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Department of Physiology, Faculty of Medicine, University of Porto, Porto, Portugal
Submitted 20 September 2005 ; accepted in final form 23 April 2006
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ABSTRACT |
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gene expression; myocardial contractility; neuroendocrine stimulation; endothelin-1; heart
The present study was undertaken to characterize the molecular and contractile phenotype of the LV myocardium of MCT-treated rats with advanced PH. After hemodynamic evaluation and morphometric characterization of PH rats 6 wk after MCT injection, we assessed in both ventricles histological features such as myocardial fibrosis and myocyte diameters, and we quantified mRNA levels of genes previously implicated in autocrine/paracrine activation during HF progression such as ET-1, angiotensin-converting enzyme (ACE), type B natriuretic peptide (BNP), angiotensinogen, and aldosterone synthase. We further evaluated the contractile phenotype of the LV myocardium of MCT rats in vitro, performing force-frequency relationships (FFR) in LV muscle strips. Because a considerable increase in myocardial ET-1 mRNA levels was observed, we assessed to greater extent both ET-1 myocardial production by immunostaining and its myocardial actions by acutely blocking ETA receptors with BQ-123 in LV muscle strips and chronically treating MCT-injected rats with the dual endothelin receptor antagonist bosentan.
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METHODS |
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Hemodynamic assessment.
As previously reported (8), animals (MCT treated and sham, n = 7 each) were anesthetized with an intraperitoneal injection of pentobarbital sodium (6 mg/100 mg), mechanically ventilated (Harvard Rodent Ventilator model 683), and compensated for per operative fluid losses. The pericardium was widely opened after a median sternotomy was performed. A silk thread was passed around the ascending aorta to transiently occlude it during the experimental protocol. RV and LV pressures were measured with high-fidelity micromanometers (Millar Instruments SPR-407) inserted into the ventricular cavities. LV septal-lateral diameter was assessed with a sonomicrometer amplifier (Triton Electronics, San Diego, CA) after the implantation of ultrasonic crystals in the interventricular septum and epicardial surface of the LV free wall as previously described (3). After stabilization for 15 min, basal RV and LV peak rate of pressure rise (dP/dtmax), end-diastolic pressure (EDP), peak systolic pressure (SP), LV end-diastolic dimension (LVEDD), and peak systolic LV isovolumetric pressure (LVPiso) were recorded. Relaxation rate was estimated with the time constant 
by fitting the isovolumetric pressure fall to a monoexponential function, as previously described (20). Parameters were sampled with a frequency of 1,000 Hz. Recordings were made with respiration suspended at end expiration. An ECG lead (II) was recorded throughout. RV and septum plus LV free wall wet weights were determined at the end of the experimental protocol after anesthetic overdose. Transmural LV and RV free wall samples were snap frozen in liquid nitrogen and stored at –80°C for subsequent molecular analyses.
Intact muscle strip preparations. Briefly, rats were anesthetized with intraperitoneal ketamine-xylazine (50 mg/kg and 8 mg/kg, respectively), a left thoracotomy was performed, and beating hearts were quickly excised and immersed in modified Krebs-Ringer (KR) solution containing (in mM) 93 NaCl, 5 KCl, 1 MgSO4, 1.2 KH2PO4, 10 glucose, 1.25 CaCl2, 20 NaHCO3, 1 NaH2PO4, 20 NaC2H3O2, and 5 U/l insulin with cardioplegic 2,3-butanedione monoxime (BDM; 2.5%) equilibrated with 95% O2-5% CO2. Thin muscle strips (cross-sectional diameter < 400 µm) were carefully isolated from LV papillary muscles under a dissecting microscope (Leica Wilde M651), mounted vertically in a 10-ml Plexiglas organ bath, and attached to an electromagnetic length-tension transducer (University of Antwerp, Antwerp, Belgium). Twenty minutes later, bathing solutions were replaced by modified KR solution without BDM. Preload was estimated according to muscle dimensions, and electrical stimulation was set 10% above threshold and at a frequency of 0.5 Hz. Muscles were allowed to stabilize, and the experimental protocol was begun once three comparable basal isotonic and isometric contractions were separately recorded with an interval of at least 10 min. Temperature was kept at 35°C and pH at 7.4 throughout the experimental protocol. In one set of LV muscle strips from sham (n = 9), MCT (n = 9) and MCT + bosentan rats (n = 8), isometric FFR were obtained after an initial period of contraction at 0.5 Hz by stepping up the frequency of stimulation at 3-min intervals and sequentially recording five contractions at 1, 2, 3, 4, and 5 Hz. In another set of LV sham (n = 7), MCT (n = 7), and MCT + bosentan (n = 6) muscle strips, the effects of ETA antagonism were evaluated by recording several isotonic and isometric contractions before and 20–30 min after a concentration of 1 µM of the selective ETA antagonist BQ-123 (Sigma Chemical) was added to the bath. Developed tension (mN/mm2) was computed, using software, from the weight and length of the muscle strip by assuming an ellipsoid shape. Muscle maximum cross-sectional diameters measured with a microscope (Leica DM4000B) were similar (P = 0.15) in sham (252 ± 24 µm), MCT (280 ± 27 µm), and MCT + bosentan muscle strips (305 ± 41 µm), excluding the possibility that differences in muscle strip dimensions had an impact on contractile function.
