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Myocardial hypertrophy after pulmonary regurgitation and valve implantation in pigs
International Journal of Cardiology 159 (2012) 29–33
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International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d
Myocardial hypertrophy after pulmonary regurgitation and valve implantation in pigs☆ Julie Smith a,b, Jens Peter Goetze b, Lars Søndergaard c, Jesper Kjaergaard c, Kasper K. Iversen c, Niels G. Vejlstrup c, Christian Hassager c, Claus B. Andersen a,⁎ a b c
Department of Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Department Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Department Cardiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
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Article history: Received 21 December 2010 Accepted 6 February 2011 Available online 15 March 2011 Keywords: Arrhythmia Fibrosis Histology Myocardial hypertrophy Pulmonary regurgitation Valve replacement
a b s t r a c t Background: Patients may suffer from right ventricular (RV) failure and malignant cardiac arrhythmias after late pulmonary valve replacement correcting pulmonary regurgitation (PR). But the underlying mechanisms of the refractory arrhythmias are not well understood. Methods: The aim of present study was to characterize the RV myocardium after percutaneous pulmonary valve implantation (PPVI) in a porcine model after severe PR for 3 months. RV histology was evaluated with morphometric methods and RV function was assessed with electrophysiology, echocardiography, and biochemical measures: The results were compared with age-matched sham-operated animals. Results: At euthanasia, RV weight was increased compared to sham-animals, median 127 g (115–137) vs. 71 g (69.5–76.5), p = 0.0007. RV myocyte diameters corrected for individual variation with the RV/LV ratio were enlarged, 1.06 (1.02–1.13) vs. 0.84 (0.80–0.91), p = 0.0006. There were no excess collagen tissue (RV/LV ratio), p = 0.77. Electrophysiological stimulation resulted in RV arrhythmia in 67% of the animals compared to 25% in the sham-operated animals, but this difference was not statistically significant, p = 0.28. Echocardiography revealed geometrical dilation in end-systolic RV area, mean ± SD, 11.8 ± 4.9 cm2 vs. 6.0 ± 3.5 cm2, p = 0.05, and end-diastolic area, 23.3 ± 10.4 cm2 vs. 12.7 ± 2.5 cm2, p = 0.08. RV anterior free wall thickness was not increased, 0.7 ± 0.2 cm vs. 0.7 ± 0.1 cm, p = 0.66. Echocardiographic functional parameters and plasma natriuretic peptides were unchanged. Conclusions: The RV does not completely recover after three months of PR with persistent myocardial hypertrophy one month after PPVI. Future studies should address whether RV chamber and cellular hypertrophy, without fibrosis or interventional scar tissue, may be substrate for arrhythmia. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pulmonary regurgitation (PR) can be the main hemodynamic lesion after repair of tetralogy of Fallot and is well tolerated with most children reaching adulthood. Exercise intolerance, atrial and ventricular arrhythmias are however still major complications, including an increased risk of sudden death [1]. Valve replacement is able to improve right ventricular (RV) function, but artificial valves
☆ Funding: This work was supported by the Heart Centre Research Committee, Rigshospitalet; Medtronic Inc®. Minneapolis, MN, USA; NuMed®, Hopkinton, NY, USA; the Danish Heart Foundation ref. no. 05-4-B269-A534-22220; the Frimodt-Heineke Foundation; the Beckett Foundation; the Augustinus Foundation; the Villadsens Family Foundation; and the Eva & Henry Frænkel Foundation. ⁎ Corresponding author at: Department of Pathology (5444), Rigshospitalet, University of Copenhagen, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel.: +45 3545 5344; fax: +45 3545 5414. E-mail address: [email protected] (C.B. Andersen). 0167-5273/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2011.02.022
have limited durability, consequently the optimal time for replacement remains controversial [2]. After valve replacement, RV systolic function can normalise rapidly while diastolic function and the RV wall mass only recover gradually [3]. The right ventricle does, however, not always completely recover following late pulmonary valve replacement with sustained risk of refractory arrhythmia and RV dysfunction [4,5]. The cellular mechanisms of the reverse remodelling process are not well understood. Fibrosis is believed to play a role in the refractory arrhythmias [6,7]. RV dilation and fibrosis can be associated with increased release of natriuretic peptides to plasma [8–10], whereas correction of RV volume overload might normalise peptide concentrations. Minimally invasive treatment for correcting pulmonary outflow tract dysfunction has been introduced during the last five years [11]. And percutaneous pulmonary valve implantation (PPVI) can postpone open-heart surgery, but requires an existing conduit [12,13]. In the present study, young piglets were subjected to acute PR for 3 months by percutaneous stenting followed by PPVI for 1 month. The
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aim was to characterize the myocardium in a porcine model with no interventional scar tissue. The study focused on myocyte hypertrophy and collagen fraction measured with gold-standard morphometric methods. Before euthanasia, the pigs underwent programmed electrical stimulation, echocardiography, and measurement of natriuretic peptide concentrations in plasma. 2. Materials and methods 2.1. Animal procedures During RV heart catheterisation, a 22 mm CP™ stent (NuMed, USA) was introduced via the femoral vein and positioned in the pulmonary valvular annulus in six piglets, crossbreeds of Danish Landrace, Yorkshire and Duroc. After three months with free PR, a bovine jugular venous valve sutured inside a stent (Medtronic Melody™, Medtronic Inc., USA) was mounted in the CP™ stent. A lethal dose of pentobarbital (200 mg/ml) was injected intravenously one month after implantation of the stented valve. A matching group of eight controls underwent the same heart catheterisation procedures, but no stents were implanted. All pigs were sedated with midazolam (0.5 mg/kg), and general anaesthesia was induced and maintained by intramuscular injections of a zoletil mixture (1 ml/10 kg for the induction). This mixture consisted of 125 mg tiletamin and 125 mg zolazepam, added 6.5 ml xylazine (20 mg/ml), 1.5 ml ketamin (100 mg/ml) and 2.5 ml sterile water or methadone (10 mg/ml). To counter the effects, naloxone (0.4 mg/40 kg) and atipamezole hydrochloride (5 mg/40 kg) were injected. Antiarrhythmic amiodarone (5 mg/kg) and anticoagulant heparin were administered intravenously as standard therapy during cardiac catheterisation (start dose 100 IU/kg, and 50 IU/kg per half hour). On the day of euthanasia and in control animals, antiarrhythmics were not administered during catheterisation procedures. Anticoagulant enoxaparin (10 mg/10 kg twice a day) was subcutaneous injected for two days, and after valve implantation acetylsalicylic acid (0.1 mg/30 kg) for four weeks. Non-steroidal anti-inflammatory drug carprofen (50 mg/ 12 kg) and antibiotics dihydrostreptomycin (25 mg/kg) with benzylpenicillinprocain (20,000 IU/kg) in a mixture were injected intramuscularly for 3 days after the two first interventions. The Animal Experiments Inspectorate, Ministry of Justice, Denmark, approved the research project (ref. no. 2005/561-1010). 2.2. Morphometric methods for measuring myocardial hypertrophy and collagen tissue To analyse RV tissue, the porcine hearts were collected immediately after euthanasia. The width of the heart along the coronary sulcus and length from sulcus to apex was measured. By cutting with a pair of scissors along the coronary sulcus and bilateral to apex, excluding the interventricular septum (IS) and the pulmonary trunk, the RV free wall was isolated and weighed. The left ventricle (LV) was isolated and weighed together with the IS. All parts were preserved in formalin, and for estimating the degree of myocardial collagen and hypertrophy, three biased arbitrary tissue sections (1 × 1 cm) from the RV free wall, two from the LV and two from the IS were cut and embedded in paraffin. To make the basement membrane distinct, sections of 5 μm thickness were stained with periodic acid silver methenamine (PASM). The specimens were magnified 40 times with a Leica DMR microscope. An unbiased systematic uniformly random sampling (SURS) gave 8–12 fields of visions per slide each transformed with Leica DFC 420 C camera to a digital screen image by using the Leica Image Manager software version IM50. An unbiased counting frame mounted on the screen included approximately one to five profiles (Fig. 1). The shortest diameter was measured going centrally through the nucleus by using the vector application in the IM50 software (Fig. 1B) [14]. For estimating the fraction of collagen tissue, new slides from the same paraffin blocks were stained with Masson's trichrome. On a slide, 8–12 fields of view were selected unbiased with the SURS method and magnified 40 times. An unbiased grid with 100 points was mounted on the screen and points touching blue stain were counted to estimate the fraction of collagen [15,16]. Measurements were blinded and all completed by one person.
