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Matrix metalloproteinase synthesis and expression in isolated LV myocyte preparations
16
Journal of Cardiac Failure Vol. 4 No. 3 Suppl. 1 1998
Cardiovascular Structure 055
056
Increased Matrix Metalloproteinase Activity and Selective Upregulation in LV Myocardium From Patients with End-Stage Dilated Cardiomyopathy
Matrix Metalloproteinase Synthesis and Expression in Isolated LV Myocyte Preparations
C h a d w i c k V. Thomas, M y t s i L. Coker, James L. Zellner, J o h n R. H a n d y , Jackson C r u m b l e y ]71I,a n d Francis G, Spinale Medical University of S o u t h Carolina, Charleston, SC One of the hallmarks of dilated cardiomyopathy (DCM) is left ventricular (LV) remodeling. The matrix metalloproteinases (MMPs) are a family of enzymes which contribute to extracellular remodeling in several disease states. However, the types of MMPs expressed within the normal and DCM LV myocardium, and the relation to MMP activity remains unexplored. Accordingly, interstitial collagenase (MMP-1), stromelysin (MMP-3), 72 kDa gelatinase (MMP-2), and 92 kDa gelatinase (MMP-9) were measured in normal (n=8) and idiopathic (n=8) human LV myocardium using quantitative immunoblotting. Relative LV myocardial MMP activity was determined by substrate zymography. LV myocardial MMP zymographic activity increased with DCM compared to normal (984±149 vs 413=1=64 pixels, p<0.05). With DCM, LV myocardial abundance of MMP-1 decreased to 16±6% (p<0.05), MMP-3 increased to 563±212% (p<0.05), MMP-9 increased to 422±64% (p<0.05), and MMP-2 was unchanged when compared to normal.The ratio o f MMP3/MMP-1 and MMP-9/MMP-1 increased over 40 times from normal levels with DCM (p<0.05). Summary: This study demonstrated increased LV myocardial MMP activity and evidence for independent regulatory mechanisms of MMP expression with DCM. These unique findings suggest that selective inhibition of MMP species within the LV rnyocardium may provide a novel therapeutic target in patients with DCM.
Mytsi L. Coker, Melissa A. Doscher, C h a d w i c k V. Thomas, Z o r i n a S. Galls ~', Francis G. Spinale Medical U n i v e r s i t y of S o u t h Carolina, Charleston, SC a n d t E m o r y School of Medicine, Atlanta, G A In several cardiac disease states, alterations in myocyte and extracellular matrix (ECM) structure occur with LV remodeling and are associated with changes in matrix metalloproteinase (MMP) activity. While non-myocyte cell types have been implicated as sites for the 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*-~tolerant LV porcine myocytes (10s cells/ml) were plated in serum-free media on basement membrane substrates which included matrigel (l.8gg/ml), laminin (50p.~ml), collagen IV (500 nghnl) and fibronectin (50gg/mt) for 24 hours. LV myocyte conditioned media were subjected to zymography and immunoblotfing for the detection of MMP activity and the relative abundance of the 72 kDa gelatinase, MMP-2. The table summarizes results from 13 independent experiments (*p<0.05 vs Matrigel). In an additional set of experiments, immunofluorescent labelling of LV myocytes yielded a strong signal for MMP-2 within the myocyte. Matrigel Zymographic /Activity 842±83 (pixels.mma)/cell [MMP-2 [(% Matrigel)
100±0
Laminin
Collagen 1V
Fibronectin
I673±297
1329±238"
1530±28I*
-237±51"
209±27*
208±46*
Summary: Adult LV myocytes synthesize and express members of the MM family and thus, may participate in the LV remodeling process through the synthesis and secretion of MMPs.
