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Circulation. 1995;91:161-170

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(Circulation. 1995;91:161-170.)
© 1995 American Heart Association, Inc.

Articles

Myocardial Fibrosis and Stiffness With Hypertrophy and Heart Failure in the Spontaneously Hypertensive Rat

Chester H. Conrad, MD, PhD; Wesley W. Brooks, DSc; John A. Hayes, MD; Subha Sen, PhD, DSc; Kathleen G. Robinson, MT; Oscar H. L. Bing, MD

From the Department of Veterans Affairs Medical Center (C.H.C., W.W.B., J.A.H., K.G.R., O.H.L.B.), Boston, Mass; Department of Medicine (C.H.C., W.W.B., O.H.L.B.), Tufts University School of Medicine, Boston, Mass; Department of Pathology (J.A.H.), Boston University School of Medicine, Boston, Mass; and Research Division (S.S.), Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to Chester H. Conrad, MD, PhD, Boston VA Medical Center (151), 150 S Huntington Ave, Boston, MA 02130.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
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Background Fibrosis is commonly found in association with cardiac hypertrophy and failure, but the relation of the connective tissue response to the development of impaired cardiac function remains unclear. We examined passive myocardial stiffness, active contractile function, and fibrosis in the spontaneously hypertensive rat (SHR), a model of chronic pressure overload in which impaired cardiac function follows a long period of stable hypertrophy.

Methods and Results We studied the passive and active mechanical properties of left ventricular (LV) papillary muscles isolated from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at the ages of 12 months and 20 to 23 months. Seven of 15 SHR between 20 and 23 months of age had findings consistent with heart failure (SHR-F). In comparison to preparations from WKY rats and nonfailing SHR (SHR-NF), papillary muscles from the SHR-F group demonstrated increased passive stiffness (central segment exponential stiffness constant, kcs: SHR-F 95.6±19.8, SHR-NF 42.1±9.7, WKY rats 39.5±9.5 (mean±SD); SHR-F P<.01 versus SHR-NF, WKY rats). The increase in stiffness was associated with an increase in LV collagen concentration (SHR-F 8.71±3.14, SHR-NF 5.83±1.20, WKY rats 4.78±0.70 mg hydroxyproline/g dry LV wt; SHR-F P<.01 versus SHR-NF, WKY rats); an increase in interstitial fibrosis, as determined histologically (SHR-F 13.5±8.0%, SHR-NF 4.9±2.1%, WKY rats 3.6±0.8%; SHR-F P<.01 versus SHR-NF, WKY rats); and impaired tension development (SHR-F 3.18±1.27, SHR-NF 4.41±1.04, WKY rats 4.64±0.85 kdyne/mm2; SHR-F P<.05 versus SHR-NF; P<.01 versus WKY rats).

Conclusions The development of heart failure in the aging SHR is associated with marked myocardial fibrosis, increased passive stiffness, and impaired contractile function relative to age-matched nonfailing SHR and nonhypertensive control animals. These data suggest that fibrosis or events underlying the connective tissue response are important in the transition from compensated hypertrophy to failure in the SHR.


Key Words: ventricles • hypertrophy • heart failure • muscles


*    Introduction
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 up arrowAbstract
 *Introduction
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down arrowResults
down arrowDiscussion
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Adaptive left ventricular hypertrophy (LVH) may be followed by a state in which contractile performance is impaired, ultimately with the transition to overt heart failure.1 The mechanisms involved in this transition are incompletely understood, in part due to a relative paucity of models in which chronic stable hypertrophy is followed by a transition to the failure state. Studies of hemodynamic function in spontaneously hypertensive rats (SHR) have demonstrated stable function followed by impaired performance in older animals.2 3 4 Investigations of intrinsic muscle properties from the SHR have demonstrated stable performance followed by depression of contractile function in animals with evidence of heart failure.5 6 7 8 These studies document a sequence of events consistent with a transition from compensated LVH to failure and thus provide a model for the study of mechanisms involved in cardiac decompensation.

