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Cardiovascular Research 2006 69(3):688-696; doi:10.1016/j.cardiores.2005.08.023
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Copyright © 2005, European Society of Cardiology

Cardiac transgenic matrix metalloproteinase-2 expression directly induces impaired contractility

Guan-Ying Wanga,d, Marina R. Bergmanb,d, Anita P. Nguyenb,d, Sally Turcatoa,d, Philip M. Swigartd, Manoj C. Rodrigoc,d, Paul C. Simpsonb,c,d, Joel S. Karlinerb,c, David H. Lovettb,d and Anthony J. Bakera,d,*

aDepartment of Radiology, University of California, San Francisco, United States
bDepartment of Medicine, University of California, San Francisco, United States
cCardiovascular Research Institute, University of California, San Francisco, United States
dVeterans Affairs Medical Center, San Francisco, United States

* Corresponding author. University of California, San Francisco, VA Medical Center, Cardiology Division (111C), 4150 Clement St, San Francisco, CA 94121, United States. Tel.: +1 415 221 4810x4790; fax: +1 415 750 6950. Email address: ajbaker{at}itsa.ucsf.edu

Received 4 March 2005; revised 2 August 2005; accepted 31 August 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Matrix metalloproteinase-2 (MMP-2) plays a major role in dysfunctional ventricular remodeling following myocardial injury induced by ischemia/reperfusion and heart failure. To directly assess the role of MMP-2 in the absence of superimposed injury, we generated cardiac-specific, constitutively active MMP-2 transgenic mice.

Methods: Morphologic and functional studies were carried out using both intact and demembranated (skinned) right ventricular trabeculae dissected from hearts of 8-month-old MMP-2 transgenic mice and wild-type controls (WT).

Results: Electron micrographs showed that compared to WT, MMP-2 myocardium had no gross, ultrastructural changes (no myocyte dropout or gross fibrosis). However, MMP-2 myocardium contained fibroblasts with abundant rough endoplasmic reticulum, consistent with an activated synthetic phenotype, suggesting extracellular matrix remodeling in MMP-2 trabeculae. Consistent with remodeling, mechanical studies found increased stiffness of intact unstimulated trabeculae (increasing sarcomere lengths from 2 to 2.3 µm caused a greater rise of passive muscle force for MMP-2 trabeculae versus WT). With electrical stimulation, MMP-2 trabeculae generated substantially less active force at all sarcomere lengths. Moreover, inotropic responses to increases of bath [Ca2+], pacing frequency, and isoproterenol were all significantly reduced versus WT trabeculae. Skinned fiber assessment of myofilament function revealed that maximum Ca2+-activated force of skinned MMP-2 trabeculae was reduced to {approx}50% of WT, suggesting a myofilament contraction defect.

Conclusion: Cardiac-specific, constitutively active MMP-2 expression leads to impaired contraction and diminished responses to inotropic stimulation. These findings indicate that MMP-2 can directly impair ventricular function in the absence of superimposed injury.

KEYWORDS Extracellular matrix; Transgenic mouse; Remodeling; Contractile function; Trabeculae


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Matrix metalloproteinases (MMPs) are an endogenous family of proteolytic enzymes that play an important role in extracellular matrix (ECM) turnover and cardiac remodeling [1–5]. The MMP family consists to date of more than 20 species including collagenases (such as MMP-1 and MMP-13), gelatinases (such as MMP-2 and MMP-9), stromelysin (MMP-3) and membranous type MMP (such as MT1-MMP) [6].

MMP-2 is produced by cardiomyocytes, cardiac fibroblasts and endocardial cells [7–10]. MMP-2 degrades a remarkably large number of substrates including Type IV collagen, laminin, elastin, and interstitial fibrillar collagen [11–13]. MMP-2 also degrades a number of substrates not associated with the ECM, including endothelin [14], calcitonin gene-related peptide [15] and adrenomedullin [16].

Alterations in the expression and activity of MMP-2 have been demonstrated in a number of pathophysiological conditions. In the setting of myocardial ischemia/reperfusion, acutely elevated MMP-2 activity contributes to myocardial stunning in human- and rat hearts, independent of an effect on the ECM [17,18]. During post-ischemic reperfusion, activation of intracellular MMP-2 leads to cleavage of the contractile regulatory protein troponin I [19]. Conversely, myocardial stunning was attenuated by inhibition of MMP-2 activity [17,18].

