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Cardiovascular Research 2003 60(3):547-556; doi:10.1016/j.cardiores.2003.09.021
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Demonstration of altered fibroblast contractile activity in hypertensive heart disease

William A Marganskia, Vanessa M De Biasea, Maria L Burgessb and Micah Dembo*,a

aDepartment of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215, USA
bDepartment of Health Sciences, Boston University, Boston, MA, USA

*Corresponding author. Tel.: +1-617-353-1671; fax: +1-617-353-6766. Email address: mxd{at}bu.edu

Received 22 May 2003; revised 9 September 2003; accepted 9 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The aim of this study is to investigate the idea that altered fibroblast contractile activity is involved in the pathogenesis of hypertensive heart disease (HHD). Methods: Cell area and contraction are quantified using the traction force microscopy technique for cardiac fibroblasts isolated from both normotensive Wistar–Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Results: The data indicate that there are marked phenotypic differences between the two cell types. For instance, WKY fibroblasts exert an average traction stress of ~3.3 kPa and have an area of ~2640 µm2. Under identical conditions the SHR fibroblasts have an area ~1.45 times larger (p<0.01) and exert an average stress ~1.86 times higher (p<0.01). Challenging WKY fibroblasts with 1 µmol/l angiotensin II (Ang II) gradually causes a ~2-fold increase in traction after 1 h while simultaneously causing a ~28% decrease in area. In contrast, Ang II has no effect on SHR fibroblasts. The data also show that WKY and SHR cells respond in different ways when challenged with irbesartan (Irb). The addition of 1 µmol/l Irb initially causes WKY cells to decrease their average traction output by ~50% after ~10 min. Subsequently, contractile activity begins to recover and returns to normal after 1 h. The SHR cells also decrease their tractions by ~50%, but this decrease requires 30 min for completion and there is no recovery to the initial contractile state. For both cell types, Irb produces no significant effect on area and the combined effect of equimolar Irb and Ang II is the same as Irb alone. Conclusion: These in vitro data suggest that among the many factors producing hypertensive heart disease in SHR's are excessive contraction of their cardiac fibroblasts and defective control of fibroblast contraction by Ang II.

KEYWORDS Hypertension; Heart failure; Ventricular function; Angiotensin; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac fibroblasts (and also myocytes) express all components of the renin–angiotensin system (RAS) including angiotensinogen (ATN), renin, and angiotensin converting enzyme (ACE) [1] and secrete angiotensin II (Ang II) at least in vitro [2]. This data and much other evidence points to the existence of a local or autocrine RAS that exists within the heart (C-RAS) and which is independent from the classic endocrine or systemic RAS (Ref. [3], see also Ref. [4] for a recent review). It is believed that the C-RAS functions primarily to promote slow adaptive changes in organ structure and function through its effects on the cardiac fibroblasts. There are several observations that support this deduction. Cardiac fibroblasts express a high density of Ang II receptors [5] and respond in many different ways when stimulated with Ang II. For instance, Ang II stimulates cardiac fibroblast to produce more extracellular matrix (ECM) [6,7], express more alpha-smooth muscle actin ({alpha}-SM actin) [5], increase their adhesion to the surrounding ECM by upregulating their integrin expression [7,8] and increase the rate at which they compact artificial collagen gels [9,10]. All of these results are receptor specific since they can be blocked by several competitive inhibitors of the Ang II receptor.

Hypertensive heart disease (HHD) is a condition observed in patients with chronic hypertension that is characterized by massive remodeling, hypertrophy, fibrosis, and dysfunction of the ventricle (see Refs. [11,12] for reviews). A leading hypothesis concerning the pathogenesis of HHD proposes that chronic elevation of blood pressure in conjunction with genetic factors cause upregulation of the C-RAS. This upregulation then produces excessive activity of cardiac fibroblasts which causes the characteristic changes in the myocardium that occur in HHD [11,12]. We will refer to this general hypothesis for HHD pathogenesis as the so-called "C-RAS model". One should note that the exact nature of the molecular changes in the C-RAS that leads to the proposed "upregulation" is not precisely known. Possibilities include overproduction of Ang II, overexpression of the agonistic Ang II receptors (i.e., AT1 receptors), or loss of desensitizing Ang II receptors (i.e., AT2 receptors). It could also be a combination of several changes and certainly many different genes are known to be involved [13]. In support of the C-RAS model, the levels of mRNA for renin, ATN, and ACE are elevated in ventricles of rats with hypertension [14,15], but the systemic plasma levels of renin, ACE, and Ang II are identical in normotensive and hypertensive rats [16]. Furthermore, several trials have shown that ACE inhibitors and Ang II receptor blockers result in so-called "reverse remodeling" in which the ventricle returns to a more normal size and shape [11,12]. These local beneficial effects seem to be independent of any inhibition on the systemic RAS [17].

