMP, and TIMP levels were determined for both the soluble and insoluble protein fractions; and fibroblast function was evaluated.

Results: LV end-diastolic dimensions and wall thickness increased in both MA and old mice, accompanied by increased soluble protein and decreased insoluble collagen. Immunoblotting revealed differential MMP/TIMP profiles. Compared to MA levels, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-14 increased, and TIMP-3 and TIMP-4 decreased in the insoluble fraction of old mice, suggesting increased extracellular matrix (ECM) degradative capacity. Fibroblast proliferation was blunted with age.

Conclusion: This study, for the first time, identified specific differences in cellular and extracellular processes that likely contribute to age-dependent ECM remodeling.

KEYWORDS Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Extracellular matrix; Matrix remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiovascular disease is a leading cause of morbidity and mortality that occurs with increasing incidence in the elderly [1,2]. With aging, the myocardium undergoes structural remodeling and hypertrophy. An important component of structural remodeling is remodeling of the extracellular matrix (ECM). The ECM integrates cell and tissue function by providing a scaffold on which cells migrate, grow, and differentiate [3]. A major constituent of the ECM is fibrillar collagen. Under basal conditions in the young adult, collagen turnover occurs at a rate that allows for normal replacement and the maintenance of left ventricular (LV) structure and function. The determinants that regulate collagen turnover, particularly as a function of aging, remain poorly understood.

Matrix metalloproteinases (MMPs) are key enzymes involved in ECM turnover. The MMP family is comprised of more than 25 individual members divided into specific classes based on in vitro substrate specificity for various ECM components. MMP activity is inhibited by tissue inhibitors of metalloproteinases (TIMPs), a family currently composed of four members [4]. MMPs are involved in several cardiovascular disease processes, including plaque rupture [5,6]; aneurysm formation and rupture [7]; LV remodeling following pressure and/or volume overload [8+IBM-12]; and during all stages of congestive heart failure progression [13+IBM-15]. Whether age-related changes in MMPs and TIMPs provide a molecular mechanism for changes in ECM structure has not been demonstrated.

The myocardial fibroblast is a predominant cell regulating ECM turnover by altering (1) ECM synthesis and deposition, (2) ECM degradation and turnover through the production and release of MMPs, and/or (3) mechanical tension on the collagen network [16]. Based on previous observations, age-related changes in fibroblast function could provide a cellular mechanism for changes in ECM structure. Therefore, the goal of this study was to evaluate aging mice for changes in LV structure; altered levels of collagen, MMPs, and TIMPs; and differences in fibroblast function.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Mice
CB6F1 mice are the F1 hybrid of C57BL/6 and BALB/c mice and were selected because they are genetically heterogeneous and display hybrid vigor. Young (3 months old; n=11 female and 17 male), middle-aged (MA; 15 months old; n=6 female and 12 male), and old (23 months old; n=6 female and 11 male) CB6F1 mice were purchased from the Aged Rodent Colonies maintained by the National Institute of Aging. The age groups (3, 15, and 23 months old) correspond to young adult, middle-aged adult, and old but not senescent adult, respectively. All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). All studies were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.

2.2. Echocardiography and tissue collection
For echocardiographic acquisition, the mice were anesthetized with 1+IBM-2% isoflurane (depending on the individual mouse), with heart rates maintained at +ImU-400 beats per minute [17]. Echocardiographic acquisition and analysis were performed using the Sonos 5500 (Agilent Technologies) equipped with a high-band linear 15.6 MHz transducer to obtain m-mode images. LV dimensions and wall thickness were measured as previously described [18]. Echocardiography was not performed on one MA and one old mouse because the mice died before echocardiograms were acquired.

For tissue collection, the mice were anesthetized with 5% isoflurane, the coronary vasculature was flushed with saline, and the heart was excised. The LV and RV were separated and weighed individually. The LV was divided longitudinally into two halves so that each half contained septum and free wall. One half was snap frozen for immunoblotting and the other half was used immediately for myocardial fibroblast isolation.