Fig. 1. A: A field of vision from the right myocardium showed with an unbiased counting frame. Cells are not included when touching the thick “forbidden” lines. Notice the multinucleated porcine cardiac myocytes. Periodic acid silver methenamine (PASM) stain. B: In this field of vision from the porcine right ventricle five cardiac myocytes are measured based on the unbiased counting frame. PASM stain. performed on the control group for the same two age groups. In supine position a 3.5 MHz transducer transmitted four-chamber, long- and short axis views imaged by GE Vivid 7 Dimension system and analysed using GE EchoPac SWO version 6 (GE, Horten, Norway). Results on echocardiography have previously been published [17], but relevant data related to the histological studies are presented here. 2.5. Natriuretic peptides in plasma After femoral venous access before starting the actual heart catheterisation procedures on the day of euthanasia, 10 ml of blood was collected and immediately transferred into Na2-EDTA tubes (1.5 mg/ml), centrifuged, and the plasma was stored at − 80 °C. For measuring B-type natriuretic peptide (BNP) in porcine plasma, samples were sent to Phoenix Europe, Karlsruhe, Germany, where one ml of plasma per sample was used for their BNP-32 porcine Enzyme Immunoassay kit (porcine BNP EIA kit). The inter- and intra assay variations were b14% and b 5% respectively and there was a minimum detectible concentration of 0.23 ng/ml.
2.3. Electrophysiological testing 2.6. Statistics On the day of euthanasia, a 5 French quadripolar electrode catheter (Josephson, Medtronic, USA) was placed in the porcine RV apex via the femoral vein. The standard ventricular stimulation protocol for patients in the Cardiac Catheterisation Laboratory was applied comprising of 8 beats at cycle length of 400 to 500 ms and introduction of up to triple extrastimuli, delivered by a programmed electrical stimulator (EP-TRACER, Cardio Tek, Maastricht, Netherlands). The ventricles were considered arrhythmogenic when the stimulation induced ventricular fibrillation (VF) or ventricular tachycardia (VT). 2.4. Echocardiography After three months of free PR, cardiac function was assessed with echocardiographic imaging during anaesthesia before the PPVI procedures. Measurements were repeated one month later on the day of euthanasia. Comparable measurements were
To assess distributions, Bland–Altman for paired data, histograms, and residual plots, were interpreted and if not normally distributed, transformation by logarithm 10 was applied on primary data. Cell diameters were transformed and mean of one field of vision was calculated, and hence the mean of field of visions was determined per slide. Applying the RV and LV ratio reduced individual variation (Fig. 2B, C). To evaluate intraobserver reproducibility limits of agreement were calculated for the cell diameters. Data were processed with Welch's t-test, Pearson's correlation coefficient, or two-sample paired t-test, otherwise when data were not normally distributed, the nonparametric Mann–Whitney test. Fisher's exact test was used for categorical data. Two-tailed p values b0.05 was considered significant. Mean ± standard deviation presents normally distributed data, if not, the median (lower quartile–upper quartile). Statistical Analysis System (SAS) software version 9.1 was used for data analysis and Graph Pad Prism version 4 for graphic presentations.
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Fig. 2. A: Weight of right ventricles (RV) and the left ventricles weighed with the interventricular septum (LV). From pigs one month after valve implantation subsequent to three months pulmonary regurgitation – test group (T) – and from the normal pig heart — control group (C). Tested with nonparametric Mann–Whitney that only showed significant difference for the right ventricles. B: Average diameters of right (RV) and left ventricular free wall (LV) myocytes. The dotted regression lines are shown for test and control groups with slopes significant different from zero (p = 0.003 for control and p = 0.03 for test). Thus RV and LV cardiac myocyte diameters correlate well in normal pigs (r = 0.89), and the ratio, RV/LV cardiac myocyte diameter, diminishes individual myocyte size as confounding factor. C: Average of shortest diameter through cardiac myocyte nuclei in the right ventricular free wall (RV), interventricular septum (IS) and left ventricular free wall (LV). Control group RV diameters are smaller in comparison to LV: p = 0.001, and IS: p = 0.02. This is not the case in the test group, where the RV diameters are the largest in average, however, not statistically significant. The lines demonstrate measurements from the same animal, and illustrate an individual variation. D: Comparison of myocyte diameter ratios from test and control groups. A and B show RV/LV. C and D show RV/IS. IS = interventricular septum. Not shown on the figure is the IS/LV ratio, where there was no difference between control and test groups (p = 0.25).
In this porcine model, piglets were induced free PR in a normal compliant right ventricle after weaning at age 8–9 weeks with body weight 13.5 ± 1.9 kg. Three months later they had a valve implanted. One month later, they were approximately 6 months old and weighed in average 88 ± 7 kg. Danish farm pigs are sexually mature at 6 to 7 months, but can be cyclic from they are 4 months old (Pig Research Centre, Denmark), so the included pigs almost reached early adulthood. The test animals had increased statistically significant in weight (Table 1) although no difference was obvious in clinical status. All six animals in the test group had pericarditis at time of euthanasia, but none of the eight in the control group (Fig. 3A). Test group hearts were heavier than the control group hearts, p = 0.005 (Table 1). A weight difference between test and control groups was significant for right ventricles, p = 0.0007, but not for left ventricles, p = 0.85 (Fig. 2A). The coronary sulcus was wider in the test group, p = 0.005, and further from apex, p = 0.02 (Table 1).
group, where the RV diameter in general was larger than the LV and IS diameter, although not significantly, p = 0.25 and p = 0.26. In the control group, diameters of RV cardiac myocytes correlate with the LV myocytes, r = 0.89, (p = 0.003) (Fig. 2B). Thus for correction of individual variation, the ratio of RV and LV myocyte diameters was used. The ratio was significantly enlarged in test group compared to control group, p = 0.0006, suggesting RV myocytes were enlarged in the test group (Table 1, Fig. 2D). Calculating limits of agreement for intraobserver repeatability of myocyte diameters, a field of view will typically differ by less than 2 μm when measured twice. Due to shrinkage of tissue when fixated in formalin the absolute measured values are not meaningful. The greatest variation in collagen tissue from the heart chambers was in the RV myocardium where fraction per field of vision tended to be larger in control animals compared to test group, however, not statistically significant, p = 0.11, but when correcting for individual variation (RV/LV ratio) the p value increases to 0.77 (Table 1). Microscopic examination of the valved stents showed foreign giant cell reaction in the vessel wall, but this was not macroscopically visible and Doppler revealed no stenosis (Fig. 3B, C).
3.2. Microscopic measurements
3.3. Electrophysiology
In the control group, RV free wall cardiac myocyte diameter was typically 15% smaller compared to myocytes in the LV free wall and 8% smaller compared to IS myocytes. The differences were significant, p = 0.001 and p = 0.02 (Fig. 2C). This was not the case in the test
After performing the standard pacing protocol, 4 out of 6 pigs (67%) in the test group developed ventricular arrhythmia in contrast to 2 out of 8 pigs (25%) in the control group; however, the difference was not statistically significant, p = 0.28 (Table 2). The arrhythmias
3. Results 3.1. Macroscopic measurements
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Table 1 Detailed results of macro- and microscopic measures, and BNP. Variables — 4 months after baseline
Control group (sham-operated) (n = 8)
Test group (after PPVI) (n = 6)
P-value
Weight of pigs (kg) Heart weight (g) Weight of RV (g) Weight of LV + IS (g) Length of SC (cm) Length from SC to apex (cm) Myocyte diameter RV (μm) Myocyte diameter IS (μm) Myocyte diameter LV (μm) Myocyte diameter ratio RV/LV Myocyte diameter ratio RV/IS* Myocyte diameter ratio IS/LV Fibrosis RV (%) Fibrosis LV (%) Fibrosis RV/LV BNP (pg/ml)
84 ± 7 321 ± 27 71 (69.5–76.5) 170 (154–188) 10.5 (10.25–11.25) 9 (8.75–9) 13.3 (11.6–15.0) 14.3 (12.4–16.1) 15.2 (13.1–17.4) 0.84 (0.80–0.91) 0.95 (0.89–0.98) 0.91 (0.85–0.98) 11.4 (7.9–13.9) 10.8 (9.5–12.9) 1.1 (0.8–1.3) 71(62–93)
93 ± 4 426 ± 58 127 (115–137) 165 (148–214) 12.5 (12–13) 10 (10–10) 13.9 (13.8–18.0) 14.5 (12.1–15.8) 13.9 (12.7–15.7) 1.06 (1.02–1.13) 1.08 (0.97–1.15) 0.94 (0.89–1.02) 8.3 (6.2–10.4) 7.5 (6.9–9.6) 1.0 (0.80–1.3) 64 (57–68)
0.02* 0.005* 0.0007* 0.85 0.005* 0.02* 0.09 0.91 0.53 0.0006* 0.03* 0.25 0.11 0.52 0.77 0.68
Detailed results of a test group examined one month after PPVI subsequent to 3 months of pulmonary regurgitation, and compared with a sham-operated control group. Presented as mean ± standard deviation or median (lower quartile–upper quartile). PPVI = percutaneous pulmonary valve implantation, RV = right ventricular free wall, LV = left ventricular free wall, IS = interventricular septum, SC = coronary sulcus, BNP = B-type natriuretic peptide. *Statistically significant, p b 0.05.
were rapidly treated by cardioversion in all animals, except for the one pig having VT, which terminated spontaneously. 3.4. Echocardiography In the test group, geometrical parameters were significantly increased after 3 months of PR compared to sham-operated (Table 3). After PPVI, RV internal diameters in diastole and systole normalised, p = 0.2 and p = 0.33, whereas the area in systole was still increased compared to the control animals p = 0.045. And to a lesser non-significant extent in diastole, p = 0.08. There was no difference between test and control groups in RV anterior wall thickness after three months of free PR neither after one month with a functional implanted valve. In the control group, the myocardial thickening increased significantly from third to fourth month from baseline (p = 0.04). All test group functional parameters were not different from those in the control group before or after PPVI (Table 3). No correlations were found when testing RV/LV myocyte diameter ratio with echocardiographic parameters, data not shown. 3.5. Biochemical testing There was no difference between control and test groups in BNP plasma concentrations, p = 0.68 (Table 1). 4. Discussion In our study, the right ventricle did not completely recover one month after minimal invasive intervention – PPVI – subsequent to 3 months of free PR. The PR caused ventricular volume overload and eccentric hypertrophy in the pigs. One month after valve implantation, histology revealed enlarged RV cardiomyocyte diameters corresponding to the increased weight of the right ventricle, expanded heart dimensions, and RV dilation on echocardiography [17]. Echocardiography, however, disclosed a normal RV function. Furthermore, there was no excessive collagen tissue and natriuretic peptides in plasma were not increased. Four of six animals from the test group were induced VF or VT after RV pacing. Interestingly, the two right ventricles that did not develop RV arrhythmia were the ones that weighed the least (Table 2). However, no significant difference
Fig. 3. A: The base of the heart (left) and the apex area (right) from two different hearts in the test group. The outflow tract possibly gave way when implanting the baseline 22 mm CP stent causing a pericarditis demonstrated by the tweezers. B and C: The implanted valve in formalin cut from the hearts of two different pigs in test group. Only post mortal coagula were present macroscopically. Relative stenosis due to fixed stent size might have occurred in the late stages in the test pigs.
was found between groups, possibly due to the limited number of animals. Other groups have used the pig for studying PR [18–20]. Our porcine model was the first pure model, free of scar tissue, which was examined with morphometric methods and electrophysiology. A porcine electrophysiological study by Zeltser et al., young piglets Table 2 The electrophysiology study. Control group
Test group
REF REF REF REF 1 VES SVT VF VF
REFa 2 × 2 VESa VT VF VF VF
VF = ventricular fibrillation, VT = ventricular tachycardia, REF = refractory ventricle, VES = ventricular extrasystoles and SVT = supraventricular tachycardia. a Those two pigs had the lightest right ventricles (91 g and 115 g) and hearts (367 g and 370 g) within the test group.
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Acknowledgements
Table 3 The echocardiography study. Variables
RVAWd RVIDd RVIDs RVAd RVAs RVFS RVFAC TAPSE
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At 3 months after baseline
At 4 months after baseline
Control group
Test group — before PPVI
Control group
Test group — after PPVI
0.6 ± 0.1** 2.9 ± 0.3 1.8 ± 0.2 12.4 ± 2.3 5.9 ± 1.8 0.4 ± 0.1 0.5 ± 0.1 2.1 ± 0.3
0.6 ± b 0.1 4.8 ± 1.3* 2.9 ± 0.8* 19.9 ± 3.0* 8.4 ± 2.0† 0.4 ± b 0.1 0.6 ± 0.1 2.4 ± 0.5
0.7 ± 0.1** 3.2 ± 0.4 2.2 ± 0.3 12.7 ± 2.5 6.0 ± 3.5 0.3 ± 0.1 0.5 ± 0.2 2.3 ± 0.3
0.7 ± 0.2 4.0 ± 1.4 2.6 ± 0.9 23.3 ± 10.4† 11.8 ± 4.9* 0.3 ± 0.1 0.5 ± 0.1 2.5 ± 0.5
Control group includes the age-matched sham-operated pigs. Left: Test group: three months of pulmonary regurgitation on the day of PPVI, before the implantation. Right: On day of euthanasia — one month after PPVI. Previously published by Kjaergaard et al. in a different manner [17]. PPVI = percutaneous pulmonary valve implantation, RVAWd = right ventricular anterior wall thickness in diastole (cm), RVIDd = right ventricular internal diameter in diastole (cm), RVIDs = right ventricular internal diameter in systole (cm), RVAd = right ventricular end diastolic area (cm2), RVAs = right ventricular end systolic area (cm2), RVFS = right ventricular fractional shortening (cm), RVFAC = right ventricular fractional area change (cm2), TAPSE = tricuspid annular plane systolic excursion (cm).*Statistically significant, p b 0.05 (test group vs. control group of the same age). **Control group RVAWd increases significantly from 3 to 4 months of baseline. This increase must have been part of normal porcine development with a total increase in body weight of 25–30 kg. †P ≤ 0.09 (test group vs. control group of the same age).