One of the main predictions of the C-RAS model is that changes in local Ang II production and fibroblast phenotype occur early in the pathogenic process, well before clinical HHD is evident. This has led to several studies of the phenotypic differences between primary cultures of cardiac fibroblasts established from the left ventricle of both normotensive Wistar–Kyoto (WKY) rats and those of a related strain of spontaneously hypertensive (SHR) rats. The SHR develops chronic high blood pressure and compensated hypertrophy during its first ~3 months of life, but at this stage the ventricles are identical to those of the WKY rat in terms of percent myocardial fibrosis, myocardial stiffness, and active tension [18]. It is only much later (~18–24 months) that the SHR begins to show evidence of dysfunction and heart failure. At this later point the percent myocardial fibrosis and the myocardial stiffness are about double the WKY values and the active tension is lower by ~25% [18]. Therefore, primary cultures of WKY and SHR fibroblasts obtained from animals before the ~3-month stage represent an excellent system for testing the C-RAS model. As predicted by this model, SHR cells show elevated levels of mRNA for ATN, ACE, and {alpha}-SM actin, lower AT1 receptor density, enhanced expression of integrins, and increased collagen production compared to WKY cells [5,8,14,15]. Moreover, as discussed previously, all of these changes can be observed in cultured WKY cells by treating them with exogenous Ang II [5–8].

Histological staining reveals that under culture conditions, both SHR and WKY cells (and most other fibroblasts) have an extensive contractile apparatus. They express abundant cytoplasmic (β and {gamma}) actin as well as myosin IIa and IIb. These are organized into well known structures like filopodia, ruffling lamellae and stress fibers that originate and end at focal adhesions and focal contacts. As mentioned above, SHR cells express more {alpha}-SM actin than WKY cells and furthermore, Ang II increases this expression in both WKY and SHR cells [5]. The abundance of {alpha}-SM actin, the isoform typically associated with myofibroblasts and smooth muscle cells, suggests that one might expect higher contractile activity in SHR cells and that this higher activity might be associated with the pathogenesis of HHD. In this study, we investigate this possibility by measuring the cell–substratum traction forces generated by cultured WKY and SHR fibroblasts that are isolated from ~3-month-old rats. We also test whether the tractions of these fibroblasts are regulated by the C-RAS by challenging these cells with exogenous Ang II and irbesartan (Irb), a compound that blocks the AT1 receptor. The measurements of cellular traction forces are conducted using a well developed method called traction force microscopy or "TFM" (see Refs. [19–21] for recent applications). The basic idea is to grow cells on a flexible material of known mechanical properties and to observe any resulting deformations of the substratum in the vicinity of a single isolated cell. These deformations are then used as a basis for making detailed deductions about cellular force production.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Preparation and characterization of the substratum
Elastic polyacrylamide (PA) substrata are prepared as previously described [22]. Briefly, a 20 µl droplet containing 5% acrylamide (Sigma), 0.05%1 N,N-methylene bis-acrylamide (Sigma), and fluorescent latex beads (1:50 dilution; 0.80 µm diameter; Polysciences) is placed on top of an activated coverslip and then covered with a 18-mm diameter coverslip. The coverslip assembly is inverted and incubated overnight at 4 °C to allow the majority of the beads to settle to the top of the substratum during polymerization. Since cells cannot adhere directly to polyacrylamide, the upper surface of each substratum is coated with type I collagen (2.0 mg/ml) using a photoactivatable heterobifunctional linker sulfosuccinimidyl 6 (4-azido-2-nitrophenyl-amino) hexanoate (sulfo-SANPAH; Pierce).

