Matrix metalloproteinases mediate {beta}-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Bindu Menon, Mahipal Singh, and Krishna Singh

Department of Physiology, James H. Quillen College of Medicine, James H. Quillen Veterans Affairs Medical Center, East Tennessee State University, Johnson City, Tennessee

Submitted 10 December 2004 ; accepted in final form 16 February 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in {beta}-adrenergic receptor ({beta}-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). {beta}-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed {beta}-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited {beta}-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with {beta}1-integrins after {beta}-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of {beta}1-integrin signaling using laminin inhibited the increased association of MMP-2 with {beta}1-integrins. {beta}-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) {beta}-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits {beta}-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with {beta}1 integrins and poly-ADP-ribose-polymerase cleavage.

integrins; poly-ADP-ribose-polymerase


MATRIX METALLOPROTEINASES (MMPs), a large family of endopeptidases, have the ability to degrade extracellular matrix (ECM) proteins, and therefore, play a fundamental role in tissue remodeling, including the heart (12, 36, 44). MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are increased in a variety of experimental heart failure models as well as in the failing human heart (5, 36, 44, 47). MMP-2 and -9 are considered to play a major role in ECM remodeling because of their ability to initiate and continue degradation of fibrillar collagen (12). Membrane type-MMPs (MT-MMPs), a family of membrane-associated MMPs, are implicated in pericellular proteolysis (21). In vitro studies demonstrate that MT1-MMP activates pro-MMP (23). Tissue inhibitors of MMPs (TIMPs) inhibit activated MMPs by binding to the active site (28). MT1-MMP and TIMPs are expressed in the heart (7, 27). TIMP-2 is suggested to have maximum affinity for MMP-2, whereas TIMP-1 forms a specific complex with pro-MMP-9 (15, 16, 35).

Apoptosis occurs in the myocardium of patients with end-stage heart failure and myocardial infarction, and in animal models of myocardial hypertrophy and failure (2, 41, 45). Stimulation of {beta}-adrenergic receptor ({beta}-AR) induces apoptosis in cardiac myocytes in vitro and in vivo (43). {beta}-AR-stimulated apoptosis in cardiac myocytes involves activation of JNKs-dependent mitochondrial death pathway and caspase (37). MMPs are implicated in the induction as well as inhibition of apoptosis in various cell types. Inhibition of MMPs enhanced apoptosis of cancer cells induced by ligands of the TNF receptor superfamily (34). Activated MMP-2 elicited survival signals in melanoma cells (1). In contrast, upregulation of MMP-2 is associated with increased apoptosis in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (26, 33). In HUVECs, interaction of MMP-2 with {beta}1-integrins is proposed to be a mechanism by which MMP-2 stimulated apoptosis (26).

Within the heart, cardiac fibroblasts are considered to be the main source of synthesis and secretion of MMPs (29). However, other heart cell types, including cardiac myocytes, are also suggested to secrete a variety of MMPs (6, 19). Porcine cardiac myocytes are shown to synthesize and secrete MMP-2 in culture (6, 7). Here, we studied the expression of MMPs (MMP-2 and -9), MT1-MMP, and TIMPs (TIMP-1, -2, and -4), measured activity of MMPs (MMP-2 and -9), and tested the hypothesis that increased MMP activity plays a proapoptotic role in {beta}-AR-stimulated apoptosis of ARVMs. To gain an insight into the mechanism by which MMP-2 may play an apoptotic role, we examined the physical association of MMP-2 with {beta}1-integrins and measured poly-ADP-ribose-polymerase (PARP) cleavage.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell isolation and culture. Calcium-tolerant ARVMs were isolated from the hearts of adult male Sprague-Dawley rats (240–280 g) as described in a study by Communal et al. (9). Briefly, the hearts were perfused retrogradely with Ca2+-free Krebs-Henseleit bicarbonate buffer for 5 min. The hearts were then perfused with Krebs-Henseleit bicarbonate buffer containing 0.04% collagenase type II for 20 min. After the atria and great vessels were removed, the hearts were minced and dissociated in the same buffer containing trypsin (0.02 mg/ml) and DNase (0.02 mg/ml). The cell mixture was filtered and sedimented through a 6% bovine serum albumin cushion to remove nonmyocyte cells. The cell pellet was resuspended in DMEM supplemented with creatine (5 mM), L-carnitine (2 mM), taurine (5 mM), and 0.1% penicillin-streptomycin, and plated at a density of 30–50 cells/mm2 on 100-mm tissue culture dishes (Fisher Scientific) or coverslips precoated with laminin (1 µg/cm2). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). The animal protocol was approved by the University Committee on Animal Care.

Cell treatment. ARVMs, cultured for 24 h, were treated with isoproterenol (Iso; 10 µM, Sigma) in the presence of ascorbic acid (100 µM) for 24 h to study expression of MMP-2, MMP-9, TIMPs, MT1-MMP, activity of MMPs and TIMP-2. To measure PARP cleavage, ARVMs were treated for 6 h. To study apoptosis, cells were pretreated with GM-6001 (2 µM) or its negative control for 60 min, SB3CT (1 nM, Calbiochem) or TIMP-2 (50 ng/ml, Calbiochem) for 30 min, followed by treatment with Iso (10 µM) or active MMP-2 (1 nM; Calbiochem) for 24 h. To stimulate {beta}1-integrin signaling, cells were pretreated with laminin (10 µg/ml) for 30 min.

