INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IN THE NORMAL STATE, the left ventricular (LV) myocardial fibrillar collagen matrix serves several important functions, including the maintenance of LV shape and function. A delicate balance between matrix deposition and degradation must occur to preserve LV myocyte alignment, force transmission, and overall contraction and relaxation (4, 5, 18). In instances that lead to LV dysfunction such as myocardial infarction and the development of heart failure, extensive LV remodeling occurs. In these cardiac disease states, the composition of the myocardial collagen matrix may be compromised by a family of enzymes that preferentially degrade extracellular matrix components termed matrix metalloproteinases or MMPs. Past studies (12, 20, 22, 24) demonstrated the existence of several MMP species in the myocardium and showed that increased MMP activity was associated with LV remodeling. Furthermore, a recent in vitro study (7) demonstrated that LV myocytes release MMPs, which suggests that the LV myocyte itself may play a role in the LV remodeling process. However, specific extracellular stimuli that influence LV myocyte MMP synthesis and release remain to be defined.

Irrespective of etiology, a fundamental event that occurs during the progression of LV dysfunction is increased sympathetic stimulation (1). Specifically, the myocardial -adrenergic receptor system is activated during the progression of LV failure. As LV pump function deteriorates further, specific bioactive peptides such as angiotensin II and endothelin-1 are released (1). However, chronic myocardial exposure to these bioactive molecules and activation of myocardial receptors may contribute to the progression of LV failure and remodeling. Accordingly, the goal of the present study was to examine the potential effect of these neurohormonal receptor systems on LV myocyte MMP synthesis and release. Specifically, LV myocytes were incubated in vitro with a -receptor agonist, angiotesin II, or endothelin-1 and following these treatments, MMP expression and activity were measured.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Isolated porcine LV myocytes have been used in several past studies performed by this laboratory in which myocyte contractile function, protein synthesis, and ultrastructure were examined (7, 8, 17, 23, 25). In addition, previous experiments using this porcine LV myocyte preparation demonstrated that LV myocytes synthesize MMPs (7). Accordingly, similar LV myocyte preparations were used in the present study to determine whether external stimuli relevant to the progression of LV dysfunction influenced LV myocyte MMP synthesis and release.

