Titin is a Target of Matrix Metalloproteinase-2

Molecular Cardiology Titin is a Target of Matrix Metalloproteinase-2 Implications in Myocardial Ischemia/Reperfusion Injury Mohammad A.M. Ali, MSc; Woo Jung Cho, MSc; Bryan Hudson, PhD; Zamaneh Kassiri, PhD; Henk Granzier, PhD; Richard Schulz, PhD 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. (Circulation. 2010;122:2039-2047.) Key Words: contractile dysfunction 䡲 ischemia 䡲 matrix metalloproteinase-2 䡲 sarcomere 䡲 titin Downloaded from http://ahajournals.org by on May 29, 2020 M atrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease that is best known for its ability to degrade the extracellular matrix in both physiological and pathological conditions. MMP-2 is synthesized as a zymogen by a variety of cells, including cardiac myocytes, and is activated either by proteases1 (such as by action of MMP-14) or by posttranslational modifications to the full-length enzyme caused by enhanced oxidative stress. For example, peroxynitrite, which is generated in early reperfusion after ischemia,2 directly activates several MMPs,3 including MMP-2,4 via a nonproteolytic mechanism involving the S-glutathiolation of a critical propeptide cysteine in its autoinhibitory domain. ogenesis, wound healing,5 atherosclerosis,6 aortic aneurysm,7 and myocardial infarction.8 More recent studies, however, show that MMP-2 is involved in several acute biological processes independently of its actions on extracellular matrix proteins. This includes platelet activation,9 regulation of vascular tone,10 and myocardial stunning injury immediately after reperfusion of the ischemic heart.11 Indeed, several reports indicate that MMP-2 does not exclusively degrade extracellular matrix components.12,13 In normal cardiac myocytes, MMP-2 is found in discrete subcellular compartments, including the thin and thick myofilaments of the cardiac sarcomere,14,15 cytoskeleton,16,17 nuclei,18 mitochondria,14 and caveolae19 (see Schulz20). MMP-2 is activated in rat hearts subjected to myocardial oxidative stress injury and is responsible for the degradation of specific sarcomeric and cytoskeletal proteins, including troponin I,14,21 myosin light chain-1,15 and ␣-actinin.17 Inhibition of MMP activity reduced both the loss of contractile Ediotrial see p 2002 Clinical Perspective on p 2047 MMPs are best recognized for their role in tissue remodeling by proteolyzing various components of the extracellular matrix in both health and disease, ie, in angiogenesis, embry- Received December 8, 2009; accepted August 23, 2010. From the Departments of Pharmacology (M.A.M.A., R.S.), Cell Biology (W.J.C.), Physiology (Z.K.), and Pediatrics (R.S.), Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada, and Department of Physiology, University of Arizona, Tucson (B.H., H.G.). The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.930222/DC1. Correspondence to Richard Schulz, Cardiovascular Research Centre, University of Alberta, 4 – 62 HMRC, Edmonton, AB T6G 2S2, Canada. E-mail [email protected] © 2010 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.109.930222 2039 2040 Circulation November 16, 2010 Figure 1. Colocalization of MMP-2 and titin at the Z-disk region of the left ventricular cardiac sarcomere in rat hearts aerobically perfused for 10 minutes (longitudinal sections). MMP-2 shows better colocalization with T12 than the M8 epitope of titin in the sarcomere of the left ventricular myocardium. MMP-2immunoreactivity reveals at Z lines with high density and M lines with low density. T12 epitope reveals at only Z lines, and M8 epitope reveals at only M-lines. A through C, High density of MMP-2 (green) colocalizes (yellow) with T12 epitope (red) at the Z lines. D through F, Low density of MMP-2 (red) colocalizes (yellow) with M8 epitope (green) at M lines. Scale bar⫽5 ␮m for all images except the enlarged portion of C illustrating the Z and M lines. Downloaded from http://ahajournals.org by on May 29, 2020 function and the degradation of these substrates, to which MMP-2 was colocalized. Furthermore, transgenic mice with myocardium-specific expression of a mutant, constitutively active MMP-2, in the absence of additional injury, show significantly impaired cardiac contractile function, disrupted sarcomeres, profound myofilament lysis, breakdown of Z-band registration, and reduced