Author + information
- Received July 20, 2010
- Accepted August 24, 2010
- Published online February 22, 2011.
- Maxim Hardziyenka, MD, PhD⁎,§,
- Maria E. Campian, MD⁎,
- Herre J. Reesink, MD, PhD†,
- Sulaiman Surie, MD†,
- Berto J. Bouma, MD, PhD‡,
- Maarten Groenink, MD, PhD‡,
- Christine A. Klemens, BSc⁎,§,
- Leander Beekman, BSc⁎,
- Carol A. Remme, MD, PhD⁎,
- Paul Bresser, MD, PhD† and
- Hanno L. Tan, MD, PhD⁎,‡,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Hanno L. Tan, Department of Cardiology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands
Objectives We sought to study whether patients with right ventricular failure (RVF) secondary to chronic thromboembolic pulmonary hypertension (CTEPH) have reduced left ventricular (LV) mass, and whether LV mass reduction is caused by atrophy.
Background The LV in patients with CTEPH is underfilled (unloaded). LV unloading may cause atrophic remodeling that is associated with diastolic and systolic dysfunction.
Methods We studied LV mass using cardiac magnetic resonance imaging (MRI) in 36 consecutive CTEPH patients (before/after pulmonary endarterectomy [PEA]) and 11 healthy volunteers selected to match age and sex of patients. We studied whether LV atrophy is present in monocrotaline (MCT)-injected rats with RVF or controls by measuring myocyte dimensions and performing in situ hybridization.
Results At baseline, CTEPH patients with RVF had significantly lower LV free wall mass indexes than patients without RVF (35 ± 6 g/m2 vs. 44 ± 7 g/m2, p = 0.007) or volunteers (42 ± 6 g/m2, p = 0.006). After PEA, LV free wall mass index increased (from 38 ± 6 g/m2 to 44 ± 9 g/m2, p = 0.001), as right ventricular (RV) ejection fraction improved (from 31 ± 8% to 56 ± 12%, p < 0.001). Compared with controls, rats with RVF had reduced LV free wall mass and smaller LV free wall myocytes. Expression of atrial natriuretic peptide was higher, whereas that of α-myosin heavy chain and sarcoplasmic reticulum calcium ATPase-2 were lower in RVF than in controls, both in RV and LV.
Conclusions RVF in patients with CTEPH is associated with reversible reduction in LV free wall mass. In a rat model of RVF, myocyte shrinkage due to atrophic remodeling contributed to reduction in LV free wall mass.
Right ventricular failure (RVF) secondary to chronic pressure overload determines survival in patients with chronic thromboembolic pulmonary hypertension (CTEPH) and other forms of pulmonary arterial hypertension (PAH) (1). The mechanisms underlying the development of heart failure in these patients are not fully understood. Both right ventricular (RV) and left ventricular (LV) dysfunction occur in patients with CTEPH (2–4) and other forms of chronic PAH (5–8). This may be based on the fact that RV and LV function are closely interdependent (9). In particular, because diastolic LV peak filling rate relates directly to RV ejection fraction (7), LV diastolic filling is diminished in patients with CTEPH (3,10) and other forms of chronic PAH (6–8). This may cause LV unloading and atrophy. As the diastolic and systolic function of fully (11) or partially (12,13) unloaded LV is impaired due to atrophic remodeling (14–16), we hypothesized that LV atrophy occurs in pressure overload–associated RVF, and that it contributes to heart failure pathophysiology. The aim of the present study was to provide evidence for reduction in LV mass in CTEPH patients with RVF with the use of cardiac magnetic resonance imaging (MRI). Moreover, we sought to establish whether reduction in LV mass is caused by atrophic remodeling by studying rats in which RVF secondary to pulmonary hypertension was induced by monocrotaline (MCT) injection (17). This model is suitable to study LV remodeling, because we recently found that underfilled LV of these rats exhibits electrophysiologic changes (18) that resemble those reported in atrophic LV (19).
