Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: LV myocardial extracts from WT, MT1-MMPexp, and MT1-MMP+/− mice with no MI and at 14 days post-MI (n = 5/group) were used to measure a common Smad, Smad-3 in the classical TGF signaling pathway, by immunoblotting. Both total Smad-3 and the phosphorylated form of Smad-3 (pSmad-3) were quantified, and the ratio was computed. The ratio of phospho-to-total Smad-3 was increased to the greatest degree in the MT1-MMP post-MI group and was reduced substantially in the MT1-MMP+/− group. P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: A: post-myocardial infarction (MI) 14-day survival curves for wild-type (WT) mice, cardiac-restricted overexpression of membrane type 1 matrix metalloproteinase [MT1-MMP (MT1-MMPexp)] mice, and mice heterozygous for MT1-MMP (MT1-MMP+/−). Post-MI survival was significantly lower in the MT1-MMPexp mice following MI compared with WT (P = 0.045). In marked contrast, post-MI survival was higher in the MT1-MMP+/− group compared with WT (P = 0.019) or MT1-MMPexp (P = 0.001). B: representative left ventricular (LV) long-axis echocardiographic views at end diastole before MI induction and at 14 days post-MI for the 3 groups. Significant LV dilation and posterior wall thinning at the site of the MI were readily evident in all groups (scale = 2 mm). C: changes in LV geometry as defined by end-diastolic volume (EDV) were computed on an individual basis from pre-MI values as was LV function as defined as ejection fraction. In the WT and MT1-MMPexp groups (n = 53 and 32, respectively), a significant and equivalent degree of LV dilation occurred, which was attenuated in the MT1-MMP+/− group (n = 18). LV ejection fraction fell in all groups post-MI but was lower in the MT1-MMPexp group and higher in the MT1-MMP+/− group. P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: Over Expression

Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: A: representative in vivo localization of MT1-MMP (40 MHz, 30 μm resolution) in a WT mouse without MI and in a WT mouse at 14 days post-MI. Images were obtained immediately following injection (First Pass) and at 15 min following tail vein injection of MT1-MMP antibody-conjugated echodense microbubbles (3 μm diameter, 3.6 × 107 microbubbles). Contrast retention at 15 min (green) was quantified. Schematics for orientation of these contrast echocardiograms are shown on the far right (scale = 2 mm). B: MT1-MMP-targeted contrast microbubble binding was quantified as contrast intensity within a region of interest (ROI) within the viable myocardium remote from the MI zone. Results were normalized by dividing the total intensity measured within each ROI by the area of the ROI. Values were computed for each non-MI group (n = 3/group) and post-MI groups (n = 5/group). In the viable myocardial region, microbubble retention was higher than respective non-MI values in all MI groups but was highest in the MT1-MMPexp group and lowest in the MT1-MMP+/− group. C: LV sections taken from the MI region and the remote region (septal wall) were stained with picro-sirius red and imaged under polarized light in WT mice (n = 5), MT1-MMPexp mice (n = 6), and MT1-MMP+/− mice (n = 4). As expected, increased collagen accumulation occurred within the MI region in all groups. However, increased collagen staining was observed in the MT1-MMPexp mice in both the remote and MI regions and appeared reduced in the MT1-MMP+/− mice. A minimum of 6 random fields from each region for each mouse was digitized and analyzed and compared with referent non-MI WT values (n = 5). This analysis is summarized in results (scale = 100 μm). P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: In Vivo, Injection, Binding Assay, Staining

Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: Top: LV myocardial extracts from WT, MT1-MMPexp, and MT1-MMP+/− mice with no MI and at 14 days post-MI (n = 8/group) were used to measure MT1-MMP-specific proteolysis using a LTBP-1 fluorogenic substrate. In the LV myocardial extracts, significantly higher MT1-MMP-mediated LTBP-1 proteolytic activity was obtained in the post-MI samples, necessitating the values being plotted using 2 different scales. Bottom: with the use of the MT1-MMP catalytic domain-LTBP-1 fluorescent standard curve, MT1-MMP-mediated LTBP-1 proteolytic activity was computed and was increased following MI in all groups. However, LTBP-1 proteolytic activity was highest in the MT1-MMPexp groups compared with WT and the lowest in the MT1-MMP+/− groups. P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: Activity Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: Top: LV myocardial extracts from WT, MT1-MMPexp, and MT1-MMP+/− mice with no MI and at 14 days post-MI (n = 8/group) were used to directly measure MT1-MMP catalytic activity using a calibrated and validated fluorogenic substrate. The reactions were monitored continuously for specific fluorescence emission, reflective of MT1-MMP proteolytic activity for up to 20 h. Significantly greater MT1-MMP activity occurred in the post-MI vs. non-MI samples; therefore, the fluorescence values are plotted using different scales. Bottom: time-averaged fluorescence (10–20 h) for each sample and actual MT1-MMP activity computed. At 14 days post-MI, LV myocardial MT1-MMP activity increased from respective non-MI values in all groups. However, MT-MMP activity was the highest in the MT1-MMPexp groups with and without an MI, whereas MT1-MMP activity was lowest in the MT1-MMP+/− groups. P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: Activity Assay, Fluorescence

Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: LV myocardial extracts from WT, MT1-MMPexp, and MT1-MMP+/− mice with no MI and at 14 days post-MI (n = 8/group) were subjected to gelatin zymography, which provides a measure of relative MMP-9 and MMP-2 levels. Relative MMP-9 levels were increased equivalently in the post-MI groups (data not shown). The molecular weight fractionation of the MMP-2 zymographic bands revealed a pronounced increase in the active form of MMP-2 (64 kDa) in the MT1-MMPexp groups, which was further increased post-MI. The active form of MMP-2 was reduced in the MT1-MMP+/− group post-MI. P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: Zymography, Molecular Weight, Fractionation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

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

doi: 10.1152/ajpheart.00141.2011

Figure Lengend Snippet: The relative mRNA levels for the endogenous mouse MT1-MMP, human MT1-MMP, and the mouse latency-associated transforming growth factor-binding protein-1 (LTBP-1) were computed using real-time PCR (rtPCR) from myocardial RNA extracted from WT, MT1-MMPexp, and MT1-MMP+/− mice with no MI and at 14 days post-MI (n = 6/group). The levels were normalized to 18S mRNA, and the degree of change was computed from the relative cycle time (Ct) values obtained for the mRNA of interest and 18S mRNA. In the post-MI WT group, a significant increase in the endogenous mouse MT1-MMP mRNA levels occurred, whereas these levels were reduced in both the MT1-MMPexp and MT1-MMP+/− post-MI groups. In the MT1-MMP+/− group, relative levels were reduced from WT values with no MI as well (P < 0.05). As expected, a robust signal for human MT1-MMP mRNA was obtained in both MT1-MMPexp groups and not detected (ND) in the WT or MT1-MMP+/− groups. LTBP-1 mRNA levels were reduced in both MT1-MMPexp groups compared with WT values. In contrast, LTBP-1 mRNA levels were increased in the MT1-MMP+/− group. P < 0.05 vs. pre-MI values (#), vs. WT MI values (*), and vs. MT1-MMPexp MI values (+).

Article Snippet: Increasing concentrations of a recombinant active MT1-MMP construct (MT1-MMP Catalytic Domain, catalog no. 475935, 8–125 ng/m; Calbiochem) with a known catalytic activity resulted in a linear relationship with respect to fluorescence emission ( y = 343 x , r 2 = 0.98, P = 0.001).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction