Hessel, M.H.M.
Citation
Hessel, M. H. M. (2008, February 7). Release characteristics of cardiac proteins after reversible or irreversible myocardial damage. Retrieved from
https://hdl.handle.net/1887/12593
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/12593
Note: To cite this publication please use the final published version (if applicable).
Chapter 6
Characterization of right ventricular function after monocrotaline-induced pulmonary hypertension
in the intact rat
M.H.M. Hessel, P. Steendijk, B. den Adel, C.I. Schutte and A. van der Laarse
Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands
Am J Physiol Heart Circ Physiol 2006;291:2424-2430
Abstract
Background: We characterized hemodynamics and systolic and diastolic right ventricular (RV) function in relation to structural changes in the rat model of monocrotaline (MCT)-induced pulmonary hypertension.
Methods: Rats were treated with MCT at 30 mg/kg body wt (MCT30, n=15) or 80 mg/kg body wt (MCT80, n=16) MCT to induce compensated RV hypertrophy or RV failure, respectively. Saline-treated rats served as control (CONT, n=13). After 4 wk, a pressure- conductance catheter was introduced into the RV to assess pressure-volume relations.
Subsequently, rats were killed, hearts and lungs were rapidly dissected, and RV, left ventricle (LV) and interventricular septum (IVS) were weighed and analyzed histochemically.
Results: RV-to-(LV+IVS) weight ratio was 0.29±0.05 in CONT, 0.35±0.05 in MCT30 and 0.49±0.10 in MCT80 (P<0.001 vs. CONT and MCT30) rats, confirming MCT-induced RV hypertrophy. RV ejection fraction was 49±6% in CONT, 40±12% in MCT30 (P<0.05 vs.
CONT) and 26±6% in MCT80 (P<0.05 vs. CONT and MCT30) rats. In MCT30 rats, cardiac output was maintained, but RV volumes and filling pressures were significantly increased compared with CONT (all P<0.05), indicating RV remodeling. In MCT80 rats, RV systolic pressure, volumes and peak wall stress were further increased, and cardiac output was significantly decreased (all P<0.05). However, RV end-systolic and end- diastolic stiffness were unchanged, consistent with the absence of interstitial fibrosis.
Conclusion: MCT-induced pressure overload was associated with a dose-dependent development of RV hypertrophy. The most pronounced response to MCT was an overload-dependent increase of RV end-systolic and end-diastolic volumes, even under nonfailing conditions.
Keywords
Monocrotaline, right ventricular hypertrophy, right ventricular failure, pressure-volume relations
Introduction
Myocardial hypertrophy is a compensatory mechanism whereby cardiac tissue adapts to increased workload. Depending on the degree or duration of increased workload, ventricular hypertrophy may progress from a compensatory state to impaired systolic or diastolic function and heart failure. This transition is characterized by alterations in extracellular matrix composition, energy metabolism, ß-adrenergic responsiveness, myofilament proteins, Ca2+ handling, signal transduction pathways and gene-expression profiles1-9.
A model that is frequently used for study of functional, structural and molecular changes associated with right ventricular (RV) compensated hypertrophy and RV failure is treatment of rats with monocrotaline (MCT), a pyrrolizidine alkaloid10-13. MCT selectively injures the vascular endothelium of the lung and induces pulmonary vasculitis6. Muscularization and hypertrophy of media in pulmonary arteries lead to increased vascular resistance and increased pulmonary arterial pressure14-18. MCT-induced pulmonary hypertension is associated with development of compensated RV hypertrophy, which progresses to failure within weeks, depending on the dose of MCT and the age of the animals7;19;20. The functional consequences of MCT treatment have been studied in isolated papillary muscles and trabeculae2;21;22, in the isolated working heart20 and in vivo by echocardiography23-26.
Previous studies have shown selective induction of RV hypertrophy or RV failure after 4 wk of treatment with a low dose (30 mg/kg body wt) or a high dose (80 mg/kg body wt) of MCT, respectively7. The purpose of this study was to accurately determine RV function in normal rats and rats treated with a low dose (30 mg/kg body wt) or high dose (80 mg/kg body wt) of MCT in vivo, using a combined pressure-conductance catheter.
RV systolic and diastolic characteristics were quantified in the three groups of rats. The advantages of this technique are the rapid acquisition time of time-dependent pressure and volume data in the RV in vivo and the acquisition of pressure-volume loops during preload alterations to record RV end-systolic and end-diastolic pressure-volume relations (ESPVR and EDPVR) as load-independent measures of intrinsic ventricular function.
Material and Methods
Animal model
All animals were treated in accordance with the national guidelines and with permission of the Animal Experiments Committee of the Leiden University Medical Center. A total of 44 male Wistar rats (200-250 g body wt; Harlan, Zeist, the Netherlands) were randomly assigned to three groups. The animals received a single subcutaneous injection of MCT (Sigma, Zwijndrecht, the Netherlands) diluted in PBS in a low dose (MCT30, 30 mg/kg body wt, n=15) or in a high dose (MCT80, 80 mg/kg body wt, n=16). control (CONT) rats (n=13) were injected with an equal volume of PBS. The animals were housed, five animals per cage, with a 12:12-h light-dark cycle and an unrestricted food supply. The rats were weighed three times a week. After 4 wk, RV function was assessed by pressure-conductance catheter, and the rats were killed.
Hemodynamic Measurements
Instrumentation. The rats were sedated by inhalation of a mixture of isoflurane (4%) and oxygen. Subsequently, general anaesthesia was administered by injection of fentanyl- fluanison-midazolam (0.25 ml/100 g body wt ip). The mixture consisted of two parts Hypnorm (0.315 mg/ml fentanyl + 10 mg/ml fluanison; VitalPharma, Maarheeze, The Netherlands), one part Dormicum (5 mg/ml midazolam; Roche, Mijdrecht, The Netherlands) and one part saline. The animals were placed on a controlled warming pad to keep body temperature constant. A tracheotomy was performed, a 25-gauge cannula was inserted and the animals were mechanically ventilated using a pressure-controlled respirator and a mixture of air and oxygen. The animals were placed under a stereomicroscope (Zeiss, Hamburg, Germany) and a midline cervical incision was made for cannulation of the left jugular vein for infusion of hypertonic saline (10%) to determine parallel conductance27. A midsternal thoracotomy was performed, and a combined pressure-conductance catheter (model SPR-878, Millar Instruments, Houston, TX) was introduced via the apex into the RV and positioned along the long axis of the RV. The catheter was connected to a Sigma signal processor (CD Leycom, Zoetermeer, The Netherlands) and RV pressures and volumes were recorded digitally. All data were
acquired using Conduct-NT software (CD Leycom) at a sample rate of 2,000 Hz and analyzed off-line by custom-made software.
Calibration of the conductance catheter. Thevolume signal of the conductance catheter was calibrated for parallel conductance and slope factor (α). The parallel conductance volume was estimated by the hypertonic saline technique27: a 20-µL bolus of hypertonic saline (10%) was injected via the cannula in the jugular vein, and parallel conductance was calculated as previously described28;29. α, was determined bymeasurement of the aortic flow using an ultrasonic flow probe (Transonic Systems, Maastricht, The Netherlands) around the ascending aorta; α was calculated as the cardiac output (CO) determined by the uncalibrated conductance catheter divided by the CO determined by aortic flow measurement.
Hemodynamics. RV pressure and volume signals were recorded to quantify general hemodynamic conditions. Heart rate, stroke volume, CO, RV end-diastolic volume, RV end-systolic volume, RV ejection fraction, RV end-diastolic pressure, RV peak systolic pressure, and RV end-systolic pressure were determined from pressure-volume loops.
Stroke work was obtained as the area of the pressure-volume loop, and the maximal rates of RV pressure upstroke and fall (dP/dtmax and dP/dtmin, respectively) were calculated. The relaxation time constant (τ) was assessed as the time constant of monoexponential decay of RV pressure during isovolumic relaxation27;30. Effective pulmonary arterial elastance, as a measure of RV afterload, was calculated as end- systolic pressure divided by stroke volume.
RV ESPVR and EDPVR. The RV ESPVR and EDPVR were determined from pressure- volume loops recorded during transient occlusion of the inferior vena cava by external compression of the vessel. Slopes and intercepts of these relations are considered relatively load-independent parameters of intrinsic myocardial function31-34. The slopes, end-systolic and end-diastolic elastance (EES and EED, respectively), were determined by linear regression. The positions of the pressure-volume relations were quantified by calculating of the intercepts of the relations at an end-systolic pressure of 37 mmHg and an end-diastolic pressure of 5 mmHg. These pressure values were determined retrospectively as the mean end-systolic and end-diastolic pressures of all animals in the study.
RV end-systolic, end-diastolic and peak wall stress. Time-varying RV wall stress [WS(t)], was calculated from RV cavity pressure and volume [P(t) and V(t), respectively] and RV wall volume (Vwall) according the formula, WS(t)=P(t)·{1+3[V(t)/Vwall]} 35. RV wall volume was approximated as the sum of the RV free wall mass and 0.5 times the mass of the interventricular septum (IVS).
Tissue Preparation
After hemodynamic measurements, hearts and lungs were rapidly dissected and weighed. The RV, left ventricle (LV) and IVS were cut free, weighed, and fixed in 4%
formaline for >24 hours. Lung tissue was freeze-dried for 12 h and weighed again.
Histochemistry
Sirius red staining. After fixation, heart tissue was embedded in paraffin. RV blocks were embedded in upright position to distinguish the endocardium, the midwall and the epicardium of the RV free wall in cross-sections. Tissue was cut into 4-µm-thick sections and deparafinized in Ultraclear (Klinipath, Duiven, the Netherlands) for 5 min. The sections were rehydrated in decreasing graded alcohols (from 100 to 25%) and washed twice for 5 min each in distilled water and TBS (150 mM NaCl, 10 mM Tris-HCl, pH 8.0).
For determination of the amount of interstitial, perivascular, and/or replacement fibrosis, RV sections were stained with sirius red 36 and photographed using a microscope (Nikon Eclipse, Nikon Europe, Badhoevedorp, the Netherlands) equipped with a X40 objective and a digital camera (model Nikon, DXM1800). Quantitative image analysis was performed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).
The extent of fibrosis was quantified as the sirius red-stained percentage of the total area, measured per image. In each section, four images were acquired and analyzed to compensate for variations within a section. A total of 132 images of 33 animals (11 CONT, 12 MCT30, and 10 MCT80) were quantified.