troponin I level.22 Titin, the largest known mammalian protein (3000 to 4000 kDa), forms an intrasarcomeric elastic filament that is thought to serve as a framework for the ordered assembly of other myofilament proteins.23 In the sarcomere, the titin molecule spans the distance from the Z-disk to the M-line region (half the length of the sarcomere). Moreover, the I-band region of titin comprises distinct elastic segments that allow titin to behave as a molecular spring, contributing to the passive tension of myofibrils and maintaining the structural and functional stability of the sarcomere. Titin is an important determinant of both systolic and diastolic function and the Frank-Starling mechanism of the heart.24 Although loss and/or disorganization of titin in ischemic and failing human hearts has been reported,25,26 the mechanism of titin degradation has not been extensively studied in hearts subjected to ischemia/reperfusion (I/R) injury. Because MMP-2 is localized to sarcomeric and cytoskeletal proteins and is activated in myocardial I/R injury, we address here whether MMP-2 targets titin to contribute to the pathogenesis of myocardial I/R injury. Methods Titin Isolation and Purification Titin was prepared as described previously.27,28 See the online-only Data Supplement. Skinned Cardiomyocyte Isolation Skinned cardiomyocytes were isolated as described in the onlineonly Data Supplement. Cleavage of Native Titin in Skinned Cardiomyocytes Skinned cardiomyocytes were incubated with human recombinant MMP-2 catalytic domain (4 to 120 nmol/L; Enzo Life Sciences, Plymouth Meeting, Pa) with or without MMP inhibitors (10 ␮mol/L o-phenanthroline or ONO-4817) at 37°C for 60 minutes. This concentration of o-phenanthroline inhibits MMP-2 activity under similar in vitro conditions.29 The samples were denatured with 2⫻ urea sample buffer (8 mol/L urea, 2 mol/L thiourea, 3% SDS, 75 mmol/L DTT, 0.03% bromophenol blue, and 0.05 mol/L TrisHCl, pH 6.8) at 60°C for 10 minutes, and the proteins were electrophoresed by 1% SDS-agarose and stained with Coomassie brilliant blue. Isolated Working Rat Heart: Ex Vivo Model of I/R Male Sprague-Dawley rats (300 to 350 g) were anesthetized with sodium pentobarbital (60 mg/kg IP). Hearts were isolated and paced at 300 bpm during perfusion at 37°C as working hearts30 with 100 mL recirculating Krebs–Henseleit solution containing 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 25 mmol/L NaHCO3, 11 mmol/L glucose, 100 ␮U/mL insulin, 2.5 mmol/L Ca2⫹, 0.5 mmol/L EDTA, and 0.1% BSA continuously gassed with 95% O2/5% CO2 (pH 7.4). The perfusate enters the left atrium at a hydrostatic preload pressure of 9.5 mm Hg, and the left ventricle ejects it against a hydrostatic afterload of 70 mm Hg. Cardiac work (cardiac output times peak systolic pressure) was used as an index of mechanical function. After 25 minutes of equilibration, hearts were either aerobically perfused for 85 minutes (control; n⫽6) or subjected to 25 minutes of global, no-flow ischemia followed by 60 minutes aerobic reperfusion without (I/R; n⫽7) or with 50 ␮mol/L ONO-4817 (I/R⫹ONO-4817; n⫽8). ONO-4817, a Ali et al Titin Degradation in Ischemic/Reperfused Heart 2041 selective MMP inhibitor (Ki in the nanomolar range for MMP-2 and almost no inhibitory activity up to 100 ␮mol/L against several other proteases of different classes31), was added to the perfusion buffer 10 minutes before the induction of ischemia. All hearts were perfused for a total of 110 minutes. At the end of perfusion, the ventricles were rapidly frozen in liquid nitrogen and processed for titin analysis in ventricular extracts as described below. Additional series of hearts (control, n⫽5; I/R, n⫽5; and I/R⫹ONO-4817, n⫽4) were perfused and processed for immunohistochemistry and confocal microscopy analysis for assessment of titin immunostaining. Another 6 hearts were briefly perfused for 10 minutes at 37°C with Krebs-Henseleit buffer at a constant hydrostatic pressure of 70 mm Hg to clear them of blood, followed by processing for immunohistochemistry as described below to investigate the colocalization of titin and MMP-2 in the left ventricle. This investigation conforms to the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care. In Vivo Model of I/R Downloaded from http://ahajournals.org by on May 29, 2020 I/R was induced in vivo by