Structural and Mechanical Adaptations of Right Ventricle Free Wall

Myocardium to Pressure Overload

MICHAEL R. HILL,1 MARC A. SIMON,2 DANIELA VALDEZ-JASSO,3 WILL ZHANG,1 HUNTER C. CHAMPION,4

and MICHAEL S. SACKS1

1Center for Cardiovascular Simulation, Institute for Computational Engineering and Sciences, Department of BiomedicalEngineering, The University of Texas at Austin, 201 East 24th Street, University Station, C0200, POB 5.236, Austin, TX 78712-0027, USA; 2Departments of Cardiology and Bioengineering, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh,PA, USA; 3Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA; and 4Southeastern Cardiology

Associates, Columbus, GA, USA

(Received 6 May 2014; accepted 13 August 2014; published online 28 August 2014)

Associate Editor Jane Grande-Allen oversaw the review of this article.

Abstract—Right ventricular (RV) failure in response topulmonary hypertension (PH) is a severe disease that remainspoorly understood. PH-induced pressure overload leads tochanges in the RV free wall (RVFW) that eventually resultsin RV failure. While the development of computationalmodels can benefit our understanding of the onset andprogression of PH-induced pressure overload, detailedknowledge of the underlying structural and biomechanicalevents remains limited. The goal of the present study was toelucidate the structural and biomechanical adaptations ofRV myocardium subjected to sustained pressure overload ina rat model. Hemodynamically confirmed severe chronic RVpressure overload was induced in Sprague–Dawley rats viapulmonary artery banding. Extensive tissue-level biaxialmechanical and histomorphological analyses were conductedto assess the remodeling response in the RV free wall.Simultaneous myofiber hypertrophy and longitudinalre-orientation of myo- and collagen fibers were observed,with both fiber types becoming more highly aligned. Trans-mural myo- and collagen fiber orientations were co-aligned inboth the normal and diseased state. The overall tissuestiffness increased, with larger increases in longitudinal vs.circumferential stiffness. The latter was attributed to longi-tudinal fiber re-orientation, which increased the degree ofanisotropy. Increased mechanical coupling between the twoaxes was attributed to the increased fiber alignment. Inter-estingly, estimated myofiber stiffness increased while thecollagen fiber stiffness remained unchanged. The increasedmyofiber stiffness was consistent with clinical results showingtitin-associated increased sarcomeric stiffening observed inPH patients. These results further our understanding of the

underlying adaptive and maladaptive remodeling mecha-nisms and may lead to improved techniques for prognosis,diagnosis, and treatment for PH.

Keywords—Hypertrophy, Tissue-level biomechanics, Pul-

monary hypertension, Myofiber orientation, Collagen fiber

orientation.

ABBREVIATION

AnatomyRV Right ventricleRVFW RV free wallPA Pulmonary artery

Hemodynamic ParametersRVESP RV end-systolic pressureRVEDV RV end-diastolic volumeSV Stroke volumeSW Stroke workMaximumdP/dt Maximal first time-derivative of pressure (a

measure of systolic function)Tau RV diastolic time constant (a measure of

diastolic function)Ees RV elastance (a measure of contractility)Ea PA elastance (a measure of afterload)Ees/Ea RV–PA coupling

Constitutive Model Measures and ParametersELL Green’s strain in longitudinal directionECC Green’s strain in circumferential directionSLL 2nd Piola–Kirchhoff (PK) stress in longi-

tudinal direction

Address correspondence to Michael S. Sacks, Center for Car-

diovascular Simulation, Institute for Computational Engineering

and Sciences, Department of Biomedical Engineering, The Univer-

sity of Texas at Austin, 201 East 24th Street, University Station,

C0200, POB 5.236, Austin, TX 78712-0027, USA. Electronic mail:

msacks@ices.utexas.edu

Annals of Biomedical Engineering, Vol. 42, No. 12, December 2014 (� 2014) pp. 2451–2465

DOI: 10.1007/s10439-014-1096-3

0090-6964/14/1200-2451/0 � 2014 Biomedical Engineering Society

2451

SCC 2nd PK stress in circumferential directionb0 Model scaling parameterbL Model parameter, representing longitudi-

nal stiffnessbC Model parameter, representing circumfer-

ential stiffnessbLC Model parameter, representing coupling,

between longitudinal and circumferentialresponse

Sens Combined myofiber-collagen effective fiberensemble stress

Eens Combined myofiber-collagen effective fiberensemble strain

Um Mass fraction of myofibersUc Mass fraction of collagen fibersgm Intrinsic myofiber modulus�gc Intrinsic collagen fiber modulus, that

accounts for the effects of gradual fiberrecruitment

Elb Lower bound on recruitment strain for thecollagen fiber ensemble

Eub Upper bound on recruitment strain for thecollagen fiber ensemble

PTTM Post-transition tangent modulus

INTRODUCTION

Right ventricular (RV) failure is a major cause ofmortality for patients of pulmonary hypertension(PH), with a mortality rate of 37.2% at 3 years post-diagnosis.1,2 RV function is the most critical determi-nant of patient longevity in PH.57 Mechanistically, theRV is the end organ, in that PH leads to RV failure,which is the cause of a majority of deaths (directly orindirectly) from PH.16,25,54 While several therapieshave been developed which address the pressure andflow in the pulmonary vascular bed,2,25 little is knownregarding their effect on RV function. Moreover, nocurative treatments have been made available exceptlung transplantation.4 Following transplantation,dramatic improvements in mean pulmonary arterypressure, pulmonary vascular resistance, and RVejections fractions have been observed,27 although theexact mechanisms by which the RV can revert back tonormal function (reversibility) are yet to be defined.