developed RV volume overload due to pulmonary valvotomy and transannular patching. Five to six months postoperatively the growing pigs had a significantly higher incidence of inducible RV arrhythmias compared to control animals [20]. Unfortunately, the hearts were not examined pathologically like in the present study. Nevertheless, Lambert et al. also made a porcine 4 months PR model using the surgical methods of Zeltser et al. [18]. They examined RV histology and cellular electrical properties. Their histology results were based on a biased random investigation, where we chose to use an approach to the unbiased stereological methods (Fig. 1). Lambert et al.'s results were cellular hypertrophy like in our study, but they also found excess collagen. Cellular action potentials were prolonged. In comparison, our pigs were only subjected to PR for 3 months and had the valve implantation for one month. Perhaps we did not find excess collagen due to the shorter period of PR or due to our pigs having a different race. This was particularly evident in their growth, where our pigs were almost twice as heavy but younger at euthanasia. In a review on 19 studies, long term results on pulmonary valve replacement in tetralogy of Fallot late after repair varied, but implanted valves overall improved clinical outcome and RV function [21]. Some studies however had limited improvement in survival rate and incidences of ventricular tachycardia [4,5]. Progressive fibrosis is suspected to play a pathological role in development of ventricular arrhythmias on long-term follow-up after primary repair [6,7]. Our porcine electrophysiological results warrant further investigation — perhaps RV hypertrophy also can increase the arrhythmogenicity in the absence of excessive collagen.
5. Conclusions In conclusion we showed, that a normal porcine compliant right ventricle, induced free PR for 3 months, developed eccentric hypertrophy without fibrosis. And one month after implantation of a pulmonary valve the dilation and cellular hypertrophy did not fully recover. RV arrhythmogenicity increased although the tendency was not statistically significant. Further studies are needed to understand the reverse remodelling mechanisms of the arrhythmogenic right ventricle.
The authors thank the Danish Heart Foundation; Frimodt-Heineke Foundation; Beckett Foundation; Augustinus Foundation; Villadsens Family Foundation; and the Eva & Henry Frænkel Foundation for financial support. Professor Hans Jørgen Gundersen is kindly thanked for his invaluable comments to the morphological estimates and Anne Andersenfor her excellent technical assistance. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [22]. References [1] Gatzoulis MA, Balaji S, Webber SA, et al. Risk factors for arrhythmia and sudden cardiac death late after repair of tetralogy of Fallot: a multicentre study. Lancet 2000;356:975–81. [2] Oosterhof T, Vliegen HW, Meijboom FJ, Zwinderman AH, Bouma B, Mulder BJ. Long-term effect of pulmonary valve replacement on QRS duration in patients with corrected tetralogy of Fallot. Heart 2007;93:506–9. [3] Romeih S, Kroft LJ, Bokenkamp R, et al. Delayed improvement of right ventricular diastolic function and regression of right ventricular mass after percutaneous pulmonary valve implantation in patients with congenital heart disease. Am Heart J 2009;158:40–6. [4] Miyazaki A, Yamamoto M, Sakaguchi H, et al. Pulmonary valve replacement in adult patients with a severely dilated right ventricle and refractory arrhythmias after repair of tetralogy of Fallot. Circ J 2009;73:2135–42. [5] Harrild DM, Berul CI, Cecchin F, et al. Pulmonary valve replacement in tetralogy of Fallot: impact on survival and ventricular tachycardia. Circulation 2009;119: 445–51. [6] Gouw SC, Le TN, Sreeram N. Tetralogy of Fallot. Curr Treat Options Cardiovasc Med 2001;3:361–9. [7] Babu-Narayan SV, Kilner PJ, Li W, et al. Ventricular fibrosis suggested by cardiovascular magnetic resonance in adults with repaired tetralogy of Fallot and its relationship to adverse markers of clinical outcome. Circulation 2006;113: 405–13. [8] Walther T, Klostermann K, Heringer-Walther S, Schultheiss HP, Tschope C, Stepan H. Fibrosis rather than blood pressure determines cardiac BNP expression in mice. Regul Pept 2003;116:95–100. [9] Liang F, Wu J, Garami M, Gardner DG. Mechanical strain increases expression of the brain natriuretic peptide gene in rat cardiac myocytes. J Biol Chem 1997;272: 28050–6. [10] Smith J, Goetze JP, Andersen CB, Vejlstrup N. Practical application of natriuretic peptides in paediatric cardiology. Cardiol Young 2010;20:353–63. [11] Khambadkone S, Coats L, Taylor A, et al. Percutaneous pulmonary valve implantation in humans: results in 59 consecutive patients. Circulation 2005;112:1189–97. [12] Lurz P, Gaudin R, Taylor AM, Bonhoeffer P. Percutaneous pulmonary valve implantation. Semin Thorac Cardiovasc Surg Pediatr Card Surg Annu 2009:112–7. [13] Schievano S, Taylor AM, Capelli C, et al. First-in-man implantation of a novel percutaneous valve: a new approach to medical device development. EuroIntervention 2010;5:745–50. [14] Fuster V, Danielson MA, Robb RA, Broadbent JC, Brown Jr AL, Elveback LR. Quantitation of left ventricular myocardial fiber hypertrophy and interstitial tissue in human hearts with chronically increased volume and pressure overload. Circulation 1977;55:504–8. [15] Hughson MD, Samuel T, Hoy WE, Bertram JF. Glomerular volume and clinicopathologic features related to disease severity in renal biopsies of African Americans and Whites in the southeastern United States. Arch Pathol Lab Med 2007;131:1665–72. [16] Thilagarajah R, Witherow RO, Walker MM. Quantitative histopathology can aid diagnosis in painful bladder syndrome. J Clin Pathol 1998;51:211–4. [17] Kjaergaard J, Iversen KK, Vejlstrup NG, et al. Effects of chronic severe pulmonary regurgitation and percutaneous valve repair on right ventricular geometry and contractility assessed by tissue Doppler echocardiography. Echocardiography 2010;27:854–63. [18] Lambert V., Capderou A., Le B.E. et al. Right ventricular failure secondary to chronic overload in congenital heart disease: an experimental model for therapeutic innovation. J Thorac Cardiovasc Surg 2010; 139:1197–204, 1204. [19] Thambo JB, Roubertie F, De GM, et al. Validation of an animal model of right ventricular dysfunction and right bundle branch block to create close physiology to postoperative tetralogy of Fallot. Int J Cardiol (2010), doi:10.1016/j.ijcard.2010.08.063. [20] Zeltser I, Gaynor JW, Petko M, et al. The roles of chronic pressure and volume overload states in induction of arrhythmias: an animal model of physiologic sequelae after repair of tetralogy of Fallot. J Thorac Cardiovasc Surg 2005;130:1542–8. [21] Adamson L, Vohra HA, Haw MP. Does pulmonary valve replacement post repair of tetralogy of Fallot improve right ventricular function? Interact Cardiovasc Thorac Surg 2009;9:520–7. [22] Shewan LG, Coats AJ. Ethics in the authorship and publishing of scientific articles. Int J Cardiol 2010;144:1–2.
