Deficiency of Ataxia Telangiectasia Mutated Kinase Modulates Cardiac Remodeling Following Myocardial Infarction: Involvement in Fibrosis and Apoptosis

Ataxia telangiectasia mutated kinase (ATM) is a cell cycle checkpoint protein activated in response to DNA damage. We recently reported that ATM plays a protective role in myocardial remodeling following β-adrenergic receptor stimulation. Here we investigated the role of ATM in cardiac remodeling using myocardial infarction (MI) as a model. Methods and Results: Left ventricular (LV) structure, function, apoptosis, fibrosis, and protein levels of apoptosis- and fibrosis-related proteins were examined in wild-type (WT) and ATM heterozygous knockout (hKO) mice 7 days post-MI. Infarct sizes were similar in both MI groups. However, infarct thickness was higher in hKO-MI group. Two dimensional M-mode echocardiography revealed decreased percent fractional shortening (%FS) and ejection fraction (EF) in both MI groups when compared to their respective sham groups. However, the decrease in %FS and EF was significantly greater in WT-MI vs hKO-MI. LV end systolic and diastolic diameters were greater in WT-MI vs hKO-MI. Fibrosis, apoptosis, and α-smooth muscle actin staining was significantly higher in hKO-MI vs WT-MI. MMP-2 protein levels and activity were increased to a similar extent in the infarct regions of both groups. MMP-9 protein levels were increased in the non-infarct region of WT-MI vs WT-sham. MMP-9 protein levels and activity were significantly lower in the infarct region of WT vs hKO. TIMP-2 protein levels similarly increased in both MI groups, whereas TIMP-4 protein levels were significantly lower in the infarct region of hKO group. Phosphorylation of p53 protein was higher, while protein levels of manganese superoxide dismutase were significantly lower in the infarct region of hKO vs WT. In vitro, inhibition of ATM using KU-55933 increased oxidative stress and apoptosis in cardiac myocytes.     

Accelerated LV remodeling after myocardial infarction in TIMP-1-deficient mice: effects of exogenous MMP inhibition

Alterations in matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been implicated in adverse left ventricular (LV) remodeling after myocardial infarction (MI). However, the direct mechanistic role of TIMPs in the post-MI remodeling process has not been completely established. The goal of this project was to define the effects of altering endogenous MMP inhibitory control through combined genetic and pharmacological approaches on post-MI remodeling in mice. This study examined the effects of MMP inhibition (MMPi) with PD-166793 (30 mg.kg(-1).day(-1)) on LV geometry and function (conductance volumetry) after MI in wild-type (WT) mice and mice deficient in the TIMP-1 gene [TIMP-1 knockout (TIMP1-KO)]. At 3 days after MI (coronary ligation), mice were randomized into four groups: WT-MI/MMPi (n = 10), TIMP1-KO-MI/MMPi (n = 10), WT-MI (n = 22), and TIMP1-KO-MI (n = 23). LV end-diastolic volume (EDV) and ejection fraction were determined 14 days after MI. Age-matched WT (n = 20) and TIMP1-KO (n = 28) mice served as reference controls. LVEDV was similar under control conditions in WT and TIMP1-KO mice (36 +/- 2 and 40 +/- 2 microl, respectively) but was greater in TIMP1-KO-MI than in WT-MI mice (48 +/- 2 vs. 61 +/- 5 microl, P < 0.05). LVEDV was reduced from MI-only values in WT-MI/MMPi and TIMP1-KO-MI/MMPi mice (42 +/- 2 and 36 +/- 2 microl, respectively, P < 0.05) but was reduced to the greatest degree in TIMP1-KO mice (P < 0.05). LV ejection fraction was reduced in both groups after MI and increased in TIMP1-KO-MI/MMPi, but not in WT-MI/MMPi, mice. These unique results demonstrated that myocardial TIMP-1 plays a regulatory role in post-MI remodeling and that the accelerated myocardial remodeling induced by TIMP-1 gene deletion can be pharmacologically "rescued" by MMP inhibition. These results define the importance of local endogenous control of MMP activity with respect to regulating LV structure and function after MI.     