LV myocyte isolation/stimulation. Twelve age- and weight-matched pigs (castrated male Yorkshire pigs, 5 mo old, 25 kg body wt) were the source of myocytes used in the present study. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, 1996). LV myocytes were isolated under sterile conditions as described previously (7, 8, 17, 23, 25). Briefly, the LV free wall encompassing the left circumflex coronary artery (5 × 5 cm) was dissected free and cannulated. The LV section was then perfused with a modified Krebs solution containing bacterial collagenase (0.5 mg/ml, type II; 273 U/mg; Worthington Biochemical; Freehold, NJ) for 20 min. After perfusion, the LV myocardial tissue was then minced and added to an oxygenated solution containing bovine serum albumin (2%, Sigma Chemical; St. Louis, MO), deoxyribonuclease (51 Kunitz U/ml, type IV, Sigma), 400 µM CaCl₂, and bacterial collagenase (0.5 mg/ml, Worthington). The LV myocardial tissue and myocyte-trituration solution were transferred to a centrifuge tube and gently agitated. After 15 min, the trituration solution was drawn off and the myocyte pellet was vigorously washed three times. First, the myocyte pellet was resuspended in Krebs solution and allowed to settle for 10 min. After the myocyte pellet had reformed, the Krebs solution was discarded, and the myocytes were resuspended in fresh serum-free culture medium (medium 199:F-12, GIBCO Life Technologies; Grand Island, NY) containing 2 mM Ca²⁺ (Sigma), 1% insulin-transferrin-selenium (ITS) medium (GIBCO), and 0.2% protease inhibitor cocktail (as prepared by manufacturer's instructions; Sigma). This washing step in fresh serum-free medium was then repeated twice. These vigorous wash and resuspension steps were performed to remove any residual bacterial collagenase. After the final wash step, the myocytes were resuspended in fresh serum-free medium at ~1 × 10⁵ cells/ml and incubated for 12 h (37°C, 95% O₂ and 5% CO₂), washed, and plated in fresh serum-free medium at 5 × 10⁴ cells/ml. After the 12-h incubation period, LV myocytes remained calcium tolerant and viable, exhibited a normal myofibrillar architecture upon -actinin immunostaining, and responded to electrical stimulation. The isolated LV myocyte preparations were then incubated for 6 h with isoproterenol (10 nM), endothelin-1 (50 pM), angiotensin II (1 µM), or phorbol 12-myristate 13-acetate (50 ng/ml). These concentrations were chosen based on the results of preliminary studies (Fig. 1) and past studies performed by this laboratory and others (10, 16, 17, 19-21, 23, 25). The LV myocyte-conditioned media and the LV myocytes were then collected and stored at 70°C until used in the following experiments.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Initial zymographic experiments were performed using conditioned media from untreated left ventricular (LV) myocyte preparations (circle) and those incubated with phorbol 12-myristate 13-acetate (PMA, square), endothelin-1 (ET-1, triangle), angiotensin II (ANG II, inverted triangle), or isoproterenol (Iso; diamond) to define appropriate experimental conditions (n = 3 experiments). Untreated LV myocyte preparations as well as those incubated with specific stimuli were incubated for up to 8 h to determine temporal mycardial matrix metalloproteins (MMP) zymographic profiles. After 2 and 4 h of incubation with PMA, a small increase in MMP gelatinase activity occurred (*P < 0.05 vs. time-matched untreated LV myocytes). After 6 and 8 h, a more robust increase in MMP gelatinase activity occurred in LV myocytes incubated with PMA, which suggests a time-dependent effect on proteolytic activity with PMA stimulation (*P < 0.05 vs. time-matched untreated LV myocytes, +P < 0.05 vs. 4 h PMA stimulated). Parallel sets of studies were performed with LV myocytes incubated with ET-1, ANG II, or Iso, which yielded similar results. Given the increased amount of MMP gelatinase activity observed in the conditioned media of LV myocytes incubated for 6 h with each stimulus, this time point was chosen for the experimental procedures executed in the present study. With respect to the concentrations chosen for PMA, ET-1, ANG II, and Iso, a separate set of initial zymography studies were performed using the conditioned media from LV myocyte preparations incubated with different concentrations of these stimuli.

MMP zymography. Before electrophoresis, the LV myocyte media were concentrated using ultrafiltration (Centricon, Amicon; Beverly, MA). Briefly, the media were centrifuged (3,000 rpm for 30 min), and the protein concentration of the LV myocyte media were then determined using a standardized colorimetric assay (Bio-Rad Protein Assay, Bio-Rad; Richmond, CA). The LV myocyte media (0.50 µg total protein) were then directly loaded onto electrophoretic gels (SDS-PAGE) containing 1 mg/ml of gelatin (Novex; San Diego, CA) under nonreducing conditions as described previously (7, 20, 22, 24). After SDS-PAGE, the gels were washed and incubated for 18 h in a substrate buffer (7, 20, 22, 24). After incubation, the gels were stained using 0.1% amido black, destained, and analyzed using densitometric methods (7, 20, 22, 24). MMP zymographic activity was then normalized with respect to the LV myocyte cell density in each preparation and then expressed as a percent change from values obtained for untreated LV myocyte preparations.

MMP-2 abundance by proteolytic antibody capture assay. To more carefully characterize the MMP activity observed in the LV myocyte-conditioned media, additional series of studies were performed in which specific MMP-2 content was measured by an antibody capture method (Amersham Pharmacia Biotech) as described previously (7). Briefly, the pro-MMP-2 in the LV myocyte-conditioned media (50 µg total protein) was allowed to bind to a monoclonal MMP-2 antibody (12 h; 4°C), which was immobilized on a 96-well microtiter plate. After the wells were washed vigorously, the captured pro-MMP-2 was activated with 1 mM p-aminophenylmercuric acetate, and an enzyme substrate solution and a chromogenic peptide substrate S-2444 were added. The reaction was allowed to proceed at 37°C for 3 h and the absorbance at 405 nm recorded. The absorbance from the cleaved chromogenic substrate was linear with increasing concentrations of the purified MMP-2 standards, y = 18x  6; r = 0.99, P < 0.01. LV myocyte MMP-2 content was computed based on the linearized results of the purified MMP-2 standards and normalized with respect to the original protein content and cell density of the LV myocyte preparations. MMP-2 content was then expressed as a percent change from values obtained for the untreated LV myocyte conditioned media.

