Mytsi L. Coker

A Matrix Metalloproteinase Induction/Activation System Exists in the Human Left Ventricular Myocardium and Is Upregulated in Heart Failure

BackgroundMatrix metalloproteinases (MMPs) contribute to matrix remodeling in disease states such as tumor metastases. Extracellular matrix metalloproteinase inducer (EMMPRIN) has been reported to increase MMP expression, and membrane-type MMP or MT1-MMP has been implicated to activate MMPs. The present study examined whether and to what degree EMMPRIN and MT1-MMP were expressed in human left ventricular (LV) myocardium as well as the association with MMP activity and expression in dilated cardiomyopathy (DCM). Methods and ResultsLV myocardial zymographic MMP activity increased by >2-fold with both nonischemic DCM (n=21) and ischemic DCM (n=16) compared with normal (n=13). LV myocardial abundance of MMP-9 was increased with both forms of DCM. MMP-2 and MMP-3 were increased with nonischemic DCM. MMP-1 levels were decreased with both forms of DCM. EMMPRIN increased by >250% and MT1-MMP increased by >1000% with both forms of DCM. ConclusionsIncreased LV myocardial MMP activity and selective upregulation of MMPs with nonischemic and ischemic forms of DCM occurred. Moreover, a local MMP induction/activation system was identified in isolated normal human LV myocytes that was upregulated with DCM. The control of MMP activation and expression in the failing human LV myocardium represents a new and potentially significant therapeutic target for this disease process.

Increased Matrix Metalloproteinase Activity and Selective Upregulation in LV Myocardium From Patients With End-Stage Dilated Cardiomyopathy

Background —One of the hallmarks of dilated cardiomyopathy (DCM) is left ventricular (LV) remodeling. The matrix metalloproteinases (MMPs) are a family of enzymes that contribute to extracellular remodeling in several disease states. Additionally, a family of inhibitors called tissue inhibitors of MMPs (TIMPs) has been shown to exist and to tightly regulate MMP activity. However, the types of MMPs and TIMPs expressed within the normal and DCM LV myocardium and the relation to MMP activity remain unexplored. Methods and Results —Relative LV myocardial MMP activity was determined in the normal (n=8) and idiopathic DCM (n=7) human LV myocardium by substrate zymography. Relative LV myocardial abundance of interstitial collagenase (MMP-1), stromelysin (MMP-3), 72 kD gelatinase (MMP-2), 92 kD gelatinase (MMP-9), TIMP-1, and TIMP-2 were measured with quantitative immunoblotting. LV myocardial MMP zymographic activity increased with DCM compared with normal (984±149 versus 413±64 pixels, P 500% with DCM. A high-molecular-weight immunoreactive band for both TIMP-1 and TIMP-2, suggesting a TIMP/MMP complex, was increased >600% with DCM. Conclusions —This study demonstrated increased LV myocardial MMP activity and evidence for independent regulatory mechanisms of MMP and TIMP expression with DCM. These findings suggest that selective inhibition of MMP species within the LV myocardium may provide a novel therapeutic target in patients with DCM.

Time-Dependent Changes in Matrix Metalloproteinase Activity and Expression During the Progression of Congestive Heart Failure: Relation to Ventricular and Myocyte Function

The development of congestive heart failure (CHF) is associated with left ventricular (LV) dilation and myocardial remodeling. However, fundamental mechanisms that contribute to this remodeling process with the progression of CHF remain unclear. The matrix metalloproteinases (MMPs) have been demonstrated to play a significant role in tissue remodeling in a number of pathological processes. The present project tested the hypothesis that the LV dilation and remodeling during the progression of CHF is associated with early changes in MMP expression and zymographic activity. LV and myocyte function, collagen content, and MMP expression and zymographic activity were serially measured during the progression of CHF caused by pacing-induced supraventricular tachycardia (SVT) in pigs. After 7 days of SVT, LV end-diastolic dimension and myocyte length both increased by 15% from control values, and LV fractional shortening fell by 20%. At the level of the myocyte, percent shortening fell by 16% after 7 days of SVT, with no change in the steady-state velocity of shortening. Longer durations of SVT caused progressive LV dilation, LV pump failure, and myocyte contractile dysfunction. Specifically, 21 days of SVT resulted in a >50% increase in LV dimension, a 56% fall in LV fractional shortening, and a 33% decline in myocyte velocity of shortening. The decline in LV and myocyte function with 21 days of SVT was accompanied by signs and symptoms of CHF. Thus, SVT causes time-dependent changes in LV geometry and function and the subsequent development of CHF. LV myocardial collagen content and confluence fell by >25% after 7 days of SVT and were accompanied by an 80% increase in LV myocardial MMP zymographic activity against the substrate gelatin. After 14 days of SVT, total LV myocardial collagen content was reduced by 24%, and LV myocardial MMP zymographic activity increased by >100% from control values. Interstitial collagenase (MMP-1), stromelysin (MMP-3), and 72-kD gelatinase (MMP-2) were increased by approximately 2-fold after 7 days of SVT. LV MMP zymographic activity and abundance remained elevated with longer durations of SVT. The results of the present study demonstrated that in this model of CHF, early changes in LV myocardial MMP zymographic activity and protein levels occurred with the initiation and progression of LV dilation and dysfunction. These findings suggest that an early contributory mechanism for the initiation of LV remodeling that occurred in this model of developing CHF is enhanced expression and potentially increased activity of LV myocardial MMPs.