Extracellular matrix components have been demonstrated to be increased in cardiac hypertrophy, and it has been suggested that alterations in the cardiac interstitium may contribute to changes in diastolic function of hypertrophied hearts.9 It has also been suggested that myocardial fibrosis may restrict myofibrillar motion and thereby impair overall cardiac function.10 Many studies have examined hypertrophy in the absence of failure and observed variable changes in cardiac fibrosis and myocardial passive properties.11 12 13 14 15 16 17 18 19 20 21 Increases in collagen have been observed with aging in the SHR.3 22 Fibrosis has also been described with experimental heart failure.23 Thus, although fibrosis is recognized to occur with both hypertrophy and failure states, its relation to the pathophysiology of heart failure with chronic pressure overload remains incompletely understood. The present study was carried out in the SHR model, where it is possible to compare SHR with heart failure (SHR-F) with age-matched SHR without failure (SHR-NF). Marked differences in the connective tissue response in these two groups of animals suggest a role of fibrosis, or events underlying the connective tissue response, in the transition to failure.


*    Methods
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 *Methods
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Animal Model
Male SHR and nonhypertensive Wistar-Kyoto (WKY) control rats were purchased as retired breeders at the age of 6 to 9 months (Taconic) and boarded at the animal facility at the Boston VA Medical Center until the time of the study. All experiments were conducted in accordance with institutional guidelines and the "Guide for the Care and Use of Laboratory Animals" (US Department of Health and Human Services, NIH Publication 86-23).

To analyze the effect of age on myocardial function in this model, groups of WKY rats and SHR were studied at 12 and 20 months of age (WKY-12: age 12.3±0.5 months, n=10; WKY-20: age 20.1±1.1 months, n=12; SHR-12: age 12.3±0.5 months, n=12; SHR-20: age 20.5±1.1 months, n=15). Beginning at 18 months of age, all animals were observed twice per week and studied if tachypnea and labored respiration were observed. These findings were not noted in any of the WKY rats. On the basis of clinical and pathological data, a group of SHR-F was defined (age 21.1±1.5 months, n=7) (see "Results"); age-matched WKY rats (WKY rats: age 20.1±1.1 months, n=12) and SHR-NF (age 20.0±0.0 months, n=8) were studied for comparison to the SHR-F group. It should be noted that the WKY rat group was identical to the WKY rat-20 group; the SHR-NF and SHR-F groups were subsets of the SHR-20 group.

Isolated Muscle Preparation
At the time of study, rats were killed by decapitation, and their hearts were quickly removed and placed in oxygenated Krebs-Henseleit solution24 at 28°C. The LV anterior and posterior papillary muscles were dissected free, mounted between two spring clips, placed vertically in a 100-mL acrylic chamber containing Krebs-Henseleit solution at 28°C, and oxygenated with a mixture of 95% O2-5% CO2 (pH 7.38). The thinner, more uniform preparation was chosen for study. The muscles were stimulated at a rate of 12 min-1 by parallel platinum electrodes delivering 5-millisecond pulses at a voltage 10% above threshold. The spring clip on the upper end of the papillary muscle was attached to a low-inertia DC pen motor (G100-PD, General Scanning) and the lower clip to a semiconductor strain gauge tension transducer (DSC-3, Kistler-Morse). A digital computer with an analog-digital interface allowed control of either tension or length of the preparation. Tension and length data were sampled at a rate of 1 kHz and stored on disk for later analysis.

After they were mounted, papillary muscles were allowed to equilibrate by contracting isotonically at a light load (on the order of 0.4 kdyne/mm2) for a period of 30 minutes. After this equilibration period, muscles were gradually stretched to the peak of the active tension versus length curve (Lmax, defined as the muscle length resulting in peak active tension), and equilibrated for 15 minutes. Physiologically sequenced contractions25 were performed with a preload equal to 50% of the preload at Lmax and an afterload of 25% of isometric active tension at Lmax. After this, several determinations of Lmax were made. Once a stable Lmax was determined, the muscle was made to contract isometrically at Lmax for 5 minutes, and the resultant isometric contraction parameters (average of five isometric contractions) were determined, which included resting tension (RT, kdyne/mm2), active tension (AT, kdyne/mm2, defined as peak isometric tension minus resting tension), peak rate of isometric tension development [peak (+)dT/dt, kdyne/mm2 per second], electromechanical delay (EMD, milliseconds, defined as the time from stimulation to the onset of tension development), time to peak tension (TPT, milliseconds, defined as the time from the onset of tension development to the time of peak tension), and time to 50% relaxation (RT1/2, milliseconds, defined as the time from peak active tension to 50% of active tension).

