Mohammad A. M. Ali Department of Pharmacology Supervisor ... · Mohammad A.M. Ali, MSc; Woo Jung Cho, MSc; Bryan Hudson, PhD; Zamaneh Kassiri, PhD; Henk Granzier, PhD; Richard Schulz,

Mohammad A. M. Ali

Department of Pharmacology

Supervisor: Dr. Richard Schulz

I did my B.Sc. degree in Pharmaceutical Sciences at Alexandria University, Egypt. In 2002, I was invited to work as an instructor in the Department of Pharmacology, Alexandria University where I completed my M.Sc. studies.

In the fall of 2006 I joined the Department of Pharmacology, University of Alberta as a PhD graduate student under the supervision of Dr. Richard Schulz. The Schulz lab studies ischemic and inflammatory heart diseases and investigates the role of specific molecules contributing to oxidative stress injury of the heart. Being in the first laboratory to discover that matrix metalloproteinase (MMP)-2 proteolyses specific proteins inside cardiomyocytes, my main project is to investigate further novel intracellular targets of MMP-2 in myocardial cell injury and/or death.

My paper in Circulation showed the unique role of MMP-2 in the degradation of titin, the largest known protein (3000-4000 kD) inside the heart responsible for heart muscle contraction. This effect particularly explains the poor contractile function of the heart following ischemia and reperfusion injury and suggests that increased MMP-2 activity inside heart muscle may be a fundamental problem in ischemic heart disease. Accordingly, the results from this research could form the basis for the development of novel therapies (e.g. specifically targeted MMP-2 inhibitors) which may ultimately prevent or reduce ischemic heart disease including reperfusion injury.

Finally I would like to acknowledge all the co-authors in this paper and members of the Schulz lab and Granzier lab, University of Arizona. I would also like to thank my supervisor, Dr. Richard Schulz, for the continuous positive mentoring I have received during my program.

ISSN: 1524-4539 Copyright © 2010 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online

72514Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX

DOI: 10.1161/CIRCULATIONAHA.109.930222 2010;122;2039-2047; originally published online Nov 1, 2010; Circulation

Granzier and Richard Schulz Mohammad A.M. Ali, Woo Jung Cho, Bryan Hudson, Zamaneh Kassiri, Henk

Ischemia/Reperfusion InjuryTitin is a Target of Matrix Metalloproteinase-2: Implications in Myocardial

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Molecular Cardiology

Titin is a Target of Matrix Metalloproteinase-2Implications 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 molecularspring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has beenreported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) islocalized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilamentproteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation duringischemia/reperfusion contributes to cardiac contractile dysfunction.

Methods and Results—Immunohistochemistry and confocal microscopy in rat and human hearts showed discretecolocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin nearthe Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed whenincubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rathearts 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-4817prevented 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 vivocompared with wild-type controls.

Conclusion—MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradationin myocardial ischemia/reperfusion injury. (Circulation. 2010;122:2039-2047.)

Key Words: contractile dysfunction � ischemia � matrix metalloproteinase-2 � sarcomere � titin

Matrix metalloproteinase-2 (MMP-2) is a zinc-dependentprotease that is best known for its ability to degrade the

extracellular matrix in both physiological and pathologicalconditions. MMP-2 is synthesized as a zymogen by a varietyof cells, including cardiac myocytes, and is activated eitherby proteases1 (such as by action of MMP-14) or by posttrans-lational modifications to the full-length enzyme caused byenhanced oxidative stress. For example, peroxynitrite, whichis generated in early reperfusion after ischemia,2 directlyactivates several MMPs,3 including MMP-2,4 via a nonpro-teolytic mechanism involving the S-glutathiolation of a crit-ical propeptide cysteine in its autoinhibitory domain.

Ediotrial see p 2002Clinical Perspective on p 2047

MMPs are best recognized for their role in tissue remod-eling by proteolyzing various components of the extracellularmatrix in both health and disease, ie, in angiogenesis, embry-

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 biologicalprocesses independently of its actions on extracellular matrixproteins. This includes platelet activation,9 regulation ofvascular tone,10 and myocardial stunning injury immediatelyafter reperfusion of the ischemic heart.11 Indeed, severalreports indicate that MMP-2 does not exclusively degradeextracellular matrix components.12,13

In normal cardiac myocytes, MMP-2 is found in discretesubcellular compartments, including the thin and thick myo-filaments 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 myocardialoxidative stress injury and is responsible for the degradationof specific sarcomeric and cytoskeletal proteins, includingtroponin I,14,21 myosin light chain-1,15 and �-actinin.17 Inhi-bition of MMP activity reduced both the loss of contractile

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

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function and the degradation of these substrates, to whichMMP-2 was colocalized. Furthermore, transgenic mice withmyocardium-specific expression of a mutant, constitutivelyactive MMP-2, in the absence of additional injury, showsignificantly impaired cardiac contractile function, disruptedsarcomeres, profound myofilament lysis, breakdown ofZ-band registration, and reduced troponin I level.22

Titin, the largest known mammalian protein (3000 to 4000kDa), forms an intrasarcomeric elastic filament that is thoughtto serve as a framework for the ordered assembly of othermyofilament proteins.23 In the sarcomere, the titin moleculespans the distance from the Z-disk to the M-line region (halfthe length of the sarcomere). Moreover, the I-band region oftitin comprises distinct elastic segments that allow titin tobehave as a molecular spring, contributing to the passivetension of myofibrils and maintaining the structural andfunctional stability of the sarcomere. Titin is an importantdeterminant of both systolic and diastolic function and theFrank-Starling mechanism of the heart.24 Although lossand/or disorganization of titin in ischemic and failing humanhearts has been reported,25,26 the mechanism of titin degra-dation has not been extensively studied in hearts subjected toischemia/reperfusion (I/R) injury. Because MMP-2 is local-ized to sarcomeric and cytoskeletal proteins and is activatedin myocardial I/R injury, we address here whether MMP-2targets titin to contribute to the pathogenesis of myocardialI/R injury.

MethodsTitin Isolation and PurificationTitin was prepared as described previously.27,28 See the online-onlyData Supplement.

Skinned Cardiomyocyte IsolationSkinned cardiomyocytes were isolated as described in the online-only Data Supplement.

Cleavage of Native Titin inSkinned CardiomyocytesSkinned cardiomyocytes were incubated with human recombinantMMP-2 catalytic domain (4 to 120 nmol/L; Enzo Life Sciences,Plymouth Meeting, Pa) with or without MMP inhibitors (10 �mol/Lo-phenanthroline or ONO-4817) at 37°C for 60 minutes. Thisconcentration of o-phenanthroline inhibits MMP-2 activity undersimilar 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 Tris-HCl, pH 6.8) at 60°C for 10 minutes, and the proteins wereelectrophoresed by 1% SDS-agarose and stained with Coomassiebrilliant blue.