Histology and immunohistochemistry. Transverse 4-µm-thick sections of paraffin-embedded, formalin-fixed specimens encompassing the RV, interventricular septum, and LV free wall were stained and photographed with a digital camera (Leica DFC320) in a group of additional sham (n = 4) and MCT rats (n = 4). The shortest diameter of 50 transversally cut, randomly selected cardiomyocytes from the RV, septum, and LV free wall myocardium was measured with image analyzer software (Leica IM-1000) at the level of the nucleus in hematoxylin-eosin-stained sections. Two independent, blinded observers ranked histological sections stained with Masson's trichrome for collagen as having no fibrosis (grade 0); a localized small amount of fibrosis (grade 1); mild, patchy fibrosis (grade 2); moderate, diffuse fibrosis (grade 3); or severe, diffuse fibrosis (grade 4). Immunohistochemical staining of ET-1 was performed with a 1:800 dilution of rabbit ET-1 antiserum (T-4050; Peninsula Laboratories) at 4°C for 16 h after initial incubation with 0.3% H2O2 and blocking solution (TR-004-HD; Labvision) at room temperature. The sample was then sequentially exposed at room temperature to biotinylated goat anti-rabbit (TR-004-HD; Labvision) for 15 min, to streptavidin peroxidase (TR-004-HD; Labvision) for 15 min, and, finally, to 4% 3,3-diaminobenzidine (TR-004-HD; Labvision) for 10 min. Counterstaining was performed with hematoxylin.
Relative quantification of mRNA.
Two-step real-time RT-PCR was performed as previously described (8). Briefly, after total mRNA extraction (no. 74124; Qiagen), standard curves were obtained for each gene correlating (R 
0.98) the mRNA quantities in graded dilutions of a rat cardiac tissue sample with the respective threshold cycles (second derivative maximum method). Equal amounts of mRNA from every sample underwent three separate two-step real-time RT-PCR experiments for each gene, using SYBR green as marker (no. 204143; Qiagen). GAPDH was used as internal control, because its mRNA levels were similar in the studied groups. Results are relative to the mean obtained for the sham group (set as arbitrary unit) and normalized for GAPDH. Specific PCR primer pairs for the studied genes are presented in Table 1.
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RESULTS |
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85% in MCT and 50% in MCT + bosentan rats. All MCT rats presented clinical signs of overt HF, namely, lethargy, labored breathing, cachexia, vein and liver engorgement, pleural effusion, and ascites. Although MCT + bosentan rats also presented these clinical signs, they distinguished themselves from MCT rats because they showed less lethargy on a qualitative evaluation. The body weight (BW) of sham rats was similar to the BW of MCT and MCT + bosentan rats (266 ± 16, 240 ± 13, and 250 ± 12 g, respectively; P = 0.37).
Cardiac hemodynamics, morphometry, and histology.