Matrix Metalloproteinase Inhibition During the Development of Congestive Heart Failure Effects on Left Ventricular Dimensions and Function

The development of congestive heart failure (CHF) is associated with left ventricle (LV) dilation and myocardial remodeling. The matrix metalloproteinases (MMPs) play a significant role in extracellular remodeling, and recent studies have demonstrated increased MMP expression and activity with CHF. Whether increased MMP activity directly contributes to the LV remodeling with CHF remains unknown. Accordingly, this study examined the effects of chronic MMP inhibition (MMPi) on LV size and function during the progression of CHF. Pigs were assigned to the following groups: (1) CHF, rapid pacing for 3 weeks at 240 bpm (n=12); (2) CHF/MMPi, rapid pacing and concomitant MMPi (PD166793, 20 mg/kg per day [n=10]), and (3) control (n=11). With pacing CHF, LV fractional shortening was reduced (19+/-1 versus 45+/-1%), and end-diastolic dimension increased (5.67+/-0.11 versus 3.55+/-0.05 cm), compared with baseline values (P<0.05). In the CHF/MMPi group, LV endocardial shortening increased (25+/-2%) and the end-diastolic dimension was reduced (4.92+/-0.17 cm) compared with CHF-only values (P<0.05). LV midwall shortening was reduced to a comparable degree in the CHF-only and CHF/MMPi groups. LV peak wall stress increased 3-fold with pacing CHF compared with controls and was significantly reduced in the CHF/MMPi group. LV myocardial stiffness was unchanged with CHF but was increased in the CHF/MMPi group. LV myocyte length was increased with pacing CHF compared with controls (180+/-3 versus 125+/-4 microm, P<0.05) and was reduced in the CHF/MMPi group (169+/-4 microm, P<0.05). Basal-state myocyte shortening velocity was reduced with pacing CHF compared with controls (33+/-2 versus 66+/-1 microm/s, P<0.05) and was unchanged in the CHF/MMPi group (31+/-2 microm/s). Using an ex vivo assay system, myocardial MMP activity was increased with pacing CHF and was reduced with chronic MMPi. In summary, concomitant MMPi with developing CHF limited LV dilation and reduced wall stress. These results suggest that increased myocardial MMP activity contributes to LV myocardial remodeling in developing CHF.

Matrix Metalloproteinase Inhibition Attenuates Left Ventricular Remodeling and Dysfunction in a Rat Model of Progressive Heart Failure

BackgroundMatrix metalloproteinase (MMP) activation contributes to tissue remodeling in several disease states, and increased MMP activity has been observed in left ventricular (LV) failure. The present study tested the hypothesis that MMP inhibition would influence LV remodeling and function in developing LV failure. Methods and ResultsLV size and function were measured in 5 groups of rats: (1) obese male spontaneously hypertensive heart failure rats (SHHF) at 9 months (n=10), (2) SHHF at 13 months (n=12), (3) SHHF rats treated with an MMP inhibitor during months 9 to 13 (PD166793 5 mg · kg−1 · d−1 PO; n=14), (4) normotensive Wistar-Furth rats (WF) at 9 months (n=12), and (5) WF at 13 months (n=12). Plasma concentrations of the MMP inhibitor (116±11 &mgr;mol/L) reduced in vitro LV myocardial MMP-2 activity by ≈100%. LV function and geometry were similar in WF rats at 9 and 13 months. LV peak +dP/dt was unchanged at 9 months in SHHF but by 13 months was reduced in the SHHF group compared with WF (3578±477 versus 5983±109 mm Hg/s, P ≤0.05). LV volume measured at an equivalent ex vivo pressure (10 mm Hg) was increased in SHHF at 9 months compared with WF (443±12 versus 563±33 mL, P ≤0.05) and increased further by 13 months (899±64 mL, P ≤0.05). LV myocardial MMP-2 activity was increased by ≈2-fold in SHHF at 9 and 13 months. With MMP inhibition, LV peak +dP/dt was similar to WF values and LV volume was reduced compared with untreated SHHF values (678±28 mL, P ≤0.05). ConclusionsMMP activity contributes to LV dilation and progression to LV dysfunction in a rodent HF model, and direct MMP inhibition can attenuate this process.

Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target.

A fundamental structural event in the progression of heart failure due to dilated cardiomyopathy is left ventricular (LV) myocardial remodeling. The matrix metalloproteinases (MMPs) are an endogenous family of enzymes which contribute to matrix remodeling in several disease states. The goal of this report is to summarize recent findings regarding the myocardial MMP system and the relation to matrix remodeling in the failing heart. In both experimental and clinical forms of dilated cardiomyopathy (DCM), increased expression of certain species of myocardial MMPs have been demonstrated. Specifically, increased myocardial levels of the gelatinase, MMP-9 has been identified in both ischemic and non-ischemic forms of human DCM. In addition, stromelysin or MMP-3 increased by over four-fold in DCM. The increased levels of MMP-3 in DCM may have particular importance since this MMP degrades a wide range of extracellular proteins and can activate other MMPs. In normal human LV myocardium, the membrane type 1 MMP (MT1-MMP) was detected. These MT-MMPs may provide important sites for local MMP activation within the myocardium. In a pacing model of LV failure, MMP expression and activity increased early and were temporally associated with LV myocardial matrix remodeling. Using a broad-spectrum pharmacological MMP inhibitor in this pacing model, the degree of LV dilation was attenuated and associated with an improvement in LV pump function. Thus, increased LV myocardial MMP expression and activity are contributory factors in the LV remodeling process in cardiomyopathic disease states. Regulation of myocardial MMP expression and activity may be an important therapeutic target for controlling myocardial matrix remodeling in the setting of developing heart failure.

Myocardial matrix metalloproteinase activity and abundance with congestive heart failure