Isolated Working Rat Heart: Ex Vivo Modelof I/RMale Sprague-Dawley rats (300 to 350 g) were anesthetized withsodium pentobarbital (60 mg/kg IP). Hearts were isolated and pacedat 300 bpm during perfusion at 37°C as working hearts30 with 100mL recirculating Krebs–Henseleit solution containing 118 mmol/LNaCl, 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 continuouslygassed with 95% O2/5% CO2 (pH 7.4). The perfusate enters the leftatrium at a hydrostatic preload pressure of 9.5 mm Hg, and the leftventricle ejects it against a hydrostatic afterload of 70 mm Hg.Cardiac work (cardiac output times peak systolic pressure) was usedas an index of mechanical function. After 25 minutes of equilibra-tion, hearts were either aerobically perfused for 85 minutes (control;n�6) or subjected to 25 minutes of global, no-flow ischemiafollowed by 60 minutes aerobic reperfusion without (I/R; n�7) orwith 50 �mol/L ONO-4817 (I/R�ONO-4817; n�8). ONO-4817, a

Figure 1. Colocalization of MMP-2 andtitin at the Z-disk region of the left ven-tricular cardiac sarcomere in rat heartsaerobically perfused for 10 minutes (lon-gitudinal sections). MMP-2 shows bettercolocalization with T12 than the M8epitope of titin in the sarcomere of theleft ventricular myocardium. MMP-2-immunoreactivity reveals at Z lines withhigh density and M lines with low den-sity. 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 T12epitope (red) at the Z lines. D through F,Low density of MMP-2 (red) colocalizes(yellow) with M8 epitope (green) at Mlines. Scale bar�5 �m for all imagesexcept the enlarged portion of C illus-trating the Z and M lines.

2040 Circulation November 16, 2010



selective MMP inhibitor (Ki in the nanomolar range for MMP-2 andalmost no inhibitory activity up to 100 �mol/L against several otherproteases of different classes31), was added to the perfusion buffer 10minutes before the induction of ischemia. All hearts were perfusedfor a total of 110 minutes. At the end of perfusion, the ventricleswere rapidly frozen in liquid nitrogen and processed for titin analysisin ventricular extracts as described below.

Additional series of hearts (control, n�5; I/R, n�5; andI/R�ONO-4817, n�4) were perfused and processed for immuno-histochemistry and confocal microscopy analysis for assessment oftitin immunostaining. Another 6 hearts were briefly perfused for 10minutes at 37°C with Krebs-Henseleit buffer at a constant hydro-static pressure of 70 mm Hg to clear them of blood, followed byprocessing for immunohistochemistry as described below to inves-tigate the colocalization of titin and MMP-2 in the left ventricle.

This investigation conforms to the Guide to the Care and Use ofExperimental Animals published by the Canadian Council on AnimalCare.

In Vivo Model of I/RI/R was induced in vivo by modifying a previously describedprotocol.32 Briefly, MMP-2 knockout and age-matched wild-typemale C57BL/6 mice were anesthetized with isoflurane, intubated,and kept on a heating pad to maintain body temperature at 37°C. Theheart was exposed, and the left anterior descending coronary arterywas temporarily ligated with a 7-0 silk suture, with a piece of 4-0 silkplaced between the left anterior descending coronary artery and the7-0 silk. After 30 minutes of left anterior descending coronary arteryocclusion, reperfusion was initiated by releasing the ligature andremoving the 4-0 silk. The loosened suture was left in place to helpidentify the ischemic area of the left ventricle. After 30 minutes ofreperfusion, the hearts were excised, and the ischemic and nonische-mic regions of the left ventricle were dissected out under a magni-fying glass and flash frozen in liquid nitrogen for titin analysis.

Analysis of Titin by Gel ElectrophoresisTitin was analyzed in ventricular extracts using 1% vertical SDS–agarose gel electrophoresis as previously described.33 See the online-only Data Supplement for details.

Immunohistochemistry and Confocal Microscopy

Colocalization of Titin and MMP-2Rat hearts perfused aerobically for 10 minutes to flush them of bloodor left ventricular tissue from the explanted heart of a heart transplantpatient was fixed with 4% paraformaldehyde in 0.1 mol/L sodiumphosphate buffer (pH 7.4) and cryoprotected with 30% sucrose in 0.1mol/L sodium phosphate buffer. Details of the double immunolabel-ing are provided in the online-only Data Supplement.

Titin 9D10 ImmunostainingAt the end of the 110 minutes of working heart perfusion protocol,some control, I/R, or I/R�ONO-4817 hearts were fixed and cryo-protected (as described above) for 9D10 immunostaining as detailedin the online-only Data Supplement.

Overlay Assay to Determine MMP-2 Bindingto TitinSkinned muscle fibers were incubated with trypsin to increase titindegradation (intact T1 to T2 fragment). The proteins in the sampleswere separated by gel electrophoresis and transferred to a polyvi-nylidene difluoride membrane. These membranes were used in anoverlay assay in which the binding of human recombinant MMP-2 totitin T1 and T2 on the membrane was assessed. For details, see theonline-only Data Supplement.

Statistical AnalysisResults are expressed as mean�SEM for n hearts. As appropriate,1-way ANOVA or repeated-measures 2-way ANOVA followed by

the Tukey posthoc test was used. Differences were consideredsignificant at P�0.05.

ResultsColocalization of MMP-2 and Titin Near theZ-Disk Region of the Cardiac SarcomereWe first investigated whether MMP-2 is localized to titin inthe cardiac sarcomere. In this regard, we used 2 differentanti-titin antibodies that target specific epitopes (Figure IA inthe online-only Data Supplement). The T12 antibody labelstitin near the Z-disk region of titin, and the M8 antibodyrecognizes an epitope at the M-line region of titin. Imagesobtained by immunohistochemistry followed by confocalmicroscopy showed that T12 immunoreactivity distributesnear Z lines and M8 immunoreactivity is alternatively dis-tributed at M lines without overlapping (Figure IB in theonline-only Data Supplement). Images obtained with anti–MMP-2 and anti–titin T12 in left ventricle sections from rathearts aerobically perfused for 10 minutes showed clearcolocalization of MMP-2 to the Z-disk region of titin (Figure1). When using anti–titin M8, we observed a weaker local-ization of MMP-2 to this region of titin (Figure 1). These datasuggest that MMP-2 localizes mainly near the Z-disk regionof the sarcomere, with a secondary and weaker localizationnear the M-line portion in the titin molecule.

MMP-2 Binds and Cleaves Titin in aConcentration-Dependent MannerIn silico mapping of MMP-2 cleavage sites in both humanand mouse N2B titin revealed multiple putative sites in both

Figure 2. In vitro incubation (60 minutes at 37°C) of skinnedcardiomyocytes with MMP-2. A, MMP-2 cleaved cardiac titin(T1) in a concentration-dependent manner (4 to 120 nmol/L) asshown by the appearance of the titin degradation product (T2).B, The cleavage of titin by MMP-2 was prevented by inhibitingthe activity of MMP-2 with MMP inhibitors o-phenanthroline(o-ph) or ONO-4817. The T2/T1 band density ratio is indicatedbelow each lane. Myosin heavy chain (MHC) is used as loadingcontrol.

