279:H1534-H1539, 2000. ;Am J Physiol Heart Circ Physiol Joseph S. JanickiLuiz S. Matsubara, Beatriz B. Matsubara, Marina P. Okoshi, Antonio C. Cicogna andpapillary muscle functionAlterations in myocardial collagen content affect rat

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1522-1539. Visit our website at http://www.the-aps.org/. Pike, Bethesda MD 20814-3991. Copyright © 2000 the American Physiological Society. ISSN: 0363-6135, ESSN:molecular levels. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville cardiovascular function at all levels of organization ranging from the intact animal to the cellular, subcellular, andphysiology of the heart, blood vessels, and lymphatics, including experimental and theoretical studies of

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Alterations in myocardial collagen contentaffect rat papillary muscle function

LUIZ S. MATSUBARA,1 BEATRIZ B. MATSUBARA,1 MARINA P. OKOSHI,1

ANTONIO C. CICOGNA,1 AND JOSEPH S. JANICKI2

1Departamento de Clınica Medica, Faculdade de Medicina de Botucatu, Universidade EstadualPaulista, Botucatu, Sao Paulo, Brazil 18618-000; and 2Department of Anatomy, Physiology,and Pharmacology, Auburn University, Auburn, Alabama 36849-5517Received 15 June 1999; accepted in final form 19 April 2000

Matsubara, Luiz S., Beatriz B. Matsubara, Marina P.Okoshi, Antonio C. Cicogna, and Joseph S. Janicki.Alterations in myocardial collagen content affect rat papil-lary muscle function. Am J Physiol Heart Circ Physiol 279:H1534–H1539, 2000.—We investigated the influence of myo-cardial collagen volume fraction (CVF, %) and hydroxypro-line concentration (mg/mg) on rat papillary muscle function.Collagen excess was obtained in 10 rats with unilateral renalischemia for 5 wk followed by 3-wk treatment with ramipril(20 mg zkg21 zday21) (RHTR rats; CVF 5 3.83 6 0.80, hy-droxyproline 5 3.79 6 0.50). Collagen degradation was in-duced by double infusion of oxidized glutathione (GSSG rats;CVF 5 2.45 6 0.52, hydroxyproline 5 2.85 6 0.18). Nineuntreated rats were used as controls (CFV 5 3.04 6 0.58,hydroxyproline 5 3.21 6 0.30). Active stiffness (AS;g zcm22 z%Lmax

21) and myocyte cross-sectional area (MA;mm2) were increased in the GSSG rats compared with con-trols [AS 5.86 vs. 3.96 (P , 0.05); MA 363 6 59 vs. 305 6 28(P , 0.05)]. In GSSG and RHTR groups the passive tension-length curves were shifted downwards, indicating decreasedpassive stiffness, and upwards, indicating increased passivestiffness, respectively. Decreased collagen content induced byGSSG is related to myocyte hypertrophy, decreased passivestiffness, and increased AS, and increased collagen concen-tration causes myocardial diastolic dysfunction with no effecton systolic function.

renovascular hypertension; fibrosis; oxidized glutathione; ac-tive stiffness; passive stiffness

MYOCARDIAL COLLAGEN CONCENTRATION is elevated inchronic arterial hypertension, aortic stenosis, experi-mental renovascular hypertension, and genetic hyper-tension (2, 6, 17, 28). In view of the mechanicalstrength and inextensibility of collagen (19), an in-creased concentration of this material within the myo-cardium would be expected to have a significant influ-ence on left ventricular (LV) chamber and myocardialstiffness. Studies in spontaneously hypertensive rats(SHR), which show a marked increase in myocardialstiffness and fibrosis, appear to suggest that a changein intrinsic myocardial function may be caused at leastin part by alterations in the extracellular matrix (5).

However, significant hypertrophy also occurs in thesevarious models of LV pressure overload, and one couldargue that myocyte enlargement also contributes tothe abnormal stiffness.