MMP immunoblotting. Before immunoblotting, the LV myocyte media were concentrated as stated in the previous section. LV myocyte extracts were prepared by incubating LV myocytes with RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0; BD Pharmingen; Franklin Lakes, NJ). LV myocyte media or extracts (2.0 µg total protein) were loaded onto an 8% SDS-PAGE and subjected to electrophoretic separation (7, 20, 22, 24). The separated proteins were then transferred to a nitrocellulose membrane. Membranes were blocked, washed, and incubated overnight at 4°C in a polyclonal antibody corresponding to MMP-2 or MT1-MMP (1.0 µg/ml, Chemicon International; Temecula, CA). Membranes were then washed, incubated in horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000 dilution, Chemicon), washed again, and conjugated with an activated secondary antibody (ECL Western blotting reagents, Amersham Life Science; Arlington Heights, IL). The chemiluminescent signal was detected by exposure to X-ray film (Eastman Kodak; Rochester, NY) for exactly 15 min. Purified MMP-2 or MT1-MMP (Chemicon) was included in all immunoblots as a positive control, and prestained molecular weight markers (Bio-Rad) were used to assess molecular weight and to ensure adequate protein separation and transfer. The relative abundance of MT1-MMP in the LV myocyte preparations was analyzed using densitometric methods (7, 24), and values were expressed as a percentage of the MT1-MMP present in untreated LV myocyte sarcolemmal preparations.

MMP immunohistochemistry. The LV myocyte primary cultures were plated on microscope slides that were previously coated with 1.0 µg/ml poly-L-lysine (7). The myocytes were fixed using 3.7% formaldehyde for 10 min and stored in a buffer [65 mM piperazine-N,N'-bis(ethanesulfonic acid), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 10 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 3 mM MgCl₂] at 4°C. Immediately before being immunostained, the myocytes were permeabilized with 1% Triton-X-100 for 10 min at room temperature and then washed with phosphate-buffered saline. Myocyte preparations were incubated with 10% goat serum or mouse serum for 1.5 h at 4°C and washed with phosphate-buffered saline. The myocyte preparations were then flooded with primary antisera for MT1-MMP (1:300) and incubated overnight at 4°C. After the overnight incubation, myocytes were washed with phosphate-buffered saline and then incubated for 30 min at room temperature with a 1:60 dilution of goat anti-mouse fluorescein-labeled secondary antibody. In the LV myocytes, nuclei were identified by propidium iodide as described previously (8). The immunostained LV myocytes were then examined by confocal microscopy. The confocal microscope (MRC-1024, Bio-Rad) was equipped with dual lasers (krypton/argon) and excitation/emission wavelengths of 488/510 nm for FITC and 562/588 nm for the propidium iodide. Images were collected using ×60 magnification, and a running average of sequentially scanned images was performed using the Kalman process software, which was integrated into the confocal microscope. Negative controls for the immunostaining procedure included substitution of the primary antisera with nonimmune serum.

MMP data analysis. Densitometric analysis was performed to quantitate MMP zymographic activity and MT1-MMP abundance as described previously (7, 24). MMP zymographic activity, specific MMP-2 content, and MT1-MMP levels were compared using ANOVA using a two-way design. Specifically, external stimuli and time were considered independent treatment variables. If the ANOVAs performed on the MMP activity, content, and abundance data revealed significant differences, pairwise tests of individual group means were compared using Bonferroni probabilities. To improve data presentation, results were also reported as a percent change from values measured in untreated, time-matched LV myocyte preparations. All statistical procedures were performed using statistical software (BMDP Statistical Software). Results are presented as means ± SE. Values of P < 0.05 were considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

