Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Am j physiol heart circ physiol 2000-matsubara-h1534-9


Published on

colageno miocardico

Published in: Healthcare
  • Be the first to comment

  • Be the first to like this

Am j physiol heart circ physiol 2000-matsubara-h1534-9

  1. 1. 279:H1534-H1539, 2000. ;Am J Physiol Heart Circ Physiol Joseph S. Janicki Luiz S. Matsubara, Beatriz B. Matsubara, Marina P. Okoshi, Antonio C. Cicogna and papillary muscle function Alterations in myocardial collagen content affect rat You might find this additional info useful... 24 articles, 11 of which you can access for free at:This article cites 15 other HighWire-hosted articles:This article has been cited by including high resolution figures, can be found at:Updated information and services can be found at:Physiology American Journal of Physiology - Heart and CirculatoryaboutAdditional material and information This information is current as of July 25, 2013. 1522-1539. Visit our website at 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, and physiology of the heart, blood vessels, and lymphatics, including experimental and theoretical studies of publishes original investigations on theAmerican Journal of Physiology - Heart and Circulatory Physiology byguestonJuly25,2013
  2. 2. Alterations in myocardial collagen content affect rat papillary muscle function LUIZ S. MATSUBARA,1 BEATRIZ B. MATSUBARA,1 MARINA P. OKOSHI,1 ANTONIO C. CICOGNA,1 AND JOSEPH S. JANICKI2 1 Departamento de Clı´nica Me´dica, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, Sa˜o Paulo, Brazil 18618-000; and 2 Department of Anatomy, Physiology, and Pharmacology, Auburn University, Auburn, Alabama 36849-5517 Received 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 (␮g/mg) on rat papillary muscle function. Collagen excess was obtained in 10 rats with unilateral renal ischemia for 5 wk followed by 3-wk treatment with ramipril (20 mg⅐kgϪ1 ⅐dayϪ1 ) (RHTR rats; CVF ϭ 3.83 Ϯ 0.80, hy- droxyproline ϭ 3.79 Ϯ 0.50). Collagen degradation was in- duced by double infusion of oxidized glutathione (GSSG rats; CVF ϭ 2.45 Ϯ 0.52, hydroxyproline ϭ 2.85 Ϯ 0.18). Nine untreated rats were used as controls (CFV ϭ 3.04 Ϯ 0.58, hydroxyproline ϭ 3.21 Ϯ 0.30). Active stiffness (AS; g⅐cmϪ2 ⅐%Lmax Ϫ1 ) and myocyte cross-sectional area (MA; ␮m2 ) were increased in the GSSG rats compared with con- trols [AS 5.86 vs. 3.96 (P Ͻ 0.05); MA 363 Ϯ 59 vs. 305 Ϯ 28 (P Ͻ 0.05)]. In GSSG and RHTR groups the passive tension- length curves were shifted downwards, indicating decreased passive stiffness, and upwards, indicating increased passive stiffness, respectively. Decreased collagen content induced by GSSG is related to myocyte hypertrophy, decreased passive stiffness, and increased AS, and increased collagen concen- tration causes myocardial diastolic dysfunction with no effect on systolic function. renovascular hypertension; fibrosis; oxidized glutathione; ac- tive stiffness; passive stiffness MYOCARDIAL COLLAGEN CONCENTRATION is elevated in chronic arterial hypertension, aortic stenosis, experi- mental renovascular hypertension, and genetic hyper- tension (2, 6, 17, 28). In view of the mechanical strength 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 myocardial stiffness. Studies in spontaneously hypertensive rats (SHR), which show a marked increase in myocardial stiffness and fibrosis, appear to suggest that a change in intrinsic myocardial function may be caused at least in part by alterations in the extracellular matrix (5). However, significant hypertrophy also occurs in these various models of LV pressure overload, and one could argue that myocyte enlargement also contributes to the 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 concluded that increased collagen accumulation, but not hyper- trophy, was responsible for an abnormal diastolic stiff- ness in the SHR. Schraeger et al. (25) used ventricular strips from SHR with and without hypertrophy to obtain tension-length curves and