Targeted overexpression of tissue inhibitor of matrix metalloproteinase-4 modifies post-myocardial infarction remodeling in mice.

RATIONALE: Myocardial infarction (MI) causes an imbalance between matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (TIMPs) and is associated with adverse left ventricular (LV) remodeling. A uniform reduction in TIMP-4 post-MI has been observed. OBJECTIVE: To examine post-MI remodeling with cardiac-restricted overexpression of TIMP-4, either through a transgenic or viral delivery approach. METHODS AND RESULTS: MI was induced in mice and then randomized to targeted injection of an adenoviral construct (10 µL; 8×10(9) plaque forming units/mL) encoding green fluorescent protein (GFP) and the full-length human TIMP-4 (Ad-GFP-TIMP4) or GFP. A transgenic construct with cardiac-restricted overexpression TIMP-4 (hTIMP-4exp) was used in a parallel set of studies. LV end-diastolic volume, an index of LV remodeling, increased by >60% from baseline at 5 days post-MI and by >100% at 21 days post-MI in the Ad-GFP only group. However, LV dilation was reduced by ≈50% in both the Ad-GFP-TIMP4 and hTIMP-4exp groups at these post-MI time points. LV ejection fraction was improved with either Ad-GFP-TIMP-4 or hTIMP-4exp. Fibrillar collagen expression and content were increased within the MI region with both TIMP-4 interventions, suggestive of matrix stabilization. CONCLUSIONS: This study is the first to demonstrate that selective myocardial targeting for TIMP-4 induction through either a viral or transgenic approach favorably altered the course of adverse LV remodeling post-MI. Thus, localized induction of endogenous matrix metalloproteinase inhibitors, such as TIMP-4, holds promise as a means to interrupt the progression of post-MI remodeling.

Discordant activation of gene promoters for matrix metalloproteinases and tissue inhibitors of the metalloproteinases following myocardial infarction.

BACKGROUND: Left ventricular (LV) remodeling following myocardial infarction (MI) is associated with increased levels of specific matrix metalloproteinases (MMPs) and relative reduction of endogenous tissue inhibitors of the MMPs (TIMPs). However, transcriptional mechanisms for the disparate post-MI MMP/TIMP expression remain unknown. Using murine constructs designed to report gene promoter activation, this study tested the hypothesis that distinctly different temporal profiles of MMP-2, MMP-9, and TIMP-1 transcription occurs post-MI. METHODS/RESULTS: Transcriptional activity (ß-galactosidase (ß-gal) reporter constructs) of MMP-2 (n = 49), MMP-9 (n = 62), or TIMP-1 (n = 40) was assayed at 1 h (acute), and 1-28 d after MI (coronary ligation) in transgenic reporter mice. At 7 d post-MI, the area of promoter activation normalized to LV area was increased from acute values for MMP-2 (63.4 ± 5.8 versus 1.1% ± 1.0%, P < 0.05) and MMP-9 (53.1 ± 6.1 versus 1.3% ± 0.9%, P < 0.05). While TIMP-1 promoter activation at 7 d post-MI increased from acute values (3.6 ± 1.3 versus 0.3% ± 0.5%, P < 0.05), this increase was smaller than that for MMP-2 or MMP-9 (both P < 0.05). MMP-2 promoter activation peaked in the MI region at 7 d post-MI and MMP-9 promoter activation was highest in the border region at 7 and 14 d post-MI. TIMP-1 promoter activation peaked within the MI region at 7 d post-MI and within the remote region at 14 d post-MI. CONCLUSIONS: These findings provided direct in vivo evidence that discordant changes in temporal and spatial patterns of MMP/TIMP transcription occurs with MI. Restoration of TIMP-1 promoter activation may represent a molecular therapeutic target to attenuate/prevent adverse post-MI LV remodeling.

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.

Cardiac restricted overexpression of membrane type-1 matrix metalloproteinase causes adverse myocardial remodeling following myocardial infarction.

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.

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.

Alterations in membrane type-1 matrix metalloproteinase abundance after the induction of thoracic aortic aneurysm in a murine model.