Predicting RV failure resulting from PH is difficultsince some patients survive years, while others rapidlydevelop RV failure. Current clinical techniques aresuccessfully used to diagnose and predict RV function,such as right heart catheterization30 and echocardiog-raphy-derived tricuspid annular plane systolic excur-sion, or TAPSE,21 respectively. Clinical predictors of

mortality include stroke volume index, pulmonaryartery capacitance, and estimated globular filtrationrate.7 Several parameters, including the size and massof the RV,10 the right atrial pressure,10 and conditionssuch as hypnoatremia,22 reflect the functional status ofthe RV and are correlated with advanced RV failureand survival. However, some techniques recommendedfor screening for PH are frequently inaccurate. Forexample, Doppler echocardiography gives inaccurateestimates of pulmonary artery systolic pressure in 48%of cases, according to a recent study of 65 PHpatients.20

At the organ level PH imposes a pressure overloadon the RV leading to ventricular enlargement.6 Thisoverload affects the RV free wall (RVFW), resulting inincreased transmural wall stress and wall weakening,ultimately resulting in ventricular failure.4 Hypertro-phy of the associated myofibers occurs via an additionof sarcomeres, with evidence of fibrosis and changes tothe extracellular matrix (ECM) both in humans and inrat models.4 However, the specific tissue-level com-pensatory adaptations of the main structural constit-uents of the myocardium (myofibers and collagenfibers) to pressure overload remain unknown.57

Although the effects of myocardial stress on cardiacremodeling have been studied for decades,28 the precisemechanisms by which cardiomyocytes sense the forceimposed by ventricular pressure and volume overloadand respond with sarcomerogenesis remainsunknown.29 Knowledge of the underlying adaptivemechanisms is essential for developing multi-scalemodels of pressure-induced growth and remodeling(G&R). Such modeling efforts may ultimately be usedto improve diagnosis and prognosis, as well as providea framework to develop therapies specifically targetingimproving RV response to disease.

Our long-term goal is to quantify and model the cel-lular- and tissue- level G&R of the RV myocardium inPH. While the development of such models can benefitour understanding of the onset and progression of PH-induced pressure overload, detailed biomechanical dataof the underlying events remains limited. Thus, theobjective of the present study was to elucidate thehypertrophic andmicro-architectural remodeling eventsthat occur in response to organ-level chronic pressureoverload in an animalmodel of PH.A pulmonary artery(PA) banding model was utilized to create a pulmonaryhypertensive condition in a rat. In vivo hemodynamicmeasurements were made to evaluate cardiac perfor-mance. Microstructural analysis was performed toquantify micro-architectural remodeling, and the tissue-level biomechanical response of the myocardium wasassessed using a novel experimental approach in whichthe isolated tissue maintains viability.56

HILL et al.2452

MATERIALS AND METHODS

A total of 55 male Sprague–Dawley rats, 8 weeksold at the start of the experiment, were used in thisstudy. Note that 8 of the rats were used for the 1-weekstudy as described in the ‘‘Discussion’’ section. Theexperimental protocol was approved by the Universityof Pittsburgh IACUC (Protocol #13021226) and con-formed to the Guide for the Care and Use of Labo-ratory Animals. There were several factors that enteredinto this choice of animal model, which have beendiscussed previously.56 Briefly, the RVFW thickness isappropriate for our biomechanical studies, being suf-ficiently thin to maintain viability after dissection. Inaddition, the mass is sufficiently large for analyzingtissue constituents, as well as allowing for the quanti-fication of myocardial remodeling due to pressureoverload. In the following, the number of rats from thetotal is provided for each section.

Pulmonary Artery Banding Procedure and In VivoHemodynamic Analysis

Of the 55 total rats, RV pressure overload was in-duced surgically by restriction of the pulmonary artery(Fig. 1) on 20 animals. Animals were anesthetized with5% isoflurane and placed on a heated table to maintaina core temperature of 37�C, measured by a rectalprobe. The chest was entered via a lateral incision toexpose the mid-thoracic aorta. A surgical clip wasplaced around the PA with a radius such that a uni-form right ventricular pressure of 45–50 mmHg wasgenerated. Alternatively, a suture was placed aroundthe PA and brought taught around a 27-gauge needle,which was removed after tying the suture. The chestwas closed and sutured, and the animal was extubatedand observed continuously for 2 h post-procedure anddaily afterwards for the first week.

Terminal invasive hemodynamics was performed onthe normotensive (n = 10) and hypertensive (n = 10;noting that the same hypertensive rats were used intissue-level biomechanical studies) rats to generatepressure–volume loops, from which relevant hemody-namic parameters were derived using methodsdescribed previously,18,38 Ees was obtained by stan-dard methods utilizing vena cava occlusion, e.g.,Refs. 10,18,50. Volume calibration was performed in 2steps (relative and then absolute). First, a saline vol-ume calibration was performed in which the catheterrests in saline for at least 30 min followed a 2-pointcalibration to obtain a relative volume unit using theMPVS-400 transducer box (Millar Instruments,Houston, TX). Next, absolute volume calibration wasperformed using volume calibration cuvettes (ADIn-struments, Colorado Springs, CO). At 3 weeks post-surgery, control (normotensive) and PA banded(hypertensive) rats (weighing 310 ± 9.15 g) wereeuthanized via inhalation of isoflurane, and the heartwas removed along with proximal sections of the greatvessels and arrested by placing in cardioplegic solu-tion.40 The RVFW was dissected from each heart andanalyzed with the following techniques.

Tissue Level Measurements

Myofibers and collagen fibers in the extracellularmatrix (ECM) were assumed to be the dominantmechanical constituents comprising the myocardium.RVFW constituent content was quantified by using alyophilization and decellularization technique on nor-motensive (n = 5) and hypertensive (n = 3) specimens.To determine the changes in constituent contentbetween specimens, density was computed as mass ofeach constituent divided by total specimen volume.Mass fractions Um and Uc were computed as myo- orcollagen fiber density divided by dry tissue density,respectively, for normo- and hypertensive specimens.

Histomorphology

Comprehensive transmural orientation angle andorientation index (OI) data were determined by ana-lyzing histologically stained sections from normoten-sive (n = 2) and hypertensive (n = 5) specimens usingthe techniques described below. A total of 10 sectionswere taken of each normotensive specimen, and 15–20sections were taken of the hypertensive specimens.Entire RVFWs were dissected and fixed in 10% buf-fered neutral formalin. Specimens were embedded inparaffin and 5 lm thick sections were obtained with amicrotome en face from epi- to endocardium at 50 lmintervals, and histological staining was performed withGomori One-Step Trichrome. This stain was chosen so

FIGURE 1. Pulmonary hypertension is induced via bandingthe pulmonary artery with a surgical clip. Before surgery, theRV is a thin shell like wall sitting on the LV chamber, whereasafter 2–3 weeks, the RV has enlarged both its wall and thick-ened compared to the LV.