The hemodynamic features of the experimental groups are summarized in Table 2. MCT rats showed a marked increase in RVSP compared with sham rats (P < 0.001). This was accompanied by other modifications in hemodynamic profile, such as increased values of RV dP/dtmax (P = 0.014), (P < 0.001), and RVEDP (P = 0.003), but also by morphometric (Fig. 1) and histological changes (Table 3 and Figs. 1C and 2), such as increased ratio of RV weight to BW (P < 0.001), increased RV cardiomyocyte diameters (P < 0.001), and significantly higher grades of myocardial fibrosis (P = 0.014). In contrast to the RV myocardium, the grade of myocardial fibrosis observed in the LV of MCT rats did not differ from that of sham rats (Table 3 and Fig. 2), and despite the tendency toward an increase in the ratio of LV and septum as a whole to BW (Fig. 1), only septal (P < 0.001) and not LV free wall cardiomyocytes presented increased diameters (Fig. 1C). Likewise, the LV filling pressures as estimated by LVEDP and LVEDD were unaltered. The maximum developed pressures (LVSP), however, were reduced (P < 0.001) as well as the indexes of contractility, dP/dtmax (P < 0.001) and LVPiso (P < 0.001), whereas the relaxation time constant was increased (P = 0.03) in the LV of MCT rats. The heart rate of MCT rats was significantly decreased as well (sham = 272 ± 14 beats/min vs. MCT = 200 ± 17 beats/min; P = 0.008). Chronic therapy with bosentan after MCT injection attenuated RV hypertrophy (P < 0.001) as assessed by a reduction in the ratio of RV free wall to LV free wall plus interventricular septum weight (Fig. 3).
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was studied in the LV of MCT-treated rats to assess a possible role of hypoxia on neuroendocrine activation. No differences were observed (sham = 1.0 ± 0.1 arbitrary units vs. MCT = 1.3 ± 0.2 arbitrary units; P = 0.22). Contractile properties of intact LV muscle strips. Isometrically contracting muscle strips from sham and MCT + bosentan LV showed a steady increase in developed tension between 1 and 5 Hz, indicating positive FFR, whereas MCT LV muscle strips responded in the opposite way (P < 0.001), presenting overall negative FFR (Fig. 6). Baseline force development was not different (P = 0.16) among the three groups and averaged 2.2 ± 0.4 mN/mm2. Adding the selective ETA antagonist BQ-123 to in vitro contracting LV intact muscle strips induced different responses (P = 0.005) in sham compared with MCT and MCT + bosentan muscle strips: a negative inotropic effect was elicited in sham muscle strips (P < 0.032), whereas the tension developed by MCT and MCT + bosentan muscle strips remained unaltered (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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MCT-treated rats developed PH, RV myocardial hypertrophy, and fibrosis. A higher expression of genes that take part in the processes of molecular remodeling was observed in the chronically overloaded RV myocardium but also in the LV myocardium. Despite the pronounced upregulation of ACE and ET-1 without counterregulatory activation of BNP gene expression in the LV of MCT-treated rats, neither myocardial hypertrophy nor fibrosis was observed, underscoring the primordial role of overload on their development (17).
Opposite to BNP and ACE, ET-1 gene expression and peptide production were similar in the RV and LV of MCT-treated rats. Several explanations can be advanced to substantiate the magnitude of LV production of ET-1. First, neurohumoral activation typically accompanies unloading (21), but because neither the LV filling pressures nor LVEDD of MCT-treated rats were lowered and their LV were atrophic, we believe unloading cannot fully explain ET-1 upregulation. Second, some degree of hypoxemia also was acknowledged in MCT-treated rats (23, 37); hence, we studied the mRNA levels of HIF-1, a central marker of hypoxia-induced ET-1 expression, (15) having observed no differences among groups. Third, various circulating mediators (2, 24) and the sympathetic nervous system (31) are likely inducers of ET-1 gene expression in MCT-treated rats, although their influence would be expected to take place earlier in the development of PH (18). Finally, because ET-1 is an autocrine/paracrine factor in angiotensin II-induced hypertrophy (6, 12), we must point out the possibility that angiotensin II generated by the overactive ACE may contribute to ET-1 activation.
