cellularmechanisms captopril-induced matrix …circ.ahajournals.org/content/90/3/1334.full.pdf ·...

1334

Cellular Mechanisms of Captopril-Induced MatrixRemodeling in Syrian Hamster Cardiomyopathy

Glenn Davison, MD; Christopher S. Hall, MA; James G. Miller, PhD;Michael Scott, BS; Samuel A. Wickline, MD

Background Although angiotensin-converting enzyme(ACE) inhibitors have become a mainstay of treatment forchronic congestive heart failure (CHF), it is not knownwhether the cardiac remodeling effects are a secondary phe-nomenon, resulting from ACE inhibitors' hemodynamic ac-tions of afterload reduction, or occur through an independentmechanism.Methods and Results We used ultrasonic tissue character-

ization to define potentially salutary effects of treatment withACE inhibitors on the material properties of the heart and itspotential influence on cardiac remodeling at the cellular level.Ten 1-month-old, cardiomyopathic (CM) Syrian hamsters and6 normal (NL) hamsters were treated with captopril (2 g/Lwater ad libitum), and 10 CM hamsters and 10 NL hamsterswere maintained untreated for 3 months. Hearts were excised,and backscattered radiofrequency data were acquired from1200 independent sites from each specimen with a high-resolution 50-MHz acoustic microscope for calculation ofintegrated backscatter (IB). Treatment with captopril reducedleft ventricular mass, calcium concentration, and IB in CM

Angiotensin-converting enzyme (ACE) inhibitors; .4 have become a mainstay of treatment forL XL chronic congestive heart failure (CHF) basedon compelling evidence that they improve exercisetolerance",2 and hemodynamic status3 and decreaseheart size and mortality.4'5 ACE inhibitors also induce aregression of left ventricular hypertrophy (LVH) inpatients with hypertensive cardiomyopathy.6 Despitethese salutary effects, the cellular events responsible forthese actions remain incompletely defined. Whether thecardiac remodeling effects of ACE inhibitors representa secondary phenomenon that results from their hemo-dynamic actions of afterload reduction or whether re-modeling occurs through a primary mechanism, such asdirect inhibition of the local tissue renin-angiotensinaxis,7-9 has not been resolved. Furthermore, determina-tion of the specific mechanisms responsible for im-proved systolic and diastolic functions are confoundedby inherently imprecise measurements of global ventric-ular function that are highly dependent on preload andafterload.A principal objective of this research was to define

potentially beneficial effects of treatment with ACE

Received November 29, 1993; revision accepted May 18, 1994.From the Department of Physics, Washington University School

of Medicine, St Louis, Mo.Reprint requests to Samuel A. Wickline, MD, Division of

Cardiology, Jewish Hospital at the Washington University Schoolof Medicine, 216 South Kingshighway, St Louis, MO 63110.

X) 1994 American Heart Association, Inc.

hearts without affecting myofiber size or collagen concentra-tion. The IB from grossly normal regions of myocardium in NLhamsters, treated CM hamsters, and untreated CM hamsterswas not significantly different. The IB from the microscopicregions of scar tissue in treated CM hamsters was significantlyless (P=.0004) than that from scar tissue in untreated CMhamsters.

Conclusions The reduced IB from treated scar tissue com-ponents reflects specific alterations in the material properties(elastic stiffness, density) of fibrous regions in CM heartsinduced by captopril. This is the first report that definesspecific cellular effects of ACE inhibitors on the materialproperties of isolated components of cardiac tissue in experi-mental cardiomyopathy. These alterations in material proper-ties of scar tissue components represent a potential mecha-nism for the salutary actions of ACE inhibitors in heart failure.(Circulation. 1994;90:1334-1342.)Key Words * captopril * cardiac remodeling * backscatter

* cardiomyopathy

inhibitors on the material properties of heart tissue andto elucidate cellular mechanisms of improved cardiacremodeling. Our laboratory has previously reportedthat quantitative ultrasonic characterization of myocar-dial tissue based on analysis of frequency-dependentbackscatter and attenuation can delineate fundamentalphysical properties and structural constituents of thehuman left ventricle10 under conditions of ischemia,"1infarction,12 and cardiomyopathy.13 We have shown thatultrasonic tissue characterization can sensitively depictpathological changes in the content14 and organization15of the myocardial connective tissue matrix. Recent workfrom our laboratory has demonstrated the validity ofultrasonic analysis for quantification of the elastic prop-erties of heart tissue.16 Ultrasonic methods can be usedto determine values for Young's modulus for hearttissue, which closely approximate those obtained byconventional methods based on deformation stress-strain relations. Because the interaction of ultrasoundwith the heart depends on the material properties ofcardiac tissue'7'18 such as density and elastic stiffness,ultrasonic tissue characterization provides a uniqueapproach for detection of the effects of ACE inhibitorson cardiac remodeling. The use of high-resolution,high-frequency acoustic microscopy permits delineationof these material properties at the microscopic level.'9We hypothesized that ultrasonic tissue characteriza-