Toll-like receptor-4 modulates survival by induction of left ventricular remodeling after myocardial infarction in mice

Left ventricular (LV) remodeling is known to contribute to morbidity and mortality after myocardial infarction (MI). Because LV remodeling is strongly associated with an inflammatory response, we investigated whether or not TLR-4 influences LV remodeling and survival in a mice model of MI. Six days after MI induction, TLR4 knockout (KO)-MI mice showed improved LV function 32 and reduced LV remodeling as indexed by reduced levels of atrial natriuretic factor and total collagen as well as by a reduced heart weight to body weight ratio when compared with WT-MI mice. This was associated with a reduction of protein levels of the intracellular TLR4 adapter protein MyD88 and enhanced protein expression of the anti-hypertrophic JNK in KO-MI mice when compared with wild-type (WT)-MI mice. In contrast, protein activation of the pro-hypertrophic kinases protein kinase Cdelta and p42/44 were not regulated in KO-MI mice when compared with WT-MI mice. Improved LV function, reduced cardiac remodeling, and suppressed intracellular TLR4 signaling in KO-MI mice were associated with significantly improved survival compared with WT-MI mice (62 vs 23%; p < 0.0001). TLR4 deficiency led to improved survival after MI mediated by attenuated left ventricular remodeling.     

Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction

Matrix metalloproteinase-2 (MMP-2) is prominently overexpressed both after myocardial infarction (MI) and in heart failure. However, its pathophysiological significance in these conditions is still unclear. We thus examined the effects of targeted deletion of MMP-2 on post-MI left ventricular (LV) remodeling and failure. Anterior MI was produced in 10- to 12-wk-old male MMP-2 knockout (KO) and sibling wild-type (WT) mice by ligating the left coronary artery. By day 28, MI resulted in a significant increase in mortality in association with LV cavity dilatation and dysfunction. The MMP-2 KO mice had a significantly better survival rate than WT mice (56% vs. 85%, P < 0.05), despite a comparable infarct size (50 +/- 3% vs. 51 +/- 3%, P = not significant), heart rate, and arterial blood pressure. The KO mice had a significantly lower incidence of LV rupture (10% vs. 39%, P < 0.05), which occurred within 7 days of MI. The KO mice exerted less LV cavity dilatation and improved fractional shortening after MI by echocardiography. The LV zymographic MMP-2 level significantly increased in WT mice after coronary artery ligation; however, this was completely prevented in KO mice. In contrast, the increase in the LV zymographic MMP-9 level after MI was similar between KO and WT mice. MMP-2 activation is therefore considered to contribute to an early cardiac rupture as well as late LV remodeling after MI. The inhibition of MMP-2 activation may therefore be a potentially useful therapeutic strategy to manage post-MI hearts.     

Deficiency of TIMP-1 exacerbates LV remodeling after myocardial infarction in mice

Recent studies have been directed at modulating the heart failure process through inhibition of activated matrix metalloproteinases (MMPs). We hypothesized that a loss of MMP inhibitory control by tissue inhibitor of MMP (TIMP)-1 deficiency alters the course of postinfarction chamber remodeling and induced chronic myocardial infarction (MI) in wild-type (WT) and TIMP-1(-/-) mice. Left ventricular (LV) pressure-volume loops obtained from WT and TIMP-1(-/-) mice demonstrated that LV end-diastolic volume [52 +/- 4 (WT) vs. 71 +/- 6 (TIMP-1(-/-)) microl] and LV end-diastolic pressure [9.0 +/- 1.2 (WT) vs. 12.7 +/- 1.4 (TIMP-1(-/-)) mmHg] were significantly increased in the TIMP-1(-/-) mice 2 wk after MI. LV contractility was reduced to a similar degree in the WT and TIMP-1(-/-) groups after MI, as indicated by a significant fall in the LV end-systolic pressure-volume relationship. Ventricular weight and cross-sectional areas of LV myocytes were significantly increased in TIMP-1(-/-) mice, indicating that the hypertrophic response was more pronounced. The observed significant loss of fibrillar collagen in the TIMP-1(-/-) controls may have been an important contributory factor for the observed LV alterations in the TIMP-1(-/-) mice after MI. These findings demonstrate that TIMP-1 deficiency amplifies adverse LV remodeling after MI in mice and emphasizes the importance of local endogenous control of cardiac MMP activity by TIMP-1.     

