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peckle-tracking 2-dimensional strain echocardiography:new noninvasive imaging tool to evaluate acute

ejection in cardiac transplantation

alen M. Pieper, PhD,a,b Akash Shah, BA,a Leanne Harmann, BA, RDCS, RDMS, RVT,c

rian C. Cooley, PhD,d Irina A. Ionova, MD,a and Raymond Q. Migrino, MDb,c,e

rom the aDepartment of Surgery (Transplant Surgery), bCardiovascular Research Center, cCardiovascular Medicine,Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, and ePhoenix Veterans Affairs Health Care

ystem, Phoenix, Arizona.

BACKGROUND: There remains no reliable non-invasive method to detect cardiac transplant rejection.Recently, speckle-tracking 2-dimensional strain echocardiography (2DSE) was shown to be sensitive inthe early detection of myocardial dysfunction in various models of cardiomyopathy. We aim todetermine if 2DSE-derived functional indices can detect cardiac transplant rejection.METHODS: Heterotopic rat cardiac transplantation was performed in histocompatible isografts orhistoincompatible allografts. Histologic rejection scores were determined. Short-axis, mid-left ventric-ular (LV) echocardiography was performed on Day 6 after transplantation. Conventional measures offunction were measured, (including LV fractional shortening and ejection fraction) as well as 2DSEparameters.RESULTS: Despite class IIIB rejection in allografts and no rejection in isografts, there was nodifference between isografts vs allografts in fractional shortening (15% � 3% vs 12% � 3%) orejection fraction (36% � 5% vs 26% � 6%; both not significant). In contrast, 2DSE revealed decreasesbetween isografts and allografts in global radial strain (12.6% � 5.6% vs 1.1% � 0.2%, p � 0.05), peakradial systolic strain rate (3.10 � 0.74/s vs 0.54 � 0.13/s, p � 0.001), and peak circumferential systolicstrain rate (–1.99 � 0.55 vs –0.43 � 0.11/s; p � 0.01).CONCLUSIONS: Systolic strain imaging using 2DSE differentiates myocardial function between ex-perimental cardiac transplant rejection in allografts and non-rejection in isografts. Therefore, 2DSE maybe useful in early non-invasive detection of transplant rejection.J Heart Lung Transplant 2010;xx:xxx © 2010 International Society for Heart and Lung TransplantationAll rights reserved.

KEYWORDS:cardiac rejection;speckle-trackingechocardiography;strain imaging;noninvasive

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Cardiac transplant rejection is associated with adverserognosis if untreated. Currently, rejection in cardiac trans-lants is diagnosed by right ventricular endomyocardialiopsy specimens, which is considered the “gold standard”ethod for identifying cardiac transplant rejection. Endo-yocardial biopsy, an invasive procedure for the on-going

Reprint requests: Galen M. Pieper, PhD, Division of Transplant Sur-ery, Medical College of Wisconsin, 9200 W Wisconsin Ave, Milwaukee,I 53226. Telephone: 414-456-5899. Fax: 414-456-6222.

bE-mail address: gmpieper@mcw.edu

053-2498/$ -see front matter © 2010 International Society for Heart and Lungoi:10.1016/j.healun.2010.04.009

urveillance of cardiac rejection, is usually performed sev-ral times during the first year after transplantation and isssociated with potential complications, such as tricuspidalve damage, cardiac perforation, and arrhythmias.1,2 Aore recent study demonstrated that the development of

ricuspid regurgitation correlated with the number of endo-yocardial biopsies.3

Alternative non-invasive techniques for the detection ofardiac transplant rejection, such as echocardiography, nu-lear imaging,4 and magnetic resonance imaging,5–8 have

een tested. However, no reliable alternative to endomyo-

Transplantation All rights reserved.

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ardial biopsy has emerged. Thus, there continues to be anngoing search for new methods and technologies thatould be used as a non-invasive alternative.

Echocardiography is a non-invasive technique that showedarly promise in correlating functional changes associated withndomyocardial specimen-proven rejection in human car-iac transplant recipients.9 However, conventional echocar-iography methods using M-mode or 2-dimensional (2D)chocardiography, such as fractional shortening or ejectionraction, are limited to assessing changes in ventricularavity size during the cardiac cycle. Furthermore, results ofmore recent large population of heart transplant patients

evealed that no single parameter or combination of param-ters emerged as sufficiently able to eliminate surveillancendomyocardial biopsy to detect cardiac rejection.10

The traditional cavity measurement-based echocardio-raphic parameters are indirect means of assessing myocar-ial function, and our group has found that they are insen-itive to early changes in cardiomyopathy. In contrast,peckle-tracking echocardiography that directly assessesyocardial mechanics was more sensitive and accurate in

he early detection of doxorubicin or ischemic cardiomyop-thy than conventional echocardiographic measure of sys-olic dysfunction such as fractional shortening and ejectionraction.11,12 Similarly strain rate imaging, but not conven-ional echocardiography, was found to detect left ventricularLV) dysfunction in aged mice.13 Strain represents defor-ation, an intrinsic mechanical property, and is a more

irect measure of systolic function12 vs conventional cavity-ased echocardiographic measures.

Diagnosis of acute sub-clinical rejection in cardiac trans-lant recipients is important. Given that some patients maye asymptomatic despite histologic evidence of rejection, its important to consider the potential that the newer speckle-racking technology might be more useful than conventionalchocardiography to determine rejection. We hypothesizedn the present study that 2D strain echocardiography (2DSE)an detect alloimmune-induced LV dysfunction in experi-ental cardiac transplantation under conditions of histo-

ogic rejection.

ethods

ll animal procedures in this study were approved by theocal Institutional Animal Care and Use Committee. Allnimals received humane care in compliance with the Prin-iples of Laboratory and Animal Care, formulated by theational Society for Medical Research, and the Guide for

he Care and Use of Laboratory Animals, prepared by thenstitute of Laboratory Animal Resources and published byhe National Institutes of Health (NIH Publication No. 86-3, revised 1996).

ransplantation

ewis (Lew:RT1l) and Wistar-Furth (WF:RT1u) rat strains

Harlan Labs, Indianapolis, IN) were chosen for genetic r

isparity at both the major and minor histocompatibility locior donor-to-recipient combination of Lewis into Lewis (forsograft) or Wistar-Furth into Lewis (for allograft rats).sogeneic (n � 6) or allogeneic (n � 8) heterotopic cardiacransplantation was performed aseptically in pentobarbital50 mg/kg, intraperitoneal) anesthetized animals as earlierescribed.14 Donor hearts were arrested in ice-cold Univer-ity of Wisconsin preservation solution and transplantednto anesthetized (50 mg/kg pentobarbital intraperitoneal)ecipient animals.

istologic rejection scoring

issue was fixed in 10% phosphate-buffered formalin. Par-ffin-embedded sections were stained with hematoxylin andosin. Histologic rejection was scored blinded using criteriastablished by the International Society for Heart and Lungransplantation (ISHLT).

chocardiography

chocardiography was performed on Day 6 after transplanta-ion using a Vivid 7 echocardiograph (General Electric,

igure 1 Example shows normal histology in rat cardiac isograftut not in the allograft. Histologic rejection in the allograft is indicatedy multi-focal inflammatory cell infiltrate, myocyte necrosis, hemor-

hage, and vasculitis. Hematoxylin and eosin (200�).

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aukesha, WI) and an 11-MHz M12L linear array transducer.he standard parasternal short-axis mid LV view was usedith the papillary muscles as anatomic landmarks. Imageepth was 2 to 2.5 cm, and frame rates of 234 to 256 frames/ec and second harmonic imaging were used. The images wereent to a separate workstation for analysis using EchoPAC with

analysis software (General Electric, Waukesha WI). Thenvestigators who performed the echocardiographic analysisere blinded to the transplant allocation group.LV size according to internal diameter in diastole

LVIDD and systole (LVIDS), and thickness measured by

igure 2 Echocardiographic evidence of increased left ventric-lar (LV) mass in 8 allografts compared with 6 isograft controls.ata are shown with the standard error of the mean. ***p � 0.001.