MMP zymography in LV myocyte preparations. Initial zymographic experiments were performed to define appropriate experimental conditions. Untreated LV myocyte preparations (n = 3) as well as those incubated with specific stimuli were incubated for up to 8 h to determine temporal MMP zymographic profiles (Fig. 1). Conditioned media from LV myocytes were sampled as early as 30 min of incubation, and at this time slight zymographic bands began to appear. The zymographic activity observed at 30 min of incubation was unchanged in media from untreated LV myocytes and those treated with each of the stimuli were used in the present study. After 2 and 4 h of incubation with phorbol 12-myristate 13-acetate, a small increase in MMP gelatinase activity occurred compared with that of the time-matched untreated LV myocytes. After 6 and 8 h, a more robust increase in MMP gelatinase activity occurred in LV myocytes incubated with phorbol 12-myristate 13-acetate, which suggests a time-dependent effect on proteolytic activity with phorbol 12-myristate 13-acetate stimulation. Parallel sets of studies were performed with LV myocytes incubated with endothelin-1, angiotensin II, or isoproterenol, which yielded similar results. Given the increased amount of MMP gelatinase activity observed in the conditioned media of LV myocytes incubated for 6 h with each stimulus, this time point was chosen for the experimental procedures executed in the present study. A slight change in zymographic activity was observed in unstimulated cells in relation to time as shown in Fig. 1. There were no significant differences in the zymographic activity observed at each time point for unstimulated LV myocytes, and this slight increase in intensity may be due to the autolytic activation of MMPs, the activation of one MMP species by another species, or a combination of both. With respect to the concentrations chosen for phorbol 12-myristate 13-acetate, endothelin-1, angiotensin II, and isoproterenol, a separate set of initial zymography studies was performed using the conditioned media from LV myocyte preparations incubated with different concentrations of these stimuli.

View larger version (73K): [in this window] [in a new window]   Fig. 2.   Representative gelatin zymograms of MMP activity in the conditioned media taken from untreated LV myocytes and those incubated with PMA, ET-1, ANG II, and Iso (105 total cells; n = 6). MMP gelatinolytic activity was observed between 100 and 50 kDa, which is consistent with several MMP species (14). LV myocyte MMP gelatinase activity appeared to be increased in the conditioned media taken from LV myocytes incubated with each treatment. MMP gelatinase activity for untreated LV myocytes and those incubated with PMA, ET-1, ANG II, and Iso is summarized in Fig. 3.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Top: total MMP gelatinase activity was increased in LV myocyte-conditioned media after treatment with PMA. In addition, total MMP gelatinase activity was also increased in LV myocyte-conditioned media after incubation with both ET-1 and ANG II. Whereas conditioned media from LV myocytes incubated with Iso exhibited higher MMP gelatinase activity compared with untreated LV myocytes, this did not reach statistical significance (P = 0.06). Middle: MMP gelatinase activity from the LV myocyte-conditioned media localized to ~90 kDa, which likely reflects the MMP-9 (14). MMP gelatinase activity at 90 kDa was increased after treatment with PMA, ET-1, and ANG II (*P < 0.05 vs. untreated LV myocytes). Bottom: MMP gelatinase activity was also localized to ~70 kDa, which may represent MMP-2 (17). MMP gelatinase activity at 70 kDa in the LV myocyte-conditioned media was increased by over 47% after treatment with PMA, ET-1, and ANG II (*P < 0.05 vs. untreated LV myocytes).

Specific MMP-2 content in LV myocyte preparations. From the robust MMP gelatinase activity observed at ~70 kDa in the conditioned LV myocyte media, a specific MMP-2 antibody capture assay was performed. MMP-2 content in the conditioned media taken from LV myocytes following incubation with phorbol 12-myristate 13-acetate, endothlin-1, angiotesin II, or isoproyerenol is summarized in Fig. 4. MMP-2 content was increased in the conditioned media of LV myocytes incubated with phorbol 12-myristate 13-acetate, endothelin-1, angiotensin II, or isoproterenol.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Relative MMP-2 content in the LV myocyte-conditioned media of untreated LV myocytes as well as those incubated with PMA, ET-1, ANG II, and Iso was examined using a specific MMP-2 antibody capture assay. Specific MMP-2 content based on the MMP-2 activity detected by the antibody capture assay was increased by over 40% after treatment with PMA, ET-1, ANG II, and Iso (*P < 0.05 vs. untreated LV myocytes).