Osteopontin inhibits interleukin-1beta-stimulated increases in matrix metalloproteinase activity in adult rat cardiac fibroblasts: role of protein kinase C-zeta

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1beta (IL-1beta), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1beta-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)-pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat beta3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1beta increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1beta-stimulated increases in translocation of PKC-zeta from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-zeta were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-zeta using PKC-zeta pseudosubstrate inhibited IL-1beta-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via beta3 integrins, inhibits IL-1beta-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-zeta. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.     

ET(A)-receptor blockade prevents matrix metalloproteinase activation late postmyocardial infarction in the rat

Endothelin (ET) A (ET(A)) receptors activate matrix metalloproteinases (MMP). Since endothelin-1 (ET) is increased in myocardium late postmyocardial infarction (MI), we hypothesized that stimulation of ET(A) receptors contributes to activation of myocardial MMPs late post-MI. Three days post-MI, rats were randomized to treatment with the ET(A)-selective receptor antagonist sitaxsentan (n = 12) or a control group (n = 12). Six weeks later, there were rightward shifts of the left ventricular (LV) end-diastolic and end-systolic pressure-volume relationships, as measured ex vivo by the isovolumic Langendorff technique. Both shifts were markedly attenuated by sitaxsentan. In LV myocardium remote from the infarct, the activities of MMP-1, MMP-2, and MMP-9 were increased in the post-MI group, and the increases were prevented by sitaxsentan treatment. Expression of tissue inhibitor of MMP-1 was decreased post-MI, and the decrease was prevented by sitaxsentan treatment. Chronic post-MI remodeling is associated with activation of MMPs in myocardium remote from the infarct. Inhibition of ET(A) receptors prevents MMP activation and LV dilation, suggesting that ET, acting via the ET(A) receptor, contributes to chronic post-MI remodeling by its effects on MMP activity.     

Inhibition of tumor necrosis factor receptor-1-mediated pathways has beneficial effects in a murine model of postischemic remodeling

The aim of the present study was to investigate the importance of tumor necrosis factor (TNF)-alpha receptor-1 (TNFR1)-mediated pathways in a murine model of myocardial infarction and remodeling. One hundred and ninety-four wild-type (WT) and TNFR1 gene-deleted (TNFR1KO) mice underwent left coronary artery ligation to induce myocardial infarction. On days 1, 3, 7, and 42, mice underwent transesophageal echocardiography. Hearts were weighed, and the left ventricle (LV) was assayed for matrix metalloproteinase (MMP)-2 and -9 activity and for tissue inhibitor of MMP (TIMP)-1 and -2 expression. Deletion of the TNFR1 gene substantially improved survival because no deaths were observed in TNFR1KO mice versus 56.4% and 18.2% in WT males and females, respectively (P < 0.002). At 42 days, LV remodeling, assessed by LV function (fractional area change of 31.9 +/- 7.9%, 32.2 +/- 7.7%, and 21.6 +/- 7.1% in TNFR1KO males, TNFR1KO females, and WT females, respectively, P < 0.04), and hypertrophy (heart weight-to-body weight ratios of 5.435 +/- 0.986, 5.485 +/- 0.677, and 6.726 +/- 0.704 mg/g, P < 0.04) were ameliorated in TNFR1KO mice. MMP-9 activity was highest at 3 days postinfarction and was highest in WT males (1.9 +/- 0.4 4, 3.6 +/- 0.24, 1.15 +/- 0.28, and 1.3 +/- 1.2 ng/100 microg protein, respectively, in TNFR1KO males, WT males, TNFR1KO females, and WT females, respectively, P < 0.002), whereas at 3 days TIMP-1 mRNA fold upregulation compared with type- and sex-matched controls was lowest in WT males (138.32 +/- 13.05, 46.74 +/- 5.43, 186.09 +/- 28.07, and 101.76 +/- 22.48, respectively, P < 0.002). MMP-2 and TIMP-2 increased similarly in all infarcted groups. These findings suggest that the benefits of TNFR1 ablation might be attributable at least in part to the attenuation of cytokine-mediated imbalances in MMP-TIMP activity.     

Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction

Myocardial remodeling after myocardial infarction (MI) is associated with increased levels of the matrix metalloproteinases (MMPs). Levels of two MMP species, MMP-2 and MMP-9, are increased after MI, and transgenic deletion of these MMPs attenuates post-MI left ventricular (LV) remodeling. This study characterized the spatiotemporal patterns of gene promoter induction for MMP-2 and MMP-9 after MI. MI was induced in transgenic mice in which the MMP-2 or MMP-9 promoter sequence was fused to the beta-galactosidase reporter, and reporter level was assayed up to 28 days after MI. Myocardial localization with respect to cellular sources of MMP-2 and MMP-9 promoter induction was examined. After MI, LV diameter increased by 70% (P < 0.05), consistent with LV remodeling. beta-Galactosidase staining in MMP-2 reporter mice was increased by 1 day after MI and increased further to 64 +/- 6% of LV epicardial area by 7 days after MI (P < 0.05). MMP-2 promoter activation occurred in fibroblasts and myofibroblasts in the MI region. In MMP-9 reporter mice, promoter induction was detected after 3 days and peaked at 7 days after MI (53 +/- 6%, P < 0.05) and was colocalized with inflammatory cells at the peri-infarct region. Although MMP-2 promoter activation was similarly distributed in the MI and border regions, activation of the MMP-9 promoter was highest at the border between the MI and remote regions. These unique findings visually demonstrated that activation of the MMP-2 and MMP-9 gene promoters occurs in a distinct spatial relation with reference to the MI region and changes in a characteristic time-dependent manner after MI.     

Clinical significance of matrix metalloproteinases activity in acute myocardial infarction

Matrix metalloproteinases (MMP) degrade myocardial fibrillar collagen in acute myocardial infarction (MI) patients. Their activity is tightly controlled in normal myocardium by a family of closely related tissue inhibitors known as TIMP. An imbalance in their activity might contribute to post-MI remodeling. Plasma levels of MMP-1, TIMP-1 and MMP-1/TIMP-1 complex were measured, using relevant ELISA kits, in 24 (22 males-2 females), acute MI patients with a mean age 59 +/- 14 years. Blood samples were taken on admission (0 h), and 3 h, 6 h, 9 h, 18 h, 24 h, 36 h, 48 h, 3rd, 4th, 5th, 7th, 15th, 30th days after MI. All patients underwent coronary arteriography with ventriculography for estimation of left ventricular ejection fraction (LVEF) and extent of coronary artery diseases, and echocardiographic study for measuring end-diastolic diameter (EDD). Ten patients with an LVEF < 45%, an EDD > 47.5 mm, and heart failure symptoms were included in group A and compared against 12 patients with an LVEF > 45% an EDD < 47.5 mm in group B. Mean plasma concentrations of MMP-1 were higher by 21% in group A (1.3 +/- 0.2 ng/mL) compared to group B (1 +/- 0.1 ng/mL) over the total study period. TIMP-1 plasma concentrations showed very little difference between the 2 groups, (704 +/- 213 ng/mL versus 691 +/- 165 ng/mL, (6%)). Finally, plasma concentrations of MMP-1/TIMP-1 complex were lower by -36% in group A with a mean value of 2.7 +/- 0.6 ng/mL versus 3.7 +/- 0.5 ng/mL in group B. Mean values for the differences were significant at time points 0, 6, 18, 24 and 48 hours for MMP-1 (p < 0.036), and on 48 h and the 4th day for MMP-1/TIMP-1 complex (p < 0.031). Moreover, a good correlation was found between plasma concentrations of creatine kinase (CK) and MMP-1 at 18 h (r = 0.422, p = 0.041) and on the 4th day (r = 0.67, p = 0.046), and TIMP-1 on the 4th day (r = 0.67, p = 0.047). Additionally, mean values for LVEF were 35.8 +/- 8.8% in group A versus 51.2 +/- 1.8% (p = 0.00014) in group B. Also, the EDD in-group A was 52.1 +/- 6.9 mm versus 42.9 +/- 3.2 mm in group B (p = 0.00013). In acute MI patients, increased MMP-1, with no change in TIMP-1, is associated with left ventricular dysfunction and dilatation, suggesting that increased collagenolytic activity contributes to loss of LV function.     

