Standard and Strain Measurements by

Echocardiography Detect Early Overloaded Right

Ventricular Dysfunction: Validation against
Hemodynamic and Myocyte Contractility Changes
in a Large Animal Model
Amir Hodzic, MD, Pierre Bobin, PhD, Delphine Mika, PhD, Mohamed Ly, MD, Florence Lefebvre, BSc,
Patrick Lech^ene, BSc, Emmanuel Le Bret, MD, PhD, Elodie Gouadon, PhD, Mathieu Coblence, MD,
Gregoire Vandecasteele, PhD, Andre Capderou, MD, PhD, Jer^
ome Leroy, PhD, Catherine Rucker-Martin, PhD,
and Virginie Lambert, MD, PhD, Le Plessis-Robinson and Ch^
atenay-Malabry, France

Background: Early detection of right ventricular (RV) failure is required to improve the management of patients
with congenital heart diseases. The aim of this study was to validate echocardiography for the early detection
of overloaded RV dysfunction, compared with hemodynamic and myocyte contractility assessment.

Methods: Using a porcine model reproducing repaired tetralogy of Fallot, RV function was evaluated over
4 months using standard echocardiography and speckle-tracking compared with hemodynamic parameters
(conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myo-
cytes. Contractile reserve (DEmax) was assessed by b-adrenergic stimulation in vivo (dobutamine 5 mg/kg)
and ex vivo (isoproterenol 100 nM).

Results: Six operated animals were compared with four age- and sex-matched controls. In the operated
group, hemodynamic RV efficient ejection fraction was significantly decreased (29.7% [26.2%34%] vs
42.9% [40.7%48.6%], P < .01), and inotropic responses to dobutamine were attenuated (DEmax was 51%
vs 193%, P < .05). Echocardiographic measurements of fraction of area change, tricuspid annular plane sys-
tolic excursion, tricuspid annular peak systolic velocity (S0 ) and RV free wall longitudinal systolic strain and
strain rate were significantly decreased. Strain rate, S0 , and tricuspid annular plane systolic excursion were
correlated with DEmax (r = 0.75, r = 0.78, and r = 0.65, respectively, P < .05). These alterations were associated
in RV isolated myocytes with the decrease of sarcomere shortening in response to isoproterenol and pertur-
bations of calcium homeostasis assessed by the increase of spontaneous calcium waves.

Conclusions: In this porcine model, both standard and strain echocardiographic parameters detected early
impairments of RV function and cardiac reserve, which were associated with cardiomyocyte excitation-
contraction coupling alterations. (J Am Soc Echocardiogr 2017;-:---.)

Keywords: Tetralogy of Fallot, Right ventricular function, Echocardiography, Speckle-tracking, Excitation-

contraction coupling, Animal model

Right ventricular (RV) performance is a major factor in the long-term dysfunction, repaired tetralogy of Fallot (TOF) is one of the most
survival of patients with congenital heart diseases. A reliable noninva- frequent. Although cardiac magnetic resonance imaging (CMR) pro-
sive technique for RV evaluation is essential in managing these pa- vides a good estimation of RV volumes and geometry, CMR could
tients. Among congenital heart diseases that result in long-term RV not accurately assess clinical status impairment in adult patients

From the UMR-S 999, Inserm, Ho ^ pital Marie Lannelongue, Universite

 Paris-Sud, Universitaire Thorax Innovation, and Laboratoire dExcellence en Recherche sur
Universite Paris-Saclay, Le Plessis-Robinson (A.H., E.G., A.C., C.R.-M, V.L.); dicament et lInnovation The
le Me rapeutique.
UMR-S 1180, Inserm, Universite  Paris-Sud, Universite  Paris-Saclay, Drs. Hodzic and Bobin contributed equally to this work.
Cha^tenay-Malabry (P.B., D.M., F.L., P.L., G.V., J.L.); and Po^ le des Cardiopathies
Conflicts of Interest: None.
Conge nitales, Ho^ pital Marie Lannelongue (M.L., E.L.B.), and Laboratoire de
gional et Universitaire de
Reprint requests: Amir Hodzic, MD, Centre Hospitalier Re
Recherche Chirurgicale, Ho ^ pital Marie Lannelongue, Universite  Paris-Sud,
^ te de Nacre, 14033 Caen Cedex 9,
Caen, Service de Cardiologie, Avenue de la Co
Universite Paris-Saclay (M.C.), Le Plessis Robinson, France.
France (E-mail: hodzic-a@chu-caen.fr).
This work was supported by Fe de
ration Francaise de Cardiologie (to A.H.), the
gion Ile-de-France (to P.B. and D.M.), Fondation pour 0894-7317/$36.00
CORDDIM program of Re
la Recherche Me dicale (FDT20140931029 to P.B.), De partement Hospitalo- Copyright 2017 by the American Society of Echocardiography.
http://dx.doi.org/10.1016/j.echo.2017.07.003
1
2 Hodzic et al Journal of the American Society of Echocardiography
- 2017

