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Journal of the American College of Cardiology Vol. 51, No. 4, 2008© 2008 by the American College of Cardiology Foundation ISSN 0735-1097/08/$34.00P

PRECLINICAL RESEARCH

Mitral Regurgitation AugmentsPost-Myocardial Infarction RemodelingFailure of Hypertrophic Compensation

Ronen Beeri, MD,* Chaim Yosefy, MD,* J. Luis Guerrero, BS,* Francesca Nesta, MD,*Suzan Abedat, MSC,§ Miguel Chaput, MD,* Federica del Monte, MD, PHD,†Mark D. Handschumacher, BS,* Robert Stroud, MS,� Suzanne Sullivan, BS,* Thea Pugatsch, PHD,§Dan Gilon, MD, FACC,§ Gus J. Vlahakes, MD, FACC,‡ Francis G. Spinale, MD, FACC,�Roger J. Hajjar, MD, FACC,† Robert A. Levine, MD, FACC*

Boston, Massachusetts; Jerusalem, Israel; and Charleston, South Carolina

Objectives We examined whether mitral regurgitation (MR) augments post-myocardial infarction (MI) remodeling.

Background MR doubles mortality after MI, but its additive contribution to left ventricular (LV) remodeling is debated and hasnot been addressed in a controlled fashion.

Methods Apical MIs were created in 12 sheep, and 6 had an LV-to-left atrial shunt implanted, consistently producing re-gurgitant fractions of �30%. The groups were compared at baseline, 1, and 3 months.

Results Left ventricular end-systolic volume progressively increased by 190% with MR versus 90% without MR (p �

0.02). Pre-load–recruitable stroke work declined by 82 � 13% versus 25 � 16% (p � 0.01) with MR, with de-creased remote-zone sarcoplasmic reticulum Ca2�-ATPase levels (0.56 � 0.03 vs. 0.76 � 0.02, p � 0.001), anddecreased isolated myocyte contractility. In remote zones, pro-hypertrophic Akt and gp130 were upregulated inboth groups at 1 month, but significantly lower and below baseline in the MR group at 3 months. Pro-apoptoticcaspase 3 remained high in both groups. Matrix metalloproteinase (MMP)-13 and membrane-type MMP-1 wereincreased in remote zones of MR versus infarct-only animals at 1 month, then fell below baseline. The MMPtissue inhibitors rose from baseline to 3 months in all animals, rising higher in the MI � MR–group borderzone.

Conclusions In this controlled model, moderate MR worsens post-MI remodeling, with reduced contractility. Pro-hypertrophicpathways are initially upregulated but subsequently fall below infarct-only levels and baseline; with sustainedcaspase 3 elevation, transformation to a failure phenotype occurs. Extracellular matrix turnover increases in MRanimals. Therefore, MR can precipitate an earlier onset of dilated heart failure. (J Am Coll Cardiol 2008;51:476–86) © 2008 by the American College of Cardiology Foundation

ublished by Elsevier Inc. doi:10.1016/j.jacc.2007.07.093

cv

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xpansion of infarcted tissue begins acutely after myocardialnfarction (MI). A more gradual remodeling process, how-ver, also involves the noninfarcted areas (1,2); initiallyompensatory, this process subsequently becomes mal-daptive, generating a larger, more spherical, and poorly

rom the *Cardiac Ultrasound Laboratory, †Cardiovascular Research Center, andCardiac Surgery Department, Massachusetts General Hospital and Harvard Medicalchool, Boston, Massachusetts; §Heart Institute and Cardiovascular Research Lab-ratory, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; and theCardiothoracic Surgery Department, Medical University of South Carolina,harleston, South Carolina. Supported, in part, by a Grant-in-Aid (0350422N)

f the American Heart Association; grants R01HL38176, R01HL72265, and24HL67434 from the NIH/NHLBI; and a grant from the USA-Israel Binationalcience Foundation (no. 2001037).

pManuscript received May 3, 2007; revised manuscript received July 9, 2007,

ccepted July 16, 2007.

ontracting ventricle (3–7), associated with reduced sur-ival (2,8,9).