2.3. Protein isolation and collagen content
A highly reproducible sample fractionation protocol based on differential protein solubility was used to extract proteins from LV samples. To extract easily soluble proteins (e.g., cytoplasmic proteins), the LV half was homogenized in soluble lysis buffer (0.25 M sucrose and 1 mM EDTA) and centrifuged at 13,000 +ANcg for 10 min [19]. To extract insoluble proteins (e.g., membrane proteins), the insoluble pellet was resuspended in chaotropic membrane extraction reagent (7 M urea, 2 M thiourea, and the detergent amidosulfobetaine-14; Sigma). Fractionation purity was confirmed by immunoblotting for thioredoxin (>90% in the soluble fraction) and SERCA2 (>95% in the insoluble fraction). Protein concentrations were determined using the Bradford assay. Because of the high urea content, insoluble protein extracts were diluted 1:40 to ensure compatibility with the Bradford assay. All samples (5 +A7w-g) were run on gels to confirm the accuracy of protein concentrations.

Collagen volume fractions were determined in protein extracts using the microplate picrosirius red assay [20,21]. Soluble collagen was measured in the soluble fraction; insoluble collagen was measured in the insoluble fraction. Equal amounts of myocardial extracts (10 +A7w-g total protein) were added to triplicate wells of a 96-well microtiter plate. The samples were dried in the incubator and stained for 1 h with 100 +A7w-l of 0.1% picrosirius red in saturated picric acid (w/v). The dye was solubilized in 100 +A7w-l of 0.1 M NaOH, and the plates were read by spectrophotometry (Multiskan MCC/340) at an absorbance of 540 nm. Vitrogen 100 purified collagen (Collagen Biomaterials) was used as a positive control and to generate a standard curve. The amount of collagen per 10 +A7w-g total protein was obtained from the standard curve and multiplied by the total protein to give total collagen levels. Total collagen levels were divided by the initial LV wet weight to obtain microgram collagen per milligram LV wet weight.

2.4. Immunoblotting
Immunoblotting was performed as described previously [22] using antibodies for MMP-2, MMP-3, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, and TIMP-4 (Chemicon), MMP-7, MMP-8, and TIMP-2 (Oncogene), TIMP-3 and Mac-3 (Cedarlane), and the discoidin domain receptor (DDR2; Genex Bioscience). With the exception of MMP-2, the MMP antibodies recognized both pro and active forms (Table 1). A sample of breast cancer homogenate was run on each gel as a positive control and to verify the correct molecular weight size band. Recombinant proteins were also used as positive controls when further confirmation was required. All samples for each set were run on one of two 26-well 4+IBM-20% Criterion Tris+IBM-HCl gels (Bio-Rad). The two gels and two blots were handled identically and simultaneously to minimize intergel variability. Ponceau staining of the blots confirmed the successful transfer, and the positive control sample, which was run on both gels, served to confirm consistency between the two blots.


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  		Table 1 MMP and TIMP antibodies used for immunoblotting

 
2.5. Myocardial fibroblast isolation
Myocardial fibroblast isolation from mice is technically challenging, and yields vary tremendously depending on which strain of mice is used (unpublished observation). Several mouse myocardial fibroblast isolation protocols have been published, from which we developed the following protocol [23+IBM-25]. The LV half was placed into cold Dulbecco's modified eagle media (DMEM; Gibco-BRL/Invitrogen; Grand Island, NY) with 10% fetal bovine serum (FBS) and 1% antibiotic+IBM-antimycotic solution (penicillin, streptomycin, amphotericin B; Cellgro) and used immediately for fibroblast isolation using the outgrowth technique. Each LV was used to establish separate primary cultures. The cells displayed characteristic fibroblast morphology, and cell cultures were confirmed to be pure fibroblasts by immunocytochemistry using a composite of antibodies. The antibodies used were against vimentin (Sigma; positive), desmin (Sigma; negative), factor VIII (Sigma; negative), the discoidin domain receptor 2 (DDR2; Genex Bioscience; positive), +A7E smooth muscle actin (Sigma; positive), and proly-4-hydroxylase (Chemicon; positive) [26,27]. Passage 1+IBM-4 cells were used for the functional assays.