Abbreviations
with TOF repair.1 These observa- The objective of this study was to validate echocardiographic
tions highlighted the need for standard and strain parameters against hemodynamic and cellular
b-AR = b-adrenergic receptor more sensitive parameters to contractility changes for detection of early overloaded RV
CMR = Cardiac magnetic detect an early decrease in RV dysfunction.
resonance imaging performance to initiate treat-
ments that can affect the progres-
eEF = Efficient ejection sion of RV dysfunction and
fraction METHODS
prevent the development of de-
FAC = Fractional area change compensated RV failure.
Experimental Design
In clinical practice, echocardi-
FW = Free wall A surgical procedure mimicking repaired TOF was performed on
ography is the modality of choice
GLS = Global longitudinal for RV assessment and screening. six Landrace piglets (operated group) that were between 50 and
strain Developments in echocardio- 67 days of age. Six age-matched animals were used as controls (con-
graphic techniques with speckle- trol group). Control animals did not undergo sham surgery. All ani-
GLSR = Global longitudinal
tracking imaging that assesses mals were male to avoid bias related to hormonal variations.
strain rate
strain and strain rate may poten- Echocardiographic and hemodynamic assessment of RV function
ICC = Intraclass correlation tially provide a more sensitive were performed at baseline just before the surgical procedure and
coefficient 4 months later. Clinical status was evaluated daily during the study.
detection of RV dysfunction.2
Iso = Isoproterenol Although speckle-tracking has After completing the 4-month follow-up period, animals were
demonstrated its clinical utility euthanatized for a cellular study on isolated RV myocytes. All exper-
LV = Left ventricular iments were carried out according to the European Community guid-
for the assessment of left ventric-
RV = Right ventricular ular (LV) dysfunction at an early ing principles on the care and use of animals (2010/63/UE,
2+ stage,3,4 the accuracy for RV September 22, 2010), the local ethics committee (CEEA26
SR-Ca = Sarcoplasmic CAPSud) guidelines, and French decree 2013-118 on the protection
reticulum calcium content evaluation in pathologic conditions
remains questionable. Considering of animals used for scientific purposes (JORF 0032, February 7,
TAPSE = Tricuspid annular the characteristics of the right 2013, p. 2199, text no. 24). Authorization to perform animal experi-
plane systolic excursion ventricle in terms of geometry ments according to this decree was obtained from Ministere Francais
TOF = Tetralogy of Fallot and mode of contraction, de lAgriculture, de lAgroalimentaire et de la For^et (agreement
speckle-tracking measurements B92-019-01).
cannot be achieved with the same level of confidence for the right
ventricle as for the left ventricle. The complexity of RV morphology, Experimental Model of Combined RV Overload
including geometry and numerous trabeculations, makes it chal- As previously described by our group,12 after premedication with
lenging to accurately define endocardial borders for the application ketamine hydrochloride (15 mg/kg intramuscularly), general anes-
of the speckle-tracking algorithm. In recent clinical studies, patients thesia was induced with propofol 1% and cisatracurium (0.3 mg/
with repaired TOF had decreased RV myocardial deformation, which kg every 2 hours), allowing endotracheal intubation, which was
seemed to be independently associated with CMR-derived RV ejec- maintained with isoflurane in 100% oxygen (Servo 900; Siemens-
tion fraction.5-7 These authors have defined thresholds of Elema, Solna, Sweden). Through a left thoracotomy approach, a
longitudinal strain for predicting low RV ejection fraction in side-biting vascular clamp was longitudinally placed across the pul-
pediatric and adult populations with TOF repair.6,7 Furthermore, monary valve annulus without obstruction of the RV outflow tract.
speckle-tracking may be helpful to identify the optimal timing for pul- A pulmonary valve leaflet was excised and the pulmonary infundib-
monary valve replacement in patients with TOF repair, which should ulum, annulus, and trunk were enlarged by a 2-cm-long, elliptically
be performed early to increase the likelihood of complete recovery.8 shaped polytetrafluoroethylene patch to ensure the loss of valve
To date, speckle-derived parameters assessing RV systolic function integrity. This chronic pulmonary valve regurgitation led to RV vol-
have not been validated using the conductance catheter. The conduc- ume overload. Branch pulmonary arterial obstruction is not uncom-
tance technique is the gold-standard method for the evaluation of RV mon after surgical correction of TOF related to native hypoplasia or
myocardial contractility and function. This was previously demon- surgical complications and is known to increase pulmonary valve
strated in experimental models of RV chronic volume and pressure regurgitation.15 In our model, RV pressure overload was achieved
overload9,10 and for patients with TOF repair.11 by pulmonary artery banding, made of umbilical tape, placed
To establish correlations between speckle-tracking echocardiog- around the artery truncus and secured for a final diameter of approx-
raphy and hemodynamic parameters, an animal model of RV imately 1 cm to ensure progressive pulmonary stenosis with animal
dysfunction is required. Our group validated a porcine model of maturation.
repaired TOF that presented with early RV dysfunction by chronic
combined overload.12 We used this model to evaluate the accuracy
of echocardiographic standard and strain parameters in the detec- Echocardiographic Analysis
tion of early alterations of RV myocardial performance.13,14 Echocardiography was performed on closed-chest animals under
Because echocardiographic evaluation of RV function is general anesthesia in the dorsal decubitus position. We used a
influenced by load conditions, we investigated functional commercially available Vivid E9 ultrasound machine (GE Medical
(myocyte contractility and calcium homeostasis) changes at the Systems, Milwaukee, WI) equipped with a 2.5-MHz transducer.
cardiomyocyte level to support the validity of these The values of all echocardiographic parameters were obtained as
echocardiographic parameters to detect myocardial alteration at the average value of three consecutive cardiac cycles during transient
a subclinical stage. apnea and were analyzed on a comprehensive workstation
Journal of the American Society of Echocardiography
Volume - Number -
(EchoPAC 110.1.2; GE Vingmed Ultrasound, Horten, Norway).
Measures indexed to body surface area were calculated using the
formula16 body surface area (m2) = (weight [kg]  body
length [cm])0.5/60.
In the parasternal long-axis view, RV anterior wall thickness was
measured, and the RV/LVend-diastolic diameter ratio was calculated.
The tricuspid/mitral annular diameter ratio was measured at the
maximal valve opening during diastole in the apical four-chamber
view. RV end-diastolic and end-systolic areas were measured in the
apical four-chamber view. This allowed the calculation of RV frac-
tional area change (FAC), defined as FAC = [RV end-diastolic
area  RV end-systolic area]/RV end-diastolic area. Tricuspid annular
plane systolic excursion (TAPSE) was measured as the maximal excur-
sion of the lateral annulus in the apical four-chamber view using
M-mode recording through the apex of the right ventricle and the
junction between the RV free wall (FW) and the tricuspid annulus.
Peak systolic velocity (S0 ) was measured at the lateral tricuspid
annulus using pulsed Doppler tissue imaging. From a parasternal
short-axis view, pulmonary annular diameter and transpulmonary
gradient through the pulmonary band were both measured using
continuous-wave Doppler flow at the maximal pulmonary valve
opening during systole. The presence of diastolic flow reversal of color
Doppler flow in the main or the branch pulmonary arteries was used
to predict severe pulmonary regurgitation.17
Regarding the speckle-tracking analysis, RV longitudinal deforma-
tion was measured using standard two-dimensional grayscale acquisi-
tions in the apical four-chamber view. All images were recorded at a
frame rate of 50 to 80 frames/sec and stored for postprocessing
analysis. RV longitudinal peak systolic strain and strain rate were
assessed using semiautomatic contouring of the endocardium of the
right ventricle with two methods. The first method consisted of con-
touring of the endocardium of the entire right ventricle (free and
septal walls) using six points placed on the image that divided the right
ventricle into six standard segments (at the basal, middle, and apical
levels). The peak global longitudinal strain (GLS) and global longitudi-
nal strain rate (GLSR) were calculated as the average of the six seg-
ments. The second method used a contouring of the endocardium
of the FW, excluding the interventricular septum to minimize the in-
fluence of the interventricular dependence. The peak global RV FW
GLS and GLSR were defined as the average of the three FW segments
(Video 1 available at www.onlinejase.com). Intra- and interobserver
variability was assessed for both FW and RV GLS and GLSR measure-
ments on a random subset of eight pigs. Intraobserver variability was
assessed $1 week after the initial measurements. Two blinded oper-
ators assessed interobserver variability. Echocardiographic assessment
of RV myocardial deformation was not studied under stress condi-
tions. Because of the important chronotropic effect obtained with a
low dose of dobutamine infusion in both groups, RV speckle-
tracking was not feasible.
Hemodynamic Analysis
Under general anesthesia, the conductance catheter (CD Leycom,
Zoetermeer, The Netherlands) was inserted into the right ventricle
through the femoral vein under fluoroscopic guidance; a balloon was
inflated in the caudal vena cava to obtain inflow occlusion.18 The refer-
ence cardiac output was measured using the transpulmonary thermo-
dilution technique (PiCCO; Pulsion Medical Systems, Munich,
Germany). This technique allowed us to obtain systemic cardiac output
reflecting the efficient pulmonary output independently of the pulmo-
nary regurgitation effect.12 Data were acquired using dobutamine infu-
Hodzic et al 3
sion (5 mg/kg/min) after 10 min of hemodynamic stability. The
acquisitions were made during 10 sec of apnea. The pulmonary regur-
gitation volume was measured from the pressure-volume loops as the
increase in RV volume during isovolumic relaxation. Although this
measured volume does not represent the total volume of diastolic pul-
monary regurgitation, which continues during RV filling, it could pro-
vide an accurate estimation of pulmonary regurgitation in patients
with TOF.11 A pulmonary regurgitation volume of >5 mL/beat/m2
was considered significant.11 The pulmonary regurgitation fraction
was calculated as the ratio between the pulmonary regurgitation vol-
ume and the total RV stroke volume. The efficient stroke volume
was calculated as the difference between the total stroke volume and
the measured volume of the pulmonary regurgitation. The efficient
ejection fraction (eEF) was calculated as the ratio between efficient
stroke volume and end-diastolic volume. The stroke work corre-
sponded to the pressure-volume loop area. RV contractility was
examined using the end-systolic pressure volume relationship. Slopes
of end-systolic pressure volume relationship (Emax) were computed
during vena cava occlusion. Measurements were acquired at rest and
during dobutamine infusion (5 mg/kg/min). The variation of Emax slope
(DEmax) represented the myocardial contractility reserve. In other
words, the percentage increase in Emax in response to b-adrenergic
receptor (b-AR) stimulation reflected the ability of the right ventricle
to improve its contractility under stress conditions. An illustration of
the changes in shape and characteristics of the RV pressure-volume
loop with volume and pressure overload is provided in Supplemental
Figure 1 (available at www.onlinejase.com).
Cellular Study
A detailed methodologic description of the cellular analysis is pro-
vided in the supplementary material (available at www.onlinejase.
com). Briefly, animals from both groups were sacrificed at the end
of the study by exsanguination. The heart was excised, and the RV
myocytes were isolated by enzymatic digestion. Excitation-
contraction coupling was characterized on isolated myocytes using
the IonOptix system (IonOptix, Milton, MA). This system offers
simultaneous measurements of sarcomere length variations using a
high-speed contractility camera and cytosolic free Ca2+ concentration
changes using a ratiometric Ca2+ indicator (Fura-2). Sarcomere short-
ening and Ca2+ transient amplitudes and relaxation kinetics were re-
corded before and after b-AR stimulation by isoproterenol (Iso).
Sarcoplasmic reticulum Ca2+ content (SR-Ca2+) was measured by
application of caffeine on isolated myocytes, inducing cytoplasmic
Ca2+ release by activation of reticulum sarcoplasmic ryanodine
receptors.19
Statistical Analysis
GraphPad Prism 6 (GraphPad Software, La Jolla, CA) and R version
3.1.1 (R Foundation for Statistical Computing, Vienna, Austria) were
used for statistical analysis. Clinical, echocardiographic, and hemody-
namic data are expressed as medians and interquartile ranges.
Nonparametric two-way analysis of variance with paired values using
bootstrapping with unrestricted permutation of observations was
used to test the significance of changes with time and between groups
(D.C. Howel: Manlys approach). Cellular data were normally distrib-
uted (Shapiro-Wilk test) and are expressed as mean 6 SEM. Students
paired t test was used to test the significance of changes between base-
line and Iso conditions in each group, and Students unpaired t test
was used to test the significance of changes between groups.
4 Hodzic et al Journal of the American Society of Echocardiography
- 2017