RV Adaptation to Pressure Overload 2453

that the myofibers and collagen fibers were easily dis-cernable, with the former appearing pink and the lattergray. The sections were imaged with a Nikon SuperCoolscan 900 ED slide scanner running Nikon Scan4.0 software, with resolution of 6.35 lm/pixel. Lengthratios were computed as the length of the specimenalong the longitudinal direction (Ll) divided by thelength along the circumferential direction (Lc). Mea-surements were taken from each histological sectionimage and averaged.

The orientations of myo- and collagen fibers werequantified from each histologically stained section,using gradient methods similar to those used previ-ously in our lab15 (Figs. 3a and 3b). A custom programwritten in MATLAB was used to quantify fiber ori-entation from the scanned images of size n 9 m pixels.First, pixels (given by location i, j) representingmyofibers (pink) and collagen (gray) were separatedinto 8-bit gray scale images by manual selection ofappropriate ranges of RGB values in the color images.Second, for each image, gradients in the x and ydirections, G

yi;j and Gx

i;j, were determined by convolvingmasks over each pixel in the color-separated gray scaleimages, by following previous methods15 and settings = 3 and r = 2.5. At each pixel, the magnitude of thegradient and the orientation angle were computed by

Gi;j ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

Gxi;j

� �2

þ Gyi;j

� �2r

ð1Þ

and

/i;j ¼ tan�1G

yi;j

Gxi;j

!

ð2Þ

respectively. We follow our previous convention byassigning 0� aligned to the outflow tract (OT), withpositive increasing angles denoting counter-clockwise(right-handed) rotation from the perspective of anobserver looking from outside the heart (epicardium)(Fig. 3a). From the orientation distribution function, astandard 2nd order structural tensor H (e.g., seeRefs. 23,26,43), was computed by

H ¼P

i;j;2½1:n;m�

12Gi;j cos

2 /i;j

P

i;j;2½1:n;m�

12Gi;j sin

2 /i;j cos2 /i;j

P

i;j;2½1:n;m�

12Gi;j sin

2 /i;j cos2 /i;j

P

i;j;2½1:n;m�

12Gi;j sin

2 /i;j

2

6

6

4

3

7

7

5

ð3Þ

The eigenvalues, m1 and m2, (m1 ‡ m2), and eigenvectors,e1 and e2, ofHwere determined using standard methods,following the normalization of the structure tensor by itsdeterminant. We subsequently defined preferred direc-tion angle hk, for each individual histological section k, as

defined as the direction of the principal eigenvector m1(Fig. 3b). Circular statistics were used to compare themean orientation angles between the two groups, sincestandard statistics are not applicable for angular data.41

Finally, themeanorientation angle for each specimenwascomputed, by taking an average over the total number, p,of transmural images of each set of sections per specimen,using

�h ¼ tan�1�Y�X

ð4Þ

where �X ¼ 1p

P

p

k¼1cos hk, �Y ¼ 1

p

P

p

k¼1sin hk, and angular

SEM for each specimen was computed from theangular variance, rvar ¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

2 1� rð Þp

, as

SEM ¼ rvarffiffiffi

pp ð5Þ

where r ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

�X2 þ �Y2p

is the length of the mean vector.Note that the mean orientation angle and angularSEM were analogous to the standard mean and SEMin standard summary statistics (see ‘‘Statistical Ana-lysis’’ section). The degree of alignment, along thedirection of the principal eigenvector, was computedusing OI = 100 (%)*(1 2 m2/m1). Thus, an OI of 100%indicated perfect alignment, and an OI of 0% repre-sented random fiber distribution. The transmurallocation of each section was expressed as a normalizedthickness as a percentage of total thickness, with 0%representing the endocardium and 100% the epicar-dium. Mean orientation angle was plotted againstnormalized thickness. For comparison, interpolationwas performed on the hypertensive specimens todetermine values on the same evenly spaced grid, at10% thickness intervals, as the normotensive speci-mens.

Tissue-Level Biomechanical Studies

Biaxial mechanical studies was performed on iso-lated myocardium using protocols described previ-ously.56 Briefly, square specimens of RVFWmyocardium from normotensive (n = 10) and hyper-tensive (n = 12, with 10 of these from in vivo hemo-dynamic analysis) rats were dissected and mountedtrampoline-fashion in a biaxial testing device (Cell-Scale Biomaterials Testing BioTester 5000, Waterloo,Ontario, Canada), in modified Kreb’s solution withBDM and oxygen. Multi-protocol displacement-con-trolled testing was performed by varying the ratio ofmaximum displacements in each axial direction. TheGreen–Lagrange strain tensor (E) and the 2nd Piola–Kirchhoff stress tensor (S) were computed for each testprotocol using standard methods,31,45 with longitudi-nal and circumferential components given respectively

HILL et al.2454

by ELL, SLL, and ECC, SCC. While shear stresses SCL

were measured, their magnitudes were very small andwere neglected.

To statistically compare the degree of anisotropybetween the normo- and hypertensive states, the dif-ferences between maximum values of SLL and SCC

were computed from the interpolated equi-biaxialresponse, in which ELL = ECC.

19,46 A larger differencewould indicate a higher degree of anisotropy, i.e., thestress–strain responses along the longitudinal and cir-cumferential directions would follow distinctly differ-ent paths. Since the mechanical tests were performedunder displacement control, the equi-biaxial stress–strain response was interpolated from the multi-pro-tocol experimental dataset with a custom MATH-EMATICA program (Wolfram Research Corp.).19

Biomechanical Modeling

As a first step in determining the intrinsic fiber-levelchanges, the effective fiber ensemble stress–strainresponses were determined from the interpolated equi-biaxial strain. We note that this response represents theeffective mechanical behavior of the intrinsic combinedmyofiber–collagen fiber structural unit, i.e., the effec-tive fiber ensemble, defined as a set of fibers with acommon orientation that undergoes an equi-biaxialstrain (Eens = ELL = ECC, ELC = 0) for a given fiberensemble direction. The ensemble stress was computedby adding the stress components, Sens = SLL + SCC,as shown previously.46 With this method, it was pos-sible to obtain the stress–strain relationship (Sens–Eens)for the effective myo- and collagen fiber ensemble,from which characteristics of the individual constitu-ents may be derived, as described in the following.

In the present work, we took a quasi-structuralapproach to characterize the tissue mechanicalresponses. We assumed the myofibers to be solelyresponsible for low stress response, followed byincreasing contributions of the collagen fibers due totheir gradual recruitment with strain. Recruitmentmodels have been utilized to represent the behavior ofmyo- and collagen fibers in myocardium,33 in whichcollagen is gradually recruited at higher strains to bearload. Justification for the assumption that collagen isinitially crimped and is gradually uncrimped to bearload with increasing tissue strain arises from scanningelectron microscopy analysis of fixed myocardium, inwhich collagen fibers appeared wavy in diastole andtaut in systole.9 From the interpolated equi-biaxial(Sens–Eens) data set, the myofiber-dominated responsewas determined from the tangent modulus of the low-strain region, while the collagen-dominated responsewas determined from the tangent modus of the high-strain region, both obtained via linear regression.

Applying a basic rule-of-mixtures approach,34 thetotal second Piola–Kirchhoff stress was given as

S ¼ UmSm þ ð1� UmÞSc ð6Þ

where Um is the mass fraction of myofibers. Althoughwe do not model recruitment directly, we took thefollowing phenomenological approach to estimaterecruitment characteristics.

We observed that low-stress portion of the Sens–Eens

curve (Fig. 7) exhibited a linear relation. We thusassumed that no collagen fibers were recruited in thisregion, and that it was entirely due to the response ofthe myofibers with an associated tangent modulusTMm. The intrinsic myofiber stiffness gm can bedetermined using gm ¼ TMm=Um: Next, the strain atwhich collagen fiber recruitment initiated was definedas the lower transition from the initial linear to thenonlinear portions of the curve. The ‘‘upper bound’’strain at which collagen fibers were assumed to be fullyrecruited was defined as the upper transition from thenonlinear to the linear portion of the curve. Weassumed all collagen fibers were recruited beyond thisupper bound, characterized by a post-transition tan-gent modulus PTTM (Fig. 7). Assuming that the col-lagen fibers are linear elastic, we note that

PTTM ¼ Umgm þ Uc�gc ð7Þ

where �gc is the effective collagen fiber modulus. Sincethe collagen fiber recruitment was not explicitly mod-eled �gc � gc, where gc is the true collagen fiber elasticmodulus. The transitions strains that encompass thefiber recruitment Elb and Eub, were determined directlyfrom tangent modulus-Eens curves, as in Fata et al.19

We note that these transitions were always marked andidentifiable.

Next, to explore the responses of the entire mea-sured stress–strain response, the biomechanicalresponse of the myocardium specimens was repre-sented by the following in-plane Kirchhoff stress–strain relations,12 which have been derived through atwo-dimensional strain energy function (see Ref. 47 fordetails).

SCC ¼ b0 bCECCe12bcE

2CCþbLCELLe

bLLELLECC

� �

SLL ¼ b0 bLELLe12bLE

2LLþbLCECCe

bLCELLECC

� � ð8Þ

Equations (8) were simultaneously fit to the exper-imental data to determine the material parametersusing a nonlinear regression algorithm in MATLAB(The MathWorks, Inc.). Goodness of fit was deter-mined by computing the standard r2 value. This modelform was chosen over others, e.g., Fung-type expo-nential,24 because it allows explicit decoupling of theresponse extensional and coupling responses, reducing

RV Adaptation to Pressure Overload 2455

the parameter covariance. Specifically, the first term onthe right hand side of Eq. (8) for SCC is independent ofELL. The second term in Eq. (8) represents the degree ofmechanical coupling between the tissue axes. We alsonote that Fung-type constitutive models can have con-vexity issues unless certain restrictions are imposed (see,e.g., Refs. 32,52), and therefore are not as desirable.Moreover, in the present study our purpose for usingEq. (8) was to provide insight into the effects of tissueremodeling from mechanical behavior over the entirestrain range. We note that future work will involveusing full structural models (e.g., Ref. 19) to directlyaccount for the myo- and collagen fiber architecture.

Tissue Viability Studies

While we have shown viability and confirmed stableactive contraction for healthy rat RVFW tissue for atleast 3 h after harvesting,56 we evaluated viability forthe PH specimens, since they had undergone adaptiveremodeling to the sustained pressure overload, e.g.,they became thicker. To ensure we were testing living,viable myocardium, tissue viability was assessed.Mitochondrial function was determined for the timecourse of biaxial mechanical evaluation, approxi-mately 1 h. Since mitochondrial respiratory control isthe best general measure of function in isolated mito-chondria, we used a Clark-type oxygen electrode,5 atechnique that has been used for over 50 years,11 tomeasure respiratory control ratios (RCRs) on n = 3specimens at baseline and at 1.5 h post dissection.

Statistical Analysis

All data, excluding histomorphology, were reportedas sample mean ± sample standard error of the mean(SEM. Statistical comparisons were made using a two-sample Student’s t test in MATLAB (ttest2.m) witha = 0.05. For histomorphological data, circular sta-tistics41 were used to compute mean and SEM, anal-ogous to standard summary statistics, for the angulardata, and comparisons were made using a circularanalysis of variance (aov.circular) in the ‘circular’package35 in R software (R Project for StatisticalComputing, http://www.r-project.org).

RESULTS

In Vivo Hemodynamic Analysis

Changes in hemodynamics and tissue properties(Table 1) were evident. The P–V loops shifted to theright and doubled in size (Fig. 2), indicating RVenlargement during pressure overload. Pressure-derivedmeasurements were available for n = 10 specimens,

while volume measures were only obtained from n = 5specimens due to difficulties in volume measurement.Changes in hemodynamics reflected significant altera-tions to the RV in the hypertensive specimens (Table 2).Pressure and volume were higher, while a small increasein SV and large increase in SWwere observed. MaximaldP/dt and Ees, measures of contractility, significantlyincreased. Diastolic function worsened as evidenced bythe increase in tau. Ea, a measure of vascular load,increased, and the ratio Ees/Ea increased, indicating ade-coupling of the RV function from the vascular load.