The left ventricular (LV) myocardial collagen matrix has been proposed to participate in the maintenance of LV geometry. Thus alterations in the composition of the LV myocardial collagen matrix may influence LV function. The matrix metalloproteinases (MMPs) are a family of enzymes that contribute to extracellular remodeling in several disease states. However, the types of MMPs expressed in the normal and congestive heart failure (CHF) state and the relation to MMP activity remained unclear. Accordingly, after 3 wk of pacing (240 beats/min), changes in LV function, substrate-specific MMP activity, and MMP subclass abundance were measured in comparison with control pigs (n = 6). Changes in LV function and geometry were measured by echocardiography; LV end-diastolic dimension increased (3.6 +/- 0.1 vs. 6.0 +/- 0.1 cm, P < 0.05) and LV fractional shortening decreased (47 +/- 1 vs. 15 +/- 1%, P < 0.05) compared with controls. Degradation of fibrillar collagen is achieved through the combined action of interstitial collagenase (MMP-1), gelatinase A (MMP-2), and stromelysin (MMP-3) (He, C., S. Wilheilm, A. Pentland, B. Marmer, G. Grant, A. Eisen, and G. Goldberg. Proc. Natl. Acad. Sci. USA 86:2632-2636, 1989; Woessner, J. FASEB J. 5: 2145-2154, 1991). Accordingly, the relative abundance of specific MMPs (MMP-1, MMP-2, and MMP-3) was examined by immunoblotting. With pacing CHF, the relative abundance for MMP-1 increased to 319 +/- 94%, MMP-2 increased to 194 +/- 31%, and MMP-3 increased to 493 +/- 159% (all P < 0.05). With pacing CHF, LV myocardial zymographic activity for the substrate gelatin increased by 119% (P < 0.05) and for the substrate collagen III by 153% (P < 0.05) over controls. Caseinolytic activity also increased with pacing CHF by 139% (P < 0.05) over controls. In conclusion, LV myocardial MMP activity and abundance increased with pacing-induced CHF. These findings demonstrate that pacing-induced CHF leads to changes in myocardial MMP activity and expression that may be responsible for LV remodeling in CHF.The left ventricular (LV) myocardial collagen matrix has been proposed to participate in the maintenance of LV geometry. Thus alterations in the composition of the LV myocardial collagen matrix may influence LV function. The matrix metalloproteinases (MMPs) are a family of enzymes that contribute to extracellular remodeling in several disease states. However, the types of MMPs expressed in the normal and congestive heart failure (CHF) state and the relation to MMP activity remained unclear. Accordingly, after 3 wk of pacing (240 beats/min), changes in LV function, substrate-specific MMP activity, and MMP subclass abundance were measured in comparison with control pigs ( n = 6). Changes in LV function and geometry were measured by echocardiography; LV end-diastolic dimension increased (3.6 ± 0.1 vs. 6.0 ± 0.1 cm, P < 0.05) and LV fractional shortening decreased (47 ± 1 vs. 15 ± 1%, P < 0.05) compared with controls. Degradation of fibrillar collagen is achieved through the combined action of interstitial collagenase (MMP-1), gelatinase A (MMP-2), and stromelysin (MMP-3) (He, C., S. Wilheilm, A. Pentland, B. Marmer, G. Grant, A. Eisen, and G. Goldberg. Proc. Natl. Acad. Sci. USA 86: 2632-2636, 1989; Woessner, J. FASEB J. 5: 2145-2154, 1991). Accordingly, the relative abundance of specific MMPs (MMP-1, MMP-2, and MMP-3) was examined by immunoblotting. With pacing CHF, the relative abundance for MMP-1 increased to 319 ± 94%, MMP-2 increased to 194 ± 31%, and MMP-3 increased to 493 ± 159% (all P < 0.05). With pacing CHF, LV myocardial zymographic activity for the substrate gelatin increased by 119% ( P < 0.05) and for the substrate collagen III by 153% ( P < 0.05) over controls. Caseinolytic activity also increased with pacing CHF by 139% ( P < 0.05) over controls. In conclusion, LV myocardial MMP activity and abundance increased with pacing-induced CHF. These findings demonstrate that pacing-induced CHF leads to changes in myocardial MMP activity and expression that may be responsible for LV remodeling in CHF.

Matrix metalloproteinase synthesis and expression in isolated LV myocyte preparations.

In several cardiac disease states, alterations in myocyte and extracellular matrix (ECM) structure occur with left ventricular (LV) remodeling and are associated with changes in matrix metalloproteinase (MMP) activity. Although nonmyocyte cell types have been implicated as sites for synthesis and expression of MMPs within the ECM, whether the LV myocyte itself expresses specific types and active forms of MMPs remains unknown. Accordingly, isolated Ca2+-tolerant LV porcine myocytes (105 cells/ml) in which selective disaggregation and resuspension was performed (13 independent experiments) were plated on basement membrane substrates including Matrigel, collagen IV, laminin, and fibronectin as well as poly-l-lysine. After 24-h incubation, LV myocyte conditioned media were subjected to zymography, a specific MMP-2 proteolytic capture assay, immunoblotting, and ELISA for detection of MMP activity and relative content of the 72-kDa gelatinase MMP-2. Although robust zymographic activity [(pixels ⋅ mm2)/cell] was observed in conditioned media from LV myocytes plated on collagen IV (1,673 ± 297), fibronectin (1,530 ± 281), and poly-l-lysine (2,545 ± 560), proteolytic activity appeared to be lower in conditioned media from LV myocytes plated on Matrigel (842 ± 83) and laminin (1,329 ± 238). MMP-2 proteolytic activity was increased by approximately eightfold in conditioned media taken from LV myocytes plated on poly-l-lysine compared with that of Matrigel. With respect to each of the adhesion substrates, MMP-2 content was at least 50% lower in LV myocyte conditioned media taken from Matrigel and laminin. Immunofluorescent labeling of LV myocytes yielded a strong signal for MMP-2 within the myocyte and along the sarcolemmal surface. In conclusion, this study demonstrated for the first time that adult LV myocytes synthesize and express members of the MMP family and thus may potentially participate in the LV remodeling process through synthesis and secretion of MMPs.In several cardiac disease states, alterations in myocyte and extracellular matrix (ECM) structure occur with left ventricular (LV) remodeling and are associated with changes in matrix metalloproteinase (MMP) activity. Although nonmyocyte cell types have been implicated as sites for synthesis and expression of MMPs within the ECM, whether the LV myocyte itself expresses specific types and active forms of MMPs remains unknown. Accordingly, isolated Ca(2+)-tolerant LV porcine myocytes (10(5) cells/ml) in which selective disaggregation and resuspension was performed (13 independent experiments) were plated on basement membrane substrates including Matrigel, collagen IV, laminin, and fibronectin as well as poly-L-lysine. After 24-h incubation, LV myocyte conditioned media were subjected to zymography, a specific MMP-2 proteolytic capture assay, immunoblotting, and ELISA for detection of MMP activity and relative content of the 72-kDa gelatinase MMP-2. Although robust zymographic activity [(pixels. mm(2))/cell] was observed in conditioned media from LV myocytes plated on collagen IV (1,673 +/- 297), fibronectin (1,530 +/- 281), and poly-L-lysine (2,545 +/- 560), proteolytic activity appeared to be lower in conditioned media from LV myocytes plated on Matrigel (842 +/- 83) and laminin (1,329 +/- 238). MMP-2 proteolytic activity was increased by approximately eightfold in conditioned media taken from LV myocytes plated on poly-L-lysine compared with that of Matrigel. With respect to each of the adhesion substrates, MMP-2 content was at least 50% lower in LV myocyte conditioned media taken from Matrigel and laminin. Immunofluorescent labeling of LV myocytes yielded a strong signal for MMP-2 within the myocyte and along the sarcolemmal surface. In conclusion, this study demonstrated for the first time that adult LV myocytes synthesize and express members of the MMP family and thus may potentially participate in the LV remodeling process through synthesis and secretion of MMPs.