Thoracic aortic aneurysms (TAAs) develop as a result of dysregulated extracellular matrix remodeling mediated by several matrix metalloproteinases (MMPs). Membrane type-1 MMP (MT1-MMP) is the prototypical member of a unique family of membrane-bound MMPs, possessing multiple substrates and functions. The present study tested the hypothesis that MT1-MMP expression, abundance, and activity would be elevated during TAA development and that this protease is produced primarily by mesenchymal cells within the thoracic aorta. Descending thoracic aortas were harvested from C57BL/6J mice at multiple time points (2, 4, 8, and 16 wk, n = 15 each) post-TAA induction (0.5M CaCl(2), 15 min) and compared with reference controls (n = 15). The expression and abundance of MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase (TIMP)-2 were assessed by quantitative PCR and immunoblot analysis. MT1-MMP activity was determined by fluorescent peptide assay. MT1-MMP was localized within the aortic wall by immunohistochemistry. MT1-MMP abundance and localization in live animals (8 wk post-TAA induction vs. control) was determined by micro-ultrasound imaging with an MT1-MMP-targeted microbubble contrast agent. Aortic diameter was increased 172 +/- 7% at 16 wk post-TAA induction (P < 0.05). MT1-MMP and MMP-2 mRNA levels were elevated at 2 wk post-TAA induction (P < 0.05). MT1-MMP protein abundance increased progressively to a maximum of 178 +/- 26% at 16 wk post-TAA induction, whereas MMP-2 and TIMP-2 peaked at 2 wk post-TAA induction (526 +/- 93% and 376 +/- 48%, respectively, P < 0.05). MT1-MMP colocalized with fibroblasts, and MT1-MMP-targeted contrast binding was elevated in 8-wk TAA-induced mice versus control mice (217 +/- 53% vs. 81 +/- 8%, P < 0.05). In conclusion, these novel results suggest that MT1-MMP plays a dynamic multifunctional role in TAA development and, therefore, may provide a significant target for therapeutic strategies.

Cellular phenotype transformation occurs during thoracic aortic aneurysm development.

OBJECTIVE: Thoracic aortic aneurysms result from dysregulated remodeling of the vascular extracellular matrix, which may occur as a result of altered resident cellular function. The present study tested the hypothesis that aortic fibroblasts undergo a stable change in cellular phenotype during thoracic aortic aneurysm formation. METHODS: Primary murine aortic fibroblasts were isolated from normal and thoracic aortic aneurysm-induced aortas (4 weeks post induction with 0.5 mol/L CaCl(2) 15 minutes) by the outgrowth method. Normal and thoracic aortic aneurysm cultures were examined using a focused polymerase chain reaction array to determine fibroblast-specific changes in gene expression in the absence and presence of biological stimulation (endothelin-1, phorbol-12-myristate-13-acetate, angiotensin-II). The relative expression of 38 genes, normalized to 4 housekeeping genes, was determined, and genes displaying a minimum 2-fold increase/decrease or genes with significantly different normalized cycle threshold values were considered to have altered expression. RESULTS: At steady state, thoracic aortic aneurysm fibroblasts revealed elevated expression of several matrix metalloproteinases (Mmp2, Mmp11, Mmp14), collagen genes/elastin (Col1a1, Col1a2, Col3a1, Eln), and other matrix proteins, as well as decreased expression of Mmp3, Timp3, and Ltbp1. Moreover, gene expression profiles in thoracic aortic aneurysm fibroblasts were different than normal fibroblasts after equivalent biological stimuli. CONCLUSIONS: This study demonstrated for the first time that isolated primary aortic fibroblasts from thoracic aortic aneurysm-induced mice possess a unique and stable gene expression profile, and when challenged with biological stimuli, induce a transcriptional response that is different from normal aortic fibroblasts. Together, these data suggest that aortic fibroblasts undergo a stable phenotypic change during thoracic aortic aneurysm development, which may drive the enhancement of extracellular matrix proteolysis in thoracic aortic aneurysm progression.

Cardiac-restricted overexpression of membrane type-1 matrix metalloproteinase in mice: effects on myocardial remodeling with aging.