We retrospectively studied 36 consecutive CTEPH patients who were referred for pulmonary endarterectomy (PEA), and who underwent cardiac MRI at preoperative assessment. CTEPH was diagnosed as reported previously (20). PEA was performed using standardized surgical techniques (21). Preoperatively, all patients underwent pulmonary angiography and RV catheterization. Coronary angiography was routinely performed in all patients older than 50 years of age, and in patients older than 40 years of age if they had a history of smoking. Plasma brain natriuretic peptide levels were measured in all patients, and 6-min walking distance (22) was determined in 26 patients. All patients who survived PEA were reassessed by cardiac MRI at 3 to 18 months post-PEA (median, 8 months). Eleven healthy volunteers served as controls for the RV and LV volumes and mass. Particular care was taken to match control subjects for age and sex (mean age 52 ± 10 years, 5 males). All subjects included gave written informed consent. Investigations were approved by the local institutional review board.
Determination of cardiac volumes and mass by MRI
RVF was defined as MRI-derived RV ejection fraction <45% (23). Masses of LV free wall and interventricular septum (IVS) were assessed from the stack of parallel short-axis images by manual detection of endocardial and epicardial borders on each slice; the papillary muscles were excluded from analysis of masses of LV and free wall (24) (Fig. 1). The LV free wall extends from the RV-LV junction in the anterior wall to the RV-LV junction in the inferior wall, as shown in Figure 1. Cardiac volume and mass were normalized to body surface area. Additionally, the following parameters were calculated: RV and LV end-diastolic volume indexes (assessment of RV and LV diastolic function); RV and LV stroke volume indexes and ejection fractions (assessment of systolic function); and LV peak filling rate (assessment of LV preload and diastolic function).
The study protocol was approved by the institutional animal use committee and was in line with European Union directives on the care and use of experimental animals. Eight-week-old male Wistar rats were injected intraperitoneally with a single dose of 60 mg/kg monocrotaline (MCT) (n = 18) to induce heart failure secondary to PAH (17). The control group (n = 18) was injected with a comparable amount (3 ml/kg) of the MCT vehicle. The animals were serially monitored clinically and by transthoracic echocardiography (17,25). MCT-injected rats and control animals were sacrificed. The heart, lungs, and liver were immediately dissected, blotted dry, and then weighed. The RV was separated from atria, IVS, and LV free wall in 10 MCT-treated animals and 10 controls, and the RV was weighed separately. Body weight and tibia length were measured (26) and used for normalization of RV, IVS, and LV free wall masses (Table 1). Hearts from MCT-injected (n = 13) and control (n = 12) animals were either enzymatically dissociated to measure cell dimensions or fixed in paraffin for in situ hybridization, or used for quantitative RT-PCR and Western blot analysis.
The statistical analysis was performed using SPSS version 16.0 (SPSS Inc., Chicago, Illinois). Assumptions of normality and homogeneity of variance for parametric testing were assessed using the Shapiro-Wilk test. Data are mean ± SD or median with interquartile range unless otherwise indicated. Paired and unpaired Student t test and nonparametric Mann-Whitney U test were used to compare means or medians of normally and non-normally distributed values, respectively. The multiple groups were compared using 1-way analysis of variance with Bonferroni post hoc correction or Kruskal-Wallis analysis of variance by rank. Chi-square test was performed to compare proportions. p < 0.05 was considered statistically significant.
For detailed methods see the Online Appendix, Supplemental Methods.
Baseline patient characteristics
On average, CTEPH patients with RVF had significantly higher mean pulmonary artery pressure and total pulmonary resistance, and significantly lower cardiac index and 6-min walking distance than patients with preserved RV systolic function (Table 2). Patients with RVF also had significantly lower LV peak filling rate and LV ejection fraction than either patients without RVF or volunteers (Table 3). No patient had coronary artery disease at coronary angiography.