Table 1 Clinical and hemodynamic characteristics in control and operated groups at each stage

At baseline At 4 months of follow-up

Controls (n = 4) Operated (n = 6) Controls (n = 4) Operated (n = 6)

Clinical characteristics
Age (d) 51 (50 to 67) 55 (50 to 59) 191 (180 to 197)* 181 (158 to 190)*
Weight (kg) 21.5 (19.5 to 23.6) 22.8 (19.7 to 25) 38.9 (36.9 to 39.9)* 33 (29 to 52)*
Body length (cm) 68.5 (67 to 73.8) 70 (63.9 to 73.3) 90 (88.5 to 91.5)* 85 (83.5 to 101.3)*
BSA (m2) 0.64 (0.6 to 0.69) 0.68 (0.59 to 0.71) 0.98 (0.96 to 1)* 0.89 (0.82 to 1.2)*
Heart rate (beats/min) 92 (85 to 110) 95 (86 to 96) 100 (84 to 122) 102 (85 to 126)
Systemic pressures (mm Hg)
Systolic BP 101.5 (83 to 107.3) 96.5 (88.3 to 110.3) 109 (104.3 to 119.3) 96.5 (92.5 to 121.5)
Mean BP 85.5 (62.3 to 92.3) 77.5 (72 to 92.5) 97 (90.8 to 100.3) 83.5 (79.8 to 101)
SVC pressure 1 (0.3 to 2.5) 2 (1 to 4.3) 3.5 (2.3 to 4)* 4.5 (2.8 to 7)*
RV pressures (mm Hg)
End-diastolic 1.5 (1.5 to 3.8) 3.5 (1.8 to 4.3) 1 (0.8 to 4.3) 6 (4 to 7)
End-systolic 11 (7.3 to 13.3) 13 (11.8 to 13.3) 10 (7.3 to 15) 21.5 (13 to 34)
Systolic peak 17.5 (17 to 23) 21 (20.8 to 25.3) 18.5 (15 to 24.3) 29.5 (22.3 to 40.8),
Systolic peak/systolic BP 0.19 (0.17 to 0.22) 0.23 (0.19 to 0.27) 0.17 (0.15 to 0.2) 0.27 (0.24 to 0.39),
RV volumes
End-diastolic/BSA (mL/m2) 131.4 (107 to 134) 119.7 (109 to 128) 107.6 (99 to 119) 148.9 (135 to 155),
2
End-systolic/BSA (mL/m ) 75.1 (51 to 80) 63.7 (52 to 74) 56.4 (53 to 58) 95.8 (80 to 104)*,,
Effective SV (mL) 34.5 (30.3 to 37.3) 34 (31.3 to 41) 47.5 (43.3 to 48.8) 41 (32 to 53)*
RV stroke work (mL $ mm Hg) 562 (498 to 663) 596 (483 to 731) 732 (496 to 803) 847 (663 to 1,813)
BP, Blood pressure; BSA, body surface area; SV, stroke volume; SVC, superior vena cava.
Data are expressed as median (interquartile range).
*P < .05 versus corresponding group at baseline.

P < .05, operated group versus control group at same step.

P < .05, operated group versus control group, analysis of variance.

The relationship between echocardiographic and hemodynamic vari-
ables was assessed using Spearman correlation. A c2 test was used to
compare the percentage of cells exhibiting spontaneous calcium
waves in RV myocytes. All tests were two tailed, and their level of
significance was defined as P < .05.
RESULTS
Clinical Status
The duration of the study was similar between operated and control
groups (121 6 11 and 133 6 11 days, respectively). Regarding age,
weight, body length, heart rate, systemic blood pressure, and central
venous pressure (Table 1), groups were similar at each stage. No
animals showed clinical signs of heart failure during the study
follow-up.
Echocardiographic and Hemodynamic Data Confirmed RV
Overload
Four months after surgery, operated animals showed significant thick-
ening of the RV FW related to moderate but significant pulmonary ste-
nosis (Table 2). All operated animals had diastolic flow reversal in the
branch pulmonary arteries and pronounced RV dilatation, indicating
severe pulmonary regurgitation (as shown in Videos 2 and 3, available
at www.onlinejase.com). The significant increase in the tricuspid/
mitral annular diameter ratio, the RV/LV end-diastolic diameter ratio,
and both RV end-diastolic and end-systolic areas reflected the RV
dilatation (Table 2). The control group displayed normal echocardio-
graphic dimensions during the study (Table 2).
Regarding RV pressure and volume (Table 1), the two groups were
hemodynamically similar at baseline. After 4 months, the hemody-
namic assessment confirmed severe pulmonary regurgitation in the
operated animals. The median pulmonary regurgitation volume was
12.2 mL/beat/m2 (interquartile range, 10.616.4 mL/beat/m2), and
the median pulmonary regurgitation fraction was 25% (interquartile
range, 22%28%). Consequently, end-systolic and end-diastolic vol-
umes were significantly higher at 4 months in the operated animals
than in the control group (Table 1). In the operated group, RV end-
systolic pressure significantly increased, and peak pressure rose to a level
of one third of systemic pressure at 4 months (Table 1). The pulmonary
stenosis was moderate under basal conditions, but it was accentuated
after dobutamine infusion, evidence of the increasing RV end-systolic
pressure that remained at an infrasystemic level (Table 3).
Predominant Volumetric Overload Resulted in Early
Alteration of RV Systolic Function
In operated animals, assessment of RV systolic function at 4 months
showed a constant cardiac index at rest and under stress (Figure 1).
Despite RV dilatation, efficient stroke volume failed to increase
(Table 1). The eEF significantly decreased at rest and remained infe-
rior to controls after dobutamine infusion (Figure 1). Stroke work
increased 4 months after surgery, without reaching a significant value
Journal of the American Society of Echocardiography Hodzic et al 5
Volume - Number -

Table 2 Standard echocardiographic parameters of RV morphology and systolic function

At baseline At 4 months follow-up

Controls (n = 4) Operated (n = 6) Controls (n = 4) Operated (n = 6)

RV dimensions
RV end-diastolic area/BSA (cm/m2) 8.8 (8.110) 8.9 (8.310.6) 11 (10.911.8)* 19.9 (18.822.2)*,,
2
RV end-systolic area/BSA (cm/m ) 5 (4.25.8) 4.9 (4.55.8) 6.2 (66.8)* 12.9 (12.114.3)*,,
RV/LV end-diastolic diameter ratio 0.41 (0.380.46) 0.43 (0.410.45) 0.42 (0.390.45) 0.79 (0.650.81)*,,
T/M diameter ratio 0.83 (0.820.85) 0.83 (0.790.86) 0.77 (0.730.84) 1 (0.971.1)*,,
PA annular diameter (cm) 1.6 (1.51.7) 1.7 (1.51.8) 2 (1.92.2) 2.6 (2.32.8)*,,
RV wall thickness (mm) 3 (2.93.3) 2.9 (2.33) 3.3 (33.4) 5.1 (55.4)*,,
Transpulmonary gradient (mm Hg)
Maximal 2.8 (2.32.9) 3.7 (2.44.3) 3.7 (24.3) 28.3 (25.234)*,,
Mean 1.5 (1.21.6) 1.7 (1.32) 1.8 (1.22.3) 16.9 (12.422.5)*,,
RV systolic function indices
FAC (%) 43.6 (41.447) 44.9 (44.745.6) 43.9 (42.745.1) 35.4 (34.636.4)*,,
TAPSE (mm) 17 (16.318.5) 19 (16.519.3) 21 (2021.8)* 17.5 (15.519)*,
0
Peak S (cm/sec) 7.5 (78) 8.5 (7.89.3) 9.5 (910.8) 7 (6.57),
BSA, Body surface area; PA, pulmonary artery; TAPSE, tricuspid annular plane systolic excursion; T/M, tricuspid/mitral.
Data are expressed as median (interquartile range).
*P < .05 versus corresponding group at baseline.

P < .05, operated group versus control group at same step.

P < .05, operated group versus control group, analysis of variance.

Table 3 Dobutamine stress-induced hemodynamic changes in control and operated groups at each stage

At baseline At 4 months follow-up

Controls (n = 4) Operated (n = 6) Controls (n = 4) Operated (n = 6)

(Variation %) (Variation %) (Variation %) (Variation %)

Heart rate (beats/min) 151 (140171) 153 (143163) 138 (129152) 147 (136152)
(+64.1) (+61.1) (+38) (+44.1)
Systemic pressures (mm Hg)
Systolic BP 125 (115.5136) 139.5 (120155.8) 160 (122182.3) 156.5 (90.5183)
(+23.2) (+44.6) (+46.8) (+62.2)
Mean BP 105 (89.8117.3) 115 (100130.8) 132 (100.8149.8) 133 (74144.3)
(+22.8) (+48.4) (+36.1) (+59.3)
RV pressures (mm Hg)
End-systolic 14.5 (12.321.3) 12 (1118) 12.5 (6.518.5) 49 (29.581.8)*,,
(+27.3) (-7.7) (+25) (+128)
Peak/systolic BP 0.21 (0.180.27) 0.21 (0.190.22) 0.21 (0.150.2) 0.43 (0.291),
(+10.5) (-8.7) (+23.5) (+59.3)
RV stroke work 1,011 (9711,149) 954 (7211,288) 1,411 (1,3401,610) 3,598 (1,7855,664)*,
(mL $ mm Hg) (+80) (+60.1) (+93) (+325)

BP, Blood pressure.

