William T Rivers

Targeted Myocardial Microinjections of a Biocomposite Material Reduces Infarct Expansion in Pigs

BACKGROUND Left ventricular (LV) remodeling after myocardial infarction (MI) commonly causes infarct expansion (IE). This study sought to interrupt IE through microinjections of a biocompatible composite material into the post-MI myocardium. METHODS MI was created in 21 pigs (coronary ligation). Radiopaque markers (2-mm diameter) were placed for IE (fluoroscopy). Pigs were randomized for microinjections (25 injections; 2- x 2-cm array; 200 microL/injection) at 7 days post-MI of a fibrin-alginate composite (Fib-Alg; fibrinogen, fibronectin, factor XIII, gelatin-grafted alginate, thrombin; n = 11) or saline (n = 10). RESULTS At 7 days after injection (14 days post-MI), LV posterior wall thickness was higher in the Fib-Alg group than in the saline group (1.07 +/- 0.11 vs 0.69 +/- 0.07 cm, respectively, p = 0.002). At 28 days post-MI, the area within the markers (IE) increased from baseline (1 cm2) in the saline (1.71 +/- 0.13 cm2, p = 0.010) and Fib-Alg groups (1.44 +/- 0.23 cm2, p < 0.001). However, the change in IE at 21 and 28 days post-MI was reduced in the Fib-Alg group (p=0.043 and p=0.019). Total collagen content within the MI region was similar in the saline and Fib-Alg groups (12.8 +/- 1.7 and 11.6 +/- 1.5 microg/mg, respectively, p = NS). However, extractable collagen, indicative of solubility, was lower in the Fib-Alg group than the saline group (59.1 +/- 3.5 vs 71.0 +/- 6.1 microg/mL, p = 0.020). CONCLUSIONS Targeted myocardial microinjection of the biocomposite attenuated the post-MI decrease in LV wall thickness and infarct expansion. Thus, intraoperative microinjections of biocompatible material may provide a novel approach for interrupting post-MI LV remodeling.

Calpain inhibition preserves myocardial structure and function following myocardial infarction.

Cardiac pathology, such as myocardial infarction (MI), activates intracellular proteases that often trigger programmed cell death and contribute to maladaptive changes in myocardial structure and function. To test whether inhibition of calpain, a Ca(2+)-dependent cysteine protease, would prevent these changes, we used a mouse MI model. Calpeptin, an aldehydic inhibitor of calpain, was intravenously administered at 0.5 mg/kg body wt before MI induction and then at the same dose subcutaneously once per day. Both calpeptin-treated (n = 6) and untreated (n = 6) MI mice were used to study changes in myocardial structure and function after 4 days of MI, where end-diastolic volume (EDV) and left ventricular ejection fraction (EF) were measured by echocardiography. Calpain activation and programmed cell death were measured by immunohistochemistry, Western blotting, and TdT-mediated dUTP nick-end labeling (TUNEL). In MI mice, calpeptin treatment resulted in a significant improvement in EF [EF decreased from 67 + or - 2% pre-MI to 30 + or - 4% with MI only vs. 41 + or - 2% with MI + calpeptin] and attenuated the increase in EDV [EDV increased from 42 + or - 2 microl pre-MI to 73 + or - 4 microl with MI only vs. 55 + or - 4 microl with MI + calpeptin]. Furthermore, calpeptin treatment resulted in marked reduction in calpain- and caspase-3-associated changes and TUNEL staining. These studies indicate that calpain contributes to MI-induced alterations in myocardial structure and function and that it could be a potential therapeutic target in treating MI patients.

Cardiac-restricted overexpression of extracellular matrix metalloproteinase inducer causes myocardial remodeling and dysfunction in aging mice.