Two studies designed to determine the separate in-fluences of hypertrophy and abnormal collagen concen-tration on myocardial stiffness have resulted in con-flicting conclusions (23, 25). Narayan et al. (23)assumed a spherical LV to calculate myocardial stiff-ness from LV pressure and volume data and concludedthat increased collagen accumulation, but not hyper-trophy, was responsible for an abnormal diastolic stiff-ness in the SHR. Schraeger et al. (25) used ventricularstrips from SHR with and without hypertrophy toobtain tension-length curves and reported that in-creased collagen concentration does not affect musclestiffness.

Others suggested that increased connective tissuewould be responsible for the increased passive stiffnessof hypertrophied trabecular and papillary muscles;however, they did not experimentally rule out the po-tential contribution of muscle hypertrophy (4, 14). Itwas further suggested that myocardial fibrosis mayrestrict myofibrillar motion and thereby impair systolicand diastolic function (29). Conrad et al. (12) observedin SHR failing hearts a reduction in tension develop-ment in association with an increased LV hydroxypro-line concentration, but they did not conclude whetherthe myocardial dysfunction was caused by fibrosis or bya relative reduction in the number of myocytes.

On the other hand, few studies have addressed theeffects of decreased collagen content without ischemiaon myocardial function. Caulfield et al. (10) observedthat the loss of collagen struts that interconnect myo-cytes had no effect on either myocyte contractility orforce delivery to the ventricle. However, they did findthis loss to cause a marked dilation of the ventricle andincreased distensibility. Thus the purpose of this studywas to analyze the relationship between LV myocar-dial collagen content and papillary muscle passive andactive stiffness. To this end, LV papillary muscles from

Address for reprint requests and other correspondence: L. S. Mat-subara, Departamento de Clınica Medica, Faculdade de Medicina deBotucatu, 18618-000 Botucatu, Sao Paulo, Brazil (E-Mail: lsmatsu@fmb.unesp.br).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Heart Circ Physiol279: H1534–H1539, 2000.

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groups of rats with different amounts of myocardialcollagen were studied.

METHODS

Experimental procedure. Thirty-three male Wistar ratswere used in the study. Their care and use conformed withNational Institutes of Health guidelines and the protocol wasapproved by the University Animal Care and Use Commit-tee. In the first group, 10 rats (6 wk old) were anesthetizedwith pentobarbital sodium (50 mg/kg ip), and renal hyper-tension was produced by placing a silver clip around the leftrenal artery to constrict it to an external diameter of 0.25mm; the contralateral kidney remained normally perfused.After a 5-wk follow-up, the rats were treated for 3 wk withthe angiotensin-converting enzyme (ACE) inhibitor ramipril(20 mg zkg21 zday21 in drinking water; RHTR group). In thesecond group (GSSG group, n 5 14), myocardial collagendegradation was induced using the method described byCaulfield and Wolkowicz (11). Briefly, 10 wk-old rats wereanesthetized and received two intravenous infusions over 3 h(0.11 ml/min), 1 wk apart, of 20 ml of a 2 mM solution ofoxidized glutathione. The animals were killed 3 wk after thesecond infusion, when myocardial hydroxyproline is expectedto be at a minimum (11). A third group (control, n 5 9)consisted of unoperated and untreated normotensive ratsthat were the same age as the other two groups at the end ofthe experiment (i.e., 14 wk old). All rats were housed in atemperature-controlled room (24°C) with 12-h light:dark cy-cles, and food and water were supplied ad libitum. At the endof the experiment, tail cuff systolic arterial pressure (SAP)was measured in all rats.

Isolated papillary muscle study. The animals were anes-thetized with pentobarbital sodium (50 mg/kg ip), and thebody weight (BW) was recorded at the time of death. Thechest was opened by median sternotomy, and the heart wasremoved and placed in oxygenated Krebs-Henseleit solutionat 28°C. The LV and septal wall were separated from theright ventricle, and their weights were determined. Onepapillary muscle was dissected from the LV, mounted be-tween two spring clips, and placed vertically in a bathingchamber. The lower spring clip was attached to a Kyowamodel 120T-20B force transducer by a thin (1/15,000 in.)steel wire. The upper spring clip was connected by a thin wireto a rigid lever arm above which was mounted an adjustablemicrometer stop for the adjustment of unstimulated musclelength. Oxygenated (95% O2-5% CO2) bathing medium con-sisted of (in mM) 118.5 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16MgSO4, 1.18 KH2PO4, 5.50 glucose, and 25.88 NaHCO3 dis-solved in deionized water. The temperature of the bathingmedium was maintained at 28°C.