Real-time PCR. Total RNA was isolated as described previously by Singh et al. (42). The RNA was reverse transcribed using superscript reverse transcriptase kit (Invitrogen). The mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, and TIMP-4 were quantified using real-time PCR (icycler, BioRad). The forward and reverse primers used were 5'-CTGATAACCTGGATGCAGTCGT-3' and 5'-CCAGCCAGTCCGATTTGA-3' (MMP-2); 5'-TTCAAGGACGGTCGGTATT-3' and 5'-CTCTGAGCCTAGACCCAACTTA-3' (MMP-9); 5'-GCAGTGGACAGCGAATA-3' and 5'-TTCCCTTTGTAGAAGTATGTGA-3' (MT1-MMP); 5'-TCTGGCATCCTCTTGTTGCTAT-3' and 5'-CCACAGCGTCGAATCCTT-3' (TIMP-1); 5'-GGATTCCGGGAATGACATCTAT-3' and 5'-CGCCTTCCCTGCAATTAGATA-3' (TIMP-2); 5'-GTCTACACGCCATTTGACTCTT-3' and 5'-GTACACGGCACTGCATAGC-3' (TIMP-4) and 5'-TGCACCACCAACTGCTTA-3' and 5'-GGATGCAGGGATGATGTTC-3' (GAPDH). The PCR conditions for MMP-2, MMP-9, TIMP-2, and TIMP-4 were 50 cycles of denaturation (94°C, 18 s), annealing and elongation (68°C for 45 s), and for MT1-MMP and GAPDH were 50 cycles of denaturation (94°C for 18 s), annealing (65°C for 20 s) and elongation (72°C for 18 s). Reactions are characterized by comparing threshold cycle (CT) values. Samples with a high starting copy number show an increase in the fluorescence early in the PCR process resulting in a low CT number, whereas a lower starting copy number results in higher CT numbers. Initial characterization of GAPDH expression using RT-PCR followed by agarose gel electrophoresis and real-time PCR indicated no significant change in the intensity of the GAPDH signal in ARVMs after {beta}-AR stimulation. Therefore, mRNA levels were normalized relative to GAPDH values.

In-gel zymography. The conditioned media were lyophilized to dryness and the pellet was resuspended in water (referred as concentrated conditioned media), and protein content was measured with the use of Bradford assay (Bio-Rad). MMP activity in the concentrated conditioned media containing 10 µg of protein was measured using gelatin in-gel zymography (51). Clear and digested regions representing MMPs activity were quantified using a Kodak documentation system, and molecular weights were estimated using prestained molecular weight markers.

MMP-2 activity assay. The levels of active MMP-2 in the concentrated conditioned media containing 20 µg of protein were measured using MMP-2 activity assay kit according to the manufacturer's instructions (Amersham Biosciences).

Immunofluorescent labeling. ARVMs were fixed in 3.7% formaldehyde and permeabilized using 1% Triton X-100. The cells were then incubated with 10% goat serum for 1 h. After being washed with phosphate-buffered saline, the cells were incubated overnight with monoclonal anti-MMP-2 antibodies (1:100, Chemicon, Temecula, CA). After incubation with FITC-conjugated secondary antibody, the coverslips were mounted, visualized with the use of a fluorescent microscope, and photographed.

Western blot analysis. To study PARP cleavage, cells were lysed in 150 µl of extraction buffer (100 µl of 25 mM Tris·HCl, pH 8, containing 50 mM glucose, 10 mM EDTA, 1 mM PMSF and 50 µl of 50 mM Tris·HCl, pH 6.8, containing 6 M urea, 6% 2-mercaptoethanol, 3% SDS, and 0.003% bromophenol blue). The lysates were then sonicated for 60 s at 180 V and incubated at 65°C for 15 min before loading on a 7.5% gel. For MMP-2 protein, concentrated conditioned media (50–100 µg) were resolved by 10% SDS-PAGE (Bio-Rad). Proteins from the gels were electrophoretically transferred to a PVDF membrane (Hybond-P, Amersham Biosciences). The membranes were stained with Ponceau S to confirm equal loading of proteins in the samples. After being destained, the membranes were incubated overnight in the TBST blocking buffer composed of 50 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20, containing 5% nonfat dry milk. The membranes were then incubated with primary antibodies diluted in blocking buffer. After being washed with TBST, the membranes were incubated with a peroxidase-conjugated secondary antibody. The immune complexes were detected using chemiluminescence reagents (Pierce Biotechnology).

TdT-mediated dUTP nick end labeling assay. TdT-mediated dUTP nick end labeling (TUNEL) staining was performed on ARVMs plated on glass coverslips using in situ death detection kit according to the manufacturer's instructions (Roche Molecular Biochemicals). The percentage of TUNEL-positive cells (relative to total ARVMs) was determined by counting ~200 cells in 10 randomly chosen fields per coverslip for each experiment.

Coimmunoprecipitation of MMP-2 and {beta}1-integrins. Cells were lysed in Tris buffer (20 mM Tris·HCl, pH 7.5, 137 mM NaCl, 20 mM NaF, 5 mM EDTA, 1 mM PMSF, 10 mM sodium pyrophosphate, 0.2 M sodium orthovanadate, and 2 µg/ ml leupeptin) containing digitonin (0.05%), and centrifuged for 15 min at 13,000 g. The pellet (membrane fraction) was extracted in above buffer containing 1% Triton X-100. Proteins (400 µg) from the membrane fraction were incubated overnight with polyclonal anti-{beta}1-integrin (Santa Cruz, CA) antibodies. The immunoprecipitates were collected using 10 µg of protein A-agarose beads and analyzed with Western blots using monoclonal anti-MMP-2 antibodies.

Statistical analyses. All data are expressed as means ± SE. Statistical analysis was performed using Student's t-test or one-way ANOVA and a post hoc Tukey's test. P < 0.05 values were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{beta}-AR stimulation increases MMP-2 expression and activity. To study whether {beta}-AR stimulation increases mRNA levels for MMPs (MMP-2 and -9), ARVMs were treated with Iso for 24 h. Real-time PCR analysis of total RNA demonstrated that {beta}-AR stimulation (Iso; 10 µM; 24 h) increases mRNA levels of MMP-2 by 1.6 ± 0.1 fold (P < 0.05; n = 3; Fig. 1A) compared with control. In contrast to MMP-2 mRNA, MMP-9 mRNA levels remained unchanged after {beta}-AR stimulation [1.3 ± 0.4-fold vs. control; P = not significant (NS); n = 3].