Data are expressed as median (interquartile range).
*P < .05 versus corresponding group at baseline.

P < .05, operated group versus control group at same step.

P < .05, operated group versus control group, analysis of variance.

at rest, but progressed after dobutamine infusion and was significantly
higher than in the control group (Table 3). In accordance with our he-
modynamic findings of operated animals, echocardiographic mea-
surements of RV FAC, TAPSE, and peak S0 significantly decreased
at 4 months and were positively correlated with hemodynamic eEF
(Tables 2 and 4).
Echocardiography Assessed Myocardial Deformation
Abnormalities Related to an Alteration of Myocardial
Reserve
In the operated group, the significant leftward shift of the end-systolic
pressure-volume relationship slope (Emax) at 4 months, compared
with baseline and the control group, suggested a positive inotropic
6 Hodzic et al Journal of the American Society of Echocardiography
- 2017

Figure 1 Hemodynamic parameters issued from pressure-volume loop measurements at rest (open circle) and under dobutamine
infusion (solid circle) in control (blue) and operated (red) animals: cardiac index (A), RV eEF (B), Emax (C), and contractility reserve
(DEmax) (D). Medians and interquartile ranges are presented. *P < .05 and **P < .01, operated group versus control group, analysis
of variance.

Table 4 Echocardiographic indices of RV systolic function: (r = 0.85 and r = 0.95, respectively). Because of poor tracking of RV
load dependency and relationship with hemodynamic myocardium in two animals (one in each group), paired strain values
functional and contractile parameters were available for only eight animals (technical feasibility of 80%).
The peak FW GLS and peak FW GLSR were both significantly reduced
Conductance catheter technique Echocardiography
Coefficient of in the operated group at 4 months (Table 5 and Figure 2). The mean
correlation r RV EDV RV ESP eEF DEmax FW GLS FW GLSR reduction in FW GLSR (25.9%) was more pronounced than for
FW GLS (11.7%). Peak RV GLS was not significantly different be-
Echocardiography
tween groups (Table 5). Although septal strain rate remained compara-
FAC 0.6 0.82* 0.7* 0.54 0.81* 0.78* ble between groups, operated animals exhibited a significant reduction
TAPSE 0.42 0.68* 0.67* 0.65* 0.9* 0.76* in peak RV GLSR (Table 5). Both peak FW GLS and FW GLSR signifi-
Peak S0 0.44 0.72* 0.68* 0.78* 0.88* 0.92* cantly correlated with FAC, TAPSE, and S0 (Table 4). Hemodynamically,
FW GLS 0.37 0.83* 0.5 0.46 only peak FW GLSR was significantly correlated with eEF (Table 4). In
response to dobutamine, DEmax showed the best correlation with FW
FW GLSR 0.55 0.63* 0.67* 0.75*
GLSR and S0 (Table 4). Intra- and interobserver reproducibility was
EDV, End-diastolic volume; ESP, end-systolic pressure. acceptable for both FW and RV GLS and GLSR. For peak FW GLS,
*P < .05. the coefficients of variation were 3.6 6 1.02% and 6 6 1.7%, respec-
tively, the intraclass correlation coefficients (ICCs) were 0.95 (95%
adaptation of RV myocardium to the overload. Despite this, the CI, 0.770.99) and 0.93 (95% CI, 0.670.99), respectively. The intra-
response to dobutamine was blunted, as shown by the significant and interobserver variability was greater for peak FW GLSR, with coef-
drop of variation of Emax slope (DEmax) compared with baseline and ficients of variation of 10.4 6 1.7% and 8.5 6 1.4%, respectively, and
controls, indicating a weak myocardium contractile reserve (Figure 1). ICCs of 0.94 (95% CI, 0.720.99) and 0.95 (95% CI, 0.760.99),
To study the impact of load influence on echocardiographic param- respectively. Similarly, for peak RV GLS, the coefficient of variation
eters, we considered that RVend-systolic pressure and end-diastolic vol- was 3.4 6 0.8% and the ICC was 0.96 (95% CI, 0.820.99) for intra-
ume reflected afterload and preload, respectively. As expected, observer variability and 3.5 6 0.8% and 0.96 (95% CI, 0.810.99),
standard and strain parameters were significantly correlated with load respectively, for interobserver variability. Finally, intraobserver vari-
conditions (Table 4). Echocardiographic measures of RV end-diastolic ability of peak RV GLSR was 6.5 6 0.7%, with an ICC of 0.95 (95%
and end-systolic areas strongly correlated with both RV end-diastolic CI, 0.790.99), and interobserver variability was 8.8 6 1%, with an
and end-systolic volumes measured by conductance catheter ICC of 0.92 (95% CI, 0.660.98).
Journal of the American Society of Echocardiography
Volume - Number -
Table 5 RV systolic longitudinal deformation parameters
At baseline
Controls (n = 3) Operated (n = 5)
Strain (%)
RV GLS 25.2 (21.5 to 30.1) 26.5 (23.6 to 29.4)
IVS 18.6 (17.4 to 25.2) 20.4 (18.9 to 23.2)
FW GLS 29.8 (29.1 to 35.1) 29.9 (28.1 to 33.9)
Strain rate (sec1)
RV GLSR 1.55 (1.34 to 1.91) 1.56 (1.28 to 1.65)
IVS 1.09 (0.96 to 1.36) 1.14 (0.9 to 1.46)
FW GLSR 1.93 (1.8 to 2.55) 2.01 (1.77 to 2.24)
IVS, Interventricular septum.
Data are expressed as median (interquartile range).
*P < .05 versus corresponding group at baseline.
P < .05, operated group versus control group, analysis of variance.
Figure 2 RV FW GLS (A) and GLSR (B) measurements in control (blue circle) and operated (red triangle) animals. FW GLS and GLSR
were significantly decreased 4 months after surgery in the operated animals (longitudinal strain and strain rate are conventionally rep-
resented as negative values). *P < .05, operated group versus control group, analysis of variance.
Our results confirmed that in addition to standard echocardio-
graphic parameters, strain imaging provides an accurate assessment
of RV systolic function and contractile capacities and allows early
detection of alterations before a decompensated stage.
Excitation-Contraction Coupling Alterations in the
Repaired TOF Model
RV myocytes isolated from the heart of operated animals were signifi-
cantly hypertrophied compared with control animals. The mean cell
length and width were both significantly increased by 14.9% and
17.6%, respectively (Supplemental Figure 2, available at www.
onlinejase.com). The cellular changes observed in the operated animals
were consistent with the RVremodeling. Under basal conditions, diastolic
sarcomere length, sarcomere shortening, and Ca2+ transient amplitude
were identical (Figure 3). The low cell shortening obtained in our exper-
iments under basal conditions can be explained by the fact that the cells
were plated on laminin-coated dishes and loaded with the ratiometric
Fura-2 probe, which chelates the intracellular Ca2+. Sarcomere relaxation
kinetics were similar in both groups. The time to 50% decay of Ca2+ tran-
sient was significantly prolonged in cells obtained from the operated an-
imals, suggesting a lower capacity to recapture released Ca2+ during
cellular relaxation (P < .01 vs control; Figure 3). Application of 100 nM
of the nonselective b-AR agonist Iso strongly increased sarcomere short-
ening in both groups. However, this inotropic effect was significantly
Hodzic et al 7
At 4 months follow-up
Controls (n = 3) Operated (n = 5)
24.5 (22.8 to 24.8) 22.5 (22.2 to 24.8)
19 (17 to 20.6) 18.8 (18.1 to 20.8)
31.8 (28.7 to 36.8) 26.4 (25.6 to 28.2)*,
1.39 (1.26 to 1.65) 1.03 (0.87 to 1.06)*
1.15 (0.87 to 1.19) 1.15 (0.92 to 1.15)
2.2 (1.77 to 2.34) 1.49 (1.33 to 1.55)*,
blunted for the operated group related to alterations in myocyte contrac-
tile reserve (increased by 27-fold for operated vs 38-fold for controls,
P < .05). Upon Iso treatment, a similar sixfold increase in the Ca2+ tran-
sient amplitude was observed in both groups, and time to 50% decay
was significantly shortened for sarcomere shortening and Ca2+ transients,
with no difference between groups. These results suggest only partial al-
terations of SR-Ca2+ uptake in our model. However, Ca2+ homeostasis
perturbations in RV myocytes from operated animals were further
demonstrated by the increased frequency of proarrhythmic spontaneous
calcium waves recorded during the last 20 sec of Iso treatment and the
greater number of cells exhibiting these proarrhythmic events
(Supplemental Figure 3, available at www.onlinejase.com).
DISCUSSION
In a large animal model mimicking congenital heart disease, speckle-
tracking provides sensitive detection of early RV dysfunction. To the
best of our knowledge, this study is the first to demonstrate the accu-
racy of echocardiographic standard and speckle-tracking parameters
to detect RV myocardial contractility impairment compared with he-
modynamic and cellular evaluation.
In congenital heart diseases, detection of RV dysfunction before the
decompensated stage is of paramount importance for practitioners to
8 Hodzic et al Journal of the American Society of Echocardiography
- 2017

