Richard Schulz

Myocardial matrix metalloproteinase-2: inside out and upside down

Since their inaugural discovery in the early 1960s, matrix metalloproteinases (MMPs) have been shown to mediate multiple physiological and pathological processes. In addition to their canonical function in extracellular matrix (ECM) remodeling, research in the last decade has highlighted new MMP functions, including proteolysis of novel substrates beyond ECM proteins, MMP localization to subcellular organelles, and proteolysis of susceptible intracellular proteins in those subcellular compartments. This review will provide a comparison of the extracellular and intracellular roles of MMPs, illustrating that MMPs are far more interesting than the one-dimensional view originally taken. We focus on the roles of MMP-2 in cardiac injury and repair, as this is one of the most studied MMPs in the cardiovascular field. We will highlight how understanding all dimensions, such as localization of activity and timing of interventions, will increase the translational potential of research findings. Building upon old ideas and turning them inside out and upside down will help us to better understand how to move the MMP field forward.

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.

Activation of intracellular matrix metalloproteinase-2 by reactive oxygen–nitrogen species: Consequences and therapeutic strategies in the heart

Reactive oxygen-nitrogen species play important roles in physiological and pathological processes in the heart. This review will focus on the activation of matrix metalloproteinases (MMPs) as a result of oxidative stress, and the consequences of this on heart function. Although the MMPs are considered to be secreted proteases acting on the extracellular matrix to effect tissue remodeling, it is now recognized that MMPs also rapidly act on intracellular protein targets to cause intracellular protein remodeling. Of the 23 known human MMPs, MMP-2 is widely expressed in almost all cell types, is one of the most abundant MMPs in cardiac tissue, and recent evidence has revealed mechanisms by which it is a bona fide intracellular protein. This review will discuss the intracellular localization and novel substrates of MMP-2 within the heart, how intracellular protein proteolysis leads to cardiac dysfunction, as well as the potential of MMPs inhibitors as therapy for cardiovascular diseases caused by enhanced reactive oxygen-nitrogen species.

MMP-2 is localized to the mitochondria-associated membrane of the heart

Matrix metalloproteinase-2 (MMP-2) has been extensively studied in the context of extracellular matrix remodeling but is also localized within cells and can be activated by prooxidants to proteolyze specific intercellular targets. Although there are reports of MMP-2 in mitochondria, a critical source of cellular oxidative stress, these studies did not take into account the presence within their preparations of the mitochondria-associated membrane (MAM), a subdomain of the endoplasmic reticulum (ER). We hypothesized that MMP-2 is situated in the MAM and therefore investigated its subcellular distribution between mitochondria and the MAM. Immunogold electron microscopy revealed MMP-2 localized in mitochondria of heart sections from mice. In contrast, immunofluorescence analysis of an MMP-2:HaloTag fusion protein expressed in HL-1 cardiomyocytes showed an ER-like distribution, with greater colocalization with an ER marker (protein disulfide isomerase) relative to the mitochondrial marker, MitoTracker red. Although MMP-2 protein and enzymatic activity were present in crude mitochondrial fractions, once these were separated into purified mitochondria and MAM, MMP-2 was principally associated with the latter. Thus, although mitochondria may contain minimal levels of MMP-2, the majority of MMP-2 previously identified as mitochondrial is in fact associated with the MAM. We also found that calreticulin, an ER- and MAM-resident Ca(2+) handling protein and chaperone, could be proteolyzed by MMP-2 in vitro. MAM-localized MMP-2 could therefore potentially impact mitochondrial function by affecting ER-mitochondrial Ca(2+) signaling via its proteolysis of calreticulin.

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

Graphical abstract

Phosphorylation Status of 72 kDa MMP-2 Determines Its Structure and Activity in Response to Peroxynitrite

Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO−) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO− treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1–1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO−. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO− activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.

Remodeling of Aorta Extracellular Matrix as a Result of Transient High Oxygen Exposure in Newborn Rats: Implication for Arterial Rigidity and Hypertension Risk

Neonatal high-oxygen exposure leads to elevated blood pressure, microvascular rarefaction, vascular dysfunction and arterial (aorta) rigidity in adult rats. Whether structural changes are present in the matrix of aorta wall is unknown. Considering that elastin synthesis peaks in late fetal life in humans, and early postnatal life in rodents, we postulated that transient neonatal high-oxygen exposure can trigger premature vascular remodelling. Sprague Dawley rat pups were exposed from days 3 to 10 after birth to 80% oxygen (vs. room air control) and were studied at 4 weeks. Blood pressure and vasomotor response of the aorta to angiotensin II and to the acetylcholine analogue carbachol were not different between groups. Vascular superoxide anion production was similar between groups. There was no difference between groups in aortic cross sectional area, smooth muscle cell number or media/lumen ratio. In oxygen-exposed rats, aorta elastin/collagen content ratio was significantly decreased, the expression of elastinolytic cathepsin S was increased whereas collagenolytic cathepsin K was decreased. By immunofluorescence we observed an increase in MMP-2 and TIMP-1 staining in aortas of oxygen-exposed rats whereas TIMP-2 staining was reduced, indicating a shift in the balance towards degradation of the extra-cellular matrix and increased deposition of collagen. There was no significant difference in MMP-2 activity between groups as determined by gelatin zymography. Overall, these findings indicate that transient neonatal high oxygen exposure leads to vascular wall alterations (decreased elastin/collagen ratio and a shift in the balance towards increased deposition of collagen) which are associated with increased rigidity. Importantly, these changes are present prior to the elevation of blood pressure and vascular dysfunction in this model, and may therefore be contributory.