MCT animals presented significant reductions of both LVSP and heart rate. This has been interpreted previously as consequence of continuous severe sickness (24). Tachycardia would be expected as a reflex response to a lower blood pressure, but MCT-treated rats presented an impaired response to the sympathetic nervous system as manifested by reductions of 
-adrenoceptor and norepinephrine neuronal transporter densities and of norepinephrine neuronal transporter and G protein-coupled receptor kinase activity (18).
Reduction of LVSP in MCT-treated rats was accompanied by disturbed hemodynamic contractility and relaxation indexes, as previously described (14). Albeit, LV fibrosis and atrophy were ruled out, and even though invasive hemodynamic indexes were obtained with a widely open pericardium, thus reducing ventricular interdependence (33), the majority of studies carried out in PH patients (22, 29) attribute LV dysfunction fundamentally to ventricular interdependence and impaired LV filling (22) or geometric modifications (4). This is supported by the development of septal hypertrophy in our group of MCT-treated rats but not by the evidence of unaltered LVEDP and LVEDD. Likewise, conserved LV volumes have been reported despite LV functional deterioration in PH (7). However, it should be stressed that because we evaluated LV dimensions through a simple septal-lateral diameter, which does not allow a thorough evaluation of LV volume, and therefore an accurate determination of LV preload, preload-dependent indexes such as dP/dtmax and peak isovolumetric pressures are insufficient to definitely establish impaired contractility.
To further elucidate the myocardial contractile phenotype of the LV in MCT-treated rats, safeguarding the possibility that our findings were not due to ventricular interdependence, we conducted experiments with LV myocardium on intact muscle strip preparations. The FFR is an important intrinsic regulatory mechanism of cardiac contractility; normal myocardium increases developed force with higher frequencies of stimulation over the frequency range close to the physiological heart rate, showing normal contractile reserve, but failing or dysfunctional myocardium loses this reserve, and therefore FFRs are excellent indicators for evaluating the severity of cardiac contractile dysfunction and cardiac reserve capacity as well as for the evaluation of the effectiveness of therapeutic agents in congestive HF (5). The normal myocardium from sham rat LV muscle strips presented positive FFR as expected (5, 16), whereas the muscle strips from the LV of MCT-treated rats showed overall negative FFR indicative of reduced contractile reserve (5), resembling the hypertrophied RVs of MCT-treated rats in earlier stages of PH (16, 34).
The strong local activation of ET-1 in the LV of MCT-treated rats could straightforwardly lead one to presume that this peptide might be partly underlying the disturbed contractile properties of MCT LV myocardium. Chronic effects of ET-1 have been previously studied. ET-1 administration augments basal force of contraction, prolongs relaxation, and significantly blunts responses to Ca2+ and isoprenaline (38). In the present study, negative FFRs were observed in the ET-1-overexpressing LV myocardium of MCT rats. Chronic ET-1 overactivity in heart disease has been associated with slower relaxation and impaired contractility through dysfunctional Ca2+ homeostasis and myosin heavy chain (MHC) isoform switch (10, 13, 26, 28). Curiously, the LV of rats with PH induced by either MCT (11) or hypoxia (32) undergoes MHC isoform switch, whereas ETA receptor antagonists have noticeably been reported to partly prevent this switch in MCT RV myocardium (10). The acute effects of ETA blockade were studied in vitro to ascertain whether there was a functional change in the acute myocardial effects of endogenous ET-1. We had previously reported a negative inotropic effect of the selective ETA antagonist BQ-123, per se, on undissected rabbit RV papillary muscles with intact endothelium in vitro (19). This also was observed in the present study in sham muscle strips. The contractility of LV muscle strips from MCT-treated rats, on the contrary, was unaffected by acute in vitro exposure to BQ-123, which might denote an abnormal functioning of the overactive endothelin system compared with the normal myocardium. Chronic therapy with bosentan did not restore the normal effects of BQ-123 on muscle strips of LV myocardium, but it restored the positivity of FFR compared with MCT-injected PH rats without subsequent treatment with bosentan. Indeed, although bosentan blunted MCT-induced PH as previously reported (9), reducing RV hypertrophy with concomitant attenuation of local neuroendocrine gene expression, the marked change in FFR in the LV of MCT-injected rat myocardium after chronic therapy with bosentan favors the hypothesis of a direct action of chronic ET-1 antagonism on the molecular and functional LV myocardial phenotype. Overall, these results suggest that the beneficial effects of chronic endothelin antagonism in MCT-treated rats (14) may not be dependent exclusively on attenuated PH and improved pulmonary blood flow but also on the myocardial effects of chronic ET-1 blockade.