In the setting of heart failure, MMP-2 has been implicated in myocardial fibrosis, increased myocardial stiffness and impaired contractility of hearts from spontaneously hypertensive rats [20,21]. In human heart failure, increased levels of MMP-2 were suggested to be responsible for long-term cardiac remodeling processes and cardiac dysfunction [22,23]. Together these studies suggest a role for MMP-2 activation in myocardial dysfunction and cardiac remodeling in the setting of disease.

Notwithstanding reports detailing the activities of MMP-2 in cardiac dysfunction under pathophysiological conditions, there is little direct evidence demonstrating that in the absence of superimposed injury, MMP-2 can directly lead to the development of cardiac dysfunction. To test this, we used transgenic mice that expressed constitutively active MMP-2 targeted to the heart. The present study provides the first demonstration that transgenic expression of enzymatically active MMP-2 in cardiac muscle leads to cardiac remodeling and dysfunction in the absence of superimposed disease.


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

2.1 Generation of cardiac-specific MMP-2 transgenic mice
Neonatal rat cardiac fibroblast cells (NRCF) were isolated and cultured as reported [24]. The MMP-2 cDNA was prepared by RT-PCR from poly-A RNA isolated using standard methodology. The murine and rat cDNAs for MMP-2 share 100% homology within the coding region [25]. The cDNA was synthesized from poly-A RNA by RT-PCR using SuperscriptTM One Step RT-PCR with Platinum Taq kit (Gibco-BRL). The 5' primer was: 5'-CGGGAGCGCGCAACGATGGAGG-3'. The 3' primer was: 5'-GGGAACAGGGCCAGCTCAGCAG-3'. The 2 kb MMP-2 cDNA was cloned into TOPO-TA vector (PCR® II-TOPO 3.9 kb, Invitrogen). The validity of the MMP-2 expression construct was confirmed by sequencing. To generate a constitutively active MMP-2, Val 107 in the prodomain was mutagenized to Gly107, a step that results in the unfolding of the prodomain from the catalytic site. The 5' mutagenesis primer was 5'-GTGGCAACCCAGATGGGGCCAACTACAACTTC-3'; the 3' primer: 5'-GAAGTTGTAGTTGGCCCCATCGGGTTGCCAC-3'. Confirmation of site-specific mutagenesis was confirmed by sequencing. The MMP-2 expression cassette was recovered from the PCR® II-TOPO plasmid using primers that contain an Xho-I restriction site and a Kozak consensus sequence at the 5– end and encode the c-myc epitope tag at the C-terminus. The 5' primer was 5'-ACGTCTCGAGCCACCATGGAGGCACGGTGGCCTGGG-3'. The 3' primer was 5'-TCACAGGTCTTCCTCGGGAGATCAGTTTCTGCTCGCAGCCCAGCCAGT CTG-3'. The MMP-2/c-myc expression cassette was isolated by PCR using a 5' primer to add a Sal I restriction site: (5'-ACGTGTCGACCACCATGGAGGCACGAGTGGCCTGGG-3'), and a 3' primer to add a Hind III restriction site (5'-ACGTAAGCTTTTACTTGTACAGC-TCGTCCATGCC-3'). This fragment was subsequently cloned into the Sal I and Hind III restriction sites of the {alpha}-myosin heavy chain promoter vector (kind gift of Dr. Jeffrey Robbins, Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center). A 5.5 kb fragment comprising the {alpha}-MHC promoter/MMP-2/c-myc cDNA was excised with Not I, purified by CsCl centrifugation and micro-injected into mouse eggs [F1[129Sv]XF1[CD1]]. Twenty-four pups were generated and four founders identified by PCR of tail genomic DNA using a 5' (5'-AAACCTCAGGCACCCTTACC-3') primer for the {alpha} myosin heavy chain promoter and a 3' primer (5'-CCTCCCCATCATGGATTCGA-3') for the MMP-2 cDNA. The same PCR product amplified from the {alpha}-MHC promoter/MMP-2/c-myc construct was used to generate a probe for Southern blot analysis of genomic DNA using standard methodology. Four individual transgenic lines were established with 4–6 integrated transgene copies. Each individual line manifested a similar cardiac phenotype. For this study, littermates from line 712 were used to provide controls and transgenics. Transgenic animals were maintained as heterozygotes within the outbred CD-1 background. Eight-month-old transgenic mice were used. At this time transgenic mice exhibited significant elevation of MMP-2 expression whereas MMP-9, MMP-13 and MMP-14 were unchanged [26].