The matrix metalloproteinases (MMPs) play a pivotal role in adverse left ventricular (LV) myocardial remodeling. The transmembrane protein extracellular MMP inducer (EMMPRIN) causes increased MMP expression in vitro, and elevated levels occur in patients with LV failure. However, the direct consequences of a prolonged increase in the myocardial expression of EMMPRIN in vivo remained unexplored. Cardiac-restricted EMMPRIN expression (EMMPRINexp) was constructed in mice using the full-length human EMMPRIN gene ligated to the myosin heavy chain promoter, which yielded approximately a twofold increase in EMMPRIN compared with that of the age/strain-matched wild-type (WT) mice; EMMPRINexp (n=27) and WT (n=33) mice were examined at 3.2+/-0.1 or at 13.3+/-0.5 mo of age (n=43 and 26, respectively). LV end-diastolic volume (EDV) was similar in young EMMPRINexp and WT mice (54+/-2 vs. 57+/-3 microl), but LV ejection fraction (EF) was reduced (51+/-1 vs. 57+/-1%; P<0.05). In old EMMPRINexp mice, LV EDV was increased compared with WT mice values (76+/-3 vs. 58+/-3 microl; P<0.05) and LV EF was significantly reduced (45+/-1 vs. 57+/-2%; P<0.05). In EMMPRINexp old mice, myocardial MMP-2 and membrane type-1 MMP levels were increased by >50% from WT values (P<0.05) and were accompanied by a twofold higher collagen content (P<0.05). Persistent myocardial EMMPRINexp in aging mice caused increased levels of both soluble and membrane type MMPs, fibrosis, and was associated with adverse LV remodeling. These findings suggest that EMMPRIN is an upstream signaling pathway that can play a mechanistic role in adverse remodeling within the myocardium.

Direct regulation of membrane type 1 matrix metalloproteinase following myocardial infarction causes changes in survival, cardiac function, and remodeling

The membrane type 1 matrix metalloproteinase (MT1-MMP) is increased in left ventricular (LV) failure. However, the direct effects of altered MT1-MMP levels on survival, LV function, and geometry following myocardial infarction (MI) and the proteolytic substrates involved in this process remain unclear. MI was induced in mice with cardiac-restricted overexpression of MT1-MMP (MT1-MMPexp; full length human), reduced MT1-MMP expression (heterozygous; MT1-MMP(+/-)), and wild type. Post-MI survival was reduced with MT1-MMPexp and increased with MT1-MMP(+/-) compared with WT. LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT post-MI and was higher in the MT1-MMP(+/-) mice. In vivo localization of MT1-MMP using antibody-conjugated microbubbles revealed higher MT1-MMP levels post-MI, which were the highest in the MT1-MMPexp group and the lowest in the MT1-MMP(+/-) group. LV collagen content within the MI region was higher in the MT1-MMPexp vs. WT post-MI and reduced in the MT1-MMP(+/-) group. Furthermore, it was demonstrated that MT1-MMP proteolytically processed the profibrotic molecule, latency-associated transforming growth factor-1-binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by over fourfold in the post-MI MT1-MMPexp group and reduced in the MT1-MMP(+/-) group, which was directionally paralleled by phospho-Smad-3 levels, a critical signaling component of the profibrotic transforming growth factor pathway. We conclude that modulating myocardial MT1-MMP levels affected LV function and matrix structure, and a contributory mechanism for these effects is through processing of profibrotic signaling molecules. These findings underscore the diversity of biological effects of certain MMP types on the LV remodeling process.

Inhibition of class I histone deacetylase activity represses matrix metalloproteinase-2 and -9 expression and preserves LV function postmyocardial infarction

Left ventricular (LV) remodeling, after myocardial infarction (MI), can result in LV dilation and LV pump dysfunction. Post-MI induction of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated as causing deleterious effects on LV and extracellular matrix remodeling in the MI region and within the initially unaffected remote zone. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We assessed the efficacy of both class I/IIb- and class I-selective HDAC inhibitors on MMP-2 and MMP-9 abundance and determined if treatment resulted in the attenuation of adverse LV and extracellular matrix remodeling and improved LV pump function post-MI. MI was surgically induced in MMP-9 promoter reporter mice and randomized for treatment with a class I/IIb HDAC inhibitor for 7 days post-MI. After MI, LV dilation, LV pump dysfunction, and activation of the MMP-9 gene promoter were significantly attenuated in mice treated with either the class I/IIb HDAC inhibitor tichostatin A or suberanilohydroxamic acid (voronistat) compared with MI-only mice. Immunohistological staining and zymographic levels of MMP-2 and MMP-9 were reduced with either tichostatin A or suberanilohydroxamic acid treatment. Class I HDAC activity was dramatically increased post-MI. Treatment with the selective class I HDAC inhibitor PD-106 reduced post-MI levels of both MMP-2 and MMP-9 and attenuated LV dilation and LV pump dysfunction post-MI, similar to class I/IIb HDAC inhibition. Taken together, these unique findings demonstrate that selective inhibition of class I HDACs may provide a novel therapeutic means to attenuate adverse LV remodeling post-MI.

