Mohammad A.M. Ali

Titin is a target of matrix metalloproteinase-2: implications in myocardial ischemia/reperfusion injury.

Background— Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results— Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion— MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury. # Clinical Perspective {#article-title-54}Background— Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results— Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion— MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury.

Mechanisms of cytosolic targeting of matrix metalloproteinase-2

Matrix metalloproteinase‐2 (MMP‐2) is best understood for its biological actions outside the cell. However, MMP‐2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP‐2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP‐2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP‐2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP‐2 signal sequence that predominantly determines MMP‐2 targeting. However, we also found that human cardiomyocytes express an MMP‐2 splice variant which entirely lacks the signal sequence. Like the fraction of ER‐excluded, full‐length MMP‐2, this variant MMP‐2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia‐reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP‐2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP‐2. J. Cell. Physiol. 227: 3397–3404, 2012.

Cardiac Sarcomeric Proteins: Novel Intracellular Targets of Matrix Metalloproteinase-2 in Heart Disease

Matrix metalloproteinases (MMPs) have been almost exclusively thought to be secreted proteases (with the exception of the membrane-type MMPs) that exert diverse biological actions in health and disease via proteolyzing substrates outside the cell. However, recent evidence has demonstrated that the role of MMPs goes far beyond their proteolytic activity in the extracellular matrix. MMP-2 is arguably the most ubiquitous member of the 23 member MMP family and is expressed in all cells of the heart and vasculature. In the past 10 years, MMP-2 was shown to change the bioactivity of a growing list of specific, non-extracellular matrix proteins both outside and inside the cell. There is clear evidence of its intracellular localization to the cardiac sarcomere, nucleus, and mitochondria and that during early phases of oxidative stress injury to the heart, MMP-2 proteolyzes specific sarcomeric and cytoskeletal proteins to cause contractile dysfunction. In this review we discuss this novel intracellular biology of MMP-2 and the potential use of MMP inhibitors for the therapy of heart injury caused by oxidative stress.

Calpain inhibitors exhibit matrix metalloproteinase-2 inhibitory activity.

Matrix metalloproteinase (MMP)-2 is a zinc-dependent endopeptidase which, alongside its known extracellular actions, plays fundamental roles in oxidative stress-induced injury to the heart. Intracellular cleavage targets of MMP-2 selectively mediating this injury include the sarcomeric proteins troponin I, myosin light chain-1 and titin; some of these are also targeted by calpains. In myocardial ischemia and reperfusion injury, inhibitors of MMP-2 and some calpain inhibitors were shown to improve the recovery of contractile function. We hypothesized that the protective effects of calpain inhibitors may be due in part to their ability to inhibit MMP-2. Four calpain inhibitors (calpain inhibitor III, ALLM, ALLN, and PD-150606) were tested for their ability to inhibit MMP-2 in comparison to the selective MMP inhibitor ONO-4817. At 100 μM, all calpain inhibitors, except ALLM, showed significant inhibition of MMP-2 gelatinolytic activity. When assessed by the troponin I proteolysis assay, both ALLN and PD-150606, but neither ALLM nor calpain inhibitor III (at 20 μM), significantly inhibited MMP-2 activity. Using a fluorogenic MMP substrate peptide OmniMMP in a kinetic assay the rank order of IC(50) values against MMP-2 were: PD-150606<ALLN<calpain inhibitor III <<< ALLM. These experiments show that the calpain inhibitors PD-150606 and ALLN have significant additional pharmacological activity as MMP-2 inhibitors. This suggests that the protective effect of some calpain inhibitors is due in part to their ability to inhibit MMP activity.

Hydrogen peroxide-induced necrotic cell death in cardiomyocytes is independent of matrix metalloproteinase-2

Matrix metalloproteinase-2 (MMP-2) is well known to proteolyse both extracellular and intracellular proteins. Reactive oxygen species activate MMP-2 at both transcriptional and post-translational levels, thus MMP-2 activation is considered an early event in oxidative stress injury. Although hydrogen peroxide is widely used to trigger oxidative stress-induced cell death, the type of cell death (apoptosis vs. necrosis) in cardiomyocytes is still controversial depending on the concentration used and the exposure time. We carefully investigated the mode of cell death in neonatal rat cardiomyocytes induced by different concentrations (50-500 μM) of hydrogen peroxide at various time intervals after exposure and determined whether MMP-2 is implicated in hydrogen peroxide-induced cardiomyocyte death. Treating cardiomyocytes with hydrogen peroxide led to elevated MMP-2 level/activity with maximal effects seen at 200 μM. Hydrogen peroxide caused necrotic cell death by disrupting the plasmalemma as evidenced by the release of lactate dehydrogenase in a concentration- and time-dependent manner as well as the necrotic cleavage of PARP-1. The absence of both caspase-3 cleavage/activation and apoptotic cleavage of PARP-1 illustrated the weak contribution of apoptosis. Pre-treatment with selective MMP inhibitors did not protect against hydrogen peroxide-induced necrosis. In conclusion hydrogen peroxide increases MMP-2 level/activity in cardiomyocytes and induces necrotic cell death, however, the later effect is MMP-2 independent.