See page 487

Myocardial infarction can also cause mitral regurgitationMR) by altering ventricular geometry and function (10–2). Such ischemic MR doubles the risk of death after MI13,14). Severe nonischemic MR has been shown to pro-ote left ventricular (LV) remodeling and reduce survival

15–19). However, whether more typically moderate isch-mic MR does so remains controversial. A recent paperaintained that “ischemic MR is a consequence, not a

ause, of post-infarction remodeling,” because remodeling

ersisted despite annuloplasty with apparent MR reduction

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477JACC Vol. 51, No. 4, 2008 Beeri et al.January 29, 2008:476–86 MR Augments Post-MI Remodeling

20). Practically, “MR might not represent a target forherapy if indeed it is not the major culprit” (21), adding tohe uncertainty regarding the risk versus benefit of repairingschemic MR (22).

In patients, ischemic myocardial changes and regurgita-ion develop concurrently, precluding the ability to separateheir effects and analyze their interaction directly (23). Thisequires a model in which MR can be varied independentlyn the presence of MI, but without interventions such asnfarct patching (24–27) that might themselves influenceemodeling. Most experimental studies of post-MI remod-ling have used an infero-posterior MI model (27,28); thisas the disadvantage of linking the MI-induced remodelingo the development of MR. We therefore tested thencremental contribution of MR in a controlled mannersing a model in which the MI by itself does not cause MR,nd the MR can be created in a standardized and fixedashion (29,30).

In such a study, it must be recalled that remodeling is aomplex process, involving changes not only in ventriculareometry (3) and in global and regional myocardial function31), but also in calcium handling, molecular signals pro-oting hypertrophy, cardiomyocyte viability (32–34), and

xtracellular matrix turnover (35). Intracellular pathwayshat promote compensatory hypertrophy also tend, if chron-cally activated, to drive cells down pathways to cell death bypoptosis and thus to loss of contractile elements, withurther deterioration of cardiac function (Fig. 1). Accord-ngly, it is important to correlate evidence of remodeling athe molecular, cellular, and whole-heart levels (36). Weherefore tested the hypothesis that MR accompanying MIroduces greater remodeling than a comparable infarctlone, and may be associated with a different progression ofolecular events, with transition from signals promoting

ypertrophy to those inducing failure.

Figure 1 The Vicious Cycle of Remodeling

EC � extracellular; LV � left ventricle; MR � mitral regurgitation.

o

ethods

R model. A modification ofhe chronic MR sheep model ofankin et al. (29) was imple-ented in conjunction with Luisuerrero, a surgeon experienced

n physiological modeling, usingn 8-cm long, 8-mm diametereinforced Teflon (PTFE) graftEdwards Lifesciences, Irvine,alifornia) (cross-sectional areaf 0.50 cm2) implanted underterile conditions into the LVase and left atrial (LA) append-ge with intramuscular portionstiffened with epoxy resin (Fig.). The regurgitant flow wasonfirmed during each thoracot-my (see the following text) us-ng a transonic flow probe andolor Doppler echocardiography imaging. The standardizedhunt diameter and length have produced consistent levelsf MR (regurgitant fractions of approximately 30%, corre-ponding to moderate MR [37]). Animals were treated witheparin (3 days) and then oral aspirin.nimal studies. A total of 12 Dorsett hybrid sheep (20 to0 kg) were loaded for 3 days with amiodarone (200 mgrally twice a day) then anesthetized with thiopentothalodium (0.5 ml/kg), intubated, and ventilated at 15 ml/kgith a 2% isoflurane-oxygen mixture. All received glyco-yrrolate (0.4 mg intravenously) and prophylactic vancomy-in (0.5 g intravenously) and amiodarone (150 mg intrave-ous drip over the course of the operation). Surfacelectrocardiogram (ECG) was monitored, and a sterile lefthoracotomy performed with pericardial incision and cre-tion of a cradle. A high-fidelity micromanometer-tippedatheter (Millar Instruments Inc., Houston, Texas) waslaced into the LV. After baseline 2-dimensional (2D) and-dimensional (3D) echocardiography imaging, an antero-pical MI was produced by ligating the mid- to distal leftnterior descending coronary artery, known to produce aubstantial MI without MR (38). An immediate 2D echo-ardiography apical image was performed to confirm thateptal wall motion abnormality extended approximatelyne-third of the way from LV apex to base for standard-zation. Mitral regurgitation equivalent volume overloadas created for 90 days in 6 sheep with an open LV-to-LA

hunt (referred to as the infarct � MR group); in the other, the shunt was closed (infarct-only group). In addition tonalgesia, propranolol, 1 mg intravenously in 2 dividedoses, was given for evident stress and sinus tachycardia�150) upon extubation. Antibiotics (cephapirin, 0.5 gntravenously) and analgesics (buprenorphine, 0.3 mgwice a day) were administered for the next 5 days, and

Abbreviationsand Acronyms

EF � ejection fraction

LA � left atrium/atrial

LV � leftventricle/ventricular

MI � myocardial infarction

MMP � matrixmetalloproteinase

MR � mitral regurgitation

MT-MMP � membrane-typematrix metalloproteinase

SERCA2a � sarcoplasmicreticulum Ca2�-ATPase

TIMP � tissue inhibitor ofmatrix metalloproteinase

2D � 2-dimensional

3D � 3-dimensional

ral amiodarone (200 mg twice a

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478 Beeri et al. JACC Vol. 51, No. 4, 2008MR Augments Post-MI Remodeling January 29, 2008:476–86

During repeat sterile thoracotomy at day 30, 3D echo-ardiography was performed to evaluate LV remodeling andunction, with directed biopsies of the noninfarcted myo-ardium near and remote from the border zone using aruCut needle (Cook, Winston-Salem, North Carolina).t day 90, 3D echocardiography and blood sampling were

epeated at thoracotomy, followed by euthanasia and whole-eart pathological and myocyte size and contractility studieshat require considerable tissue. The animal studies con-ormed to National Institutes of Health Guidelines fornimal Research (Guide for the Care and Use of Labora-

ory Animals, National Research Council, Washington,C, 1996) and were approved by the institutional animal

are committee.D echocardiography and LV function. Three-imensional quantitation is necessary because LV shapehanges with chronic MR, as well as MI (39). Rotatedpical images were obtained at 10° intervals with an epicar-ial 5-MHz transesophageal echocardiography probe (So-os 7500, Philips Medical Systems, Andover, Massachu-etts), rotated by software and gated to ECG andespiration. Digital images were analyzed on a workstationith custom programs (40). Endocardial surfaces were

raced to calculate LV volumes and ejection fraction (EF),nd coded for wall motion abnormality to produce auantitative 3D wall motion map validated against a 36-rystal sonomicrometer array (41). Remodeling was quan-ified in terms of increasing LV volumes (42). Regurgitantraction was calculated as transonic MR-equivalent flowivided by LV ejection volume, with cross-correlation byulsed Doppler time–velocity integral of shunt flow multi-lied by shunt cross-sectional area. At thoracotomy, thenitial and final pressure–volume loops were obtained using

illar catheters and 4 sterile subendocardial crystals (Sono-etrics Corp., London, Ontario, Canada), with an inferior

ena cava occlusion to obtain the end-systolic pressure–olume relation for contractility (43) and pre-load–ecruitable stroke work (44). Crystals were located at the LVpex and base (between aorta and mitral annulus) and

Figure 2 Schematic and 2D EchocardiographicImage of the LA-LV Shunt Creating MR

Single arrows indicate flow direction; paired arrows indicate shunt exit fromthe LV. LA � left atrial; MI � myocardial infarction; 2D � 2-dimensional; otherabbreviations as in Figure 1.

nteriorly and posteriorly along a midventricular short-axis. s

rystals were not placed at day 30 to maximize sterility andurvival. dP/dt was obtained by the high-fidelity Millaratheter (Millar Instruments, Houston, Texas).

yocyte size and contraction. Small (5 to 10 g) areas ofissue were removed at euthanasia from the noninfarctedV and perfused via the corresponding coronary artery for 5in with low-magnesium Krebs-Henseleit (KH) cardiople-

ia solution. Myocardial cells were isolated using a well-stablished method (45,46). The LV sections were perfusedith Ca2�-free KH containing collagenase (Worthingtoniochem Corp., Lakewood, New Jersey) and hyaluronidase

Sigma, St. Louis, Missouri). Ventricular muscle tissue wasinced into small pieces and placed in enzyme buffer I

Sigma) in the presence of trypsin and deoxy-ribonuclease.he tissue was washed in a 3:1 mixture of Ca2�-free KH inulbecco’s phosphate-buffered saline (GIBCO, Gaithers-

urg, Maryland). Cells were loaded with the Ca2� fluores-ent indicator Fura-2 for 15 min in a light-sealed chamberounted on an inverted microscope. Myocyte length andidth were measured offline on 50 consecutive cells using

ommercial image analysis software to establish proportionsf normal short cells versus elongated cells characteristic ofccentric hypertrophy in the failing phenotype. The cellsere excited to contract using platinum electrodes in theath, and a dual excitation spectrofluorometer recordeduorescence emissions (505 nm from exciting wavelengths40 mm and 380 mm) to calculate intracellular calciumoncentrations. Cell motion was measured by a video-edgeotion detector (Crescent Electronics, Sandy, Utah) (45).olecular assays. We measured levels of several molecular

pecies that determine cell contractility, that modulate cellypertrophy and death, and that are responsible for extra-ellular matrix turnover. All protein assays were performednd specimens pooled from the same treatment group andtage.alcium cycling proteins: sarcoplasmic reticulum Ca2�-TPase (SERCA2a) and phospholamban. Sarcoplasmic

eticulum membranes were obtained from the myocardialiopsy specimens using a sucrose gradient centrifugationethod (47). Myocardium was homogenized and centri-

uged at 7,700 g for 20 min, and the supernatant filtered andentrifuged at 136,000 g for 60 min. The pellets, resus-ended and rehomogenized in 10% sucrose buffer, wereentrifuged at 55,000 g in a 10% to 40% sucrose gradient,nd the bands between 30% and 40% enriched in sarcoplas-ic reticulum were collected, centrifuged at 136,000 g for

0 min, and the pellet resuspended in freezing buffer.rotein was determined by a modified Bradford procedure

using bovine serum albumin for the standard curve).roteins were separated from the sarcoplasmic reticulumesicle preparation from tissue frozen in liquid nitrogen, andn immunoblot using monoclonal anti-SERCA2 and phos-holamban performed. Proteins levels were normalizedgainst total myocardial protein.ro-hypertrophic and anti-apoptotic cascades. We mea-

ured levels of Akt (protein kinase B) and gp130, which are

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479JACC Vol. 51, No. 4, 2008 Beeri et al.January 29, 2008:476–86 MR Augments Post-MI Remodeling

oth at their respective levels (cytosol and membrane)mportant crossroads in pro-hypertrophic signaling; andaspase 3, the final common pathway for intracellularignaling of apoptosis. Western blot analysis was performedn cell lysates from biopsies at baseline and days 30 and 90.ntibodies binding for anti-gp130, anti-phospho Akt, and

ntiactivated caspase 3 (48) were detected with aeroxidase-conjugated antimouse IgG and chemilumines-ence. �-Actin was used as a housekeeping control. Densi-ometry of the blots was assessed using commercially avail-ble software (Photoshop 5, Adobe Systems Inc., San Jose,alifornia).odulators of extracellular matrix turnover: matrix met-

lloproteinases (MMPs). Assays were performed forembrane-type matrix metalloproteinase-1 (MT1-MMP),MP-13, tissue inhibitor of matrix metalloproteinase

TIMP)-1, and TIMP-4 by immunoblotting using methodsreviously described (35). Left ventricular myocardial sam-les were homogenized and concentrated to prevent MMProteolytic activation and degradation. Trypsin (2.5 �M; 5in) was used to cleave nascent MMPs to fully activate

nzyme moieties and/or facilitate the removal of endoge-ous inhibitors. Relative TIMP-1, TIMP-4, MT1-MMP,nd MMP-13 levels were then measured by immunoblot-ing. A positive control was included in all immunoblotsMMP-13: CC068; MT1-MMP: CC1042) (Chemicon,emecula, California).tatistics. Statistical analysis was performed using a Stu-ent t test for continuous variables and a chi-square test forategorical variables (Stat View 5, SAS Institute Inc., Cary,orth Carolina). A value of p � 0.05 was considered

ignificant. Multiple comparisons were performed usingnalysis of variance (for repeated measurement whereeeded) and a Student-Neumann Keuls post-hoc test.nterobserver and intraobserver variability for 3D echo-easured LV volumes and EF in our laboratory is 3.5%.

esults

emodeling: LV volumes. Left ventricular end-diastolicolume relative to the acute post-MI value increased by anverage of 26% in the infarct-only group versus 130% inhose with volume overload (p � 0.02). Left ventricularnd-systolic volume progressively increased by 190% with

R-type volume overload versus 90% with MI only (p �.02) (Figs. 3 and 4).emodeling: LV function. Pre-load–recruitable strokeork from pressure-volume loops declined more in the

nfarct � MR group by 82 � 13% versus 25 � 16% ofaseline in the infarct-only group (p � 0.01). Left ventric-lar dP/dt also declined progressively in the MR group to 52

9% of baseline, but only by 22 � 12% in the shams (p �.04) (Fig. 4). These results were corroborated by reducedontractility of isolated cells with reduced calcium transientsnd increased diastolic intracellular calcium concentration

Tables 1 and 2). The diastolic relaxation constant tau was s

ore prolonged in the infarct � MR group (78 � 23 ms vs.4 � 13 ms, p � 0.01).alcium cycling proteins. As shown in Figure 5,ERCA2a abundance was downregulated in the infarctegion and border zone compared with the remote region inoth infarct � MR and infarct-only sheep LVs. There waslower level of SERCA2a in the remote zone of infarct �R than of infarct-only sheep. Phospholamban was un-

hanged in all regions. These changes are typical foremodeling myocardium.ntracellular signaling pathways. Akt, a pro-hypertrophicnzyme (49), was upregulated in the remote zones of infarct

MR and infarct-only animals at 1 month, but was lowern animals with than without MR at 3 months (Fig. 6). Inhe infarct � MR group at 3 months, Akt also fell belowaseline of the same group. The same pattern was observedor the interleukin-6-family receptor subunit gp130 (50).xpression of activated caspase 3 (32–34,51), the final

ommon pathway of apoptosis, remained elevated in allegions of both groups at both 1 and 3 months.

ell morphology. Myocytes in the MR group were elon-ated, characteristic of myocardial failure (45), comparedith cells from the MI-only animals (Fig. 7).xtracellular matrix and MMPs. In the remote myocar-ium of infarct � MR sheep, at initial follow-up, there wassignificant increase in MMP-13 and MT1-MMP, fol-

owed by a decrease at the time of sacrifice (Fig. 8, “shuntemote” bars): MMP-13 increased to 238 � 29% of baselinet follow-up, and then fell to 79 � 13% of baseline at 3onths sacrifice; MT1-MMP increased to 165 � 23% of

aseline at follow-up, and then fell to 85 � 16% of baselinet sacrifice. In the remote myocardium of infarct-only

Figure 3 Echocardiographic 3D Reconstructionsof the Endocardial Surface of the LV

Echocardiographic 3-dimensional (3D) reconstructions of the endocardial sur-face of infarct (MI) only (A and B) and infarct � MR (C and D) animals at dias-tole (top) and systole (bottom). Note the enlarged and spherical infarct � MRventricles. Abbreviations as in Figures 1 and 2.

heep, the rise and fall of MMP-13 did not reach statistical

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480 Beeri et al. JACC Vol. 51, No. 4, 2008MR Augments Post-MI Remodeling January 29, 2008:476–86

ignificance (170 � 85% of baseline at initial follow-up; 7522% at sacrifice), and MT1-MMP did not significantly

Figure 4 Measurement of LV Volumes and Function

End-diastolic volume (EDV) (A) and end-systolic volume (ESV) (B) of infarct-only(yellow bars) and infarct � MR (red bars) sheep at baseline (pre), 1 monthfollow-up (f/u), and 3 months sacrifice. (C and D) dP/dt and pre-load–recruit-able stroke work (PRSW) of infarct-only (broken lines) and infarct � MR (solidlines) sheep at pre, f/u, and 3 months sacrifice. *p � 0.05. Abbreviations asin Figure 1.

ncrease at 1 month (Fig. 8, “sham remote” bars). In ther

order zones of the infarct-only sheep, there was a signifi-ant decrease in MT1-MMP at sacrifice (to 60 � 8% ofaseline), with levels maintained in the infarct � MRroup. In contrast, TIMP-4 showed progressive rises fromaseline to initial follow-up and then to sacrifice in theorder and remote zones of both groups, with significantlyigher levels at sacrifice in the infarct � MR group borderone (207 � 9% vs. 152 � 10% of baseline, p � 0.05).issue inhibitor of matrix metalloproteinase-1 showed a

imilar pattern of progressive increases in the infarct � MRroup remote zone, with a significant late rise at sacrifice.

iscussion

yocardial infarction, with loss of contracting myocytes,eads to a sequence of events aimed at preserving cardiacutput, including increased LV end-diastolic volume tougment pre-load. Cells in noninfarcted areas hypertrophyy addition of contractile elements in series, creating eccen-ric hypertrophy without increased wall thickness. Thisllows the LV to dilate, a process that is also promoted byltered MMP activation with extracellular matrix degrada-ion (52), and acceleration of cell death by apoptosis (32,53).his process eventually culminates in systolic enlargement,

xtensive fibrosis, and LV failure, the extent of whichredicts further cardiac events (2,9). Volume and pressureverload increase wall stress, which can aggravate remodel-ng (54) and promote eccentric hypertrophy through in-reased fiber stretch (Fig. 1). Conversely, hemodynamicallynloading the LV reverses the functional and cellulartigmata of cardiac failure as well as the remodeling process55,56). Neurohumoral activation and activation of inflam-atory mediators and cytokines initially compensate for

educed cardiac output but, in the long term, mediatencreased load, cell death, and cardiac fibrosis (50,57–59).ncreased wall stress also up-regulates the production and

solated Cardiomyocyteontractility in the Different Groups

Table 1 Isolated CardiomyocyteContractility in the Different Groups

ContractilityMeasurements

NormalControl Sheep Infarct � MR Infarct Only

Cell shortening (%) 5.7 � 0.4 2.6 � 0.7* 3.5 � 0.3*†

t90% (ms) 390 � 82 676 � 68* 510 � 59*†

p � 0.01 compared with normal control sheep; †p � 0.01 compared with infarct � mitralegurgitation (MR) group.

t90% � time to 90% relaxation.

ntracellular Calciumeasurements in the Different Groups

Table 2 Intracellular CalciumMeasurements in the Different Groups

CalciumMeasurements

NormalControl Sheep Infarct � MR Infarct Only

Diastolic (Ca2�) (nM) 230 � 41 320 � 21* 275 � 22*†

Systolic (Ca2�) (nM) 796 � 24 535 � 44* 714 � 43*‡

t90% (ms) 361 � 34 542 � 38* 455 � 43*†

p � 0.01 compared with normal control sheep; †p � 0.03 compared with infarct � mitral

egurgitation (MR) group; ‡p � 0.005 compared with infarct � MR group.

t90% � time to 90% relaxation of the calcium signal.

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481JACC Vol. 51, No. 4, 2008 Beeri et al.January 29, 2008:476–86 MR Augments Post-MI Remodeling

elease of MMPs, which are believed to be pivotal inltering the extracellular matrix in ways that promoteentricular dilatation (60). Their transgenic activation in-uces congestive heart failure (61), and their inhibitionttenuates post-MI remodeling (62,63).

Mitral regurgitation, caused by alterations in ventriculareometry and function after MI (10,12), can itself initiatehe remodeling cascade, and causes progressive deteriorationf ventricular function at a cellular and molecular level15–17). Mitral regurgitation considerably alters the load onhe LV. It increases diastolic wall stress, which can induceccentric LV hypertrophy and subsequent dilatation andailure (18), and it thereby increases early systolic wall stress.lthough MR permits LV emptying during systole into the

ower-pressure LA, it has been shown actually to increasend-systolic wall stress and, hence, afterload in patients withhronic MR (64,65). Moreover, MR induces further LVilatation due to activation of neurohumoral and cytokineomponents of the remodeling cascade (66–68). As MR isoth a cause and a result of LV remodeling, it canotentially exacerbate the vicious cycle spiraling down toardiac failure unless the remodeling or the MR is reversed69–71).

Regarding clinical implications, knowing whether thearallel occurrence of MR and MI causes more pronouncedemodeling is critical, because at least MR can be eliminatedy repairing or replacing the valve, and thus relieving theolume overload induced by it. Separating these 2 dynamic

Figure 5 SERCA2a and Phospholamban Quantification

(A) Quantification of sarcoplasmic reticulum Ca2�-ATPase (SERCA2a) activity in reImmunoblots from different regions of the myocardium in different groups for SERCdium that was homogenized and protein extracted. Abbreviations as in Figures 1 a

rocesses in an experimental model is a challenge, because o

n most existing models they are linked. One recent study ofnferior MIs, in fact, concluded that prevention of ischemic

R does not influence the outcome of remodeling (20),espite benefits in nonischemic models (18) and concerns inonischemic patients (72). In our model, we therefore

mplanted an LV-to-LA shunt, which created a moderatend standardized MR-like regurgitant flow, independent ofmedium-sized antero-apical MI that by itself does not

ause MR.In this controlled model, moderate MR worsened

ost-MI remodeling. Specifically, it induced large diastolicnd systolic LV volumes reflecting volume overload andeduced global function. Reduced contractility was alsopparent in less pre-load–dependent physiological measuresuch as pre-load–recruitable stroke work. These changesaralleled decreased single-cell contractility in the nonin-arcted myocardium with reduced SERCA2a levels, whichirectly correlate with cell contraction (46).In contrast to progressive changes in the LV, severalediators of the hypertrophic process underwent biphasic

hanges in the noninfarcted myocardium of animals withR-type volume overload. These include gp130, a glyco-

rotein that forms heterodimers to produce different recep-ors, notably of the interleukin 6/CT1/LIF family. Thectivation of these receptors has been linked to cardiomyo-yte hypertrophy (50,73), and their reduced abundance,anifested by reduced gp130 levels, has been associatedith the transition from hypertrophy to failure (74,75). Akt,

border, and infarct regions of experimental and normal control sheep hearts. (B)nd phospholamban. Note that in the infarct region, there was residual myocar-

mote,A2a and 2.