modifying a previously described protocol.32 Briefly, MMP-2 knockout and age-matched wild-type male C57BL/6 mice were anesthetized with isoflurane, intubated, and kept on a heating pad to maintain body temperature at 37°C. The heart was exposed, and the left anterior descending coronary artery was temporarily ligated with a 7-0 silk suture, with a piece of 4-0 silk placed between the left anterior descending coronary artery and the 7-0 silk. After 30 minutes of left anterior descending coronary artery occlusion, reperfusion was initiated by releasing the ligature and removing the 4-0 silk. The loosened suture was left in place to help identify the ischemic area of the left ventricle. After 30 minutes of reperfusion, the hearts were excised, and the ischemic and nonischemic regions of the left ventricle were dissected out under a magnifying glass and flash frozen in liquid nitrogen for titin analysis. Analysis of Titin by Gel Electrophoresis Titin was analyzed in ventricular extracts using 1% vertical SDS– agarose gel electrophoresis as previously described.33 See the onlineonly Data Supplement for details. Immunohistochemistry and Confocal Microscopy Colocalization of Titin and MMP-2 Rat hearts perfused aerobically for 10 minutes to flush them of blood or left ventricular tissue from the explanted heart of a heart transplant patient was fixed with 4% paraformaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4) and cryoprotected with 30% sucrose in 0.1 mol/L sodium phosphate buffer. Details of the double immunolabeling are provided in the online-only Data Supplement. Titin 9D10 Immunostaining At the end of the 110 minutes of working heart perfusion protocol, some control, I/R, or I/R⫹ONO-4817 hearts were fixed and cryoprotected (as described above) for 9D10 immunostaining as detailed in the online-only Data Supplement. Overlay Assay to Determine MMP-2 Binding to Titin Skinned muscle fibers were incubated with trypsin to increase titin degradation (intact T1 to T2 fragment). The proteins in the samples were separated by gel electrophoresis and transferred to a polyvinylidene difluoride membrane. These membranes were used in an overlay assay in which the binding of human recombinant MMP-2 to titin T1 and T2 on the membrane was assessed. For details, see the online-only Data Supplement. Figure 2. In vitro incubation (60 minutes at 37°C) of skinned cardiomyocytes with MMP-2. A, MMP-2 cleaved cardiac titin (T1) in a concentration-dependent manner (4 to 120 nmol/L) as shown by the appearance of the titin degradation product (T2). B, The cleavage of titin by MMP-2 was prevented by inhibiting the activity of MMP-2 with MMP inhibitors o-phenanthroline (o-ph) or ONO-4817. The T2/T1 band density ratio is indicated below each lane. Myosin heavy chain (MHC) is used as loading control. the Tukey posthoc test was used. Differences were considered significant at P⬍0.05. Results Colocalization of MMP-2 and Titin Near the Z-Disk Region of the Cardiac Sarcomere We first investigated whether MMP-2 is localized to titin in the cardiac sarcomere. In this regard, we used 2 different anti-titin antibodies that target specific epitopes (Figure IA in the online-only Data Supplement). The T12 antibody labels titin near the Z-disk region of titin, and the M8 antibody recognizes an epitope at the M-line region of titin. Images obtained by immunohistochemistry followed by confocal microscopy showed that T12 immunoreactivity distributes near Z lines and M8 immunoreactivity is alternatively distributed at M lines without overlapping (Figure IB in the online-only Data Supplement). Images obtained with anti– MMP-2 and anti–titin T12 in left ventricle sections from rat hearts aerobically perfused for 10 minutes showed clear colocalization of MMP-2 to the Z-disk region of titin (Figure 1). When using anti–titin M8, we observed a weaker localization of MMP-2 to this region of titin (Figure 1). These data suggest that MMP-2 localizes mainly near the Z-disk region of the sarcomere, with a secondary and weaker localization near the M-line portion in the titin molecule. Statistical Analysis MMP-2 Binds and Cleaves Titin in a Concentration-Dependent Manner Results are expressed as mean⫾SEM for n hearts. As appropriate, 1-way ANOVA or repeated-measures 2-way ANOVA followed by In silico mapping of MMP-2 cleavage sites in both