reported that in- creased collagen concentration does not affect muscle stiffness. Others suggested that increased connective tissue would be responsible for the increased passive stiffness of hypertrophied trabecular and papillary muscles; however, they did not experimentally rule out the po- tential contribution of muscle hypertrophy (4, 14). It was further suggested that myocardial fibrosis may restrict myofibrillar motion and thereby impair systolic and diastolic function (29). Conrad et al. (12) observed in SHR failing hearts a reduction in tension develop- ment in association with an increased LV hydroxypro- line concentration, but they did not conclude whether the myocardial dysfunction was caused by fibrosis or by a relative reduction in the number of myocytes. On the other hand, few studies have addressed the effects of decreased collagen content without ischemia on myocardial function. Caulfield et al. (10) observed that the loss of collagen struts that interconnect myo- cytes had no effect on either myocyte contractility or force delivery to the ventricle. However, they did find this loss to cause a marked dilation of the ventricle and increased distensibility. Thus the purpose of this study was to analyze the relationship between LV myocar- dial collagen content and papillary muscle passive and active stiffness. To this end, LV papillary muscles from Address for reprint requests and other correspondence: L. S. Mat- subara, Departamento de Clı´nica Me´dica, Faculdade de Medicina de Botucatu, 18618-000 Botucatu, Sa˜o Paulo, Brazil (E-Mail: lsmatsu The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Am J Physiol Heart Circ Physiol 279: H1534–H1539, 2000. 0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society http://www.ajpheart.orgH1534 byguestonJuly25,2013
  3. 3. groups of rats with different amounts of myocardial collagen were studied. METHODS Experimental procedure. Thirty-three male Wistar rats were used in the study. Their care and use conformed with National Institutes of Health guidelines and the protocol was approved by the University Animal Care and Use Commit- tee. In the first group, 10 rats (6 wk old) were anesthetized with pentobarbital sodium (50 mg/kg ip), and renal hyper- tension was produced by placing a silver clip around the left renal artery to constrict it to an external diameter of 0.25 mm; the contralateral kidney remained normally perfused. After a 5-wk follow-up, the rats were treated for 3 wk with the angiotensin-converting enzyme (ACE) inhibitor ramipril (20 mg⅐kgϪ1 ⅐dayϪ1 in drinking water; RHTR group). In the second group (GSSG group, n ϭ 14), myocardial collagen degradation was induced using the method described by Caulfield and Wolkowicz (11). Briefly, 10 wk-old rats were anesthetized and received two intravenous infusions over 3 h (0.11 ml/min), 1 wk apart, of 20 ml of a 2 mM solution of oxidized glutathione. The animals were killed 3 wk after the second infusion, when myocardial hydroxyproline is expected to be at a minimum (11). A third group (control, n ϭ 9) consisted of unoperated and untreated normotensive rats that were the same age as the other two groups at the end of the experiment (i.e., 14 wk old). All rats were housed in a temperature-controlled room (24°C) with 12-h light:dark cy- cles, and food and water were supplied ad libitum. At the end of 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 the body weight (BW) was recorded at the time of death. The chest was opened by median sternotomy, and the heart was removed and placed in oxygenated Krebs-Henseleit solution at 28°C. The LV and septal wall were separated from the right ventricle, and their weights were determined. One papillary muscle was dissected from the LV, mounted be- tween two spring clips, and placed vertically in a bathing chamber. The lower spring clip was attached to a Kyowa model 120T-20B force transducer by a thin (1/15,000 in.) steel wire. The upper spring clip was connected by a thin wire to a rigid lever arm above