View larger version (65K): [in this window] [in a new window]   Fig. 5.   To ensure that the MMP antibody capture assay was specific for MMP-2, immunoblotting for MMP-2 was performed using the conditioned media from untreated (U) LV myocytes and those incubated with PMA (P), ET-1 (E), ANG II (A), and Iso (I). LV myocyte sarcolemmal preparations made following each of the above treatments were also subjected to immunoblotting for MMP-2. In addition to the substitution of nonimmune antisera, which abolished all staining, a positive control for MMP-2 (+) obtained from a human epithelial cell line (AG771; Chemicon) was used. A positive immunoreactive signal was observed at ~70 and 60 kDa, which is consistent for this MMP species (14).

MT1-MMP abundance and localization in LV myocyte preparations. Past studies have demonstrated that a local activation mechanism exists for MMP-2, which is mediated by MT1-MMP (15). Accordingly, immunoblotting for MT1-MMP was performed and the results are summarized in Fig. 6. A positive immunoreactive signal for MT1-MMP was observed at ~60, 55, and 40 kDa, which is consistent with past reports concerning the proform, the active form, and a potential activation byproduct of this MMP species (15). Total MT1-MMP abundance was increased following exposure to phorbol 12-myristate 13-acetate, endothelin-1, angiotensin-II, or isoproterenol. MT1-MMP was localized by immunofluorescence to porcine LV myocytes by laser scanning confocal microscopy (Fig. 7). Localization of MT1-MMP to the LV myocyte sarcolemma was observed, and ~90% of the cardiocytes analyzed exhibited positive immunostaining for MT1-MMP, which provided further evidence for the existence of MT1-MMP in adult porcine LV myocytes.

View larger version (57K): [in this window] [in a new window]   Fig. 6.   Immunoblotting for a recently discovered MMP, membrane type-1 MMP (MT1-MMP), was performed using sarcolemmal preparations made from untreated LV myocytes and those incubated with PMA, ET-1, ANG II, and Iso. Purified recombinant MT1-MMP (CC1043; Chemicon) was also used as a positive control (+) for immunoblotting. A positive immunoreactive signal was observed at ~60, 50, and 40 kDa (arrows), which is consistent with past reports (15). Quantitation of LV myocyte MT1-MMP was performed using densitometric methods. Total LV myocyte MT1-MMP was increased after treatment with PMA, ET-1, and Iso (*P < 0.05 vs. untreated LV myocytes). In sarcolemmal preparations made from LV myocytes incubated with ANG II, an even greater increase in MT1-MMP levels approximating 60% was measured.

View larger version (64K): [in this window] [in a new window]   Fig. 7.   Isolated porcine LV myocytes were subjected to immunofluorescent staining for MT1-MMP as well as nuclear DNA. A: under routine phase-contrast microscopy, the isolated myocytes used for immunostaining demonstrated a normal rod-shaped morphology with regular sarcomeric registration. B: same myocyte was then examined under fluorescence and a strong signal was detected for MT1-MMP. C: laser scanning confocal microscopy revealed a uniform staining pattern with robust intensity apparent in a sarcomeric banding orientation. D: higher power magnification revealed a heavy distribution of MT1-MMP in a sarcomeric banding pattern in LV myocytes. E: substitution of nonimmune antisera abolished the specific fluorescent staining for MT1-MMP.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The MMPs are a family of enzymes that degrade a wide variety of extracellular matrix components (26). Different MMP species have been subdivided based on substrate specificity and amino acid sequence homology (15, 26). A subgroup of MMPs that preferentially degrade basement membrane components is the gelatinases, or MMP-9 and MMP-2 (26). A past in vitro study demonstrated that LV myocytes synthesize and release this subgroup of MMPs (7). Because the basement membrane forms the interface between the myocyte and the extracellular matrix, changes in the synthesis and release of MMP-2 and/or MMP-9 by the myocyte would probably affect myocyte shape and alignment in vivo (4, 5, 18). The first important finding of the present study was that specific extracellular stimuli influenced the synthesis and release of MMP-2 in isolated LV myocytes. MT1-MMP is a member of one of the more recently discovered subgroups of MMPs, termed membrane-type MMPs, that degrades extracellular matrix proteins as well as proteolytically converts pro-MMP-2 to the active form (15). A second unique finding of the present study was that LV myocytes express MT1-MMP and that the abundance of MT1-MMP was also influenced by specific extracellular factors. The results of the present study indicate that LV myocytes release MMP species that may be activated by MT1-MMP (15) and that this local LV myocyte proteolytic system can be influenced by neurohormonal stimuli relevant to LV dysfunction.