r protein kinase B, is a serine-threonine kinase activated by

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482 Beeri et al. JACC Vol. 51, No. 4, 2008MR Augments Post-MI Remodeling January 29, 2008:476–86

everal pro-survival and -hypertrophic factors, among othersardiotrophin-1 (76), acting through the gp130-containingeceptor, or growth factor receptors activatinghosphoinositide-3 kinase (77).The constitutive activationf Akt in a model of rat ischemia-reperfusion injury reducedell death and improved function (78). Like gp130, reducedevels of Akt are related to increased apoptosis and aransition from compensatory hypertrophy to a failurehenotype (49). In our model, while both infarct � MR andnfarct-only sheep had elevated expression of Akt and gp130fter 1 month, the MR sheep demonstrated a notableeduction in these protein levels after 3 months, comparedith the no-MR animals and with baseline. The pro-ypertrophic process, which begins in both groups at 1onth, continues in the animals without MR but is down-

egulated at 3 months in the MR group, in association withontinued adverse remodeling and LV dysfunction. Thisecline may represent earlier termination of the hypertro-

Figure 6 Protein Levels of Akt, gp130, and Caspase 3

Protein expression of Akt, gp130, and activated caspase 3 from infarct-only(broken lines) and infarct � mitral regurgitation (solid lines) sheep at base-line, 1 month follow-up (f/u), and 3 months sacrifice. Note the biphasic changein infarct � mitral regurgitation animals for gp130 and Akt expression at fol-low-up (high) and sacrifice, while no significant change is noted in caspase 3levels. Values are � standard error of the mean, *p � 0.01.

hic process or transition to a failure phenotype specific to

he persistent-MR situation; either way, it represents failuref the hypertrophic process. The ongoing drive to cell death,ith the suppressed pro-hypertrophic drive, further pro-otes the transition to failure. These findings are consistentith the emerging view of LV remodeling in general as a

ailed attempt at compensation for initial stresses on theyocardium—in this case, exacerbated by the MR volume

verload (7,74); in others, it reflects the transition fromypertensive hypertrophy to failure (79,80).There was also excessive activation of MMPs, notablyMP-13 and MT-MMP1, in the noninfarcted remote

one of the MR sheep. This could induce excess extracel-ular matrix turnover and potentially exacerbate remodeling.

owever, these same MMP levels fell at sacrifice, with theall in remote-zone MT-MMP1 being significantly greatern the MR sheep, and the fall in MMP-13 being significantnly for the MR group. As this change parallels theown-regulation of the pro-hypertrophic signaling process,t could be also part of the failure of compensation byimiting the adaptive geometrical changes in myocardialypertrophy. Matrix metalloproteinase inhibitors showed aifferent pattern, with a progressive rise from baseline toacrifice that was significantly greater with MR. Thisuggests a possible shift from matrix turnover to matrixccumulation and stabilization over time, a response typicalf remodeling myocardium (81). However, it is clear thateither the MMP nor the TIMP changes adequatelyompensate for the remodeling process, as LV volumerogressively rose while systolic function fell.This study has several limitations. Ischemic MR affectingnative valve often progressively increases (28,69–71), but

s inherently linked to the underlying MI and is nottandardized. Based on the motivation for this study, it was

Figure 7 Photomicrographs of Separated Cardiomyocytes

Elongated myocytes from sheep with MI and MR shunt (A) versus more normalcells from MI-only animals (B). Note the different scales used to accommodatethe elongated cells of the MI � MR sheep within the field. Abbreviations as inFigures 1 and 2.

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483JACC Vol. 51, No. 4, 2008 Beeri et al.January 29, 2008:476–86 MR Augments Post-MI Remodeling

ritical to separate the 2 processes of infarction and regur-itation to determine the incremental role of MR mostirectly, and to do so with a standardized orifice, whichrovided stable regurgitant fractions of �30% throughouthe study. Although the LV also fills through this valvelessrifice, based on prior experience, this should not impor-antly alter LV filling (29).

