1. Quantitative Histomorphological Analysis of Right Ventricular Myocardium Under
Chronic Pressure Overload
S. M. Siegel1
, U. A. Dar1
, M. Rahman1
, M. R. Hill1
, M. A. Simon2
, and M. S. Sacks1
1
The University of Texas at Austin, Austin, TX, 2
The University of Pittsburgh, Pittsburgh, PA
Introduction: Pulmonary hypertension (PH) imparts a pressure overload on the right ventricle (RV)
leading to structural and functional changes in the RV myocardium. Eventually, the RV cannot maintain
cardiac output against the increased pressure overload, resulting in fatal RV failure (1). Though the
organ-level response to pressure overload caused by PH is generally understood, little is currently known
about the underlying tissue-level response of the myocardium. An understanding of the changes
undergone by the structural components of the myocardium in the prelude to RV failure and death could
lead to better techniques for diagnosis and the development of new treatments or therapies to prevent such
failure. The purpose of this study is to gain insight into the pathological tissue-level remodeling process
undergone by the RV due to PH by assessing the myofiber and collagen fiber remodeling response in the
RV myocardium of rats with induced chronic pressure overload.
Materials and Methods: A pressure-overloaded state was induced in the RV of Sprague-Dawley rats (n
= 5) through surgical banding of the pulmonary artery, while control rats (n = 2) were left unaltered. Both
the pulmonary artery banded
rats (hypertensive) and the
control rats (normotensive) were
sacrificed three weeks after the
banding surgery, and the hearts
removed. Remodeling of RV
myocardium was assessed
through histomorphological
analysis of the RV free wall
(RVFW). The entire RVFW
was dissected from each heart,
fixed in 10% neutral buffered
formalin, marked to maintain
orientation, embedded in
paraffin and sectioned
transmurally into 5 μm thicknesses every 50 μm from the endocardium to the epicardium. Gomori’s
Trichrome stain was used to distinguish between myo- (pink) and collagen (grey) fibers in each section of
RVFW tissue. A slide scanner was used to take color images of each tissue section in a specific
anatomical orientation, with the longitudinal (vertical) axis defined by the outflow tract at top and the
apex at the bottom, and the circumferential (horizontal) axis defined by the free wall to the left and the
septum to the right (Fig. 1). The point of view in this set orientation is from outside the heart, with the
more-epicardial side of each tissue section facing the viewer. Raw section images were processed with a
custom MATLAB (The MathWorks, Inc.) program in which pink and grey pixels were separated,
creating one 8-bit myofiber image and one 8-bit collagen fiber image per section. These separate fiber
images were then analyzed by a second custom MATLAB program with the purpose of quantifying the
fiber orientation angles and orientation index (OI), a measure of the degree of alignment, as well as
counting the total number of pixels (n), the pink pixels (m), and the grey pixels (c) in each set of RVFW
sample section images to estimate the volumes of myo- and collagen fibers. Relative volume ratios were
computed as percentages by 100*m/n and 100*c/n for myofibers and collagen fibers, respectively. To
determine the orientation angles the input fiber images were converted to grey scale and 7 × 7 pixel
masks were convolved over each pixel yielding x and y direction gradient components, and , similar
to previous methods (2). The gradient components were used to determine the gradient vector
magnitude, , , and orientation angle, , ⁄ , values for each
Figure 1. Dissected rat heart with RVFW tissue and anatomical
orientation guide locations indicated, in (A) ventral view, (B) dorsal view,
and (C) the epicardium of the fully dissected RVFW sample with the
prescribed longitudinal and circumferential axes shown. (Tic mark = 1
mm)

2. pixel, where the coordinates (i, j) represent the pixel location within the image. These data were then
used to compute the structure tensor matrix for each image, formed by the equation:
∑ , ,, ∑ , , ,,
∑ , , ,, ∑ , ,,
The eigenvectors and eigenvalues of the structure
tensor were calculated, and the predominant fiber
orientation angle for each image was determined from
the principal eigenvector. When the raw gradient
magnitude and orientation angle pixel data are
displayed in polar plot form (Fig. 2) the principal
eigenvector of the structure tensor is oriented at an
angle along the long axis of the ellipse. The OI value
was determined from the ratio of the
eigenvalues, 1 λ λ⁄ , where λ is the
principal eigenvalue. Values for OI closer to zero
indicate more random orientation of fibers, while
values closer to one indicate highly uniform
orientation. T-tests (α = 0.05) were used for all
statistical comparisons except histomorphological
fiber orientation analysis, for which circular statistics
(R Circular Statistics package) were applied over
interpolated normalized RVFW section thicknesses (n
= 10). Circular statistics were used to accommodate
the fiber orientation angle values, which vary over the
full [-180°, 180°] range.