Myocardial and interstitial matrix metalloproteinase activity after acute myocardial infarction in pigs

A structural event during the evolution of a myocardial infarction (MI) is left ventricular (LV) remodeling. The mechanisms that contribute to early changes in LV myocardial remodeling in the post-MI period remain poorly understood. Matrix metalloproteinases (MMPs) contribute to tissue remodeling in several disease states. Whether and to what degree MMP activation occurs within the myocardial interstitium after acute MI remains to be determined. Adult pigs (n = 15) were instrumented to measure regional myocardial function and interstitial MMP levels within regions served by the circumflex and left anterior descending arteries. Regional function was measured by sonomicrometry, and interstitial MMP levels were determined by selective microdialysis and zymography as well as by MMP interstitial fluorogenic activity. Measurements were performed at baseline and sequentially for up to 3 h after ligation of the obtuse marginals of the circumflex artery. Regional fractional shortening fell by over 50% in the MI region but remained unchanged in the remote region after coronary occlusion. Release of soluble MMPs, as revealed by zymographic activity of myocardial interstitial samples, increased by 2 h post-MI. The increased zymographic activity after MI was consistent with MMP-9. Myocardial interstitial MMP fluorogenic activity became detectable within the ischemic region as early as 10 min after coronary occlusion and significantly increased after 1 h post-MI. MMP fluorogenic activity remained unchanged from baseline values in the remote region. The present study demonstrated that myocardial MMP activation can occur within the MI region in the absence of reperfusion. These unique results suggest that MMP release and activation occurs within the ischemic myocardial interstitium in the early post-MI period.     

Ghrelin对心肌梗死后心肌重塑及心脏功能的影响及其机制研究

目的:观察ghrelin对心肌梗死(MI)大鼠心肌重塑和心脏功能的影响,并探讨其可能的机制。方法:应用冠状动脉结扎术创建大鼠MI模型,并设立假手术组作为对照;造模成功后每天2次注射ghrelin(100μg/kg),持续4周,以此作为MI-ghrelin组,并以每天注射生理盐水的MI大鼠作为MI-生理盐水组。检测和比较各组大鼠左心室重塑和血流动力学的改变情况;非梗死心肌中白介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶(MMP)-2、MMP-9 mRNA和蛋白的表达;梗死边界心肌细胞的凋亡情况。结果:Ghrelin可使心肌梗死后的MI大鼠降低的缩短分数(FS)、左室内压最大变化率均显著下降(dP/dtmax)、疤痕厚度明显升高,增加左室舒张末压(LVEDP)、左室收缩末内径(LVESD)、左室舒张末期内径(LVEDD)、梗死边界心肌细胞的凋亡指数显著降低。此外,ghrelin可抑制心肌梗死后的MI大鼠非梗死心肌中白介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、质金属蛋白酶(MMP)-2和MMP-9的mRNA和蛋白的表达。结论:Ghrelin可缓解MI后大鼠LV功能紊乱及心室重塑,这可能与其抑制炎症反应及基质金属蛋白酶的表达有关。     

Mice lacking the gene encoding for MMP-9 and resistance artery reactivity

OBJECTIVES: To define the link between the deletion of gene encoding for metalloproteinase 9 and resistance artery reactivity, we studied in vitro smooth muscle and endothelial cell function in response to pressure, shear stress, and pharmacological agents. BACKGROUND: Matrix metalloproteinases play a crucial role in the regulation of extracellular matrix turnover and structural artery wall remodeling. METHODS: Resistance arteries were isolated from mice lacking gene encoding for MMP-9 (KO) and their control (WT). Hemodynamic, pharmacology approaches, and Western blot analysis were used in this study. RESULTS: The measurement of blood pressure in vivo was similar in KO and WT mice. Pressure-induced myogenic tone, contractions to angiotensin-II and phenylephrine were similar in both groups. The inhibition of MMP2/9 ((2R)-2-[(4-biphenylylsulfonyl) amino]-3-phenylpropionic acid) significantly decreased myogenic tone in WT and had no effect in KO mice. Relaxation endothelium-dependent (flow-induced- dilation 41.3+/-0.6 vs. 21+/-1.6 at 10 microl/min in KO and WT mice, respectively, P<0.05) and eNOS expression were increased in KO compared to WT mice. The inhibition of eNOS with L-NAME significantly decreased endothelium response to shear stress, which was more pronounced in KO mice resistance arteries (-26.83+/-2.5 vs. -15.84+/-2.3 at 10 microl/min in KO and WT, respectively, P<0.05). However, the relaxation to exogenous nitric oxide-donor was similar in both groups. CONCLUSION: Our study provides evidence of a selective effect of MMP-9 on endothelium function. Thus, MMP-9 gene deletion specifically increased resistance artery dilation endothelium-dependent and eNOS expression. Based on our results, MMP-9 could be a potential therapeutic target in cardiovascular disease associated with resistance arteries dysfunction.     