Selective Matrix Metalloproteinase Inhibition With Developing Heart Failure: Effects on Left Ventricular Function and Structure

Abstract— The matrix metalloproteinases (MMPs) are an endogenous family of proteolytic enzymes implicated to contribute to LV remodeling. However, broad-spectrum MMP inhibition (MMPi), particularly inhibition of interstitial collagenase (MMP-1), may not be clinically applicable. This study examined the effects of selective MMPi (sparing MMP-1) in a model of developing congestive heart failure. Pigs were randomly assigned to 3 groups: (1) rapid pacing for 3 weeks (240 bpm, n=10); (2) selective MMPi (20 mg/kg per day-PO;PGE7113313) and rapid pacing (n=12); and (3) controls (n=10). LV peak wall stress increased from controls with rapid pacing (140±6 versus 319±18 g/cm2;P <0.05) and was reduced with selective MMPi (208±9 g/cm2;P <0.05. Preload recruitable stroke work was reduced with rapid pacing (4.3±0.4 versus 1.2±0.2 dyne · cm/mm Hg;P <0.05) and was increased with selective MMPi (2.6±0.3 dyne · cm/mm Hg;P <0.05). Plasma norepinephrine increased by 6-fold in the rapid pacing group (P <0.05) and was reduced from untreated values with selective MMPi (P <0.05). At the myocardial level, myocyte cross-sectional area was increased with selective MMPi but fibrillar collagen volume fraction remained unchanged relative to control values. These results suggest that targeting a selective portfolio of myocardial MMP species for inhibition may provide a more rational therapeutic strategy in the setting of congestive heart failure.

Defects in Matrix Metalloproteinase Inhibitory Stoichiometry and Selective MMP Induction in Patients with Nonischemic or Ischemic Dilated Cardiomyopathy

The left ventricular (LV) myocardial collagen matrix has been proposed to participate in the maintenance of LV geometry. With several cardiac disease states such as dilated cardiomyopathy (DCM), alterations in extracellular matrix composition and structure have been reported to occur that may facilitate LV remodeling. 1–3 An important family of enzymes responsible for collagen remodeling are the matrix metalloproteinases (MMPs). 4,5 Several species of MMPs have been detected in the LV myocardium as well as the tissue inhibitors of MMPs, or TIMPs. 6 However, the molecular basis for changes in MMP/TIMP expression, MMP/TIMP stoichiometry, and the relation to MMP activity with nonischemic DCM and ischemic DCM remains unknown. Accordingly, the goal of the present study was to examine MMP/ TIMP species expression and MMP activity in patients with end-stage nonischemic and ischemic DCM.