tion could detect the potentially beneficial effects ofACE inhibitors on microscopic cardiac structure andmaterial properties in experimental cardiomyopathy.

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Davison et al Captopril Effects on Remodeling in Syrian Hamster Cardiomyopathy 1335

Selected strains of Syrian hamsters exhibit a heritablehypertrophic cardiomyopathy that progresses to a di-lated stage according to a predictable time course.20,21The cardiomyopathy is characterized by focal myocar-dial myolysis and necrosis beginning at approximately 30days of age and subsequent replacement of myocyteswith fibrotic connective tissue. As the fibrosis increases,there is a compensatory hypertrophy followed by cham-ber dilation and finally death occurring from CHF atapproximately 280 days of age.20,2'The focality of the myocardial lesions seen in this

cardiomyopathy has led many investigators to postulatethat microvascular spasm may be the primary etiologyof the cardiomyopathy. Factor et a122 demonstrated thatverapamil treatment caused histological resolution ofthe myocardial lesions of cardiomyopathic (CM) Syrianhamsters. Other investigators have also shown thattherapy with verapamil23,24 or diltiazem25 can signifi-cantly improve or prevent the pathological myocardialchanges of CM Syrian hamsters. We propose that thepresence of multiple discrete areas of myocardial fibro-sis and hypertrophy in these hearts would afford aninvaluable model for studying cellular effects of ACEinhibitors on cardiac scar tissue remodeling. BecauseACE inhibitors have been shown to reduce globalmyocardial chamber stiffness26 without a concomitantimprovement in the passive stiffness of the noninfarctedmyocardium,27 we hypothesized that captopril treat-ment would elicit specific changes in material propertiesof isolated scar tissue components. Accordingly, wereport the use of quantitative ultrasound to characterizethe specific effects of ACE inhibitor therapy on thematerial properties of isolated scar tissue componentsin cardiomyopathy.

MethodsAnimal PreparationsA total of 46 1-month-old, male Syrian hamsters were used.

The hearts of 5 normal (NL) Syrian hamsters (Bio Fl-B strain,Biobreeders Inc) and 5 CM Syrian hamsters (Bio TO-2 strain,Biobreeders Inc) were examined at 1 month of age to obtainbaseline morphological and ultrasonic heart measurementsbefore the institution of ACE inhibitor therapy. All hamsterswere housed 3 to a cage with free access to laboratory rodentchow (Purina) and were maintained in Hepa-filtered "stayclean" units with a 12-hour light/dark cycle. Ten CM and 6 NLhamsters were treated with captopril (2 g/L water ad libitum).Ten CM and 10 NL hamsters were maintained untreated for 3months. Body mass was measured every 2 weeks. At the end ofthe study, the 4-month-old hamsters were first anesthetizedwith methoxyflurane, then killed by pentobarbital overdose(>120 mg/kg IP). The study protocol was approved by theAnimal Care Committee of the Jewish Hospital at WashingtonUniversity Medical Center.

Morphometric AnalysisAfter fixation in formalin, the hearts were sectioned. The

right ventricle was removed, the left atrium and great vesselswere trimmed away, and the left ventricle was weighed. Theleft ventricular (LV) apex was used for analysis of collagencontent by the method of Woessner.28 A 500-,um section fromthe midmyocardium was used for determination of calciumcontent by quantitative photometric absorption.29 A section ofthe LV posterior wall was stained with hematoxylin-eosin,Masson's trichrome stain, and Von Kossa's stain for calcium.Collagen and calcium content also were calculated with com-puter-assisted planimetry of stained sections according to

methods previously described.13 Calcium content was deter-mined by planimetry in all scar tissue regions of both CMgroups and expressed as percentage of total scar. Myofiberdiameter was determined to the nearest hundredth of a micronwith computer-assisted planimetry by measuring approxi-mately 140 fibers from each specimen within 25 high-powerfields.

Acquisition and Analysis of Ultrasonic DataA vibratome (model Pelco 101, Ted Pella, Inc) was used to

cut a 500-,um-thick section from the midmyocardium of eachheart. These sections were then mounted in a holding devicewith the endocardial side facing the transducer. The tissueholder and the mounted specimen were then immersed indegassed distilled water at room temperature for ultrasonicinsonification. A custom-designed acoustic microscope wasused to collect ultrasonic data as described previously by ourlaboratory.'3 Briefly, a 50-MHz, broadband, focused, piezo-electric delay-line transducer (¼/4-in. diameter, 1/2-in. focallength, model V390, Panametrics Co) was operated in thepulse-echo mode. A Tektronix DSA 601 digitizing oscilloscopewas used to digitize backscattered radiofrequency data at 500megasamples per second with eight-bit resolution. Radiofre-quency data were acquired from approximately 1200 indepen-dent sites from each specimen.The radiofrequency data were stored in a low-resolution,

raster scan format and analyzed with custom software. Seg-ments of the radiofrequency lines 500 nanoseconds in durationand 100 nanoseconds below the cut surface of the section weregated for analysis to avoid the front and back wall specularechoes from the specimen. The gated data were multiplied bya Hamming window, and their power spectra were determinedby fast-Fourier transformation. The power spectra from themyocardial specimens were referenced to the power spectrumreturned from a near-perfect steel planar reflector, accordingto methods previously described.10"2 Integrated backscatter(IB), a measure of the relative efficiency of ultrasonic scatter-ing from tissue, was computed from the average of thefrequency-dependent backscatter transfer function across theuseful bandwidth of the transducer (30 to 50 MHz). IB wasexpressed in decibels relative to scattering from the steel plate.The IB data at every independent site within each tissue slicewere converted into a gray scale map to allow visual inspectionand quantitative analysis of backscatter from regions of grosslynormal or fibrotic tissue (Fig 1). The gray scale map allowed usto use the grossly normal regions as control data.

Statistical AnalysisUltrasonic, morphological, and biochemical measurements

were compared with the use of commercially available statis-tical software (STATVIEw 4.0, Abacus Concepts). Two groupsof 1-month-old hamsters (NL and CM) were used to deter-mine the baseline comparability of IB, LV mass %, andcollagen content (based on hydroxyproline analysis), with theuse of an unpaired t test. Three groups of 4-month-oldhamsters (NL, treated, and untreated CM) were used todetermine the effects of 3 months of captopril therapy on IB,LV mass %, collagen content (based on hydroxyproline analy-sis), weekly weight gain, and body mass, with the use ofANOVA methods to test the significance of differences, whichwas assigned at the P<.05 level. Testing of significance differ-ences among these three groups was performed with theFisher's protected least squares difference t test. All data areexpressed as mean+SE unless otherwise indicated.

ResultsBaseline Measurements for Untreated 1-Month-OldNL and CM Hamsters

Because all hamsters were approximately 1 month ofage at the start of the study, we sought to establish



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1336 Circulation Vol 90, No 3 September 1994

FIG 1. Integrated backscatter gray scale map from a section of myocardium from a cardiomyopathic Syrian hamster. The darker areasrepresent higher scattering and correspond to fibrotic bands; surrounding lighter areas represent normal tissue.

baseline measurements for both NL and CM hamsterhearts. There were no statistically significant differencesin LV mass % (normalized for body mass) or collagenconcentration (mg/g LV dry mass) between the1-month-old NL and CM hamsters (Fig 2). Fig 2 alsoshows that 1-month-old NL hamsters exhibited slightlygreater myocardial scattering than did the CM hamsters(P=.005).

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Body WeightFig 3 shows the growth curves for NL, untreated, and

treated CM hamsters over the 3-month study period.Baseline weights among the three groups were notsignificantly different at the beginning of the study.After 3 months, however, the NL hamster group exhib-ited a significantly higher mean weight than did either ofthe two CM hamster groups, which were equivalent(P<.0001). These data indicate that captopril treatmentdid not affect the body weights for the CM hamsters.Both treated and untreated CM hamsters gained lessweight than did the NL hamsters despite starting atequivalent weights at 1 month of age (see above).

LVMass and Myofiber DiameterFig 4 shows the LV mass % (normalized for body

mass) for 4-month-old NL, untreated CM, and treatedCM hamsters. The hearts of the NL and treated CMhamsters exhibited a similar LV mass %, which wassignificantly less than that of the untreated CM ham-sters (P<.0001). Grossly, both groups of CM hamstersmanifested whitish bands of fibrous tissue runningthroughout the myocardium, whereas hearts of NLhamsters had none. Microscopic analysis of the grossly

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FIG 2. Graphs show baseline data for 1-month-old hamsters. LVindicates left ventricular; NL, normal hamsters; and CM, cardio-myopathic hamsters. *P= .005.

FIG 3. Growth curves for normal hamsters, treated cardiomyo-pathic hamsters, and untreated cardiomyopathic hamsters. CMindicates cardiomyopathic hamsters.

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FIG 4. Graph shows effect of therapy with captopril for 3 monthson left ventricular (LV) mass % of cardiomyopathic hamsters(CM) (all values normalized for body mass). No significantdifference in LV mass % was observed between treated cardio-myopathic hamsters and untreated normal hamsters (NL). TheLV masses for treated cardiomyopathic hearts were significantlyless than those for untreated cardiomyopathic hearts(*P< .0001).

normal tissue in each heart revealed that myofiberdiameters differed minimally among the three groups:9.8±0.1 gm for untreated NL hamsters, 9.3±0.1 ,um fortreated NL hamsters, 8.9±0.1 gm for untreated CMhamsters, and 9.7±0.1 ,um for treated CM hamsters.Thus, treatment with captopril elicited a 23% decreasein LV mass in treated as compared with untreated CMhamsters, with minimal changes in the size of grosslynormal myofibers.Normal hamsters were treated to determine the gen-

eral effect that ACE inhibitors have on LV mass. After3 months of captopril therapy, the treated NL hamstersmanifested an 18% decrease in LV mass % as comparedwith the untreated NL hamsters (0.197±0.007 versus0.240±0.005, P=.0004). These data show that captopriltherapy starting at 1 month of age can inhibit heartgrowth in both NL and CM hamsters.

Collagen ConcentrationFig 5 shows the LV collagen concentration (mg/g left

ventricle) for 4-month-old NL, untreated CM, andtreated CM hamsters. After 3 months, both treated anduntreated CM hamsters exhibited similar increases inLV collagen to levels significantly greater than that forthe NL hamsters (P=.0002). Computer-assisted planim-etry of histological sections stained with Masson's tri-chrome also corroborated the increased LV collagenconcentration of the CM hamsters as compared with NLhamster hearts. Both the hydroxyproline assay and thecomputer-assisted planimetry yielded CM collagen con-centrations approximately 1.5 to 2 times control levels inNL animals. The collagen concentrations measured bythe two methods were highly correlated (r=.86). Thus, 3months of captopril treatment does not appear tosignificantly reduce the average concentration of colla-

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FIG 5. Graph shows effect of therapy with captopril for 3 monthson left ventricular (LV) collagen concentration. Treated anduntreated cardiomyopathic hamsters had similar increases incollagen (P=NS), both of which were significantly greater thanthe normal hamsters (NL) (*P=.0002).

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FIG 6. Graph shows effect of therapy with captopril for 3 monthson left ventricular (LV) calcium concentration. Therapy withcaptopril for 3 months decreased the LV calcium concentrationin the treated cardiomyopathic hamster (CM) hearts (tP<.0001,*P<.0001, **P=.001). NL indicates normal hamsters.

gen in the CM hamsters despite an overall reduction inLV mass (compare with Fig 4).

Cakium ConcentrationFig 6 shows the LV calcium concentration (mg/g left

ventricle) for 4-month-old NL, untreated CM, andtreated CM hamsters. After 3 months, the whole heartsof both treated and untreated CM hamsters exhibitedLV calcium concentrations significantly greater thanthat for the NL hamsters (P<.0001). Treatment withcaptopril resulted in a 37% decrease in calcium concen-tration for the treated as compared with the untreatedCM hearts (P=.001). Examination of histological sec-tions stained with Von Kossa's demonstrated little grosscalcification in the normal myocardium. Computer-assisted planimetry of scar tissue regions corroboratedthe increased calcification of scar tissue in the untreatedCM hearts as compared with the captopril-treated CMhearts (56±1% versus 43±2%, P<.0001). Thus, 3months of captopril treatment significantly reduced theaverage concentration of calcium in the scar tissueregions of CM hamsters. There was also a strongcorrelation between LV mass % and planimetry scarcalcium content (r=.85, P=.0005).

Quantitative Ultrasonic IndexesFig 7 illustrates the effect of 3 months of captopril

therapy on IB. Both treated and untreated CM hamsterhearts demonstrated substantially greater IB as com-pared with the NL hearts. Captopril therapy, however,resulted in a 2.1-dB (1.6-fold) decrease in IB for thetreated as compared with the untreated CM hearts(P<.02). The hearts of the 4-month-old untreated CMhamsters exhibited a 6.5±0.7-dB (4.5-fold) increase inIB as compared with the 1-month-old untreated CMhamsters (P<.0001). The hearts of the 4-month-old

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FIG 7. Graph shows comparison of integrated backscatter fromhearts of normal hamsters (NL), treated cardiomyopathic ham-sters, and untreated cardiomyopathic hamsters. Therapy withcaptopril for 3 months caused a decrease in integrated back-scatter for the treated cardiomyopathic hamster hearts(tP<.0001, *P<.02, **P<.007). CM indicates cardiomyopathichamsters.

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FIG 8. Correlation of integrated backscatter gray scale map on left with corresponding gross tissue section on right. Lighter areas

represent greater scattering.

treated CM hamsters exhibited a 4.4±0.8-dB (2.7-fold)increase in IB as compared with the 1-month-old un-treated CM hamsters (P=.0006). Thus, the effect oftreatment with captopril produces a 2.1-dB (1.6-fold)decrease in IB in CM hamsters. The values of IB fromthe 1-month-old (see Fig 2) and 4-month-old NL heartswere not significantly different. Thus, aging did not alterthe ultrasonic scattering from NL hearts but signifi-cantly augmented IB from CM hearts. This progressiveincrease in IB from CM hearts was mitigated bycaptopril.The IB data reported above were acquired from

unselected regions of myocardial tissue, comprisingadmixed grossly normal and fibrotic tissue in the case ofCM hamsters. To address the question of which com-ponent of the myocardial tissue in CM hearts might bemost affected by captopril therapy, we reanalyzed thedata by partitioning the heart into zones of high and lowscattering according to the IB gray scale map (see Fig1). By comparing the histological sections from the sameregions on which backscatter analysis was performed,we determined that the areas of high scattering corre-sponded precisely to the whitish bands of fibrous tissueobserved in the gross specimens (Fig 8). Therefore, foreach CM specimen, all radiofrequency lines lying withina high scattering zone were counted as fibrous scar, andall radiofrequency lines lying within low scattering zoneswere counted as grossly normal myocardium. For anygiven sample of CM myocardium, the scarred areasmade up no more than 10% to 20% of the totalradiofrequency data (see Fig 1).

Fig 9 illustrates that the grossly normal (low scatter-ing) zones exhibited no significant difference in IBamong the NL, treated, and untreated CM hamster


Fin 9. Graph shows comparison of integrated backscatter fromgrossly normal regions in hearts of normal hamsters (NL), treatedcardiomyopathic hamsters, and untreated cardiomyopathic ham-sters (P=NS). CM indicates cardiomyopathic hamsters.

hearts (P=NS). Fig 10 shows that the magnitude of IBfrom the fibrous (high scattering) zones in CM animalswas significantly less in the treated than in the untreatedCM hamsters (P=.0004). The IB from these high scat-tering fibrous zones correlated linearly with scar tissuecalcium content measured by planimetry (r=.60,P=.01). These data indicate that over the 3 months ofthe study, IB increased in both treated and untreatedCM hamster hearts due primarily to the presence ofbandlike areas of fibrosis. The magnitude of IB in thefibrous scar zones reflected in part calcium content ofscar tissue. Captopril therapy resulted in a specificreduction of IB from fibrous scar tissue in treatedanimals but had no effect on IB from grossly normalareas in any hearts.

Additional evidence of this specific effect of captoprilon scar tissue is provided by comparing grossly normaland fibrotic regions from individual animals. This analy-sis uses each animal as its own control. The differencebetween IB from normal and fibrotic regions was13.0±0.4 dB in the treated CM hamsters (P<.0001) and15.4±0.8 dB in the untreated CM hamsters, a 2.4-dB(1.7-fold) increase (P<.0001). These data verify that IBwas significantly greater in fibrotic as compared withgrossly normal regions in CM hearts. Therefore, therapywith captopril resulted in reduced backscatter in scartissue components.

DiscussionIn this study, we used ultrasonic tissue characteriza-

tion methods to elucidate the cellular effects of capto-pril on remodeling in a heritable cardiomyopathy. These

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data indicate that captopril exerts a prominent remod-eling effect on the material properties of the scar tissuecomponent of cardiomyopathic hearts, whereas the ma-terial properties of the interspersed grossly normalmyocardium remain unchanged. The use of cardiomyo-pathic Syrian hamsters permitted accurate control ofexperimental conditions necessary to elucidate specificfeatures of ACE inhibitors in cardiac remodeling. Theuse of high-frequency, high-resolution acoustic micros-copy was crucial for defining the material properties ofintact cardiac tissue with microscopic precision.Four predictable morphological/histological and clin-

ical stages evolve in this heritable CM disease process:prenecrotic, necrotic or myolytic, hypertrophic, andterminal.20,30'31 Grossly visible white streaks are causedby fibrosis and calcification of the degenerating muscleand follow the direction of the myocardial fibers at alllevels of the hypertrophied left ventricle.3' The CMhamster proved to be an ideal model for studying themicroscopic process of cardiac remodeling by providingmultiple discrete areas of fibrosis for examination. IBincreased in both treated and untreated CM hamsters,but captopril therapy significantly limited the increasein IB in the treated CM hamsters. These differences inultrasonic scattering were due to the effects of captoprilon the fibrous scar regions because the IB from thegrossly normal (nonscarred) myocardium was not dif-ferent among the NL, treated, and untreated CManimals. Therefore, captopril induced a specific remod-eling effect primarily on the material properties of thescar tissue component, resulting in a 3.8-dB (37.2-dBversus 33.4-dB for untreated versus treated CM fibrousareas) or 2.4-fold decrease in IB from these regions.The material properties of the grossly normal, non-scarred myocardium, however, were unaffected by cap-topril treatment.Other investigators have demonstrated salutary ef-

fects of ACE inhibitors in CM Syrian hamsters. Haleenet a132 showed that administration of quinapril to CMhamsters from 180 to 300 days of age prevented thedecline of LV function in vitro and coronary flow invitro, reduced the age-dependent increases in LV vol-ume, and increased median survival time 33%, ascompared with vehicle-treated CM Syrian hamsters.Kato et a133 observed that administration of captopril to5-week-old CM Syrian hamsters ameliorated the ex-pected elevations of creatine kinase, aldolase, V3 myo-sin, and malondialdehyde (MDA) typically observed inuntreated CM hamsters. They attributed the rise ofMDA in untreated CM Syrian hamsters to an increasein active oxygen species as a consequence of damage tothe active oxygen scavenger systems after cell mem-brane damage.33 Nascimben et a134 recently showed thatenalapril treatment restores intracellular energy re-serve systolic performance in CM Syrian hamsters.Our data indicate that captopril elicited an antihyper-

trophic effect on CM hamsters by causing a 23% de-crease in LV mass as compared with the untreated CMhamster group. Treatment with captopril caused a de-crease in calcium concentration in the scar tissue re-gions but did not affect myofiber diameter or relativecollagen concentration (normalized for LV mass). Onlya small difference in myofiber diameter of approxi-

was observed in both NL and CM animals after treat-ment with captopril. Thus, ACE inhibitors appeared toinduce a proportionate reduction in the amount of bothgrossly normal myofibrils and fibrous tissues for NL andCM animals. Therefore, this effect appears generalizedin contrast to the specific effect on the material prop-

erties of scar tissue components.The hypothesis that remodeling of scar tissue ele-

ments alters the material properties of the fibrous tissueis supported by previous data from our laboratory. Weand others have shown that one of the primary deter-minants of scattering from myocardial tissue is colla-gen.18 Hoyt et a135 and Wong et al'3 also demonstrateda close correspondence between the magnitude of IBand collagen concentration. In the normal myocardium,IB is greater in the right ventricle than in the leftventricle, a difference corresponding to a higher colla-gen content in the right ventricle.36 However, the phys-ical determinants of backscatter are more complex.O'Donnell et al14 showed that the organization ofcollagen represents an important parameter of scatter-ing behavior in infarct scar tissue. They demonstratedthe magnitude of IB increases dramatically as scar tissueages and contracts. Mimbs et al'5 further defined therole of collagen organization in scattering from rabbitmyocardium. They showed that perfusion of infarctregions with collagenase resulted in a substantial de-crease in IB, although the concentration of collagenfragments measured in the infarct region remainedunchanged. These data demonstrate that the organiza-tion of the complex structure of intact collagen is a moreimportant determinant of scattering than merely theamount of its substrate present in the tissue.