Figure 3 Sarcomere shortening, Ca2+ transient amplitude, and associated decay kinetics in RV myocytes (RVMs) isolated from con-
trol and operated animals. (A) Representative traces of sarcomere shortening (left) and Ca2+ transients (right) in Fura-2-loaded RVMs
paced at 1 Hz at baseline (gray) and upon Iso (100 nmol/L) application (black). (B) Mean 6 SEM amplitude of sarcomere shortening
(percentage of resting sarcomere length) (left) and time to 50% decay for sarcomere shortening (T1/2 off) (right) under basal condi-
tions and upon Iso (control: N = 4, n = 12; operated: N = 3, n = 12). (C) Mean 6 SEM amplitude of Ca2+ transients (percentage of
diastolic ratio) (left) and time to 50% decay for Ca2+ transient (T1/2 off) (right) in control conditions and upon Iso (control: N = 4,
n = 17; operated: N = 3, n = 22). ***P < .001 (paired t test), #P < .05 (unpaired t test), and ##P < .01.

initiate the necessary interventional procedures that could potentially
allow complete recovery. That is particularly important in patients
with repaired TOF. These patients present with surgical RVoutflow tract
diseases such as pulmonary valve insufficiency and pulmonary branch
arterial stenosis, leading to RV chronic overload and dysfunction that
may be reversed if the correction is performed early enough.
Currently, the indications for pulmonary valve replacement in asymp-
tomatic patients are controversial. Detecting the first signs of RV
dysfunction could help indicate the optimal timing for a reinterven-
tion.20 This experimental model of RV chronic combined overload
was previously studied using RV hemodynamic, electrophysiologic,
and histologic approaches.12 As observed in the most severe repaired
TOF patients, animals presented an important pulmonary regurgitation
and an increased RV/LV pressure ratio reproduced in our model by
placing banding just before the bifurcation of the pulmonary branch ar-
teries to mimic branch stenosis. That led to an early stage of RV contrac-
tile and functional impairment.12 Color Doppler showed an excellent
ability to detect severe pulmonary regurgitation consistent with hemo-
dynamic measurements. A similar chronic swine model was compared
with symptomatic adults with repaired TOF.21 These investigators
demonstrated that hemodynamic and dyssynchrony parameters
mimicked those observed in patients with TOF. In our study, the oper-
ated animals exhibited greater values of Emax at 4 months compared
with controls. Lambert et al.12 previously observed in the same model
that during the first 3 months after surgery, Emax was increased because
of a transitory RV positive inotropic change. The investigators noted a
significant rightward shift of Emax at 4 months after surgery, suggesting
a turning point from transitory RV homeometric autoregulation to RV
maladaptation. In our study, we investigated the variations of Emax be-
tween rest and stressful conditions (DEmax) to detect early alterations
in contractile reserve.22 We did not analyze the kinetics of Emax between
baseline and the end of the study, to limit the number of invasive pro-
cedures. In stressful conditions, operated animals had a minor capacity
to increase contractility, suggesting altered contractile reserve.
Furthermore, they showed a smaller increase in eEF, despite the incre-
mental stroke work. These animals preserved cardiac index because of
an important positive chronotropic effect of dobutamine. In this model
with predominant volumetric overload, the hemodynamic results
confirmed early alterations of RV function and contractile reserve.
Standard echocardiographic parameters such as FAC, TAPSE, and S0
are recommended for RV systolic function assessment.13,14 These
parameters have been well correlated with RV ejection fraction
assessed by CMR.23 Although CMR provides an interesting evaluation
of RV function, it fails to assess RV contractility, and it is limited by the
complexity of RV morphology. Only one study compared standard echo-
cardiography with hemodynamic parameters using the gold-standard
conductance catheter technique in a porcine model of RV pure pressure
overload.24 We also did not find a direct correlation between the absolute
value of Emax assessing the inotropic change and FAC, TAPSE, and S0 .
However, DEmax was significantly correlated with both TAPSE and S0 .
In addition, FAC, TAPSE, and S0 were closely correlated with hemody-
namic eEF. Our study demonstrated the accuracy of these standard
Journal of the American Society of Echocardiography
Volume - Number -
echocardiographic parameters used in routine clinical practice to detect
the early alteration of overloaded RV performance.
In our model, these standard echocardiographic indices of global RV
systolic function were strongly correlated with speckle-tracking-derived
strain and strain rate measurements, as observed in patients.25 In previ-
ous research, speckle-tracking showed significant alterations of RV lon-
gitudinal deformation in adults with repaired TOF who were mildly
symptomatic, whereas the ejection fraction measured by CMR was pre-
served.26 However, there is no evidence reported of the accuracy of
speckle-tracking to assess RV systolic function and contractility in com-
parison with hemodynamic evaluation by catheter conductance. In our
study, RV strain measures were able to detect early impairment of RV
systolic function. We found a significant correlation between FW
GLSR and both RV eEF and RV contractile reserve DEmax. Similarly
to standard parameters, spckle-tracking indices were not correlated
with the absolute value of Emax. FW GLS significantly decreased but
did not show any correlation with hemodynamic parameters. This
discrepancy between strain and strain rate measurements may be due
to a high baseline heart rate of the pigs (about 100 beats/min regardless
of group), strain rate being superior to strain for the assessment of
myocardial function at higher heart rates.27 RV FWand RV global strain
indices showed similar and acceptable intra- and interobserver repro-
ducibility. Alterations of myocardial deformation being more marked
on the RV FW than the septal wall, which remained unchanged in the
operated animals, FW GLS seemed to be more sensitive compared
with RV GLS to detect early RV dysfunction.
In our study, both speckle-tracking and standard parameters were
negatively correlated with RV end-systolic pressure. Ventricular pressure
is not the only determinant of afterload. In our model, we did not measure
end-systolic stress, and we did not study arterial-ventricular coupling,
which would be inaccurate within the context of pulmonary arterial
banding and pulmonary regurgitation.28 On the basis of pressure-
volume loops, we made the approximation that at constant volume,
end-systolic pressure mainly reflected afterload. Our results confirmed
the load dependency of echocardiographic parameters, which raised
questions about their accuracy in early detection of cardiomyocytes con-
tractile alterations. To answer this question, sarcomere shortening and
Ca2+ transients were evaluated in RV isolated myocytes. This approach
showed a significant reduction of the sarcomere shortening amplitude
in response to b-AR stimulation in operated animals. These results, consis-
tent with the alteration of the myocardial reserve assessed by DEmax, sug-
gest the beginning of the mismatch stage of the overloaded RV
contractility.29 Alterations in echocardiographic standard and strain pa-
rameters, beyond the impact of the overload, may reflect these cardio-
myocyte dysfunction in the operated animals. Although speckle-
tracking appeared to perform nearly as well as conventional indices for
the assessment of RV function, deformation variables might have added
value over standard indices in the management of early RV dysfunction.
Speckle-tracking is independent of ventricular geometry and therefore
may be useful for assessment of RV function with variable morphology,
especially in congenital heart diseases. In addition to the clinical signifi-
cance of RV strain assessment of patients with TOF repair as previously
discussed, RV strain showed strong associations with adverse clinical out-