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1. {beta}-Adrenergic receptor ({beta}-AR) stimulation increases matrix metalloproteinase-2 (MMP-2) mRNA and protein expression. A: adult rat ventricular myocytes (ARVMs) were treated with isoproterenol (Iso; 10 µM) for 24 h. Total RNA was analyzed by real time PCR using primers specific for MMP-2 and -9. mRNA levels were normalized to GAPDH and are expressed as fold increase vs. control. *P < 0.05; n = 3. B: ARVMs were treated with 10 µM Iso for 24 h. Concentrated conditioned media were analyzed by Western blot analysis using monoclonal anti-MMP-2 antibodies. Bottom, intensity of MMP-2 as fold increase vs. control (CTL). *P < 0.05 vs. CTL; n = 8. C: ARVMs treated with 10 µM Iso for 24 h were immunostained using anti-MMP-2 antibodies. Negative control performed with the omission of primary antibodies exhibited no staining (data not shown). The experiments were repeated three times with similar results.

 
Western blot analysis of conditioned media using anti-MMP-2 antibodies demonstrated increased MMP-2 protein levels (1.9 ± 0.1-fold vs. control; P < 0.05; n = 8; Fig. 1B) after 24 h of {beta}-AR stimulation. To confirm that ARVMs are indeed the source of MMP-2 protein, immunocytochemical analysis was performed. This analysis showed positive immunoreactivity for MMP-2 in untreated rod-shaped ARVMs (Fig. 1C). {beta}-AR stimulation induced morphological changes from rod-shaped to rounded, and the cells exhibited increased immunostaining for MMP-2 protein (Fig. 1C). Negative control in the absence of primary antibodies showed no fluorescence (data not shown).

Gelatin in-gel zymographic analysis of conditioned media demonstrated increased MMP-2 activity (1.7 ± 0.1-fold vs. control; P < 0.05; n = 12; Fig. 2A) after 24 h of {beta}-AR stimulation. In contrast to MMP-2 activity, MMP-9 activity remained unchanged after {beta}-AR stimulation. Analysis of levels of active MMP-2 in the conditioned media using MMP-2 activity assay indicated increased MMP-2 activity (levels of active MMP-2, ng/20 µg of total protein; CTL, 1.6 ± 0.2; Iso, 2.5 ± 0.3; n = 3; P < 0.05; Fig. 2B).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. {beta}-AR stimulation increases MMP-2 activity. A: ARVMs were treated with 10 µM Iso for 24 h. Concentrated conditioned media were analyzed by gelatin in-gel zymography. Bottom, the intensity of MMP-2 as fold increase vs. CTL. *P < 0.05 vs. CTL; n = 12. B: levels of active MMP-2 in the concentrated conditioned media were measured using MMP-2 activity assay. *P < 0.05 vs. CTL; n = 3.

 
{beta}-AR stimulation reduces TIMP-2 mRNA levels and protein expression. MT1-MMP activates MMP-2 (23), whereas TIMP-2 is suggested to inhibit MMP-2 activity (28). TIMP-4 is predominantly expressed in the heart (18), whereas TIMP-1 forms a specific complex with MMP-9 (16). Therefore, we next studied the expression of MT1-MMP and TIMPs (TIMP-1, -2, and -4) at mRNA level using real-time PCR. MT1-MMP mRNA levels remained unchanged after 24 h of {beta}-AR stimulation (1.2 ± 0.3-fold vs. control, P = NS; n = 3; Fig. 3A). Interestingly, {beta}-AR stimulation significantly reduced TIMP-2 mRNA levels (0.4 ± 0.1-fold; P < 0.05; n = 3; Fig. 3B) compared with control. {beta}-AR stimulation increased TIMP-1 mRNA levels (2.5 ± 0.3-fold vs. control; P < 0.05; n = 3; Fig. 3B), whereas there was no significant change in TIMP-4 mRNA levels. Analysis of conditioned media using Western blot analysis demonstrated a significant decrease in TIMP-2 protein levels after 24 h of {beta}-AR stimulation (0.45 ± 0.05-fold vs. control, P < 0.05; n = 3; Fig. 3C).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3. Effect of Iso on expression of membrane type 1 (MT1)-MMP and tissue-inhibitor MMPs (TIMPs). A: ARVMs were treated with Iso (10 µM) for 24 h. Total RNA was analyzed by real-time PCR using primers specific for MT1-MMP (A) or TIMPs (B). B: mRNA levels were normalized to GAPDH and are expressed as fold increase vs. control. *P < 0.05; n = 3. C: ARVMs were treated with Iso (10 µM) for 24 h. Concentrated conditioned media underwent Western blot analysis using anti-TIMP-2 antibodies. Bottom, intensity of TIMP-2 as fold increase vs. CTL. *P < 0.05 vs. CTL; n = 3.

 
Inhibition of MMP-2 inhibits {beta}-AR-stimulated apoptosis. To study the role of MMPs in {beta}-AR-stimulated apoptosis, ARVMs were pretreated with GM-6001 (a broad spectrum inhibitor of MMPs) and its negative control for 60 min, followed by treatment with Iso for 24 h. As reported previously (10), Iso increased the percentage of TUNEL-positive myocytes (CTL, 5.03 ± 0.6%; Iso, 18.03 ± 0.9%; P < 0.05; n = 4). Pretreatment with GM-6001 (2 µM, 60 min) almost completely inhibited {beta}-AR-stimulated apoptosis (Iso+GM-6001, 7.5 ± 1.2%; P < 0.05 vs. Iso; n = 4; Fig. 4). GM-6001(–), a structurally similar compound, in which the metal binding site hydroxamic acid is replaced by butoxycarbonyl group used as a negative control, failed to inhibit {beta}-AR-stimulated apoptosis (Iso+GM-6001 negative control, 15.8 ± 0.9; P = NS vs. Iso; n = 4; Fig. 4).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. GM-6001, not its structurally similar negative control, inhibits {beta}-AR-stimulated increases in apoptosis. ARVMs were pretreated with GM-6001 [GM(+); 2 µM] or its negative control [GM(–); 2 µM] for 60 min, followed by treatment with Iso for 24 h. The number of apoptotic cells was measured with the use of TUNEL-staining assay. *P < 0.05 vs. CTL; #P < 0.05 vs. Iso; {dagger}P = not significant (NS) vs. Iso; @P < 0.05 vs. GM(+)+Iso; n = 4.