Results and Discussion: When viewed from
outside the heart, myofibers and collagen
fibers of the normotensive control RVFW
tissue rotated clockwise from a longitudinal
(vertical) alignment at the endocardium to a
circumferential (horizontal) alignment at the
epicardium, demonstrating a spiral transmural
orientation angle variation (Fig. 3) which has
been seen previously in canine (3) and rat
myocardium (4). A dramatic transmural re-
alignment of both myofibers and collagen
fibers along the longitudinal axis was shown
in two of the five hypertensive RVFW
samples. Statistically significant (p < 0.05)
differences in mean orientation angle occurred
at normalized RVFW thickness intervals of
20% for the normotensive state and at > 30%
for the hypertensive state. Myofiber OI
increased from 31.7 ± 1.6% in the
normotensive state to 50.0 ± 1.3% (p < 10-7
) in
the hypertensive state, and collagen fiber OI
increased from 30.2 ± 1.5% to 49.5 ± 2.3% (p
< 0.001), indicating an increased degree of
Figure 3. Comparison of normo- and hypertensive RVFW
samples, with (A) Gomori-stained mid-thickness sections
with vector indicating primary fiber orientation, and (B)
polar plots depicting transmural variation in myofiber
orientation angle from endo- (red) to epi-cardium (purple).
OI = length of each orientation vector, with 1 indicating
completely uniform fiber alignment and 0 indicating
random fiber alignment within each section.
Figure 2. Polar plot depicting raw gradient
magnitude versus orientation angle pixel data for
myofibers (pink circles) and collagen fibers (grey
circles), showing the orientation angle of the
corresponding principal eigenvector (green),
which is aligned along the long axis of the ellipse.

3. fiber alignment in the direction of the predominant orientation angle for each section. The average total
number of pink pixels for all RVFW sample sections increased from 7.18x106
± 4.76x105
for
normotensive tissue to 1.97x107
± 3.96x106
(p < 0.039) for hypertensive tissue, representing an increase
in RVFW myofiber volume. Similarly, the average total number of grey pixels increased from 1.70x106
±
2.49x105
for normotensive tissue to 5.61x106
± 5.54x105
(p < 0.009) for hypertensive tissue, representing
an increase in RVFW collagen fiber volume. Relative volume ratios for myo- and collagen fibers,
respectively, were 80.9% and 19.1% for normotensive RVFW samples and were 77.8% and 22.2% for
hypertensive RVFW samples, though this decrease in the relative proportion of myofiber volume is not
significant (p > 0.05).
Conclusions: Chronic pressure overload due to pulmonary artery banding led to tissue-level remodeling
of RVFW myocardium, in which fibers re-oriented longitudinally and became more highly aligned. Both
myofiber and collagen fiber volume significantly increased, likely as results of hypertrophy and fibrosis,
which occur in clinical PH cases (5). Interestingly, the relative proportion of myofibers to collagen fibers
decreased slightly in the hypertensive state, contrary to expectations, though the finding was not
statistically significant. The focus of future work will be to determine the structural changes which occur
at the cellular and extracellular matrix levels of myofibers and collagen fibers as a result of PH.
Acknowledgements: Thanks to funding sources at the National Institutes of Health (1F32 HL117535,
P01 HL103455 & U01 HL108642-01) and the American Heart Association (13POST14720047 &
10BGIA3790022).
References: (1) Voelkel NF, et al., Circ, 2006, 114:1883-91, (2) Courtney T, et al., Biomaterials, 2006,
27:3631-8, (3) Streeter DD, Jr, et al., Circ Res, 1969, 24:339-47, (4) Chuong CJ, et al., Am J Physiol,
1991, 260: H1224-35, (5) Bogaard HJ, et al., Chest, 2009, 135:794-804.