comes in patients with diverse pathologic states, including systemic right
ventricle,30 pulmonary arterial hypertension,31 and left heart diseases.32
Limitations
The small number of animals enrolled in the study is the first limita-
tion. Abiding by European and French legislations on animal experi-
mentations, our experimental model was approved for a limited
Hodzic et al 9
number of animals. Four animals died, which was consistent with
the mortality rate previously observed in our pilot study.12 Two piglets
died before the baseline assessment and were excluded from the
study; two additional animals died during the follow-up period and
were excluded from the final paired comparison analysis. That being
the case, we used a large animal model sharing cardiovascular similar-
ities with the human heart, and our experimental follow-up was
longer than similar studies that assessed the accuracy of cardiac imag-
ing in large animal models with heart failure.9,10,24 Also, the control
group did not undergo the same surgical procedure as the operated
group, which could raise the question of a possible effect of opened
pericardium on RV functional indices. This is justified because a
thoracotomy is a painful and risky procedure (risk for RV
perforation and postsurgical infections or bleedings), and it did not
result in significant changes in RV performance in a similar model
of RV pressure overload.24 Finally, the important chronotropic effect
obtained with dobutamine infusion did not allow us to accurately
assess the RV speckle-tracking strain variations under stress condi-
tions. However, the primary aim of the study was to investigate the
validity of speckle-tracking to detect early ventricular dysfunction at
rest. To date, no experimental study has validated RV speckle-
tracking during stress test.
CONCLUSIONS
Echocardiographic standard parameters used in routine clinical prac-
tice are hemodynamically validated to assess RV systolic function in
this model of RV dysfunction at an early stage. Among speckle-
tracking indices, FW strain rate appeared to be the most sensitive index
to detect early regional and global impairments of contractility, consis-
tent with hemodynamic study and cellular evaluation. Because the
impairment of overloaded RV contractility exists long before heart fail-
ure signs become evident, subclinical alterations of these echocardio-
graphic parameters should be considered as the first signs of RV
systolic dysfunction and should require specific management of these
patients.
ACKNOWLEDGMENTS
We thank Dr. Boulate for his blind duplicate analysis of echocardiographic
data and the surgical research staffBenoit Decante, Bruno Baudet,
Frederic Seccatore, and Anaelle Le Bretonfor technical assistance and
animal care. We greatly acknowledge the Surgical Research Laboratory
and Microscopy Facility of Centre Chirurgical Marie Lannelongue.
SUPPLEMENTARY DATA
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.echo.2017.07.003.
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SUPPLEMENTAL METHODS
Myocyte Isolation Procedure
After heart explantation, ventricular myocytes were enzymatically
isolated from the RV FW. The right coronary artery ostium was can-
nulated, and the tissue was perfused with a constant flow of approx-
imately 200 mL/min; temperature was maintained at 37 C. After
10 min washing with a Ca2+-free Krebs-Ringer solution, tissue diges-
tion was made by adding 0.354 UI/mL collagenase A (Roche
Diagnostic). After 15 to 20 min of enzymatic perfusion, the right
ventricle was removed, and myocytes from endocardial and myocar-
dial layers were mechanically collected, filtered, washed with a buffer
solution (HEPESbovine serum albumin 2%), and resuspended in
increasing Ca2+ concentrations to reach 1.2 mmol/L. Finally, isolated
myocytes were plated on laminin coated glass-bottom-dishes in
Dulbeccos modified Eagles medium supplemented with 5% fetal
calf serum for 1 hour and maintained at 37 C. Fetal calf serum was
removed, and Ca2+-tolerant cells were used within 12 hours after
isolation. Myocyte length and width were determined using ImageJ
(ImageJ-win32; National Institutes of Health, Bethesda, MD) on
20 photographs taken with an optical microscope (Z1 Axio
Observer; Carl Zeiss; 1015 fields/animal).
Assessment of Sarcomere Shortening and Ca2+ Transient
Using the IonOptix System
All experiments were performed at 30 6 2 C. Isolated RV myocytes
were loaded with 1 mmol/L Fura-2 AM (Invitrogen) for 15 min in a
Ringer solution containing 5.4 mmol/L KCl, 121.6 mmol/L NaCl,
5 mmol/L Na-pyruvate, 4.013 mmol/L NaHCO3, 0.8 mmol/L
NaH2PO4, 1.8 mmol/L CaCl2, 1.8 mmol/L MgCl2, 5 mmol/L glucose,
and 10 mmol/L HEPES (pH = 7.4 with NaOH). Sarcomere shortening
and the Fura-2 ratio (measured at 512 nm upon excitation at 340 and
380 nm) were simultaneously recorded in Ringer solution, using a
double excitation spectrofluorimeter coupled with a video detection
system (IonOptix). Myocytes were electrically stimulated with biphasic
field pulses (5 V, 4 msec) at a frequency of 1 Hz.1
Ca2+ transient amplitude was measured by dividing the twitch
amplitude (the difference between the end-diastolic and the peak sys-
tolic ratios) by the end-diastolic ratio, thus corresponding to the per-
Supplemental Figure 1 Example of RV pressure-volume loops obtained from an animal at baseline before the creation of the model
(A) and 4 months after surgery (B). After 4 months, the operated animal showed an important rise of end-diastolic volume and end-
systolic pressure secondary to RV overload. The triangular normal RV pressure-volume loop (A) changed to a square shape (B) char-
acterized by an increase in RV volume during isovolumetric relaxation secondary to pulmonary regurgitation.
Hodzic et al 10.e1
centage of variation in the Fura-2 ratio. Similarly, sarcomere
shortening was assessed by its percentage of variation, which is ob-
tained by dividing the twitch amplitude (the difference between the
end-diastolic and the peak systolic sarcomere length) by the end-
diastolic sarcomere length. Relaxation was assessed by measuring
the time to 50% relaxation from the time to peak shortening, and
the Ca2+ transient decay was evaluated by measuring the time to
50% decay of the Fura-2 ratio from the time to peak ratio. The frac-
tional Ca2+ release was calculated as the ratio between the Ca2+ tran-
sient amplitude and the caffeine Ca2+ transient amplitude (SR-Ca2+)
measured in the same RV myocyte.2 Myocyte contractility and cal-
cium were measured under basal condition and upon b-AR stimula-
tion (Iso, 100 nmol/L).
SUPPLEMENTAL RESULTS
The sarcoplasmic reticulum calcium content was similar in RV myo-
cytes from control and operated animals under control conditions
but also upon b-AR stimulation (Supplemental Figure 4). However,
the fractional Ca2+ release was significantly reduced in RV myocytes
obtained from the operated group, suggesting early alterations in the
Ca2+-induced Ca2+ release process (Supplemental Figure 4). The
diminished fractional Ca2+ release and the increased frequency of
spontaneous calcium waves (Supplemental Figures 3 and 4) could
be related to the diminished T-tubule density associated with an
altered spatial distribution of the pore-forming subunit of L-type cal-
cium channels (CaV1.2; data not shown).
Although b-AR stimulation of Ca2+ transient amplitude remained
unchanged in operated animals, Iso did not increase sarcomere short-
ening as efficiently as it did for cardiomyocytes isolated in healthy pigs.
Myocytes from operated animals exhibited a prolonged relaxation
kinetic of Ca2+ transients. That agrees with the decreased velocity
of SR-Ca2+ uptake observed in RV- and LV-sided heart failure and
associated with a decreased myocardial gene expression of SR-
Ca2+ adenosine triphosphatase and contractile proteins.3 However,
Ca2+ transient amplitude remained normal, and relaxation kinetics
were enhanced with b-AR stimulation, suggesting only partial alter-
ations of SR-Ca2+ uptake in our model of early RV dysfunction.
10.e2 Hodzic et al Journal of the American Society of Echocardiography
- 2017