 
To study the role of MMP-2 in {beta}-AR-stimulated apoptosis, ARVMs were pretreated with SB3CT (1 nM). The Ki value of SB3CT for MMP-2 is 13 nM, whereas it is 600 nM for MMP-9 (3, 22). Measurement of levels of active MMP-2 demonstrated that SB3CT at 1 nM concentration completely inhibits {beta}-AR-stimulated increases in active MMP-2 (levels of active MMP-2, ng/20 µg of total protein; Iso, 2.5 ± 0.3; Iso+SB3CT, 0.98 ± 0.24; P < 0.05 vs. Iso; n = 3). Analysis of apoptosis using TUNEL-staining assay indicated that pretreatment with SB3CT almost completely inhibits {beta}-AR-stimulated apoptosis (Iso, 18.03 ± 0.9%; Iso+SB3CT, 6.85 ± 0.3%; P < 0.05 vs. Iso; n = 4; Fig. 5A). Pretreatment of ARVMs with purified TIMP-2 protein (50 ng/ml) also significantly inhibited {beta}-AR-stimulated apoptosis (Iso, 18.03 ± 0.9%; Iso+TIMP-2, 7.2 ± 1.8; P < 0.05 vs. Iso; n = 4; Fig. 5B). Furthermore, treatment of ARVMs with purified active MMP-2 significantly increased the number of apoptotic cells compared with control (CTL, 6.4 ± 1.0; MMP-2, 18.2 ± 1.3; P < 0.05 vs. CTL; n = 6; Fig. 5C). MMP's inhibitors, GM-6001 and SB3CT, inhibited MMP-2-mediated increases in apoptosis.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Inhibition of MMP-2 inhibits {beta}-AR-stimulated apoptosis. ARVMs were pretreated with SB3CT (1 nM; A) or TIMP-2 protein (50 ng/ml; B) for 30 min, followed by treatment with Iso for 24 h. C: ARVMs were pretreated with GM-6001 [GM(+); 2 µM; 60 min] or SB3CT (1 nM; 30 min), followed by treatment with active MMP-2 (1 nM) for 24 h. The number of apoptotic cells was measured using TdT-mediated dUTP nick end labeling (TUNEL)-staining assay. *P < 0.05 vs. CTL; #@P < 0.05 vs. Iso; n = 3–6.

 
{beta}-AR stimulation increases association of MMP-2 with {beta}1-integrins. Altered cellular localization of MMP-2 and its physical association with {beta}1-integrins is suggested to be a mechanism by which MMPs induce apoptosis in HUVECs (26). Because ARVMs predominantly express {beta}1-integrins (39), therefore, we next examined the physical association of MMP-2 with {beta}1-integrins using coimmunoprecipitation assay. This analysis demonstrated that MMP-2 interacts with {beta}1-integrins at basal levels (Fig. 6). {beta}-AR stimulation increased the level of interaction between {beta}1-integrins and MMP-2 protein by 1.7 ± 0.1 fold (P < 0.05, n = 6; Fig. 6). Pretreatment with SB3CT significantly inhibited {beta}-AR-stimulated interaction of MMP-2 with {beta}1-integrins.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6. {beta}-AR stimulation increased physical association of MMP-2 with {beta}1-integrins. ARVMs were pretreated with SB3CT (1 nM) for 30 min, followed by treatment with 10 µM Iso for 24 h. The proteins from the membrane fractions were immunoprecipitated using polyclonal anti-{beta}1-integrin antibodies. The immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot using monoclonal anti-MMP-2 antibodies. Immunoprecipitation of {beta}1-integrins was confirmed by probing the membrane with anti-{beta}1-integrin antibodies. Bottom, intensity of MMP-2 as fold increase vs. CTL. *P < 0.05 vs. CTL; n = 6, #P < 0.05 vs. Iso; n = 3.

 
Activation of {beta}1-integrin signaling inhibits association of MMP-2 and {beta}1-integrins. Previously, we have shown that stimulation of {beta}1-integrin signaling using laminin pretreatment protects ARVMs against {beta}-AR-stimulated apoptosis (11). To determine whether association of MMP-2 with {beta}1-integrins is regulated by the activation state of the {beta}1-integrins, we pretreated ARVMs with laminin (10 µg/ml, 30 min). Coimmunoprecipitation analysis of proteins from the membrane fractions demonstrated that stimulation of {beta}1-integrin signaling decreases the amount of MMP-2 protein coimmunoprecipitated with {beta}1-integrins (fold increase vs. control, Iso, 1.7 ± 0.1; laminin +Iso, 1.1 ± 0.02; P < 0.05 vs. Iso; n = 3–6; Fig. 7).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7. Activation of {beta}1-integrin signaling inhibits interaction of MMP-2 with {beta}1-integrins. ARVMs were pretreated with laminin (LN; 10 µg/ml) for 30 min, followed by treatment with 10 µM Iso for 24 h. The proteins from the membrane fractions were immunoprecipitated using polyclonal anti-{beta}1-integrin antibodies. The immunoprecipitates were resolved by SDS-PAGE and underwent Western blot analysis using monoclonal anti-MMP-2 antibodies. Immunoprecipitation of {beta}1-integrins was confirmed by probing the membrane with anti-{beta}1-integrin antibodies. Bottom, intensity of MMP-2 as fold increase vs. CTL. *P < 0.05 vs. CTL; n = 6, #P < 0.05 vs. Iso; n = 3.