LV free wall mass index in CTEPH patients with RVF
At baseline, CTEPH patients with RVF had lower LV free wall mass index than patients without RVF or volunteers. Conversely, their IVS mass index was higher than in volunteers. LV mass index was not different between the groups (Table 3). There was no significant difference between patients without RVF and volunteers with regard to RV ejection fraction, or mass indexes of LV free wall or IVS (Table 3). PEA resulted in significant improvement in RV ejection fraction (from 31 ± 8% to 56 ± 12%, p < 0.001) in patients with RVF at baseline (Table 4). In parallel, LV free wall mass index increased (from 38 ± 6 g/m2 to 44 ± 9 g/m2, p = 0.001), whereas IVS mass index declined (from 28 ± 8 g/m2 to 22 ± 5 g/m2, p = 0.001) compared with baseline. In contrast, patients without RVF at baseline exhibited no significant changes in RV ejection fraction (from 54 ± 9% to 56 ± 20%, p = 0.89) or LV free wall mass index (from 46 ± 10 g/m2 to 50 ± 12 g/m2, p = 0.21) (Table 4).
Morphometric and echocardiographic parameters
Rats with RVF had significantly reduced RV contractility (low tricuspid annular plane systolic excursion), and impaired LV early diastolic relaxation and diastolic filling (low LV early diastolic relaxation velocity E′, LV early diastolic filling velocity E, LV end-diastolic area, and LV end-diastolic diameter) (Table 5). These rats also had reduced LV free wall mass (lower LV free wall weight and LV free wall/tibia length index) (Table 1). Moreover, they had smaller LV free wall myocytes, as evidenced by diminished cell length (Table 1).
Regional gene expression pattern, quantitative RT-PCR, and Western blot analysis
In situ hybridization revealed an altered gene expression profile: increased expression of ANP, and reduced expression of α-MHC and SERCA2 (Fig. 2). These changes were present both in RV and LV, and they were more pronounced in RV. Similarly, SERCA2 mRNA levels appeared to be reduced in RVF ventricular tissue compared with control, but these differences were not significant (Fig. 3A). ANP mRNA expression levels showed an increase in RVF versus control tissue (p = 0.07). These results may be consistent with the findings from the in situ hybridization experiments (Fig. 2), with regional heterogeneity of the ANP, α-MHC, and SERCA2 transcripts within the LV precluding accurate quantification. Western blot analysis did not show a significant difference in α-MHC or SERCA2 protein expression levels between control and RVF rats, although there was a tendency towards lower SERCA2 protein levels in RVF rats (Fig. 3B).
We found that CTEPH patients with RVF had significantly lower LV free wall mass than either patients with preserved RV contractility or volunteers. In these patients, PEA restored LV free wall mass to values comparable with volunteers. Using a rat model of RVF secondary to PAH, we found that reduction in LV free wall mass can be, at least in part, explained by myocyte shrinkage due to atrophic remodeling.
LV mass in RVF due to chronic pressure overload
In line with previous studies (10), we found that the sum of LV free wall mass index and IVS mass index was not significantly different between patients (either with or without RVF) and controls (10). However, separate analysis of IVS and LV free wall masses revealed that LV free wall mass was significantly reduced, whereas IVS was hypertrophic, in patients with RVF. We reasoned that LV free wall mass is a better reflection of LV remodeling than IVS, because IVS is composed of both LV and RV, and IVS hypertrophy may be largely explained by RV hypertrophy (10). Thus, our findings support the hypothesis that LV atrophic remodeling occurs in RVF. Accordingly, reduction in LV mass was also reported in end-stage pulmonary emphysema, another disorder that is associated with chronic RV pressure overload, dysfunction, and altered LV diastolic filling (27,28). In the present study, reduction in LV free wall volume (mass) of patients with RVF may have been underestimated because LV interstitial edema was also present (29). Interestingly, in patients with mitral stenosis (30) and end-stage pulmonary emphysema (31), disorders that are both associated with reduced LV pre-load, normalization of RV function and LV diastolic filling after mitral valvuloplasty and orthotopic single-lung transplantation, respectively, led to significant increase in LV mass.
In the rat model, absolute LV free wall mass in rats with RVF was significantly lower than in controls. When we normalized LV free wall weight to body weight, we found no significant difference between RVF animals and controls, in line with several previous studies, using a similar model of heart failure that described unchanged (32) or augmented (33) LV mass. However, the body weight of MCT rats declines significantly as early as 3 to 7 days prior to overt heart failure (17). Accordingly, normalization of LV mass to tibia length may be a more accurate method to reveal changes in LV mass in rats (26). This analysis showed that LV free wall mass in RVF rats was reduced. Similarly, reduction in absolute LV mass in MCT-treated rats with heart failure and rats with pulmonary artery banding was reported by other investigators (34).