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International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d
Myocardial hypertrophy after pulmonary regurgitation and valve implantation in pigs☆ Julie Smith a,b, Jens Peter Goetze b, Lars Søndergaard c, Jesper Kjaergaard c, Kasper K. Iversen c, Niels G. Vejlstrup c, Christian Hassager c, Claus B. Andersen a,⁎ a b c
Department of Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Department Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark Department Cardiology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
a r t i c l e
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Article history: Received 21 December 2010 Accepted 6 February 2011 Available online 15 March 2011 Keywords: Arrhythmia Fibrosis Histology Myocardial hypertrophy Pulmonary regurgitation Valve replacement
a b s t r a c t Background: Patients may suffer from right ventricular (RV) failure and malignant cardiac arrhythmias after late pulmonary valve replacement correcting pulmonary regurgitation (PR). But the underlying mechanisms of the refractory arrhythmias are not well understood. Methods: The aim of present study was to characterize the RV myocardium after percutaneous pulmonary valve implantation (PPVI) in a porcine model after severe PR for 3 months. RV histology was evaluated with morphometric methods and RV function was assessed with electrophysiology, echocardiography, and biochemical measures: The results were compared with age-matched sham-operated animals. Results: At euthanasia, RV weight was increased compared to sham-animals, median 127 g (115–137) vs. 71 g (69.5–76.5), p = 0.0007. RV myocyte diameters corrected for individual variation with the RV/LV ratio were enlarged, 1.06 (1.02–1.13) vs. 0.84 (0.80–0.91), p = 0.0006. There were no excess collagen tissue (RV/LV ratio), p = 0.77. Electrophysiological stimulation resulted in RV arrhythmia in 67% of the animals compared to 25% in the sham-operated animals, but this difference was not statistically significant, p = 0.28. Echocardiography revealed geometrical dilation in end-systolic RV area, mean ± SD, 11.8 ± 4.9 cm2 vs. 6.0 ± 3.5 cm2, p = 0.05, and end-diastolic area, 23.3 ± 10.4 cm2 vs. 12.7 ± 2.5 cm2, p = 0.08. RV anterior free wall thickness was not increased, 0.7 ± 0.2 cm vs. 0.7 ± 0.1 cm, p = 0.66. Echocardiographic functional parameters and plasma natriuretic peptides were unchanged. Conclusions: The RV does not completely recover after three months of PR with persistent myocardial hypertrophy one month after PPVI. Future studies should address whether RV chamber and cellular hypertrophy, without fibrosis or interventional scar tissue, may be substrate for arrhythmia. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Pulmonary regurgitation (PR) can be the main hemodynamic lesion after repair of tetralogy of Fallot and is well tolerated with most children reaching adulthood. Exercise intolerance, atrial and ventricular arrhythmias are however still major complications, including an increased risk of sudden death [1]. Valve replacement is able to improve right ventricular (RV) function, but artificial valves
☆ Funding: This work was supported by the Heart Centre Research Committee, Rigshospitalet; Medtronic Inc®. Minneapolis, MN, USA; NuMed®, Hopkinton, NY, USA; the Danish Heart Foundation ref. no. 05-4-B269-A534-22220; the Frimodt-Heineke Foundation; the Beckett Foundation; the Augustinus Foundation; the Villadsens Family Foundation; and the Eva & Henry Frænkel Foundation. ⁎ Corresponding author at: Department of Pathology (5444), Rigshospitalet, University of Copenhagen, 9 Blegdamsvej, DK-2100 Copenhagen, Denmark. Tel.: +45 3545 5344; fax: +45 3545 5414. E-mail address: [email protected] (C.B. Andersen). 0167-5273/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2011.02.022
have limited durability, consequently the optimal time for replacement remains controversial [2]. After valve replacement, RV systolic function can normalise rapidly while diastolic function and the RV wall mass only recover gradually [3]. The right ventricle does, however, not always completely recover following late pulmonary valve replacement with sustained risk of refractory arrhythmia and RV dysfunction [4,5]. The cellular mechanisms of the reverse remodelling process are not well understood. Fibrosis is believed to play a role in the refractory arrhythmias [6,7]. RV dilation and fibrosis can be associated with increased release of natriuretic peptides to plasma [8–10], whereas correction of RV volume overload might normalise peptide concentrations. Minimally invasive treatment for correcting pulmonary outflow tract dysfunction has been introduced during the last five years [11]. And percutaneous pulmonary valve implantation (PPVI) can postpone open-heart surgery, but requires an existing conduit [12,13]. In the present study, young piglets were subjected to acute PR for 3 months by percutaneous stenting followed by PPVI for 1 month. The
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aim was to characterize the myocardium in a porcine model with no interventional scar tissue. The study focused on myocyte hypertrophy and collagen fraction measured with gold-standard morphometric methods. Before euthanasia, the pigs underwent programmed electrical stimulation, echocardiography, and measurement of natriuretic peptide concentrations in plasma. 2. Materials and methods 2.1. Animal procedures During RV heart catheterisation, a 22 mm CP™ stent (NuMed, USA) was introduced via the femoral vein and positioned in the pulmonary valvular annulus in six piglets, crossbreeds of Danish Landrace, Yorkshire and Duroc. After three months with free PR, a bovine jugular venous valve sutured inside a stent (Medtronic Melody™, Medtronic Inc., USA) was mounted in the CP™ stent. A lethal dose of pentobarbital (200 mg/ml) was injected intravenously one month after implantation of the stented valve. A matching group of eight controls underwent the same heart catheterisation procedures, but no stents were implanted. All pigs were sedated with midazolam (0.5 mg/kg), and general anaesthesia was induced and maintained by intramuscular injections of a zoletil mixture (1 ml/10 kg for the induction). This mixture consisted of 125 mg tiletamin and 125 mg zolazepam, added 6.5 ml xylazine (20 mg/ml), 1.5 ml ketamin (100 mg/ml) and 2.5 ml sterile water or methadone (10 mg/ml). To counter the effects, naloxone (0.4 mg/40 kg) and atipamezole hydrochloride (5 mg/40 kg) were injected. Antiarrhythmic amiodarone (5 mg/kg) and anticoagulant heparin were administered intravenously as standard therapy during cardiac catheterisation (start dose 100 IU/kg, and 50 IU/kg per half hour). On the day of euthanasia and in control animals, antiarrhythmics were not administered during catheterisation procedures. Anticoagulant enoxaparin (10 mg/10 kg twice a day) was subcutaneous injected for two days, and after valve implantation acetylsalicylic acid (0.1 mg/30 kg) for four weeks. Non-steroidal anti-inflammatory drug carprofen (50 mg/ 12 kg) and antibiotics dihydrostreptomycin (25 mg/kg) with benzylpenicillinprocain (20,000 IU/kg) in a mixture were injected intramuscularly for 3 days after the two first interventions. The Animal Experiments Inspectorate, Ministry of Justice, Denmark, approved the research project (ref. no. 2005/561-1010). 2.2. Morphometric methods for measuring myocardial hypertrophy and collagen tissue To analyse RV tissue, the porcine hearts were collected immediately after euthanasia. The width of the heart along the coronary sulcus and length from sulcus to apex was measured. By cutting with a pair of scissors along the coronary sulcus and bilateral to apex, excluding the interventricular septum (IS) and the pulmonary trunk, the RV free wall was isolated and weighed. The left ventricle (LV) was isolated and weighed together with the IS. All parts were preserved in formalin, and for estimating the degree of myocardial collagen and hypertrophy, three biased arbitrary tissue sections (1 × 1 cm) from the RV free wall, two from the LV and two from the IS were cut and embedded in paraffin. To make the basement membrane distinct, sections of 5 μm thickness were stained with periodic acid silver methenamine (PASM). The specimens were magnified 40 times with a Leica DMR microscope. An unbiased systematic uniformly random sampling (SURS) gave 8–12 fields of visions per slide each transformed with Leica DFC 420 C camera to a digital screen image by using the Leica Image Manager software version IM50. An unbiased counting frame mounted on the screen included approximately one to five profiles (Fig. 1). The shortest diameter was measured going centrally through the nucleus by using the vector application in the IM50 software (Fig. 1B) [14]. For estimating the fraction of collagen tissue, new slides from the same paraffin blocks were stained with Masson's trichrome. On a slide, 8–12 fields of view were selected unbiased with the SURS method and magnified 40 times. An unbiased grid with 100 points was mounted on the screen and points touching blue stain were counted to estimate the fraction of collagen [15,16]. Measurements were blinded and all completed by one person.