Wickline et al'2 recently showed that the three-dimen-sional organization of scar tissue elements profoundlyinfluences scattering behavior in human myocardial in-farct scars. Furthermore, the angle dependence of inson-ification or ultrasonic anisotropy of grossly normal andinfarcted tissue also influences scattering behavior,'0'12'16as IB is maximal in a direction perpendicular to the fibersand minimal in a direction parallel to or along the fiberaxis. These anisotropic effects are even more pronouncedin tissue composed primarily of collagen. Hoffmeister eta137 showed a 39-dB difference in IB when insonifyingeither parallel or perpendicular to human Achilles tendon(80% to 90% type I collagen by dry weight), whereasnormal myocardium displayed only 15 dB of anisotropy.Because we insonified all tissue sections perpendicular tothe ultrasonic beam, the effect of anisotropy on themagnitude of IB was minimized for these data. Theseobservations stress the importance of the organization andtertiary structure of collagen as additional critical deter-minants of the magnitude of IB.Our laboratory has demonstrated that backscatter

depends on the intrinsic material properties of thetissue.38 These material properties, elastic stiffness (E)and density (p), determine the intrinsic acoustic imped-ance, Z, of the tissue, where Z=(Ep)°s. Local (micro-scopic) variations in acoustic impedance (or elasticstiffness and density) elicit marked changes in scatteringbehavior. The differences observed in IB between thescar tissue and grossly normal myocardial regions there-fore confirm that scar tissue is either stiffer or denser

mately 0.7 gm (7%) was noted in the untreated CMhamster. A generalized downregulation of heart growth

than normal tissue. These alterations in material prop-erties might represent increased cross-linking of colla-



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gen,39 increased scar tissue calcification, or both, whichcould increase elastic stiffness and/or density. Thishypothesis is further corroborated by the significantcorrelation between IB in fibrotic regions and thecontent of calcium in scar tissue measured by planime-try. Regardless of the particular mechanism, it is clearthat the decrease in the magnitude of IB induced bycaptopril reflects specific alterations in the materialproperties of the scar tissue elements. These dataprovide the first evidence to document changes inmaterial properties of scar tissue at the cellular level.

Potential Mechanisms Responsible forCaptopril-Induced RemodelingThe mechanisms responsible for the downregulation

of LV mass and the reorganization of scar tissue ele-ments remain unknown, although several hypothesesseem plausible. ACE inhibitors have long been recog-nized to cause regression of LVH in animal models40-44and patients.6,45 Previously, this antihypertrophic effecthad mostly been considered a secondary phenomenonattributed to hemodynamic changes brought about byACE inhibitors. However, angiotensin II is now knownto be a potent cardiac myocyte growth factor in vitro,46,47which can be blocked by ACE inhibitors.46 Evidence ofa dissociation between antihypertrophic and systemichemodynamic effects was provided by studies from Linzet a148 and Baker et al,49 which demonstrated thattreatment with "subtherapeutic" doses of ACE inhibi-tors did not affect blood pressure48 or afterload49 butresulted in prevention and regression of LVH in aortic-banded rats.Our data are compatible with these in vitro observa-

tions. Captopril caused a reduction in LV mass %(normalized for body mass) in treated NL hamsters ascompared with untreated NL controls. Thus, the lowerLV mass observed in the captopril-treated CM hamstergroup may have been due to inhibition of the growth-promoting effects of angiotensin II. However, this effectmay be unrelated to scar tissue formation, since it wasobserved in both NL and CM myocardium. Despitereducing LV mass %, captopril treatment did not resultin any significant changes in myofiber diameter in bothNL and CM groups, which suggests an antihyperplasticeffect. Thus, treatment with captopril would seem toelicit a nonspecific downregulation of growth in alltissue components.

Angiotensin II also may induce fibroblast prolifera-tion,50 in vitro collagen synthesis,51 and in vivo myocar-dial fibrosis.50,52 Tan et a153 showed that chronic infusionof subpressor doses of angiotensin II resulted in afibrotic response in rat hearts. ACE inhibitors couldfunction to decrease or prevent fibrogenic propensity ofangiotensin II.