The muscle preparation was placed between two parallelplatinum electrodes and stimulated at a frequency of 0.2 Hz,using square-wave pulses of 5-ms duration. Voltage was setto a value 10% greater than the minimum required to pro-duce a maximal mechanical response. After 60 min, duringwhich the preparation stabilized, the muscle was loaded tocontract isometrically and stretched to the peak length of itstension-length curve (Lmax).

Once a stable Lmax was determined, the muscle was madeto contract isometrically at Lmax and the resultant isometriccontraction parameters were determined, which includedpeak developed active tension (AT, g/mm2), resting tension(RT, g/mm2), peak rate of isometric tension development(1dT/dt, g zmm22 zs21), peak rate of tension decrease (2dT/dt, g zmm22 zs21), time to peak tension (TPT, ms), and timefrom peak tension to 50% relaxation (RT1/2, ms). Active and

passive tension-length curves were derived from data ob-tained at lengths corresponding to 90%, 92%, 94%, 96%, 98%,and 100% of Lmax. The muscle length was measured with aGaertner cathetometer and telescope. At the end of the ex-periment, the muscle between the spring clips was weighedand its cross-sectional area (CSA) was calculated, assumingcylindrical uniformity and a specific gravity of 1.00. All val-ues of force were normalized for muscle CSA.

Biochemical study. It has been demonstrated that hy-droxyproline concentration in the LV free wall is similar tothat in the papillary muscle (15). Therefore, we assumed thatthe hydroxyproline observed in the apex of the LV is repre-sentative of that in the entire ventricle, including the papil-lary muscle. We measured hydroxyproline in tissue obtainedfrom the LV apex according to the method described bySwitzer (27). Briefly, the tissue was dried for 4 h using aSpeedVac Concentrator SC 100 attached to a refrigeratedcondensation trap TR 100 and vacuum pump VP 100 (SavantInstruments, Farmingdale, NY). Tissue dry weight was de-termined, and the samples were hydrolyzed overnight at110°C with 6 N HCl (1 ml/10 mg dry tissue). An aliquot of 50ml of hydrolysate was transferred to an Eppendorf tube anddried in the SpeedVac Concentrator. One milliliter of deion-ized water was added, and the sample was transferred to atube. One milliliter of potassium borate buffer (pH 8.7) wasadded to maintain stable pH, and the sample was oxidizedwith 0.3 ml of chloramine T solution at room temperature forexactly 20 min. The oxidation was stopped by the addition of1 ml of 3.6 M sodium thiosulfate with thorough mixing for10 s. The solution was then saturated with 1.5 g of KCl, andthe tubes were capped and heated in boiling water for 20 min.After the tubes cooled to room temperature, 2.5 ml of toluenewere added and the tubes were shaken over 5 min. The tubeswere briefly centrifuged at low speed, and 1 ml of tolueneextract was transferred to a 12 3 75 mm test tube. In thenext step, 0.4 ml of Ehrlich’s reagent was added to allow thecolor to develop for 30 min. Absorbencies were read at 565 nmwith a double-beam spectrophotometer (A-160 spectropho-tometer, Shimadzu) against a reagent blank. Deionized wa-ter and 20 mg/ml hydroxyproline were used as blank andstandard, respectively.

Histology and morphometry. Transverse sections of LVwere fixed in 10% buffered Formalin and embedded in par-affin. Five-micrometer-thick sections were cut from theblocked tissue and stained with hematoxylin-eosin and withthe collagen-specific stain picrosirius red (Sirius red F3BA inaqueous saturated picric acid). Myocyte CSA (MA) was de-termined for at least 100 myocytes per slide stained withhematoxylin-eosin. The measurements were performed usinga Leica microscope (340 magnification lens) attached to avideo camera and connected to a personal computer equippedwith image analyzer software (Image-Pro Plus 3.0, MediaCybernetics, Silver Spring, MD). MA was measured with adigitizing pad, and the selected cells were transversely cutwith the nucleus clearly identified in the center of the myo-cyte. Interstitial collagen volume fraction (CVF) was deter-mined for the entire section of the heart stained with picro-sirius red using an automated image analyzer (Image-ProPlus 3.0, Media Cybernetics). The components of the cardiactissue were identified according to their color level: red forcollagen fibers, yellow for myocytes, and white for interstitialspace. The digitized profiles were sent to a computer thatcalculated collagen volume fraction as the sum of all connec-tive tissue areas divided by the sum of all connective tissueand myocyte areas. On the average, 35 microscopic fieldswere analyzed with a 320 lens. Perivascular collagen wasexcluded from this analysis.

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Statistics. All grouped data were expressed as means 6 SDand compared by one-way ANOVA and post hoc Tukeys test.Statistical analyses were performed with SigmaStat statisti-cal software (Jandel Scientific Software, San Rafael, CA).Differences with P # 0.05 were considered significant.Straight lines were fit to the systolic tension-length relationsusing linear regression analysis (22). The resulting slopescorresponded to AS, and the means among the groups werecompared by ANOVA. Before the diastolic tension-lengthrelationship was compared for the three groups, the restingtension at the muscle length corresponding to 90% of Lmax(L90) was subtracted from all subsequent tension data in eachexperiment to have all tension-length curves intercepting they-axis origin at L90.

The diastolic tension-length curves for the three groupswere fit to monoexponential relations of the form RT 5A[eB(L 2 L0) 2 1], where A and B are fitting parameters and L0is the muscle length corresponding to zero resting tension.These nonlinear relations were compared by constructing anF ratio from the residual sum of squares. This test deter-mines whether separate fits to three groups are significantlybetter than the fit to data pooled from all groups. Accord-ingly, a significant F ratio indicates that the two sets of databeing compared were significantly different from one an-other. For all comparisons, statistical significance was takento be P , 0.05/k where k is the number of comparisons (24).

RESULTS

Average group values for BW, LV weight (LVW),right ventricular weight, papillary muscle CSA, SAP,and LVW normalized to BW (LVW/BW) are shown inTable 1. In the RHTR group, treatment with an ACEinhibitor for 3 wk significantly reduced systolic bloodpressure from an average value of 202 6 31 mmHg to111 6 11 mmHg (P , 0.001) and regressed LVW to avalue comparable to the control and GSSG groups. MAwas significantly higher in the GSSG group comparedwith control and RHTR groups (Fig. 1). CVF and hy-droxyproline (Fig. 2) were statistically higher in RHTRthan in the other two groups. The difference betweenGSSG and control groups reached a level of signifi-cance of 10% (Fig. 2A), whereas hydroxyproline wasstatistically lower in the GSSG compared with thecontrol group (Fig. 2B).

The isolated papillary muscle functional parametersRT, Lmax, AT at Lmax, AT at L90, 1dT/dt, 2dT/dt, TPT,

TR1/2, and AS are shown in Table 2. RT was signifi-cantly higher in the RHTR group (0.64 6 0.08 g/mm2)compared with control (0.47 6 0.14 g/mm2) and GSSG(0.35 6 0.10 g/mm2) groups. AT at L90 and at Lmax werenot different among the groups.

In all experiments the relation between peak devel-oped active tension and muscle length was linear, asevidenced by the coefficient of determination (r2),which was typically .0.94. This finding means that atleast 94% of the sum of squares of deviations of ATvalues about their means is attributable to the linearrelation between AT and muscle length (22). The slopeof these linear regressions corresponds to the myocar-dial AS, which was significantly increased in the GSSGgroup compared with the control group (5.86 6 1.14 vs.3.96 6 1.33 g zmm22 z%Lmax

21; P 5 0.008). The differ-ences between GSSG and RHTR groups and betweencontrol and RHTR groups were not statistically signif-icant (Fig. 3).

