Bryan G. Hughes

Targeting MMP-2 to treat ischemic heart injury

Matrix metalloproteinase (MMPs) are long understood to be involved in remodeling of the extracellular matrix. However, over the past decade, it has become clear that one of the most ubiquitous MMPs, MMP-2, has numerous intracellular targets in cardiac myocytes. Notably, MMP-2 proteolyzes components of the sarcomere, and its intracellular activity contributes to ischemia–reperfusion injury of the heart. Together with the well documented role played by MMPs in the myocardial remodeling that occurs following myocardial infarction, this has led to great interest in targeting MMPs to treat cardiac ischemic injury. In this review we will describe the expanding understanding of intracellular MMP-2 biology, and how this knowledge may lead to improved treatments for ischemic heart injury. We also critically review the numerous preclinical studies investigating the effects of MMP inhibition in animal models of myocardial infarction and ischemia–reperfusion injury, as well as the recent clinical trials that are part of the effort to translate these results into clinical practice. Acknowledging the disappointing results of past clinical trials of MMP inhibitors for other diseases, we discuss the need for carefully designed preclinical and clinical studies to avoid mistakes that have been previously made. We conclude that inhibition of MMPs, and in particular MMP-2, shows promise as a therapy to prevent the progression from ischemic injury to heart failure. However, it is critical that the full breadth of MMP-2 biology be taken into account as such therapies are developed.

Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

Graphical abstract

Nuclear matrix metalloproteinase-2 in the cardiomyocyte and the ischemic-reperfused heart

Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in intra- and extra-cellular matrix remodeling resulting from oxidative stress injury to the heart. MMP-2 was the first MMP to be localized to the nucleus; however, its biological functions there are unclear. We hypothesized that MMP-2 is present in the nucleus under normal physiological conditions but increases during myocardial ischemia-reperfusion (I/R) injury-induced oxidative stress, proteolyzing nuclear structural proteins. Lamins are intermediate filament proteins that provide structural support to the nucleus and are putative targets of MMP-2. To identify lamin susceptibility to MMP-2 proteolysis, purified lamin A or B was incubated with MMP-2 in vitro. Lamin A, but not lamin B, was proteolysed by MMP-2 into an approximately 50kDa fragment, which was also predicted by in silico cleavage site analysis. Immunofluorescent confocal microscopy and subcellular fractionation showed MMP-2 both in the cytosol and nuclei of neonatal rat ventricular myocytes. Rat hearts were isolated and perfused by the Langendorff method aerobically, or subjected to I/R injury in the presence or absence of o-phenanthroline, an MMP inhibitor. Nuclear fractions extracted from I/R hearts showed increased MMP-2 activity, but not protein level. The level of troponin I, a known sarcomeric target of MMP-2, was rescued in I/R hearts treated with o-phenanthroline, demonstrating the efficacy of MMP inhibition. However, lamin A or B levels remained unchanged in I/R hearts. MMP-2 has a widespread subcellular distribution in cardiomyocytes, including a significant presence in the nucleus. The increase in nuclear MMP-2 activity seen during stunning injury here, indicates yet unknown biological actions, other than lamin proteolysis, which may require more severe ischemia to effect.

Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis.

Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.

Matrix metalloproteinase-2 in oncostatin M-induced sarcomere degeneration in cardiomyocytes

Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration.

Doxorubicin induces de novo expression of N-terminal truncated MMP-2 in cardiac myocytes

Anthracyclines, such as doxorubicin, are commonly prescribed antineoplastic agents that cause irreversible cardiac injury. Doxorubicin cardiotoxicity is initiated by increased oxidative stress in cardiomyocytes. Oxidative stress enhances intracellular matrix metalloproteinase-2 (MMP-2) by direct activation of its full-length isoform and (or) de novo expression of an N-terminal-truncated isoform (NTT-MMP-2). As MMP-2 is localized to the sarcomere, we tested whether doxorubicin activates intracellular MMP-2 in neonatal rat ventricular myocytes (NRVM) and whether it thereby proteolyzes two of its identified sarcomeric targets, α-actinin and troponin I. Doxorubicin increased oxidative stress within 12 h as indicated by reduced aconitase activity. This was associated with a twofold increase in MMP-2 protein levels and threefold higher gelatinolytic activity. MMP inhibitors ARP-100 or ONO-4817 (1 μM) prevented doxorubicin-induced MMP-2 activation. Doxorubicin also increased the levels and activity of MMP-2 secreted into the conditioned media. Doxorubicin upregulated the mRNA expression of both full-length MMP-2 and NTT-MMP-2. α-Actinin levels remained unchanged, whereas doxorubicin downregulated troponin I in an MMP-independent manner. Doxorubicin induces oxidative stress and stimulates a robust increase in MMP-2 expression and activity in NRVM, including NTT-MMP-2. The sarcomeric proteins α-actinin and troponin I are, however, not targeted by MMP-2 under these conditions.

Intracellular Matrix Remodeling and Cardiac Function in Ischemia–Reperfusion Injury

The effects of ischemia on animal and human myocardium have been extensively studied during the last four decades. Myocardial ischemia followed by subsequent reperfusion can cause profound damage to cardiac myocytes through enhanced oxidative stress and intracellular Ca2+ overload. Ischemia–reperfusion injury leads to structural and functional remodeling of multiple intracellular matrix components in the cardiac myocyte. The intracellular matrix of cardiac myocytes includes major cytosolic components that include the cytoskeleton, contractile myofibrils, and subcellular organelles such as mitochondria, sarcoplasmic reticulum, and the nucleus. There are several proteolytic pathways inside the cell which may participate in cell injury and/or cell repair upon reperfusion of ischemic heart muscle. These include matrix metalloproteinases, calpains, lysosomal proteases, and the proteasome system, which are a major focus of research in ischemia–reperfusion injury. The discovery of intramyocyte matrix metalloproteinase-2 (MMP-2) and biologically relevant protein substrates of it in the intracellular matrix has shaped a new paradigm of the pathophysiological role of MMP-2 during myocardial ischemia–reperfusion injury. Emerging evidence indicates that oxidative stress can efficiently activate intracellular MMP-2 which rapidly mediates intracellular matrix remodeling of injured myocytes. This chapter will focus on the structural and functional remodeling of the intracellular matrix including the sarcomere, cytoskeleton, mitochondria, and nucleus, by proteolytic and other processes, in the context of ischemia–reperfusion (I/R) injury, with a particular emphasis on the rapidly expanding knowledge of the role of intracellular MMP-2.