057
O58
The Effects of the Calcium Channel Antagonist Mibefradil on Interstitial and Perivascular Fibrosis in Myocardial InfarctionInduced Cardiac Failure in Rats
Altered Expression and Distribution of Intercalated DiskAssociated Proteins in Guinea Pigs with Pressure-Overloaded Cardiac Hypertrophy and Failure
St. S a n d m a n n , R. M. Bohle*, Th. Dreyer*, Th. Unger; Institute of P h a r m a c o l o g y , University of Kiel a n d *Dept. of Pathology, U n i v e r s i t y of Giel~en, G e r m a n y
Xuejun W a n g , A. M a r t i n Gerdes, University of S o u t h Dakota, Vermillion, SD
Structural alterations of the heart following cardiac failure are associated with the accumulation of fibrillar collagen that ~xzurs within the extracellular matrix and around intramyocardial coronary arteries. This interstitial and perivascular fibrosis is responsible for abnormal myocardial stiffness and reduced coronary blood flow associated with impaired cardiac function. We characterized the effects of a long term treatment with the preferentially T-channel blocking calcium chamael antagonist mibefradil on the remodeling of interstifium and structural alterations of coronary arteries 6 weeks after chronic myocardial infarction (MI). After pernuanent ligation of the left coronary artery, Wistar rats were divided in sham-operated (group 1), placebo- (group 2) or mibefradil-treated (gronp 3-7) MI groups. Mibeffadil treatment (10 mg/kg/d p.o.) was started 7d pre, , 3h post, 24h post, 3d post and 7d post Ivfl-induction and continued for 6 weeks after MI. At this time point, the hearts were stained with picrosirius red and subjected to morphometrical determinations of interstitial collagen volume fraction (ICVF) of the right ventricle (RV) and the septal endocardium and Lepicardium of the left ventricle (LV). Structural parameters of coronary arteries of RV and LV were perivascular collagen volume fraction 0PCVF), vascular perimeter (VP), inner vascular diameter 0VD) and media thickness (MT). Six weeks after MI, total heart weight (THW), ICVF of RV, septal epicardium and endocardium, PCVF of RV and LV and MT of RV and LV were increased. Mibefradil treatment reduced ICVF of RV (group 3) and of septal epicardium and endocardinm (,groups 3 and 4), PCVF of RV and LV in groups 3-6 and MT of RV and LV in groups 3-5 compared to the placebo-treated MI group. Ivl'I' of RV and LV of 7d pro MI mibefradil-treated rats (group 3) did not differ from shamoperated rats anywere. Under mibefradil treatment, VP and IVD of RV and LV were increased in groups 3 and 4 compared to placebo-treated group. We conclude, that chronic treatment with mibefradil exerted antifihrofic actions ' evidenced by the reduction of interstitial and perivascular fibrosis. The structural improvement of coronary arteries may give rise to increased myocardial perfnsion in post MI heart failure. Cardiac remodeling was best prevented when treatment was begun either prior to or within honrs after the acute isehemic event.
To investigate the cellular mechanisms and structural correlates of congestive heart failure caused by pressure overload, chronic pressure overload was produced in young male guinea pigs by constriction of the descending thoracic aorta. Hemodynamies and echeeardiography demonstrated compensated pressure-overloaded cardiac hypertrophy at 4 weeks (4w) and congestive heart failure 6 months (6m) after surgery. Gap junctions-, fascia adberentes- and desmosomes-associated proteins in the intercalated disk (ID) components of left ventricular (LV) myocytes isolated from both time points were examined with iminunofluorescent confocal microscopy and inmaanoblotting. Compared to agematched sham controls, it was shown that: (1) counexin43 was unchanged at 4w but decreased 37 % (p < 0,01 ) at 6m while double labeled pan-cadherin remained unchanged, and connexin45 did not show a compensated increase; (2) although cateains a and y showed 11odiscernible changes at both 4w and 6m, the staining of catenin 13at the ID sites was substantially reduced at 6m but not 4w; (3) the overall abundance of vinculin was unchanged as determined by western blots at both time points, but reduced staining at the ID sites was evident in about 25% of LV myccytes when LV failure occurred; (4) the expression of desmin protein and the density of desmin filaments were progressively increased during development of the cardiac hypertrophy and failure, but the distribution of desmin filaments followed a normal pattern; and (5) desmoplakin showed no qualitatively discernible changes at both time points. Thus, a substantial remodeling of intercalated disks is associated with the progression from compensated pressure overloaded hypertrophy to congestive heart failure. This remodeling may underlie myocardial dysfunction and maladaption of myocyte shape in heart failure.