RV thickness was measured in an overview image of CONT (n=11), MCT30 (n=12) and MCT80 (n=10) sections at three levels to compensate for variations in wall thickness within a section. To derive absolute thickness (mm), measurements were corrected
using a microscopic ruler (Leitz, Wetzlar, Germany). No correction was made for tissue shrinkage by the fixation procedure, inasmuch as this factor is equal in all samples.
Statistical Analysis
The effect of treatment (CONT, MCT30 or MCT80) was evaluated by one-way ANOVA followed by Bonferroni's post hoc test. Correlations were determined using a parametric test (Pearson). Differences were considered significant at P<0.05. SPSS11 for Windows (SPSS Inc, Chicago, IL) was used for statistical analysis. Values are means ± SD.
Results
Body, Lung and Heart Weights
The time curves of body weights of the three experimental groups over 4-wk period after MCT injection indicate a slightly reduced weight gain in MCT30 group (Fig. 1). The MCT80 animals showed a more pronounced growth retardation, resulting in a significantly lower (P<0.05) body weight after day 11, than CONT rats. The MCT80 animals started to loose weight at around day 25, which is a sign of heart failure comparable to cachexia in patients with chronic heart failure. In contrast to CONT and MCT30 animals, MCT80 rats showed an obvious lack of physical activity.
Table 1 summarizes characteristics of the animals on the day they were killed. Body weight of MCT80 rats was significantly lower than that of CONT and MCT30 rats (P<0.001 vs. CONT and MCT30). Wet and dry lung weights were significantly higher in MCT80 rats than in CONT and MCT30 rats, confirming the presence of an extensive proliferative pulmonary response in MCT80 rats. These increases were not related to pulmonary edema, because dry-to-wet weight ratios of lung tissue did not differ significantly between the groups.
RV weight of MCT80 animals was significantly higher than that of CONT and MCT30 rats, whereas LV and IVS weights were unaffected by MCT treatment. The degree of RV hypertrophy was determined as RV-to-(LV+IVS) weight ratio (Fig. 2). This hypertrophy parameter tended to increase (+21%) in MCT30 and was significantly higher in MCT80 rats (+69% compared with CONT and +40% compared with MCT30, both P<0.001).
Corresponding results were obtained for RV wall thickness (Table 1).
Figure 1. Time-dependent body weights of control (CONT, n=13) rats and rats treated with monocrotaline (MCT) at 30 mg/kg body wt (MCT30, n=15) or 80 mg/kg body wt (MCT80, n=16). Values are mean ± SD.
Body weight differences between MCT80 and CONT rats were significantly different from day 11 and thereafter (P<0.05).
Table 1. General , and cardiac characteristics of CONT, MCT30 , and MCT80 animals
CONT (n=13) MCT30 (n=15) MCT80 (n=16)
BW (g) 379 ± 31 362 ± 21 300 ± 31***,###
HW (g) 1.35 ± 0.13 1.40 ± 0.22 1.44 ± 0.20
HW/BW (mg/g) 3.53 ± 0.38 3.88 ± 0.47 5.00 ±1.29**, # RV weight (g) 0.24 ± 0.03 0.29 ± 0.05 0.37 ± 0.08***,###
LV weight (g) 0.50 ± 0.07 0.50 ± 0.04 0.49 ± 0.07
IVS weight (g) 0.31 ± 0.06 0.30 ± 0.04 0.28 ± 0.04
RV/(LV+IVS) 0.29 ± 0.05 0.35 ± 0.05 0.49 ± 0.10***,###
RV/BW (mg/g) 0.63 ± 0.01 0.74 ± 0.47 1.24 ± 0.23***,###
RV thickness (mm) 0.98 ± 0.25 1.26 ± 0.23 1.43 ± 0.41**
LW wet (g) 1.32 ± 0.21 1.66 ± 0.77 2.25 ± 0.49***,#
LW dry (g) 0.27 ± 0.05 0.34 ± 0.15 0.55 ± 0.13***,###
LW dry/wet 0.20 ± 0.04 0.21 ± 0.04 0.24 ± 0.04
LW wet/BW (mg/g) 3.47 ± 0.44 4.58 ± 2.02 7.08 ± 2.62***,#
Abbreviations; BW, body weight; HW, heart weight, RV, right ventricle; LV, left ventricle; IVS, interventricular septum; LW, lung weight. * P<0.05, ** P<0.01, *** P<0.001 vs. CONT, # P<0.05, ##
Figure 2. Right ventricular (RV)-to- [left ventricular (LV) + interventricular septum (IVS)] weight ratio in CONT (n=13), MCT30 (n=15) and MCT80 (n=16) animals on the day the animals were killed (* P< 0.001 vs. CONT; # P<0.001 vs. MCT30).
RV Function
RV function was determined by recording pressure-volume loops. Results are summarized in Table 2. In MCT30 rats, RV peak pressure was significantly increased (+30%), whereas RV ejection fraction was significantly reduced (-18%), compared with CONT (both P<0.05). CO and stroke volume were maintained, indicating a compensatory state at the expense of increased RV end-diastolic and end-systolic volumes and increased RV filling pressure (all P<0.05 vs. CONT).
In MCT80 rats, RV peak pressure was further increased (+69%, P<0.05 vs. CONT), consistent with the increased pulmonary arterial elastance, which was twice as high as that in CONT and MCT30 rats (both P<0.01). RV ejection fraction decreased further (- 47% compared with CONT and -35% compared with MCT30, both P<0.05) and CO was significantly reduced by 31% compared with MCT30 rats (P<0.05). RV dilatation was evident from progressive increases of RV end-systolic and end-diastolic volumes (roughly twice the corresponding values of CONT). τ, was significantly increased in MCT80 rats compared with CONT and MCT30 rats, indicating prolonged early relaxation. RV peak wall stress of MCT80 rats was increased by 90% (P<0.05) compared with CONT rats, and by 29% compared with MCT30 rats.
Table 2. Cardiac, and right ventricular function of CONT, MCT30 , and MCT80 animals
CONT (n=12) MCT30 (n=15) MCT80 (n=9)
HR (beats/min) SV (µL) CO (mL/min) VES (µL) VED (µL) PPEAK (mmHg) PES (mmHg) PED (mmHg) dP/dtMAX (mmHg/s) dP/dtMIN (mmHg/s) EF (%)
SW (mmHg µL) τ (ms)
EA (mmHg/µL) WSMAX (mmHg) WSED (mmHg)
399 ± 30 208 ± 74 82 ± 27 245 ± 92 431 ± 152
33 ± 6 29 ± 7 3.9 ± 2.5 2709 ±790 1668 ± 428
49 ± 6 5381 ± 2323
14 ± 4 0.15 ± 0.06
144 ± 53 14 ±11
381 ± 48 237 ± 82 89 ± 27 441 ± 270*
649 ± 323*
42 ± 15*
35 ± 14 6.1 ± 2.4*
2473 ± 778 1781 ± 675 40 ±12*
7384 ± 4423 12 ± 3 0.16 ± 0.05
213± 112 36 ± 26
320 ± 85*,#
196 ± 66 61 ± 22# 634 ± 325*
804 ± 368*
55 ± 20*
50 ± 17*,#
5.4 ± 2.4 2332 ± 903 1959 ± 881 26 ± 6*,#
7901 ± 3189*
27 ± 12*,#
0.28 ± 0.15**,##
274 ±111*
31 ± 16
Abbreviations; HR, heart rate; SV, stroke volume; CO, cardiac output; VES, RV end-systolic volume; VED, RV end-diastolic volume; PPEAK, RV peak pressure; PES, RV end-systolic pressure; PED, RV end-diastolic pressure; EF, RV ejection fraction; SW, RV stroke work; dP/dtMAX, maximal rate of pressure increase;
dP/dtMIN, maximal rate of pressure decline; τ, relaxation time constant; EA, effective arterial elastance;
WSMAX, RV peak wall stress; WSED, RV end-diastolic wall stress. * P<0.05, ** P<0.01 vs. CONT , and
# P<0.05, ## P<0.01 vs. MCT30
Pressure-Volume Relations
Slopes of ESPVR and EDPVPR, EES and EED, were not significantly different between CONT, MCT30 and MCT80 (Table 3). To correct for the influence of RV muscle mass, we also performed an univariate analysis of variance with RV muscle mass as co-factor;
the results did not show significant differences between groups for the normalized parameters. Both relations were shifted towards larger volumes in MCT30 rats and were even more pronounced in the MCT80 rats (Fig. 3). However, despite the clear trends, because of large within-group variability, only the shift in the EDPVR for the MCT80
Figure 3. Typical examples of RV pressure-volume loops during vena cava occlusion and RV end-systolic pressure-volume relations (ESPVR) of CONT, MCT30 and MCT80 rats.
Myocardial Fibrosis
RV myocardial sections were stained with sirius red for assessment of interstitial, perivascular, and replacement fibrosis. Quantification of volume percentages showed no significant differences between groups (Fig. 4), indicating no interstitial nor for replacement fibrosis in MCT-treated rats. Similarly, perivascular fibrosis in MCT-treated rats did not differ from that in CONT rats (data not shown).
Table 3. Pressure-volume indexes in CONT, MCT30 , and MCT80 animals
CONT (n=12) MCT30 (n=15) MCT80 (n=9)
EES (mmHg/µL) EES
N (mmHg/µL) EED (mmHg/µL) EEDN
(mmHg/µL) VES,37 (µL) VED,5 (µL)
0.082 ± 0.053 0.093 ± 0.20 0.0074 ± 0.0091
0.0082 ±0.0019 391 ± 185 541 ± 344
0.100 ± 0.084 0.082 ± 0.015 0.0062 ± 0.0028 0.0061 ± 0.0014
478 ± 321 614 ± 377
0.094 ± 0.087 0.079 ± 0.022 0.0067 ± 0.0028 0.0055 ± 0.0021
722 ± 550 1025 ± 386*,#
Abbreviations; EES, end-systolic elastance; EESN
, normalized end-systolic elastance; EED, end-diastolic stiffness; EED
N, normalized end-diastolic stiffness; VES,37 , end-systolic volume at a mean end-systolic pressure of 37 mmHg (position of end-systolic pressure volume relationship, ESPVR), VED,5, end-diastolic volume at a mean end-diastolic pressure of 5 mmHg (position of end-diastolic pressure-volume relationship, ESPVR). EES
N , and EED
N,were obtained by adding RV muscle mass as a cofactor in the univariate analysis of variance , and evaluating EES , and EED in the three groups at a common RV muscle mass (440 mg). * P<0.05, ** P<0.01, *** P<0.001 vs. CONT and # P<0.05, ## P<0.01, ### P<0.001 vs.