Age-related differences in postinfarct left ventricular rupture and remodeling

Cardiac rupture is more prevalent in elderly patients with first onset of acute myocardial infarct (MI), but the mechanism remains unexplored. We investigated the differences in the incidence of cardiac rupture and early left ventricular (LV) remodeling following coronary artery ligation between old (12-mo) and young (3-mo) C57Bl/6 male mice and explored responsible mechanisms. The incidence of rupture within 1 wk after MI was significantly higher in old than in young mice (40.7 vs. 18.3%, P = 0.013) despite a similar infarct size in both age groups. Old mice dying of rupture had more severe infarct expansion than young counterparts. Echocardiography and catheterization at day 7 revealed more profound LV chamber dilatation and dysfunction as well as higher blood pressures in aged mice. At day 3 after MI immediately before the peak of rupture occurrence, we observed significantly higher content of type I and III collagen, a greater density of macrophage and neutrophil, and markedly enhanced mRNA expression of inflammatory cytokines in the infarcted myocardium in old than in young mice. Furthermore, a more dramatic increment of matrix metalloproteinase (MMP)-9 activity was found in old than in young infarcted hearts, in keeping with enhanced inflammatory response. Collectively, these results revealed that old mice had a higher risk of post-MI cardiac rupture despite a higher level of collagen content and cross-linking. Enhanced inflammatory response and subsequent increase in MMP-9 activity together with higher blood pressure are important factors responsible for the higher risk of cardiac rupture and more severe LV remodeling in the aged heart following acute MI.     

Serum MMP-8: A Novel Indicator of Left Ventricular Remodeling and Cardiac Outcome in Patients after Acute Myocardial Infarction

Objective

Left ventricular (LV) remodeling following myocardial infarction (MI) is characterized by progressive alterations of structure and function, named LV remodeling. Although several risk factors such as infarct size have been identified, LV remodeling remains difficult to predict in clinical practice. Changes within the extracellular matrix, involving matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are an integral part of left ventricular (LV) remodeling after myocardial infarction (MI). We investigated the temporal profile of circulating MMPs and TIMPs and their relations with LV remodeling at 1 year and clinical outcome at 3 years in post-MI patients.

Methods

This prospective multicentre study included 246 patients with a first anterior MI. Serial echocardiographic studies were performed at hospital discharge, 3 months, and 1 year after MI, and analysed at a core laboratory. LV remodeling was defined as the percent change in LV end-diastolic volume (EDV) from baseline to 1 year. Serum samples were obtained at hospital discharge, 1, 3, and 12 months. Multiplex technology was used for analysis of MMP-1, -2, -3, -8, -9, -13, and TIMP-1, -2, -3, -4 serum levels.

Results

Baseline levels of MMP-8 and MMP-9 were positively associated with changes in LVEDV (P = 0.01 and 0.02, respectively). When adjusted for major baseline characteristics, MMP-8 levels remained an independent predictor LV remodeling (P = 0.025). By univariate analysis, there were positive relations between cardiovascular death or hospitalization for heart failure during the 3-year follow-up and the baseline levels of MMP-2 (P = 0.03), MMP-8 (P = 0.002), and MMP-9 (P = 0.03). By multivariate analysis, MMP-8 was the only MMP remaining significantly associated with clinical outcome (P = 0.02).

Conclusion

Baseline serum MMP-8 is a significant predictor of LV remodeling and cardiovascular outcome after MI and may help to improve risk stratification.     

