Rupak Mukherjee

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.

Myocardial Infarct Expansion and Matrix Metalloproteinase Inhibition

Background—A potential mechanism for left ventricular (LV) remodeling after myocardial infarction (MI) is activation of the matrix metalloproteinases (MMPs). This study examined the effects of MMP inhibition (MMPi) on regional LV geometry and MMP levels after MI. Methods and Results—In pigs instrumented with radiopaque markers to measure regional myocardial geometry, MI was created by ligating the obtuse marginals of the circumflex artery. In the first study, pigs were randomized to MMPi (n=7; PD166793, 20 mg · kg−1 · d−1) or MI only (n=7) at 5 days after MI, and measurements were performed at 2 weeks. Regional MI areas were equivalent at randomization and were increased in the MI-only group at 2 weeks after MI compared with the MMPi group. In the second study, pigs randomized to MMPi (n=9) or MI only (n=8) were serially followed up for 8 weeks. At 8 weeks after MI, LV end-diastolic dimension was lower with MMPi than in the MI-only group (4.7±0.1 versus 5.1±0.1 cm, P <0.05). Regional MI area was reduced with MMPi at 8 weeks after MI (1.3±0.1 versus 1.7±0.1 cm2, P <0.05). MMPi reduced ex vivo MMP proteolytic activity. In the MI region, membrane-type MMP levels were normalized and levels of the endogenous tissue inhibitor of MMPs (TIMP-1) were increased compared with normal levels with MMPi. These effects were not observed in the MI-only group. Conclusions—MMPi attenuated the degree of post-MI LV dilation and expansion of the infarct during the late phase of MI healing. In addition, exogenous MMPi caused region-specific modulation of certain MMP and TIMP species.

Region- and Type-Specific Induction of Matrix Metalloproteinases in Post-Myocardial Infarction Remodeling

Background—Induction of matrix metalloproteinases (MMPs) contributes to adverse remodeling after myocardial infarction (MI). Whether a region- and type-specific distribution of MMPs occurs within the post-MI myocardium remained unknown. Methods and Results—Ten sheep were instrumented with a sonomicrometry array to measure dimensions in 7 distinct regions corresponding to the remote, transition, and MI regions. Eight sheep served as reference controls. The relative abundance of representative MMP types and the tissue inhibitors of the MMPs (TIMPs) was quantified by immunoblotting. Segment length increased from baseline in the remote (24.9±5.4%), transition (18.0±2.9%), and MI (53.8±11.0%) regions at 8 weeks after MI (P <0.05) and was greatest in the MI region (P <0.05). Region- and type-specific changes in MMPs occurred after MI. For example, MMP-1 and MMP-9 abundance was unchanged in the remote, fell to 3±2% in the transition, and was undetectable in the MI region (P <0.05). MMP-13, MMP-8, and MT1-MMP increased by >300% in the transition and MI regions (P <0.05). TIMP abundance decreased significantly in the transition region after MI and fell to undetectable levels within the MI region. Conclusions—The unique findings of this study were 2-fold. First, changes in regional geometry after MI were associated with changes in MMP levels. Second, a region-specific portfolio of MMPs was induced after MI and was accompanied by a decline in TIMP levels, indicative of a loss of MMP inhibitory control. Targeting the regional imbalance between specific MMPs and TIMPs within the post-MI myocardium holds therapeutic potential.

