Novel intracellular role of matrix metalloproteinase-2 in cardiac cell injury

Abstract

Despite originally being described as a secreted protease, matrix metalloproteinase‐2 (MMP‐2) was recently revealed to have targets within cardiomyocytes. The biological mechanism(s) that describes the intracellular localization of MMP‐2 is unknown and is thus the subject of this thesis. Additionally, activation and inhibition of MMP-2 as well as novel intracellular target(s) of it that are involved in cardiomyocyte injury were investigated. The cytosolic targeting of MMP-2 was examined and it was found that the signal sequence of MMP-2 led to its incomplete targeting to the endoplasmic reticulum for secretion. Moreover, an MMP-2 splice variant which lacks the signal sequence and is enriched in the cytosol was discovered. Thus, intracellular MMP-2 is explained by the expression of a splice variant and by the inefficient targeting of canonical MMP-2 to the secretory pathway. Intracellular MMP-2 was found to play a pathological role in myocardial ischemia/reperfusion injury by proteolyzing the giant sarcomeric protein, titin. Discrete co-localization between MMP-2 and titin was found at the sarcomeric Z-disc region. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin, whereas inhibition of MMP-2 prevented titin proteolysis and improved the contractile function. To explore whether oxidative stress affects intracellular MMP-2 activity, isolated cardiomyocytes were treated with various concentrations of hydrogen peroxide. Treatment with 200 μM hydrogen peroxide led to elevated MMP-2 level/activity in cardiomyocyte lysates. Hydrogen peroxide primarily caused necrotic and not apoptotic cell death, however, pretreatment with selective MMP inhibitors did not protect against necrosis. In myocardial ischemia/reperfusion injury, MMP-2 or calpain inhibitors were shown to improve the myocardial contractile function. In order to investigate whether calpain inhibitors target intracellular MMP-2, the inhibitory effect of some calpain inhibitors on MMP-2 activity was tested. The IC50 values of calpain inhibitors, PD- 150606 and ALLN, against MMP-2 were determined to be 9.3 and 21.9 μM, respectively, revealing that some calpain inhibitors have significant pharmacological activity as inhibitors of MMP-2. In summary, these studies describe a set of mechanisms that cells utilize to equilibrate MMP-2 in both extracellular and intracellular locations. These results suggest that MMP-2 inhibitors should be rigorously tested as a therapeutic strategy to alleviate myocardial ischemia/reperfusion injury.