Supplemental Figure 2 Distribution of RV isolated myocyte length and width in control and operated animals. Cell length (153.4 6 4.3
vs 133.5 6 3.3 mm for controls, P < .001) and width (18.7 6 0.6 vs 15.9 6 0.5 mm for controls, P < .001) shift toward higher values,
suggesting an adaptive hypertrophy of RV myocytes in response to chronic combined overload.
Journal of the American Society of Echocardiography Hodzic et al 10.e3
Volume - Number -

Supplemental Figure 3 Effect of chronic overload on spontaneous calcium wave (SCW) occurrence under b-adrenergic stimulation
in RV isolated myocytes (RVMs) isolated from control and operated animals. (A) Representative trace of SCWs (arrows) recorded in a
Fura-2-loaded RVM isolated from an operated pig paced at 1 Hz during Iso (100 nmol/L) application. (B) Mean 6 SEM frequency of
SCWs in RVMs paced at 1 Hz under basal conditions and upon Iso (control: N = 4, n = 17; operated: N = 3, n = 22). (C) Percentage of
cells exhibiting SCWs in RVMs in control conditions and in Iso (control: N = 4, n = 17; operated: N = 3, n = 22). *P < .05, ***P < .001,
#
P < .05, and ##P < .01 (c2 test).

Supplemental Figure 4 Effect of chronic overload on SR-Ca2+ and the fractional release in isolated RV myocytes (RVMs) isolated
from control and operated animals. (A) Representative traces of Ca2+ transients (five upon electrical pacing and the last one induced
by a pulse of caffeine) recorded in a Fura-2-loaded control RVM paced at 1 Hz in basal and Iso (100 nmol/L) stimulation. (B) Mean
amplitude 6 SEM of Ca2+ transients induced by caffeine (10 mmol/L) estimating SR-Ca2+ in control (N = 4, n = 17) and operated
(N = 3, n = 22) animals before and during Iso application. (C) Mean 6 SEM amplitude of fractional release after caffeine pulse.
***P < .001 (paired t test) and #P < .05 (unpaired t test).
10.e4 Hodzic et al
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