Modulation of the Renin-Angiotensin Pathway Through Enzyme Inhibition and Specific Receptor Blockade in Pacing-Induced Heart Failure I. Effects on Left Ventricular Performance and Neurohormonal Systems

BACKGROUND The goal of this study was to determine the effects of ACE inhibition (ACEI) alone, AT1 angiotensin (Ang) II receptor blockade alone, and combined ACEI and AT1 Ang II receptor blockade on LV function, systemic hemodynamics, and neurohormonal system activity in a model of congestive heart failure (CHF). METHODS AND RESULTS Pigs were randomly assigned to each of 5 groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) ACEI (benazeprilat, 0.187 mg x kg(-1) x d(-1)) and rapid pacing (n=9), (3) AT1 Ang II receptor blockade (valsartan, 3 mg x kg(-1) x d(-1)) and rapid pacing (n=9), (4) ACEI and AT1 Ang II receptor blockade (benazeprilat/valsartan, 0.05/3 mg x kg(-1) d(-1)) and rapid pacing (n=9), and (5) sham controls (n=10). In the pacing group, LV fractional shortening (LVFS) fell (13.4+/-1.4% versus 39.1+/-1.0%) and end-diastolic dimension (LVEDD) increased (5.61+/-0.11 versus 3.45+/-0.07 cm) compared with control (P<.05). With AT1 Ang II blockade and rapid pacing, LVEDD and LVFS were unchanged from pacing-only values. ACEI reduced LVEDD (4.95+/-0.11 cm) and increased LVFS (20.9+/-1.9%) from pacing-only values (P<.05). ACEI and AT1 Ang II blockade reduced LVEDD (4.68+/-0.07 cm) and increased LVFS (25.2+/-0.9%) from pacing only (P<.05). Plasma norepinephrine and endothelin increased by more than fivefold with chronic pacing and remained elevated with AT1 Ang II blockade. Plasma norepinephrine was reduced from pacing-only values by more than twofold in the ACEI group and the combination group. ACEI and AT1 Ang II receptor blockade reduced plasma endothelin levels by >50% from rapid-pacing values. CONCLUSIONS These findings suggest that the effects of ACEI in the setting of CHF are not solely due to modulation of Ang II levels but rather to alternative enzymatic pathways and that combined ACEI and AT1 Ang II receptor blockade may provide unique benefits for LV pump function and neurohormonal systems in the setting of CHF.

Concomitant Endothelin Receptor Subtype-A Blockade During the Progression of Pacing-Induced Congestive Heart Failure in Rabbits Beneficial Effects on Left Ventricular and Myocyte Function

BACKGROUND Plasma levels of endothelin-1 (ET-1) are increased in patients and animals with severe congestive heart failure (CHF). It remains unknown, however, whether ET-1 plays a direct and contributory role in the progression of CHF. Accordingly, the present project tested the hypothesis that chronic blockade of the ETA receptor would have direct and beneficial effects on left ventricular (LV) and myocyte function in a model of CHF. METHODS AND RESULTS Global LV and isolated myocyte function were examined in rabbits in the following groups (12 per group): chronic rapid ventricular pacing (RVP; 400 bpm, 3 weeks), RVP and concomitant administration of the selective ETA receptor antagonist (PD 156707 24 mg/d), and sham controls. LV fractional shortening decreased after RVP (17 +/- 5 versus 42 +/- 3%) and end-diastolic dimension increased (2.36 +/- 0.44 versus 1.24 +/- 0.18 cm) compared with controls (P < .05). With RVP plus ETA blockade, LV fractional shortening was increased (33 +/- 6%) and end-diastolic dimension decreased (2.02 +/- 0.30 cm) compared with RVP-only values (P < .05). Plasma norepinephrine and endothelin increased twofold in the RVP group. In the RVP plus ETA blockade group, plasma endothelin increased threefold compared with RVP values. Isolated myocyte shortening velocity declined after RVP (42 +/- 13 versus 72 +/- 10 microns/s, P < .05) compared with controls but was normalized with RVP plus ETA blockade (77 +/- 16 microns/s). Myocyte inotropic response to extracellular Ca2+, beta-receptor stimulation, and ET-1 was reduced in the RVP group and returned to control levels with RVP and concomitant ETA receptor blockade. CONCLUSIONS The results from this study suggest that chronically elevated ET-1 levels and subsequent activation of the ETA receptor play a direct and contributory role in the progression of the CHF process. Thus, specific ETA receptor blockade may provide a new and useful therapeutic modality in the setting of CHF.