Tissue Samples
After the papillary muscles were dissected and mounted, atria were removed, and the right ventricle was dissected free from the left ventricle. Samples of both ventricles were taken, blotted, and weighed. Tissue dry weight was determined after drying to a constant weight (60°C for 24 hours). Tissue water content (g/g dry wt) was determined as [(W/D)-1] where W/D is the ratio of wet weight to dry weight. Left and right ventricular wet weight normalized by body weight (LV/BW and RV/BW, respectively) and dry weight by tibial length (LV/TL and RV/TL)26 were used as indexes of ventricular hypertrophy. A sample of LV free wall was taken for measurement of hydroxyproline (see "Hydroxyproline Determinations"). At the conclusion of each experiment, papillary muscles were fixed for histological analysis (see "Histological Studies").

Central Segment Measurements
After these baseline determinations were made, two central segment markers, spaced approximately 1 to 2 mm apart, were applied. These markers consisted of 10-0 silk (Deknatel) gently tied around the papillary muscle using a single overhand knot.27 In each case, baseline isometric, isotonic, and passive stretch measurements were repeated after marker placement. Application of the markers had no significant effect on active tension development. Central segment dimensions were measured at three levels in both the frontal and lateral projections, and cross-sectional area calculated assuming an elliptical cross section with measured major and minor axes. There was no significant difference in papillary muscle cross-sectional area among groups (see "Results").

The central segment scanning system used for these studies is similar to that used previously in this laboratory.27 The preparation is scanned longitudinally by a laser beam. When the beam traverses the silk markers, the resulting decrease in reflected light is detected by a photodiode, and the time between marker detection events converted to a distance signal. The scanning rate, 150 Hz in the original system, has been increased to 1000 Hz. Resolution (1.6 µm) and RMS noise (on the order of 6.5 µm, or approximately 0.4% of central segment length for a typical 2.0-mm segment) are comparable to the original system.

Stress-Strain Analysis
The analysis of myocardial stiffness was based on central segment measurements, to avoid potential errors due to "damaged end" effects. Passive tension-length relations were determined by applying length ramps to the whole papillary muscle at a rate of 1.0 mm/s, corresponding to a normalized rate of length change on the order of 0.1 muscle length per second. Stretches were applied over a physiological range (from a load of approximately 0.1 kdyne/mm2 to a load approximately equal to the preload at Lmax). Tension and central segment lengths were sampled in real time (Fig 1Down); central segment stress-strain relations were derived from these measurements (Fig 2Down). Because of the large deformations involved, Eulerian stress (tension/instantaneous area) was used, as opposed to Lagrangian stress (tension/reference area). Central segment stress ({varsigma}cs) was defined as tension normalized by instantaneous cross-sectional area, calculated from the measured cross-sectional area assuming incompressibility:



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  		Figure 1. Examples of central segment length (upper traces) and tension (lower traces) versus time for passive stretches in typical preparations from Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR)-NF (without heart failure), and SHR-F (with heart failure). A constant-velocity stretch (1.0 mm/s) was applied to the whole muscle (whole muscle length not shown). "Zero" on the strain axis represents a near-slack length at a stress of 0.1 kdyne/mm2 (see text); preparations were stretched to a length approximately equal to Lmax (arrows indicate resting tension at Lmax).