2.6. Fibroblast functional assays and protein levels
Proliferation, migration, and adhesion indexes were evaluated using protocols published for mice, rat, and/or dog and modified as described below. For proliferation assays, myocardial fibroblasts were plated at 1+ANc-104 per well in quadruplicate on 0.2% gelatin-coated 24-well plates and incubated for 24 h. The wells were washed with phosphate-buffered saline, and the cells were fixed for 20 min with zinc+IBM-formalin (Anatech). The plates were stained with 1% methylene blue. After eluting stain with acid alcohol (0.05 M HCl in 50% ethanol), the plates were read at an absorbance of 620 nm [28+IBM-30].

To compare migration properties, fibroblasts were maintained in serum-free media (DMEM supplemented with a 1+ANc solution of insulin, transferrin, and selenium, 0.1% bovine serum albumin, 10 +A7w-g/mL ascorbate, and 1+ANc antibiotic/antimycotic solution) for 24 h and then passaged. Serum starved cells were seeded (3+ANc-104 per well in duplicate wells) into the upper compartment of a transwell tissue-culture-treated insert (Costar Brand, Corning, Acton, MA) of 24-well tissue culture plates. Preliminary studies were performed to determine optimal cell densities and incubation times. FBS (10%) served as a chemoattractant and was placed in the lower chamber of each well. Following 4 h incubation, fibroblasts that had migrated to the underside of the membrane were fixed and stained with 1% methylene blue as described above. The migration index was calculated as a percentage of the average optical density for the young fibroblast group.

For adhesion rates, myocardial fibroblasts were plated at 3+ANc-104 per well in duplicate on ECM-coated 24-well plates (BD BioCoat Cellware; Becton Dickinson, Franklin Lakes, NJ). Substrates analyzed for fibroblast adhesion characteristics were used at concentrations recommended [31] and included plastic, gelatin (0.2 mg/mL), laminin (2 +A7w-g/mL), fibronectin (2.5 +A7w-g/mL), collagen I (5 +A7w-g/mL), and collagen IV (5 +A7w-g/mL). Plates were incubated for 10 min and then washed with PBS to remove nonadherent cells. Initial experiments using 30- and 60-min incubations demonstrated that saturation was already reached by 10 min. Fibroblasts were fixed and stained with 1% methylene blue as described above.

To analyze protein levels in fibroblast cell pellets, confluent fibroblasts were incubated in serum-free media for 48 h, and the cell pellets were homogenized in chaotropic membrane extraction reagent to obtain total protein. Immunoblotting was performed using antibodies for +A7E smooth muscle actin (Sigma) and MMP-9.

2.7. Data analysis
Data are presented as mean+ALE-standard error of the mean (S.E.M.). Statistical analyses were performed using Intercooled Stata 8.0 for Windows (Stata, College Station, TX). Echocardiographic, proliferation, and adhesion data were analyzed by repeated measures analysis of variance, with comparisons between individual groups made using the Bonferroni-corrected t-test. A two-tailed value of p<0.05 was considered statistically significant. The migration data were normalized as a percentage of the average value for the young or MA group and analyzed using a one-sample t-test to compare MA and old values to young or MA levels set at 100%. Immunoblotting data were analyzed as total MMP data (which included all forms of the enzyme in the pooled soluble and insoluble fractions) and for each MMP form individually for each of the two fractions. A one-sample t-test was used to compare MA and old values to the average young value. Linear regressions were performed to determine relationships between groups, using age as the independent variable. Changes that occurred with aging were divided into four types: (1) changes that continually progressed in the same direction from the young to MA to old groups; (2) changes that occurred from the young to MA group and then remained stable in the old group; (3) changes that occurred only in the old group; and (4) changes that occurred in one direction in MA and then reversed direction in the old group [32].


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effect of aging on LV morphometrics
LV mass, end-diastolic dimensions, and posterior wall thickness increased in the MA and old groups, while fractional shortening was preserved (Table 2). The LV mass calculated from echocardiographic measurements correlated significantly with LV mass taken at necropsy (r=0.85; p<0.001; n=61).