The passive tension-length curve from the RHTRgroup was shifted upward from that of the controlgroup (F 5 14.25; P , 0.01) and that of the GSSG group(F 5 38.8; P , 0.01), reflecting an increased passivestiffness. The GSSG curve was shifted downwardsfrom the control group (F 5 9.95; P , 0.01), indicatingdecreased passive stiffness (Fig. 3).

DISCUSSION

In a previous study (21), we showed that renovascu-lar hypertension induces marked myocardial hypertro-phy and interstitial fibrosis. Treatment with ramiprilfor 3 wk did not reverse perivascular and interstitialfibrosis but fully treated the arterial hypertension andpromoted regression of myocardial hypertrophy.Therefore, we used that experimental model to studymyocardial function in papillary muscle from rat heartwith increased collagen concentration without myocar-dial hypertrophy. In the present study, collagen

Table 1. Group comparisons of morphometricparameters and tail cuff systolic arterial pressurein control, GSSG, and RHTR rats

Control GSSG RHTR

BW, g 329617 332620 346630SAP, mmHg 136614 129618 111611*LVW, g 0.6660.07 0.6560.05 0.6960.14LVW/BW, mg/g 2.0160.16 1.9660.09 1.9060.17RVW, g 0.2160.03 0.1960.05 0.2160.04CSA, mm2 0.8460.18 0.7860.17 0.8660.22

Data are presented as means 6 SD. BW, body weight; SAP,systolic arterial pressure; LVW/BW, left ventricle weight (LVW) toBW ratio; RVW, right ventricle weight; CSA, papillary muscle cross-sectional area. *P , 0.05 vs. control. See METHODS for description ofthe control, oxidized glutathione (GSSG), and ramipril-treated rat(RHTR) groups.

Fig. 1. Myocyte cross-sectional area in control, oxidized glutathione(GSSG), and ramipril-treated rat (RHTR) groups. Data are means 6SD analyzed by one-way ANOVA with Tukey’s posttest procedure.

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amount was measured with CVF and hydroxyproline.It has been shown that total volume fraction is closelyrelated to hydroxyproline concentration in the LV (28),and in our study both measurements indicated that theinterstitial collagen was altered in the treated groupsrelative to the control rats. However, we observed thatthe CVF measurement was associated with a greatervariation than the measurement of hydroxyproline,and, consequently, the decrease in CVF in the GSSGgroup came close but did not reach the level of statis-tical significance. The variability of CVF might becaused in part by the measurement method used. Inthe present investigation we used a 320 microscopeobjective to obtain a large field. This magnificationwould detect only large perimysial collagen fibers,thereby decreasing the sensitivity of the measurement.Even so, a power analysis indicated that the differencebetween the GSSG and control groups would havereached the level of significance if but a few additionalhistological samples were available.

Despite similar LVW, the papillary muscles weresignificantly stiffer in the group with greater collagenconcentration. This result is similar to that obtained byNarayan et al. (23) using hydralazine to prevent myo-cyte hypertrophy but not abnormal collagen accumula-tion in SHR. The collagen excess resulted in abnor-mally elevated passive myocardial stiffness. Incontrast, Schraeger and co-workers (25) concluded thatACE inhibitor-induced regression of LV hypertrophy in

Fig. 3. Left ventricular papillary muscle active and passive tension-length curves obtained from control, GSSG, and RHTR groups.Results are presented as means 6 SD. The active stiffness (AS)obtained from the GSSG group was statistically higher than thatfrom controls (P 5 0.008). Statistically, no differences were observedbetween GSSG and RHTR groups (P 5 0.493) and between controland RHTR groups (P 5 0.085). AS was analyzed by ANOVA and posthoc Tukey’s test. The RHTR passive tension-length curve was shiftedupwards compared with either control (F 5 14.25; P , 0.01) or GSSG(F 5 38.8; P , 0.01) groups. The curve from the GSSG group wasshifted downwards compared with the control group (F 5 9.95; P ,0.01). The passive tension-length curves were fitted to a monoexpo-nential relation, and comparisons were made by constructing an Fratio from the residual sum of squares. Statistical significance wastaken to be P # 0.05/k where k is the number of comparisons.