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  		Figure 2. Plots of examples of stress-strain relations for passive stretches in WKY, SHR-NF, and SHR-F animals. Top, Central segment stress (vertical axis) vs central segment strain (horizontal axis). Bottom, log(stress) (vertical axis) vs central segment strain (horizontal axis). Note the linear nature of the log(stress) vs strain relations, indicative of an exponential stress-strain relation. Slope of these plots represents the exponential stiffness constant, kcs. Abbreviations as in Fig 1Up.


where CSAcs(inst) is instantaneous central segment cross-sectional area, CSAcs(ref) is the cross-sectional area at the reference length, Lcs(inst) is instantaneous central segment length, and Lcs(ref) is the reference central segment length. Natural strain ({varepsilon}) is generally defined as {varepsilon}=ln (L/L0), where L is length and L0 is length at zero stress (or "slack length"). Because of the exponential nature of the stress-strain relation, and therefore the shallow slope at low loads, the determination of true slack length (as used in the traditional definition of strain) is subject to considerable experimental error. Therefore, a modified natural strain definition was used in the present study:


where Lcs is instantaneous central segment length, and L0.1 is central segment length at a load of 0.1 kdyne/mm2. With this definition, {varepsilon}=0 at a near-slack "reference length" at which {varsigma}=0.1 kdyne/mm2.

If we assume that passive myocardial stress({varsigma})-strain({varepsilon}) relations are exponential in nature,28 the relation can be expressed as {varsigma}=ce(k{varepsilon}). With a log transformation, log({varsigma})=log(c)+ k{varepsilon}. Thus, k can be determined from the slope of the log({varsigma}) versus {varepsilon} relation. The central segment stiffness constant, kcs, was derived from the slope of the log({varsigma}cs) versus {varepsilon}cs relation.

Note that the use of the modified definition of strain might conceivably alter the value of k determined by this method. For a stress-strain relation that can be represented by a single exponential, the exponential stiffness constant, k (or kcs , in the case of the central segment), is independent of the choice of "slack length." It has been noted that a single exponential (with a linear tangent elastic modulus versus stress relation) is not adequate to characterize the overall stress-strain relation in all situations.28 In the present study, however, it was found that the relations were almost invariably well described by a single exponential (with the log[stress] versus strain relation being quite linear), so that the choice of this definition should not significantly alter the determination of k.

Hydroxyproline Determinations
Hydroxyproline content of LV wall samples was determined by using a modified Stegemann procedure.29 A sample of LV myocardial tissue weighing 200 to 300 mg was homogenized, and hydrolysis of the sample solution was carried out with 6 N HCl at 100°C for 24 hours. The hydrolyzed samples were dried using a flash evaporator. Hydroxyproline standard solutions of 2.0, 4.0, 6.0, 8.0, and 10.0 µg/mL were made. A reagent blank was included in the procedure by substituting water for the hydroxyproline solution; the absorbance was corrected accordingly. Then, 0.5 mL of hydroxyproline standard solutions of different strengths and homogenates of heart samples were placed in glass tubes, and 1.0 mL of isopropanol was added to each. The tubes were then vortexed. To this solution, 0.5 mL of oxidant (0.35 g chloramine T in 5.0 mL water and 20.0 mL citrate buffer) was added, vortexed, and allowed to stand for 4 minutes. Next, 3.25 mL of Ehrlich's reagent (3.0 mL Ehrlich's reagent in 15.0 mL isopropanol) was added. The tubes were kept at 25°C for 18 hours, and the intensity of red coloration was measured using a spectrophotometer (model DU-50, Beckman Instruments). The amount of hydroxyproline in unknown samples was calculated using the standard curve, and expressed as milligrams per 100 mg tissue dry wt.

Histological Studies
After the completion of the physiological measurements, papillary muscles were fixed for 24 hours in Karnovsky's fixative (1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer at pH 7.4) at a load corresponding to the preload at Lmax. The fixed papillary muscles were washed in cacodylate buffer and postfixed in 2% osmic acid in cacodylate buffer. The blocks were then processed for embedding in Epon (EM Sciences). Sections 1 µm thick were cut and stained with a differential stain using methylene blue, azure II, and basic fuchsin,30 following Humphrey and Pittman.31 The stain facilitates morphometry by permitting a clear distinction between connective tissue (red) and muscle fibers (blue). A quantitative estimate of fibrosis was obtained by counting the frequency of occurrence of red-staining material at the intersections of an optical grid.32 The grid had 36 points; at least 10 fields of each slide were counted using a 40x objective. Fractional area of fibrosis was expressed as the ratio of points with fibrosis to total points counted.