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  		Table 2 LV geometry, necropsy data, and total protein levels in aging mice

 
3.2. Effect of aging on total protein and collagen levels
Protein from young (n=24), MA (n=15), and old LV samples (n=14) were differentially extracted into soluble and insoluble fractions. The percentage of total protein obtained for the combined fractions per initial wet weight was 14.7+ALE-0.5%, 14.2+ALE-0.7%, and 14.0+ALE-0.7% for young, MA, and old groups (p=0.68), indicating that equal yields were recovered.

Concordant with increases in LV mass, total protein and collagen levels increased in the MA and old groups. Protein levels were increased in the soluble and insoluble fractions, while collagen levels increased in the soluble fraction only. When normalized to the initial LV wet weight, protein levels remained elevated in the soluble fraction, indicating that the increase in soluble protein was greater in proportion to the increase in LV mass (Table 2). The increase in soluble protein contributed to an increased soluble to insoluble protein ratio. The ratios were 0.27+ALE-0.02, 0.36+ALE-0.03, and 0.36+ALE-0.02 for young, MA, and old groups, respectively (p<0.05 for young vs. MA and young vs. old). In contrast, collagen levels decreased in the insoluble fraction, indicating that insoluble collagen levels were not maintained for the given increase in LV mass (Table 2). The decrease in insoluble collagen contributed to an increased soluble to insoluble collagen ratio. The ratios were 0.28+ALE-0.03, 0.38+ALE-0.03, and 0.41+ALE-0.04 for young, MA, and old groups, respectively (p<0.05 for young vs. old).

3.3. Effect of aging on MMP and TIMP profiles
MMP and TIMP levels were determined by immunoblotting. Representative immunoblots are shown in Fig. 1. Total MMP and TIMP levels were analyzed as a percent change from young and MA levels (Table 3). MMP-2, MMP-7, MMP-8, and TIMP-2 levels were not changed between the young and MA or old groups or between MA and old groups. Compared to young levels, MMP-3, MMP-9, MMP-12, MMP-14, and TIMP-1 were decreased and TIMP-3 increased in the MA group. MMP-13, MMP-14, and TIMP-4 were decreased in the old group. Compared to MA levels, MMP-3, MMP-9, and MMP-14 were increased and TIMP-4 decreased in the old group.


Figure 1
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  		Fig. 1 Aging differentially regulates MMP-8 and TIMP-4 expression. Soluble MMP-8 levels were unchanged (A), while insoluble MMP-8 levels increased in the old age group (B)+-breast cancer homogenate positive control sample. Soluble TIMP-4 levels were unchanged (C), while insoluble TIMP-4 levels decreased in the old age group (D). All immunoblots are representative. All samples were run on one of two gels. For each set, two lanes from each of the two gels are shown for each age group.

 

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  		Table 3 Total myocardial MMP and TIMP levels in middle-aged and old mice, as a percentage of young or middle-aged levels

 
MMP and TIMP levels from each fraction were compared to young levels (Table 4). MMP-7, MMP-13, TIMP-1, and TIMP-2 were detected in the soluble fraction only. MMP-2, MMP-7, TIMP-2, and TIMP-4 levels were not changed between the young and MA or old groups. In the soluble fraction, MMP-3, MMP-9, MMP-12, MMP-13, MMP-14, and TIMP-1 levels decreased in the MA group. Soluble MMP-3, MMP-12, MMP-13, and MMP-14 levels remained decreased in the old group. In the insoluble fraction, MMP-3 levels increased and MMP-9 levels decreased in the MA group. MMP-3, MMP-8, and MMP-14 levels were increased, while TIMP-3 levels were decreased in the insoluble fraction of the old group.


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  		Table 4 Myocardial MMP and TIMP levels in young, middle-aged, and old LV

 
MMP and TIMP levels in the old group were also compared to MA levels (Table 4). MMP-2, MMP-7, TIMP-1, and TIMP-2 were not different between the MA and old groups. In the soluble fraction of the old group, MMP-3, MMP-12, and MMP-13 levels decreased, and MMP-14 levels increased. In the insoluble fraction of the old group, MMP-3, MMP-8, MMP-9, MMP-12, and MMP-14 levels increased, and the levels of TIMP-3 and TIMP-4 decreased. Changes between the groups are summarized and characterized in Fig. 2.