There are a number of neurohormonal systems that become activated with LV dysfunction (1, 2). During the progression of LV dysfunction, increased circulating levels of norepinephrine occur, which result in LV myocyte ₁-adrenergic receptor activation (1, 2). Furthermore, the increased production and release of the bioactive peptide angiotensin II has been implicated to promote LV remodeling in both clinical and experimental LV dysfunction (1, 21). In a clinical study, increased circulating plasma levels of endothelin-1 were associated with the severity of LV dysfunction (2). Endothelin-1 has been demonstrated to influence a number of biosynthetic processes within the myocardium (13, 16). Whereas these neurohormonal axes have been implicated in the development of LV dysfunction, the number of cellular and extracellular sequelae caused by chronic activation of these neurohormonal pathways with respect to LV remodeling and dysfunction are not fully understood. Recent studies (12, 20, 22, 24) that used MMP inhibitors demonstrated that increased MMP activation can contribute to the LV remodeling, which occurs with LV dysfunction. The results of the present study suggest that chronic activation of specific neurohormonal pathways such as the ₁-adrenergic, endothelin-1, and angiotensin II receptor systems, may result in increased myocardial MMP activational states. MT-MMPs are membrane-associated MMPs that have been implicated in pericellular matrix degradation (15). MT-MMPs are thought to provide a specific localized activation site for other MMP species at the surface of the cell membrane, and these enzymes have the capacity to directly degrade extracellular matrix components (15). Currently, four MT-MMPs have been discovered with the most characterized MT-MMP being MT1-MMP (15). Low levels of MT1-MMP have been detected in normal tissue (15), but in lung and breast carcinoma MT1-MMP expression is present in greater amounts (15), suggesting that this MMP species is important in tissue remodeling. Past in vitro studies (15) demonstrated that pro-MMP-2 is activated by MT1-MMP. Because previous studies performed by this laboratory have shown that LV myocytes release MMP-2 (7) and because the present study demonstrated increased MMP-2 activity in LV myocyte-conditioned media following specific neurohormonal stimulation, MT1-MMP expression was also examined in isolated LV myocyte preparations under similar conditions. MT1-MMP was observed at ~60 and 55 kDa, which is consistent with the reported pro- and active forms of this MMP species (15). A lower molecular mass band for MT1-MMP was also detected at ~40 kDa, which may represent an MT1-MMP fragment released following the activation of other MMP species (15). MT1-MMP was specifically localized to the sarcolemma of isolated porcine LV myocytes, and MT1-MMP abundance was increased in LV myocytes following exposure to a -receptor agonist, endothelin-1, and angiotensin II. Furthermore, these specific treatments caused an increase in 70-kDa zymographic activity in LV myocyte-conditioned media, suggesting a heightened activational state of MMP-2, which may have been due to the local activation of MT1-MMP. Therefore, MT1-MMP is expressed by LV myocytes and may constitute a local MMP activational system, which is influenced by specific neurohormonal stimuli.