2.2 RV trabecula preparation and solutions
The right ventricular (RV) trabecula preparation is widely used for physiology studies because of the simple unbranched uniform geometry along with ultra-thin dimensions that minimize problems due to diffusion. Our experiments using intact and skinned trabeculae were performed at 22 °C to avoid rapid run-down of the preparation. Male and female mice were anesthetized with sodium pentobarbital (100 mg/kg, intraperitoneal) and heparinized (100 U, Sigma-Aldrich, St Louis, MO, USA) [27]. Hearts were removed and promptly perfused through the aorta with a modified Krebs–Henseleit solution containing (in mM): NaCl, 112; KCl, 15; MgCl2, 1.2; glucose, 10; NaHCO3, 24; Na2SO4, 1.2; NaH2PO4, 2.0; 2, 3-butanedione monoxime (BDM), 30; and CaCl2, 1. The perfusate was oxygenated with 95% O2/5% CO2 to give a pH of 7.4 at 22 °C. The RV was removed and weighed, and a trabecula that was free-running between the RV wall and the tricuspid valve was dissected. Only a single trabecula was used from each mouse heart. Trabeculae were approximately 1 mm long and with an elliptical cross-section. The width, thickness and cross-sectional area of trabeculae from wild type mice (148 ± 19 µm, 95 ± 8 µm, 0.012 ± 0.002 mm2, n=12, respectively) were not statistically different (p>0.05) from those in MMP-2 mice (179 ± 24 µm, 107 ± 10 µm, 0.017 ± 0.004 mm2, n=13).

Trabeculae were placed in a muscle chamber (3 x 5 x 15 mm) and attached to the apparatus by mounting the ends on stainless steel pins (100 µm diameter). One end consisted of a remnant of the tricuspid valve that was attached to a micro-manipulator to vary muscle length. The other end was a cube of ventricular wall that was attached to a force transducer (SensoNor, Model AE-80, Norway). Trabeculae were superfused at 22 °C with Krebs–Henseleit solution (5 ml/min, composition as described above, but with 5 mM KCl and no BDM).

Sarcomere length was measured from the diffraction of light from a He/Ne laser (Melles Griot). The diffracted light was monitored with a scanning photodiode array that was coupled to an analog computer (Biotechnical Support Centre, University of Calgary). The diastolic sarcomere length was monitored periodically during the experiment and adjusted to 2.1 µm. Trabeculae were field stimulated using platinum wire electrodes at a frequency of 0.5 Hz and supramaximal voltage.

2.3 Skinned trabeculae experiments
For some experiments, trabeculae were demembranated (skinned) to study myofilament function. Mounted trabeculae were skinned for 30 min at 22 °C in a relaxing solution containing 1% Triton X-100. Sarcomere length was then set to 2.1 µm and trabeculae underwent contraction/relaxation cycles by sequential exposure to 3 solutions: relaxing solution, preactivating solution, and activating solution; trabeculae were then returned to relaxing solution. Relaxing solution contained (in mM) 20 EGTA, 7.12 MgCl2, 6.1 Na2ATP, 10 creatine phosphate, 100 N,N-bis[2-hydroxyethyl]2-aminoethane sulfonic acid (BES), pH was adjusted to 7.1 with KOH, ionic strength was adjusted to 180 mM with KCl. Preactivating solution was identical except calcium buffering was reduced by replacing 19.5 mM of EGTA with HDTA (hexamethylenediamine-N,N,N',N'-tetraacetate) (Fluka). For activating solution, EGTA was replaced by Ca-EGTA and MgCl2 was 6.21 mM. Relaxing and activating solutions were mixed to obtain solutions with intermediate Ca2+ levels. All solutions contained 1% (v/v) protease inhibitor cocktail P-8340 and 10 IU/mL creatine kinase (Sigma, St. Louis, MO), and were designed to have 5 mM MgATP, 0.5 mM free magnesium and 180 mM ionic strength at 22 °C as determined from the methods of Fabiato and Fabiato [28,29].