Heterogeneity in MT1-MMP activity with ischemia-reperfusion and previous myocardial infarction: relation to regional myocardial function

After a myocardial infarction (MI), an episode of ischemia-reperfusion (I/R) can result in a greater impairment of left ventricular (LV) regional function (LVRF) than that caused by an initial I/R episode in the absence of MI. Membrane type-I matrix metalloproteinase (MT1-MMP) proteolytically processes the myocardial matrix and is upregulated in LV failure. This study tested the central hypothesis that a differential induction of MT1-MMP occurs and is related to LVRF after I/R in the context of a previous MI. Pigs with a previous MI [3 wk postligation of the left circumflex artery (LCx)] or no MI were randomized to undergo I/R [60-min/120-min left anterior descending coronary artery (LAD) occlusion] or no I/R as follows: no MI and no I/R (n = 6), no MI and I/R (n = 8), MI and no I/R (n = 8), and MI and I/R (n = 8). Baseline LVRF (regional stroke work, sonomicrometry) was lower in the LAD region in the MI group compared with no MI (103 ± 12 vs. 188 ± 26 mmHg·mm, P < 0.05) and remained lower with peak ischemia (35 ± 8 vs. 88 ± 17 mmHg·mm, P < 0.05). Using a novel interstitial microdialysis method, MT1-MMP was directly measured and was over threefold higher in the LCx region and over twofold higher in the LAD region in the MI group compared with the no MI group at baseline. MT1-MMP fluorogenic activity was persistently elevated in the LCx region in the MI and I/R group but remained unchanged in the LAD region. In contrast, no changes in MT1-MMP occurred in the LCx region in the no MI and I/R group but increased in the LAD region. MT1-MMP mRNA was increased by over threefold in the MI region in the MI and I/R group. In conclusion, these findings demonstrate that a heterogeneous response in MT1-MMP activity likely contributes to regional dysfunction with I/R and that a subsequent episode of I/R activates a proteolytic cascade within the MI region that may contribute to a continued adverse remodeling process.

Long-Term Localized High-Frequency Electric Stimulation Within the Myocardial Infarct: Effects on Matrix Metalloproteinases and Regional Remodeling

Background— Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contributes to left ventricular wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high-frequency stimulation (LHFS) using low-amplitude electric pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning. Methods and Results— At 3 weeks after MI, pigs were randomized for LHFS (n=7; 240 bpm, 0.8 V, 0.05-ms pulses) or were left unstimulated (UNSTIM; n=10). At 4 weeks after MI, left ventricular wall thickness (echocardiography; 0.89±0.07 versus 0.67±0.08 cm; P<0.05) and regional stiffness (piezoelectric crystals; 14.70±2.08 versus 9.11±1.24; P<0.05) were higher with LHFS than in UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage; 943±59 versus 1210±72 U; P<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower and TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all P<0.05). Transforming growth factor-&bgr; receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electric stimulation (4 Hz) of isolated fibroblasts resulted in reduced MMP-2 and MT1-MMP levels but increased TIMP-1 levels compared with unstimulated fibroblasts. Conclusions— These unique findings demonstrate that LHFS of the MI region altered left ventricular wall thickness and material properties, likely as a result of reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify left ventricular remodeling after MI.

Hemodynamics and myocardial blood flow patterns after placement of a cardiac passive restraint device in a model of dilated cardiomyopathy.