Ali et al Titin Degradation in Ischemic/Reperfused Heart 2041



I-band and A-band titin regions, including near the Z-lineterminus of titin. These sites show �60% homology to the 3MMP-2 cleavage motifs (Figure II in the online-only DataSupplement). Moreover, human recombinant MMP-2 wasable to bind to titin prepared from skinned muscle fibers asshown by the overlay assay method (Figure III in theonline-only Data Supplement). Next we tested the suscepti-bility of purified titin to proteolytic degradation by MMP-2 invitro. Incubation of titin with MMP-2 (60 minutes at 37°C) atincreasing molar ratios of MMP-2 to titin (1:500, 1:50, and1:5) caused titin degradation in a concentration-dependentmanner (Figure IVA in the online-only Data Supplement).Inhibition of MMP-2 activity with GM-6001 or ONO-4817prevented titin cleavage by MMP-2 (Figure IVB in theonline-only Data Supplement). To determine whetherMMP-2 directly cleaves cardiac titin in situ, we incubatedskinned mouse cardiomyocytes with increasing concentra-tions of MMP-2 (60 minutes at 37°C). This resulted inconcentration-dependent cleavage of cardiac titin (T1) asshown by the increased level of the degradation product oftitin (T2) with increasing MMP-2 concentration (Figure 2A).Inhibition of MMP-2 activity with o-phenanthroline or ONO-4817 prevented titin cleavage by MMP-2 (Figure 2B).

Effect of ONO-4817 on Functional Recovery ofI/R HeartsIsolated working rat hearts were perfused for 110 minutesunder 1 of 3 conditions: aerobic perfusion (control); 25minutes of global, no-flow ischemia and 60 minutes of

aerobic reperfusion (I/R); or I/R in the presence of a selectiveMMP inhibitor, ONO-4817 (I/R�ONO-4817; Figure 3A).Control hearts showed no significant loss of mechanicalfunction over 110 minutes of aerobic perfusion (Figure 3). I/Rhearts showed markedly reduced recovery of mechanicalfunction during reperfusion compared with control hearts(Figure 3B). The recovery of cardiac work during reperfusionwas significantly improved after MMP inhibition with ONO-4817, compared with the I/R group (Figure 3B).

Myocardial I/R Causes Titin Cleavage, an EffectDiminished by an MMP InhibitorTo investigate whether MMP-2 can cleave titin in the intactheart under pathophysiological conditions, titin content wasassessed with 1% vertical SDS–agarose gels in ventricularextracts prepared from the control, I/R, or I/R�ONO-4817hearts. Ventricular extracts from control hearts revealed atitin band at �3000 kDa (Figure 4A). I/R caused a significantincrease in T2 band density, an effect abolished in theI/R�ONO-4817 hearts (Figure 4A). Quantification of theratio of total titin to myosin heavy chain (MHC) showed thatI/R did not significantly change this ratio compared withcontrol hearts (Figure 4B), whereas the ratio of T2 to MHCwas significantly increased in I/R hearts. ONO-4817 abol-ished the I/R-induced increase in the T2/MHC ratio (Figure4C). These observations were further confirmed by immuno-histochemistry experiments using the anti–titin 9D10 anti-body, raised against the proline-glutamate-valine-lysine(PEVK) domain in the spring region of titin. Titin immuno-

Figure 3. Mechanical recovery of iso-lated perfused working rat hearts sub-jected to 25 minutes of global, no-flowischemia followed by 60 minutes ofreperfusion without (I/R) or with50 �mol/L ONO-4817 (I/R�ONO-4817)vs aerobically perfused control hearts. A,Schematic representation of the perfu-sion protocols for control (n�6), I/R(n�7), and I/R�ONO-4817 (n�8) groups.B, Time course of changes in cardiacwork of isolated working rat hearts.**P�0.001, *P�0.05 vs correspondingvalues of I/R group, repeated-measures2-way ANOVA.




staining was significantly reduced by I/R, whereas ONO-4817 treatment preserved titin immunostaining to a levelcomparable to control (Figure 4D).

Titin Degradation Is Reduced in Hearts FromMMP-2 Knockout Mice Subjected to I/R InjuryIn VivoWe next determined whether genetic ablation of MMP-2could influence titin degradation in cardiac muscle. Mousehearts subjected in vivo to left anterior descending coronaryartery ligation for 30 minutes followed by 30 minutes ofreperfusion exhibited titin degradation, which was signifi-cantly less in MMP-2 knockout hearts than in wild-typecontrol hearts (Figure 5).

MMP-2 Localizes Near the Z-Disk Region of Titinin the Human HeartImmunostaining of sections prepared from the left ventricleof an explanted heart from a patient undergoing heart trans-plantation 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 thehuman heart was more diffuse yet still showed a sarcomericstaining pattern (Figure 6).

DiscussionIn this study, we demonstrated that titin, the giant sarcomericprotein, is a target of the proteolytic activity of MMP-2 in thesetting of acute myocardial I/R injury. Immunohistochemicalanalysis shows that MMP-2 clearly colocalizes with titin nearthe Z-disk region of the sarcomere in both rat and humanhearts. We established that under in vitro conditions MMP-2is able to bind to and cleave titin in a concentration-dependentmanner. The proteolytic action of MMP-2 is blocked by theselective MMP inhibitors GM-6001 and ONO-4817, verify-ing that the cleavage is indeed due to MMP activity. ONO-4817 not only improves the functional recovery after I/R inisolated rat hearts but also prevents the significant increase inthe titin degradation product T2 caused by I/R injury, indi-cating that titin degradation is reduced when MMP activity is

Figure 4. Titin degradation in I/R rathearts. A, Representative SDS–agarosegel for analysis of titin in ventricularextracts. Titin (T1) and titin degradationproduct (T2) in ventricular homogenatesfrom control, I/R, and I/R�ONO-4817hearts analyzed with a 1% vertical SDS–agarose gel. Bovine left ventricle (BLV)was used as a standard and showsN2BA and N2B isoforms of titin; notethat the majority of rat heart titin is theN2B isoform. Each lane is an extractfrom individually perfused hearts. B,Ratio of total titin (T1�T2) to MHC con-tent (n�6 in each group). C, Ratio of T2titin to MHC content (n�6 in eachgroup). *P�0.05 vs control (1-wayANOVA, Tukey posthoc test). D, Repre-sentative left ventricular cryosectionsimmunostained against titin epitope9D10. Titin immunostaining with the9D10 antibody (raised against the PEVKdomain) was decreased in I/R hearts vscontrol, whereas staining intensity wascomparable between I/R�ONO-4817and control hearts. Scale bar�10 �m forall images. Images are representative ofat least 4 individual hearts investigatedunder each condition.




inhibited. Furthermore, hearts from MMP-2 knockout micesubjected to in vivo I/R injury show less titin degradationcompared with wild-type controls. Titin proteolysis has beenobserved in various human heart diseases associated withincreased myocardial oxidative stress, including dilated car-diomyopathy, the terminally failing heart, and Chagas car-diomyopathy25,26,34; however, the proteases responsible forthis were not identified.

MMPs are best known as proteases responsible for thedegradation and remodeling of extracellular matrix proteinsin both physiological and pathological conditions, includingvarious cardiac pathologies. However, the discovery of theintracellular localization14,16,18,22 and functions of MMP-2 toproteolyze troponin I,14,22 myosin light chain-1,15 and�-actinin17 during myocardial oxidative stress injury chal-lenged the canonical notion of extracellular-only actions ofthis enzyme. In previous studies, we showed that peroxyni-trite biosynthesis in I/R rat hearts peaks within the firstminute of reperfusion2 and that the peak in MMP-2 activityfollows at 2 to 5 minutes of reperfusion.11 Infusion ofperoxynitrite into isolated perfused rat hearts35 or isolatedcardiomyocytes36 caused a time- and concentration-dependent contractile dysfunction that was abrogated withMMP inhibitors. In vitro, peroxynitrite was shown to directlyactivate MMP-2 via a nonproteolytic mechanism involvingS-glutathiolation of the propeptide cysteine sulfydryl group.4

Indeed, this intracellular activity of MMP-2 on I/R injurycaused proteolytic degradation of specific sarcomeric (tropo-nin I14 and myosin light chain-115) and cytoskeletal (�-actinin17) proteins that are susceptible to its proteolyticactivity.