Table 2. Papillary muscle isometric contraction datafor the control, GSSG, and RHTR groups

Control GSSG RHTR

RT, g/mm2 0.4760.14 0.3560.10 0.6460.08*†AT at Lmax, g/mm2 8.0561.59 9.4461.86 8.5261.72AT at L90, g/mm2 5.6461.26 5.9561.64 5.2361.58RT1/2, ms 290654 272623 280647Lmax, mm 6.2360.99 6.0760.50 6.3160.811dT/dt, g zmm22 zs21 75.8621.1 84.3621.0 72.8616.52dT/dt, g zmm22 zs21 19.065.5 21.864.9 18.864.1AS, g zmm22 z%Lmax

21 3.9661.33 5.8661.14* 5.2161.18TPT, ms 201616 193612 204617

Data are reported as means 6 SD. RT, resting tension; AT, activetension; 1dT/dt, peak rate of isometric tension development; 2dT/dt,peak rate of tension decrease; TPT, time to peak tension; Lmax,muscle length at peak of the tension-length curve; L90, muscle lengthat 90% of Lmax; RT1/2, time from peak tension to 50% relaxation; AS,active stiffness. *P , 0.05 vs. control; *†P , 0.05 vs. GSSG.

Fig. 2. Collagen volume fraction (A) and hydroxyproline concentra-tion (B) in control, GSSG, and RHTR groups. Data are means 6 SDanalyzed by one-way ANOVA with Tukey’s posttest procedure.

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SHR significantly decreased the passive stiffness ofskinned trabecular muscle despite abnormally ele-vated hydroxyproline levels. It is not clear to whatextent, if any, the 48-h incubation in the skinningsolution at 0°C influenced their observations. The dis-crepancies observed between the studies may becaused by the animal strains as well as by the differentexperimental models used to produce hypertrophy andfibrosis.

In our study, using the model of presumed collage-nase activation by oxidized glutathione described byCaulfield and Wolkowicz (11), it was possible to induce,in vivo, an 11% reduction of myocardial collagen con-centration measured by hydroxyproline concentrationand a 19% reduction in the interstitial CVF. Theseresults are less expressive than the 30–35% reductionin collagen as reported by Caulfield et al. (10). Theauthors have shown that the double infusion of GSSGresulted in no visible myocyte damage at any time asexamined by light microscopy and scanning electronmicroscope (SEM). The collagen matrix alteration wasnot visible by light microscopy; SEM revealed damageto the endomysium with loss of the weave that sur-rounds groups of myocytes and the struts that inter-connect myocyte to myocyte and myocyte to adjacentcapillaries, with no change in coiled perimysial fibers.These changes in the fibrillar collagen network re-sulted in increased ventricular volume and compliance,suggesting that damage to the intermyocyte struts andto the weave complex might be more important thanthe decrease in myocardial collagen. Other studieshave shown that the double infusion of GSSG in ratspromotes a reduction in CVF, ventricular dilatation,and a shift to the right of the diastolic pressure-volumecurve of the entire LV (18, 20). However, a similareffect in the papillary muscle preparation has not beenstudied previously. The main advantage of this prepa-ration is that the muscle force and length are directlymeasured and that the mathematical assumptions re-quired when myocardial mechanical characteristicsare evaluated in the LV chamber are unnecessary.