which was mounted an adjustable micrometer stop for the adjustment of unstimulated muscle length. Oxygenated (95% O2-5% CO2) bathing medium con- sisted of (in mM) 118.5 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 5.50 glucose, and 25.88 NaHCO3 dis- solved in deionized water. The temperature of the bathing medium was maintained at 28°C. The muscle preparation was placed between two parallel platinum electrodes and stimulated at a frequency of 0.2 Hz, using square-wave pulses of 5-ms duration. Voltage was set to a value 10% greater than the minimum required to pro- duce a maximal mechanical response. After 60 min, during which the preparation stabilized, the muscle was loaded to contract isometrically and stretched to the peak length of its tension-length curve (Lmax). Once a stable Lmax was determined, the muscle was made to contract isometrically at Lmax and the resultant isometric contraction parameters were determined, which included peak developed active tension (AT, g/mm2 ), resting tension (RT, g/mm2 ), peak rate of isometric tension development (ϩdT/dt, g⅐mmϪ2 ⅐sϪ1 ), peak rate of tension decrease (ϪdT/ dt, g⅐mmϪ2 ⅐sϪ1 ), time to peak tension (TPT, ms), and time from 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 a Gaertner cathetometer and telescope. At the end of the ex- periment, the muscle between the spring clips was weighed and its cross-sectional area (CSA) was calculated, assuming cylindrical 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 to that in the papillary muscle (15). Therefore, we assumed that the 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 obtained from the LV apex according to the method described by Switzer (27). Briefly, the tissue was dried for 4 h using a SpeedVac Concentrator SC 100 attached to a refrigerated condensation trap TR 100 and vacuum pump VP 100 (Savant Instruments, Farmingdale, NY). Tissue dry weight was de- termined, and the samples were hydrolyzed overnight at 110°C with 6 N HCl (1 ml/10 mg dry tissue). An aliquot of 50 ␮l of hydrolysate was transferred to an Eppendorf tube and dried in the SpeedVac Concentrator. One milliliter of deion- ized water was added, and the sample was transferred to a tube. One milliliter of potassium borate buffer (pH 8.7) was added to maintain stable pH, and the sample was oxidized with 0.3 ml of chloramine T solution at room temperature for exactly 20 min. The oxidation was stopped by the addition of 1 ml of 3.6 M sodium thiosulfate with thorough mixing for 10 s. The solution was then saturated with 1.5 g of KCl, and the tubes were capped and heated in boiling water for 20 min. After the tubes cooled to room temperature, 2.5 ml of toluene were added and the tubes were shaken over 5 min. The tubes were briefly centrifuged at low speed, and 1 ml of toluene extract was transferred to a 12 ϫ 75 mm test tube. In the next step, 0.4 ml of Ehrlich’s reagent was added to allow the color to develop for 30 min. Absorbencies were read at 565 nm with a double-beam spectrophotometer (A-160 spectropho- tometer, Shimadzu) against a reagent blank. Deionized wa- ter and 20 ␮g/ml hydroxyproline were used as blank and standard, respectively. Histology and morphometry. Transverse sections of LV were fixed in 10% buffered Formalin and embedded in par- affin. Five-micrometer-thick sections were cut from the blocked tissue and stained with hematoxylin-eosin and with the collagen-specific stain picrosirius red (Sirius red F3BA in aqueous saturated picric acid). Myocyte CSA (MA) was de- termined for at least 100 myocytes per slide stained with hematoxylin-eosin. The measurements were performed using a Leica microscope (ϫ40 magnification lens) attached to a video camera and connected to a personal computer equipped with image analyzer software (Image-Pro Plus 3.0, Media Cybernetics, Silver Spring, MD). MA was measured with a digitizing pad, and the selected cells were transversely cut with 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-Pro Plus 3.0, Media Cybernetics). The components of the cardiac tissue were identified according to their color level: red for collagen fibers, yellow for myocytes, and white for interstitial space. The digitized profiles were sent to a computer that calculated collagen volume fraction as the sum of all connec- tive tissue areas divided by the sum of all connective tissue and myocyte areas. On the average, 35 microscopic fields were analyzed with a ϫ20 lens. Perivascular collagen was excluded from this analysis. H1535COLLAGEN AND MYOCARDIAL MECHANICS byguestonJuly25,2013