Skinned trabeculae were subjected to 8 consecutive contractions using activating solutions with different [Ca2+]. The order was randomized between experiments. For each experiment, the sigmoidal relationship between steady state force (F) and [Ca2+] was fit to the Hill equation: F=Fmax x [Ca2+]nH/([Ca2+]nH+Ca50nH), where Fmax is the maximum Ca2+-activated force, Ca50 is the [Ca2+] at which F is 50% of Fmax, and nH is Hill coefficient reflecting the slope of the Ca2+–force relationship at Ca50.

2.4 β-AR stimulation
The β-AR agonist isoproterenol (Abbott Labs, Chicago, IL) was added in a dose-dependent manner to the Krebs–Henseleit solution. For these experiments low extracellular [Ca2+] was used (0.3 mM) to avoid saturation of the force response at the higher doses of isoproterenol. Forskolin (20 µM) (Sigma-Aldrich, St. Louis, MO) was used to stimulate adenylate cyclase directly.

2.5 Electron microscopy
MMP-2 and WT trabeculae were pinned at their working length, fixed overnight in buffered 4% glutaraldehyde at 4 °C, rinsed, osmicated, dehydrated and embedded in Epon. Ultrathin transverse sections were stained with lead citrate/uranyl acetate using standard methodology. Serial sections from all trabeculae were examined.

2.6 In situ zymography
To assess MMP-2 activity, in situ zymography using 5 µm frozen sections from WT- and MMP-2 transgenic hearts was performed according to Mook et al. [30] with quenched fluorogenic DQ-gelatin as the substrate (Molecular Probes, Eugene, OR). Proteolytic cleavage of the gelatin substrate results in loss of quenching and yields a fluorescent signal. Specificity of MMP-2 enzymatic activity was confirmed by co-incubation with the cyclic peptide MMP-2 inhibitor CTTHWGFTLCGG at 25 µmol/L.

2.7 Ribonuclease protection assay (RPA)
{alpha}- and β-myosin heavy chain (MHC) mRNA abundance was detected by ribonuclease protection assay (RPA III kit, Ambion Inc., Austin, Texas, USA) using total ventricular RNA (TRIzol, Invitrogen Life Technologies, Carlsbad, California, USA) isolated from WT and MMP-2 transgenic hearts (n=3). RNA (3 µg) was hybridized with radio labeled probes for MHC and β-actin (Ambion Inc., Austin, Texas, USA), digested with RNase, and protected fragments resolved by gel electrophoresis. Autoradiographic bands for {alpha}- and β-MHC isoform mRNAs were quantified using ImageQuant (Molecular Dynamics, Sunnyvale, California, USA) and normalized to β-actin [31].

2.8 Heart rate and blood pressure
Systolic blood pressure and heart rate were measured using a noninvasive computerized tail cuff system (BP-2000; Visitech Systems, Apex, North Carolina, USA) [31]. Mice were trained for 3 days, and recordings were made on the next 6 days, with at least 15 of 20 successful readings each day.

2.9 Statistical analysis
Data are presented as mean ± S.E. Comparisons between WT and MMP-2 transgenic mice were made using Students t-test and two-way repeated measures ANOVA where values of p<0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Table 1 shows that for wild type (WT) versus MMP-2 transgenic mice, there were no significant differences in body weight, left ventricle (LV) weight, right ventricle (RV) weight, heart rate or blood pressure.


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  		Table 1 Ventricular weights, heart rate and blood pressure for wild type and MMP-2 transgenic mice

 
3.1 Increased MMP-2 activity in MMP-2 transgenic hearts
In situ zymography resulted in a low level of fluorescence for WT myocardium indicating low levels of gelatinolytic activity (Fig. 1A). In contrast, fluorescence, reflecting gelatinolytic activity, was greatly increased for MMP-2 transgenic myocardium (Fig. 1B). To confirm the specificity of gelatinolytic activity due to MMP-2 activity, inclusion of a specific cyclic peptide MMP-2 inhibitor (CTTHWGFTLCGG) reduced the fluorescence for MMP-2 myocardium to background levels (Fig. 1C).