We did not assess whether any one of the molecularhanges reported in the present study is causative. Wentended to test whether differences in remodeling based on

R are associated with molecular changes described in theemodeling process. Determining which factors are caus-

Figure 8 MMP Abundance

Levels of membrane-type (MT) matrix metalloproteinase (MMP)1 and tissue inhibitinfarct � mitral regurgitation (red bars) animals at first follow-up (T2) and sacrifice� mitral regurgitation relative to myocardial infarction only animals for the same z

tive versus concomitants, and studying all possible factors, c

s beyond the scope of this study. Nevertheless, we analyzedathways extensively linked to the pro-hypertrophic andemodeling processes, and causally implicated throughransgenic and pharmacologic modification (61,74,78).

While the impact of isolated MR is important to explore,he study specifically addresses the challenge posed by theecent literature (20), which can be stated as an alternativeypothesis that remodeling in ischemic MR is caused only orredominantly by the underlying infarct, not the MR. Thisequired comparing infarct-only versus infarct � MRroups. The data do show important effects of the infarctlone, with end-systolic volume nearly doubling, and in-

atrix metalloproteinase (TIMP)-4 in the infarct-only (“sham”, green bars) andime points. *p � 0.05 relative to baseline; #p � 0.05 for myocardial infarctiond stage; $p � 0.05 for sacrifice relative to initial follow-up.

or of m(T3) t

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reases in gp130, Akt, and caspase. The changes with infarct

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484 Beeri et al. JACC Vol. 51, No. 4, 2008MR Augments Post-MI Remodeling January 29, 2008:476–86

lus MR, however, were greater, with end-systolic volumeearly tripling, and a different pattern of activation followedy exhaustion of hypertrophic- and apoptotic-signalingolecules gp130 and Akt. The findings, therefore, suggest

n additive rather than exclusive role of MR. This is sup-orted by our data (unpublished results) from sheep with andentical shunt and no MI, which did not cause significantemodeling in the same 3-month time-frame in terms ofither volume or contractility changes.

Recently, contribution of MR to post-MI remodeling haseen challenged by Guy et al. (20) in an inferior MI modeln which either MR was reduced using an undersizednnuloplasty ring repair, or MI was restrained using a mesh.he MR repair group remodeled more, leading to the

uggestion that the MR is not causative, but may be aonsequence of post-MI remodeling. The main caveat tohese conclusions is that undersized ring annuloplasty mayy itself alter LV mechanics, as recently shown (82), so as toxacerbate remodeling. Thus, the excess remodeling ob-erved may have been caused, at least partially, by the repairtself, which was performed 2 weeks before infarct creation.he difficulty in eliminating MR as a potential contributor

o continued remodeling by annuloplasty has also beeneported (72). Another recent paper (83) has suggested thatepairing MR in a subgroup of patients with LV dysfunc-ion has no survival benefit. While this is intriguing, theatient population in that study had prominent LV dys-unction at baseline (�30% EF), and thus it is possible thatepair was performed too late for many to benefit in termsf LV function or outcome. Based on their inclusionriteria, the patients also had at least moderate-to-severe

R—more severe than in our model. Further, unlike thisI model, roughly 40% of the patients did not have

oronary artery disease as the basis for their LV dysfunction.hus, these clinical data cannot readily be compared withur relatively mild MI model with normal baseline LVunction and moderate MR. There is definitely a need for aandomized controlled study to clinically address the issuef mitral valve repair in moderate ischemic MR.

onclusions

his study demonstrates that MR accompanying MI pro-uces greater ventricular remodeling than a comparablenfarction alone, with an earlier transition to a failurehenotype. This is the case even for only moderate MR,ost typical of the ischemic situation, for which the greatest

ncertainty exists regarding impact on the heart and needor intervention. The changes at the level of the whole heartarallel cellular and molecular abnormalities in the nonin-arcted myocardium that reflect the complex remodelingrocess. These molecular events also progress differentlyith MR than with comparable infarction alone, with an

nitial rise in pro-hypertrophic and antiapoptotic signals,ollowed by their exhaustion. Recognizing this pattern can

otivate a search for the responsible molecular “switch,” so

1

hat decompensation might be limited or reversed, avertinghe earlier onset of dilated heart failure precipitated by MRhat may portend a worse prognosis.

eprint requests and correspondence: Dr. Robert A. Levine,ardiac Ultrasound Laboratory YAW5, Massachusetts Generalospital, 55 Fruit Street, Boston, Massachusetts 02114. E-mail:

levine@partners.org.

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