Introduction

Right ventricular (RV) function may be impaired in various conditions in association with pressure or volume overload in patients with congenital heart disease (CHD) [14]. The pressure-overload RV dysfunction can be a sequela of Eisenmenger syndrome, pulmonary stenosis (PS), or tetralogy of Fallot (TOF). In some cases, RV dysfunction also results when the right ventricle works as a systemic ventricle, as occurs in congenitally corrected transposition of the great arteries (TGA) and postoperative atrial switch TGA. When the RV pressure overload remains uncorrected for a long period, RV failure results, which is closely associated with poor outcomes [6].

Although the assessment of RV function is important in the clinical management of children with CHD, the available imaging techniques are limited due to the complex geometry of the right ventricle, irregularities in the ventricular cavities, and abnormalities of wall motion of patients with congenital heart lesions [16, 19].

The myocardial performance index (MPI) has been described as a noninvasive Doppler-derived measurement of ventricular function by echocardiography [9, 12, 21–23, 26] that is less limited by the geometric shape of the ventricle and shown to be independent of heart rate [10, 22]. The MPI has been shown to correlate well with other invasive and noninvasive measurements of left ventricular (LV) function in adults [24]. The MPI is defined as the sum of the isovolumic contraction and relaxation time divided by the ejection time [21]. The RV MPI is calculated from tricuspid valve inflow and pulmonary valve outflow Doppler images.

The RV MPI also has been implicated as an indicative index of RV function in patients with CHD. The index usually is increased in the presence of RV systolic or diastolic dysfunction [15, 20]. The RV MPI also is increased in the pressure-overload condition, such as with increased pulmonary artery pressure [13].

However, disagreement exists about the implications of the RV MPI in CHD patients with pressure-overload RV dysfunction [1, 11]. The RV MPI in patients with isolated PS does not differ from that in normal children [11]. Paradoxically, a noncompliant right ventricle may shorten the RV isovolumetric relaxation time, resulting in a low RV MPI in patients with RV dysfunction after corrective surgery for TOF with residual RV outflow tract obstruction [1]. This lower RV MPI seems to be caused by alteration of global RV function secondary to myocardial hypertrophy or fibrosis [1]. However, these investigations were cross-sectional studies undertaken at a certain point in time during which echocardiographic evaluation was done without corresponding invasive RV hemodynamic functional studies. Also, those studies did not investigate the differences in RV MPI according to the severity of pressure overload.

Therefore, in the current study, we aimed to define the time course of changes in the RV MPI according to the severity of pressure load. To do this, we evaluated serial changes in the RV MPI and also recorded invasive RV hemodynamic measurements in a rat model that had mild to severe RV pressure overload induced by pulmonary arterial banding (PAB).

Methods

Animals

For the study, 3-week-old male Sprague–Dawley rats were purchased from Daehan Biolink (Daejeon, Korea) and housed individually in plastic cages in a temperature-controlled room. The investigation conformed to the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health: NIH Publication No. 85-23, revised 1996). The study design was approved by the Chonnam National Medical School Research Institutional Animal Care and Use Committee. All surgical procedures and echocardiography were performed with the animals under anesthesia induced by ketamine (16.65 mg/kg) administered intramuscularly (IM) and xylazine (7.77 mg/kg IM).

RV Pressure-Overload Model

To evaluate the changes in the MPI according to the severity of RV pressure overload, we divided the rats into two groups: rats with mild supravalvar PS induced by PAB (mild PS group, n = 20) and rats with severe supravalvar PS induced by PAB (severe PS group, n = 28).

For PAB, the midline sternum was opened with the rat under anesthesia. Partial pulmonary arterial constriction was performed by ligation of the main pulmonary artery using a 4-0 braided polyester suture with 18-gauge needles for mild stenosis or 22-gauge needles for severe stenosis. Supravalvar PS was assessed by two-dimensional echocardiography and Doppler echocardiography. The sham-treated animals (sham group, n = 30) underwent the same surgical procedure without constriction of the pulmonary artery.