The Alberta Heart Failure Etiology and Analysis Research Team (HEART) study

BackgroundNationally, symptomatic heart failure affects 1.5-2% of Canadians, incurs

Doxycycline reduces cardiac matrix metalloproteinase-2 activity but does not ameliorate myocardial dysfunction during reperfusion in coronary artery bypass patients undergoing cardiopulmonary bypass.

3 billion in hospital costs annually and the global burden is expected to double in the next 1–2 decades. The current one-year mortality rate after diagnosis of heart failure remains high at >25%. Consequently, new therapeutic strategies need to be developed for this debilitating condition.Methods/DesignThe objective of the Alberta HEART program (http://albertaheartresearch.ca) is to develop novel diagnostic, therapeutic and prognostic approaches to patients with heart failure with preserved ejection fraction. We hypothesize that novel imaging techniques and biomarkers will aid in describing heart failure with preserved ejection fraction. Furthermore, the development of new diagnostic criteria will allow us to: 1) better define risk factors associated with heart failure with preserved ejection fraction; 2) elucidate clinical, cellular and molecular mechanisms involved with the development and progression of heart failure with preserved ejection fraction; 3) design and test new therapeutic strategies for patients with heart failure with preserved ejection fraction. Additionally, Alberta HEART provides training and education for enhancing translational medicine, knowledge translation and clinical practice in heart failure. This is a prospective observational cohort study of patients with, or at risk for, heart failure. Patients will have sequential testing including quality of life and clinical outcomes over 12 months. After that time, study participants will be passively followed via linkage to external administrative databases. Clinical outcomes of interest include death, hospitalization, emergency department visits, physician resource use and/or heart transplant. Patients will be followed for a total of 5 years.DiscussionAlberta HEART has the primary objective to define new diagnostic criteria for patients with heart failure with preserved ejection fraction. New criteria will allow for targeted therapies, diagnostic tests and further understanding of the patients, both at-risk for and with heart failure.Trial registrationClinicalTrials.gov NCT02052804.

Matrix metalloproteinase inhibition attenuates right ventricular dysfunction and improves responses to dobutamine during acute pulmonary thromboembolism

Objectives:Matrix metalloproteinase-2 proteolyzes intracellular proteins in the heart and induces acute myocardial contractile dysfunction in ischemia-reperfusion injury. Doxycycline, a matrix metalloproteinase inhibitor, prevented matrix metalloproteinase-2-induced troponin I cleavage in rat hearts and improved contractile function following ischemia-reperfusion. In patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass, increased atrial matrix metalloproteinase-2 activity was inversely correlated with cardiac mechanical function at 3 hours reperfusion. We performed a study in patients with coronary artery disease undergoing primary elective coronary artery bypass graft surgery with cardiopulmonary bypass to determine whether doxycycline reduces cardiac mechanical dysfunction, matrix metalloproteinase activity, and troponin I degradation after reperfusion. Design:Randomized, double-blinded, placebo-controlled study. Setting:University of Alberta Hospital. Patients:Forty-two patients with coronary artery disease undergoing coronary artery bypass graft surgery with cardiopulmonary bypass. Interventions:Patients were randomized to receive either oral administration of 20 mg of doxycycline or matching placebo pill twice a day at least 2 days prior to surgery, on the day of surgery, and for the first 3 postoperative days. Measurements and Main Results:Left ventricular stroke work index was examined prior to cardiopulmonary bypass and at 24 hours reperfusion. Right atrial biopsies were collected before cardiopulmonary bypass and 10 minutes after aortic cross-clamp release to determine matrix metalloproteinase-2 activity and troponin I level. Blood was collected to determine matrix metalloproteinase activity and interleukin-6, C-reactive protein, and troponin I levels. Cardiac 72-kDa matrix metalloproteinase-2 activity was lower upon reperfusion in biopsies from the doxycycline group (p = 0.01), and the increase of matrix metalloproteinase-2 activity in the placebo group due to reperfusion did not appear in the doxycycline group (p = 0.05). Doxycycline, however, did not ameliorate cardiac mechanical dysfunction following reperfusion or the cardiopulmonary bypass-coronary artery bypass graft-induced increased plasma matrix metalloproteinase-9, interleukin-6, and C-reactive protein levels. Cardiopulmonary bypass-coronary artery bypass graft or doxycycline did not change tissue or plasma troponin I levels at 10 minutes reperfusion. Conclusions:Although doxycycline did not improve myocardial stunning following coronary artery bypass graft surgery with cardiopulmonary bypass, it reduced cardiac matrix metalloproteinase-2 activity in these patients. A larger trial and/or higher dose of doxycycline may yet be warranted.