Since PA substrata are isotropic elastomers, they are characterized by two constants: the Poisson's ratio {nu} and the Young's Modulus E. The Poisson's ratio for polyacrylamide has been estimated to be ~0.30 [23]. E is determined using a method based on the Hertz theory [24] and involves deforming the substratum with a spherical ball of radius r and density {rho}b. Furthermore, a fluid with density {rho}f covers the substratum so that it is solvated. The resulting indentation d is measured by following the vertical deflection of surface fluorescent beads underneath the center of the ball using a calibrated microscope. E is then calculated as:


Formula (1)

The parameter f is the buoyancy corrected weight of the ball and is equal to:

Formula (2)
where g is the gravitational acceleration. Using this method, E~9 kPa for all substrata utilized in this study.

2.2. Cell isolation and culture
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, 1996). Primary cultures of cardiac fibroblasts are established from young (~2.5–3 months) WKY and SHR rats using a tissue digestion method [25]. After isolation, the fibroblasts are allowed to grow to ~70% confluency (~1 week) while incubated at 37 °C and 5% CO2 in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and the standard antibiotics (0.5 U/ml penicillin, 0.5 mg/ml streptomycin, and 2 pg/ml amphotericin B).

For all TFM experiments, the cardiac fibroblasts are trypsinized (0.25% trypsin in 0.1% ethylene diaminetetraacetic acid), centrifuged, and resuspended in the same medium as described above. The cells are then plated onto the substratum at a density of ~3000 cells/cm2 to avoid cell-to-cell contacts. After adhesion (~18–24 h), the cells are washed with phosphate-buffered saline (PBS) and serum-free conditions are maintained for ~18–24 h. All experiments are conducted under serum-free conditions to keep the fibroblasts quiescent and to prevent any external factors such as growth hormones from influencing the contraction of the fibroblasts. Finally, since the phenotype of both WKY and SHR cells has been shown to remain stable for at least six passages in culture [5], this study only used cells from passage 2–4.

2.3. Microscopy and application of chemical agents
The substratum is mounted to a Zeiss Axiovert S 100 microscope equipped with a Zeiss 20X, NA 0.60 phase objective and a stage incubator. Phase and fluorescent images are collected using a cooled CCD camera (Roper Scientific) and the ISee imaging software (Inovision). An individual cell is selected that is well spread with a polar morphology, has normal ruffling activity, is not undergoing mitosis, and is isolated from neighboring cells. Each fibroblast is then followed for several minutes to obtain baseline fluorescent images of the substratum and phase images of the cell. The serum-free media (SFM) is then supplemented with either 1 µmol/l Ang II (Sigma), 1 µmol/l Irb (kindly provided by Bristol-Myers-Squibb), or 1 µmol/l of both Ang II and Irb. Control experiments are also performed by adding only SFM. The effects of these treatments on cellular contraction are followed by acquiring further phase and fluorescent images over 1 h. The full extent of the substratum deformation is obtained by trypsinizing the cell and acquiring a final fluorescent image of the cell-free, relaxed substratum.

2.4. Measuring substratum deformations
Deformations of the substratum in the region surrounding a cell are determined using a correlation-based optical flow algorithm [26]. Essentially, a small patch that contains a number of marker beads is defined within the fluorescent image of the relaxed, cell-free substratum. Then a search is performed within the fluorescent image of the deformed substratum for a patch with the most similar intensity pattern. Deformation vectors are then generated from the center of the patch in the relaxed, cell-free image to the center of the patch in the deformed image that is determined to be the best match. Multiple measurements are performed in a similar manner to provide a robust estimation of the substratum deformation field. Interpolation methods are then used to refine the measurements to an accuracy of ~0.10 pixel.

2.5. Calculation of cellular traction forces
The theory for interpreting deformations of elastic substratum in terms of cellular traction forces has been previously described [27,28]. Briefly, the substratum is assumed to behave as a linear elastic half-space with all tractions being confined within the boundary of the cell, acting tangentially on the free surface, and constrained such that the net forces and torques are negligible. The spatial coordinates of the tractions are defined by tessellating the region enclosed by the cell boundary into a mesh of quadrilaterals. The vertices of each quadrilateral specify discrete spatial locations called nodes at which the traction stresses are defined. The most-likely traction field is then computed by maximizing a Bayesian likelihood function that provides the best approximation to the substratum deformations while maintaining minimal complexity (i.e., either by constraining the amplitudes or the spatial transitions of the tractions).

The overall contraction of a cell can be objectively quantified by several measures. One such measurement is the integral of the absolute value of the traction field:


Formula (3)

In this expression T=[Tx(x,y),Ty(x,y)] is the continuous field of traction vectors defined at any spatial location (x,y) within the cell. Another useful measure is the average traction magnitude (Formula), which is simply the ratio of |F| and the cell area (|A|). Note that |F| and |A| are computed by numerical integration over the quadrilateral mesh that tessellates the cell interior.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Traction force microscopy
In this study, we selected random samples of 27 WKY cells and 24 SHR cells. Each cell was followed for ~5–10 min to get baseline data and then was randomized to a treatment protocol as described subsequently. Fig. 1 shows the results of a TFM experiment in the case of a WKY fibroblast under control conditions during the initial observation period. The particular cell shown is closest to the sample mean in terms of both projected area and the various overall measures of contraction. As seen in the phase image (Fig. 1a), the fibroblast is polarized with a classic "hand-mirror" morphology and leading lamella at the lower right. Also evident in the phase image are a few of the marker beads that are embedded in the substratum below the cell.


Figure 1
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  		Fig. 1 Traction force microscopy of the most typical WKY fibroblast. (a) Phase contrast image used to determine the cell outline. (b) Vector plot of the cellular traction field. The length and traction vector scales are equal to 20 µm and 100 kPa, respectively, and are shown in the upper right corner.

 
Fig. 1b shows the cellular traction field in terms of a vector plot that is computed from the substratum deformations (data not shown). Each arrow in this plot has length proportional to the traction stress acting at a node of the quadrilateral mesh. Nodes with no significant tractions are represented by small dots. For all traction calculations reported in this study, at least several hundred such nodes are used, which are more or less evenly spaced throughout the interior of the cell. The main cell–substratum tractions within this particular WKY fibroblast are produced at three localized zones. These include two protrusions at the leading edge and one at the tip of the tail. Also note that traction vectors generally have a centripetal orientation (i.e., they point towards the nucleus). The contractile strength of this most typical WKY fibroblast can be appreciated by the overall measures Formula and |F|, which equal ~4 kPa and ~7.5 µN, respectively. Finally, |A| for this cell equals ~1900 µm2.

By way of comparison, Fig. 2 shows the most typical SHR fibroblast drawn from the sample of 24 cells studied. All conditions, methods, and scale factors are the same as in Fig. 1. The distribution of tractions for this particular SHR cell is somewhat different from that seen in the WKY cell. In particular, it has several advancing and actively ruffling lamellae (one to the left and three at the top). The bottom edge of the cell is retracting with strong retraction forces in the two tails, which are located at the lower right and left. The contractile strength of this most typical SHR fibroblast can be appreciated by the overall measures Formula and |F|, which equal ~5 kPa and ~20 µN, respectively. Finally, |A| for this cell equals ~3400 µm2.


Figure 2
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  		Fig. 2 Traction force microscopy of the most typical SHR fibroblast. (a) Phase contrast image used to determine the cell outline. (b) Vector plot of the cellular traction field. The length and traction vector scales are equal to 20 µm and 100 kPa, respectively, and are shown in the upper right corner.

 
3.2. Cell-to-cell variability
Cell-to-cell differences in the spatial distribution of tractions within the SHR and WKY populations are quite large and as a result it was not possible for us to recognize any particular qualitative feature of the traction field that would allows us to uniquely distinguish between the two cell types. Nevertheless, it is possible to recognize statistically significant differences in the contractile activity of SHR and WKY populations using quantitative measures such as Formula, |F|, and |A|. These measures show the typical cell-to-cell variability characteristic of biological systems and are summarized in Table 1 in terms of their minimum, mean±S.E.M., and maximum values during the initial observation period. Note that in general the WKY fibroblasts show greater variability as compared to SHR cells. For instance, |F| varies by a factor of ~60 for the WKY fibroblasts, but only ~20 for the SHR cells.


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  		Table 1 Population variability

 
Obviously since |F| involves an area integral and Formula=|F|/|A|, the three quantities Formula, |F|, and |A| are not likely to vary independently. It is unclear however, as to how strongly they are correlated and what is the most statistically efficient choice for comparative studies of contraction. To answer these questions, Fig. 3a and b shows scatter plots of |F| versus |A| for the two sample populations. In the case of WKY cells, the results indicate a statistically significant linear correlation between |F| and |A| with intercept close to the origin (R~0.79, p~0.0001). This can be interpreted in two ways, either larger WKY cells tend to exert more total force or more contractile WKY cells tend to spread over a larger area. In the case of SHR cells, any correlation between variations of |F| and |A| are below the level of statistical significance (R~0.22, p~0.32). Nevertheless, it is possible that some weak linkage might emerge if a much larger sample was available. We conclude that as a pure statistical measure of mechanical activity |F| is unsatisfactory since it is influenced by cell size in a least one population of this study.


Figure 3
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  		Fig. 3 Covariation of projected cell area (|A|), total force (|F|), and average traction magnitude (Formula) in randomly selected cardiac fibroblasts. (a) |F| versus |A| for 26 WKY fibroblasts. (b) |F| versus |A| for 24 SHR fibroblasts. (c) Formula versus |A| for 26 WKY fibroblasts. (d) Formula versus |A| for 24 SHR fibroblasts. Solid lines show the best linear regression. The correlation coefficient and p-value for each covariation are 0.79 and 0.0001 (a), 0.22 and 0.32 (b), 0.05 and 0.80 (c), and –0.14 and 0.53 (d).

 
In contrast to the situation with |F|, the scatter plots of Formula versus |A| for both WKY and SHR fibroblasts (Fig. 3c and d) indicate that cell-to-cell variations in these two quantities occur independently (R~0 and p>0.50 for both scatter plots). This means that a change in Formula can be unambiguously interpreted as a change in the intensive level of cellular mechanical activity that is independent of cell size. Likewise |A| is a pure extensive measure of cell size independent of the intensive mechanical activity. Furthermore, the paired statistics |A| and Formula show the minimum degree of variability within a given population, which means that fewer measurements are needed to determine statistically significant changes. Denoting population averages of these two quantities as ||A|| and ||Formula||, it can be seen from Table 1 that SHR fibroblasts are on average ~1.45 larger than WKY fibroblasts and also that the average traction stress exerted by SHR cells is about ~1.86 times higher. Since the sample sizes are large, these results are statistically significant and indicate that extreme phenotypic differences exist between the two types of fibroblasts under the control conditions (p<0.01).

3.3. Effects of Ang II and Irb on area and traction
After the initial period of observation, the sampled WKY and SHR fibroblasts were randomized to one of four treatment groups as follows: group #1 (six WKY cells and six SHR cells) was the control which received only sham treatment with SFM, group #2 (10 WKY cells and six SHR cells) received 1 µmol/l Ang II, group #3 (six WKY cells and six SHR cells) received 1 µmol/l Irb, and group #4 (six WKY cells and six SHR cells) received both 1 µmol/l Ang II and 1 µmol/l Irb. The effects of the various treatments were followed by measuring |A| and Formula at various time points up to 1 h.

Fig. 4a and b show the temporal changes in |A| and Formula for some typical cells from the control group (three of each type). Note that some cells undergo substantial decreases or increases in |A| and Formula over the course of 1 h, but even the largest variations are less than 50% and the direction of the changes has no apparent pattern. There are also very few cases where individual curves cross over one another. This means that if conditions are kept constant, temporal changes in |A| and Formula for a given cell are slow, random, and much smaller then the systematic cell-to-cell differences described previously. Thus, for a given cell, size and contractile activity are conserved attributes reflective of phenotypic properties that persist at least 1 h.