SPARC mediates early extracellular matrix remodeling following myocardial infarction

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that functions in the extracellular processing of newly synthesized collagen. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in the early post-MI setting have not been defined, we examined age-matched wild-type (WT; n=22) and SPARC-deficient (null; n=25) mice at day 3 post-MI. Day 0 WT (n=28) and null (n=20) mice served as controls. Infarct size was 52 ± 2% for WT and 47 ± 2% for SPARC null (P=NS), indicating that the MI injury was comparable in the two groups. By echocardiography, WT mice increased end-diastolic volumes from 45 ± 2 to 83 ± 5 μl (P < 0.05). SPARC null mice also increased end-diastolic volumes but to a lesser extent than WT (39 ± 3 to 63 ± 5 μl; P < 0.05 vs. day 0 controls and vs. WT day 3 MI). Ejection fraction fell post-MI in WT mice from 57 ± 2 to 19 ± 1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65 ± 2 to 28 ± 2%). Fibroblasts isolated from SPARC null left ventricle (LV) showed differences in the expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including fibronectin, connective tissue growth factor (CTGF; CCN2), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-2 (TIMP-2). The change in fibroblast gene expression levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2. Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3 post-MI but may be detrimental for the long-term response due to impaired fibroblast activation.     

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.     

Lack of osteopontin improves cardiac function in streptozotocin-induced diabetic mice

The purpose of this study was to investigate the role of osteopontin (OPN) in diabetic hearts. Diabetes was induced in wild-type (WT) and OPN knockout (KO) mice by using streptozotocin (150 mg/kg) injection. Left ventricular (LV) structural and functional remodeling was studied 30 and 60 days after induction of diabetes. Induction of diabetes increased OPN expression in cardiac myocytes. Heart weight-to-body weight ratio was increased in both diabetic (D) groups. Lung wet weight-to-dry weight ratio was increased only in the WT-D group. Peak left ventricular (LV) developed pressures measured using Langendorff perfusion analyses were reduced to a greater extent in WT-D versus KO-D group. LV end-diastolic pressure-volume curve exhibited a significant leftward shift in WT-D but not in KO-D group. LV end-diastolic diameter, percent fractional shortening, and the ratio of peak velocity of early and late filling (E/A wave) were significantly reduced in WT-D mice as analyzed by echocardiography. The increase in cardiac myocyte apoptosis and fibrosis was significantly higher in the WT-D group. Expression of atrial natriuretic peptide and transforming growth factor-beta1 was significantly increased in the WT-D group. Induction of diabetes increased protein kinase C (PKC) phosphorylation in both groups. However, phosphorylation of PKC-betaII was significantly higher in the WT-D group, whereas phosphorylation of PKC-zeta was significantly higher in the KO-D group. Levels of peroxisome proliferator-activated receptor-gamma were significantly decreased in the WT-D group but not in the KO-D group. Thus increased expression of OPN may play a deleterious role during streptozotocin-induced diabetic cardiomyopathy with effects on cardiac fibrosis, hypertrophy, and myocyte apoptosis.     

Gender differences in cardiac function during early remodeling after acute myocardial infarction in mice

There are conflicting data about gender differences in cardiac function after myocardial infarction (MI), including cardiac rupture and mortality. Using a mouse model of MI, we recently found that the cardiac rupture rate during the first week after MI was significantly lower in females than in males, suggesting that females have attenuated structural remodeling. Thus in this study, we attempted to determine whether: a) females have attenuated remodeling and faster healing during the early phase post-MI, and b) females have better cardiac function and outcome during the chronic phase compared to males. MI was induced in 12-week-old male and female C57BL/6J mice. Signs of early remodeling, including cardiac rupture, infarct expansion, inflammatory response, and collagen deposition, were studied during the first 2 weeks post-MI. Left ventricular remodeling and function were followed for 12 weeks post-MI. We found that males had a higher rate of cardiac rupture, occurring mainly at 3 to 5 days of MI and associated with a higher infarct expansion index. Neutrophil infiltration at the infarct border was more pronounced in males than females during the first days of MI, which were also characterized by increased MMP activity. However, the number of infiltrating macrophages was significantly higher in females at day 4. During the chronic phase post-MI, males had significantly poorer LV function, more prominent dilatation and significant myocyte hypertrophy compared to females. In conclusion, males have delayed myocardial healing, resulting in cardiac rupture, and the survivors have poorer cardiac function and pronounced maladaptive remodeling, whereas females show a better outcome during the development of HF.     

The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

Background

Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47^phox -/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway.

Methods

Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.

Results

BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47^phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.

Conclusions

These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis.