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
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ABSTRACT |
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integrins; poly-ADP-ribose-polymerase
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 -adrenergic receptor (
-AR) induces apoptosis in cardiac myocytes in vitro and in vivo (43).
-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
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 -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
1-integrins and measured poly-ADP-ribose-polymerase (PARP) cleavage.
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MATERIALS AND METHODS |
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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 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 -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 (50100 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 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-
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.
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RESULTS |
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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 -AR stimulation. In contrast to MMP-2 activity, MMP-9 activity remained unchanged after
-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).
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DISCUSSION |
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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 -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
-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 -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 -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
-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 -AR-stimulated myocyte hypertrophy in rats (31). Our results show that inhibition of MMPs, specifically MMP-2, plays a protective role in
-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
1-integrin signaling plays a protective role in
-AR-stimulated apoptosis in ARVMs (11). Here, we demonstrate physical association of MMP-2 with
1 integrins on the surface of ARVMs.
-AR stimulation significantly increased the level of interaction between MMP-2 and
1-integrins. The increased association of MMP-2 with
1-integrins was inhibited by activation of
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
1-integrins. Interestingly, activation of
1-integrin signaling pathway using laminin inhibited the interaction of MMP-2 with
1-integrins. Similar observations have been made in HUVECs (26), where recruitment and binding of MMP-2 with
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 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
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
-AR stimulation does not affect expression of
1-integrins (11), and analysis of total cell lysates (prepared using RIPA buffer) exhibited no change in the levels of intact
1-integrin or 55-kDa fragment after 24 h of
-AR stimulation (data not shown). Therefore, another possibility is that recruitment and interaction of MMP-2 with
1-integrins disrupt
1-integrin-mediated intracellular survival signal/s. In support of this possibility, we found that inhibition of MMP-2 using SB3CT inhibits
-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
-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 -AR stimulation increases MMP-2 expression and activity while inhibiting TIMP-2 expression. Inhibition of MMP-2 activity inhibits
-AR-stimulated apoptosis. The apoptotic effects of MMP-2 may be mediated via its interaction with
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
1-integrins affects
-AR-stimulated apoptosis in cardiac myocytes may have important implications for the regulation of myocyte survival.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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