Figure 2
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  		Fig. 2 Summary of matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) changes with aging. Four types of changes occurred: (1) progressive change from young to middle-aged to old groups; (2) change from young to middle-aged and old groups but not between middle-aged and old groups; (3) change only in old group; or (4) change in one direction between young and middle-aged groups then a reverse in direction between middle-aged and old groups. +IZE increased; +IZM decreased; NC+IBQ-no change; ND+IBQ-not detected.

 
3.4. Effect of aging on cell numbers in vivo
Levels of Mac-3, a macrophage marker, were not different between the groups, suggesting that increased MMP levels were not due to increased macrophage numbers. Levels of DDR2, a fibroblast marker, decreased in the MA and old groups to 64+ALE-13% and 74+ALE-8% of young levels, respectively (p<0.05 for both).

3.5. Effect of aging on fibroblast function and MMP-9 expression
Fibroblasts are the principal cell regulators of ECM turnover. Given the changes in collagen, MMP, and TIMP levels, we characterized the age-related fibroblast phenotype. We successfully established primary cultures from 58 of the 63 LV samples used for fibroblast isolation. Of the five samples that did not establish, all were from young LV samples (four female and one male). The cell counts at first passage were 39+ALE-5+ANc-104 cells for young (n=14), 29+ALE-6+ANc-104 cells for MA (n=11), and 33+ALE-8+ANc-104 cells for old mice (n=10; p=0.48), indicating the equal and adequate yields were obtained for all three age groups. For these mice, the time from isolation to first passage was 19+ALE-1 days for the young, 20+ALE-3 days for MA, and 21+ALE-1 days for the old mice (p=0.72), indicating that the rate of establishing culture was similar between the three groups. All fibroblasts were positive for +A7E smooth muscle actin, indicating that some differentiation into a myofibroblast phenotype occurred in culture.

Fibroblasts from old mice displayed lower proliferative capacity than fibroblasts from young and MA mice (Fig. 3). There was no correlation between proliferation and passage number(r=0.02; p=0.88), indicating that proliferation rates did not change with passaging. The migration index, normalized to the average of young values (n=13), was 61+ALE-6% for MA (n=12; p<0.001) and 81+ALE-10% for old myocardial fibroblasts (n=9; p=0.11). Adhesion to plastic (n=8+IBM-12), gelatin (n=13+IBM-19), fibronectin (n=13+IBM-19), laminin (n=8+IBM-14), collagen I (n=13+IBM-19), and collagen IV (n=13+IBM-19) were similar for all age groups (p=n.s.). Changes in adhesive characteristics therefore did not explain the decrease in migration in the MA group. Immunoblotting of cell pellets demonstrated equal levels of +A7E smooth muscle actin between the three groups (p=0.48 and 0.41 for young vs. MA and old, respectively). MMP-9 levels, normalized to +A7E smooth muscle actin levels, were lower in fibroblasts isolated from the MA and old groups (Figs. 3 and 4Go).


Figure 3
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  		Fig. 3 Fibroblasts isolated from old (n=13) mice displayed lower proliferative capacity compared to fibroblasts isolated from young (n=19) and middle-aged (n=14) mice (*p<0.05 for young vs. old and +ICAp<0.05 for middle-aged vs. old).

 

Figure 4
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  		Fig. 4 MMP-9 levels were decreased in fibroblasts isolated from the MA (n=11) and old (n=10) groups compared with young (n=14). (Top) Representative immunoblots for MMP-9 and +A7E smooth muscle actin. (Bottom) The ratio of MMP-9 to +A7E smooth muscle actin relative to young levels (*p<0.05 for young vs. middle-aged and old).