Statistical Methods
Data are expressed as mean±SD. Data from SHR with heart failure (SHR-F) were compared with those from age-matched nonfailing SHR (SHR-NF) and from WKY rats using a one-way ANOVA with replications33 and the Tukey (a) procedure for multiple comparisons.34 For the analysis of data from 12- and 20-month-old animals, a two-way ANOVA with replications was used to test for strain and age effects, and the unpaired t test33 was used to localize differences where appropriate.


*    Results
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*Results
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Clinical and Pathological Data
Criteria for heart failure were based on previous findings, in which animals with evidence of heart failure had findings that included labored respiration, left atrial thrombi, pleural and pericardial effusions, and right ventricular hypertrophy.8 In studies of the isolated perfused heart, we have found marked depression of systolic function in the SHR with these findings.35 The SHR-F group (seven animals) included five SHR that were studied when they exhibited tachypnea and labored respiration; two additional animals were included on the basis of right ventricular hypertrophy.8 In this group of seven animals, two had left atrial thrombi, three had pleural and/or pericardial effusions, all had evidence of right ventricular hypertrophy (RV/TL >=0.020 g/cm), and six had grossly visible endocardial fibrosis. Animals in the SHR-NF group exhibited grossly visible endocardial fibrosis but none of the other features suggestive of heart failure. None of the WKY rats exhibited any of these clinical or pathological features.

Cardiac chamber weights (raw and normalized) for the analysis of data from 12- and 20-month-old animals are presented in Table 1aDown. LV weight was greater in the 20-month SHR group than in the 20-month WKY group or the 12-month SHR group. LV wet weight normalized by body weight was greater in the SHR than in WKY rats groups at both ages; in the SHR an increase with age was noted as well. Similar results were noted for LV dry weight normalized by tibial length. Right ventricular wet weight, as well as right ventricular wet weight normalized by body weight or dry weight normalized by tibial length, were greater in the 20-month than the 12-month SHR group. Normalized right ventricular weight was greater in the SHR than in the WKY groups (pooled ages).


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  		Table 1. Cardiac Chamber Weight Data

Data for the SHR-F versus SHR-NF versus WKY rats analyses are presented in Table 1bUp. LV weight, both raw and normalized, was greater in both the SHR-NF and SHR-F than in the WKY group. Right ventricular weight was greater in the SHR-F than in either the WKY rats and SHR-NF group; the same was true for right ventricular weight normalized by tibial length.

Isometric Contraction Parameters
Mean data for isometric contraction parameters are presented in Table 2Down. There was no significant difference in cross-sectional area among the groups studied. In the analysis of data from 12- and 20-month-old groups (Table 2aDown), there was no significant difference in resting tension among groups. Active tension was greater in the 12-month SHR group than in the 12-month WKY group and the 20-month SHR group. The peak rate of tension development [(+)dT/dt] was greater in the 12-month SHR group than in the 12-month WKY group but reduced in the 20-month SHR group compared with both the 12-month SHR and the 20-month WKY groups. Both electromechanical delay time (EMD) and time to peak tension (TPT) were greater in the SHR than in WKY groups (pooled ages), and both parameters increased with age (pooled strains). Time to 50% relaxation (RT1/2) was abbreviated in the SHR compared with WKY groups (pooled ages).


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  		Table 2. Isolated Muscle Parameters

In the SHR-F versus SHR-NF versus WKY rats analyses (Table 2bUp), there was no significant difference in resting tension at Lmax among groups. Active tension (Fig 3Down, left) and (+)dT/dt were reduced in the SHR-F compared with SHR-NF and WKY rats. EMD was increased in the SHR-F compared with WKY rats and SHR-NF, whereas TPT was increased in both SHR-NF and SHR-F compared with WKY rats. RT1/2 was reduced in the SHR-F group compared with both the SHR-NF and WKY groups.



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  		Figure 3. Bar graphs of active tension (left) and central segment myocardial stiffness constant (kcs) (right) for WKY, SHR-NF, and SHR-F groups. Data are mean±SD; **P<.01. Abbreviations are as in Fig 1Up.