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.

Inhibitory effects of caspase inhibitors on the activity of matrix metalloproteinase-2.

Matrix metalloproteinase (MMP)-2, a zinc-dependent endopeptidase, plays a detrimental role in several diseases including ischemia and reperfusion (I/R) injury of the heart. Caspases are a group of cysteine-dependent, aspartate-directed proteases which regulate cellular apoptosis. Interestingly, protective effects of caspase inhibitors independent of apoptosis have been shown in I/R injury of the heart. The cardioprotective actions of both these classes of protease inhibitors led us to hypothesize that caspase inhibitors may also reduce MMP-2 activity. Five known caspase inhibitors (Z-IE(OMe)TD(OMe)-fmk, Ac-DEVD-CHO, Ac-LEHD-cmk, Z-VAD-fmk and Ac-YVAD-cmk) were tested for their possible inhibitory effects on MMP-2 activity in comparison to the MMP inhibitors ONO-4817 and ARP-100 (which themselves were unable to inhibit caspase-3 activity). MMP-2 activity was assessed by an in vitro troponin I (TnI) proteolysis assay and a quantitative kinetic fluorescence assay using a fluorogenic peptide substrate (OmniMMP). TnI proteolysis was also measured by western blot in neonatal cardiomyocytes subjected to hypoxia-reoxygenation injury. Using human recombinant MMP-2 and TnI as its substrate, the caspase inhibitors, in comparison with ONO-4817, significantly inhibited MMP-2-mediated TnI degradation in a concentration-dependent manner. The kinetic assay using OmniMMP revealed that these caspase inhibitors blocked MMP-2 activity in a concentration-dependent manner with similar IC50 values. TnI degradation in neonatal cardiomyocytes was enhanced following hypoxia-reoxygenation and this was blocked by ARP-100 and Ac-LEHD-cmk. Inhibition of MMP-2 activity is an additional pharmacological action which contributes to the protective effects of some caspase inhibitors.

Titin is a Target of Matrix Metalloproteinase-2

Background— Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results— Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion— MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury. # Clinical Perspective {#article-title-54}Background— Titin is the largest mammalian (≈3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results— Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion— MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury.

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.

Postresuscitation administration of doxycycline preserves cardiac contractile function in hypoxia-reoxygenation injury of newborn piglets*.

Objective:Cardiac injury is common in asphyxiated neonates and is associated with matrix metalloproteinase-2 activation. Although studies have demonstrated the cardioprotective effects of matrix metalloproteinase inhibition, this has not been tested in clinically translatable models of hypoxia-reoxygenation injury. We aimed to elucidate the effect of doxycycline, a matrix metalloproteinase inhibitor, on cardiac injury and functional recovery in a swine model of neonatal hypoxia-reoxygenation. Design:Thirty-three newborn piglets were acutely instrumented for continuous monitoring of cardiac output and systemic arterial pressure. After stabilization, normocapnic alveolar hypoxia (10–15% oxygen) was instituted for 2 hours followed by 4 hours of normoxic reoxygenation. Piglets were blindly, block randomized to receive IV boluses of normal saline (control) and doxycycline at 5 minutes of reoxygenation (n = 7/group). Sham-operated piglets (n = 5) received no hypoxia-reoxygenation. Markers of myocardial injury (plasma and myocardial tissue troponin I; myocardial lactate) and oxidative stress (lipid hydroperoxides) were measured by enzyme-linked immunosorbent assay and Western blot. Myocardial matrix metalloproteinase-2 activity was quantified by gelatin zymography and immunoprecipitation. Setting:University animal laboratory. Subjects:Piglets (1–4 d old, weighing 1.4–2.5 kg). Interventions:IV doxycycline (3, 10, or 30 mg/kg) given during resuscitation. Measurements and Main Results:Hypoxic piglets had cardiogenic shock (cardiac output 58% ± 1% of baseline), hypotension (systemic arterial pressure 31 ± 1 mm Hg), and acidosis (pH 7.02 ± 0.02). Doxycycline improved cardiac and stroke volume index with no chronotropic effect in doxycycline-treated piglets compared with controls. Systemic arterial pressure was higher and the pulmonary artery pressure/systemic arterial pressure ratio was lower in doxycycline groups, with reduced levels of markers of myocardial injury and oxidative stress in doxycycline-treated piglets compared with controls. Negative correlations were found between markers of myocardial injury (plasma troponin I, myocardial lactate) and functional recovery and between myocardial tissue and plasma troponin I. Doxycycline-treated piglets had lower myocardial matrix metalloproteinase-2 activity compared with controls. Conclusions:Postresuscitation administration of doxycycline attenuates cardiac injury and improves functional recovery in newborn piglets with hypoxia-reoxygenation.