In each group, the ratio of RV wall weight to body weight (RV/BW) and the ratio of RV free wall weight to LV free wall and interventricular septal (IVS) wall weight (RV/[LV + IVS]) were evaluated 1, 2, 3, 5, and 8 weeks after PAB. Hematoxylin-eosin and Masson’s trichrome staining also were performed 1, 2, 3, 5, and 8 weeks after PAB as described previously [18]. Images were obtained and digitized with an Olympus CX31 microscope equipped with an Infinity 1 camera (Lumenera Scientific, Ottawa, Canada). The density of collagen, shown as blue or green, was calculated by using an image analysis system (NIS-Elements AR 3.0; Nikon, Melville, NY, USA), and the sum of all the collagen areas was divided by the total area [5].

Confirmation of RV hypertrophy or RV fibrosis secondary to RV pressure overload was provided by the following indexes: (1) parameters from echocardiographic evaluation, (2) RV/(LV + IVS), (3) RV/BW, (4) hematoxylin-eosin staining (Sigma, St. Louis, MO, USA) and Masson’s trichrome staining (Sigma, St. Louis, MO, USA) for fibrosis, and (5) expressions of genes related to hypertrophy or fibrosis by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) [5].

Echocardiography

Echocardiographic studies were performed with a 15-MHz linear array transducer system (iE33 system; Philips Medical Systems, Bothell, WA, USA) 1, 2, 3, 5, and 8 weeks after PAB [7, 18]. Two-dimension (2D)-guided M-mode of the RV was obtained from the parasternal view. The RV cavity dimension and thickness of the RV free and IVS walls were measured.

Using the apical four-chamber view, we traced the endocardial borders of the RV free wall and septum from base to apex and determined the respective RV areas. The RV fractional area change (RVFAC) was defined using the following formula (end-diastolic area–end-systolic area)/end-diastolic are × 100 [2].

Doppler Measurements

The RV outflow velocity pattern was recorded from the parasternal short-axis view with the Doppler sample volume positioned just below the pulmonary valve. The size of the Doppler sample volume was set at an axial length of 2–4 mm with a wall filter setting of 400 Hz. Care was taken to perform these studies with the transducer beam as close as possible to the Doppler beam at 20° or lower in selected planes. Angle correction of the Doppler signal was made.

Measurements of the MPI

The RV MPI was calculated from tricuspid valve inflow and pulmonary valve outflow Doppler images. The time from cessation to the beginning of tricuspid inflow (a) and the RV ejection time (b) were measured. The RV MPI was calculated as follows:

$$ {\text{RV}}\,{\text{MPI}} = \left( {{\text{ICT}} + {\text{IRT}}} \right)/{\text{ejection}}\,{\text{time}} = \left( {{\text{a}} - {\text{b}}} \right)/{\text{b}} $$

where ICT is the RV isovolumic contraction time and IRT is the isovolumic relaxation time. Doppler-derived intervals were measured offline from stored data, and five consecutive cardiac cycles were averaged.

To evaluate interobserver variability in RV MPI measurement, two independent observers blindly calculated the Doppler-derived intervals at different times in 72 randomly selected rats. Each observer individually selected the frames to measure and had no knowledge of the results obtained by the other observer [16]. Intraobserver variability analyses also were performed several months after the primary measurements in 72 randomly selected rats.

Intracardiac Pressure Monitoring

After echocardiography, hemodynamic evaluation was performed as described previously 8 weeks after PAB [3]. After the induction of general anesthesia, a sterile left thoracotomy was performed with pericardial cradle creation, and then an arterial catheter (PE 50 tube; Clay Adams, Parsippany, NJ, USA) was advanced into the LV and RV through the apical lateral myocardium. The pressure curve of the LV and RV was recorded and monitored continuously. Pressure variables were averaged for three consecutive cardiac cycles by an experienced technician blinded to the group. Exclusively, ventricular systolic blood pressure, maximal rate of ventricular pressure rise (dP/dt), and end-diastolic pressure (EDP) were recorded and statistically analyzed.