MMP-2 is localized within the cardiac sarcomere, includ-ing near the Z disks.14–16 These previous observations are

supplemented by the present data showing clear colocaliza-tion of MMP-2 near the Z-disk region of titin using the T12clone in rat (Figure 1) and human (Figure 6) hearts. Severalstudies show that titin interacts with �-actinin at the Z disk ofthe sarcomere and that this interaction plays a crucial role inZ-disk assembly and sarcomeric integrity.37–39 Interestingly,MMP-2 was found not only to colocalize with �-actinin in theZ disk of cardiac sarcomeres16,17 but also to degrade it afterperoxynitrite infusion into isolated rat hearts.17 The M8 titinantibody (raised against the M-line region of titin) shows aweaker localization of MMP-2 to this region of titin. Al-though our data do not rule out the possible localization ofMMP-2 also to other region(s) of titin, they do suggest that amain MMP-2 anchoring site is at/near the Z disk of thesarcomere.

Titin is the third myofilament (in addition to thick and thinmyofilaments) of the sarcomere that plays an important rolein sarcomere integrity and cardiac muscle contraction.23 Anyalterations in its structure could severely affect the contractileperformance of the heart. The increase in T2 titin and thedecrease in titin immunostaining after I/R injury observedhere (Figure 4) were associated with poor cardiac mechanicalrecovery during reperfusion (Figure 3). These effects arelikely due at least in part to titin degradation by MMP-2,given the colocalization of MMP-2 with titin near the Z diskof cardiac sarcomeres, the susceptibility of titin to degrada-tion by MMP-2, and the reduction in I/R-induced titindegradation in hearts from MMP-2 knockout mice or in rathearts in which MMP activity was selectively blocked withONO-4817. A significant increase in MMP-2 activity wasseen in the heart after experimental Trypanosoma cruziinfection (the parasite responsible for Chagas disease), andmortality was markedly reduced upon treatment with anMMP inhibitor, suggesting a role of MMP-2 in mediatingacute Chagas cardiomyopathy.40 Putative titin degradationproducts were detected in the plasma of patients with Chagasdisease,34 further supporting a role of MMP-2 in titin degra-dation. Moreover, myocardial infarction is associated with asignificant right shift in the left ventricle pressure-volumerelation (an observation consistent with titin degradation inthe heart), and the broad-spectrum MMP inhibitor PD-166793 was shown to protect against this shift.41 Althoughcardiac mechanical function at the end of perfusion isinversely related to ratios of T2 to MHC in hearts (Figures 3Band 4C), caution is needed in relating this effect exclusivelyto titin degradation. As mentioned, other sarcomeric/cy-toskeletal proteins, including troponin I, myosin light chain-1,and �-actinin, are also susceptible to degradation by MMP-2under conditions of myocardial oxidative stress injury. How-ever, our work clearly suggests that titin proteolysis is animportant factor that negatively affects myocardial contrac-tility on I/R injury.

Titin content in rat ventricles was investigated here bySDS–agarose gel electrophoresis or immunofluorescencestaining against titin epitopes at the PEVK domain. Ourelectrophoresis results showed the �60% elevation in theratio of T2 to MHC in the I/R group compared with aerobiccontrol hearts. Immunofluorescence data also showed areduction of titin immunostaining in the I/R group using the

Figure 5. Titin degradation in MMP-2 knockout (KO) and wild-type (WT) mouse hearts subjected to I/R in vivo. A, Representa-tive 1% vertical SDS–agarose gel shows titin (T1) isoforms(N2BA and N2B) and titin degradation product T2 in the leftventricle from sham or in ischemic regions from I/R groups ineither WT or MMP-2 KO mice. B, Quantification of ratios of T2titin to total titin (n�6 in each group). *P�0.01 vs sham control(1-way ANOVA, Tukey posthoc test).




9D10 antibody. In addition to degradation, posttranslationalmodifications of titin may have occurred upon I/R that led todiminished binding of titin antibodies to the specific epitopes.Posttranslational modifications of many cardiac myofilament/cytoskeletal proteins during I/R, including actin42 and myosinlight chain-1,43 have been reported in previous studies.

Our study does not rule out the possible action of otherproteases in titin degradation. Calpains are most likelyinvolved in sarcomeric protein degradation after ischemicepisodes more severe than that observed in our model.44

Indeed, calpain was shown to be able to cleave titin only after24 hours of doxorubicin treatment of rat cardiomyocytes.45

The ubiquitin-proteasome system is another proteolytic path-way that may be involved in titin degradation. Increasedproteasome activities have been reported in various models ofI/R injury.46–48 Moreover, the E3 ubiquitin-ligase MURF1 isknown to be associated with the M-line region of titin49 andubiquitinates titin in yeast 2-hybrid screens.50 In a rat heartfailure model, both a loss of titin51 and an increase in MMP-2gene expression52 were observed in diaphragm muscle. How-ever, in our short-term experiments, we did not observe asignificant loss of intact titin on I/R injury. We speculate thatMMP-2 activation not only results in titin cleavage but alsomay trigger a cascade of proteolytic events leading to titinloss several hours after reperfusion.

ConclusionsThe present results indicate that MMP-2 cleaves titin duringeither ex vivo or in vivo I/R injury. Furthermore, MMP-2inactivation by pharmacological or genetic approaches pro-tects against titin degradation. Our previous findings oftroponin I,14 myosin light chain-1,15 and �-actinin17 cleavageby MMP-2, in addition to our present results with titin,suggest that MMP-2 plays a crucial role in the pathogenesisof acute I/R injury at the level of the sarcomere andcytoskeleton. Whether MMP-2 causes contractile proteinalterations in other cardiac pathologies needs further investi-gation. Pharmacological inhibition of MMP activity couldrepresent a useful strategy for the prevention and/or treatmentof myocardial I/R injury.

AcknowledgmentsWe acknowledge Tiffany Pecor, William Rogers, Sike Pan, andChanrasekhar Saripalli for technical assistance. We thank Dr XiuhuaWang for technical assistance with mouse heart experiments, DrCostas Schulze for help in procuring human heart tissue, and DrEliana Lucchinetti for help with the in silico analysis. We thankDawne Colwell for help with the illustrations. We thank Dr ElisabethEhler (King’s College, London UK) for anti-titin antibodies (T12and M8 clones).

Figure 6. Colocalization of MMP-2 andtitin near the Z disk in diseased humanheart. Left ventricle sections were usedfrom the explanted failing heart from apatient receiving a heart transplant. Athrough D, High density of MMP-2(green) colocalizes (yellow) with T12epitope (red) at the Z lines. E through H,Low density of MMP-2 (red) colocalizes(yellow) with M8 epitope (green) at Mlines. BF indicates bright-field images.Scale bar�10 �m.