Fig. 1. A: A field of vision from the right myocardium showed with an unbiased counting frame. Cells are not included when touching the thick “forbidden” lines. Notice the multinucleated porcine cardiac myocytes. Periodic acid silver methenamine (PASM) stain. B: In this field of vision from the porcine right ventricle five cardiac myocytes are measured based on the unbiased counting frame. PASM stain. performed on the control group for the same two age groups. In supine position a 3.5 MHz transducer transmitted four-chamber, long- and short axis views imaged by GE Vivid 7 Dimension system and analysed using GE EchoPac SWO version 6 (GE, Horten, Norway). Results on echocardiography have previously been published [17], but relevant data related to the histological studies are presented here. 2.5. Natriuretic peptides in plasma After femoral venous access before starting the actual heart catheterisation procedures on the day of euthanasia, 10 ml of blood was collected and immediately transferred into Na2-EDTA tubes (1.5 mg/ml), centrifuged, and the plasma was stored at − 80 °C. For measuring B-type natriuretic peptide (BNP) in porcine plasma, samples were sent to Phoenix Europe, Karlsruhe, Germany, where one ml of plasma per sample was used for their BNP-32 porcine Enzyme Immunoassay kit (porcine BNP EIA kit). The inter- and intra assay variations were b14% and b 5% respectively and there was a minimum detectible concentration of 0.23 ng/ml.
2.3. Electrophysiological testing 2.6. Statistics On the day of euthanasia, a 5 French quadripolar electrode catheter (Josephson, Medtronic, USA) was placed in the porcine RV apex via the femoral vein. The standard ventricular stimulation protocol for patients in the Cardiac Catheterisation Laboratory was applied comprising of 8 beats at cycle length of 400 to 500 ms and introduction of up to triple extrastimuli, delivered by a programmed electrical stimulator (EP-TRACER, Cardio Tek, Maastricht, Netherlands). The ventricles were considered arrhythmogenic when the stimulation induced ventricular fibrillation (VF) or ventricular tachycardia (VT). 2.4. Echocardiography After three months of free PR, cardiac function was assessed with echocardiographic imaging during anaesthesia before the PPVI procedures. Measurements were repeated one month later on the day of euthanasia. Comparable measurements were
To assess distributions, Bland–Altman for paired data, histograms, and residual plots, were interpreted and if not normally distributed, transformation by logarithm 10 was applied on primary data. Cell diameters were transformed and mean of one field of vision was calculated, and hence the mean of field of visions was determined per slide. Applying the RV and LV ratio reduced individual variation (Fig. 2B, C). To evaluate intraobserver reproducibility limits of agreement were calculated for the cell diameters. Data were processed with Welch's t-test, Pearson's correlation coefficient, or two-sample paired t-test, otherwise when data were not normally distributed, the nonparametric Mann–Whitney test. Fisher's exact test was used for categorical data. Two-tailed p values b0.05 was considered significant. Mean ± standard deviation presents normally distributed data, if not, the median (lower quartile–upper quartile). Statistical Analysis System (SAS) software version 9.1 was used for data analysis and Graph Pad Prism version 4 for graphic presentations.
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Fig. 2. A: Weight of right ventricles (RV) and the left ventricles weighed with the interventricular septum (LV). From pigs one month after valve implantation subsequent to three months pulmonary regurgitation – test group (T) – and from the normal pig heart — control group (C). Tested with nonparametric Mann–Whitney that only showed significant difference for the right ventricles. B: Average diameters of right (RV) and left ventricular free wall (LV) myocytes. The dotted regression lines are shown for test and control groups with slopes significant different from zero (p = 0.003 for control and p = 0.03 for test). Thus RV and LV cardiac myocyte diameters correlate well in normal pigs (r = 0.89), and the ratio, RV/LV cardiac myocyte diameter, diminishes individual myocyte size as confounding factor. C: Average of shortest diameter through cardiac myocyte nuclei in the right ventricular free wall (RV), interventricular septum (IS) and left ventricular free wall (LV). Control group RV diameters are smaller in comparison to LV: p = 0.001, and IS: p = 0.02. This is not the case in the test group, where the RV diameters are the largest in average, however, not statistically significant. The lines demonstrate measurements from the same animal, and illustrate an individual variation. D: Comparison of myocyte diameter ratios from test and control groups. A and B show RV/LV. C and D show RV/IS. IS = interventricular septum. Not shown on the figure is the IS/LV ratio, where there was no difference between control and test groups (p = 0.25).
In this porcine model, piglets were induced free PR in a normal compliant right ventricle after weaning at age 8–9 weeks with body weight 13.5 ± 1.9 kg. Three months later they had a valve implanted. One month later, they were approximately 6 months old and weighed in average 88 ± 7 kg. Danish farm pigs are sexually mature at 6 to 7 months, but can be cyclic from they are 4 months old (Pig Research Centre, Denmark), so the included pigs almost reached early adulthood. The test animals had increased statistically significant in weight (Table 1) although no difference was obvious in clinical status. All six animals in the test group had pericarditis at time of euthanasia, but none of the eight in the control group (Fig. 3A). Test group hearts were heavier than the control group hearts, p = 0.005 (Table 1). A weight difference between test and control groups was significant for right ventricles, p = 0.0007, but not for left ventricles, p = 0.85 (Fig. 2A). The coronary sulcus was wider in the test group, p = 0.005, and further from apex, p = 0.02 (Table 1).
group, where the RV diameter in general was larger than the LV and IS diameter, although not significantly, p = 0.25 and p = 0.26. In the control group, diameters of RV cardiac myocytes correlate with the LV myocytes, r = 0.89, (p = 0.003) (Fig. 2B). Thus for correction of individual variation, the ratio of RV and LV myocyte diameters was used. The ratio was significantly enlarged in test group compared to control group, p = 0.0006, suggesting RV myocytes were enlarged in the test group (Table 1, Fig. 2D). Calculating limits of agreement for intraobserver repeatability of myocyte diameters, a field of view will typically differ by less than 2 μm when measured twice. Due to shrinkage of tissue when fixated in formalin the absolute measured values are not meaningful. The greatest variation in collagen tissue from the heart chambers was in the RV myocardium where fraction per field of vision tended to be larger in control animals compared to test group, however, not statistically significant, p = 0.11, but when correcting for individual variation (RV/LV ratio) the p value increases to 0.77 (Table 1). Microscopic examination of the valved stents showed foreign giant cell reaction in the vessel wall, but this was not macroscopically visible and Doppler revealed no stenosis (Fig. 3B, C).