Despite the similarities with human PH (25), given that MCT-induced PH has no human equivalent, any implication in human pathophysiology is limited. Nevertheless, we must point out that LV myocardial abnormalities may partly underlie LV dysfunction in patients with severe PH (36). Indeed, in patients with PH, normal LV filling is only restored 1 yr after single-lung transplantation even if LV geometry and RV function are immediately restored (36), and combined heart-lung transplantation is favored if severe impairment of LV function is also present because the LV may not recover after transplantation of the lungs alone (27). A patient with severely diminished LV function, however, tolerated double-lung transplantation and fully recovered cardiac function after therapy with bosentan. The authors wisely attributed this to a reduction in ventricular interaction but also admitted that bosentan might have acted directly on the myocardium (1).
In conclusion, MCT-treated rats with severe long-standing PH develop local autocrine/paracrine system activation that is not restricted to the overloaded ventricle. The LV myocardium expresses high levels of genes involved in autocrine/paracrine systems, but despite this, it does not undergo hypertrophy or fibrosis. The LV contractile phenotype, however, is altered: LV muscle strips from MCT-treated rats present negative FFR. Acute ETA antagonism in vitro has distinct effects on contractile performance compared with sham myocardium, and chronic ET-1 blockade after MCT injection restores the positivity of LV myocardium FFR, suggesting a possible direct detrimental action of ET-1 overexpression in the LV myocardium of PH rats.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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induced for impaired myocardial energy metabolism. Circulation 103: 2387–2394, 2001. A, Pönicke K, Heinroth-Hoffmann I, and Brodde OE. Ventricular hypertrophy plus neurohumoral activation is necessary to alter the cardiac -adrenoceptor system in experimental heart failure. Circ Res 91: 1056–1062, 2002.-adrenoceptor-G-protein(s)-adenylyl cyclase system in monocrotaline treated rats. J Mol Cell Cardiol 32: 2315–2326, 2000.[CrossRef][ISI][Medline]This article has been cited by other articles:
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  S. G. Haworth The cell and molecular biology of right ventricular dysfunction in pulmonary hypertension Eur. Heart J. Suppl., December 1, 2007; 9(suppl_H): H10 - H16. [Abstract] [Full Text] [PDF]
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M. Kurzyna and A. Torbicki Neurohormonal modulation in right ventricular failure Eur. Heart J. Suppl., December 1, 2007; 9(suppl_H): H35 - H40. [Abstract] [Full Text] [PDF]
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 R. R. Lamberts, E. Caldenhoven, M. Lansink, G. Witte, R. J. Vaessen, J. A. St Cyr, and G. J. M. Stienen Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1869 - H1876. [Abstract] [Full Text] [PDF]
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I. R. Henkens, K. T. B. Mouchaers, H. W. Vliegen, W. J. van der Laarse, C. A. Swenne, A. C. Maan, H. H. M. Draisma, I. Schalij, E. E. van der Wall, M. J. Schalij, et al. Early changes in rat hearts with developing pulmonary arterial hypertension can be detected with three-dimensional electrocardiography Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1300 - H1307. [Abstract] [Full Text] [PDF]
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 F. Akhavein, E. J. St.-Michel, E. Seifert, and C. V. Rohlicek Decreased left ventricular function, myocarditis, and coronary arteriolar medial thickening following monocrotaline administration in adult rats J Appl Physiol, July 1, 2007; 103(1): 287 - 295. [Abstract] [Full Text] [PDF]
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P < 0.001).
) of developed tension at 1 Hz. Sham and MCT+bosentan LV muscle strips show positive FFR, whereas FFR are negative in MCT LV muscle strips. *P < 0.001 for interaction, as evaluated with two-way repeated-measures ANOVA. 