Possible mechanisms of reduction in LV free wall mass during RVF secondary to chronic PAH
Mechanical load plays a critical role in determining the mass of cardiac myocytes (35). Accordingly, unloaded LV undergoes atrophy (16,36). While doing so, it replicates the fetal gene expression profile also seen in hypertrophy (15). In the present study, we did not find LV hypertrophy. In contrast, LV mass was reduced in rats with RVF. This was associated with LV myocyte shrinkage, increased ANP mRNA expression, and diminished α-MHC and SERCA2 mRNA expression. Such a fetal gene expression profile was previously observed in the LV of MCT rats (33,37), and rats that developed RVF after chronic pulmonary artery banding (38). Of note, these changes in expressions of ANP, α-MHC, and SERCA were not predominant in the LV. Not surprisingly, they were more prominent in pressure-overloaded RV, in line with previous studies (33,38). Furthermore, although we found a similar trend in the difference in gene expression in LV between control and RVF rats using quantitative reverse transcriptase PCR (RT-PCR), Western blot analysis did not reveal significant differences in protein expression levels of α-MHC and SERCA2. Since, in the present study, LV samples for quantitative RT-PCR were taken separately from LV samples for Western blot analysis, this discrepancy may be explained by regional heterogeneity in the expression of fetal genes in myocardium of the LV in RVF (38). Taken together, these findings indicate that LV remodeling that occurs in RVF due to chronic pulmonary hypertension may be explained by cardiac myocyte atrophy (14–16,36,39). Similarly, LV myocyte atrophy was demonstrated in patients with end-stage pulmonary emphysema (27,28). Of note, myocyte atrophy is independent of catecholamines from LV tissue and/or systemic circulation, or neural activity (39,40). Although apoptosis and/or other mechanisms may also be responsible for the loss of myocardial mass in unloaded LV, atrophy may be the main mechanism (41). In line with this notion, we did not find enhanced cell death in LV free wall of MCT-treated rats with RVF (42).
Possible limitations of the present study are as follows. First, the number of patients studied both at baseline and at postoperative follow-up was limited. Second, cardiac MRI was performed at different time points of follow-up after PEA (ranging from 3 to 18 months with a median at 8 months). This may be important because full restoration of RV and LV systolic function after PEA requires >12 months (43). However, significant improvements in RV ejection fraction, LV end-diastolic volume, and cardiac index were documented as early as at discharge from the hospital, with further improvements being reported at 3 and 12 months of follow-up (43). Similarly, orthotopic single-lung transplantation in patients with end-stage emphysema resulted in significant improvement in RV ejection fraction and increases in LV end-diastolic volumes, stroke volumes, and LV mass as early as 3 months after surgery (31). Third, LV myocardial biopsies were not taken to assess the morphology and confirm atrophic remodeling. On the other hand, we demonstrated LV remodeling in a rat model of RVF due to chronic RV pressure overload. Although the data should be cautiously extrapolated to human disease, this experimental model is a generally accepted model to study RVF due to chronic pressure overload (17,32,44).
RVF in patients with CTEPH is associated with reduction in LV free wall mass. This reduction is reversible and can be restored after PEA. Using a rat model of RVF secondary to PAH, we found that reduction in LV free wall mass can be, at least in part, explained by myocyte shrinkage due to atrophic remodeling.
For an expanded Methods section, please see the online version of this article.
Dr. Tan was supported by the Netherlands Organization for Scientific Research (NWO, grant ZonMW Vici 918.86.616). All authors have reported that they have no relationships to disclose.
- Abbreviations and Acronyms
- chronic thromboembolic pulmonary hypertension
- interventricular septum
- left ventricle/ventricular
- magnetic resonance imaging
- pulmonary arterial hypertension
- pulmonary endarterectomy
- right ventricle/ventricular
- right ventricular failure
- Received July 20, 2010.
- Accepted August 24, 2010.
- American College of Cardiology Foundation
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