Myocardial Stiffness
Examples of typical passive stretches are shown in Fig 1Up. As noted in "Methods," constant-velocity stretches were applied to the whole papillary muscle (not shown); the figure shows central segment length and tension versus time. Note that the central segment length change with time is approximately linear. Central segment stress-strain relations (derived from the data shown in Fig 1Up) are shown in Fig 2Up (top). Note that the definition of strain used results in "zero" strain at a stress of 0.1 kdyne/mm2. Fig 2Up (bottom) shows log(stress) plotted versus strain for the same data. The linear nature of the log(stress) versus strain relations is indicative of an exponential stress-strain relation. The central segment exponential stiffness constant, kcs, was derived from the slope of the log(stress) versus strain relation. In this example, k was 35.2 for the WKY rats, 45.7 for the SHR-NF, and 80.2 for the SHR-F preparation.

Myocardial stiffness data for the 12- and 20-month SHR and WKY rats groups are presented in Table 2aUp. Central segment stiffness (kcs) was greater in the SHR than the WKY rats groups (pooled ages). Central segment data also suggest a small increase in stiffness with age (age effect P<.05 by ANOVA; borderline statistical significance by direct comparison). No age effect was demonstrable using whole muscle stiffness measurements.

Stiffness data for the WKY rats versus SHR-NF versus SHR-F analyses are presented in Table 2bUp. Central segment stiffness (kcs) was markedly increased in the SHR-F group compared with both the SHR-NF and WKY groups (Fig 3Up, right); there was no statistically significant difference between the SHR-NF and WKY groups. Whole muscle stiffness (kwm) was increased in the SHR-F compared with SHR-NF and WKY groups, but kwm was less than kcs in the SHR-F group (there was no significant difference between kwm and kcs in the WKY or SHR-NF groups).

To examine the influence of lateral translation on central segment length measurement, recordings were routinely made with the beam centered on the papillary muscle and repeated with the beam positioned laterally (both left and right). There was no significant difference in kcs derived from the three measurements, suggesting that beam position and lateral translation do not have a major impact on the stiffness data reported in this study.

Hydroxyproline
LV hydroxyproline data are presented in Fig 4Down. Hydroxyproline concentration was increased in the SHR-F group compared with both the WKY rats and SHR-NF.



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  		Figure 4. Bar graph of left ventricular (LV) hydroxyproline concentration in the WKY, SHR-NF, and SHR-F groups. Note the increase in hydroxyproline in the SHR-F compared with WKY and SHR-NF. Data are mean±SD; *P<.05, **P<.01. Abbreviations are as in Fig 1Up.

Histology
Microscopic examination showed distinct differences between the three groups of animals, illustrated in Fig 5Down. The WKY rats showed fibers that were relatively uniform in cross-sectional area and associated with only a small amount of interstitial connective tissue. In the SHR-NF, there was an increase in interstitial fibrosis, which was of patchy distribution, together with some increase in cross-sectional area of muscle fibers. In the SHR-F, there was a marked increase in interstitial fibrosis. The scarred areas contained muscle fibers with marked variation in cross-sectional area, as well as focal crowding and grouping of capillaries, suggesting muscle fiber loss. In addition, the SHR-F rats show vacuolar change within individual fibers, a feature not seen in either of the other groups.



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  		Figure 5. Photomicrographs of longitudinal sections (left) and cross sections (right) from rats showing characteristic changes in each group. Epon-embedded sections, 1 µm thick, stained with methylene blue–azure II–basic fuchsin. Magnification of each photograph is identical (approximately x250). In the WKY rats, no evidence of scarring is present. The cross section shows uniform muscle fiber size with minimal interstitial connective tissue and no prominence of capillaries. In the SHR-NF, fiber size is less uniform, and the interstitial space is more prominent. The cross section shows two areas of scarring. In the SHR-F, marked variation is seen in cross-sectional area of myocytes. Scarring is diffuse and is striking in degree. Abbreviations are as in Fig 1Up.

Morphometry showed a reduction in fractional myocyte area in the SHR-F group (Fig 6Down, left). This decline in fractional myocyte area was accompanied by an increase in the cross-sectional area of interstitial fibrous tissue in the SHR-F group (Fig 6Down, right).