BACKGROUND: The direct consequences of a persistently increased myocardial expression of the unique matrix metalloproteinase (MMP) membrane type-1 (MT1-MMP) on myocardial remodeling remained unexplored. METHODS AND RESULTS: Cardiac-restricted MT1-MMPexp was constructed in mice using the full-length human MT1-MMP gene ligated to the myosin heavy chain promoter, which yielded approximately a 200% increase in MT1-MMP when compared with age/strain-matched wild-type (WT) mice. Left ventricular (LV) function and geometry was assessed by echocardiography in 3-month ("young") WT (n=32) and MT1-MMPexp (n=20) mice and compared with 14-month ("middle-aged") WT (n=58) and MT1-MMPexp (n=35) mice. LV end-diastolic volume was similar between the WT and MT1-MMPexp young groups, as was LV ejection fraction. In the middle-aged WT mice, LV end-diastolic volume and ejection fraction was similar to young WT mice. However, in the MT1-MMPexp middle-aged mice, LV end-diastolic volume was approximately 43% higher and LV ejection fraction 40% lower (both P<0.05). Moreover, in the middle-aged MT1-MMPexp mice, myocardial fibrillar collagen increased by nearly 2-fold and was associated with approximately 3-fold increase in the processing of the profibrotic molecule, latency-associated transforming growth factor binding protein. In a second study, 14-day survival after myocardial infarction was significantly lower in middle-aged MT1-MMPexp mice. CONCLUSIONS: Persistently increased myocardial MT1-MMP expression, in and of itself, caused LV remodeling, myocardial fibrosis, dysfunction, and reduced survival after myocardial injury. These findings suggest that MT1-MMP plays a mechanistic role in adverse remodeling within the myocardium.

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.

Aprotinin modifies left ventricular contractility and cytokine release after ischemia-reperfusion in a dose-dependent manner in a murine model.

BACKGROUND: Periods of ischemia-reperfusion (I/R) during cardiac surgery are associated with transient left ventricular (LV) dysfunction and an inflammatory response. In this study, we examined the potential dose-dependent effects of aprotinin (APRO) on LV contractility and cytokine release in the setting of I/R. METHODS: An index of LV contractility, LV maximal elastance (E(max)), was measured at baseline, 30 min of ischemia, and 60 min of reperfusion by microtransducer volumetry. Mice were randomized as follows: (a) APRO 20,000 kallikrein-inhibiting units (KIU)/kg (n = 11); (b) APRO 4 x 10(4) KIU/kg (n = 10); (c) APRO 8 x 10(4) KIU/kg (n = 10); and (d) vehicle (saline; n = 10). APRO doses were calculated to reflect half, full, and twice the clinical Hammersmith dosing schedule. After I/R, plasma was collected for cytokine measurements. RESULTS: After I/R, E(max) decreased from the baseline value by more than 40% in the vehicle group as well as in the APRO 4 x 10(4) KIU/kg and APRO 8 x 10(4) KIU/kg groups (P < 0.05). However, E(max) returned to near baseline values in the APRO 2 x 10(4) KIU/kg group. Tumor necrosis factor (TNF) increased 10-fold after I/R, but it was reduced with higher APRO doses. CONCLUSIONS: This study demonstrated that a low dose of APRO provided protective effects on LV contractility, whereas higher doses suppressed TNF release. These unique findings suggest that there are distinct and independent mechanisms of action of APRO in the context of I/R.

Aprotinin exacerbates left ventricular dysfunction after ischemia/reperfusion in mice lacking tumor necrosis factor receptor I.

Aprotinin is a serine protease inhibitor with diverse biological effects; until recently, it was utilized in the context of ischemia/reperfusion (I/R). It has been hypothesized that a signaling pathway modulated by aprotinin in the context of I/R is the tumor necrosis factor-alpha receptor (TNFR) pathway. An intact mouse model of I/R (30 min ischemia and 60 min reperfusion) was used and left ventricular (LV) peak + maximal rate of left ventricular (LV) peak pressure (dP/dt) was measured in wild-type mice (WT, C57BL/6; n = 10), WT mice with aprotinin (4 mL/kg; n = 10), transgenic mice devoid of the TNFRI (TNFRI-null; n = 10), and TNFRI-null with aprotinin (n=10). Following I/R, LV peak + dP/dt decreased in both WT groups, but remained similar to baseline values in the TNFRI-null group. In contrast, aprotinin caused a marked reduction in LV peak + dP/dt in the TNFRI-null group following I/R. Soluble plasma TNF levels increased in the WT and TNFRI-null mice with I/R and was reduced with aprotinin. Soluble TNFRI and TNFRII levels, indicative of TNF activation, increased in the WT mice following I/R and remained elevated with aprotinin. Soluble TNFRII levels were increased in the TNFRI-null mice following I/R and remained elevated with aprotinin. The new and unique findings of this study were twofold. First, aprotinin failed to improve LV function after I/R despite a reduction in circulating TNF levels. Second, genetic ablation of TNFRI uncovered a negative inotropic effect of aprotinin. These findings demonstrate that complex biological pathways and interactions are affected with broad spectrum serine protease inhibition, which are relevant to myocardial function in the context of I/R.