human and mouse N2B titin revealed multiple putative sites in both 2042 Circulation November 16, 2010 Figure 3. Mechanical recovery of isolated perfused working rat hearts subjected to 25 minutes of global, no-flow ischemia followed by 60 minutes of reperfusion without (I/R) or with 50 ␮mol/L ONO-4817 (I/R⫹ONO-4817) vs aerobically perfused control hearts. A, Schematic representation of the perfusion protocols for control (n⫽6), I/R (n⫽7), and I/R⫹ONO-4817 (n⫽8) groups. B, Time course of changes in cardiac work of isolated working rat hearts. **P⬍0.001, *P⬍0.05 vs corresponding values of I/R group, repeated-measures 2-way ANOVA. Downloaded from http://ahajournals.org by on May 29, 2020 I-band and A-band titin regions, including near the Z-line terminus of titin. These sites show ⬎60% homology to the 3 MMP-2 cleavage motifs (Figure II in the online-only Data Supplement). Moreover, human recombinant MMP-2 was able to bind to titin prepared from skinned muscle fibers as shown by the overlay assay method (Figure III in the online-only Data Supplement). Next we tested the susceptibility of purified titin to proteolytic degradation by MMP-2 in vitro. Incubation of titin with MMP-2 (60 minutes at 37°C) at increasing molar ratios of MMP-2 to titin (1:500, 1:50, and 1:5) caused titin degradation in a concentration-dependent manner (Figure IVA in the online-only Data Supplement). Inhibition of MMP-2 activity with GM-6001 or ONO-4817 prevented titin cleavage by MMP-2 (Figure IVB in the online-only Data Supplement). To determine whether MMP-2 directly cleaves cardiac titin in situ, we incubated skinned mouse cardiomyocytes with increasing concentrations of MMP-2 (60 minutes at 37°C). This resulted in concentration-dependent cleavage of cardiac titin (T1) as shown by the increased level of the degradation product of titin (T2) with increasing MMP-2 concentration (Figure 2A). Inhibition of MMP-2 activity with o-phenanthroline or ONO4817 prevented titin cleavage by MMP-2 (Figure 2B). Effect of ONO-4817 on Functional Recovery of I/R Hearts Isolated working rat hearts were perfused for 110 minutes under 1 of 3 conditions: aerobic perfusion (control); 25 minutes of global, no-flow ischemia and 60 minutes of aerobic reperfusion (I/R); or I/R in the presence of a selective MMP inhibitor, ONO-4817 (I/R⫹ONO-4817; Figure 3A). Control hearts showed no significant loss of mechanical function over 110 minutes of aerobic perfusion (Figure 3). I/R hearts showed markedly reduced recovery of mechanical function during reperfusion compared with control hearts (Figure 3B). The recovery of cardiac work during reperfusion was significantly improved after MMP inhibition with ONO4817, compared with the I/R group (Figure 3B). Myocardial I/R Causes Titin Cleavage, an Effect Diminished by an MMP Inhibitor To investigate whether MMP-2 can cleave titin in the intact heart under pathophysiological conditions, titin content was assessed with 1% vertical SDS–agarose gels in ventricular extracts prepared from the control, I/R, or I/R⫹ONO-4817 hearts. Ventricular extracts from control hearts revealed a titin band at ⬇3000 kDa (Figure 4A). I/R caused a significant increase in T2 band density, an effect abolished in the I/R⫹ONO-4817 hearts (Figure 4A). Quantification of the ratio of total titin to myosin heavy chain (MHC) showed that I/R did not significantly change this ratio compared with control hearts (Figure 4B), whereas the ratio of T2 to MHC was significantly increased in I/R hearts. ONO-4817 abolished the I/R-induced increase in the T2/MHC ratio (Figure 4C). These observations were further confirmed by immunohistochemistry experiments using the anti–titin 9D10 antibody, raised against the proline-glutamate-valine-lysine (PEVK) domain in the spring region of titin. Titin immuno- Ali et al Titin Degradation in Ischemic/Reperfused Heart 2043 Downloaded from http://ahajournals.org by on May 29, 2020 Figure 4. Titin degradation in I/R rat hearts. A, Representative SDS–agarose gel for analysis of titin in ventricular extracts. Titin (T1) and titin degradation product (T2) in ventricular homogenates from control, I/R, and I/R⫹ONO-4817 hearts analyzed with a 1% vertical SDS– agarose gel. Bovine left ventricle (BLV) was used as a standard and shows N2BA and N2B isoforms of titin; note that the majority of rat heart titin is the N2B isoform. Each lane is an extract from individually perfused hearts. B, Ratio of total titin (T1⫹T2) to MHC content (n⫽6 in each group). C, Ratio of T2 titin to MHC content (n⫽6 in each group). *P⬍0.05 vs control (1-way ANOVA, Tukey posthoc test). D, Representative left ventricular cryosections immunostained against titin epitope 9D10. Titin immunostaining with the 9D10 antibody (raised against the PEVK domain) was decreased in I/R hearts vs control, whereas staining intensity was comparable between I/R⫹ONO-4817 and control hearts. Scale bar⫽10 ␮m for all images. Images are representative of at least 4 individual hearts investigated under each condition. staining was significantly reduced by I/R, whereas ONO4817 treatment preserved titin immunostaining to a level comparable to control (Figure 4D). Titin Degradation Is Reduced in Hearts From MMP-2 Knockout Mice Subjected to I/R Injury In Vivo We next determined whether genetic ablation of MMP-2 could influence titin degradation in cardiac muscle. Mouse hearts subjected in vivo to left anterior descending coronary artery ligation for 30 minutes followed by 30 minutes of reperfusion exhibited titin degradation, which was significantly less in MMP-2 knockout hearts than in wild-type control hearts (Figure 5). MMP-2 Localizes Near the Z-Disk Region of Titin in the Human Heart Immunostaining of sections prepared from the left ventricle of an explanted heart from a patient undergoing heart transplantation showed colocalization of MMP-2 and titin mainly near the Z disk, with a weaker colocalization at the M line. Compared with the rat heart, MMP-2 immunostaining in the human heart was more diffuse yet still showed a sarcomeric staining pattern (Figure 6). Discussion In this study, we demonstrated that titin, the giant sarcomeric protein, is a target of the proteolytic activity of MMP-2 in the setting of acute myocardial I/R injury. Immunohistochemical analysis shows that MMP-2 clearly colocalizes with titin near the Z-disk region of the sarcomere in both rat and human hearts. We established that under in vitro conditions MMP-2 is able to bind to and cleave titin in a concentration-dependent manner. The proteolytic action of MMP-2 is blocked by the selective MMP inhibitors GM-6001 and ONO-4817, verifying that the cleavage is indeed due to MMP activity. ONO4817 not only improves the functional recovery after I/R in isolated rat hearts but also prevents the significant increase in the titin degradation product T2 caused by I/R injury, indicating that titin degradation is reduced when MMP activity is 2044 Circulation November 16, 2010 Figure 5. Titin degradation in MMP-2 knockout (KO) and wildtype (WT) mouse hearts subjected to I/R in vivo. A, Representative 1% vertical SDS–agarose gel shows titin (T1) isoforms (N2BA and N2B) and titin degradation product T2 in the left ventricle from sham or in ischemic regions from I/R groups in either WT or MMP-2 KO mice. B, Quantification of ratios of T2 titin to total titin (n⫽6 in each group). *P⬍0.01 vs sham control (1-way ANOVA, Tukey posthoc test). Downloaded from http://ahajournals.org by on May 29, 2020 inhibited. Furthermore, hearts from MMP-2 knockout mice subjected to in vivo I/R injury show less titin degradation compared with wild-type controls. Titin proteolysis has been observed in various human heart diseases associated with increased myocardial oxidative stress, including dilated cardiomyopathy, the terminally failing heart, and Chagas cardiomyopathy25,26,34; however, the proteases responsible for this were not identified. MMPs are best known as proteases responsible for the degradation and remodeling of extracellular matrix proteins in both physiological and pathological conditions, including various cardiac pathologies. However, the discovery of the intracellular localization14,16,18,22 and functions of MMP-2 to proteolyze troponin I,14,22 myosin light chain-1,15 and ␣-actinin17 during myocardial oxidative stress injury challenged the canonical notion of extracellular-only actions of this enzyme. In previous studies, we showed that peroxynitrite biosynthesis in I/R rat hearts peaks within the first minute of reperfusion2 and that the peak in MMP-2 activity follows at 2 to 5 minutes of reperfusion.11 Infusion of peroxynitrite into isolated perfused rat hearts35 or isolated cardiomyocytes 36 caused a