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  		Figure 6. Bar graphs of myocyte fractional area (left) and fractional area of fibrosis (right) for WKY SHR-NF, and SHR-F groups. Data are mean±SD; **P<.01. Abbreviations are as in Fig 1Up.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
LV hypertrophy and failure are well recognized to be accompanied by changes in both systolic and diastolic function.36 37 38 Studies of LV diastolic chamber properties with LVH in humans and in a number of animal models have demonstrated alterations in ventricular chamber stiffness and in myocardial stiffness.36 39 It has been pointed out that alterations in chamber properties may reflect changes in intrinsic myocardial properties, as well as other factors such as wall thickness and geometry.36 Myocardial stiffness can be derived from chamber measurements using mathematical models,39 although such determinations are subject to assumptions that may be difficult to validate. Direct measurement of myocardial stiffness, in isolated muscle preparations, offers another approach to an understanding of intrinsic muscle compliance in hypertrophy and failure. Because there has been some concern that "damaged end" artifacts may introduce errors in these measurements,40 41 we have used a central segment scanning system27 to measure papillary muscle stress-strain relations.

A major finding of this study is the marked increase in myocardial stiffness in preparations from animals with heart failure, while little or no increase is seen in age-matched animals with chronic LVH alone. In addition, fibrosis is significantly increased in preparations from animals with failure compared with those with hypertrophy without evidence of heart failure. The present study demonstrates that the failure state in the SHR is associated with myocardial fibrosis, increased myocardial stiffness, and depressed systolic function; none of these findings are observed in age-matched SHR without heart failure. More recently, we have observed marked increases in the expression of genes encoding extracellular matrix components, including collagen I, collagen III, and fibronectin in SHR-F relative to age-matched WKY rats and SHR-NF.42

Many studies of LV pressure-overload hypertrophy in the absence of heart failure have been carried out; a number of these have examined the relation between the connective tissue response and myocardial mechanical properties. Early studies demonstrated an increased resting tension measured at the apex of the length-tension relation in isolated papillary muscles from rats with acute aortic constriction that was associated with an increase in endomyocardial hydroxyproline concentration.12 In a later study,13 it was found that ß-aminopropionitrile, an inhibitor of collagen cross-linking, prevented the increase in collagen and elevation of resting tension at Lmax seen after aortic constriction. Holubarsch14 found an increase in the slope of the passive stress-strain relation in LV trabeculae from rats with renal artery constriction, associated with an increase in LV wall collagen content. Thiedemann et al19 reported an increase in hydroxyproline in association with increased myocardial stiffness in both the aging (80-week-old) SHR and in the rat with renovascular hypertension. Brilla et al21 studied myocardial stiffness (derived from isolated heart measurements) and fibrosis in 14- and 26-week-old SHR, finding increased stiffness in the SHR relative to the WKY rats at both ages.

Studies in the SHR have also noted an increase in fibrosis with age.3 21 22 Isolated muscle studies in aging Wistar rats have shown an increase in resting tension in LV papillary muscles in association with an increase in endocardial hydroxyproline content.43 Capasso et al,20 using the renal artery constriction model of hypertension, showed an increase in resting tension (at Lmax) with age but no difference between normal and hypertensive rats. Yin et al15 found no change in passive stiffness with age or aortic banding in rats. In the present study, myocardial stiffness was greater in the SHR than in the WKY rats at both 12 and 20 months of age and greater at 20 months than at 12 months in the WKY rats (there is a trend suggesting an increase with age in the SHR as well, but this apparent increase did not reach statistical significance). This suggests that there may be a gradual, progressive increase in fibrosis with age in both strains. The apparent small increase in stiffness with age stands in contrast, however, to the marked increase in stiffness and fibrosis that is seen in the SHR with development of the failure state.