Figure 1
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  		Fig. 1 In situ zymographs to detect increased MMP-2 activity in myocardium from MMP-2 transgenic mice. Combined Normarski/fluorescence photomicrographs of 5 µm frozen section of myocardium from: A) Wild Type; B) MMP-2 transgenic; C) MMP-2 transgenic + cyclic peptide inhibitor (magnification 200 x).

 
3.2 Effect of transgenic MMP-2 expression on muscle ultrastructure
Serial transverse sections of WT and MMP-2 trabeculae were studied and representative electron micrographs are shown in Fig. 2. No gross ultrastructural changes were evident in MMP-2 myocardium compared to WT. There was no gross fibrosis or gross myocyte dropout. However, there were subtle changes: compared to WT, MMP-2 myocardium displayed fibroblasts with abundant rough endoplasmic reticulum, characteristic of an activated synthetic phenotype. This finding suggests some underlying fibrosis in MMP-2 trabeculae.


Figure 2
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  		Fig. 2 Representative transmission electron micrographs of transverse sections from WT (A) and MMP-2 trabeculae (C) showing no gross differences in ultrastructure (2000 x) (CM cardiac myocyte; CAP capillary). Arrows point to fibroblasts; fibroblasts at black arrows magnified in right panels. Fibroblasts in MMP-2 myocardium had abundant rough endoplasmic reticulum (indicated by black arrows in panel D) characteristic of an activated phenotype.

 
3.3 Effect of transgenic MMP-2 expression on myocardial stiffness
To investigate the effect of transgenic MMP-2 expression on myocardial stiffness, unstimulated trabeculae were bathed in a Krebs–Henseleit solution containing low extracellular [Ca2+] (1 mM) and the relaxing agent 2,3-Butanedione (BDM). Unstimulated trabeculae were stretched while measuring sarcomere length and passive force. Fig. 3 shows that MMP-2 trabeculae had greater passive force at increased sarcomere length compared with WT (p<0.01, two-way repeated measures ANOVA). This indicates that transgenic expression of MMP-2 increased myocardial stiffness.


Figure 3
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  		Fig. 3 Relationship between passive force and sarcomere length. Unstimulated trabeculae were bathed in low [Ca2+] Krebs–Henseleit solution containing the relaxing agent 2,3-Butanedione. There was a significant difference between data from MMP-2 vs. WT trabeculae (p<0.01, two way repeated measures ANOVA; mean ± S.E., n=6 per group).

 
3.4 Effect of transgenic MMP-2 expression on myocardial contractile function
To investigate the effect of transgenic MMP-2 expression on myocardial contractile function, trabeculae were electrically stimulated in a Krebs–Henseleit solution containing 1.5 mM [Ca2+] and no BDM. Fig. 4 shows that increases in sarcomere length caused increased force for both MMP-2 and WT myocardium (Starlings law). However, force development per muscle area for MMP-2 trabeculae was less than WT at all sarcomere lengths studied (mean ± S.E., n=6 per group, p<0.05 two-way repeated measures ANOVA). The time to peak of the twitch and the time to half relaxation were not significantly different between MMP-2 and WT trabeculae (not shown).


Figure 4
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  		Fig. 4 Relationship between developed force and sarcomere length (1.5 mM bath [Ca2+]). Electrically stimulated MMP-2 trabeculae had reduced force development with increasing sarcomere length compared to WT (p<0.01, two-way repeated measures ANOVA; mean ± S.E., n=6 per group).

 
To determine if transgenic MMP-2 expression affected maximum force development at high bath [Ca2+] we monitored the relationship between force development and bath [Ca2+] for MMP-2 and WT trabeculae (sarcomere length 2.1 µm). Fig. 5 shows that compared to WT, MMP-2 trabeculae had appreciably lower maximum developed force at raised bath [Ca2+] (n=6 per group, p<0.01, two-way repeated measures ANOVA). Together these findings on force development suggest that transgenic MMP-2 expression reduced contractile function.