Selective Targeting and Timing of Matrix Metalloproteinase Inhibition in Post-Myocardial Infarction Remodeling

Background—A cause-and-effect relationship exists between matrix metalloproteinase (MMP) induction and left ventricular (LV) remodeling after myocardial infarction (MI). Whether broad-spectrum MMP inhibition is necessary and the timing at which MMP inhibition should be instituted after MI remain unclear. This study examined the effects of MMP-1 and MMP-7-sparing inhibition (sMMPi) on regional and global LV remodeling when instituted before or after MI. Methods and Results—Pigs instrumented with coronary snares and radiopaque markers within the area at risk were randomized to MI only (n=11) or sMMPi (PGE-530742, 10 mg/kg PO TID) begun 3 days before MI (n=11) or 3 days after MI (n=10). Eleven weight-matched noninstrumented pigs served as reference controls. At 10 days after MI, infarct size was similar between groups (47±3% of the area at risk). Marker area increased from baseline in the MI-only group (10±3%, P <0.05) but was unchanged with sMMPi. LV end-diastolic volume increased in the MI-only group (82±3 mL) compared with controls (56±3 mL, P <0.05) but was attenuated with pre-MI and post-MI sMMPi (69±3 and 69±4 mL, respectively, P <0.05). Collagen content increased in the infarct zone of the MI-only group (34±5%) compared with control (2±1%, P <0.05) but was reduced with pre-MI and post-MI sMMPi (24±1% and 23±2%, P <0.05). Collagen content increased in the border zone (12±2%) and decreased in the remote zone (3±1%) of the pre-MI sMMPi group compared with post-MI sMMPi values (7±1% and 5±1%, P <0.05). Conclusions—Inhibition of MMP-1 and −7 is not required to favorably influence LV remodeling after MI. Moreover, a temporal difference exists with respect to the timing of sMMPi and regional and global myocardial remodeling patterns after MI.

Relation between ventricular and myocyte function with tachycardia-induced cardiomyopathy.

Chronic supraventricular tachycardia (SVT) causes left ventricular (LV) dilatation and dysfunction. Changes in myocyte function and structure may be important factors in the development of SVT cardiomyopathy. Accordingly, LV function and isolated myocyte structure and function were examined in six pigs with pacing-induced SVT cardiomyopathy (3 weeks at 240 beats per minute) and six control pigs. LV function was examined by simultaneous echocardiography and catheterization, and isolated myocyte function was studied using computer-assisted video microscopy. Indexes of isolated myocyte contractile performance were examined in the unloaded, unattached state (31 control and 24 SVT cells) and after attachment to a basement membrane substrate (65 control and 45 SVT cells). LV fractional shortening and peak +dP/dt significantly decreased in SVT cells compared with control cells (12 +/- 2% versus 28 +/- 2%, and 842 +/- 61 versus 1,216 +/- 119 mm Hg/sec, respectively; p less than 0.05). Isolated myocyte percent shortening and normalized peak velocity of shortening of SVT myocytes adherent to a basement membrane were significantly lower than attached control myocytes (1.2 +/- 0.2% versus 4.3 +/- 0.3%, and 15 +/- 2 versus 37 +/- 5% resting cell length/sec, respectively; p less than 0.05). Similarly, in the unattached state, the extent and velocity of shortening of SVT myocytes were reduced by over 50% from control values. Contractile properties of attached and unattached cardiocytes were also examined in the presence of 2-8 mM extracellular Ca2+. For both attached and unattached SVT myocytes, responsiveness to increases in extracellular Ca2+ were significantly blunted from control values. Ultrastructural examination of SVT myocytes revealed that the percent volume of myofibrils within isolated myocytes was reduced from control values (46 +/- 7% versus 65 +/- 2%, p less than 0.05). In summary, SVT cardiomyopathy is probably due to a primary defect in isolated myocyte contractile performance. The reduced contractile function of SVT cardiomyopathic myocytes was associated with abnormalities in cytoarchitecture and Ca2+ responsiveness.