3.2. Microscopic measurements
3.3. Electrophysiology
In the control group, RV free wall cardiac myocyte diameter was typically 15% smaller compared to myocytes in the LV free wall and 8% smaller compared to IS myocytes. The differences were significant, p = 0.001 and p = 0.02 (Fig. 2C). This was not the case in the test
After performing the standard pacing protocol, 4 out of 6 pigs (67%) in the test group developed ventricular arrhythmia in contrast to 2 out of 8 pigs (25%) in the control group; however, the difference was not statistically significant, p = 0.28 (Table 2). The arrhythmias
3. Results 3.1. Macroscopic measurements
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Table 1 Detailed results of macro- and microscopic measures, and BNP. Variables — 4 months after baseline
Control group (sham-operated) (n = 8)
Test group (after PPVI) (n = 6)
P-value
Weight of pigs (kg) Heart weight (g) Weight of RV (g) Weight of LV + IS (g) Length of SC (cm) Length from SC to apex (cm) Myocyte diameter RV (μm) Myocyte diameter IS (μm) Myocyte diameter LV (μm) Myocyte diameter ratio RV/LV Myocyte diameter ratio RV/IS* Myocyte diameter ratio IS/LV Fibrosis RV (%) Fibrosis LV (%) Fibrosis RV/LV BNP (pg/ml)
84 ± 7 321 ± 27 71 (69.5–76.5) 170 (154–188) 10.5 (10.25–11.25) 9 (8.75–9) 13.3 (11.6–15.0) 14.3 (12.4–16.1) 15.2 (13.1–17.4) 0.84 (0.80–0.91) 0.95 (0.89–0.98) 0.91 (0.85–0.98) 11.4 (7.9–13.9) 10.8 (9.5–12.9) 1.1 (0.8–1.3) 71(62–93)
93 ± 4 426 ± 58 127 (115–137) 165 (148–214) 12.5 (12–13) 10 (10–10) 13.9 (13.8–18.0) 14.5 (12.1–15.8) 13.9 (12.7–15.7) 1.06 (1.02–1.13) 1.08 (0.97–1.15) 0.94 (0.89–1.02) 8.3 (6.2–10.4) 7.5 (6.9–9.6) 1.0 (0.80–1.3) 64 (57–68)
0.02* 0.005* 0.0007* 0.85 0.005* 0.02* 0.09 0.91 0.53 0.0006* 0.03* 0.25 0.11 0.52 0.77 0.68
Detailed results of a test group examined one month after PPVI subsequent to 3 months of pulmonary regurgitation, and compared with a sham-operated control group. Presented as mean ± standard deviation or median (lower quartile–upper quartile). PPVI = percutaneous pulmonary valve implantation, RV = right ventricular free wall, LV = left ventricular free wall, IS = interventricular septum, SC = coronary sulcus, BNP = B-type natriuretic peptide. *Statistically significant, p b 0.05.
were rapidly treated by cardioversion in all animals, except for the one pig having VT, which terminated spontaneously. 3.4. Echocardiography In the test group, geometrical parameters were significantly increased after 3 months of PR compared to sham-operated (Table 3). After PPVI, RV internal diameters in diastole and systole normalised, p = 0.2 and p = 0.33, whereas the area in systole was still increased compared to the control animals p = 0.045. And to a lesser non-significant extent in diastole, p = 0.08. There was no difference between test and control groups in RV anterior wall thickness after three months of free PR neither after one month with a functional implanted valve. In the control group, the myocardial thickening increased significantly from third to fourth month from baseline (p = 0.04). All test group functional parameters were not different from those in the control group before or after PPVI (Table 3). No correlations were found when testing RV/LV myocyte diameter ratio with echocardiographic parameters, data not shown. 3.5. Biochemical testing There was no difference between control and test groups in BNP plasma concentrations, p = 0.68 (Table 1). 4. Discussion In our study, the right ventricle did not completely recover one month after minimal invasive intervention – PPVI – subsequent to 3 months of free PR. The PR caused ventricular volume overload and eccentric hypertrophy in the pigs. One month after valve implantation, histology revealed enlarged RV cardiomyocyte diameters corresponding to the increased weight of the right ventricle, expanded heart dimensions, and RV dilation on echocardiography [17]. Echocardiography, however, disclosed a normal RV function. Furthermore, there was no excessive collagen tissue and natriuretic peptides in plasma were not increased. Four of six animals from the test group were induced VF or VT after RV pacing. Interestingly, the two right ventricles that did not develop RV arrhythmia were the ones that weighed the least (Table 2). However, no significant difference
Fig. 3. A: The base of the heart (left) and the apex area (right) from two different hearts in the test group. The outflow tract possibly gave way when implanting the baseline 22 mm CP stent causing a pericarditis demonstrated by the tweezers. B and C: The implanted valve in formalin cut from the hearts of two different pigs in test group. Only post mortal coagula were present macroscopically. Relative stenosis due to fixed stent size might have occurred in the late stages in the test pigs.
was found between groups, possibly due to the limited number of animals. Other groups have used the pig for studying PR [18–20]. Our porcine model was the first pure model, free of scar tissue, which was examined with morphometric methods and electrophysiology. A porcine electrophysiological study by Zeltser et al., young piglets Table 2 The electrophysiology study. Control group
Test group
REF REF REF REF 1 VES SVT VF VF
REFa 2 × 2 VESa VT VF VF VF
VF = ventricular fibrillation, VT = ventricular tachycardia, REF = refractory ventricle, VES = ventricular extrasystoles and SVT = supraventricular tachycardia. a Those two pigs had the lightest right ventricles (91 g and 115 g) and hearts (367 g and 370 g) within the test group.
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Acknowledgements
Table 3 The echocardiography study. Variables
RVAWd RVIDd RVIDs RVAd RVAs RVFS RVFAC TAPSE
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At 3 months after baseline
At 4 months after baseline
Control group
Test group — before PPVI
Control group
Test group — after PPVI
0.6 ± 0.1** 2.9 ± 0.3 1.8 ± 0.2 12.4 ± 2.3 5.9 ± 1.8 0.4 ± 0.1 0.5 ± 0.1 2.1 ± 0.3
0.6 ± b 0.1 4.8 ± 1.3* 2.9 ± 0.8* 19.9 ± 3.0* 8.4 ± 2.0† 0.4 ± b 0.1 0.6 ± 0.1 2.4 ± 0.5
0.7 ± 0.1** 3.2 ± 0.4 2.2 ± 0.3 12.7 ± 2.5 6.0 ± 3.5 0.3 ± 0.1 0.5 ± 0.2 2.3 ± 0.3
0.7 ± 0.2 4.0 ± 1.4 2.6 ± 0.9 23.3 ± 10.4† 11.8 ± 4.9* 0.3 ± 0.1 0.5 ± 0.1 2.5 ± 0.5
Control group includes the age-matched sham-operated pigs. Left: Test group: three months of pulmonary regurgitation on the day of PPVI, before the implantation. Right: On day of euthanasia — one month after PPVI. Previously published by Kjaergaard et al. in a different manner [17]. PPVI = percutaneous pulmonary valve implantation, RVAWd = right ventricular anterior wall thickness in diastole (cm), RVIDd = right ventricular internal diameter in diastole (cm), RVIDs = right ventricular internal diameter in systole (cm), RVAd = right ventricular end diastolic area (cm2), RVAs = right ventricular end systolic area (cm2), RVFS = right ventricular fractional shortening (cm), RVFAC = right ventricular fractional area change (cm2), TAPSE = tricuspid annular plane systolic excursion (cm).*Statistically significant, p b 0.05 (test group vs. control group of the same age). **Control group RVAWd increases significantly from 3 to 4 months of baseline. This increase must have been part of normal porcine development with a total increase in body weight of 25–30 kg. †P ≤ 0.09 (test group vs. control group of the same age).
