Abstract 362: Proteomic Analysis Identifies In vivo Matrix Metalloproteinase-9 Substrates in the Left Ventricle Post-Myocardial Infarction
Background: A key component of left ventricular (LV) remodeling following myocardial infarction (MI) is the accumulation of extracellular matrix (ECM) proteins, which can predict morbidity and mortality. ECM turnover is regulated by matrix metalloproteinases (MMPs), but global MMP inhibition strategies have not shown a benefit post-MI in humans. Of the 25 MMPs identified, MMP-9 is elevated post-MI, and MMP-9 gene deletion attenuates LV remodeling. Before MMP-9 specific strategies can be applied clinically, however, a more complete catalogue of MMP-9 substrates is needed to fully understand the net effect of MMP-9 activity in the post-MI LV.
Methods and Results: We used a proteomic strategy to detect known and novel MMP-9 substrates by comparing the total protein in the infarct region of wild-type (wt) and MMP-9 null (null) male mice 7 days post-MI. Both groups showed similar infarct sizes and LV-to-body weight ratios (4.4±0.1mg/g in wt and 4.0±0.1 mg/g in null), indicating equal injury. LV infarct tissue was separated from non-infarct tissue, homogenized, and analyzed by two-dimensional (2-D) gel electrophoresis and mass spectrometry. Of 31 spot intensity differences, the intensities of 9 spots increased and 22 spots decreased in null mice compared to wt (all p<0.05). Several ECM proteins were identified by mass spectrometry, including collagen types I and type III, fibronectin, fibrinogen, tenascin, thrombospondin-1 (tsp-1), and laminin. The following were observed at lower than expected molecular weights indicative of substrate cleavage and were decreased in null compared to wt: collagen type III (25%), fibronectin (19%), tenascin (13%), thrombospondin (25%) and laminin (21%). For 4 of the 5 proteins further analyzed by immunoblotting, reduced proteolysis was confirmed in the null samples. Additionally, we identified tsp-1 as a novel substrate for MMP-9.
Conclusions: This is the first time that proteomics has been used to demonstrate in vivo cleavage of MMP-9 substrates in the LV post-MI and the first report of LV ECM proteins being resolved on a 2-D gel. We conclude that this powerful strategy provides a unique method to detect novel MMP substrates.