Sources of FundingThis work was supported by the Canadian Institutes of Health Research(MOP-77526 to Dr Schulz, MOP-84279 to Dr Kassiri) and the NationalInstitutes of Health (HL062881 to Dr Granzier and T-31 HL07249-31 toDr Hudson. M. Ali is supported by an Alberta Heritage Foundation forMedical Research (AHFMR) studentship award. Dr Schulz was anAHFMR scientist.

DisclosuresNone.

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CLINICAL PERSPECTIVEIn addition to the well-known extracellular effects of matrix metalloproteinases (MMPs), we provide evidence that MMP-2 islocalized inside the cardiac myocyte, near the Z-disk region of the sarcomere. We also show that upon acute ischemia/reperfusioninjury, MMP-2 is activated and proteolyses titin, the largest known protein that plays a crucial role in both the diastolic andsystolic function of the heart. Titin contains several cleavage motifs for MMP-2, and its proteolysis is reduced in heartsprotected by pharmacological inhibition of MMP activity and in MMP-2–deficient hearts. This study provides new insightsinto the pathophysiological mechanism of ischemia/reperfusion injury and suggests that MMP inhibitors might be a usefulstrategy for reducing reperfusion injury.




A l i e t a l . S u p p l e m e n t a l m a t e r i a l - CIRCULATIONAHA/2009/930222 Page S1

SUPPLEMENTAL MATERIAL

Supplemental Methods

Titin isolation and purification

Titin was prepared as described1, 2. Briefly, myofibrils were prepared from chilled fresh rabbit longissimus dorsi muscle

by homogenisation in 3 volumes of 50 mmol/L KCl, 5 mmol/L EGTA, 1 mmol/L NaHCO3 and 5 µmol/L E64 (Sigma);

(pH 7.0, 4°C). This was followed by three cycles of centrifugation (2000 g) and resuspension in buffer without E64

inhibitor. After the fourth spin the myofibrils were resuspended and extracted on ice for 5 min with stirring in 2 volumes

of 0.9 mol/L KCl, 2 mmol/L MgCl2, 10 mmol/L imidazole, 2 mmol/L EGTA, 1 mmol/L PMSF, 10 µg/ml trypsin

inhibitor, 0.5 mmol/L dithiothreitol, 5 µmol/L E64; pH 7.0. The extract was clarified at 20,000 g for 30 min, diluted

three times with water (final ionic strength ~0.2 mol/L) and after 1 hr for precipitation, myosin was removed by

spinning for 30 min at 20,000 g. The supernatant was diluted five times more (final ionic strength 0.05 mol/L), left for

40 min, and then spun at 11,000 g for 30 min. The crude titin pellet was resuspended in 0.6 mol/L KCl, 30 mmol/L

potassium phosphate (pH 7.0), clarified for 30 min at 25,000 g, and then chromatographed in this buffer in a 90 cm × 1

cm Sepharose CL2B column maintained at 1°C. On occasion the titin at this stage was also passed through a Q-

Sepharose column as described by Nave et al.3 The purification was monitored by SDS-polyacrylamide electrophoresis

in gradient slab gels (4% to 15%). Samples for these were dissociated at 56°C for 20 min in SDS/urea, as described by

Fritz et al.4

Skinned cardiomyocyte preparation

Skinned cardiomyocytes were isolated as described previously5. Briefly, mice were anesthesitized via isoflurane

inhalation, sacrificed via cervical dislocation, and the heart was rapidly (< 90 seconds) cannulated via the aorta. The

heart was then perfused for 4 min with perfusion buffer (113 NaCl, 4.7 KCl, 0.6 Na2HPO4, 1.2 MgSO4, 12 NaHCO3, 10

KHCO3, 10 HEPES, and 30 taurine, all in mmol/L) and then switched to digestion buffer (perfusion buffer plus 0.148

mg/ml Liberase blendzyme 2, 0.13 mg/ml of trypsin, 12.5 µmol/L CaCl2) for 8-10 min. When the heart was flaccid,

digestion was halted and the heart was placed in myocyte stopping buffer 1 (perfusion buffer plus 0.04 ml bovine calf

serum (BCS)/ml buffer and 5 µmol/L CaCl2). The left ventricle was cut into small pieces, triturated several times with a




transfer pipet, filtered through a 300 µm nylon mesh filter and the filtered cells were gravity pelleted. We added 10 ml

of myocyte stopping buffer 2 (perfusion buffer plus 0.05 ml BCS/ml buffer and 12.5 µmol/L CaCl2) and then skinned

the cells in 1X relaxing solution (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonate (BES) 40 mmol/L, EGTA 10

mmol/L, MgCl2 6.56 mmol/L, ATP 5.88 mmol/L, dithiothreitol 1 mmol/L, K-propionate 46.35 mmol/L, creatine

phosphate 15 mmol/L, pH 7.0) (chemicals from Sigma-Aldrich, MO, USA) with 1% Triton-X-100 (Pierce, IL, USA) for

6 min and then quickly washed in 1X relaxing solution without Triton-X-100. Finally, skinned cells were stored in 50%

glycerol/50% relaxing solution at -20oC.

Analysis of titin by gel electrophoresis

Titin was analyzed in rat ventricular extracts at the end of the 110 min working heart perfusion protocol using 1%

vertical SDS-agarose gel electrophoresis as previously described6. Briefly, the frozen ventricles were pulverized under

liquid nitrogen and homogenized in urea sample buffer (8 mol/L urea, 2 mol/L thiourea, 3% SDS, 75 mmol/L DTT,

0.05 mol/L Tris–HCl, 0.03% bromophenol blue, 25% glycerol and 10 µmol/L leupeptin, 10 µmol/L E64 and 0.5

mmol/L phenylmethylsulfonylfluoride, pH adjusted to 6.8) (20:1 v/w buffer to tissue ratio). Samples were thoroughly

vortexed and then heated at 60°C for 10 min. The samples were again vortexed and subsequently centrifuged (12,000g

for 5 min) at 4°C. The supernatant was removed and stored at -80°C until use. The integrated optical density of T1 titin

(full-length titin), T2 titin (degradation product) and myosin heavy chain

(MHC) were determined as a function of the

volume of the solubilized protein sample that was loaded (a range of volumes was loaded

onto each gel). The slope of

the linear range of the relation between integrated optical density and loaded volume was obtained

for each protein. The

total titin (T1+T2):MHC and titin’s degradation product T2:MHC ratios were calculated as the slope of titin (either T1

+ T2 or T2 alone) divided by the slope of MHC.

Immunohistochemistry and confocal microscopy

a- Co-localization of titin and MMP-2

Rat hearts perfused aerobically for 10 min 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. The cryoprotected hearts were cryosectioned

into 6 µm thick sections which were attached to glass slides coated with poly-L-lysine (Cat. No. 63410, Electron

Microscopy Sciences, Hatfield, PA, USA) and dried at room temperature. The dried cryosections were rinsed twice (5

min each) in 0.5% Triton-X 100 in phosphate buffered saline (PBS, pH 7.4) and rinsed once in PBS for 5 min. Double

immunolabeling was accomplished by sequential staining of each primary antibody (mouse anti-MMP-2 IgG, 1:200

final dilution, Cat. No. 3308, Chemicon International; mouse anti-titin T12 IgG, 1:10 final dilution; rabbit anti-titin M8

IgG, 1:200 final dilution, both titin antibodies provided by Dr. Elisabeth Ehler, King’s College London, UK) for 16 hr.