igure 3 M-mode echocardiography revealed no significant change

nternal diameter in systole, end-systolic volume) in allografts compared with

nterior (AT) and inferior (IT) diastolic thickness, wereeasured using anatomic M-mode of the 2D B-mode im-

ges through the plane of the anterior and inferior segments.ractional shortening (FS) was calculated using the formulaS � (LVIDD – LVIDS)/LVIDD � 100%. LV ejectionraction was calculated using the Teicholz method.15 LVass was derived using the formula: 0.8 [1.04{(IT � LVID �T)3 – LVID3}] � 0.6.16

Diastolic measurements were performed at the R wave oflectrocardiographic gating that corresponded to the donoreart rhythm in cases where 2 distinct R waves were seen (ie,rom the native and heterotopically-transplanted donor heart).n cases where the donor heart rhythm could not be distin-uished from the native heart on electrocardiography, diastoliceasurements were done at the largest diameter of the heart

uring the cardiac cycle.Radial and circumferential strain and strain rate were mea-

ured as previously published.11 In brief, the middle portion ofhe LV was divided into 6 segments as defined by the Amer-can Society of Echocardiography.17 The endocardial borderas traced, while the outer border was adjusted to fit the

picardial contour. The software automatically selected stablecoustic objects within the myocardium to track and computetrain and strain rate in radial and circumferential directions inhe segments of the middle ventricle throughout the cardiacycle. Peak systolic and peak early diastolic strain and strainate were obtained for each segment, and the average was usedo compute global radial or circumferential strain and strainates.

stolic function (fractional shortening, ejection fraction, left ventricular

s in sy isograft controls. Error bars indicate the standard error of the mean.

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4 The Journal of Heart and Lung Transplantation, Vol xx, No x, Month 2010ARTICLE IN PRESS

ata and statistical analysis

ata are expressed as means � standard error of means.tatistical analysis was performed using the unpaired t-testr the Mann-Whitney U-test, where appropriate, with sig-ificance set at p � 0.05.

esults

istologic evidence of grade IIIB rejection in rat heartllografts on Day 6 after transplant was evidenced by theresence of inflammatory cell infiltrate, myocyte necrosis,nterstitial edema, hemorrhage, and vasculitis (Figure 1).one of these features (ie, grade 0) were exhibited in

sograft controls or in native hearts of either isograft orllograft recipients. Graded scoring revealed a significant

Figure 4 Images show qualitative decreases in radial strain in

ncrease in rejection over isograft controls or native heartsf allograft recipients.

Echocardiography indicated that LV mass was signifi-antly (p � 0.001) elevated in rat allografts compared withsograft controls (Figure 2). LV ejection fraction, fractionalhortening, internal diameter in systole, and end-systolicolume were not significantly different between allograftnd isograft controls (Figure 3).

An example of radial strain is shown throughout theardiac cycle in an allograft vs isograft control (Figure 4).lobal radial strain in the allograft rats was significantly

p � 0.05) decreased from that seen in the isograft controlsFigure 5A). Global circumferential strain showed a similarrend (Figure 5B); however, this was not statistically sig-ificant.

Decreases in both the peak systolic and peak early dia-tolic radial strain rate were seen in allograft recipients

(lower panel) allograft vs (upper panel) isograft control.

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ompared with isograft controls (Figure 6). Quantitatively,lobal peak systolic and peak early diastolic radial strainates were decreased in allograft recipients compared withsograft controls (Figure 7A and B). Similarly, the globaleak systolic and peak early diastolic circumferential strainates were significantly (p � 0.01 and p � 0.001, respec-ively) decreased in allograft recipients vs isograft controlsFigure 7C and D).

iscussion

n the present study, we examined the potential usefulnessf speckle tracking-based 2DSE in the evaluation of cardiacransplant rejection in an experimental rat transplant model.

major finding in this study is that radial strain, the peakystolic radial and circumferential strain rate, and the peakarly diastolic radial and circumferential strain rate areeduced in cardiac transplants undergoing alloimmune re-ection vs transplants without rejection.