Modulation of the Renin-Angiotensin Pathway Through Enzyme Inhibition and Specific Receptor Blockade in Pacing-Induced Heart Failure: II. Effects on Myocyte Contractile Processes

BACKGROUND The goal of this study was to determine the effects of ACE inhibition alone, AT1 angiotensin (Ang) II receptor blockade alone, and combined ACEI and AT1 Ang II receptor blockade in a model of congestive heart failure (CHF) on isolated LV myocyte function and fundamental components of the excitation-contraction coupling process. METHODS AND RESULTS Pigs were randomly assigned to one of five groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) concomitant ACEI (benazeprilat, 0.187 mg x kg(-1) x d(-1)) and rapid pacing (n=9), (3) concomitant AT1 Ang II receptor blockade (valsartan, 3 mg/kg/d) and rapid pacing (n=9), (4) concomitant ACEI and AT1 Ang II receptor blockade (benazeprilat/valsartan, 0.05/3 mg x kg(-1) x d(-1)) and rapid pacing (n=9), and (5) sham controls (n=10). LV myocyte shortening velocity was reduced with chronic rapid pacing compared with control (27.2+/-0.6 versus 58.6+/-1.2 microm/s, P<.05) and remained reduced with AT1 Ang II receptor blockade and rapid pacing (28.0+/-0.5 microm/s, P<.05). Myocyte shortening velocity increased with ACEI or combination treatment compared with rapid pacing only (36.9+/-0.7 and 42.3+/-0.8 microm/s, respectively, P<.05). Myocyte beta-adrenergic response was reduced by >50% in both the rapid pacing group and the AT1 Ang II blockade group and improved by 25% with ACEI and increased by 54% with combined treatment. Both L-type Ca2+ channel density and the relative abundance of sarcoplasmic reticulum Ca2+ ATPase density were reduced with rapid pacing and returned to control levels in the combined ACEI and AT1 Ang II blockade group. CONCLUSIONS The unique findings of this study were twofold. First, basic defects in specific components of the myocyte excitation-contraction coupling process that occur with CHF are reversible. Second, combined ACEI and AT1 Ang II blockade may provide unique benefits on myocyte contractile processes in the setting of CHF.

Matrix Metalloproteinase-7 Affects Connexin-43 Levels, Electrical Conduction, and Survival After Myocardial Infarction

Background— Matrix metalloproteinases (MMPs) contribute to left ventricular remodeling after myocardial infarction (MI). Specific causative roles of particular MMPs, however, remain unclear. MMP-7 is abundant in cardiomyocytes and macrophages, but MMP-7 function after MI has not been defined. Methods and Results— Wild-type (WT; n=55) and MMP-7–null (MMP-7−/−; n=32) mice underwent permanent coronary artery ligation for 7 days. MI sizes were similar, but survival was greatly improved in MMP-7−/− mice. The survival difference could not be attributed to differences in left ventricular dilation because end-diastolic volumes increased similarly. ECG analysis revealed a prolonged PR interval in WT but not in MMP-7−/− post-MI mice. Post-MI conduction velocity, determined by optically mapping electrical wavefront propagation, decreased to 78±6% of control for WT and was normalized in MMP-7−/− mice. In WT mice, slower conduction velocity correlated with a 53% reduction in the gap junction protein connexin-43. Direct binding of MMP-7 to connexin-43, determined by surface plasmon resonance technology, occurred in a dose-dependent manner. Connexin-43 processing by MMP-7 was confirmed by in silico and in vitro substrate analyses and MMP-7 infusion induced arrhythmias in vivo. Conclusions— MMP-7 deletion results in improved survival and myocardial conduction patterns after MI. This is the first report to implicate MMP-7 in post-MI remodeling and to demonstrate that connexin-43 is a novel MMP-7 substrate.