Articles in PresS. Am J Physiol Cell Physiol (September 7, 2005). doi:10.1152/ajpcell.00235.2005
1 β-adrenergic receptor-stimulated apoptosis in adult cardiac myocytes involves MMP-2mediated disruption of β1 integrin signaling and mitochondrial pathway
Bindu Menon1, Mahipal Singh1, Robert S. Ross2, Jennifer N. Johnson1, Krishna Singh1*
1
Department of Physiology, James H Quillen College of Medicine James H Quillen Veterans Affairs Medical Center East Tennessee State University, Johnson City, TN 37614 2
Department of Medicine, UCSD School of Medicine and
Veterans Administration San Diego Healthcare System, San Diego, CA 92161
Running Title: Role of MMP-2 in cardiac myocyte apoptosis
*Correspondence:
Krishna Singh, Ph.D. Dept of Physiology James H Quillen College of Medicine East Tennessee State University PO Box 70576, Johnson City, TN 37614 Ph: 423-439-2049 Fax: 423-439-2052 E-mail:
[email protected]
Copyright © 2005 by the American Physiological Society.
2 Abstract Stimulation of beta-adrenergic receptors (β-AR) induces apoptosis in adult rat ventricular myocytes (ARVMs) via the JNK-dependent activation of mitochondrial death pathway. Recently, we have shown that inhibition of MMP-2 inhibits β-AR-stimulated apoptosis and that the apoptotic effects of MMP-2 are possibly mediated via its interaction with β1 integrins. Here we tested the hypothesis that MMP-2 impairs β1 integrin-mediated survival signal/s such as activation of focal adhesion kinase (FAK), and activates JNK-dependent mitochondrial death pathway. Inhibition of MMP-2 using SB3CT, a selective gelatinase inhibitor, significantly increased FAK phosphorylation (Tyr-397 and Tyr-576). TIMP-2, tissue inhibitor of MMP-2, produced a similar increase in FAK phosphorylation, while treatment of ARVMs with purified active MMP-2 significantly inhibited FAK phosphorylation. Inhibition of MMP-2 using SB3CT inhibited β-AR-stimulated activation of JNK pathway and cytosolic cytochrome c release. Treatment of ARVMs with purified MMP-2 increased cytosolic cytochrome c release. Furthermore, inhibition of MMP-2 using SB3CT and TIMP-2 attenuated β-AR-stimulated decreases in mitochondrial membrane potential. Over-expression of β1 integrins using adenoviruses expressing the human beta (1A) integrin (Adβ1A) decreased β-AR-stimulated cytochrome c release and apoptosis. Over-expression of β1 integrins also inhibited apoptosis induced by purified active MMP-2. These data suggest that MMP-2 interferes with the β1 integrin survival signals and activates JNK-dependent mitochondrial death pathway leading to apoptosis.
Key Words: MMPs, FAK, JNK, Cytochrome c
3 Introduction A variety of pathological states of the heart are associated with myocyte apoptosis including heart failure, myocardial infarction, and myocarditis (2). Stimulation of β-adrenergic receptors (β-AR) increases cardiac myocyte apoptosis in vivo and in vitro (25). β-AR-stimulated apoptosis in adult rat ventricular myocytes (ARVMs) is demonstrated to occur via JNK-dependent mitochondrial death pathway (20). Matrix metalloproteinases (MMPs), a large family of endopeptidases, are capable of degrading a wide variety of the extracellular matrix proteins (26). MMPs are present in the heart, and changes in the levels of MMPs have been described in a variety of animal models of heart failure (26). Inhibition of activity of MMPs through pharmacological interventions is demonstrated to attenuate the process of ventricular remodeling after infarction in mouse and rat (21,30), and in the rapid cardiac pacing model of heart failure (27). Adult cardiac myocytes express MMP-2 and neurohormonal stimulation increases its expression and activity (6,16,18). Integrins play a crucial role in the organization of the extracellular matrix, which undergoes extensive reorganization during disease. Integrins serve as adhesion receptors for extracellular matrix proteins, transduce biochemical signals into the cell and regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis (4). ARVMs predominantly express β1 integrins (23). Upon integrin occupancy and clustering, focal adhesion kinase (FAK), an important mediator of β1 integrin signaling pathway, undergoes autophosphorylation and associates with other intracellular signaling molecules (1). Inhibition of FAK phosphorylation by adenovirally mediated over-expression of FRNK (FAK related nonkinase) has been shown to induce apoptosis in neonatal cardiac myocytes (11).
4 We have previously shown that stimulation of β1 integrins plays an anti-apoptotic role in βAR stimulated apoptosis of ARVMs (9). Recently, we have shown that MMP-2 plays a proapoptotic role in β-AR stimulated apoptosis, possibly via its interaction with β1 integrins (18). Here, we demonstrate that MMP-2 interferes with the β1 integrin-mediated survival signal/s involving FAK and activates JNK-dependent mitochondrial death pathway.