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.

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.

Aprotinin exerts differential and dose-dependent effects on myocardial contractility, oxidative stress, and cytokine release after ischemia-reperfusion.

BACKGROUND: Cardiac surgery can result in left ventricular ischemia and reperfusion (I/R), the release of cytokines such as tumor necrosis factor, and oxidative stress with release of myeloperoxidase. Although aprotinin has been used in cardiac surgery, the likely multiple effects of this serine protease inhibitor limit clinical utility. This study tested the hypothesis that different aprotinin doses cause divergent effects on left ventricular contractility, cytokine release, and oxidative stress in the context of I/R. METHODS: Left ventricular I/R (30 minutes I, 60 minutes R) was induced in mice, and left ventricular contractility (maximal end-systolic elastance) determined. Mice were randomly allocated to 2 x 10(4) kallikrein inhibitory units (KIU)/kg aprotinin (n = 11), 4 x 10(4) KIU/kg aprotinin (n = 10), and vehicle (saline, n = 10). Based upon a fluorogenic assay, aprotinin doses of 2 and 4 x 10(4) KIU/kg resulted in plasma concentrations similar to those of the half and full Hammersmith doses, respectively. RESULTS: After I/R, maximal end-systolic elastance fell by more than 40% from baseline (p < 0.05), and this effect was attenuated by 2 x 10(4) KIU/kg but not 4 x 10(4) KIU/kg aprotinin. Tumor necrosis factor increased by more than 60% from control (p < 0.05) with I/R, but was reduced with 4 x 10(4) KIU/kg aprotinin. Myeloperoxidase increased with I/R, and was reduced to the greatest degree by 2 x 10(4) KIU/kg aprotinin. CONCLUSIONS: Aprotinin influences left ventricular contractility, cytokine release, and oxidative stress, which are dose dependent. These results provide mechanistic evidence that multiple pathways are differentially affected by aprotinin in a context relevant to cardiac surgery.

Interruption of endothelin signaling modifies membrane type 1 matrix metalloproteinase activity during ischemia and reperfusion.

The matrix metalloproteinases (MMPs), in particular, membrane type 1 MMP (MT1-MMP), are increased in the context of myocardial ischemia and reperfusion (I/R) and likely contribute to myocardial dysfunction. One potential upstream induction mechanism for MT1-MMP is endothelin (ET) release and subsequent protein kinase C (PKC) activation. Modulation of ET and PKC signaling with respect to MT1-MMP activity with I/R has yet to be explored. Accordingly, this study examined in vivo MT1-MMP activation during I/R following modification of ET signaling and PKC activation. With the use of a novel fluorogenic microdialysis system, myocardial interstitial MT1-MMP activity was measured in pigs (30 kg; n = 9) during I/R (90 min I/120 min R). Local ET(A) receptor antagonism (BQ-123, 1 microM) and PKC inhibition (chelerythrine, 1 microM) were performed in parallel microdialysis probes. MT1-MMP activity was increased during I/R by 122 +/- 10% (P < 0.05) and was unchanged from baseline with ET antagonism and/or PKC inhibition. Selective PKC isoform induction occurred such that PKC-betaII increased by 198 +/- 31% (P < 0.05). MT1-MMP phosphothreonine, a putative PKC phosphorylation site, was increased by 121 +/- 8% (P < 0.05) in the I/R region. These studies demonstrate for the first time that increased interstitial MT1-MMP activity during I/R is a result of the ET/PKC pathway and may be due to enhanced phosphorylation of MT1-MMP. These findings identify multiple potential targets for modulating a local proteolytic pathway operative during I/R.