The structure of the newly synthesized MMP contains a large propeptide domain that prevents autolytic intracellular activity (15, 26). Furthermore, the nascent MMP contains a signal peptide that facilitates intracellular trafficking and ultimately secretion into the extracellular space. MMPs such as MMP-2 and MMP-9 contain a large hemopexin-like domain that allows for binding to extracellular matrix proteins. After activation, MMP-2 and MMP-9 degrade many extracellular matrix components, particularly those that make up the basements membrane (26). In the present study, increased zymographic activity consistent with MMP-2 (26) and increased MMP-2 content were observed in LV myocyte preparations following incubation with endothelin-1 and angiotensin II. A fundamental event that has been reported to occur following endothelin-1 and angiotensin II receptor activation is the activation of protein kinase C (3, 17). One of the outcomes of the present study was that incubation of LV myocytes with a phorbol ester, which activates protein kinase C, resulted in similar changes in MMP-2 expression and activity as those observed following incubated with endothelin-1 and angiotensin II. These findings suggest that one potential intracellular mechanism by which endothelin-1 and angiotensin II receptor activation influences MMP activity and expression is through the activation of protein kinase C. A study performed by our laboratory (17) also demonstrated that phorbol 12-myristate 13-acetate treatment caused intracellular translocation of protein kinase C in isolated LV myocyte preparations. Intracellular activation of protein kinase C can induce protooncogenes, which in turn, interact with response elements in MMP promoter regions (26). However, the specific MMP response elements that are involved following receptor activation warrant further study. In the present study, neurohormonal receptor stimulation and protein kinase C activation may have resulted in increased MMP abundance in LV myocytes.

In addition to MMP-2 and MT1-MMP, several MMP species have been demonstrated to exist in the myocardium (20, 24) and with continuing research even more MMP species will undoubtably be identified as well. Those that have been identified thus far include MMP species that have the capacity to degrade most, if not all, extracellular matrix components. In conditions such as dilated cardiomyopathy or hypertrophy, in which the abundance and/or activational state of these MMP species becomes altered, extensive pathological LV remodeling can occur. With dilated cardiomyopathy, the changes in MMP abundance and activation identified thus far favor matrix degradation. With respect to hypertrophy, although the changes in MMP abundance and activation have not been as extensively characterized, given the alterations in LV geometry and shape that occur with this disease process, it is likely that altered MMP abundance and activation favor matrix deposition. Further investigation is warranted to identify which particular portfolio of MMP species may contribute to these disease states and how changes in the induction and activation of these MMP species influence LV remodeling.

The composition of the LV myocardial extracellular matrix is undoubtably influenced by each of the cell types of which it is composed, i.e., fibroblasts, endothelial cells, smooth muscle cells, and myocytes. Currently, the extent to which each of these cells contributes to LV remodeling in both normal and diseased states is unknown. Whereas the goal of the present study was to identify the potential role of the LV myocyte in the remodeling process with respect to MMPs, changes in the myocardial matrix are a likely result of other nonmyocyte cellular processes as well. Past studies (6, 11) demonstrated that exposure to angiotensin II and endothelin-1 resulted in increased collagen synthesis in cardiac fibroblasts. However, these studies were performed using different times and concentrations of these external stimuli compared with the present study. The results of the present study suggest that in LV myocytes, exposure to angiotensin II and endothelin-1 may result in increased myocardial matrix degradation. Thus in the myocardium increased local matrix deposition and degradation may occur simultaneously in different cell types in response to a given stimulus.

There have been many studies performed that have examined the biological responses of isolated LV myocytes following -adrenergic, endothelin-1, and angiotensin II receptor stimulation (9, 16, 19, 21, 23, 25). To our knowledge, this is the first study that has focused on the effects of the activation of these specific receptor systems on MMP expression. However, there are limitations to this approach that must be recognized. First, in the present study, isolated adult unattached LV myocytes were exposed to fixed concentrations of bioactive compounds. Thus results regarding MMP synthesis and release cannot be directly extrapolated to in vivo receptor activation and signaling. Second, it has been demonstrated previously that LV myocytes interact with the extracellular matrix through the integrins (5, 14), and the potential effects of extracellular matrix adhesion and integrin engagement have not been examined in the present study. Third, several MMP species have been identified in the myocardium in addition to those examined in the present study. Thus future studies examining the induction of other MMP species in LV myocytes in response to external stimuli would be warranted. Nevertheless, the results of the present study provide evidence to suggest that following chronic neurohormonal system activation, increased elaboration and release of MMPs into the local extracellular matrix of the LV myocyte occurs, which in turn could contribute to LV remodeling.