 
3.6. Regression analyses
To determine which parameters correlated with age, regression analyses were performed using age as the independent variable (Table 5). Insoluble MMP-3, insoluble MMP-14, and total collagen correlated positively with age. Soluble MMP-12, soluble MMP-14, insoluble collagen, and fibroblast proliferation correlated negatively with age.


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  		Table 5 Regression analyses using age as the independent variable

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1. Major findings
LV remodeling resulting in LV hypertrophy is a common occurrence in the aging myocardium [2]. The present study hypothesized that age-related LV hypertrophy was a result of changes in ECM remodeling. Accordingly, the goal of this study was to examine the effects of aging on LV structure, collagen, MMP, and TIMP levels, and myocardial fibroblast function in mice. The unique findings of this study were as follows. First, the age-related increase in LV mass was due to increases in both LV dimensions and wall thickness. Second, soluble protein levels increased and insoluble collagen levels decreased with age. Soluble MMPs decreased, insoluble MMPs increased, and TIMPs decreased in both fractions. Third, fibroblasts have decreased functional capacity and produce less MMP-9 with age.

This study provides mechanistic insight into ECM remodeling events that occur as a function of aging. The shift in MMP localization from soluble to insoluble fractions may indicate increased recruitment of the MMP to an insoluble substrate. Increased MMP activity correlated with decreased insoluble collagen levels, suggesting that ECM degradation was stimulated in aging mice. The primary difference between MA and old mice was a further shift in MMPs from soluble to insoluble fractions, suggesting that increased MMP degradative capacity serves an important function in regulating collagen levels in old mice. The continued increase in MMP levels in old mice suggests that increased MMP activity is required to maintain increased LV size. This is the first study to correlate age-dependent changes in LV structure with changes in specific MMPs and TIMPs.

4.2. Aging effects on LV size
Age-related LV hypertrophy occurred in CB6F1 mice. Body weight and LV mass increased significantly with aging, similar to previous studies in other mice strains, rats, and humans [23,33]. None of these parameters were different between the MA and old groups, indicating that growth occurred primarily during the transition from young to MA and was maintained thereafter (a type 2 change). Increases in both LV dimension and wall thickness contributed to the increase in LV mass.

4.3. Aging effects on collagen, MMP, and TIMP levels
With age, decreased soluble MMP and TIMP levels accompanied the increase in soluble protein, and increased insoluble MMP and decreased insoluble TIMP levels accompanied the decrease in insoluble collagen. Together, the results suggest two interpretations. First, the decrease in insoluble collagen levels is likely due to increased MMP and decreased TIMP levels in the insoluble fraction. Changes in collagen solubility reflect changes in the biochemical characteristics of the collagen. Coupled with an overall increase in total protein, collagen synthesis may be increased but matched with a greater increase in collagen degradation. Alternatively, the increased soluble to insoluble ratio may indicate a change in collagen type or degree of cross-linking [34]. Second, changes in the soluble fraction (decreased MMP-12 and MMP-14 and increased protein) correlated with the increase in wall thickness and provided a mechanism for increased LV size with aging.

A major finding of this study was that changes to specific MMPs occur during aging rather than a global change in all MMP levels. In the soluble fraction, the predominant types of changes were type 1 (progressive changes with aging) and type 4 changes (directional change in MA that reversed in the old group), indicating that changes in soluble MMP levels occurred throughout the aging process. In contrast, type 3 changes (change only in the old group) characterized the insoluble fraction. A primary difference between the MA and old groups, therefore, was the increased levels of MMPs and decreased levels of TIMPs in the insoluble fraction. The changes in MMP levels between MA and old groups indicate a change in myocardial ECM composition, while LV mass is still maintained. The use of three time points allowed nonlinear changes to be assessed and the rate of change to be determined [32]. Interestingly, changes in total MMP levels from the pooled fractions mirrored changes in the soluble but not insoluble fraction. The increases in total MMP-3, MMP-9, and MMP-14 suggest increased synthesis of these particular MMP types. The differential MMP-3 expression between soluble and insoluble fractions highlights the need to use fractionated samples to obtain information on age-related distribution patterns as shifts between fractions cannot be discerned by monitoring total myocardial levels. The decrease in MMP-3, MMP-9, MMP-12, and MMP-14 in the soluble fraction was accompanied by increases of the same MMPs in the insoluble fraction, suggesting that increased binding to insoluble substrates occurred with aging.