MCT30.
Figure 4. Quantification of interstitial fibrosis in sirius red-stained RV myocardial sections from CONT (n=11), MCT30 (n=12), and MCT80 (n=10) rats. Values are means ± SD are presented.
Relations Between RV Performance and Myocardial Characteristics
In studies on the cellular/molecular transition from compensated hypertrophy to failure using the MCT-model, MCT is supposed to induce a dose-dependent increase in pulmonary vascular resistance with subsequent RV pressure-overload and increased wall stress, leading to compensatory hypertrophy and, ultimately, RV dilatation and pump failure. However, this process has not been studied in the intact animal using detailed invasive hemodynamic measurements. Data reported in Tables 1-3 generally support these assumptions but also show substantial variability in the various parameters within groups. To provide a better insight into the underlying mechanisms, we investigated the relations between the various indexes of RV pressure-overload, hypertrophy, wall stress, and RV function.
Figure 5a shows RV hypertrophy as a function of peak RV pressure. The significant overall correlation supports the concept of gradual pressure overload-induced RV hypertrophy. The functional consequences are reflected by Fig. 5b that shows a gradual decrease in ejection fraction, with lowest values in the MCT80 group, despite substantially higher hypertrophy in this group. The drop in ejection fraction is caused largely by increased end-diastolic volume, because stroke volume was reasonably maintained (Table 2). However, we did not find a significant correlation between RV end-diastolic volume and severity of hypertrophy. Finally, Fig. 5c shows that this progressive dilatation causes a gradual increase in wall stress, with the highest values in the MCT80 group, despite the compensatory increase in muscle mass, illustrating the decompensation in these animals. The significant negative correlation within the MCT80 group indicates that the MCT80 animals showing highest RV hypertrophy had the least wall stress. So, it appears that at least part of the variability within the MCT80 group is caused by the ability to adequately increase RV weight, thereby opposing RV dilatation and limiting excessive RV wall stress.
Figure 5. Peak RV pressure (A), RV ejection fraction (B), and RV peak wall stress (C) as a function of RV hypertrophy [quantified as RV-to-(LV+IVS) weight ratio] for CONT, MCT30 and MCT80 animals. Linear correlations were tested for pooled data and within groups: only relations with significant correlations are shown.
Discussion
This study was conducted to characterize changes in RV structure and function after MCT treatment in the intact rat model. Apart from RV hypertrophy, the primary response to MCT treatment was RV dilatation, i.e. increases of RV end-systolic and end-diastolic volumes and, consequently, decreases of RV ejection fraction.
The dose-dependent RV hypertrophy strongly correlated with MCT-induced pressure overload, but, despite this increased muscle mass, RV wall stress gradually increased ultimately leading to RV decompensation. Interestingly, RV end-systolic elastance and end-diastolic stiffness did not change significantly, even when we corrected for myocardial muscle mass, suggesting that intrinsic myocardial function was not importantly altered. The unchanged diastolic stiffness was consistent with the absence of changes in fibrosis and the fact that filling pressures remained relatively normal.
However, ESPVR and EDPVR showed a tendency to be shifted towards larger volumes suggesting myocyte slippage as a potential mechanism for dilatation. In addition, early active relaxation as reflected by τ was severely depressed in the MCT80 group, consistent with severe RV hypertrophy. Moreover, a recent study demonstrated reduced protein levels of sarcoplasmic reticulum Ca2+-ATPase in the RV of MCT-treated animals, which should result in a slower decline of intracellular Ca2+ concentration during relaxation and may partially explain the prolonged τ 2.
In studies in which a single dose of 40-60 mg MCT/kg body wt was administered, up to 50% of the animals progressed to RV failure after 4 wk, whereas the remaining animals developed stable compensatory RV hypertrophy without signs of heart failure 25;37-39. The failing animals showed a significantly greater degree of RV hypertrophy, reflecting a higher pulmonary arterial pressure25;39. Recently, selective induction of compensated RV hypertrophy or RV failure after 4 wk was demonstrated by using a low dose (30 mg/kg body wt) or a high dose (80 mg/kg body wt) of MCT, respectively7. In the present study, MCT30 rats developed a phenotype without clinical signs of cardiac failure, whereas MCT80 rats showed progressive body weight loss (cachexia), an increased respiratory rate, and a lack of physical activity starting at day 25. In this latter group, hemodynamic data could be obtained in only 9 of the 16 animals. The remaining 7 animals died either shortly before the measurements (n=2), during induction of anesthesia (n=2), or during
instrumentation (n=3), presumably due to very poor cardiac function. In the MCT30 group, no animals died before or during the hemodynamic studies, whereas one animal in the CONT group died during the instrumentation phase. Thus the hemodynamic data of the MCT80 group reported in our study probably reflect the animals with relatively preserved cardiac function, which may partly explain the substantial overlap of functional parameters between the groups.
