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Novel Roles of Matrix Metalloproteinase-2 in Mitochondrial and Endoplasmic/Sarcoplasmic Reticulum Dysregulation
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- Author / Creator
- Farag, Wesam M E B
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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.
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.
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.
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.
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.
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. -
- Graduation date
- Fall 2024
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.