 
SB3CT inhibits {beta}-AR-stimulated increases in PARP cleavage. Activation of caspase-3 has been suggested to cause proteolytic cleavage of PARP (13). {beta}-AR stimulated apoptosis in cardiac myocytes is mediated via JNK-dependent mitochondrial death pathway and activation of caspase. Inhibition of caspase using z-Val-Ala-Asp(OME)-CH2F inhibits {beta}-AR-stimulated apoptosis (37). To determine whether MMP-2 is involved in PARP cleavage, we analyzed total cell lysates for the presence of 89-kDa PARP fragment by Western blot analysis. This analysis indicated that {beta}-AR stimulation increases the amount of 89-kDa PARP fragment (Fig. 8). Pretreatment with SB3CT inhibited {beta}-AR-stimulated increases in PARP cleavage (fold increase vs. control, Iso, 1.9 ± 0.2; Iso+SB3CT, 0.7 ± 0.04; P < 0.05, n = 3).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8. SB3CT inhibits {beta}-AR-stimulated increases in poly-ADP-ribose polymerase (PARP) cleavage. ARVMs were pretreated with SB3CT (1 nM) for 30 min, followed by treatment with 10 µM Iso for 6 h. Total cell lysates were loaded on a 7.5% gel and analyzed by Western blot analysis using anti-PARP antibodies. Equal loading of proteins in each lane was normalized with the use of actin immunostaining. *P < 0.05 vs. CTL, n = 3; #P < 0.05 vs. Iso, n = 3.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Stimulation of {beta}-AR induces apoptosis in cardiac myocytes in vitro and in vivo (43). The present study demonstrates that {beta}-AR stimulation increases the expression and activity of MMP-2, and inhibition of MMP-2 inhibits {beta}-AR-stimulated apoptosis in ARVMs. Increased recruitment and association of MMP-2 with {beta}1-integrins may be a mechanism by which MMP-2 plays a pro-apoptotic role in {beta}-AR-stimulated apoptosis.

Coker et al. (6) were the first to demonstrate that porcine cardiac myocytes express and secrete MMPs, specifically MMP-2, in vitro. Intracellular staining for MMP-2 in cardiac myocytes in conjunction with sarcomere is also observed in human heart during dilated cardiomyopathy (40). Recently, Kwan et al. (24) demonstrated presence of MMP-2 within the nucleus of rat cardiac myocytes. Endothelin-1 and angiotensin II increased MMP-2 activity in porcine cardiac myocytes, while Iso at 10 nM concentration had no effect with the use of a gelatin in-gel zymography assay (7). Using several different techniques, we provide evidence that Iso at 10 µM concentration increases expression and activity of MMP-2 in ARVMs. Lower concentrations of Iso (1 µM or less) showed no significant change in MMP-2 activity (data not shown). Lower concentrations of Iso only slightly increase the extent of apoptosis in ARVMs (52). In fact, Iso at 1 µM concentration is shown to protect ARVMs against apoptosis (20). We and others have shown that 10 µM concentration of Iso significantly increases apoptosis in ARVMs (10, 52). Collectively, these data suggest that the apoptotic concentration of Iso (10 µM) increases MMP-2 expression and activity in cardiac myocytes. The specific increase in MMP-2, not MMP-9, expression and activity suggests that MMP-2 may be an important factor in determining the interaction of ARVMs with ECM components and their survival. The signaling mechanisms by which {beta}-AR stimulation increases MMP-2 expression are not yet clear. At the level of transcription, activation of mitogen-activated protein kinase (MAPK) superfamily, which includes ERK1/2, JNKs, and p38 kinase, plays an important role in the regulation of MMP expression (25, 32, 49). Our preliminary data suggest involvement of JNKs in the regulation of MMP-2 because SP-600125, an inhibitor of JNK pathway, inhibits {beta}-AR-stimulated increases in MMP-2 protein levels and activity (30).

MMPs are synthesized and secreted as proenzymes. MT1-MMP activates MMP-2 on the cell surface with MT1-MMP-TIMP-2 complex serving as a receptor for pro-MMP-2 (23). In porcine cardiac myocytes, Iso (10 nM) increased MT1-MMP abundance within 4 h of treatment (7). In ARVMs, we found no change in MT1-MMP mRNA levels. This may be because MT1-MMP is required for the activation of MMP-2; therefore, an early increase in MT1-MMP may be crucial for the increased activation of MMP-2 observed after 24 h of {beta}-AR stimulation.

The concentration of TIMP-2 is suggested to determine the role of TIMP-2 in the activation of MMP-2. At low concentrations, TIMP-2 may serve as a receptor for pro-MMP-2, whereas at higher concentrations, TIMP-2 may neutralize MT1-MMP and prevent MMP-2 activation (28). Increased expression and activity of MMPs with decreased expression of TIMPs is observed in many pathological situations of the heart (38, 46). The data presented here demonstrate that {beta}-AR stimulation decreases expression of TIMP-2 in ARVMs. The decreased TIMP-2 expression with increased MMP-2 suggests an increase in the MMP-2/TIMP-2 ratio. It is interesting to note that there is no significant change in the expression and activity of MMP-9, whereas the expression of TIMP-1 is significantly increased after {beta}-AR stimulation. TIMP-1 forms a complex with MMP-9 (16).

The inhibition of MMPs attenuates left ventricular remodeling events associated with chronic volume overload and postmyocardial infarction (4, 48). Inhibition of MMPs is shown to regress {beta}-AR-stimulated myocyte hypertrophy in rats (31). Our results show that inhibition of MMPs, specifically MMP-2, plays a protective role in {beta}-AR-stimulated apoptosis, suggesting that increased MMP-2 expression and activity during heart failure may induce cardiac myocyte loss due to apoptosis. Previously, we have shown that stimulation of {beta}1-integrin signaling plays a protective role in {beta}-AR-stimulated apoptosis in ARVMs (11). Here, we demonstrate physical association of MMP-2 with {beta}1 integrins on the surface of ARVMs. {beta}-AR stimulation significantly increased the level of interaction between MMP-2 and {beta}1-integrins. The increased association of MMP-2 with {beta}1-integrins was inhibited by activation of {beta}1-integrin signaling pathway using laminin and inhibition of MMP-2 activity using SB3CT. SB3CT directly binds the catalytic zinc ion of MMP-2. The novel mode of binding of SB3CT to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme (22). Therefore, the data presented here suggest that the cell-secreted activated MMP-2, not the newly synthesized intracellular MMP-2, interacts with {beta}1-integrins. Interestingly, activation of {beta}1-integrin signaling pathway using laminin inhibited the interaction of MMP-2 with {beta}1-integrins. Similar observations have been made in HUVECs (26), where recruitment and binding of MMP-2 with {beta}1-integrins is proposed to be a mechanism by which activation of MMP-2 induces apoptosis.

The intracellular mechanism/s by which interaction of MMP-2 with {beta}1-integrins affects apoptosis in ARVMs is not yet clear. Disruption of normal myocyte anchorage to adjacent ECM and cells is proposed to be a mechanism of increased myocyte apoptosis during the transition from hypertrophy to early failure in mice (14). The presence of a 55-kDa extracellular domain fragment of {beta}1-integrins is observed in the ECM of rat heart after 1 mo of aortic stenosis, and in the conditioned media of neonatal cardiac myocytes and fibroblasts (17). Previously, we have shown that {beta}-AR stimulation does not affect expression of {beta}1-integrins (11), and analysis of total cell lysates (prepared using RIPA buffer) exhibited no change in the levels of intact {beta}1-integrin or 55-kDa fragment after 24 h of {beta}-AR stimulation (data not shown). Therefore, another possibility is that recruitment and interaction of MMP-2 with {beta}1-integrins disrupt {beta}1-integrin-mediated intracellular survival signal/s. In support of this possibility, we found that inhibition of MMP-2 using SB3CT inhibits {beta}-AR-stimulated increases in proteolytic cleavage of PARP. Of note, integrin engagement is suggested to control mitochondrial function in rabbit synovial fibroblasts via Rho GTPase-dependent mechanism (50).

ARVMs are isolated in a manner to eliminate nonmyocyte cell contamination. After collagenase and trypsin digestion, the cell mixture is filtered and sedimented through a 6% BSA cushion to remove nonmyocyte cells. Using propidium iodide staining and morphological examination, we found that the myocyte culture is ~97% pure. The observation that myocytes express MMP-2 and Iso increases MMP-2 expression in cardiac myocytes is supported by our data demonstrating positive immunoreactive staining for MMP-2 in rod-shaped cells (myocytes; Fig. 1C), and increased staining after 24 h of {beta}-AR stimulation. In addition, other studies have shown that cardiac myocytes express and regulate MMPs in vitro and in vivo (6, 7, 24, 40). However, the possibility that small numbers of nonmyocytes are also contributing to the synthesis of MMPs in culture cannot be completely ruled out.

In conclusion, the data presented here demonstrate that {beta}-AR stimulation increases MMP-2 expression and activity while inhibiting TIMP-2 expression. Inhibition of MMP-2 activity inhibits {beta}-AR-stimulated apoptosis. The apoptotic effects of MMP-2 may be mediated via its interaction with {beta}1-integrins. Continued loss of viable myocytes through apoptosis in failing human hearts is proposed to be a mechanism for progressive myocardial failure (8). The results presented here suggest that inhibition of MMP-2 may inhibit or reverse pathological remodeling. Further studies aimed at determining the molecular mechanism by which interaction of MMP-2 with {beta}1-integrins affects {beta}-AR-stimulated apoptosis in cardiac myocytes may have important implications for the regulation of myocyte survival.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work is supported in part by National Heart, Lung, and Blood Institute Grant HL-071519, a Merit Review grant from the Department of Veterans Affairs (both to K. Singh), and a postdoctoral fellowship from the American Heart Association, Southeast Affiliate (to B. Menon).


    ACKNOWLEDGMENTS
 
We thank Jennifer N. Johnson for cardiac myocyte isolation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Singh, Dept. of Physiology, James H. Quillen College of Medicine, East Tennessee State Univ., PO Box 70576, Johnson City, TN 37614 (e-mail: singhk{at}etsu.edu)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Ailenberg M and Silverman M. Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals. Biochim Biophys Acta 1593: 249–258, 2003.[CrossRef][ISI][Medline]

2. Anversa P, Kajstura J, and Olivetti G. Myocyte death in heart failure. Curr Opin Cardiol 11: 245–251, 1996.[ISI][Medline]

3. Brown S, Bernardo MM, Li ZH, Kotra LP, Tanaka Y, Fridman R, and Mobashery S. Potent and selective mechanism-based inhibition of gelatinases. J Am Chem Soc 122: 6799–6800, 2000.[CrossRef][ISI]

4. Chancey AL, Brower GL, Peterson JT, and Janicki JS. Effects of matrix metalloproteinase inhibition on ventricular remodeling due to volume overload. Circulation 105: 1983–1988, 2002.[Abstract/Free Full Text]

5. Cleutjens JP, Kandala JC, Guarda E, Guntaka RV, and Weber KT. Regulation of collagen degradation in the rat myocardium after infarction. J Mol Cell Cardiol 27: 1281–1292, 1995.[ISI][Medline]

6. Coker ML, Doscher MA, Thomas CV, Galis ZS, and Spinale FG. Matrix metalloproteinase synthesis and expression in isolated LV myocyte preparations. Am J Physiol Heart Circ Physiol 277: H777–H787, 1999.[Abstract/Free Full Text]

7. Coker ML, Jolly JR, Joffs C, Etoh T, Holder JR, Bond BR, and Spinale FG. Matrix metalloproteinase expression and activity in isolated myocytes after neurohormonal stimulation. Am J Physiol Heart Circ Physiol 281: H543–H551, 2001.[Abstract/Free Full Text]

8. Colucci WS, Sawyer DB, Singh K, and Communal C. Adrenergic overload and apoptosis in heart failure: implications for therapy. J Card Fail 6: 1–7, 2000.