The T12 antibody labels titin near the Z disc region of titin and the M8 antibody recognizes an epitope at the M line

region of titin. Therefore, T12 and M8 antibodies were used to address the proximity of MMP-2 localization to either

the N-terminus or C-terminus regions of titin, respectively. Secondary antibodies conjugated with fluorescent dyes

(Cy3-donkey anti-mouse IgG, 1:25 final dilution, Cat. No. 715-165-151, Jackson ImmunoResearch Laboratories;

Alexa488-donkey anti-rabbit IgG, 1:50 final dilution, Cat. No. A-21206, Invitrogen) were applied for 2 hr. During

incubation with any of the antibodies, 2% normal donkey serum was added. A solution of 10 µmol/L DRAQ5 (Biostatus

Ltd., Leicestershire, UK) was applied to stain nuclei. To determine specificity of immunolabeling, primary or secondary

antibodies were omitted.

The immunolabeled cryosections were observed by confocal microscopy (LSM 510, Carl Zeiss Co., Jena,

Germany). Cy3 (red) was scanned with a helium/neon green laser (543 nm) with a band pass 565 - 615 nm filter (565 -

615 nm excitation). Alexa488 (green) was captured using an argon laser (488 nm) with band pass 500 - 530 filter (500 -

530 nm excitation). DRAQ5 (blue) for nuclei was obtained with a helium/neon red laser (633 nm) with a long pass 650

nm filter. All confocal images were exported as TIFF files without any modifications by LSM 510 Image.

b- Titin 9D10 immunostaining

At the end of the 110 min working heart perfusion protocol some Control, I/R or I/R + ONO-4817 hearts were fixed and

cryoprotected as described above. The cryoprotected hearts were cryosectioned and dried at room temperature. The

dried cryosections were rinsed and titin immunolabeling was accomplished using 9D10 antibody (mouse anti-titin 9D10




IgM, 1:100, developed by Dr. Marion Greaser and available at the Development Studies Hybridoma Bank at the

University of Iowa). 9D10 antibody is raised against the proline-glutamate-valine-lysine (PEVK) domain in the spring

region of titin and is used to measure titin immunostaining in Control, I/R and I/R + ONO-4817 groups. Secondary

antibody conjugated with fluorescent dyes (Alexa488-goat anti-mouse IgM, 1:40, Cat. No. 20142, Invitrogen) were

applied. DRAQ5 was applied to stain nuclei. To determine specificity of immunolabeling, primary or secondary

antibodies were omitted. The immunolabeled cryosections were observed by confocal microscopy for Alexa488 and

DRAQ5 as above.

Three dimensional surface rendered image construction

Z-stack images obtained from the LSM 510 were reconstructed to three dimensional images and surface rendered using

the Inside 4D module of AxioVison software (Version 4.6, Carl Zeiss Co., Jena, Germany). The same configuration of

the Inside 4D module was applied to all images.

In silico analysis

According to Turk et al.7 and Chen et al.

8 three MMP-2 cleavage motifs were chosen. These are PVS↓LRS, PVG↓LLA

and L/ISR↓LTA with MMP-2 cleavage site indicated by the arrow. These consensus sequences were shown to be

optimal MMP-2 cleavage motifs and, importantly, they showed high selectivity towards MMP-2 in comparison to other

MMPs including MMP-1, MMP-3, MMP-7, MMP-9 and MMP-147. These consensus sequences were aligned vs. N2B

mouse and human titin and the result was restricted to the top 20 with more than 60% homology using the SIM

Alignment tool for protein sequence.




Overlay assay to determine MMP-2 binding to titin

Skinned muscle fibers, stored in relaxing solution (40 mmol⁄ L BES pH 7.0, 10 mmol/L EGTA, 6.56 mmol/L MgCl2,

5.88 mmol/L ATP, 1 mmol/L DTT, 46.35 mmol/L potassium propionate and 15 mmol/L creatine phosphate) for 24

hours post-dissection, were incubated with 0.75 ug/ml of trypsin from bovine pancreas (Sigma, T9201) in relaxing

solution for 10 min at room temperature. The fibers were solubilized with a glass pestle, in solubilization buffer (750 ul

of 8 mol/L urea, 2 mol/L thiourea, 3% SDS, 50 mmol/L Tris-HCL pH 6.8, 0.03% bromphenol blue, 250 ul of 50%

glycerol with leupeptin, E-64, and PMSF, and 23 mg of DTT), heated for 5 min at 65ºC and centrifuged 11,000 g for 10

min to remove the particulate fraction. The proteins of the samples were separated by electrophoresis on 1% agarose gel

SDS-PAGE and run with Fairbank’s buffer (50 mmol/L Trizma base pH 7.5, 384 mmol/L glycine, 0.1% SDS, 10

mmol/L 2-mercaptoethanol) for 3 h and 20 min at 15 mA. Then the proteins were transferred to a PDVF (Millipore)

membrane, 2 h 30 min at 1.33 mA/cm2, and stained with Ponceau S to visualize the protein bands.

The overlay assay was performed using the transferred membrane as described previously9 followed by western

blot. Briefly, membranes were blocked in blocking buffer (Odyssey, 927-40000) for 1 h followed by a 4 h incubation

with recombinant MMP-2 (active human; EMD, PF023), 10 nmol/L in 10 ml blocking buffer (1.3 µl/20 ml). Both steps

were performed at room temperature on a gentle rocker. Following incubation, the blot was washed 3 X 5 min with 10

ml of blocking buffer and then incubated with primary antibody (MMP-2 (1:250), Millipore; MAB3308, mouse

monoclonal, 2 mg/ml). Following overnight incubation with the primary antibody in blocking buffer (0.05% Tween-20)

membranes were washed with PBST (0.1% Tween-20), subjected to secondary antibodies labeled with IR dyes (Goat

anti mouse 1:20,000; 800 green) for 1 h, and washed again with PBST. Membranes were scanned using a Li-Cor

Odyssey infrared imager at 700 nm and 800 nm.

In vitro degradation of titin

Two micrograms of rabbit longissimus dorsi titin were incubated with human recombinant 64 kDa MMP-2

(Calbiochem, 500:1, 50:1 and 5:1, titin:MMP-2 molar ratios) in 50 mmol/L Tris-HCl buffer (5 mmol/L CaCl2 and 150

mmol/L NaCl) at 37°C for 60 min. In additional experiments, MMP-2 was preincubated with either of the MMP




inhibitors GM-6001 (100 nmol/L) or ONO-4817 (10 µmol/L)

for 15 min at 37°C before adding to titin. This

concentration of GM-6001 was previously shown to inhibit the exogenously added MMP-2 activity under similar in

vitro conditions7, 10. The reaction mixtures were denatured with 2X urea sample buffer (8 mol/L urea, 2 mol/L thiourea,

3% SDS, 75 mmol/L dithiothreitol, 0.03% bromophenol blue, and 0.05 mol/L Tris-HCl; pH 6.8) at 100°C for 3 min, and

the proteins were separated by 2% SDS-PAGE strengthened with 0.5% agarose11. Protein bands were visualized with

silver stain (Invitrogen kit).




Fig. S1: A, Schematic representation of titin (N2B isoform) showing T12 and M8 epitopes near the Z-disc and at the M-

line regions, respectively. B, Three dimensional rendered images of T12 and M8 epitopes of titin in sarcomeres of the

left ventricular myocardium in aerobic normal control. a-c show frontal views of distributions of T12 (red) and M8

(green). d-f show clipping views of distributions of T12 (red) and M8 (green). Nuclei are blue in all images.




Fig. S2: Possible MMP-2 cleavage sites within mouse and human N2B titin. A, Titin schematic indicating putative

MMP-2 cleavage sites (*). Possible cleavage sites are located in both the I-band including near the Z-disc and the A-

band of titin. Panel B lists the putative MMP-2 cleavage sites in mouse and human titin shown in A; data is based from

three consensus cleavage motifs: PVS↓LRS, PVG↓LLA, and L/ISR↓LTA. Number correlates with the initial amino acid that is in parenthesis listed in the left column.

A

B




Fig. S3: A, Coomassie blue stain following electrophoretic separation of proteins from skinned muscle fibers treated for

10 min with trypsin, in order to increase T1 degradation to T2, and used for overlay/WB analysis seen in B. B, Overlay

assay showing MMP-2 binding to both T1 and T2. C, Quantification of the overlay/WB analysis T1. p < 0.001 (One-way ANOVA, n= 3), MHC (myosin heavy chain).

T1

T2




Fig. S4: In vitro degradation (60 min, 37°C) of rabbit longissimus dorsi titin by MMP-2. A, MMP-2 cleaved titin in a

concentration-dependent manner (1:500, 1:50 and 1:5 MMP-2:titin molar ratios). B, The cleavage of titin by MMP-2

was prevented by inhibiting the activity of MMP-2 with GM-6001 (100 nmol/L) or ONO-4817 (10 µmol/L).

Supplementary Fig. 4




Supplemental References

1. Kellermayer MS, Granzier HL. Calcium-dependent inhibition of in vitro thin-filament motility by native titin. FEBS

Lett. 1996; 380: 281-286.

2. Soteriou A, Gamage M, Trinick J. A survey of interactions made by the giant protein titin. J Cell Sci. 1993; 104 ( Pt

1): 119-123.

3. Nave R, Furst D, Vinkemeier U, Weber K. Purification and physical properties of nematode mini-titins and their

relation to twitchin. J Cell Sci. 1991; 98 ( Pt 4): 491-496.

4. Fritz JD, Swartz DR, Greaser ML. Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-

molecular-weight myofibrillar proteins. Anal Biochem. 1989; 180: 205-210.

5. O'Connell TD, Rodrigo MC, Simpson PC. Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol.

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6. Warren CM, Krzesinski PR, Greaser ML. Vertical agarose gel electrophoresis and electroblotting of high-molecular-

weight proteins. Electrophoresis. 2003; 24: 1695-1702.

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oriented peptide libraries. Nat Biotechnol. 2001; 19: 661-667.

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11. Tatsumi R, Hattori A. Detection of giant myofibrillar proteins connectin and nebulin by electrophoresis in 2%

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ISSN: 1524-4539 Copyright © 2010 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online

72514Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX

DOI: 10.1161/CIRCULATIONAHA.110.985317 2010;122;2002-2004; originally published online Nov 1, 2010; Circulation

Wolfgang A. Linke Proteins Degraded by Matrix Metalloproteinase-2

Molecular Giant Vulnerable to Oxidative Damage: Titin Joins the Club of

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Editorial

Molecular Giant Vulnerable to Oxidative DamageTitin Joins the Club of Proteins Degraded by Matrix Metalloproteinase-2

Wolfgang A. Linke, PhD

Matrix metalloproteinases (MMPs) are a family of �20enzymes that have long puzzled scientists for their

diversity in tissue location and function. True to their name,MMPs are best known for their role as degradation enzymescleaving multiple components of the extracellular matrix,including collagen, laminin, elastin, and fibronectin. How-ever, the traditional view that MMPs are proteases whoseonly function is to target matrix proteins does not hold upanymore. A large body of evidence suggests that the prote-olysis of extracellular matrix scaffold proteins causes releaseof growth factors and other bioactive molecules, indicatingthat MMPs are involved in the regulation of cell and tissuefunction.1 Moreover, recent proteomic screens for MMPsubstrates have shown that MMPs regulate a host of signalingmolecules, such as chemokines, both directly and indirectly.2

The novel functions of MMPs are also crucial to the cardio-vascular system. MMP enzymes can regulate, for instance,the bioavailability of vascular endothelial growth factor andthe potent vasodilator adrenomedullin.2 The diverse roles ofMMPs are relevant not only for physiological processes suchas heart development but also for pathological processes suchas remodeling events resulting from myocardial infarction,hypertensive heart disease, or cardiomyopathy.3

Article see p 2039Importantly, members of the MMP family can also have

intracellular localizations: MMP-2, which is abundantly ex-pressed in cardiac cells, is found in organelles like the nucleusand the mitochondria, as well as at the caveolae and thecytoskeleton and in particular at the sarcomeres (Figure).4 Thesarcomeric localization was shown to be mediated by MMP-2binding to troponin-I,5 �-actinin,6 and myosin light chain-1.7 Inthis series of studies, Schulz and coworkers4 established that the3 sarcomeric proteins are degraded by MMP-2 after activation ofthe protease during ischemia/reperfusion (I/R) damage, thuscontributing to acute contractile dysfunction under oxidativestress. The same laboratory now provides evidence in this issuethat the giant protein titin, a structural, elastic, and signalingmolecule spanning half of the sarcomere, is a novel target ofMMP-2 and is also degraded during I/R injury (Figure).8

Titin Functions and Alterations in HumanHeart Disease

Titin, the largest protein of the human body, has multipleimportant functions in the heart, providing both a scaffold formyofibril assembly and a hub for various molecules partici-pating in myocyte signaling pathways.9,10 Perhaps bestknown is the role of titin as an elastic spring contributing tototal myocardial passive (diastolic) stiffness. The titin springslocated in the I-band segment of the sarcomere (Figure) arethe predominant source of myocardial passive stiffness in thenormal human heart, even exceeding the contribution tostiffness coming from the collagen fibers of the extracellularmatrix.11 In end-stage failing hearts of patients with dilatedcardiomyopathy, the contribution of titin to total myocardialpassive stiffness decreases, whereas the contribution made bycollagen increases.11 Moreover, cardiac titin is expressed in 2main isoforms: the shorter and stiffer N2B isoform and thelonger, more compliant, N2BA isoform (which, in fact, hasmultiple splice pathways). Importantly, the N2BA:N2B iso-form ratio is increased in end-stage failing human heartscompared with donor hearts in both chronically ischemichearts of patients with coronary artery disease12 and nonis-chemic dilated cardiomyopathy hearts.11,13 The titin-isoformswitch decreases passive myocyte stiffness, probably as acompensatory mechanism counteracting an increased total pas-sive stiffness related to fibrosis.10 Dysregulation of titin stiffnessin cardiac failure could severely impair various mechanicalfunctions of the heart, including diastolic filling and earlydiastolic recoil, for which titin is held partially responsible,9

length-dependent activation as the molecular basis of the Frank-Starling mechanism,9 and contractile performance in systole,which is supported by the titin springs.10

Other alterations to cardiac titin occur in heart disease, such asreduced levels of protein kinase A– or protein kinase G–medi-ated titin phosphorylation, observed in human dilated cardiomy-opathy hearts.14,15 This titin phosphorylation deficit in end-stagefailing hearts increases myocyte passive stiffness and contributesto impaired diastolic function. Furthermore, cardiac-specificregions in titin are vulnerable to intramolecular disulfide bond-ing, which could stiffen the titin springs under oxidative stressconditions.16 These recent findings complement earlier studiesof ischemic and failing human hearts, which showed that titin isdegraded17 and appears highly disorganized in the cardiomyo-cytes when analyzed by immunofluorescence microscopy.18

Taken together, evidence for titin alterations in human heartdisease is manifold; derangement of titin in cardiac myocytesand reduced titin expression are obvious.

Proteases Targeting Cardiac TitinLittle has been known about specific proteases targeting cardiactitin in acute or chronic myocardial dysfunction. On the other

The opinions expressed in this article are not necessarily those of theeditors or of the American Heart Association.

From the Department of Cardiovascular Physiology, Institute ofPhysiology, Ruhr University Bochum, Bochum, Germany.

Correspondence to Wolfgang A. Linke, PhD, Department of Cardio-vascular Physiology, Institute of Physiology, MA 3/56, Ruhr UniversityBochum, D-44780 Bochum, Germany. E-mail [email protected]

(Circulation. 2010;122:2002-2004.)© 2010 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.orgDOI: 10.1161/CIRCULATIONAHA.110.985317

2002 at UNIVERSITY OF ALBERTA LIBRARY on January 20, 2011 circ.ahajournals.orgDownloaded from


hand, titin is notorious for rapid degradation ex vivo: Proteaseinhibitors are required to keep titin intact.12 A curious correlationhas been found between the extent of titin-isoform transitions inchronic ischemic human heart disease and the degree of cardiactroponin-I degradation or complex formation.12 A candidateenzyme potentially degrading titin is the ubiquitous Ca2�-dependent protease calpain-1, which has been implicated in thepathogenesis of myocardial dysfunction after I/R. Indeed,calpain-1 binds titin near the Z-disk19 and in vitro degradesvarious cytoskeletal and myofibrillar proteins isolated fromhuman cardiac tissue, including titin.20 Titin is also proteolyzedin cultured rat cardiomyocytes after doxorubicin treatment toinduce calpain-1.21

Similar to calpain-1, MMP-2 targets a subset of structuraland regulatory sarcomeric proteins, including titin (Figure).

The important news is that MMP-2 is activated by oxidativestress after I/R and degrades titin both in an ex vivo workingrat heart model of I/R and in an in vivo mouse model of I/R.8

However, it remains to be answered unambiguously whetherthe degradation of titin and other intracellular target proteinsunder oxidative stress in vivo is due mainly to the activity ofMMP-2 or calpain-1 or both. The authors address this issue byshowing reduced titin degradation in hearts from MMP-2 knock-out mice subjected to I/R in vivo, suggesting that MMP-2 reallydoes the job, at least in part.8 Along the same line, titin cleavageinduced by I/R damage was diminished by an MMP inhibitor. Insummary, in light of the importance of titin for diastolic andsystolic function, titin degradation by MMP-2 (and perhapscalpain-1) likely contributes to the acute contractile depressiondeveloping in stunned myocardium.

Figure. Diversity in cellular location and function of matrix metalloproteinase-2 (MMP-2), emphasizing the novel binding and cleavage targettitin. Oxidative stress, evoked by I/R injury, generates peroxynitrite (ONOO�), which activates MMP-2. The MMP is found, among others, inthe nucleus and mitochondria and at the cytoskeleton and caveolae. In the sarcomeres, activated MMP-2 degrades troponin-I (TnI) on thethin filaments, myosin light chain-1 (MLC-1) on the thick filaments, �-actinin in the Z-disk, and titin. MMP-2 binding to titin may preferentiallyoccur near the Z-disk, whereas cleavage of titin could take place at multiple sites in the Z-disk, I-band, A-band, and M-band portions. Titincleavage by MMP-2 might add to the acute myocardial contractile dysfunction in I/R. MMP-2 can also trigger signaling events leading toeither necrosis or apoptosis. In the extracellular compartment, MMP-2 can be activated by proteolytic cleavage.

Linke Titin Degradation by MMP-2 2003



Activation of MMP-2 During I/R InjuryThe intracellular activation of MMP-2 as an effector of acutemyocardial I/R injury in cardiomyocytes occurs via generationof peroxynitrite, a highly pro-oxidant species that increases tocardiotoxic levels within the first minutes of I/R.4 Peroxynitriteactivates cytosolic MMP-2 from an inactive precursor state, andMMP-2 now executes its actions on the sarcomeric proteins,troponin-I, myosin light chain-1, �-actinin, and titin (Figure). Aliet al8 propose that MMP-2 binding to titin occurs preferentiallynear the Z-disk, as suggested by colocalization of immunofluo-rescence signals for MMP-2 and Z-disk–adjacent titin antibod-ies. However, the MMP-2 binding site(s) on titin still need to bemapped precisely. Whether the binding sites on titin are also thesites of titin cleavage is another open question. From sequenceanalysis, the authors predict potential cleavage sites in variousregions along the titin filament, including the Z-disk–flanking,I-band, and A-band segments (Figure). Providing experimentalevidence in support of these predictions could be a challengingtask, given the huge size (3000 to 4000 kDa) of the titinmolecule.

Despite convincing evidence showing that MMP-2 de-grades titin, it is not clear whether this cleavage, or even thatof other sarcomeric proteins, is the principal cause of reducedcardiac work output during I/R injury. More likely, thecontractile depression after oxidative stress has additionalcauses. MMP-2 by itself probably has biological actions inother subcellular compartments, such as caveolae, mitochon-dria, and the nucleus, as well as in the extracellular space(Figure). The nature of these actions is still largely unknown,but both acute and long-term effects on contractile perfor-mance triggered by I/R damage could originate here. Finally,MMP-2 has links to signaling molecules associated with thedevelopment of apoptosis or necrosis, and it has been dis-cussed that higher levels of oxidative stress could preferen-tially induce these pathways, causing irreversible cell injury.4

ConclusionThe article by Ali et al8 is important because it demonstrates forthe first time an acute effect of a protease, MMP-2, on thestability of the giant elastic protein titin as a consequence ofoxidative stress during I/R. The finding provides another pieceof evidence for an intracellular role of MMP-2. The study alsouncovers a previously unrecognized link between the 2 compo-nents principally responsible for myocardial passive stiffness:extracellular matrix proteins (“classically” degraded by MMPs)and titin filaments. Both components are remodeled in heartdisease, with detrimental effects on cardiac performance. Inhib-iting MMP-2 activity by pharmacological intervention could bea desirable cardioprotective strategy in I/R injury and possiblyalso in chronic heart failure.

Sources of FundingThis study was supported by German Research Foundation grant Li690/7-2 (KFO155).

DisclosuresNone.

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KEY WORDS: Editorials � ischemic heart disease




Documents

Mohammad A. M. Ali Department of Pharmacology Supervisor ... · Mohammad A.M. Ali, MSc; Woo Jung Cho, MSc; Bryan Hudson, PhD; Zamaneh Kassiri, PhD; Henk Granzier, PhD; Richard Schulz,