  4. 4. Statistics. All grouped data were expressed as means Ϯ SD and 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 Յ 0.05 were considered significant. Straight lines were fit to the systolic tension-length relations using linear regression analysis (22). The resulting slopes corresponded to AS, and the means among the groups were compared by ANOVA. Before the diastolic tension-length relationship was compared for the three groups, the resting tension at the muscle length corresponding to 90% of Lmax (L90) was subtracted from all subsequent tension data in each experiment to have all tension-length curves intercepting the y-axis origin at L90. The diastolic tension-length curves for the three groups were fit to monoexponential relations of the form RT ϭ A[e B (L Ϫ L0) Ϫ 1], where A and B are fitting parameters and L0 is the muscle length corresponding to zero resting tension. These nonlinear relations were compared by constructing an F ratio from the residual sum of squares. This test deter- mines whether separate fits to three groups are significantly better than the fit to data pooled from all groups. Accord- ingly, a significant F ratio indicates that the two sets of data being compared were significantly different from one an- other. For all comparisons, statistical significance was taken to be P Ͻ 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 in Table 1. In the RHTR group, treatment with an ACE inhibitor for 3 wk significantly reduced systolic blood pressure from an average value of 202 Ϯ 31 mmHg to 111 Ϯ 11 mmHg (P Ͻ 0.001) and regressed LVW to a value comparable to the control and GSSG groups. MA was significantly higher in the GSSG group compared with control and RHTR groups (Fig. 1). CVF and hy- droxyproline (Fig. 2) were statistically higher in RHTR than in the other two groups. The difference between GSSG and control groups reached a level of signifi- cance of 10% (Fig. 2A), whereas hydroxyproline was statistically lower in the GSSG compared with the control group (Fig. 2B). The isolated papillary muscle functional parameters RT, Lmax, AT at Lmax, AT at L90, ϩdT/dt, ϪdT/dt, TPT, TR1/2, and AS are shown in Table 2. RT was signifi- cantly higher in the RHTR group (0.64 Ϯ 0.08 g/mm2 ) compared with control (0.47 Ϯ 0.14 g/mm2 ) and GSSG (0.35 Ϯ 0.10 g/mm2 ) groups. AT at L90 and at Lmax were not different among the groups. In all experiments the relation between peak devel- oped active tension and muscle length was linear, as evidenced by the coefficient of determination (r2 ), which was typically Ͼ0.94. This finding means that at least 94% of the sum of squares of deviations of AT values about their means is attributable to the linear relation between AT and muscle length (22). The slope of these linear regressions corresponds to the myocar- dial AS, which was significantly increased in the GSSG group compared with the control group (5.86 Ϯ 1.14 vs. 3.96 Ϯ 1.33 g⅐mmϪ2 ⅐%Lmax Ϫ1 ; P ϭ 0.008). The differ- ences between GSSG and RHTR groups and between control and RHTR groups were not statistically signif- icant (Fig. 3). The passive tension-length curve from the RHTR group was shifted upward from that of the control group (F ϭ 14.25; P Ͻ 0.01) and that of the GSSG group (F ϭ 38.8; P Ͻ 0.01), reflecting an increased passive stiffness. The GSSG curve was shifted downwards from the control group (F ϭ 9.95; P Ͻ 0.01), indicating decreased 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 