Abstract 547: A Unique Protein Kinase C Phosphorylation Site on Membrane Type-1 Matrix Metalloproteinase is Increased with Ischemia and Reperfusion and is Associated with Increased Activation and Trafficking
Background: Matrix metalloproteinases (MMPs), in particular membrane type-1 MMP (MT1-MMP), have been implicated in left ventricular (LV) remodeling with ischemia and reperfusion (IR). MT1-MMP induction occurs in heart failure, but signaling mechanisms for this upregulation remain unknown. In silico mapping identified a likely protein kinase C (PKC) phosphorylation site on Thr567 located within the cytoplasmic domain of MT1-MMP. This study tested the hypothesis that MT1-MMP is phosphorylated by PKC and that this phosphorylation contributes to its trafficking pattern during IR.
Methods and Results: Pigs (30 kg, n=6) underwent IR (90 minutes of circumflex occlusion and 120 minutes of R) and where interstitial in vivo MT1-MMP activity (microdialysis/fluorescence) increased from baseline by 122±10% (p<0.05). LV sections from the IR region was subjected to PKC isoform immunoblotting post IR and in controls (n=6). The PKC isoform beta II increased by 198±31% from control (p<0.05). Next, full length MT1-MMP was extracted from IR and control LV sections by immunoprecipitation followed by quantitative immunoblotting for threonine phosphorylation of MT1-MMP. MT1-MMP phosphothreonine levels increased by 121±8% in the IR myocardium indicative of PKC mediated phosphorylation (p<0.05). In the next set of studies, IR was simulated in isolated LV myocyte preparations (hypoxia/reoxygenation, n=3) and MT1-MMP was immuno-localized by confocal microscopy. With simulated IR there was an increased sarcolemmal MT1-MMP density indicative of increased trafficking to the membrane.
Conclusions: The unique findings of this study are 2-fold:
MT1-MMP activity is increased within the interstitium in vivo and is associated with a specific PKC isoform induction and site specific phosphorylation;
IR induced MT1-MMP trafficking within the LV myocyte is likely due to a PKC mediated event.
Thus, these results uncovered a novel pathway for regulation of MT1-MMP in the context of LV ischemia and reperfusion and identified new pharmacologic targets.