Tissue Constituents

Changes in bulk RVFW myocardial tissue wereobserved from the normo- to the hypertensive state(Tables 1 and 2). Statistically significant increases inboth thickness and mass of the RVFW myocardiumwere observed. Average mass of both myofibers andECM increased. The mean overall tissue density wasfound to decrease slightly. The mean density of totalmyofibers and ECM remained approximately equal,with the mean density of myofibers increasing andECM density decreasing. Mass fractions of myo- andcollagen fibers for normo- and hypertensive specimenswere Um = 44% and 63%, respectively for muscle, andUc = 56% and 37%, respectively for collagen.

Histomorphology

Histological staining revealed increases in bulk tissuein the hypertensive state (Fig. 3a). Longitudinal length(Ll) increased, though not significantly, and circumfer-ential length (Lc) significantly increased (Table 2),indicating an overall increase in cross-sectional area.The ratio Ll/Lc decreased significantly under pressureoverload, suggesting a change in ellipticity of RV shapefrom highly ellipsoidal, with the long axis oriented lon-gitudinally, towards more spherical. Within each histo-logical section, myo- and collagen fibers were orientedalong similar directions in both states (Figs. 3b and 4).In the normotensive state, there was a transmural(through-thickness) variation in the fiber orientation.Moving outward, the preferred orientation directionfollowed a helical clockwise (left-handed) rotation, fromlongitudinally in the endocardium to circumferentiallyin the epicardium. We observed a trend in the hyper-tensive specimenswhere the transmural fiber orientationclearly lost its helical through-wall variation, and dra-matically re-oriented towards the longitudinal direction(Fig. 4). For all specimens, the mean degree of align-ment, or orientation index (OI), increased significantlyfor both myo- and collagen fibers (Figs. 4 and 5a), andthe mean fiber angle was 241.8� ± 4.6� in the normo-and 221.6� ± 2.5� in the hypertensive state (Fig. 5b).

HILL et al.2456

Tissue-Level Biomechanical Behavior

Biaxial mechanical evaluation of the isolatedRVFW myocardium was very reproducible, givingqualitatively comparable responses to previous stud-ies.56 Stability in the response was confirmed by thereproducibility of the 1:1 displacement-controlledtesting performed at the beginning and at the conclu-sion of specimen testing, where we observed a similarstress–strain behavior between the two responses(results not shown). The degree of anisotropy increasedin the pressure-overloaded state, illustrated by theincrease in difference between the longitudinal andcircumferential mechanical response curves and by theincreased separation of the maximum values for SLL

and SCC in the hypertensive state (Fig. 6). Further-more, the tissue stiffness was higher in the longitudinaldirection than in the circumferential direction.

Biomechanical Modeling

There was a change in the mechanical response ofthe intrinsic fiber ensemble. Fiber recruitment initiatedat lower strain levels in the hypertensive specimens(Fig. 7). Relative recruitment increased slightly from50.2 to 54.6%. The low strain tangent modulus(TMm) was significantly greater in hyper- vs. normo-tensive specimens (p = 0.003), with no statistical dif-ferences in PPTM. The intrinsic myofiber stiffnessmodulus increased from gm = 105 kPa in the nor-motensive to 228 kPa in the hypertensive state. Theintrinsic collagen fiber stiffness modulus decreasedslightly from �gc = 1240 to 1190 kPa, from normo- tohypertensive.

The constitutive model fit the mechanical datagenerally well for both states; mean R2 values were0.9446 ± 0.0078 for normotensive and 0.8856 ±

0.0141 for hypertensive. The scaling parameter b0increased from 0.16 ± 0.02 to 0.23 ± 0.08 kPa,though the difference was not significant. Very largeand significant increases were observed in other meanparameter values by approximately 163% for bL,from 64.1 ± 8.0 to 169.0 ± 17.5; 95% for bC, from35.7 ± 4.0 to 69.6 ± 7.3; and 180% for bLC, from41.2 ± 2.3 to 115.2 ± 13.3 (Fig. 8), indicating anincreased stiffness in the longitudinal vs. circumferen-tial direction and an increase in coupling. These resultsreflect the substantial remodeling of the tissuemechanical behavior with PH.

Tissue Viability

Respiratory control ratios (RCR) at baseline were4.64 ± 0.41 and at 1.5 h were 2.22 ± 0.07. Valuesabove 2 are considered viable; thus, mitochondria wererespiring, intact, and functional, indicating living tissuethroughout the time course required for biomechanicaltesting.5

TABLE 1. Summary of multi-scale changes to the right ven-tricle under pressure overload.

Scale Change from Normo- to Hypertensive

Organ-level Hemodynamic changes

Chamber dilation

Sphericalization of RV chamber shape

Tissue-level Increased overall RVFW mass and volume

Increased myo- and collagen fiber mass

Increased myofiber mass density

Decreased ECM mass density

Longitudinal re-orientation of myo- and

collagen fibers

Increased myo- and collagen fiber

degree of alignment

Highly increased longitudinal stiffness

Increased circumferential stiffness

Increased mechanical coupling between axes

Increased combined effective fiber ensemble

(EFE) stiffness

Cellular/

Fiber-level

Increased myofiber EFE stiffness

Slightly decreased collagen-dominated

EFE stiffness

Little changes in relative recruitment strain

FIGURE 2. Right-ventricular pressure–volume (P–V) loops for (a) the control, normotensive state and for (b) the pressure-over-loaded, hypertensive state. The P–V loops shifted to the right and doubled in area, indicating the RV enlargement during PH.

RV Adaptation to Pressure Overload 2457

DISCUSSION

The objective of the current studywas to elucidate thehypertrophic and architectural remodeling events that

occur in response to organ-level chronic pressure over-load in a rat model of PH. Interestingly, we observedchanges at all length scales studied here (Table 2). This

TABLE 2. Derived hemodynamic parameters and bulk tissue properties.

Measure Normotensive Hypertensive

Hemodynamic parameter

RVESP (mmHg) 28.0 ± 1.2i 93.8 ± 5.6i

RVEDV (lL) 124.8 ± 18.1ii 223.1 ± 45.2ii

SV (lL) 131.4 ± 12.7ii 190.0 ± 22.2ii

SW (lL mmHg) 2671.8 ± 217.3iii 9354.6 ± 1986.6iii

Maximum dP/dt (mmHg/s) 2297.8 ± 124.5iv 4547.2 ± 420.3iv

Tau, 1/e (ms) 3.8 ± 0.4iv 7.0 ± 0.3iv

Ees (mmHg/lL) 0.216 ± 0.046iii 0.808 ± 0.117iii

Ea (mmHg/lL) 0.242 ± 0.085iii 0.440 ± 0.033iii

Ees/Ea 0.82 ± 0.28v 1.98 ± 0.66v

Bulk RVFW myocardium tissue property

Specimen Thickness (lm) 754 ± 10vi 1223 ± 48vi

Specimen volume (lL w.v.) 29.7 ± 1.8 73.2 ± 6.0

Specimen mass (mg w.w.) 61 ± 3vii 123 ± 7vii

Longitudinal length, Ll (mm) 11.53 ± 0.47 12.41 ± 0.36

Circumferential length, Lc (mm) 7.24 ± 0.14viii 10.13 ± 0.28viii

Ll/Lc Length ratio 1.58 ± 0.04ix 1.22 ± 0.02ix

Tissue wet density (mg/mL w.w./w.v.) 2173 ± 126 1922 ± 47

Tissue dry density (mg/mL d.w./w.v.) 434 ± 16 424 ± 9

Myofiber mass (mg d.w.) 6.3 ± 1.1 14.0 ± 1.4

ECM mass (mg d.w.) 6.1 ± 0.9 8.7 ± 2.2

Myofiber density (mg/mL d.w./w.v.) 191 ± 24 265 ± 33

Collagen fiber density (mg/mL d.w./w.v.) 243 ± 26 159 ± 24

Significant differences indicated between normotensive and hypertensive states.i p < 1029, ii p = .03, iii p < .001, iv p < 1024, v p = .009, vii p < 1027, vii p < 1028, viii p < 1025, ix p < 10211.

w.w., wet weight; w.v., wet volume; d.w., dry weight.

FIGURE 3. Methodology and results of fiber orientation angle and orientation index quantification for Gomori-stained histologicalsections. (a) Image of the rat heart, showing outline of RVFW (blue and red dots) and square slab for biaxial testing (dotted lines).Histological section shown is taken from the midwall at 53% thickness of a hypertensive rat RVFW specimen, stained with GomoriOne-Step Trichrome for myofibers (pink) and collagen (gray). Overall measured myofiber and collagen fiber orientation angles inthe illustrative image were both / 5 7.7�, indicated by the blue arrow (V1). Insets illustrate the local fiber orientation angles withvectors (blue arrows) in the separate myofiber and collagen images. (b) Polar plot depicting the variation in gradient magnitudewith angle for myofibers (pink circle) and collagen (gray circles) in a single histological section. Orientation angle is along thedirection of the principal eigenvector (V1). Orientation convention: 0� is along the +xL direction (oriented longitudinally towards theoutflow tract); 180� is along the 2xL direction (oriented longitudinally towards the apex); 90� is along the +xC direction (orientedcircumferentially towards the free wall); and 290� is along the 2xC direction (oriented circumferentially towards the septum).

HILL et al.2458

results suggests that that myocardium’s response to PHis complex and multi-scale. Interpretation of specificresults is given in the following by scale level.

RV Response to Pressure Overload

In vivo hemodynamic studies confirmed RV pressureoverload in the PA-banded group (Fig. 2; Table 2).End-diastolic pressure increased significantly, as pre-viously seen in PAB rat models18 and as expected in thehypertensive condition. Clinically, hypertrophy hasbeen described as the initial adaptive response to PH,in order to increase contractility.57 Here, higher dP/dtand Ees were indicative of an increase in RV

contraction to maintain flow against the increasedpressure afterload. In response to an increase in end-systolic pressure, we saw an increase in end-diastolicvolume (124.8 ± 18–223.1 ± 45.2 lL) as expected.End-systolic volume also increased (from 75 ± 52 to160 ± 119 lL, p = 0.07) but the increase in ESV wasnot as great as the increase in EDV, therefore theresultant stroke volume increased. The ejection frac-tion and cardiac output did not significantly change(EF: 85 ± 22–79 ± 23%, p = 0.58; CO: 53 ± 16–70 ± 23 mL/min, p = 0.12), and stroke workincreased as expected. These results are consistent withboth Bogaard et al.,4 who found similar results in thePA banding model, in which they reported no change

FIGURE 4. Transmural variation in orientation angle, depicted in the polar plots, and orientation index (OI), shown in the bargraphs, for myofibers and collagen fibers in illustrative normotensive and hypertensive specimens. Color represents positionalong thickness of the RVFW. Orientation angle is indicated by the direction of the vectors in the polar plots. The length of thesevectors represents OI and matches the ordinate values of the bar graph. An OI of 0% indicates randomly oriented fibers, with 100%being perfectly aligned. Black arrows depict mean fiber angle.

RV Adaptation to Pressure Overload 2459

in cardiac output and Maughan et al.,42 who observedthat an increase in afterload is associated with greatervolumes, including stroke volume.

There was a significant increase in both tissuethickness and mass, indicative of hypertrophy, i.e.,more bulk myocardium was being created to increasecontractility. We found the density of combinedmyofibers and extracellular matrix (ECM) remainedrelatively constant from the normo- to the hypertensivestate, and the relative density of myofibers increased(Table 2). So hypertrophy, as opposed to ECMexpansion (fibrosis), likely dominated the remodelingprocess. This hypertrophic response likely underlies theincrease in contractility seen in pressure overload priorto overt ventricular failure.

According to the Laplace relationship,4 T = Pr/h,where T is wall stress, P is intraluminal pressure, r ischamber internal radius, and h is chamber wall

thickness. In the hypertensive state, chamber pres-sure, P, must be increased to pump against increasedafterload. The RV likely responded to increase thispressure through hypertrophy, which simultaneously

a

b

FIGURE 5. (a) Mean orientation index, or OI, for myofibersand collagen in normotensive and hypertensive specimens.Statistically significant differences indicated on plot(bars 5 SEM). (b) Variation of mean orientation angle withnormalized thickness.

FIGURE 6. Mechanical response of right ventricle free wall(RVFW) myocardium: mean interpolated longitudinal (L) andcircumferential (C) stress (S) vs. mean interpolated equibiax-ial strain (E) response, determined from biquintic finite ele-ment interpolation, of normotensive (n 5 10) andhypertensive (n 5 12) specimens of isolated rat RVFW myo-cardium. Statistically significant differences indicated on plot(bars 5 SEM).

TMlowH = 143kPa

PTTM H = 535kPa

Eens

Sen

s(kPa)

TM low

N = 46kPa

PTTM N = 716kPa

FIGURE 7. Changes in the mechanical (stress–strain)response of the intrinsic combined myofiber-collagen EFEresponse from the normo- to the hypertensive state(bars 5 SEM). From left to right, dotted lines indicate thestrain at which fiber recruitment initiates and the maximumstrain for the normotensive state, EN

lb 5 0.102 andEN

ub 5 0.203, and for the hypertensive state, EHlb 5 0.053 and

EHub 5 0.097, respectively. Straight lines represent linear fit to

the upper and lower portions of the curves, with effectivetangent moduli defined as the slopes of these lines. Lowstrain tangent modulus was significantly higher (p 5 0.003) inhyper- vs. normotensive specimens.

HILL et al.2460

achieved a bulk increases in thickness, h, and anincrease in pressure, P (Table 2), via increased con-tractility.

Clinically, hypertrophy is followed by progressivecontractile dysfunction,57 along with chamber dilationto maintain cardiac output in spite of reduced con-tractility.18,57 Here, an increase in end diastolic volumewas indicative of RV chamber dilation. As the condi-tion progresses with sustained pressure overload,contractility increases are insufficient to maintain car-diac output (i.e., decoupling of ventricular function toits vascular load), resulting in progressive chamberdilation, i.e., increase in chamber internal radius, r, toincrease ejection volumes through increased pressure.Ees/Ea increased, indicative of ventricular-vasculardecoupling, as seen clinically in PH,39,48,53,55 suggest-ing that the increased contractility was not sufficient tomaintain cardiac output against the increased after-load. Moreover, sphericalization, as shown here withthe change in ratio of longitudinal to circumferentiallength towards unity, has been associated with theonset of heart failure (see Janicki et al.36 and referencestherein). Eventually, RV failure occurs and is charac-terized by increased filling pressures, diastolic dys-function, and decreased cardiac output.57 Tausignificantly increased with PA banding from 3.8 ± 0.4to 7.0 ± 0.3 ms, p< 1024, suggesting that diastolicdysfunction is occurring here in the PA banding model,and that the rats are at the onset of RV failure. Thus,we conclude that these PA-banded rats were in a stageof advanced right ventricular dysfunction, prior toovert failure.

Structural and Mechanical Adaptations to PressureOverload

We found the intrinsic mechanical properties andmicrostructure of the myocardium were altered fromthe normo- to the hypertensive state, in addition tobulk tissue accumulation. First, the overall tissuestiffness increased (Fig. 6), demonstrated quantita-tively by increases in both material parameters bL andbC (Fig. 8), which we attributed to stiffening of thecombined myofiber-collagen fiber ensemble structuralunit (Fig. 7). The increased fiber ensemble stiffness waslikely due to intrinsic stiffening of the myofibers alone,since a greater than two-fold increase in intrinsicmyofiber stiffness was observed and a slight decrease inintrinsic stiffness and slight increase in relativerecruitment strains were observed for collagen.

Second, a larger increase in bL compared to bC(Fig. 8) suggested a greater increase in longitudinalstiffness compared to circumferential (Fig. 6), whichwe attributed to longitudinal re-orientation of fibers(Figs. 4 and 5b). This fiber angle exhibits a clockwisetransmural variation through the myocardial thicknesssimilar to previous measurements in the normotensivecanine RVFW,13 with spiral variations also observedin canine left ventricle free wall49 and in mice septum.37

While this variation has been measured previously andused in biomechanical models of myocardium,17 webelieve this to be the first report of a stress-induced lossof spiral variation.

Third, the increase in the bLC parameter (Fig. 8)suggested that the coupling between the longitudinaland circumferential axes increased, indicating a morehighly anisotropic material behavior in the hyperten-sive state. We have shown in previous studies of valvetissues3 that as fiber alignment becomes more clusteredabout a given direction, coupling between this pre-ferred direction and orthogonal directions increases,suggesting a more highly anisotropic behavior. Forexample, given a fiber distribution about a singledirection, if the fiber splay is randomly distributed(e.g., an OI of 0%), the response along each axis will besimilar. Separation in the axial response (e.g., thelongitudinal and circumferential) occurs as the fibersbecome more highly aligned (e.g., as the OI increasesfrom 0 to 100%). Here, the average OI increased forthe both myo- and collagen fibers (Figs. 4 and 5b), sowe conjectured that increased myofiber alignment wasthe predominant microstructural change underlyingincreased coupling.

Further Insight into Time Rate Changes

The results we presented here demonstrateddramatic changes between normotensive rats and

FIGURE 8. Mean values for material parameters given in Eq.(8) for normotensive (n 5 10) and hypertensive (n 5 12)specimens. Statistically significant differences indicated onplot (bars 5 SEM).

RV Adaptation to Pressure Overload 2461

hypertensive rats subjected to 3 weeks of chronicpressure overload from banding. Future endeavorsshould be taken to elucidate the time-evolving G&Rresponse of the RVFW. In pilot studies, we bandedn = 8 rats for 1-week, isolated the RVFW, and per-formed biomechanical analysis on the tissue slabs asdescribed above. The low stress tangent modulus wassignificantly greater in 1-week hyper- (TMm =

104 kPa) vs. normotensive specimens (Fig. 7;p = 0.003), but less than the 3-week hypertensive,though not significantly. No significant differenceswere observed between the post transition tangentmoduli of the 1-week hypertensive (PTTM = 524 kPa)and the normo- and 3-week hypertensive specimens(Fig. 7). Therefore, we provided evidence that intrinsiccollagen properties are not altered in response topressure overload, but myofiber stiffness is increasedover time, likely to increase contractility.