ramipril for 3 wk did not reverse perivascular and interstitial fibrosis but fully treated the arterial hypertension and promoted regression of myocardial hypertrophy. Therefore, we used that experimental model to study myocardial function in papillary muscle from rat heart with increased collagen concentration without myocar- dial hypertrophy. In the present study, collagen Table 1. Group comparisons of morphometric parameters and tail cuff systolic arterial pressure in control, GSSG, and RHTR rats Control GSSG RHTR BW, g 329Ϯ17 332Ϯ20 346Ϯ30 SAP, mmHg 136Ϯ14 129Ϯ18 111Ϯ11* LVW, g 0.66Ϯ0.07 0.65Ϯ0.05 0.69Ϯ0.14 LVW/BW, mg/g 2.01Ϯ0.16 1.96Ϯ0.09 1.90Ϯ0.17 RVW, g 0.21Ϯ0.03 0.19Ϯ0.05 0.21Ϯ0.04 CSA, mm2 0.84Ϯ0.18 0.78Ϯ0.17 0.86Ϯ0.22 Data are presented as means Ϯ SD. BW, body weight; SAP, systolic arterial pressure; LVW/BW, left ventricle weight (LVW) to BW ratio; RVW, right ventricle weight; CSA, papillary muscle cross- sectional area. *P Ͻ 0.05 vs. control. See METHODS for description of the 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 Ϯ SD analyzed by one-way ANOVA with Tukey’s posttest procedure. H1536 COLLAGEN AND MYOCARDIAL MECHANICS byguestonJuly25,2013
  5. 5. amount was measured with CVF and hydroxyproline. It has been shown that total volume fraction is closely related to hydroxyproline concentration in the LV (28), and in our study both measurements indicated that the interstitial collagen was altered in the treated groups relative to the control rats. However, we observed that the CVF measurement was associated with a greater variation than the measurement of hydroxyproline, and, consequently, the decrease in CVF in the GSSG group came close but did not reach the level of statis- tical significance. The variability of CVF might be caused in part by the measurement method used. In the present investigation we used a ϫ20 microscope objective to obtain a large field. This magnification would detect only large perimysial collagen fibers, thereby decreasing the sensitivity of the measurement. Even so, a power analysis indicated that the difference between the GSSG and control groups would have reached the level of significance if but a few additional histological samples were available. Despite similar LVW, the papillary muscles were significantly stiffer in the group with greater collagen concentration. This result is similar to that obtained by Narayan 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. In contrast, Schraeger and co-workers (25) concluded that ACE 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 Ϯ SD. The active stiffness (AS) obtained from the GSSG group was statistically higher than that from controls (P ϭ 0.008). Statistically, no differences were observed between GSSG and RHTR groups (P ϭ 0.493) and between control and RHTR groups (P ϭ 0.085). AS was analyzed by ANOVA and post hoc Tukey’s test. The RHTR passive tension-length curve was shifted upwards compared with either control (F ϭ 14.25; P Ͻ 0.01) or GSSG (F ϭ 38.8; P Ͻ 0.01) groups. The curve from the GSSG group was shifted downwards compared with the control group (F ϭ 9.95; P Ͻ 0.01). The passive tension-length curves were fitted to a monoexpo- nential relation, and comparisons were made by constructing an F ratio from the residual sum of squares. Statistical significance was taken to be P Յ 0.05/k where k is the number of comparisons. Table 2. Papillary muscle isometric contraction data for the control, GSSG, and RHTR groups Control GSSG RHTR RT, g/mm2 0.47Ϯ0.14 0.35Ϯ0.10 0.64Ϯ0.08*† AT at Lmax, g/mm2 8.05Ϯ1.59 9.44Ϯ1.86 8.52Ϯ1.72 AT at L90, g/mm2 5.64Ϯ1.26 5.95Ϯ1.64 5.23Ϯ1.58 RT1/2, ms 290Ϯ54 272Ϯ23 280Ϯ47 Lmax, mm 6.23Ϯ0.99 6.07Ϯ0.50 6.31Ϯ0.81 ϩdT/dt, g⅐mmϪ2 ⅐sϪ1 75.8Ϯ21.1 84.3Ϯ21.0 72.8Ϯ16.5 ϪdT/dt, g⅐mmϪ2 ⅐sϪ1 19.0Ϯ5.5 21.8Ϯ4.9 18.8Ϯ4.1 AS, g⅐mmϪ2 ⅐%Lmax Ϫ1 3.96Ϯ1.33 5.86Ϯ1.14* 5.21Ϯ1.18 TPT, ms 201Ϯ16 193Ϯ12 204Ϯ17 Data are reported as means Ϯ SD. RT, resting tension; AT, active tension; ϩdT/dt, peak rate of isometric tension development; ϪdT/dt, peak rate of tension decrease; TPT, time to peak tension; Lmax, muscle length at peak of the tension-length curve; L90, muscle length at 90% of Lmax; RT1/2, time from peak tension to 50% relaxation; AS, active stiffness. *P Ͻ 0.05 vs. control; *†P Ͻ 0.05 vs. GSSG. Fig. 2. Collagen volume fraction (A) and hydroxyproline concentra- tion (B) in control, GSSG, and RHTR groups. Data are means Ϯ SD analyzed by one-way ANOVA with Tukey’s posttest procedure. H1537COLLAGEN AND MYOCARDIAL MECHANICS byguestonJuly25,2013