Captopril has been shown to augment intracellularcollagen degradation.54 Angiotensin II stimulates colla-gen synthesis from cardiac fibroblasts in vitro, whereasprostaglandin E2 inhibits this process.51 ACE inhibitioncould decrease levels of both circulating and tissueangiotensin II and increase myocardial prostaglandinE2, which together may ameliorate myocardial fibrosis.Furthermore, ACE inhibition may concomitantly in-crease collagenase activity, which could further de-crease the extent of myocardial fibrosis. Previous obser-

profound impact of treatment of myocardial scar tissuewith clostridial collagenase to reduce the magnitude ofIB.15 We observed a similar increase in collagen con-centration in both CM groups. Enhanced collagen deg-radation could have played a role in both the decreasein IB as well as the decreased LV mass % in treated CMhamsters.Other studies have shown that CM Syrian hamsters

demonstrate a significant increase in mitochondrial freeradicals,24,55 antioxidant activity,55 and myocardial lipidperoxides24 compared with normal age-matched controlhamsters. The upregulation of antioxidant activity isapparently compensatory although inadequate, becausetreatment with a-tocopherol, a free radical scavenger,elicits a significant decrease in myocardial damage andcalcification as compared with untreated CM controlhearts.55

It has been demonstrated that those ACE inhibitorsthat contain a sulfhydryl group (captopril and zofeno-pril) possess free radical scavenger properties,56whereas others do not.8 Many studies have shown acardioprotective effect with captopril treatment in bothstunned myocardium and injury, whereas enalapril ef-fects were somewhat more variable.8 Other theoreticalmechanisms warrant additional consideration, however,because both quinapril32 and enalapril,34 ACE inhibi-tors without a sulfhydryl group, exhibit significant salu-tary effects in Syrian hamster CM.Because captopril appears to induce a proportional

downregulation of growth in all tissue components, itmay have delayed the natural history of the cardiomy-opathy, resulting in reduced LV mass and essentially"younger" scar tissue in the treated CM hamsters at 4months of age. This hypothesis is further supported bythe strong correlation between LV mass % and scartissue calcium content: The larger the left ventricle, thefurther the progression of the natural history, and theolder the scar, the more extensive the calcification. Ourlaboratory has previously shown that the magnitude ofIB increases dramatically as scar tissue ages and con-tracts.14 If the scarred regions of the captopril-treatedCM hamster hearts were less mature or less wellorganized (either less cross-linked and/or calcified),scars would be expected to produce less backscatter ascompared with the more mature fibrotic regions of theuntreated CM hamsters. Thus, the effect of captopril todelay scar tissue calcification may be due to molecularmechanisms that may inhibit the deposition of a tissuematrix that would favor calcification.

Clinical ImplicationsIn our study, captopril induced potentially beneficial

changes in the material properties of scarred myocar-dium. The ability to detect these changes with ultra-sonic tissue characterization may have many clinicalramifications. Although ACE inhibitors have beenshown to cause regression of LVH6 and reduce mortal-ity in chronic CHF4 5 and after myocardial infarction,57it is unclear whether these results are a product of a

hemodynamic effect or operate directly on tissuethrough an independent mechanism. Our study demon-strated one of the potential reasons why ACE inhibitorsmay play a salutary role in various forms of heartdisease. As elucidated with ultrasonic tissue character-

vations from our laboratory have demonstrated the ization, the scarred myocardium from captopril-treated



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CM hamsters either manifested a delayed natural his-tory or a direct reorganization of scar tissue compo-nents, either of which could result in a more compliantscar with reduced cross-linking and calcification. Hypo-thetically, more compliant scars would result in lowerLV filling pressures, thereby improving diastolic func-tion and ameliorating the need for a compensatoryLVH or LV dilation to maintain systolic function.Our data indicate that treatment of CM hamsters

with captopril for 3 months produced an antihyper-trophic effect, evidenced by a 23% decrease in LV mass

compared with the untreated CM group. We demon-strated that ultrasonic tissue characterization can sen-

sitively detect captopril-induced changes in materialproperties of scar tissue components of the heart. Theseare the first data to show that captopril elicits specificchanges in scar tissue material properties, which mayreflect retarded maturation and calcification of scar. Todate, there are no reliable methods for monitoring theremodeling effects of ACE inhibitors at the cellularlevel. Quantitative ultrasonic tissue characterizationmay ultimately play a role in initiating and guiding thetherapy with ACE inhibitors in patients with cardiomy-opathy and other forms of CHF.

AcknowledgmentsThis study was supported in part by grants HL-42950,

HL-40302, and an Established Investigator Award from theAmerican Heart Association (Dr Wickline).

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G Davison, C S Hall, J G Miller, M Scott and S A Wicklinecardiomyopathy.

Cellular mechanisms of captopril-induced matrix remodeling in Syrian hamster

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