To our knowledge, this study is the first to evaluate dose-dependent effects of MCT injection using invasive RV hemodynamics in the intact rat model. Previous studies in MCT-treated animals in vivo have used echocardiography and showed progressive development of pulmonary hypertension and RV hypertrophy consistent with our findings23-26, but no volumetric data have been reported. More detailed functional parameters were obtained by Werchan et al.20 in the model of the isolated perfused heart with working RV: increased RV peak systolic pressures were found 3 wk after MCT injection (60mg/kg body wt), but considerable variability was present. Consistent with our findings, the authors reported significantly increased RV systolic pressure only for animals with severe RV hypertrophy (>0.5), whereas +dP/dtmax and dP/dtmin were unchanged with mild hypertrophy and increased with severe hypertrophy. Similar to our study, diastolic RV pressure was not importantly altered.
In vitro studies with RV papillary muscles and trabeculae isolated from rats 3 and 4 wk after MCT treatment (40 mg/kg body wt) demonstrated a negative force-frequency relation and reduced maximum force compared to controls21;22.
Interestingly, our findings indicate substantial differences in RV function adaptation between the MCT model and pulmonary artery banding (PAB) model, which is frequently used in RV pressure-overload research3;30;40-44. Dependent on degree/severity of pulmonary artery stenosis, most PAB studies report an almost twofold increase in RV muscle mass, comparable to our findings, but despite the even higher RV systolic pressures in most PAB studies, generally no clear signs of cardiac failure and almost no RV dilatation have been reported3;30;42;43. A recent study in rats with instrumentation similar to that used in the present study, showed that 6 wk of PAB resulted in a RV systolic pressure of ~60 mmHg and a twofold increase in RV mass,
unchanged systemic hemodynamics and RV volumes, and increased RV contractility (EES)45. A comparison with the MCT-treated rats in our study suggests that effects of pressure overload and the mechanisms underlying contractility and RV dilatation are substantially different between the two models. These differences could be related to model-specific changes of the β-adrenergic receptor system. Rats with MCT-induced RV hypertrophy showed a reduction in RV β-adrenoceptor density due to a downregulation of the β1-adrenoreceptor and showed β-adrenoceptor-G-protein-adenylyl cyclase system desensitization46;47. This reduced β-adrenoceptor responsiveness may explain why the hypertrophied RV cannot increase contractility, which is necessary to maintain cardiac function in MCT-induced pulmonary hypertension. In contrast, animals with pulmonary hypertension induced by PAB showed a two- to threefold increase of RV contractility in the absence of a changed β-adrenoceptor system45;48.
We hypothesize that MCT-induced RV remodeling is caused by cardiomyocyte slippage due to diminished adhesion of cardiomyocytes to the extracellular matrix49;50. However, future studies are needed to investigate this hypothesis. An important issue for clinicians caring for patients with pulmonary hypertension is determination of the optimal timing for surgical or catheter-based interventions. Studies indicate that RV failure may develop in patients with pulmonary hypertensive heart diseases, even in the absence of impaired contractility51. Although we did not perform a longitudinal study, our findings suggest that cardiac failure was preceded by gradual cardiac dilatation and increased wall stress, despite myocardial hypertrophy. The animals failed to adequately increase contractility and showed prolonged relaxation, ultimately leading to a decrease in CO. MCT-induced pulmonary hypertension is a useful animal model for study of the transition from compensatory RV hypertrophy to RV failure, which has important clinical relevance.
In conclusion, we characterized the chronic effects of MCT injection on myocardial structure, RV function and hemodynamics in the intact rat model. Our findings show a dose-dependent increase in RV systolic pressure, RV myocardial mass, and RV volumes. In high-dose MCT, this leads to significantly elevated wall stress, severely reduced ejection fraction, and decreased pump function. Future studies are required to investigate the molecular mechanisms.
References
1. Katz AM. Energetics and the failing heart. Hosp Pract (Off Ed). 1991;26:78-90.
2. Kögler H, Hartmann O, Leineweber K, Nguyen vP, Schott P, Brodde OE, Hasenfuss G. Mechanical load-dependent regulation of gene expression in monocrotaline-induced right ventricular hypertrophy in the rat. Circ Res. 2003;93:230-237.
3. LekanneDeprez RH, van den Hoff MJ, de Boer PA, Ruijter PM, Maas AA, Chamuleau RA, Lamers WH, Moorman AF. Changing patterns of gene expression in the pulmonary trunk-banded rat heart.
J Mol Cell Cardiol. 1998;30:1877-1888.
4. Ricchiuti V, Zhang J, Apple FS. Cardiac troponin I and T alterations in hearts with severe left ventricular remodeling. Clin Chem. 1997;43:990-995.
5. van der Laarse A. Hypothesis: troponin degradation is one of the factors responsible for deterioration of left ventricular function in heart failure. Cardiovasc Res. 2002;56:8-14.
6. Wilson DW, Segall HJ, Pan LC, Lame MW, Estep JE, Morin D. Mechanisms and pathology of monocrotaline pulmonary toxicity. Crit Rev Toxicol. 1992;22:307-325.
7. Buermans HP, Redout EM, Schiel AE, Musters RJ, Zuidwijk M, Eijk PP, van Hardeveld C, Kasanmoentalib S, Visser FC, Ylstra B, Simonides WS. Microarray analysis reveals pivotal divergent mRNA expression profiles early in the development of either compensated ventricular hypertrophy or heart failure. Physiol Genomics. 2005;21:314-323.