Figure 4
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  		Fig. 4 Temporal changes in projected area (|A|) and average traction magnitude (Formula) for three randomly selected WKY fibroblasts ({square}) and three randomly selected SHR fibroblasts ({circ}).

 
To characterize the systematic temporal changes in cell area following the various treatments, the measured value of |A| for a given cell was normalized using the starting value for that cell (|A0|). Averaging |A|/|A0| over a sample population then yields a measure of the relative change in area for an average cell since the start of the experiment (denoted ||A/A0||). We found that the only treatment to produce any statistically significant effect on area was Ang II alone and furthermore, this effect was only observed in WKY cells. After 1 h of treatment with only Ang II, there was a ~28% decrease in ||A/A0|| for WKY cells (p<0.05; Fig. 5a), but only a ~7% decrease for SHR cells (not significant; Fig. 5b). Treatment with Ang II+Irb or Irb alone had no significant effect on ||A/A0|| for either WKY or SHR cells.


Figure 5
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  		Fig. 5 Effects of 1 µmol/l Ang II (a,b), 1 µmol/l Ang II+Irb (c,d), and 1 µmol/l Irb (e,f) on ||A/A0|| for WKY fibroblasts (a,c,e) and SHR fibroblasts (b,d,f). Each value represents the mean±S.E.M. (n = 6 in all cases except for the study of WKY cells exposed to Ang II alone where n = 10). * Indicates that at the particular time-point a treatment had a statistically significant (p<0.05) effect on |A| relative to the SFM control.

 
The effects of the various treatments on traction were gauged using an analysis similar to the one described for area. In this case, the population average change in Formula/Formula0 is denoted as ||Formula/Formula0|| and the results for the various groups of WKY and SHR fibroblasts are summarized in Fig. 6. First one should note that in the control groups of both types of fibroblasts, ||Formula/Formula0|| varied by less than ~5%. For WKY cells, treatment with Ang II alone caused ||Formula/Formula0|| to increase by a factor of two relative to control (p<0.05; Fig. 6a). In contrast, Ang II alone had no significant effect on the contraction of SHR cells (not significant; Fig. 6b). The effect seen in WKY cells starts soon after the treatment and reaches a plateau after ~1000 s. Given that WKY fibroblasts begin with tractions that are approximately half those of SHR cells, these results indicate that the presence of Ang II effectively eliminates this systematic difference in contraction. Furthermore, the 2-fold increase of ||Formula/Formula0|| in WKY cells after exposure to Ang II is coupled with only a rather small ~28% decrease in ||A/A0||. This implies that the increase in contraction and tension of the cell body is usually not sufficient to cause widespread disruption and peeling of the adhesive bonds at the cell edges. Thus the cell does not retract and remains for the most part in its original fully spread configuration.


Figure 6
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  		Fig. 6 Effects of 1 µmol/l Ang II (a,b), 1 µmol/l Ang II+Irb (c,d), and 1 µmol/l Irb (e,f) on ||Formula/Formula0|| for WKY fibroblasts (a,c,e) and SHR fibroblasts (b,d,f). Each value represents the mean±S.E.M. (n = 6 in all cases except for the study of WKY cells exposed to Ang II alone where n = 10). * Indicates that at the particular time-point a treatment had a statistically significant (p<0.05) effect on Formula relative to the SFM control.

 
In the case of WKY cells, the response to Ang II+Irb (Fig. 6c) is very similar as the response to Irb alone (Fig. 6e) but different from the control response. Thus Irb not only blocks the effects of Ang II, but it causes a significant decrease in contraction below control levels. This decrease however is transient, reaching a minimum of ~50% below control after ~10 min (p<0.05) and returning to normal after 1 h.

In the case of SHR fibroblasts, the response to Ang II+Irb (Fig. 6d) is also very similar to that towards Irb alone (Fig. 6f). This is hardly surprising however since Ang II alone has no effect on this cell type. The effect of Irb on the SHR fibroblast is to cause a gradual and progressive weakening in contraction. ||Formula/Formula0|| reaches a minimum ~50% below controls after ~30 min (p<0.05). Unlike the WKY fibroblasts, this decline in traction seems to be persistent (i.e., at least for the final 30 min of the experiment there is no evidence of any substantial recovery in traction towards the controls).

