Experimental Design

A surgical procedure mimicking repaired TOF was performed on six Landrace piglets (operated group) that were between 50 and 67 days of age. Six age-matched animals were used as controls (control group). Control animals did not undergo sham surgery. All animals were male to avoid bias related to hormonal variations. Echocardiographic and hemodynamic assessment of RV function were performed at baseline just before the surgical procedure and 4 months later. Clinical status was evaluated daily during the study. After completing the 4-month follow-up period, animals were euthanatized for a cellular study on isolated RV myocytes. All experiments were carried out according to the European Community guiding principles on the care and use of animals (2010/63/UE, September 22, 2010), the local ethics committee (CEEA26 CAPSud) guidelines, and French decree 2013-118 on the protection of animals used for scientific purposes (JORF 0032, February 7, 2013, p. 2199, text no. 24). Authorization to perform animal experiments according to this decree was obtained from Ministère Français de l’Agriculture, de l’Agroalimentaire et de la Forêt (agreement B92-019-01).

Experimental Model of Combined RV Overload

As previously described by our group, after premedication with ketamine hydrochloride (15 mg/kg intramuscularly), general anesthesia was induced with propofol 1% and cisatracurium (0.3 mg/kg every 2 hours), allowing endotracheal intubation, which was maintained with isoflurane in 100% oxygen (Servo 900; Siemens-Elema, Solna, Sweden). Through a left thoracotomy approach, a side-biting vascular clamp was longitudinally placed across the pulmonary valve annulus without obstruction of the RV outflow tract. A pulmonary valve leaflet was excised and the pulmonary infundibulum, annulus, and trunk were enlarged by a 2-cm-long, elliptically shaped polytetrafluoroethylene patch to ensure the loss of valve integrity. This chronic pulmonary valve regurgitation led to RV volume overload. Branch pulmonary arterial obstruction is not uncommon after surgical correction of TOF related to native hypoplasia or surgical complications and is known to increase pulmonary valve regurgitation. In our model, RV pressure overload was achieved by pulmonary artery banding, made of umbilical tape, placed around the artery truncus and secured for a final diameter of approximately 1 cm to ensure progressive pulmonary stenosis with animal maturation.

Echocardiographic Analysis

Echocardiography was performed on closed-chest animals under general anesthesia in the dorsal decubitus position. We used a commercially available Vivid E9 ultrasound machine (GE Medical Systems, Milwaukee, WI) equipped with a 2.5-MHz transducer. The values of all echocardiographic parameters were obtained as the average value of three consecutive cardiac cycles during transient apnea and were analyzed on a comprehensive workstation (EchoPAC 110.1.2; GE Vingmed Ultrasound, Horten, Norway). Measures indexed to body surface area were calculated using the formula body surface area (m ² ) = (weight [kg] × body length [cm]) ^0.5 /60.

In the parasternal long-axis view, RV anterior wall thickness was measured, and the RV/LV end-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 fractional 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 excursion 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 (S′) 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.

Regarding the speckle-tracking analysis, RV longitudinal deformation was measured using standard two-dimensional grayscale acquisitions 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 contouring 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 longitudinal strain rate (GLSR) were calculated as the average of the six segments. The second method used a contouring of the endocardium of the FW, excluding the interventricular septum to minimize the influence 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 measurements on a random subset of eight pigs. Intraobserver variability was assessed ≥1 week after the initial measurements. Two blinded operators assessed interobserver variability. Echocardiographic assessment of RV myocardial deformation was not studied under stress conditions. 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. The reference cardiac output was measured using the transpulmonary thermodilution technique (PiCCO; Pulsion Medical Systems, Munich, Germany). This technique allowed us to obtain systemic cardiac output reflecting the efficient pulmonary output independently of the pulmonary regurgitation effect. Data were acquired using dobutamine infusion (5 μg/kg/min) after 10 min of hemodynamic stability. The acquisitions were made during 10 sec of apnea. The pulmonary regurgitation 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 pulmonary regurgitation, which continues during RV filling, it could provide an accurate estimation of pulmonary regurgitation in patients with TOF. A pulmonary regurgitation volume of >5 mL/beat/m ² was considered significant. The pulmonary regurgitation fraction was calculated as the ratio between the pulmonary regurgitation volume 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 corresponded to the pressure-volume loop area. RV contractility was examined using the end-systolic pressure volume relationship. Slopes of end-systolic pressure volume relationship (E _max ) were computed during vena cava occlusion. Measurements were acquired at rest and during dobutamine infusion (5 μg/kg/min). The variation of E _max slope (ΔE _max ) represented the myocardial contractility reserve. In other words, the percentage increase in E _max in response to β-adrenergic receptor (β-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 provided 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 Ca ²⁺ concentration changes using a ratiometric Ca ²⁺ indicator (Fura-2). Sarcomere shortening and Ca ²⁺ transient amplitudes and relaxation kinetics were recorded before and after β-AR stimulation by isoproterenol (Iso). Sarcoplasmic reticulum Ca ²⁺ content (SR-Ca ²⁺ ) was measured by application of caffeine on isolated myocytes, inducing cytoplasmic Ca ²⁺ release by activation of reticulum sarcoplasmic ryanodine receptors.

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 hemodynamic 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: Manly’s approach). Cellular data were normally distributed (Shapiro-Wilk test) and are expressed as mean ± SEM. Student’s paired t test was used to test the significance of changes between baseline and Iso conditions in each group, and Student’s unpaired t test was used to test the significance of changes between groups. The relationship between echocardiographic and hemodynamic variables was assessed using Spearman correlation. A χ ² 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.

Clinical Status

The duration of the study was similar between operated and control groups (121 ± 11 and 133 ± 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 thickening of the RV FW related to moderate but significant pulmonary stenosis ( 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 echocardiographic 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 hemodynamic assessment confirmed severe pulmonary regurgitation in the operated animals. The median pulmonary regurgitation volume was 12.2 mL/beat/m ² (interquartile range, 10.6–16.4 mL/beat/m ² ), and the median pulmonary regurgitation fraction was 25% (interquartile range, 22%–28%). Consequently, end-systolic and end-diastolic volumes 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 inferior to controls after dobutamine infusion ( Figure 1 ). Stroke work increased 4 months after surgery, without reaching a significant value at rest, but progressed after dobutamine infusion and was significantly higher than in the control group ( Table 3 ). In accordance with our hemodynamic findings of operated animals, echocardiographic measurements of RV FAC, TAPSE, and peak S′ significantly decreased at 4 months and were positively correlated with hemodynamic eEF ( Tables 2 and 4 ).

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) , E _max (C) , and contractility reserve (ΔE _max ) (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: load dependency and relationship with hemodynamic functional and contractile parameters

Coefficient of correlation r	Conductance catheter technique				Echocardiography
	RV EDV	RV ESP	eEF	ΔE max	FW GLS	FW GLSR
Echocardiography
FAC	−0.6	−0.82 ∗	0.7 ∗	0.54	0.81 ∗	0.78 ∗
TAPSE	−0.42	−0.68 ∗	0.67 ∗	0.65 ∗	0.9 ∗	0.76 ∗
Peak S′	−0.44	−0.72 ∗	0.68 ∗	0.78 ∗	0.88 ∗	0.92 ∗
FW GLS	−0.37	−0.83 ∗	0.5	0.46
FW GLSR	−0.55	−0.63 ∗	0.67 ∗	0.75 ∗

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