developed RV volume overload due to pulmonary valvotomy and transannular patching. Five to six months postoperatively the growing pigs had a significantly higher incidence of inducible RV arrhythmias compared to control animals [20]. Unfortunately, the hearts were not examined pathologically like in the present study. Nevertheless, Lambert et al. also made a porcine 4 months PR model using the surgical methods of Zeltser et al. [18]. They examined RV histology and cellular electrical properties. Their histology results were based on a biased random investigation, where we chose to use an approach to the unbiased stereological methods (Fig. 1). Lambert et al.'s results were cellular hypertrophy like in our study, but they also found excess collagen. Cellular action potentials were prolonged. In comparison, our pigs were only subjected to PR for 3 months and had the valve implantation for one month. Perhaps we did not find excess collagen due to the shorter period of PR or due to our pigs having a different race. This was particularly evident in their growth, where our pigs were almost twice as heavy but younger at euthanasia. In a review on 19 studies, long term results on pulmonary valve replacement in tetralogy of Fallot late after repair varied, but implanted valves overall improved clinical outcome and RV function [21]. Some studies however had limited improvement in survival rate and incidences of ventricular tachycardia [4,5]. Progressive fibrosis is suspected to play a pathological role in development of ventricular arrhythmias on long-term follow-up after primary repair [6,7]. Our porcine electrophysiological results warrant further investigation — perhaps RV hypertrophy also can increase the arrhythmogenicity in the absence of excessive collagen.
5. Conclusions In conclusion we showed, that a normal porcine compliant right ventricle, induced free PR for 3 months, developed eccentric hypertrophy without fibrosis. And one month after implantation of a pulmonary valve the dilation and cellular hypertrophy did not fully recover. RV arrhythmogenicity increased although the tendency was not statistically significant. Further studies are needed to understand the reverse remodelling mechanisms of the arrhythmogenic right ventricle.
The authors thank the Danish Heart Foundation; Frimodt-Heineke Foundation; Beckett Foundation; Augustinus Foundation; Villadsens Family Foundation; and the Eva & Henry Frænkel Foundation for financial support. Professor Hans Jørgen Gundersen is kindly thanked for his invaluable comments to the morphological estimates and Anne Andersenfor her excellent technical assistance. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [22]. References [1] Gatzoulis MA, Balaji S, Webber SA, et al. Risk factors for arrhythmia and sudden cardiac death late after repair of tetralogy of Fallot: a multicentre study. Lancet 2000;356:975–81. [2] Oosterhof T, Vliegen HW, Meijboom FJ, Zwinderman AH, Bouma B, Mulder BJ. Long-term effect of pulmonary valve replacement on QRS duration in patients with corrected tetralogy of Fallot. Heart 2007;93:506–9. [3] Romeih S, Kroft LJ, Bokenkamp R, et al. Delayed improvement of right ventricular diastolic function and regression of right ventricular mass after percutaneous pulmonary valve implantation in patients with congenital heart disease. Am Heart J 2009;158:40–6. [4] Miyazaki A, Yamamoto M, Sakaguchi H, et al. Pulmonary valve replacement in adult patients with a severely dilated right ventricle and refractory arrhythmias after repair of tetralogy of Fallot. Circ J 2009;73:2135–42. [5] Harrild DM, Berul CI, Cecchin F, et al. Pulmonary valve replacement in tetralogy of Fallot: impact on survival and ventricular tachycardia. Circulation 2009;119: 445–51. [6] Gouw SC, Le TN, Sreeram N. Tetralogy of Fallot. Curr Treat Options Cardiovasc Med 2001;3:361–9. [7] Babu-Narayan SV, Kilner PJ, Li W, et al. Ventricular fibrosis suggested by cardiovascular magnetic resonance in adults with repaired tetralogy of Fallot and its relationship to adverse markers of clinical outcome. Circulation 2006;113: 405–13. [8] Walther T, Klostermann K, Heringer-Walther S, Schultheiss HP, Tschope C, Stepan H. Fibrosis rather than blood pressure determines cardiac BNP expression in mice. Regul Pept 2003;116:95–100. [9] Liang F, Wu J, Garami M, Gardner DG. Mechanical strain increases expression of the brain natriuretic peptide gene in rat cardiac myocytes. J Biol Chem 1997;272: 28050–6. [10] Smith J, Goetze JP, Andersen CB, Vejlstrup N. Practical application of natriuretic peptides in paediatric cardiology. Cardiol Young 2010;20:353–63. [11] Khambadkone S, Coats L, Taylor A, et al. Percutaneous pulmonary valve implantation in humans: results in 59 consecutive patients. Circulation 2005;112:1189–97. [12] Lurz P, Gaudin R, Taylor AM, Bonhoeffer P. Percutaneous pulmonary valve implantation. Semin Thorac Cardiovasc Surg Pediatr Card Surg Annu 2009:112–7. [13] Schievano S, Taylor AM, Capelli C, et al. First-in-man implantation of a novel percutaneous valve: a new approach to medical device development. EuroIntervention 2010;5:745–50. [14] Fuster V, Danielson MA, Robb RA, Broadbent JC, Brown Jr AL, Elveback LR. Quantitation of left ventricular myocardial fiber hypertrophy and interstitial tissue in human hearts with chronically increased volume and pressure overload. Circulation 1977;55:504–8. [15] Hughson MD, Samuel T, Hoy WE, Bertram JF. Glomerular volume and clinicopathologic features related to disease severity in renal biopsies of African Americans and Whites in the southeastern United States. Arch Pathol Lab Med 2007;131:1665–72. [16] Thilagarajah R, Witherow RO, Walker MM. Quantitative histopathology can aid diagnosis in painful bladder syndrome. J Clin Pathol 1998;51:211–4. [17] Kjaergaard J, Iversen KK, Vejlstrup NG, et al. Effects of chronic severe pulmonary regurgitation and percutaneous valve repair on right ventricular geometry and contractility assessed by tissue Doppler echocardiography. Echocardiography 2010;27:854–63. [18] Lambert V., Capderou A., Le B.E. et al. Right ventricular failure secondary to chronic overload in congenital heart disease: an experimental model for therapeutic innovation. J Thorac Cardiovasc Surg 2010; 139:1197–204, 1204. [19] Thambo JB, Roubertie F, De GM, et al. Validation of an animal model of right ventricular dysfunction and right bundle branch block to create close physiology to postoperative tetralogy of Fallot. Int J Cardiol (2010), doi:10.1016/j.ijcard.2010.08.063. [20] Zeltser I, Gaynor JW, Petko M, et al. The roles of chronic pressure and volume overload states in induction of arrhythmias: an animal model of physiologic sequelae after repair of tetralogy of Fallot. J Thorac Cardiovasc Surg 2005;130:1542–8. [21] Adamson L, Vohra HA, Haw MP. Does pulmonary valve replacement post repair of tetralogy of Fallot improve right ventricular function? Interact Cardiovasc Thorac Surg 2009;9:520–7. [22] Shewan LG, Coats AJ. Ethics in the authorship and publishing of scientific articles. Int J Cardiol 2010;144:1–2.