9. Communal C, Colucci WS, and Singh K. p38 Mitogen-activated protein kinase pathway protects adult rat ventricular myocytes against {beta}-adrenergic receptor-stimulated apoptosis. Evidence for Gi-dependent activation. J Biol Chem 275: 19395–19400, 2000.[Abstract/Free Full Text]

10. Communal C, Singh K, Pimentel DR, and Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the {beta}-adrenergic pathway. Circulation 98: 1329–1334, 1998.[Abstract/Free Full Text]

11. Communal C, Singh M, Menon B, Xie Z, Colucci WS, and Singh K. beta1 integrins expression in adult rat ventricular myocytes and its role in the regulation of {beta}-adrenergic receptor-stimulated apoptosis. J Cell Biochem 89: 381–388, 2003.[CrossRef][ISI][Medline]

12. Creemers EE, Cleutjens JP, Smits JF, and Daemen MJ. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ Res 89: 201–210, 2001.[Abstract/Free Full Text]

13. Decker P and Muller S. Modulating poly (ADP-ribose) polymerase activity: potential for the prevention and therapy of pathogenic situations involving DNA damage and oxidative stress. Curr Pharm Biotechnol 3: 275–283, 2002.[CrossRef][Medline]

14. Ding B, Price RL, Goldsmith EC, Borg TK, Yan X, Douglas PS, Weinberg EO, Bartunek J, Thielen T, Didenko VV, and Lorell BH. Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure. Circulation 101: 2854–2862, 2000.[Abstract/Free Full Text]

15. Goldberg GI, Marmer BL, Grant GA, Eisen AZ, Wilhelm S, and He CS. Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteases designated TIMP-2. Proc Natl Acad Sci USA 86: 8207–8211, 1989.[Abstract/Free Full Text]

16. Goldberg GI, Strongin A, Collier IE, Genrich LT, and Marmer BL. Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J Biol Chem 267: 4583–4591, 1992.[Abstract/Free Full Text]

17. Goldsmith EC, Carver W, McFadden A, Goldsmith JG, Price RL, Sussman M, Lorell BH, Cooper G, and Borg TK. Integrin shedding as a mechanism of cellular adaptation during cardiac growth. Am J Physiol Heart Circ Physiol 284: H2227–H2234, 2003.[Abstract/Free Full Text]

18. Greene J, Wang M, Liu YE, Raymond LA, Rosen C, and Shi YE. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase. J Biol Chem 271: 30375–30380, 1996.[Abstract/Free Full Text]

19. Hanemaaijer R, Koolwijk P, le Clercq L, de Vree WJ, and van Hinsbergh VW. Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor alpha, interleukin 1 and phorbol ester. Biochem J 296: 803–809, 1993.[ISI][Medline]

20. Henaff M, Hatem SN, and Mercadier JJ. Low catecholamine concentrations protect adult rat ventricular myocytes against apoptosis through cAMP-dependent extracellular signal-regulated kinase activation. Mol Pharmacol 58: 1546–1553, 2000.[ISI][Medline]

21. Hernandez-Barrantes S, Bernardo M, Toth M, and Fridman R. Regulation of membrane type-matrix metalloproteinases. Semin Cancer Biol 12: 131–138, 2002.[CrossRef][ISI][Medline]

22. Kleifeld O, Kotra LP, Gervasi DC, Brown S, Bernardo MM, Fridman R, Mobashery S, and Sagi I. X-ray absorption studies of human matrix metalloproteinase-2 (MMP-2) bound to a highly selective mechanism-based inhibitor. Comparison with the latent and active forms of the enzyme. J Biol Chem 276: 17125–17131, 2001.[Abstract/Free Full Text]

23. Knauper V and Murphy G. Membrane-Type Matrix Metalloproteinases and Cell-Surface Associated Activation Cascades for Matrix Metalloproteinases, edited by Parks WC and Mecham RP. New York: Academic, 1998, p. 199–218.

24. Kwan JA, Schulze CJ, Wang W, Leon H, Sariahmetoglu M, Sung M, Sawicka J, Sims DE, Sawicki G, and Schulz R. Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro. FASEB J 18: 690–692, 2004.[Abstract/Free Full Text]

25. Lee KH, Hyun MS, and Kim JR. Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer: MEK/ERK and p38 MAP kinase interaction in uPA synthesis. Clin Exp Metastasis 20: 499–505, 2003.[CrossRef][ISI][Medline]

26. Levkau B, Kenagy RD, Karsan A, Weitkamp B, Clowes AW, Ross R, and Raines EW. Activation of metalloproteinases and their association with integrins: an auxiliary apoptotic pathway in human endothelial cells. Cell Death Differ 9: 1360–1367, 2002.[CrossRef][ISI][Medline]

27. Li YY, McTiernan CF, and Feldman AM. Proinflammatory cytokines regulate tissue inhibitors of metalloproteinases and disintegrin metalloproteinase in cardiac cells. Cardiovasc Res 42: 162–172, 1999.[CrossRef][ISI][Medline]

28. Li YY, McTiernan CF, and Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 46: 214–224, 2000.[CrossRef][ISI][Medline]

29. Manabe I, Shindo T, and Nagai R. Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy. Circ Res 91: 1103–1113, 2002.[Abstract/Free Full Text]