The study of cardiac function in the whole heart isbased on the pressure-volume and stress-strain rela-tionships. In that condition, myocardial stiffness isderived from chamber measurements using mathemat-ical models and assumptions regarding LV shape. Ifthe LV is assumed to be a thick-walled sphere, thestress will be underestimated (30), whereas the as-sumption of an ellipsoid shape would result in anoverestimated wall stress (7). Therefore, isolated mus-cle experiments provide descriptions of myocardial be-havior without the influence of chamber and wall ge-ometry. In our study, the diastolic tension-lengthrelations obtained for the three groups were differentfrom each other, showing that the changes in collagencontent, measured by hydroxyproline and CVF, areassociated with myocardial passive properties. Com-pared with the control group, the diastolic tension-length curves were significantly shifted upwards andto the left in the RHTR group and downwards and tothe right in the GSSG group. Therefore, our results

allow us to conclude that the decreased passive stiff-ness in the GSSG group strongly correlates with thefibrillar collagen loss and that increased collagen con-tent strongly correlates with the elevated passive stiff-ness observed in the RHTR group. Previous studieshave suggested that collagen cross-linking (9) mayaffect myocardial stiffness, regardless of collagenamount. In addition to the effect of altered collagenamounts, it is important to be mindful of the effects ofcollagen crosslink density, as well as collagen type(type I or III) and collagen distribution. At present, wecannot rule out that changes in the collagen character-istics might also have influenced myocardial stiffnessin the present study. Nevertheless, the results clearlyindicate that alterations in collagen concentration andpapillary muscle function are correlated.

Ventricular elastance and myocardial stiffness areindexes of contractility of the ventricular chamber andmyocardium, respectively (8, 26). Elastance is the ratioof the change in peak isovolumetric pressure for agiven change in volume, and stiffness is defined as theratio of the change in active force related to change inmuscle length (8). Myocardial contractility is a verycomplex property of the heart that is difficult to mea-sure directly. During the last two decades it has beenproposed that an ideal index of myocardial contractilitymust be able to measure the ability of the myocardiumto generate force independently of loading condition.The slope of the linear pressure-volume relationship inthe isolated canine heart has been shown to be rela-tively independent of preload and afterload and there-fore has been used as an index of contractility (26).

Using the slope of active tension-length (active stiff-ness) as an index of myocardial contractility, we haveshown an enhancement of active stiffness when themuscle is stretched from 90% to 100% of Lmax in theGSSG group. The mechanisms underlying the associa-tion between decreased myocardial collagen and en-hanced active stiffness are not well established, andthe results presented in this study do not answer allthe questions concerning this matter. When collagen isreduced, ventricular dilatation occurs (10) and myocytehypertrophy takes place in response to alterations inthe loading state of the ventricle (17). Therefore, myo-cyte hypertrophy might play an important role in theimprovement in contractility observed in the GSSGgroup. Another explanation would be related to theintracellular glutathione metabolisms. The glutathi-one level in the heart is ;1.2 mM/g (16), mainly in thereduced form, GSH, because of the high activity ofGSSG reductase (13). That means that, inside the cell,most of the infused GSSG was rapidly converted toGSH. The action of excess GSH or GSSG in the heart isnot completely elucidated. Bauer et al. (3), working onfiber bundles from papillary muscle of porcine rightventricle, observed an increased sensitivity of contrac-tile protein to calcium and, consequently, an increasedforce development in the presence of GSH. In ourstudy, considering that the half-life of the glutathioneis only a few minutes (1), it is doubtful that the doubleinfusion of oxidized glutathione might increase the

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glutathione level in the cardiac tissue after 3 wk.Nevertheless, this is a very complex matter that re-quires further study. Active tension and active stiff-ness in papillary muscles from RHTR rats were similarto those in the control rats, suggesting that regressionof hypertrophy by treatment with an ACE inhibitor isassociated with preserved myocardial contractility.

We conclude that decreased collagen content inducedby GSSG is associated with myocyte hypertrophy, de-creased passive stiffness, and increased active stiff-ness. Abnormally high collagen concentration corre-lates with myocardial diastolic dysfunction and has norelation with systolic function.

This study was supported by a grant from Fundacao de Amparo aPesquisa do Estado de Sao Paulo (FAPESP), Sao Paulo, Brazil, Proc.No. 92/4528–1.

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