  6. 6. SHR significantly decreased the passive stiffness of skinned trabecular muscle despite abnormally ele- vated hydroxyproline levels. It is not clear to what extent, if any, the 48-h incubation in the skinning solution at 0°C influenced their observations. The dis- crepancies observed between the studies may be caused by the animal strains as well as by the different experimental models used to produce hypertrophy and fibrosis. In our study, using the model of presumed collage- nase activation by oxidized glutathione described by Caulfield and Wolkowicz (11), it was possible to induce, in vivo, an 11% reduction of myocardial collagen con- centration measured by hydroxyproline concentration and a 19% reduction in the interstitial CVF. These results are less expressive than the 30–35% reduction in collagen as reported by Caulfield et al. (10). The authors have shown that the double infusion of GSSG resulted in no visible myocyte damage at any time as examined by light microscopy and scanning electron microscope (SEM). The collagen matrix alteration was not visible by light microscopy; SEM revealed damage to 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 adjacent capillaries, 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 and to the weave complex might be more important than the decrease in myocardial collagen. Other studies have shown that the double infusion of GSSG in rats promotes a reduction in CVF, ventricular dilatation, and a shift to the right of the diastolic pressure-volume curve of the entire LV (18, 20). However, a similar effect in the papillary muscle preparation has not been studied previously. The main advantage of this prepa- ration is that the muscle force and length are directly measured and that the mathematical assumptions re- quired when myocardial mechanical characteristics are evaluated in the LV chamber are unnecessary. The study of cardiac function in the whole heart is based on the pressure-volume and stress-strain rela- tionships. In that condition, myocardial stiffness is derived from chamber measurements using mathemat- ical models and assumptions regarding LV shape. If the LV is assumed to be a thick-walled sphere, the stress will be underestimated (30), whereas the as- sumption of an ellipsoid shape would result in an overestimated 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-length relations obtained for the three groups were different from each other, showing that the changes in collagen content, measured by hydroxyproline and CVF, are associated with myocardial passive properties. Com- pared with the control group, the diastolic tension- length curves were significantly shifted upwards and to the left in the RHTR group and downwards and to the 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 the fibrillar collagen loss and that increased collagen con- tent strongly correlates with the elevated passive stiff- ness observed in the RHTR group. Previous studies have suggested that collagen cross-linking (9) may affect myocardial stiffness, regardless of collagen amount. In addition to the effect of altered collagen amounts, it is important to be mindful of the effects of collagen crosslink density, as well as collagen type (type I or III) and collagen distribution. At present, we cannot rule out that changes in the collagen character- istics might also have influenced myocardial stiffness in the present study. Nevertheless, the results clearly indicate that alterations in collagen