8. Schott P, Singer SS, Kogler H, Neddermeier D, Leineweber K, Brodde OE, Regitz-Zagrosek V, Schmidt B, Dihazi H, Hasenfuss G. Pressure overload and neurohumoral activation differentially affect the myocardial proteome. Proteomics. 2005;5:1372-1381.
9. Spinale FG, Gunasinghe H, Sprunger PD, Baskin JM, Bradham WC. Extracellular degradative pathways in myocardial remodeling and progression to heart failure. J Card Fail. 2002;8:S332-S338.
10. Ghodsi F, Will JA. Changes in pulmonary structure and function induced by monocrotaline intoxication. Am J Physiol. 1981;240:H149-H155.
11. Ito KM, Sato M, Ushijima K, Nakai M, Ito K. Alterations of endothelium and smooth muscle function in monocrotaline-induced pulmonary hypertensive arteries. Am J Physiol Heart Circ Physiol.
2000;279:H1786-H1795.
12. Morita K, Ogawa Y, Tobise K. Effect of endothelium of pulmonary artery vasoreactivity in monocrotaline-induced pulmonary hypertensive rats. Jpn Circ J. 1996;60:585-592.
13. Nakazawa H, Hori M, Ozaki H, Karaki H. Mechanisms underlying the impairment of endothelium- dependent relaxation in the pulmonary artery of monocrotaline-induced pulmonary hypertensive rats. Br J Pharmacol. 1999;128:1098-1104.
14. Mathew R, Gloster ES, Sundararajan T, Thompson CI, Zeballos GA, Gewitz MH. Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension. J Appl Physiol.
1997;82:1493-1498.
15. Mitani Y, Maruyama K, Sakurai M. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation. 1997;96:689-697.
16. Cowan KN, Heilbut A, Humpl T, Lam C, Ito S, Rabinovitch M. Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor. Nat Med. 2000;6:698-702.
17. Rosenberg HC, Rabinovitch M. Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension. Am J Physiol. 1988;255:H1484-H1491.
18. Valdivia E, Lalich JJ, Hayashi Y, Sonnad J. Alterations in pulmonary alveoli after a single injection of monocrotaline. Arch Pathol. 1967;84:64-76.
19. Hislop A, Reid L. Arterial changes in Crotalaria spectabilis-induced pulmonary hypertension in rats.
Br J Exp Pathol. 1974;55:153-163.
20. Werchan PM, Summer WR, Gerdes AM, McDonough KH. Right ventricular performance after monocrotaline-induced pulmonary hypertension. Am J Physiol. 1989;256:H1328-H1336.
21. Korstjens IJ, Rouws CH, van der Laarse WJ, Van der ZL, Stienen GJ. Myocardial force development and structural changes associated with monocrotaline induced cardiac hypertrophy and heart failure. J Muscle Res Cell Motil. 2002;23:93-102.
22. Versluis JP, Heslinga JW, Sipkema P, Westerhof N. Contractile reserve but not tension is reduced in monocrotaline-induced right ventricular hypertrophy. Am J Physiol Heart Circ Physiol.
2004;286:H979-H984.
23. Boissiere J, Gautier M, Machet MC, Hanton G, Bonnet P, Eder V. Doppler tissue imaging in assessment of pulmonary hypertension-induced right ventricle dysfunction. Am J Physiol Heart Circ Physiol. 2005;289:H2450-H2455.
24. Cottrill CM, Johnson GL, Gillespie MN. Echocardiographic detection of pulmonary hypertension in anesthetized rats. Res Commun Chem Pathol Pharmacol. 1988;60:189-196.
25. Jones JE, Mendes L, Rudd MA, Russo G, Loscalzo J, Zhang YY. Serial noninvasive assessment of progressive pulmonary hypertension in a rat model. Am J Physiol Heart Circ Physiol.
2002;283:H364-H371.
26. Kato Y, Iwase M, Kanazawa H, Kawata N, Yoshimori Y, Hashimoto K, Yokoi T, Noda A, Takagi K, Koike Y, Nishizawa T, Nishimura M, Yokota M. Progressive development of pulmonary hypertension leading to right ventricular hypertrophy assessed by echocardiography in rats. Exp Anim.
2003;52:285-294.
27. Baan J, van der Velde ET, de Bruin HG, Smeenk GJ, Koops J, van Dijk AD, Temmerman D, Senden J, Buis B. Continuous measurement of left ventricular volume in animals and humans by conductance catheter. Circulation. 1984;70:812-823.
28. Steendijk P, Baan J. Comparison of intravenous and pulmonary artery injections of hypertonic saline for the assessment of conductance catheter parallel conductance. Cardiovasc Res. 2000;46:82-89.
29. Yang B, Larson DF, Beischel J, Kelly R, Shi J, Watson RR. Validation of conductance catheter system for quantification of murine pressure-volume loops. J Invest Surg. 2001;14:341-355.
30. Leeuwenburgh BP, Steendijk P, Helbing WA, Baan J. Indexes of diastolic RV function: load dependence and changes after chronic RV pressure overload in lambs. Am J Physiol Heart Circ Physiol. 2002;282:H1350-H1358.
31. Sagawa K. The end-systolic pressure-volume relation of the ventricle: definition, modifications and clinical use. Circulation. 1981;63:1223-1227.