Figure 5
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  		Fig. 5 Relationship between developed force and bath [Ca2+]. Electrically stimulated MMP-2 trabeculae developed less force with raised bath [Ca2+] compared to WT (p<0.01, two-way repeated measures ANOVA; mean ± S.E., n=6 per group).

 
Some studies suggest that in models of disease, contractility may appear normal at low pacing rates but that impaired function may become apparent at higher pacing rates [32]. Therefore, we monitored the contractile response of MMP-2 and WT trabeculae to increased pacing frequency (1 mM bath [Ca2+]). Fig. 6 shows that at the higher pacing rates (>1.5 Hz), MMP-2 trabeculae generated lower force than WT trabeculae (n=6 per group, p<0.05, two-way repeated measures ANOVA).


Figure 6
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  		Fig. 6 Relationship between developed force and stimulation frequency (1 mM bath [Ca2+]). MMP-2 trabeculae developed less force with increasing stimulation frequency compared to WT (p<0.01, two-way repeated measures ANOVA; mean ± S.E., n=6 per group).

 
To determine if active MMP-2 expression altered the myocardial response to β-AR stimulation, we monitored the response of trabeculae to isoproterenol. Fig. 7 showed that isoproterenol resulted in a smaller rise force for MMP-2 trabeculae compared to WT (n=6 per group, p<0.05, two-way repeated measures ANOVA). Furthermore, to study effects independent of the β-adrenergic receptor, we stimulated adenylate cyclase directly using forskolin. Forskolin increased the force development for both WT and MMP-2 trabeculae; however, the force of MMP-2 trabeculae remained considerably below that of WT (p<0.05, Students t-test).


Figure 7
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  		Fig. 7 Effect of β-AR stimulation on developed force (0.3 mM bath [Ca2+]). The response to isoproterenol was significantly lower for MMP-2 trabeculae compared to WT (p<0.05, two-way repeated measures ANOVA; mean ± S.E., n=6 per group). Forskolin was administered to stimulate adenylate cyclase directly. Forskolin increased the force development for both WT and MMP-2 trabeculae, however, the force of MMP-2 trabeculae remained considerably below that of WT (p<0.05, Students t-test).

 
To determine if decreased contraction of MMP-2 trabeculae involved impaired myofilament function we used demembranated (skinned) trabeculae. Fig. 8 shows the force–Ca2+relation for skinned WT and MMP-2 trabeculae. Similar to the results from intact trabeculae, there was {approx}50% fall in maximum force development (Fmax) for MMP-2 versus WT trabeculae (p=0.001). However, for MMP-2 versus WT trabeculae there was not a significant difference in Ca50 (1.54 ± 0.26 µmol/L, n=7 vs. 1.1 ± 0.08 µmol/L, n=6, p>0.05) or Hill coefficient nH (2.9 ± 0.6, n=7 vs. 2.7 ± 0.6, n=6, p>0.05).


Figure 8
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  		Fig. 8 Relationship between steady-state force development and [Ca2+] for skinned trabeculae. Line shows pooled data fitted to the Hill equation.

 
Trabeculae from MMP-2 transgenic hearts tended to be larger than WT which could affect force development due to a greater diffusion distance (proportional to muscle thickness) from the muscle core to the external medium. However, Fig. 9 shows that for both WT and MMP-2 trabeculae (living or skinned) there was not a relationship between force development and muscle thickness, indicating that the finding of decreased force in MMP-2 trabeculae could not be attributed to differences in muscle size.


Figure 9
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  		Fig. 9 Relationship between force development and trabecula thickness for intact- (circles) and skinned trabeculae (squares) from WT (open) and MMP-2 transgenic mice (closed).

 
To determine if changes in myofilament function for MMP-2 trabeculae were associated with altered contractile protein isoforms we monitored expression of genes for {alpha}-myosin heavy chain ({alpha}-MHC) and β-MHC. Fig. 10A shows that for MMP-2 versus WT trabeculae, mRNA levels for β-MHC were increased, mRNA levels for {alpha}-MHC were decreased, and mRNA levels for β-actin were unchanged. Fig. 10B, summarizes these changes in mRNA levels referenced to β-actin mRNA; both increased β-MHC mRNA and decreased {alpha}-MHC mRNA were statistically significant.