time- and concentrationdependent contractile dysfunction that was abrogated with MMP inhibitors. In vitro, peroxynitrite was shown to directly activate MMP-2 via a nonproteolytic mechanism involving S-glutathiolation of the propeptide cysteine sulfydryl group.4 Indeed, this intracellular activity of MMP-2 on I/R injury caused proteolytic degradation of specific sarcomeric (troponin I14 and myosin light chain-115) and cytoskeletal (␣actinin17) proteins that are susceptible to its proteolytic activity. MMP-2 is localized within the cardiac sarcomere, including near the Z disks.14 –16 These previous observations are supplemented by the present data showing clear colocalization of MMP-2 near the Z-disk region of titin using the T12 clone in rat (Figure 1) and human (Figure 6) hearts. Several studies show that titin interacts with ␣-actinin at the Z disk of the sarcomere and that this interaction plays a crucial role in Z-disk assembly and sarcomeric integrity.37–39 Interestingly, MMP-2 was found not only to colocalize with ␣-actinin in the Z disk of cardiac sarcomeres16,17 but also to degrade it after peroxynitrite infusion into isolated rat hearts.17 The M8 titin antibody (raised against the M-line region of titin) shows a weaker localization of MMP-2 to this region of titin. Although our data do not rule out the possible localization of MMP-2 also to other region(s) of titin, they do suggest that a main MMP-2 anchoring site is at/near the Z disk of the sarcomere. Titin is the third myofilament (in addition to thick and thin myofilaments) of the sarcomere that plays an important role in sarcomere integrity and cardiac muscle contraction.23 Any alterations in its structure could severely affect the contractile performance of the heart. The increase in T2 titin and the decrease in titin immunostaining after I/R injury observed here (Figure 4) were associated with poor cardiac mechanical recovery during reperfusion (Figure 3). These effects are likely due at least in part to titin degradation by MMP-2, given the colocalization of MMP-2 with titin near the Z disk of cardiac sarcomeres, the susceptibility of titin to degradation by MMP-2, and the reduction in I/R-induced titin degradation in hearts from MMP-2 knockout mice or in rat hearts in which MMP activity was selectively blocked with ONO-4817. A significant increase in MMP-2 activity was seen in the heart after experimental Trypanosoma cruzi infection (the parasite responsible for Chagas disease), and mortality was markedly reduced upon treatment with an MMP inhibitor, suggesting a role of MMP-2 in mediating acute Chagas cardiomyopathy.40 Putative titin degradation products were detected in the plasma of patients with Chagas disease,34 further supporting a role of MMP-2 in titin degradation. Moreover, myocardial infarction is associated with a significant right shift in the left ventricle pressure-volume relation (an observation consistent with titin degradation in the heart), and the broad-spectrum MMP inhibitor PD166793 was shown to protect against this shift.41 Although cardiac mechanical function at the end of perfusion is inversely related to ratios of T2 to MHC in hearts (Figures 3B and 4C), caution is needed in relating this effect exclusively to titin degradation. As mentioned, other sarcomeric/cytoskeletal proteins, including troponin I, myosin light chain-1, and ␣-actinin, are also susceptible to degradation by MMP-2 under conditions of myocardial oxidative stress injury. However, our work clearly suggests that titin proteolysis is an important factor that negatively affects myocardial contractility on I/R injury. Titin content in rat ventricles was investigated here by SDS–agarose gel electrophoresis or immunofluorescence staining against titin epitopes at the PEVK domain. Our electrophoresis results showed the ⬇60% elevation in the ratio of T2 to MHC in the I/R group compared with aerobic control hearts. Immunofluorescence data also showed a reduction of titin immunostaining in the I/R group using the Ali et al Titin Degradation in Ischemic/Reperfused Heart 2045 Figure 6. Colocalization of MMP-2 and titin near the Z disk in diseased human heart. Left ventricle sections were used from the explanted failing heart from a patient receiving a heart transplant. A through D, High density of MMP-2 (green) colocalizes (yellow) with T12 