32. Steendijk P, Baan J, Jr., van der Velde ET, Baan J. Effects of critical coronary stenosis on global systolic left ventricular function quantified by pressure-volume relations during dobutamine stress in the canine heart. J Am Coll Cardiol. 1998;32:816-826.
33. Suga H, Sagawa K, Shoukas AA. Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio. Circ Res.
1973;32:314-322.
34. van der Velde ET, Burkhoff D, Steendijk P, Karsdon J, Sagawa K, Baan J. Nonlinearity and load sensitivity of end-systolic pressure-volume relation of canine left ventricle in vivo. Circulation.
1991;83:315-327.
35. Arts T, Bovendeerd PH, Prinzen FW, Reneman RS. Relation between left ventricular cavity pressure and volume and systolic fiber stress and strain in the wall. Biophys J. 1991;59:93-102.
36. Sweat F, Puchtler H, Rosenthal SI. Sirius Red F3BA as a stain for connective tissue. Arch Pathol.
1964;78:69-72.
37. Brunner F, Wolkart G, Haleen S. Defective intracellular calcium handling in monocrotaline-induced right ventricular hypertrophy: protective effect of long-term endothelin-A receptor blockade with 2- benzo[1,3]dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl-)- 4-oxobut-2-enoate-sodium (PD 155080). J Pharmacol Exp Ther. 2002;300:442-449.
38. Seyfarth T, Gerbershagen HP, Giessler C, Leineweber K, Heinroth-Hoffmann I, Ponicke K, Brodde OE. The cardiac beta-adrenoceptor-G-protein(s)-adenylyl cyclase system in monocrotaline-treated rats. J Mol Cell Cardiol. 2000;32:2315-2326.
39. Wassen FW, Schiel AE, Kuiper GG, Kaptein E, Bakker O, Visser TJ, Simonides WS. Induction of thyroid hormone-degrading deiodinase in cardiac hypertrophy and failure. Endocrinology.
2002;143:2812-2815.
40. Borenstein N, Jian Z, Fromont G, Bruneval P, Hekmati M, Behr L, Laborde F, Montarras D, Le Bret E. Noncultured cell transplantation in an ovine model of right ventricular preparation. J Thorac Cardiovasc Surg. 2005;129:1119-1127.
41. Faber MJ, Dalinghaus M, Lankhuizen IM, Bezstarosti K, Dekkers DH, Duncker DJ, Helbing WA, Lamers JM. Proteomic changes in the pressure overloaded right ventricle after 6 weeks in young rats: correlations with the degree of hypertrophy. Proteomics. 2005;5:2519-2530.
42. Hon JK, Steendijk P, Petrou M, Wong K, Yacoub MH. Influence of clenbuterol treatment during six weeks of chronic right ventricular pressure overload as studied with pressure-volume analysis. J
43. Leeuwenburgh BP, Helbing WA, Steendijk P, Schoof PH, Baan J. Biventricular systolic function in young lambs subject to chronic systemic right ventricular pressure overload. Am J Physiol Heart Circ Physiol. 2001;281:H2697-H2704.
44. Olivetti G, Ricci R, Lagrasta C, Maniga E, Sonnenblick EH, Anversa P. Cellular basis of wall remodeling in long-term pressure overload-induced right ventricular hypertrophy in rats. Circ Res.
1988;63:648-657.
45. Faber MJ, Dalinghaus M, Lankhuizen IM, Steendijk P, Hop WC, Schoemaker RG, Duncker DJ, Lamers JM, Helbing WA. Right- and left ventricular function after chronic pulmonary artery banding in rats assessed with biventricular pressure-volume loops. Am J Physiol Heart Circ Physiol. 2006.
46. Leineweber K, Brandt K, Wludyka B, Beilfuss A, Ponicke K, Heinroth-Hoffmann I, Brodde OE.
Ventricular hypertrophy plus neurohumoral activation is necessary to alter the cardiac beta- adrenoceptor system in experimental heart failure. Circ Res. 2002;91:1056-1062.
47. Leineweber K, Seyfarth T, Abraham G, Gerbershagen HP, Heinroth-Hoffmann I, Ponicke K, Brodde OE. Cardiac beta-adrenoceptor changes in monocrotaline-treated rats: differences between membrane preparations from whole ventricles and isolated ventricular cardiomyocytes. J Cardiovasc Pharmacol. 2003;41:333-342.
48. Kuehne T, Yilmaz S, Steendijk P, Moore P, Groenink M, Saaed M, Weber O, Higgins CB, Ewert P, Fleck E, Nagel E, Schulze-Neick I, Lange P. Magnetic resonance imaging analysis of right ventricular pressure-volume loops: in vivo validation and clinical application in patients with pulmonary hypertension. Circulation. 2004;110:2010-2016.
49. Gerdes AM, Capasso JM. Structural remodeling and mechanical dysfunction of cardiac myocytes in heart failure. J Mol Cell Cardiol. 1995;27:849-856.
50. Zimmerman SD, Criscione J, Covell JW. Remodeling in myocardium adjacent to an infarction in the pig left ventricle. Am J Physiol Heart Circ Physiol. 2004;287:H2697-H2704.
51. Stein PD, Sabbah HN, Anbe DT, Marzilli M. Performance of the failing and nonfailing right ventricle of patients with pulmonary hypertension. Am J Cardiol. 1979;44:1050-1055.