Figure 10
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  		Fig. 10 A) mRNA levels for {alpha}-myosin heavy chain ({alpha}-MHC) and β-MHC in hearts from WT and MMP-2 transgenic mice. B) pooled data normalized to β-actin (mean ± S.E., n=3 per group; **p<0.01, *p<0.05 versus WT).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The most important finding in this study was that in the absence of superimposed injury, transgenic expression of active MMP-2 can directly lead to the development of cardiac contractile dysfunction.

Previous studies suggested a role for MMP-2 activation in cardiac remodeling and dysfunction in the setting of disease such as heart failure and ischemia [17,18,22,23]. However, there has been little direct evidence demonstrating that in the absence of superimposed injury, increased activity of MMP-2 can directly lead to the development of cardiac dysfunction. Furthermore, while the role of MMP-2 in extracellular matrix (ECM) remodeling is well established, its role in regulating myofilament activity has been less clear.

We found that for right ventricular trabeculae, expression of active MMP-2 resulted in increased myocardial passive stiffness consistent with the role of MMP-2 in cardiac remodeling and fibrosis. However, electron micrographs revealed that there was no evidence of gross fibrosis in MMP-2 trabeculae. Nevertheless, activated fibroblasts were present in MMP-2 trabeculae suggesting ECM remodeling. Consistent with our findings, previous studies found that even minor changes in the ECM can lead to increased myocardial stiffness [33].

A major new observation was that both intact and skinned MMP-2 trabeculae had markedly impaired active force generation compared to WT. Interestingly, despite reduced myocardial force, systolic blood pressure and heart rate were normal for MMP-2 transgenic mice, suggesting that whole heart function was able to accommodate an appreciable loss of myocardial contractility.

Compared to WT, MMP-2 intact trabeculae developed less active force with increasing sarcomere length; moreover, inotropic responses to increases of bath [Ca2+], pacing frequency, and isoproterenol were all reduced in MMP-2 trabeculae compared to WT. Results in intact trabeculae were paralleled by those in skinned trabeculae where MMP-2 trabeculae had {approx}50% reduction of force development compared to WT. These results suggest that in addition to its role in ECM remodeling, MMP-2 activity is also a potent inhibitor of contractile function. Moreover, our findings demonstrate that in the absence of superimposed injury, MMP-2 activity directly leads to a disease phenotype involving abnormal passive and active mechanical properties of the myocardium.

Inhibition of force generation in MMP-2 trabeculae was not associated with appreciable reduction of myocyte area. Moreover, use of skinned fibers eliminates influences from ec-coupling and intracellular milieu. Thus, our findings suggest that decreased force of MMP-2 trabeculae was due to dysfunction of the myofilaments. Our findings cannot rule out depressed force development due to subtle changes in the ECM; for example, alterations in the alignment and/or tethering of myocytes to the ECM [34]. However, an intriguing alternative is that elevated MMP-2 activity could directly lead to myofilament damage. Indeed, Cleutjens speculated that MMPs have biological effects in the heart separate from their action on the extracellular matrix [33]. Several previous findings are consistent with this hypothesis. The protective actions of MMP inhibitors on myocardial contractile function were independent of changes in collagen content [35]. Furthermore, an intracardiomyocyte and sarcomeric association of MMP-2 was found in hearts of patients with dilated cardiomyopathy [36]. Finally, with myocardial stunning, elevated MMP-2 activity was associated with TnI proteolysis [17,19]. Thus, consistent with our findings in skinned fibers, growing evidence supports a role for MMP-2 activity in regulating myofilament function.

MMP-2 trabeculae had decreased {alpha}-myosin heavy chain ({alpha}-MHC) mRNA and increased β-MHC mRNA. These changes are characteristic of the fetal program of gene expression that is found in pathological hypertrophy and heart failure. These changes are consistent with a cardiac disease phenotype in MMP-2 transgenic mice.

In conclusion, targeting expression of constitutively active MMP-2 to cardiac muscle resulted in abnormal passive and active mechanical properties of the myocardium; suggesting that MMP-2 can directly lead to cardiac disease in the absence of superimposed injury.


   Acknowledgements
 
This work was supported by NIH grants P01 HL68738, and by an Established Investigator Award from the American Heart Association (AJB).


   Notes
 
Time for primary review 29 days


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

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