concentration and papillary muscle function are correlated. Ventricular elastance and myocardial stiffness are indexes of contractility of the ventricular chamber and myocardium, respectively (8, 26). Elastance is the ratio of the change in peak isovolumetric pressure for a given change in volume, and stiffness is defined as the ratio of the change in active force related to change in muscle length (8). Myocardial contractility is a very complex property of the heart that is difficult to mea- sure directly. During the last two decades it has been proposed that an ideal index of myocardial contractility must be able to measure the ability of the myocardium to generate force independently of loading condition. The slope of the linear pressure-volume relationship in the 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 have shown an enhancement of active stiffness when the muscle is stretched from 90% to 100% of Lmax in the GSSG group. The mechanisms underlying the associa- tion between decreased myocardial collagen and en- hanced active stiffness are not well established, and the results presented in this study do not answer all the questions concerning this matter. When collagen is reduced, ventricular dilatation occurs (10) and myocyte hypertrophy takes place in response to alterations in the loading state of the ventricle (17). Therefore, myo- cyte hypertrophy might play an important role in the improvement in contractility observed in the GSSG group. Another explanation would be related to the intracellular glutathione metabolisms. The glutathi- one level in the heart is ϳ1.2 ␮M/g (16), mainly in the reduced form, GSH, because of the high activity of GSSG reductase (13). That means that, inside the cell, most of the infused GSSG was rapidly converted to GSH. The action of excess GSH or GSSG in the heart is not completely elucidated. Bauer et al. (3), working on fiber bundles from papillary muscle of porcine right ventricle, observed an increased sensitivity of contrac- tile protein to calcium and, consequently, an increased force development in the presence of GSH. In our study, considering that the half-life of the glutathione is only a few minutes (1), it is doubtful that the double infusion of oxidized glutathione might increase the H1538 COLLAGEN AND MYOCARDIAL MECHANICS byguestonJuly25,2013
  7. 7. 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 similar to those in the control rats, suggesting that regression of hypertrophy by treatment with an ACE inhibitor is associated with preserved myocardial contractility. We conclude that decreased collagen content induced by 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 no relation with systolic function. This study was supported by a grant from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Sa˜o Paulo, Brazil, Proc. No. 92/4528–1. REFERENCES 1. Ammon HPT, Melien MCM, and Verpohl EJ. Pharmacoki- netics of intravenously administered glutathione in the rat. J Pharm Pharmacol 38: 721–725, 1986. 2. Anversa P, Olivetti G, and Melissari M. Morphometric study of myocardial hypertrophy induced by abdominal aortic stenosis. Lab Invest 40: 341–349, 1979. 3. Bauer SF, Schwarz K, and Ruegg JC. Glutathione alters calcium responsiveness of cardiac skinned fibers. Basic Res Car- diol 84: 591–596, 1989. 4. Bing OHL, Matsushita S, Farburg BL, and Levine HJ. Mechanical properties of rat cardiac muscle during experimental hypertrophy. Circ Res 28: 234–245, 1971. 5. Boluyt MO, O’Neil E, Meredith AL, Bing OHL, Brooks WW, Conrad CH, Crow MT, and Lakatta EG. Alterations in car- diac gene expression during the transition from stable hypertro- phy to failure. Circ Res 75: 23–32, 1994. 6. Brilla CG, Janicki JS, and Weber KT. Impaired diastolic function and coronary reserve in genetic hypertension: role of interstitial fibrosis and medial thickening of intramyocardial coronary artery. Circ Res 69: 107–115, 1991. 7. Burns J, Covell J, Meyrs R, and Ross JJ. Comparison of directly measured left ventricular wall stress and stress calcu- lated from geometric references figures. Circ Res 28: 611–621, 1971. 