30. Menon B, Singh M, and Singh K. {beta}-Adrenergic receptor stimulation increases MMP-2 activity in adult rat cardiac myocytes: role in regulation of apoptosis. FASEB J 18: A238, 2004.[CrossRef]

31. Miura S, Ohno I, Suzuki J, Suzuki K, Okada S, Okuyama A, Nawata J, Ikeda J, and Shirato K. Inhibition of matrix metalloproteinases prevents cardiac hypertrophy induced by {beta}-adrenergic stimulation in rats. J Cardiovasc Pharmacol 42: 174–181, 2003.[CrossRef][ISI][Medline]

32. Moon SK, Cha BY, and Kim CH. ERK1/2 mediates TNF-{alpha}-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-{kappa}B and AP-1: involvement of the ras dependent pathway. J Cell Physiol 198: 417–427, 2004.[CrossRef][ISI][Medline]

33. Nataatmadja M, West M, West J, Summers K, Walker P, Nagata M, and Watanabe T. Abnormal extracellular matrix protein transport associated with increased apoptosis of vascular smooth muscle cells in Marfan syndrome and bicuspid aortic valve thoracic aortic aneurysm. Circulation 108, Suppl 1: II329–II334, 2003.[Medline]

34. Nyormoi O, Mills L, and Bar-Eli M. An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells. Cell Death Differ 10: 558–569, 2003.[CrossRef][ISI][Medline]

35. Olson MW, Gervasi DC, Mobashery S, and Fridman R. Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. J Biol Chem 272: 29975–29983, 1997.[Abstract/Free Full Text]

36. Peterson JT, Li H, Dillon L, and Bryant JW. Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat. Cardiovasc Res 46: 307–315, 2000.[CrossRef][ISI][Medline]

37. Remondino A, Kwon SH, Communal C, Pimentel DR, Sawyer DB, Singh K, and Colucci WS. Beta-adrenergic receptor-stimulated apoptosis in cardiac myocytes is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of the mitochondrial pathway. Circ Res 92: 136–138, 2003.[Abstract/Free Full Text]

38. Romanic AM, Burns-Kurtis CL, Gout B, Berrebi-Bertrand I, and Ohlstein EH. Matrix metalloproteinase expression in cardiac myocytes following myocardial infarction in the rabbit. Life Sci 68: 799–814, 2001.[CrossRef][ISI][Medline]

39. Ross RS and Borg TK. Integrins and the myocardium. Circ Res 88: 1112–1119, 2001.[Abstract/Free Full Text]

40. Rouet-Benzineb P, Buhler JM, Dreyfus P, Delcourt A, Dorent R, Perennec J, Crozatier B, Harf A, and Lafuma C. Altered balance between matrix gelatinases (MMP-2 and MMP-9) and their tissue inhibitors in human dilated cardiomyopathy: potential role of MMP-9 in myosin-heavy chain degradation. Eur J Heart Fail 1: 337–352, 1999.[CrossRef][ISI][Medline]

41. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, and Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation 95: 320–323, 1997.[Abstract/Free Full Text]

42. Singh K, Balligand JL, Fischer TA, Smith TW, and Kelly RA. Regulation of cytokine-inducible nitric oxide synthase in cardiac myocytes and microvascular endothelial cells. Role of extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and STAT1{alpha}. J Biol Chem 271: 1111–1117, 1996.[Abstract/Free Full Text]

43. Singh K, Xiao L, Remondino A, Sawyer DB, and Colucci WS. Adrenergic regulation of cardiac myocyte apoptosis. J Cell Physiol 189: 257–265, 2001.[CrossRef][ISI][Medline]

44. Spinale FG, Coker ML, Bond BR, and Zellner JL. Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target. Cardiovasc Res 46: 225–238, 2000.[CrossRef][ISI][Medline]

45. Teiger E, Than VD, Richard L, Wisnewsky C, Tea BS, Gaboury L, Tremblay J, Schwartz K, and Hamet P. Apoptosis in pressure overload-induced heart hypertrophy in the rat. J Clin Invest 97: 2891–2897, 1996.[Abstract/Free Full Text]

46. Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ III, and Spinale FG. Increased matrix metalloproteinase activity and selective upregulation in LV myocardium from patients with end-stage dilated cardiomyopathy. Circulation 97: 1708–1715, 1998.[Abstract/Free Full Text]

47. Tyagi SC, Campbell SE, Reddy HK, Tjahja E, and Voelker DJ. Matrix metalloproteinase activity expression in infarcted, noninfarcted and dilated cardiomyopathic human hearts. Mol Cell Biochem 155: 13–21, 1996.[CrossRef][ISI][Medline]

48. Villarreal FJ, Griffin M, Omens J, Dillmann W, Nguyen J, and Covell J. Early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling. Circulation 108: 1487–1492, 2003.[Abstract/Free Full Text]

49. Wang BW, Chang H, Lin S, Kuan P, and Shyu KG. Induction of matrix metalloproteinases-14 and -2 by cyclical mechanical stretch is mediated by tumor necrosis factor-{alpha} in cultured human umbilical vein endothelial cells. Cardiovasc Res 59: 460–469, 2003.[CrossRef][ISI][Medline]

50. Werner E and Werb Z. Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases. J Cell Biol 158: 357–368, 2002.[Abstract/Free Full Text]

51. Xie Z, Singh M, Siwik DA, Joyner WL, and Singh K. Osteopontin inhibits interleukin-1{beta}-stimulated increases in matrix metalloproteinase activity in adult rat cardiac fibroblasts: role of protein kinase C-zeta. J Biol Chem 278: 48546–48552, 2003.[Abstract/Free Full Text]

52. Zaugg M, Xu W, Lucchinetti E, Shafiq SA, Jamali NZ, and Siddiqui MA. Beta-adrenergic receptor subtypes differentially affect apoptosis in adult rat ventricular myocytes. Circulation 102: 344–350, 2000.[Abstract/Free Full Text]