Implications

We demonstrated an increase in myofiber stiffnessin response to pressure overload, with little change incollagen biomechanical properties. The increase inmyofiber stiffness was beyond that expected by simpleaddition of mass (increase in mass fraction). Therefore,not only was myofiber mass increased, the intrinsicstiffness of the myofibers also increased. Increasedcardiomyocyte cross-sectional area and increasedintrinsic stiffness has been reported in pulmonaryartery hypertension (PAH) patients by Rain et al.,44 soour findings may suggest a pressure-driven remodelingprocess that occurs clinically. Tau significantlyincreased with PA banding from 3.8 ± 0.4 to7.0 ± 0.3 ms, p< 1024. Therefore, diastolic dysfunc-tion is occurring in the PA banding model. Also, we’dlike to distinguish between the organ-level stiffness,which we represent by the time-constant tau, and theintrinsic stiffness of the myofibers, given in our man-uscript by gm. The latter is a measure of the changes inintrinsic stiffness of the myofibers, irrespective of themass accretion. In this study, we found that bothmyofiber mass and intrinsic stiffness increased. Simi-larly, and highly exciting to us, our findings of myocytehypertrophy are consistent with the clinical findings ofRain et al.

Myocardial insults often result in decreased myofi-ber numbers, with remaining myofibers becomingelongated or hypertrophied to maintain stroke volumeto compensate for their absence. Stretch-inducedmyofiber remodeling may lead to new contractileprotein synthesis and new sarcomere assembly.14 Inpressure-overload hypertrophy associated with hyper-tension, ventricular thickening is associated with par-allel deposition to sarcomeres, to increase myofiber

cross sectional area relative to cell length (see Sugdenand Clerk51 and references therein), and clinically, ithas been shown that titin phosphorylation was reducedin PH patients with sarcomeric stiffening.44 However,the mechanotransduction mechanisms by which wallstress is sensed by cardiomyocytes to activate G&Rpathways remains unknown.29 While we have dem-onstrated quantitatively here the manifestation of thesechanges to the tissue level mechanical properties of thecardiomyocytes, future studies into these sub-cellularmyofiber changes, molecular pathways, and their ef-fects on the cellular mechanical and contractile prop-erties are warranted.

Clinically, longitudinal shortening of the myocardiumhas been shown to be a greater contributor to RV strokevolume than circumferential shortening during normalcontraction.8 Regional myocardial shortening duringcontraction has been correlated with local transmuralmyocardial fiber orientations.13 Here, we hypothesizethat the tissue responds to pressure overload by re-ori-enting myo- and collagen fibers to increase longitudinalshortening, a possible mechanism of contractile reserve,beyond hypertrophy, chamber dilation, and increases infiber stiffness. Moreover, low tricuspid annular planesystolic excursion (TAPSE)values are strongly correlatedwith RV dysfunction.21 We further hypothesize that to-wards the final stages of RV failure, a loss of contractilityin the longitudinally re-oriented myofibers reduces lon-gitudinal shortening and underscores the decrease inexcursion measured with TAPSE.

Limitations

There were limitations to the methodology andapproach used in this study. First, regarding method-ology, when quantifying tissue constituent density, weassumed that our decellularization technique com-pletely isolated the ECM from the tissue by removingmyofibers, and when performing histomorphology, weassumed that the Gomori stain targeted only myofibersand collagen fibers. Although no technique is perfect,inspection of the Gomori-stained decelluarized tissuerevealed all gray with no remaining pink color, i.e., themyofibers were removed with this process, with onlyECM remaining. Therefore, we are confident theselimitations did not affect the accuracy of our results.Moreover, both normo- and hypertensive specimenswere subjected to the same processing steps, so directcomparisons could be made between the two groups.

Second, regarding our approach, a higher degree ofvariation in results was observed in the hyper- vs.normotensive rats. Although specimens were taken atthe same time point, the pathological response differedamong rats. Some rats may have been in a more ad-vanced stage of RV compensation or dysfunction than

HILL et al.2462

others, depending on their individual responses to thepressure overload. This variation may explain thehigher standard error observed in the hyper- vs. nor-motensive specimens. Here, we applied a phenomeno-logical approach to model the biomechanical responseof the myocardium, noting that fully structural models,e.g., as in Fata et al.,19 will be employed in futurestudies to fully represent the growth and remodelingresponse and to identify the specific tissue- and cellularlevel adaptations within the RVFW.

CONCLUSIONS

The RVFW myocardium responded to a sustainedpressure increase via an increase in bulk tissue amount,stiffness, and proportion of myofiber density to ECM.The initial response to pressure overload was hyper-trophic. However, hypertrophy alone was insufficientto maintain cardiac output, so the RV chamber dilatedto increase output. Overall tissue stiffness increased,which we attributed to increased effective structuralfiber unit stiffness due to myofiber hypertrophy. Lon-gitudinal stiffness increased proportionately more thancircumferential, and we attributed this increased stiff-ening to longitudinal re-orientation of the myofibersand collagen fibers. The intrinsic mechanical stiffnessof the myofibers increased, independent of mass frac-tion increases, with little changes to collagen recruit-ment or intrinsic stiffness. The biomechanical andmicrostructural changes measured in this study will beused in future computational models of the pressure-induced growth and remodeling of the RVFW myo-cardium, which may eventually be used to predict theresponse to altered hemodynamic states.

ACKNOWLEDGMENTS

This work was supported by the U.S. NationalInstitutes of Health [1F32 HL117535 to M.R.H., P01HL103455 and U01 HL108642-01 to H.C.C.]; theAmerican Heart Association [13POST14720047 toM.R.H., 11POST6950004 to D.V-J., 10BGIA3790022to M.A.S.]; and The Pittsburgh Foundation [M2010-0052 to M.A.S. and M.S.S.]. We’d like to thankSunaina Rustagi, Andrea Sebastiani, and SamanthaCarter at the University of Pittsburgh (Pitt) for per-forming the biomechanical testing; Jeffrey J. Baust atPitt for performing pulmonary artery banding proce-dures; Sruti Shiva at Pitt for performing the tissueviability study; Simone Siegel, Michelle Atkins, andJohn Lesicko at the University of Texas at Austin(UT-Austin) for performing the histomorphologicalanalysis.

CONFLICT OF INTEREST

No benefits in any form have been or will bereceived from a commercial party related directly orindirectly to the subject of this manuscript.

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