RT-PCR and Immunoblotting

Total RNA was isolated with TriZol reagent (Invitrogen, Carlsbad, CA, USA), and 1 μg of RNA was subjected to reverse transcription reaction with the Superscript first-strand synthesis system for RT-PCR kit (Invitrogen) [17]. The sequences of the primers used were as follows: Nppa sense, GCT CGA GCA GAT CGC AAA AG; Nppa anti-sense, GAG TGG GAG AGG TAA GGC CT; CTGF sense, GTT AGC CTC GCC TTG GTG CT; CTGF anti-sense, ATC TTT GGC AGT GCA CAC GC; GAPDH sense, CAT TGT TGC CAT CAA CGA CCC CTT C; GAPDH anti-sense, CCA TCA CGC CAC AGC TTT CCA GAG.

For Western blot, anti-collagen type 1 antibody (1:1000, Abcam [ab292], Cambridge, UK), anti-α-tubulin antibody (1:1,000, Zymed [A01410-40], South San Francisco, CA, USA), and GAPDH antibody (1:1,000, Santa Cruz Biotechnology Inc. [sc-166574], CA, USA) were used.

Statistical Analysis

All data are expressed as mean value ± standard error of the mean (SEM). The Kruskal–Wallis H test, followed by post hoc analysis (Mann–Whitney U test), was used to compare differences among the three groups. Correlations were tested using the simple linear regression method. A P value lower than 0.05 was considered statistically significant. Statistical analysis was performed with SPSS software for Windows (version 17.0; SPSS, Chicago, IL, USA).

Results

RV Hypertrophy and Fibrosis Induced by PAB

Body weight increased similarly in the three groups of rats 0–8 weeks after the operation (Fig. 1a; Table 1). However, the RV weight and RV/BW increased more in the severe PS group than in either the mild PS group or the sham-treataed group at 1–8 weeks (Fig. 1b; Table 1). The RV/(LV + IVS) also increased more in the severe PS group than in either the mild PS group (0.38 ± 0.06 vs 0.46 ± 0.03) or sham-treated group (0.28 ± 0.00 vs 0.33 ± 0.01): 0.61 ± 0.01 at 1 week (P < 0.001 vs sham; P = 0.003 vs mild PS), 0.54 ± 0.03 at 2 weeks (P = 0.020 vs sham), 0.56 ± 0.04 at 3 weeks, 0.63 ± 0.03 at 5 weeks (P = 0.024 vs sham), and 0.61 ± 0.02 at 8 weeks (P < 0.001 vs sham; P = 0.005 vs mild PS, Fig. 1b; Table 1). The liver/BW ratio did not differ significantly between the three groups (Table 1).

Fig. 1
figure 1

a Serial measurements of body weight. b Ratio of right ventricle (RV) weight to left ventricular free and interventricular septal (LV+IVS) weight in the sham group, mild PS group, and severe PS group 1, 2, 3, 5, and 8 weeks after pulmonary artery banding. *P < 0.05, †P < 0.01 versus sham, §P < 0.01 versus mild PS

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Table 1 Body and heart weights in rats 1, 2, 3, 5, and 8 weeks after PAB
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The gross findings of the rat hearts showed severe RV area enlargement in the severe PS group compared with either the mild PS group or the sham-treated rats. The increase was gradual with prolongation of PAB. Hematoxylin-eosin staining of rat hearts from the severe PS group showed an increase in RV free wall thickness and a flattened IVS 1–8 weeks after the operation. The severity of the increase in RV free wall thickness was aggravated by age. However, those findings were not significant in the mild PS group (Fig. 2a). PAB caused fibrosis of the myocardium, as demonstrated by Masson’s trichrome staining, and the severity of fibrosis was more prominent in the severe PS group than in the mild PS group 8 weeks after PAB (Fig. 2b).