epitope (red) at the Z lines. E through H, Low density of MMP-2 (red) colocalizes (yellow) with M8 epitope (green) at M lines. BF indicates bright-field images. Scale bar⫽10 ␮m. Downloaded from http://ahajournals.org by on May 29, 2020 9D10 antibody. In addition to degradation, posttranslational modifications of titin may have occurred upon I/R that led to diminished binding of titin antibodies to the specific epitopes. Posttranslational modifications of many cardiac myofilament/ cytoskeletal proteins during I/R, including actin42 and myosin light chain-1,43 have been reported in previous studies. Our study does not rule out the possible action of other proteases in titin degradation. Calpains are most likely involved in sarcomeric protein degradation after ischemic episodes more severe than that observed in our model.44 Indeed, calpain was shown to be able to cleave titin only after 24 hours of doxorubicin treatment of rat cardiomyocytes.45 The ubiquitin-proteasome system is another proteolytic pathway that may be involved in titin degradation. Increased proteasome activities have been reported in various models of I/R injury.46 – 48 Moreover, the E3 ubiquitin-ligase MURF1 is known to be associated with the M-line region of titin49 and ubiquitinates titin in yeast 2-hybrid screens.50 In a rat heart failure model, both a loss of titin51 and an increase in MMP-2 gene expression52 were observed in diaphragm muscle. However, in our short-term experiments, we did not observe a significant loss of intact titin on I/R injury. We speculate that MMP-2 activation not only results in titin cleavage but also may trigger a cascade of proteolytic events leading to titin loss several hours after reperfusion. Conclusions The present results indicate that MMP-2 cleaves titin during either ex vivo or in vivo I/R injury. Furthermore, MMP-2 inactivation by pharmacological or genetic approaches protects against titin degradation. Our previous findings of troponin I,14 myosin light chain-1,15 and ␣-actinin17 cleavage by MMP-2, in addition to our present results with titin, suggest that MMP-2 plays a crucial role in the pathogenesis of acute I/R injury at the level of the sarcomere and cytoskeleton. Whether MMP-2 causes contractile protein alterations in other cardiac pathologies needs further investigation. Pharmacological inhibition of MMP activity could represent a useful strategy for the prevention and/or treatment of myocardial I/R injury. Acknowledgments We acknowledge Tiffany Pecor, William Rogers, Sike Pan, and Chanrasekhar Saripalli for technical assistance. We thank Dr Xiuhua Wang for technical assistance with mouse heart experiments, Dr Costas Schulze for help in procuring human heart tissue, and Dr Eliana Lucchinetti for help with the in silico analysis. We thank Dawne Colwell for help with the illustrations. We thank Dr Elisabeth Ehler (King’s College, London UK) for anti-titin antibodies (T12 and M8 clones). 2046 Circulation November 16, 2010 Sources of Funding This work was supported by the Canadian Institutes of Health Research (MOP-77526 to Dr Schulz, MOP-84279 to Dr Kassiri) and the National Institutes of Health (HL062881 to Dr Granzier and T-31 HL07249-31 to Dr Hudson. M. Ali is supported by an Alberta Heritage Foundation for Medical Research (AHFMR) studentship award. Dr Schulz was an AHFMR scientist. Disclosures None. References Downloaded from http://ahajournals.org by on May 29, 2020 1. 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CLINICAL PERSPECTIVE In addition to the well-known extracellular effects of matrix metalloproteinases (MMPs), we provide evidence that MMP-2 is localized inside the cardiac myocyte, near the Z-disk region of the sarcomere. We also show that upon acute ischemia/reperfusion injury, MMP-2 is activated and proteolyses titin, the largest known protein that plays a crucial role in both the diastolic and systolic function of the heart. Titin contains several cleavage motifs for MMP-2, and its proteolysis is reduced in hearts protected by pharmacological inhibition of MMP activity and in MMP-2– deficient hearts. This study provides new insights into the pathophysiological mechanism of ischemia/reperfusion injury and suggests that MMP inhibitors might be a useful strategy for reducing reperfusion injury. Downloaded from http://ahajournals.org by on May 29, 2020