Second, these strain-based measures detected LV dys-unction during histologic rejection at a time when con-entional echocardiographic measures, such as fractionalhortening or ejection fraction, were not able to detectifferences in systolic function between the 2 groups.

These findings in an experimental cardiac transplantodel are consistent with prior studies showing 2DSE can

etect LV dysfunction in a variety of experimental andlinical settings under asymptomatic conditions that could

igure 5 Decreased global (A) radial (*p � 0.05) and (B)ircumferential (not significant, P � 0.108 by Mann-Whitney-test) strain in allografts vs isograft controls. Data are shown with

he standard error of the mean.

ot be detected using conventional systolic echocardio- o

raphic measures of LV function.18,19 The value of 2DSErises from its ability to directly measure wall deformation,hich is an intrinsic mechanical property vs conventional

avity-based echocardiographic analysis of LV systolicunction.

The findings in the present study confirm the traditionalnding that LV mass is increased during alloimmune rejec-

ion in experimental models20 and in human20–22 cardiacransplant rejection. This increased LV mass has been at-ributed acutely to inflammatory cell infiltration and graftdema.22 Chronically, the increase in LV mass in cardiacransplants has been attributed to hypertrophy arising fromarious factors, including repetitive rejection episodes, im-unosuppression-induced hypertension therapy, chronic

achycardia, and denervation.22

The concept of using strain imaging as a tool to evaluateardiac rejection is new. In a study published in 2009, tissueoppler imaging was able to show for the first time a

orrelation between systolic radial strain rate in the anteriorall and rejection grade. However, no correlation was

ound with ejection fraction during chronic rejection in aeterotopic rat cardiac transplant model, although ejec-ion fraction was clearly decreased at all grades of his-ologic rejection.23 This was followed by another studysing a different technique of strain magnetic resonancemaging (MRI) that showed decreased circumferentialtrain despite normal ejection fraction in a rat heart-lungransplant model.24

Our results using 2DSE of global strain determinedver 6 segments of LV wall in a model of acute rejectionre consistent with these recent findings. Collectively, theRI study and our study indicate that strain imaging

iscriminates dysfunction independent of loading condi-ions and that strain imaging may be a useful tool forcute and chronic rejection. In addition, we expand onhis promising approach by showing that both systolicnd diastolic radial and circumferential strain rate as wells radial strain are all decreased in alloimmune-inducedardiac transplant rejection. Although strain and strainate can be measured by Doppler as well as by tissuepeckle-tracking techniques, the Doppler method has theisadvantage of being angle-dependent, potentially lim-ting its versatility.19

Furthermore, our study demonstrates differences notnly in systolic strain but also in the early diastolic strainate between allograft and isograft hearts. Additional eval-ation of diastolic dysfunction by strain and strain imagings potentially important clinically because diastolic dysfunc-ion as evidenced by tissue Doppler echocardiography fre-uently occurs before development of systolic dysfunctionn cardiac transplant patients.25

tudy limitations

or technical reasons, analysis of longitudinal strain andtrain rate imaging is difficult in the rat model of cardiacransplantation. Owing to mechanical unloading as a result

f heterotopic cardiac transplantation in rodents, the echo-

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ardiographic values of conventional systolic function andtrain are typically lower than in normal hearts. Because ofhis, comparisons with pre-operative strain measurementsf donor hearts or native hearts of graft recipients wereeemed to be invalid for purposes of functional analysis. Toircumvent this limitation, we used time-matched, hetero-opic isograft controls for the alloimmune rejection group.

Furthermore, because of the short timeframe of acuteejection in this rat strain model, we avoided serial echo-ardiographic measurements that would necessitate inha-ation anesthetic to eliminate any modulation of graftunction. Inhalation anesthetics are well known to pro-uce pre-conditioning or cardioprotection by increased NO

igure 6 Example images show peak systolic and diastolic radiearts.

ynthesis that could directly affect not only myocardial g

unction but also immune function and complicate interpre-ation of the study.