Fig. 2
figure 2

a Hematoxylin-eosin staining of rat hearts from the sham, mild PS, and severe PS groups 1, 2, 3, 5, and 8 weeks after pulmonary artery banding. b Masson’s trichrome staining of rat hearts from the sham, mild PS, and severe PS groups 8 weeks after pulmonary artery banding. c Representative immunoblot analysis of α-tubulin and collagen type 1 in rat hearts from the sham, mild PS, and severe PS groups 8 weeks after pulmonary artery banding. d Representative reverse transcription-polymerase chain reaction (RT-PCR) analysis of heart mRNA from rats in the sham, mild PS, and severe PS groups 8 weeks after PAB

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The protein levels of α-tubulin, a hypertrophy marker [17], and collagen type 1, a fibrosis marker [4], were increased in the right ventricle of the severe PS group (Fig. 2c). The transcript levels of natriuretic polypeptide type A (Nppa, which encodes ANP), a hallmark of cardiac hypertrophy [17] and of connective tissue growth factor (Ctgf), a fibrosis marker [4], were increased with increasing severity of PAB (Fig. 2d).

The RV pressure in the severe PS group (124.5 ± 4.1 mmHg) was significantly higher than in the sham group (25.3 ± 0.6 mmHg; P < 0.003) or the mild PS group (32.4 ± 1.6 mmHg; P < 0.009). The LV pressure in the severe PS group (112.6 ± 4.5 mmHg) also was significantly higher than in the sham group (75.3 ± 1.8 mmHg; P = 0.01) (Fig. 3a; Table 2). The RV peak positive dP/dt, a measure of global contractility [15], was significantly higher in the severe PS group (4663.0 ± 107.8 mmHg/s) than in the sham group (727.1 ± 64.5 mmHg/s; P = 0.003) or the mild PS group (778.7 ± 146.4 mmHg/s; P = 0.009). The LV peak positive dP/dt in the severe PS group, however, did not differ significantly from the values in the sham and mild PS groups (Fig. 3b; Table 2).

Fig. 3
figure 3

Right ventricular (RV) and left ventricular (LV) pressure (a), maximal rate of RV and LV pressure rise (dP/dt) (b), and RV and LV end-diastolic pressure (c) in the sham, mild PS, and severe PS groups 8 weeks after PAB. *P < 0.05, †P < 0.01 versus sham; §P < 0.01 versus mild PS

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Table 2 Ventricular hemodynamic parameters in rats 8 weeks after PAB
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The RV EDP was significantly higher in the severe PS group (8.12 ± 0.52 mmHg) than in the sham-treated group (3.02 ± 0.22 mmHg; P = 0.005). The LV EDP also was significantly higher in the severe PS group (8.18 ± 0.77 mmHg) than in the sham-treated group (3.70 ± 0.22 mmHg; P = 0.008) (Table 2).

Echocardiographic Findings

On echocardiography, the supravalvar PS induced by PAB significantly increased main pulmonary artery flow velocity compared with that in the sham-treated rats. In addition, the flow velocity was much higher in the severe PS group than in the mild PS group.

The thickness of both the end-diastolic RV free wall and the IVS also was increased in the severe PS group, whereas the IVS was flattened. The thickness of the end-diastolic RV free wall in the severe PS group was higher than in the sham group: 0.98 ± 0.01 at 1 week (P < 0.001), 1.08 ± 0.01 at 2 weeks (P < 0.001), 1.25 ± 0.01 at 3 weeks (P < 0.001), 1.54 ± 0.02 at 5 weeks (P < 0.001), and 1.61 ± 0.04 at 8 weeks after PAB (P < 0.001).

The thickness of the end-diastolic RV free wall was mildly increased in the mild PS group (Fig. 4a; Table 3). The RV cavity was larger in the severe PS group than in the sham or mild PS group and was increased by the prolongation of PAB (Table 3). The RVFAC, an assessment of RV systolic function [2], did not differ significantly among the three groups (Fig. 4b; Table 3). The heart rate also did not differ significantly among the three groups (Table 3).

Fig. 4
figure 4

Echocardiographic end-diastolic right ventricular (RV) free wall thickness (a), RV fractional area change (b), and correlations between end-diastolic RV free wall thickness and RV pressure in rats 8 weeks after surgery (c). *P < 0.05, †P < 0.01 versus sham; §P < 0.01 versus mild PS

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Table 3 Echocardiographic parameters and heart rate in rats 1, 2, 3, 5, and 8 weeks after PAB
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Good correlation was observed between end-diastolic RV free wall thickness by echocardiogram and RV pressure in rats 8 weeks after the operation (r = 0.87; P < 0.001; Fig. 4c).

MPI

The RV MPI did not differ significantly between the mild PS group and the sham group 1–8 weeks after the operation (Fig. 5a; Table 4). At 1 week after the operation, the RV MPI of the severe PS group (0.23 ± 0.01) did not differ significantly from that of the sham group (0.27 ± 0.01). Beyond this time point, however, the RV MPI in the severe PS group was lower than in the sham group: 0.19 ± 0.01 at 2 weeks (P < 0.001), 0.16 ± 0.01 at 3 weeks (P < 0.001), 0.20 ± 0.01 at 5 weeks (P = 0.021), and 0.18 ± 0.01 at 8 weeks after PAB (P < 0.001; Fig. 5a; Table 4).

Fig. 5
figure 5

Right ventricular (RV) myocardial performance index (MPI) (a), isovolumetric relaxation time (b), and ejection time (c) of the sham, mild PS, and severe PS groups 1, 2, 3, 5, and 8 weeks after PAB, and correlations between RV pressure and RV MPI in rats 8 weeks after surgery (d). *P < 0.05, †P < 0.01 versus sham; ‡P < 0.05 versus mild PS

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Table 4 Myocardial performance index in rats 1, 2, 3, 5, and 8 weeks after PAB
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The RV ICT of the severe PS group did not differ significantly from that of the sham group except at 2 weeks after the operation (Table 4). At 2 to 3 weeks after the operation, the RV ICT in the mild PS group was lower than in the sham group. At 1, 5, and 8 weeks after the operation, the RV ICT did not differ significantly between the three groups (Table 4).

The RV IRT in the severe PS group was lower than in the sham group 2–8 weeks after the operation (Fig. 5b; Table 4). At 8 weeks after the operation, the RV IRT in the mild PS group was lower than in the sham group (Fig. 5b; Table 4).

The RV ejection time in the severe PS group was significantly lower than in the sham group 1–8 weeks after the operation (Fig. 5c; Table 4). At 1–2 weeks after the operation, the RV ejection time in the mild PS group was lower than in the sham group (Fig. 5c; Table 4).

A negative correlation was observed between RV MPI and RV pressure in the rats 8 weeks after the operation (r = −0.51; P = 0.029; Fig. 5d). Interobserver variability in the measurement of the RV MPI showed excellent correlation between the two observers (r = 0.96; P < 0.001). The mean interobserver variance was 0.02 ± 0.01. Intraobserver variability in the RV MPI also showed excellent correlation (r = 0.94; P < 0.001). The mean intraobserver variance was 0.02 ± 0.01.

Discussion

The study findings showed that the RV MPI was decreased in the rats with severe pressure-overload RV hypertrophy induced by PAB. The decrease in the RV MPI of the severe PS group was significant compared with that of the sham rats at 2–8 weeks. By contrast, the RV MPI did not change significantly in the rats with mild pressure-overload RV hypertrophy.

Recently, the RV MPI was proposed for the quantitative assessment of global RV function [21, 26]. The RV MPI has been assessed for determining the severity of RV dysfunction in patients with congestive heart failure and pressure-overload conditions such as primary pulmonary hypertension [21, 25, 26]. Many studies have assessed RV dysfunction by use of the MPI in CHD patients with various pressure-overload conditions of RV [1, 11, 16]. However, no consensus exists about the usefulness of the RV MPI in CHD [1, 11, 16].

Ishii et al. [16] reported that the average RV MPI was significantly higher in 8 TGA patients who had a Senning operation (0.58 ± 0.09) than in 150 healthy children (0.24 ± 0.04) [16]. They found that the sum of ICT and IRT was significantly prolonged and that the ejection time was significantly shortened in the patients who had undergone a Senning operation.