Although increased collagen in the SHR-F is associated with an increase in kcs, suggesting that increased myocardial stiffness in the SHR-F is due to an increase in collagen content, it is important to recognize that total collagen concentration is not the sole determinant of mechanical properties and that the chemical composition of the collagen44 45 46 and physical arrangement of the collagen10 47 are important determinants of mechanical properties. Medugorac and Jacob48 reported an increase in the proportion of type III collagen with age and with LVH (SHR, renal artery banding, and rats with aortic constriction). Mukherjee and Sen49 have found that the ratio of type I to type III collagen is altered in the aging SHR, suggesting that collagen type as well as quantity may influence myocardial function in the aging SHR. The importance of the physical structure of the collagen framework in relation to underlying pathophysiology has been stressed by Weber et al,9 who pointed out the distinction between "reparative" fibrosis, which follows myocyte necrosis, and "reactive" fibrosis, which is a more generalized perivascular and interstitial process. We were not able to clearly distinguish between these two types of fibrosis in the present study.

It is interesting to compare central segment measurements to whole muscle measurements, as are usually used in studies of myocardial stiffness. kcs (the exponential stiffness constant derived from central segment measurements) and kwm (derived from whole muscle measurements) are presented in Table 2Up. In the WKY rats and SHR-NF group, kcs was similar to kwm, whereas in the SHR-F group, kcs was substantially greater. This suggests that damaged end compliance may not substantially affect stiffness measurements in papillary muscles with normal compliance, but that added series compliance may mask increases in stiffness in pathological states.

Although the findings of the present study are consistent with the concept that fibrosis plays a role in the increase in passive stiffness in SHR-F, it is less clear that the observed depression of active tension development is due to the connective tissue response. In studies by Mirsky et al4 of the 18- and 24-month-old SHR, myocardial stiffness was found to be increased, in association with evidence of impaired LV function. It has been suggested that myocardial fibrosis may restrict myofibrillar motion and thereby impair overall cardiac function.10 In the present study, papillary muscles from SHR-F demonstrate a reduction in tension development in association with an increase in LV hydroxyproline concentration (Fig 7Down, right). A reduction in the fractional area occupied by myocytes is also noted, which might result either from myocyte loss or from an increase in nonmyocyte material (including fibrosis). Thus, the reduction in the tension generating capacity of the myocardium might be due to a relative reduction in myocyte area. If one normalizes active tension by myocyte area, as an index of the tension generating capacity of the myocytes, active tension is not significantly lower in the SHR-F group (SHR-F 5.53±0.59, SHR-NF 5.20±1.26, WKY rats 5.53±0.59 kdyne/mm2). This suggests that the reduction in active tension in the SHR-F may be explained at least in part by a relative reduction in myocytes. We have previously observed changes in energetics35 and calcium dynamics50 with heart failure in the SHR, and it is possible that these and other factors (eg, changes in the cytoskeleton51 ) may contribute directly to impairment of muscle function or indirectly, by an effect on myocyte loss and fibrosis.



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  		Figure 7. Plots of central segment stiffness (left) and active tension (right) versus left ventricular (LV) hydroxyproline concentration. WKY: circles; SHR-NF: triangles; SHR-F: squares. Open symbols are individual values; filled symbols represent mean values (±SD). Abbreviations are as in Fig 1Up.

It is unclear whether the process of hypertrophy itself plays a role in the development of fibrosis or myocardial failure. There are models of hypertrophy, in fact, in which fibrosis is not a prominent feature.52 53 The transition to failure in the SHR, however, is associated with changes in the collagen framework that may influence myocardial passive properties, and possibly active properties as well. Overall, the present findings suggest that adaptive hypertrophy alone is not necessarily associated with fibrosis and depressed function. On the other hand, hypertrophied hearts, subjected to the effects of chronic pressure overload, appear susceptible to the development of fibrosis, which is associated with increased muscle stiffness, depressed active tension development, and evidence of heart failure.


*    Acknowledgments
 
This work was supported in part by Medical Research Funds from the Department of Veterans Affairs, by US Public Health Service grant HL-27838, and by a Grant-in-Aid from the American Heart Association (Dr Sen). This work was done during the tenure of a Clinician-Scientist Award from the American Heart Association (Dr Conrad). Data analysis was performed using the PROPHET system, a national computer resource sponsored by the Division of Research Resources, National Institutes of Health.

Received July 19, 1994; accepted August 2, 1994.


*    References
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*References
 

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