BACKGROUND The present study examined a cardiac passive restraint device which applies epicardial pressure (HeartNet Implant; Paracor Medical, Inc, Sunnyvale, Calif) in a clinically relevant model of dilated cardiomyopathy to determine effects on hemodynamic and myocardial blood flow patterns. METHODS Dilated cardiomyopatht was established in 10 pigs (3 weeks of atrial pacing, 240 beats/min). Hemodynamic parameters and regional left ventricular blood flow were measured under baseline conditions and after acute placement of the HeartNet Implant. Measurements were repeated after adenosine infusion, allowing maximal coronary vasodilation and coronary flow reserve to be determined. RESULTS Left ventricular dilation and systolic dysfunction occurred relative to baseline as measured by echocardiography. Left ventricular end-diastolic dimension increased and left ventricular fractional shortening decreased (3.8 ± 0.1 vs 6.1 ± 0.2 cm and 31.6% ± 0.5% vs 16.2% ± 2.1%, both P < .05, respectively), consistent with the dilated cardiomyopathy phenotype. The HeartNet Implant was successfully deployed without arrhythmias and a computed median mid-left ventricular epicardial pressure of 1.4 mm Hg was applied by the HeartNet Implant throughout the cardiac cycle. Acute HeartNet placement did not adversely affect steady state hemodynamics. With the HeartNet Implant in place, coronary reserve was significantly blunted. CONCLUSIONS In a large animal model of dilated cardiomyopathy, the cardiac passive restraint device did not appear to adversely affect basal resting myocardial blood flow. However, after acute HeartNet Implant placement, left ventricular maximal coronary reserve was blunted. These unique results suggest that cardiac passive restraint devices that apply epicardial transmural pressure can alter myocardial blood flow patterns in a model of dilated cardiomyopathy. Whether this blunting of coronary reserve holds clinical relevance with chronic passive restraint device placement remains unestablished.

Cardiac-restricted overexpression or deletion of tissue inhibitor of matrix metalloproteinase-4: differential effects on left ventricular structure and function following pressure overload-induced hypertrophy

Historically, the tissue inhibitors of matrix metalloproteinases (TIMPs) were considered monochromatic in function. However, differential TIMP profiles more recently observed with left ventricular (LV) dysfunction and matrix remodeling suggest more diverse biological roles for individual TIMPs. This study tested the hypothesis that cardiac-specific overexpression (TIMP-4OE) or deletion (knockout; TIMP-4KO) would differentially affect LV function and structure following pressure overload (LVPO). LVPO (transverse aortic constriction) was induced in mice (3.5 ± 0.1 mo of age, equal sex distribution) with TIMP-4OE (n = 38), TIMP-4KO (n = 24), as well as age/strain-matched wild type (WT, n = 25), whereby indexes of LV remodeling and function such as LV mass and ejection fraction (LVEF) were determined at 28 days following LVPO. Following LVPO, both early (7 days) and late (28 days) survival was ~25% lower in the TIMP-4KO group (P < 0.05). While LVPO increased LV mass in all groups, the relative hypertrophic response was attenuated with TIMP-4OE. With LVPO, LVEF was similar between WT and TIMP-4KO (48 ± 2% and 45 ± 3%, respectively) but was higher with TIMP-4OE (57 ± 2%, P < 0.05). With LVPO, LV myocardial collagen expression (type I, III) increased by threefold in all groups (P < 0.05), but surprisingly this response was most robust in the TIMP-4KO group. These unique findings suggest that increased myocardial TIMP-4 in the context of a LVPO stimulus may actually provide protective effects with respect to survival, LV function, and extracellular matrix (ECM) remodeling. These findings challenge the canonical belief that increased levels of specific myocardial TIMPs, such as TIMP-4 in and of themselves, contribute to adverse ECM accumulation following a pathological stimulus, such as LVPO.

Short-term disruption in regional left ventricular electrical conduction patterns increases interstitial matrix metalloproteinase activity

Increased matrix metalloproteinase (MMP) abundance occurs with adverse left ventricular (LV) remodeling in a number of cardiac disease states, including those induced by long-standing arrhythmias. However, whether regionally contained aberrant electrical activation of the LV, with consequent dyskinesia, alters interstitial MMP activation remained unknown. Electrical activation of the LV of pigs (n = 10, 30-35 kg) was achieved by pacing (150 beats/min) at left atrial and LV sites such that normal atrioventricular activation (60 min) was followed by regional early LV activation for 60 min within 1.5 cm of the paced site and restoration of normal atrioventricular pacing for 120 min. Regional shortening (piezoelectric crystals) and interstitial MMP activity (microdialysis with MMP fluorogenic substrate) at the LV pacing site and a remote LV site were monitored at 30-min intervals. During aberrant electrical stimulation, interstitial MMP activity at the paced site was increased (122 +/- 4%) compared with the remote region (100%, P < 0.05). Restoration of atrioventricular pacing after the 60-min period of aberrant electrical activation normalized segmental shortening (8.5 +/- 0.4%), but MMP activity remained elevated (121 +/- 6%, P < 0.05). This study demonstrates that despite the restoration of mechanical function, disturbances in electrical conduction, in and of itself, can cause acute increases in regional in vivo MMP activation and, therefore, contribute to myocardial remodeling.