One may note a curious anti-symmetry in the effects of Ang II and Irb on the WKY and SHR fibroblasts. After 1 h in the presence of Ang II, the contraction of the two cell types is equalized because the WKY cells increase their tractions while the SHR cells remain the same. On the other hand, after 1 h in the presence of Irb, the contraction of the two cell types is also equalized, but in this instance it is because the SHR cells relax to a greater extent.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The C-RAS model predicts that chronic local elevation of Ang II causes SHR cells to differentiate towards a myofibroblast phenotype and furthermore, that this change long precedes clinical HHD since it is involved in the pathogenesis and is not a result of the disease. The idea is supported by studies that show that SHR cells isolated from young rats are indeed more like classical myofibroblasts, that addition of exogenous Ang II causes WKY cells to approximate the SHR phenotype, and finally that SHR cells can be induced to revert towards the WKY phenotype by exposure to antagonists of the AT1 receptor (see Introduction for details). All of these results pertain to in vitro studies comparing SHR and WKY cells. Their relevance under in vivo conditions is supported by the study of Norton et al. [29], which found an elevated left ventricular diastolic stiffness in SHR rats in vivo at ~6 months of age (~2.8 kPa versus ~2.0 kPa for WKY controls). This significant differential finding was eliminated after the SHR rats were treated with the ACE inhibitor captopril (this ACE inhibitor had no effect on the myocardial stiffness of WKY rats). To all these data we may now add our observation that SHR fibroblasts from young rats are hypercontractile when compared to age matched WKY cells and that exogenous Ang II causes WKY cells to increase their contractile activity so as to match the level found in SHR cells. Furthermore, as with other manifestations caused by addition of exogenous Ang II, the increased contractile activity of SHR cells is evident early in the disease, is stable in culture, and is reversed by blockers of the AT1 receptor. We conclude that our data support the C-RAS model and contradict an alternative idea that the differences between SHR and WKY cells are the result of HHD rather than the cause.

A surprising finding of our study is that SHR cells seem to have lost contractile response to exogenous Ang II. One possibility is that this is due to the ~50% downregulation of AT1 receptors in SHR cells when compared to WKY cells [5]. However, this is unlikely since the AT1 receptor dissociation constant is unaltered [5] and since SHR cells are still capable of other responses to Ang II (e.g., collagen production). An alternative explanation comes from an earlier observation that exogenous Ang II causes an increase of intracellular calcium in WKY cells but not in SHR cells [30]. Since this is exactly analogous to what we see for contraction, one is lead to the notion that an increase of intracellular calcium is an essential step in the pathway leading from the AT1 receptor to enhanced contraction. It may also be speculated that intracellular calcium in SHR cells is already at a maximum level and cannot be further stimulated.

The average traction stresses (~3–6 kPa) we measure for typical cardiac fibroblasts are among the highest ever recorded in any cell type (about 10 times higher than 3T3 fibroblasts [19–21] and about the same as cardiac myocytes [31]). It must be remembered that these are in vitro measurements and that there is no certainty that the contractile activity is similar in vivo. Nevertheless, one may speculate that the extraordinary tractions produced by cardiac fibroblasts are needed to compress and rearrange the ECM of the myocardium into an optimal density. During the pathogenesis of HHD this possibility takes added significance if one considers that cardiac fibroblasts are plentiful within the myocardium comprising ~20% of the total volume [32,33]. Moreover, the fibroblasts are arranged into interconnected sheets that can organize and distribute the contractile stresses [34]. Finally, the myocardium as a whole is exceedingly soft compared to the substratum used in the current study [29]. Thus, since even a single WKY fibroblast is able to visibly deform our substratum, it is likely that over time the chronic hypercontraction of the highly organized fibroblasts in the hypertensive heart is sufficient to rearrange and compact the myocardium into an abnormal configuration.


   Acknowledgements
 
Funding for this research was provided by the Computational Science Graduate Fellowship Program of the Office of Scientific Computing and Office of Defense Programs in the Department of Energy under contract DE-FG02-97ER25308 to W. A. Marganski and NIH Grant RO1 GM61806 to M. Dembo.


   Notes
 
Time for primary review 30 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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