8. Campbell KB, Taheri H, Kirkpatrick RD, Burton T, and Hunter WC. Similarities between dynamic elastance of left ventricular chamber and papillary muscle of rabbit heart. Am J Physiol Heart Circ Physiol 264: H1926–H1941, 1993. 9. Capasso JM, Robinson TF, and Anversa P. Alteration in collagen cross-linking impair myocardial contractility in mouse heart. Circ Res 65: 1657–1664, 1989. 10. Caulfield JB, Norton P, and Weaver RD. Cardiac dilatation associated with collagen alterations. Mol Cell Biochem 118: 171–179, 1992. 11. Caulfield JB and Wolkowicz PE. Myocardial connective tis- sue alterations. Toxicol Pathol 18: 488–496, 1990. 12. Conrad CH, Brooks WW, Hayes JA, Sen S, Robinson KG, and Bing OHL. Myocardial fibrosis and stiffness with hyper- trophy and heart failure in spontaneously hypertensive rats. Circulation 91: 161–170, 1995. 13. Curello S, Ceconi C, Bigoli B, Ferrari R, Albertini A, and Guarnieri C. Changes in the cardiac glutathione status after ischemia and reperfusion. Experientia 41: 42–43, 1985. 14. Holubarsch CH, Holubarsch T, Jacob R, Medugorac I, and Thiedemann K. Passive elastic properties of myocardium in different models of hypertrophy: a study comparing mechanical, chemical, and morphometric parameters. Perspect Cardiovasc Res 7: 323–336, 1983. 15. Imataka K, Naito S, Seko Y, and Fujii J. Hydroxyproline in all parts of the rabbit heart in hypertension and in its reversal. J Mol Cell Cardiol 21: 133–139, 1989. 16. Ishikawa T and Sies H. Cardiac transport of glutathione disulfide and s-conjugate. J Biol Chem 259: 3838–3843, 1984. 17. Janicki JS. Myocardial collagen remodeling and left ventricu- lar diastolic function. Braz J Med Biol Res 25: 975–982, 1992. 18. Janicki JS and Matsubara BB. Myocardial collagen and left ventricular diastolic dysfunction. In: Left Ventricular Diastolic Dysfunction and Heart Failure, edited by Gaasch W and LeWin- ter M. Philadelphia, PA: Lea and Febiger, 1994, p. 125–140. 19. MacKenna DA, Omens JH, and Covell JW. Left ventricular perimysial collagen fibers uncoil rather than stretch during diastolic filling. Basic Res Cardiol 91: 111–122, 1996. 20. Matsubara BB, Henegar JR, and Janicki JS. Structural and functional role of myocardial collagen in the normal rat heart (Abstract). Circulation 84: 212, 1991. 21. Matsubara LS, Matsubara BB, Okoshi MP, Franco M, and Cicogna AC. Myocardial fibrosis rather than hypertrophy in- duces diastolic dysfunction in renovascular hypertensive rats. Can J Physiol Pharmacol 75: 1328–1334, 1997. 22. McClave JT and Dietrich FH. Simple linear regression. In: Statistics (3rd ed.). San Francisco, CA: Dellen, 1985, p. 581–635. 23. Narayan S, Janicki JS, Shroff SG, Pick R, and Weber KT. Myocardial collagen and mechanics after preventing hypertro- phy in hypertensive rats. Am J Hypertens 2: 675–682, 1989. 24. Ratkowsky D. Comparing parameter estimates from more than one data set. In: Nonlinear Regression Modelling; a Unified and Practical Approach. New York: Dekker, 1983, p. 135–145. 25. Schraeger JA, Canby CA, Rongish BJ, Kawai M, and To- manek RJ. Normal left ventricular diastolic compliance after regression of hypertrophy. J Cardiovasc Pharmacol 23: 349– 357, 1994. 26. Suga H, Sagawa K, and Shoukas AA. Load independence of the instantaneous pressure-volume ratio of the canine left ven- tricle and effects of epinephrine and heart rate on the ratio. Circ Res 32: 314–322, 1973. 27. Switzer BR. Determination of hydroxyproline in tissue. J Nutr Biochem 2: 229- 231, 1991. 28. Weber KT, Janicki JS, Pick R, Abrahams C, Shroff SG, and Bashey RI. Collagen in the hypertrophied pressure-overloaded myocardium. Circulation 75: 140–147, 1987. 29. Weber KT, Pick R, Jalil JE, Janicki JS, and Carroll EP. Patterns of myocardial fibrosis. J Mol Cell Cardiol 21, Suppl. V: 121–131, 1989. 30. Yin FCP. Ventricular wall stress. Circ Res 49: 829–842, 1981. H1539COLLAGEN AND MYOCARDIAL MECHANICS byguestonJuly25,2013