Journal of Cardiac Failure Vol. 4 No. 3 Suppl. 1 1998
Cardiovascular Structure 055
056
Increased Matrix Metalloproteinase Activity and Selective Upregulation in LV Myocardium From Patients with End-Stage Dilated Cardiomyopathy
Matrix Metalloproteinase Synthesis and Expression in Isolated LV Myocyte Preparations
C h a d w i c k V. Thomas, M y t s i L. Coker, James L. Zellner, J o h n R. H a n d y , Jackson C r u m b l e y ]71I,a n d Francis G, Spinale Medical University of S o u t h Carolina, Charleston, SC One of the hallmarks of dilated cardiomyopathy (DCM) is left ventricular (LV) remodeling. The matrix metalloproteinases (MMPs) are a family of enzymes which contribute to extracellular remodeling in several disease states. However, the types of MMPs expressed within the normal and DCM LV myocardium, and the relation to MMP activity remains unexplored. Accordingly, interstitial collagenase (MMP-1), stromelysin (MMP-3), 72 kDa gelatinase (MMP-2), and 92 kDa gelatinase (MMP-9) were measured in normal (n=8) and idiopathic (n=8) human LV myocardium using quantitative immunoblotting. Relative LV myocardial MMP activity was determined by substrate zymography. LV myocardial MMP zymographic activity increased with DCM compared to normal (984±149 vs 413=1=64 pixels, p<0.05). With DCM, LV myocardial abundance of MMP-1 decreased to 16±6% (p<0.05), MMP-3 increased to 563±212% (p<0.05), MMP-9 increased to 422±64% (p<0.05), and MMP-2 was unchanged when compared to normal.The ratio o f MMP3/MMP-1 and MMP-9/MMP-1 increased over 40 times from normal levels with DCM (p<0.05). Summary: This study demonstrated increased LV myocardial MMP activity and evidence for independent regulatory mechanisms of MMP expression with DCM. These unique findings suggest that selective inhibition of MMP species within the LV rnyocardium may provide a novel therapeutic target in patients with DCM.
Mytsi L. Coker, Melissa A. Doscher, C h a d w i c k V. Thomas, Z o r i n a S. Galls ~', Francis G. Spinale Medical U n i v e r s i t y of S o u t h Carolina, Charleston, SC a n d t E m o r y School of Medicine, Atlanta, G A In several cardiac disease states, alterations in myocyte and extracellular matrix (ECM) structure occur with LV remodeling and are associated with changes in matrix metalloproteinase (MMP) activity. While non-myocyte cell types have been implicated as sites for the 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*-~tolerant LV porcine myocytes (10s cells/ml) were plated in serum-free media on basement membrane substrates which included matrigel (l.8gg/ml), laminin (50p.~ml), collagen IV (500 nghnl) and fibronectin (50gg/mt) for 24 hours. LV myocyte conditioned media were subjected to zymography and immunoblotfing for the detection of MMP activity and the relative abundance of the 72 kDa gelatinase, MMP-2. The table summarizes results from 13 independent experiments (*p<0.05 vs Matrigel). In an additional set of experiments, immunofluorescent labelling of LV myocytes yielded a strong signal for MMP-2 within the myocyte. Matrigel Zymographic /Activity 842±83 (pixels.mma)/cell [MMP-2 [(% Matrigel)
100±0
Laminin
Collagen 1V
Fibronectin
I673±297
1329±238"
1530±28I*
-237±51"
209±27*
208±46*
Summary: Adult LV myocytes synthesize and express members of the MM family and thus, may participate in the LV remodeling process through the synthesis and secretion of MMPs.