Proteins likely to be found in the soluble fraction include cytoplasmic and easily soluble ECM proteins. Proteins likely to be found in the insoluble fraction include membrane and insoluble ECM proteins. The fact that MMP-7 and TIMP-1, known soluble proteins, were only seen in the soluble fraction confirms this idea. MMP-14, a membrane-bound MMP type, is known to recycle intracellularly and may be found in both fractions [35]. The age-related change in MMP-14 localization suggests that degradation and signaling pathways through this MMP were altered.

MMP-3 is a well-established activator of other MMPs [36], and the increase of MMP-3 in the insoluble fraction supports the idea of increased collagen degradation with aging. The localization of MMP-12 around the vasculature supports a role for MMP-12 in regulating changes in arterial compliance and stiffening that occur with aging [37]. MMP-14 is a membrane-type MMP with putative roles in pericellular proteolysis and signaling [35,38]. The decreases in soluble MMP-12 and MMP-14 would support the accumulation of soluble ECM fragments. Matricryptins, biologically active enzymatic ECM degradation products, have been shown to stimulate signaling pathways and affect cell growth [39]. The attenuation of MMPs in the soluble fraction potentially promotes the accumulation of ECM fragments and affects downstream signaling cascades. The fact that fibroblast proliferation and migration were altered with aging suggests a role for these ECM fragments in regulating fibroblast function. The results from this study provide the foundation for future studies that evaluate the effects of ECM fragments on LV structure and ECM remodeling, particularly during aging.

4.4. Aging effects on fibroblast function
Myocardial fibroblasts exhibited decreased proliferation and migration in the MA group. Proliferation decreased further in the old age group. In the old mice, the decrease in fibroblast proliferation rates corresponded with the decrease in myocardial DDR2 levels, which together suggest a decreased number of fibroblasts in the aging myocardium. In the MA mice, the fact that DDR2 levels were decreased in vivo but fibroblast proliferation was unaltered in vitro suggests that apoptotic rates may be increased. Apoptosis was not evaluated in this study. Concordant with our results, migration and proliferation are both attenuated in dermal fibroblasts isolated from mice of increasing age [40]. The decrease in fibroblast-derived MMP-9 was consistent with the drop in myocardial MMP-9 levels in the MA group. The rebound in myocardial MMP-9 in the old group is likely due to other cell contributors. We did not observe a change in Mac-3 levels, suggesting that macrophage levels were unaltered with age. The amount of MMP-9 produced per macrophage could be higher, however. Alternatively, smooth muscle, endothelial, and mast cells also potentially contribute MMP-9 [15]. The decreased proliferative and migratory capacities in aging fibroblasts suggest that global fibroblast function may be impaired, which would provide an age-related cellular mechanism for altered ECM levels. Studies examining the response of aging myocardial fibroblasts to growth factor and cytokine stimulation would support this concept.

4.5. Clinical relevance
MMPs have demonstrated roles in several components of LV remodeling, including the inflammatory, angiogenic, and hypertrophic responses. MMP inhibition has been used to successfully attenuate LV remodeling following myocardial infarction [41,42] and during the progression to heart failure [43]. The data presented here establish age-related baseline differences in LV echocardiographic parameters, ECM and MMP levels, and fibroblast functions that may influence the effects of MMP inhibition during disease pathologies such as pressure overload and myocardial infarction. These results reveal unique targets for future interventions to modify age-dependent matrix remodeling.


   Acknowledgements
 
The authors acknowledge the following sources of support: HL-10337 (MLL), HL-75360 (MLL), HL-45024 (FGS), HL-97012 (FGS), P01-48788 (FGS), and a VA Career Development Award (FGS).


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Cheitlin M.D. Cardiovascular physiology-changes with aging. Am. J. Geriatr. Cardiol. (2003) 12:9–13.[Medline]
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