Novel Roles of Matrix Metalloproteinase-2 in Mitochondrial and Endoplasmic/Sarcoplasmic Reticulum Dysregulation
Creators: Farag, Wesam M E B
Publisher: name: University of Alberta Library
Year: 2024

<p>Matrix metalloproteinase-2 (MMP-2) is a ubiquitous multifunctional protease that cleaves both extracellular and intracellular proteins. MMP-2 is synthesized as an inactive zymogen. It is rapidly activated inside the cell in response to oxidative stress during different cardiac pathologies such as myocardial ischemia-reperfusion (IR) injury as well as doxorubicin (DXR)-induced cardiotoxicity. This occurs by the action of peroxynitrite-induced S-glutathiolation which exposes its catalytic site. Upon activation in the heart, MMP-2 induces the cleavage of several cardiac sarcomeric proteins resulting in cardiac contractile dysfunction. An important isoform of MMP-2, known as N-terminal truncated MMP-2 (MMP-2NTT76), is constitutively active and gets expressed in cardiomyocytes in response to oxidative stress. MMP-2 is localized to several intracellular compartments including the cytosol, sarcomere, cytoskeleton, nuclei, mitochondria and mitochondrial-endoplasmic reticulum (ER)-associated membrane (MAM), while MMP-2NTT76 is localized to the intermembranous space of mitochondria. However, how MMP-2 affects mitochondrial and ER functions is not well investigated. <br>
Mitochondria and ER play significant roles in the regulation of several biological processes. A specialized form of the ER, known as the sarcoplasmic reticulum (SR), found only in striated muscle, interacts with the T-tubules to regulate intracellular calcium level. As MMP-2 is localized at the MAM, I hypothesize that MMP-2, activated in response to oxidative stress, disrupts mitochondrial function, ER homeostasis and the T-tubule-SR interaction, by proteolyzing specific mitochondrial and/or ER/SR proteins localized at or near the MAM, leading to the development of cardiac pathologies. <br>
A fundamental role of mitochondria during cellular injury is the proteolytic processing of apoptosis-inducing factor (AIF), a mitochondrial flavoprotein that mediates staurosporine (STS)-induced cell death. I investigated whether MMP-2 activity is affected in response to STS and if it mediates AIF cleavage in human fibrosarcoma HT1080 cells. I found a significant increase in MMP-2 activity in response to STS treatment, prior to the induction of cell necrosis. However, mitochondrial AIF cleavage is independent of the proteolytic activity of MMP-2. <br>
Interestingly, enhanced oxidative stress which occurs during myocardial IR injury also impairs mitochondrial function which is regulated by different proteins including mitofusin-2 (Mfn-2). Oxidative stress and mitochondrial dysfunction also trigger the NLRP3 inflammasome response. In isolated hearts from mice subjected to IR injury, I found that increased MMP-2 activity and MMP-2NTT76 mRNA expression are associated with a reduction in post-ischemic recovery of cardiac contractile function, the loss in mitochondrial Mfn-2 level, enhanced inflammasome response and increased infarct size, compared to aerobically perfused hearts. All these changes were attenuated by MMP-2 inhibition. An association between MMP-2 and Mfn-2 was observed in aerobic and IR hearts. IR also impaired the rate of mitochondrial oxygen consumption in muscle fibers freshly isolated from mouse hearts, whereas MMP-2 inhibitors attenuated this reduction. <br>
Upon investigating how MMP-2 activation affects ER homeostasis using the same model of IR injury, I found that MMP-2 is not involved in the induction of the ER stress response itself. However, it affects the unfolded protein response (UPR) by targeting and cleaving the UPR sensor, inositol-requiring enzyme 1α (IRE1α). MMP-2 inhibitors are able to preserve IRE1α levels and protect against the progression of ER stress-mediated myocardial cell death. An association between MMP-2 and IRE1α was also observed in aerobic and IR hearts. Finally, the Schulz lab previously showed that junctophilin-2 (JPH-2), a membrane protein that tethers T-tubules to the SR, is proteolyzed by MMP-2 during IR injury. I investigated the involvement of MMP-2-mediated JPH-2 proteolysis in DXR-induced cardiotoxicity. I found that myocytes in hearts from DXR-treated mice had abnormally swollen T-tubules and mitochondria, accompanied by JPH-2 cleavage. These changes were also attenuated by MMP inhibitors. Loss in JPH-2 and impaired calcium transients were also observed in response to DXR in neonatal rat ventricular cardiomyocytes, while MMP-2 inhibition attenuated these changes. <br>
In conclusion, MMP-2, activated in response to oxidative stress, plays a fundamental role in MAM dysregulation by affecting different downstream pathways that are essential for cardiac function including mitochondrial and ER homeostasis and T-tubule-SR interaction. Potential MMP-2 proteolytic targets whose loss could mediate these pathophysiological changes include Mfn-2, IRE1α and JPH-2. Therefore, MMP-2 inhibition could be a promising adjunct therapy that can be used to protect against cardiac dysfunction in patients with ischemic heart disease or receiving anthracycline treatment for cancer. </p>