057
O58
The Effects of the Calcium Channel Antagonist Mibefradil on Interstitial and Perivascular Fibrosis in Myocardial InfarctionInduced Cardiac Failure in Rats
Altered Expression and Distribution of Intercalated DiskAssociated Proteins in Guinea Pigs with Pressure-Overloaded Cardiac Hypertrophy and Failure
St. S a n d m a n n , R. M. Bohle*, Th. Dreyer*, Th. Unger; Institute of P h a r m a c o l o g y , University of Kiel a n d *Dept. of Pathology, U n i v e r s i t y of Giel~en, G e r m a n y
Xuejun W a n g , A. M a r t i n Gerdes, University of S o u t h Dakota, Vermillion, SD
Structural alterations of the heart following cardiac failure are associated with the accumulation of fibrillar collagen that ~xzurs within the extracellular matrix and around intramyocardial coronary arteries. This interstitial and perivascular fibrosis is responsible for abnormal myocardial stiffness and reduced coronary blood flow associated with impaired cardiac function. We characterized the effects of a long term treatment with the preferentially T-channel blocking calcium chamael antagonist mibefradil on the remodeling of interstifium and structural alterations of coronary arteries 6 weeks after chronic myocardial infarction (MI). After pernuanent ligation of the left coronary artery, Wistar rats were divided in sham-operated (group 1), placebo- (group 2) or mibefradil-treated (gronp 3-7) MI groups. Mibeffadil treatment (10 mg/kg/d p.o.) was started 7d pre, , 3h post, 24h post, 3d post and 7d post Ivfl-induction and continued for 6 weeks after MI. At this time point, the hearts were stained with picrosirius red and subjected to morphometrical determinations of interstitial collagen volume fraction (ICVF) of the right ventricle (RV) and the septal endocardium and Lepicardium of the left ventricle (LV). Structural parameters of coronary arteries of RV and LV were perivascular collagen volume fraction 0PCVF), vascular perimeter (VP), inner vascular diameter 0VD) and media thickness (MT). Six weeks after MI, total heart weight (THW), ICVF of RV, septal epicardium and endocardium, PCVF of RV and LV and MT of RV and LV were increased. Mibefradil treatment reduced ICVF of RV (group 3) and of septal epicardium and endocardinm (,groups 3 and 4), PCVF of RV and LV in groups 3-6 and MT of RV and LV in groups 3-5 compared to the placebo-treated MI group. Ivl'I' of RV and LV of 7d pro MI mibefradil-treated rats (group 3) did not differ from shamoperated rats anywere. Under mibefradil treatment, VP and IVD of RV and LV were increased in groups 3 and 4 compared to placebo-treated group. We conclude, that chronic treatment with mibefradil exerted antifihrofic actions ' evidenced by the reduction of interstitial and perivascular fibrosis. The structural improvement of coronary arteries may give rise to increased myocardial perfnsion in post MI heart failure. Cardiac remodeling was best prevented when treatment was begun either prior to or within honrs after the acute isehemic event.
To investigate the cellular mechanisms and structural correlates of congestive heart failure caused by pressure overload, chronic pressure overload was produced in young male guinea pigs by constriction of the descending thoracic aorta. Hemodynamies and echeeardiography demonstrated compensated pressure-overloaded cardiac hypertrophy at 4 weeks (4w) and congestive heart failure 6 months (6m) after surgery. Gap junctions-, fascia adberentes- and desmosomes-associated proteins in the intercalated disk (ID) components of left ventricular (LV) myocytes isolated from both time points were examined with iminunofluorescent confocal microscopy and inmaanoblotting. Compared to agematched sham controls, it was shown that: (1) counexin43 was unchanged at 4w but decreased 37 % (p < 0,01 ) at 6m while double labeled pan-cadherin remained unchanged, and connexin45 did not show a compensated increase; (2) although cateains a and y showed 11odiscernible changes at both 4w and 6m, the staining of catenin 13at the ID sites was substantially reduced at 6m but not 4w; (3) the overall abundance of vinculin was unchanged as determined by western blots at both time points, but reduced staining at the ID sites was evident in about 25% of LV myccytes when LV failure occurred; (4) the expression of desmin protein and the density of desmin filaments were progressively increased during development of the cardiac hypertrophy and failure, but the distribution of desmin filaments followed a normal pattern; and (5) desmoplakin showed no qualitatively discernible changes at both time points. Thus, a substantial remodeling of intercalated disks is associated with the progression from compensated pressure overloaded hypertrophy to congestive heart failure. This remodeling may underlie myocardial dysfunction and maladaption of myocyte shape in heart failure.