In another study, Eidem et al. [11] reported that the RV MPI in patients who had congenitally corrected TGA with severe atrioventricular valve insufficiency (0.72 ± 0.17) was significantly higher than in healthy control subjects (0.32 ± 0.03). Both ICT and IRT were longer, and the ejection time was shorter than the corresponding control values. The RV MPI was significantly increased together with the increase in degrees of qualitative RV dysfunction. These authors postulated that a long-standing increase in the afterload would be expected to have a deleterious effect on the RV MPI [11].

Although isolated PS is similar to the condition with increased afterload, Eidem et al. [11] reported that the RV MPI in 21 patients with moderate to severe PS did not differ significantly from that in normal children. The mean RV systolic pressure was 81 ± 50 mmHg, which was more than 50 % of the measured systolic pressure. None of the 21 patients had RV dilation or dysfunction. The RV ICT was significantly longer than in healthy individuals. However, the authors did not observe significant differences in the MPI. Varying severity of pulmonary valve obstruction did not have a significant impact on the Doppler time intervals or the RV MPI [11].

The observations of Eidem et al. [11] differ in several ways from ours. In our study, the RV MPI also was not significantly changed in the group with mild RV pressure overload but no hypertrophy. However, the MPI was decreased in the group with severe RV pressure overload. This decrease was caused by a significant shortening of IRT combined with a prolonged ejection time. We observed that the RV pressure was much higher than the systolic pressure and that significant hypertrophy and fibrosis developed in the severe pressure-overload condition. By contrast, Eidem et al. [11] did not evaluate RV hypertrophy or fibrosis. In addition, we induced acute pressure overload over 8 weeks, whereas the report by Eidem et al. [11] showed isolated PS groups exposed to chronic pressure overload over 6.6 years [11].

In a contrasting study, Abd El Rahman et al. [1] performed a prospective analysis of 51 patients after corrective surgery for TOF. The average of RV MPI was paradoxically below the normal range (0.10 ± 0.19). A total of 39 patients (76.5 %) had an RV MPI below the normal range. The authors explained that this decrease was caused mainly by a significant shortening of IRT, which resulted from early opening of the pulmonary valve in late or even mid diastole even before actual closure of the tricuspid valve [1].

These phenomena have been noted previously in conditions of altered global RV function secondary to myocardial hypertrophy or fibrosis, which allow the ventricular pressure to exceed the pulmonary artery pressure before atrial contraction [8]. Abd El Rahman et al. [1] assumed that this may reduce the sensitivity of the index in recognizing patients with RV dysfunction after corrective surgery for TOF [1].

Likewise, in the current study, we also observed that the RV MPI in the severe pressure-overload condition was decreased compared with that in the sham-treated animals and in the group with mild pressure overload. We also observed that decreased IRT and increased ejection time were associated with the severity of myocardial hypertrophy.

Another report has addressed a limitation of the RV MPI in assessing RV function. Yoshifuku et al. [27] reported that the RV MPI in severe RV infarction decreased or pseudo-normalized due to significant shortening of ICT and equalization of the end-diastolic RV and pulmonary artery pressure. They proposed that the MPI may not be useful for expressing global cardiac function when ICT, ejection time, and IRT are not determined by cardiac function. It is known that arrhythmia and respiration also can influence the changes in ICT, ejection time, and IRT [27].

In our study, we observed no significant changes in the RVFAC, which is one of the parameters of RV systolic function. Therefore, the RV pressure overload induced by pulmonary artery banding did not induce the systolic dysfunction, and the RV adapted well to the severe pressure overload. However, RV diastolic dysfunction occurred in the severe pressure-overload condition, which was shown by the increased EDP in our study. Further studies investigating ventricular systolic and diastolic function in RV hypertrophy and fibrosis are warranted.

Conclusion

In this study, the RV MPI was decreased in rats with severe pressure-overload RV hypertrophy compared with that in rats with mild pressure-overload RV hypertrophy and sham animals. We also found that the severity of the RV hypertrophy was increased with the duration of pressure overload. Thus, we propose that RV hypertrophy contributes to the decreased RV MPI in the severe pressure-overload condition.