Our study also did not examine a detailed correlationetween gradations of histologic rejection score and strainnd strain rate. Rather, this study was designed specificallyo illustrate the potential of the new 2DSE technique toiscriminate LV dysfunction in rejecting vs non-rejectingardiac transplants under conditions in which global echo-ardiographic markers of systolic dysfunction were not de-ected. In future studies, careful registration between re-ional strain and histologic rejection will allow a moreefinitive correlative analysis between these 2 measures.

Because abnormal strain can occur in multiple patholo-

n rate in (lower panel) allograft and (upper panel) isograft control

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ies,11,12,26 it is a non-specific finding that may not neces-

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arily arise from transplant rejection. Therefore, the poten-ial clinical use is as a screening tool to identify transplantatients who would require an endomyocardial biopsy spec-men for confirmation of pathology, but strain echocardiog-aphy would not supplant the need for biopsy. However, theotential clinical utility remains undiminished despite thison-specificity, with strain echocardiography possibly be-ng useful for screening of patients needing a biopsy insteadf the current practice of needing an invasive biopsy spec-men as a screening method.

train imaging and clinical cardiac rejection

e acknowledge that findings in this rodent model of het-rotopic cardiac transplantation may not necessarily be ex-rapolated to the clinical situation. Nevertheless, the find-ngs here provide the experimental basis for systematicallyalidating the potential use of 2DSE in evaluating humanardiac transplant rejection because there are no publishedeports, to our knowledge, of the use of this new technologyn human cardiac transplant recipients.

In this context, a recent published report using a Dopplertrain imaging technology has demonstrated systolic strainysfunction in a small population of heart transplant pa-ients with ISHLT rejection grade �1B but not �1B.27 Theuthors found systolic strain dysfunction occurred withoutifferences in conventional echocardiographic analysis ofjection fraction, wall thickness, and other parameters. De-

igure 7 Decreased global (A) systolic and (B) diastolic radial sate in allografts vs isograft controls. *p � 0.05, **p � 0.01, ***

pite the promise of the clinical study using Doppler strain 2

maging, the study had limitations. It examined only systolictrain/strain rate and only in a single posterior wall segmentf the LV, which may limit its discriminating efficacy dueo heterogeneity in histologic rejection. Another significantoncern is that no baseline values were provided. Thus,here was no way of discerning whether variances in earlyost-transplant myocardial function existed that may haverisen due to differences in initial donor heart performancer to differences in donor heart ischemic times that mayave affected post-transplant performance and echocardio-raphic values and altered the course of rejection and theater echocardiographic measurements of strain. Finally,hat clinical study was limited to the evaluation of systolicongitudinal and radial strain and strain rate. Collectively,he present study and the recent clinical study indicate theotential promise of using 2DSE as a new tool in diagnosisf cardiac transplant rejection.

dvantages of speckle-tracking imaging overurrent imaging techniques

issue Doppler-based strain imaging is severely limited byts angle-dependency and the effects of translational motion;herefore, this technique can only estimate strain along theltrasound beam. Tissue Doppler imaging requires the usef high frame rates in contrast to lower frame rates for 2Dtrain imaging. There is less artifact and considerably lessntraobserver and interobserver variability with the newer

ate and global (C) systolic and diastolic (D) circumferential strain.001.

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D strain imaging than for Doppler-based strain imaging.19

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he far less expensive cost of 2D strain imaging comparedith techniques such as MRI is an attractive feature. Fi-ally, the advancement of sophisticated algorithms has al-owed the data analysis for 2D strain imaging to be fairlyapid, allowing a much more timely acquisition of dataompared with current tissue Doppler-based strain imagingnalysis.

isclosure statement

his work was supported by grant HL079837 from theational Institutes of Health, Heart and Lung Institute, and

rom a stipend to Akash Shah from the Friends of MCWedical Student Summer Training Grant. The assistance ofhao-Ying Chen for the cardiac transplant surgeries isreatly appreciated.

None of the authors has a financial relationship with aommercial entity that has an interest in the subject of theresented manuscript or other conflicts of interest to dis-lose.

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