5 Materials and Methods Cell isolation and culture - Calcium-tolerant ARVMs were isolated from the hearts of adult male Sprague-Dawley rats (240-280 g) as described previously (7). Briefly, hearts were perfused retrogradely with Ca++ free Krebs-Henseleit bicarbonate (KHB) buffer for 5 min. Hearts were then perfused with KHB buffer containing 0.04% collagenase type II for 20 min. After removing atria and great vessels, the hearts were minced and dissociated in the same buffer containing trypsin (0.02 mg/ml) and deoxyribonuclease (0.02 mg/ml). The cell mixture was filtered and sedimented through a 6% bovine serum albumin cushion to remove non-myocyte cells. The cell pellet was resuspended in Dulbecco’s Modification of Eagle’s medium (DMEM; Mediatech) supplemented with HEPES (25 mM), albumin (0.2%), creatine (5 mM), L-carnitine (2 mM), Taurine (5 mM) and 0.1% penicillin-streptomycin. The ARVMs were then plated in the above media at a density of 30-50 cells/mm2 on 100-mm tissue culture dishes (Fisher Scientific) or coverslips precoated with laminin (1µg/cm2). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National institutes of Health (NIH Publication No.85-23, revised 1996).
Cell treatment - ARVMs, cultured for 24 h, were treated with isoproterenol (ISO; 10 µM, Sigma) in the presence of ascorbic acid (100 µM) for 15 min to study activation of FAK and JNKs, 6h to study cytosolic cytochrome c release or 24 h for TUNEL and JC-1 staining. To inhibit MMP-2, cells were pretreated with SB3CT (1 nM; Calbiochem) or TIMP-2 (50 ng/ml; Calbiochem) for 30 min before treatment with ISO. The inhibitors were maintained in the medium during the treatment period with ISO. Cells were also treated with activated MMP-2 (1 nM; Calbiochem) to study cytochrome c release, apoptosis and FAK phosphorylation.
6
Adenovirus Infection of ARVMs - Adenoviruses expressing the human beta (1A) integrin (Adβ1A) (24) were propagated using HEK-293 cells. The adenoviral titer was determined using the end-point dilution method. ARVMs plated on laminin-coated dishes were infected with the adenovirus, with a matched multiplicity of infection of 50-100/cell, in modified DMEM for 2448 h before treating with ISO or MMP-2. Cells infected with equal multiplicity of infection of green fluorescence protein (GFP) adenoviruses served as controls.
Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay TUNEL staining was performed on ARVMs plated on thermanox 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.
Preparation of cytosolic fraction for cytochrome c release - To prepare cytosolic fraction, cells were washed with PBS and scraped in the lysis buffer (250 mM Sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 17 µg/ml PMSF, 8 µg/ml aprotinin, 2 µg/ml leupeptin) and homogenized gently using a glass homogenizer. The cell suspension was centrifuged at 3,500 g for 5 min to pellet nuclei and other cell debris. The supernatant was centrifuged at 14,000 g for 10 min to pellet mitochondrial fraction. The collected supernatant was then centrifuged at 100,000 g for 30 min at 4oC. The supernatant (cytosolic fraction) was analyzed by western blot using anti-cytochrome c antibody (Santa Cruz).
7 Immune complex kinase assay - Activation of JNK was studied using SAPK/JNK assay kit (Cell Signaling Technology). Total cell lysates (250 µg) were incubated overnight with 2 µg of c-Jun fusion protein beads. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2), and then suspended in 50 µl of kinase buffer containing 100 µM ATP. After incubation for 30 min at 300C, the reaction was terminated using 2X SDS sample buffer. The immunoprecipitates were analyzed by western blot using anti-phospho-c-Jun (ser 63) antibody.
Western Blot analysis - To analyze FAK phosphorylation, ARVMs were lysed in cell lysis buffer (10 mM Tris-HCl, pH 7.6, containing 150 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.4 mM PMSF and 1% Triton X 100). The protein content in cell lysates was measured using Bradford assay (Bio-Rad). Equal amount of proteins (50-100 µg) were resolved by SDS-PAGE (BioRad). Proteins from the gel were electrophoretically transferred to a PVDF membrane (HybondP, Amersham BioSciences). The membranes were stained with Ponceau S to confirm equal loading of proteins in the samples. After destaining, the membranes were incubated overnight in the blocking buffer; TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk. The membranes were then incubated with primary [anti FAKpY397 and anti FAK-pY576 (Biosource)] antibodies, diluted as suggested by the vendor, in blocking buffer. Following washings with TBST, the membranes were incubated with a diluted peroxidase-conjugated secondary antibody. The immune complexes were detected using chemiluminescence reagents (Pierce Biotechnology). The membranes were stripped and probed with antibodies against actin or FAK to optimize the loading.
8 JC-1 staining - To study mitochondrial membrane potential, ARVMs were incubated with JC-1 dye (Cell Technology Inc.) for 15 min at 37oC in dark. The cells were then washed in PBS twice to remove excess dye and the images were immediately captured using a fluorescent microscope (Nikon). The fluorescent images were analyzed using Bioquant Image analysis software and the ratio of red (aggregate) to green (monomer) fluorescence [F (λ585)/F (λ510)] was calculated.
Statistical Analyses - All data are expressed as mean ± SE. Statistical analysis was performed using the student’s t test or a one-way analysis of variance (ANOVA) and a post hoc Tukey’s test. Probability (p) values of
