The heart hypertrophies in response to a chronic elevation in workload, as in hypertension (1). Administration of the Crotalaria alkaloid, monocrotaline, to rats is used as a noninvasive, slowly developing, hemodynamically relevant model for pulmonary hypertension leading to right ventricular hypertrophy (2). Right ventricular performance was enhanced in these monocrotaline-treated rats, yet β1-adrenoceptor density and adenylate cyclase activity are selectively decreased in the right ventricle (3), as are isoprenaline-induced responses in isolated right ventricular myocytes (4).
Positive inotropic compounds increase force by increasing either intracellular calcium concentrations or the calcium sensitivity of the contractile proteins. Cardiac hypertrophy may lead to marked decreases in positive inotropic responses, especially to the cyclic adenosine monophosphate (cAMP) generators (5,6) and α-adrenoceptor agonists (7), whereas responses to the calcium sensitizer, EMD 57033, are unchanged (8). Vasoconstrictor responses may be changed in cardiovascular disease; α-adrenoceptor-mediated responses are increased in hyperthyroid and spontaneously hypertensive rats (7), whereas the potencies of 5-hydroxytryptamine, noradrenaline, and potassium are increased in the hypertrophied pulmonary artery of monocrotaline-treated rats (9).
The aim of this study was to determine positive inotropic, positive chronotropic, and vasoconstrictor responses in isolated tissues from rats with pulmonary hypertension after a single injection of monocrotaline. Experiments were performed either 4 weeks (hypertrophy without signs of heart failure) or 6 weeks (hypertrophy with failure) after treatment. Positive inotropic responses were measured for noradrenaline (selective β1-adrenoceptor agonist), forskolin (adenylate cyclase activator), EMD 57033 (calcium sensitizer, possibly with phosphodiesterase inhibitory activity), and calcium chloride in isolated papillary muscles from the hypertrophied right ventricle and the nonhypertrophied left ventricle. Positive chronotropic responses were measured in the hypertrophied right atrium; vasoconstrictor responses to noradrenaline, 5-hydroxytryptamine, and KCl were determined in the hypertrophied pulmonary artery and nonhypertrophied thoracic aorta. Right and left ventricular, lung, and liver β-adrenoceptors were characterized by radioligand-binding studies. These results will determine whether the ability of the heart to increase force is altered in monocrotaline-induced right ventricular hypertrophy and failure. Comparison with reported cardiac responses in humans with pulmonary hypertension may indicate whether monocrotaline-treated rats are a useful model of the human disease.
Male Wistar rats (∼8 weeks old), obtained from the Central Animal Breeding House of The University of Queensland, were given a single subcutaneous injection of monocrotaline (105 mg/kg; 9) and housed singly for 4 or 6 weeks (n = 40 and 35, respectively). Food and water consumption and body weight were measured daily; rats were killed by chloroform overdose.
Isolated, contracting cardiac tissues
The heart was rapidly removed, and the right atria and papillary muscles from both the left and right ventricles were dissected free and prepared for measurement of force of contraction (10). Tissues were suspended in organ baths at a resting tension of 5-10 mN, adjusted to give the maximal twitch response. Muscles were bathed in physiological solution containing (in mM): NaCl, 136.9; KCl, 5.4; MgCl2, 1.05; CaCl2, 1.8; NaHCO3, 22.6; NaH2PO4, 0.42; glucose, 5.5; ascorbic acid, 0.28; and sodium edetate, 0.1; maintained at 35°C, bubbled with 95% O2/5% CO2, and stimulated at 1 Hz. Concentration-response curves were obtained by cumulative addition of either noradrenaline, forskolin, or EMD 57033, and then, after washout for ≥1 h, to calcium chloride by addition of drug at apparent equilibration of the response to the preceding concentration. Maximal responses were determined either by no further increase in force of contraction at higher concentrations or by the development of toxicity, usually as ectopic beats.
Isolated vascular rings
A length of thoracic aorta and the common pulmonary artery near the right ventricle were removed. Rings from the aorta (∼4 mm long) and pulmonary artery (∼3 mm long) were suspended with a resting tension of 10 mN. Rings were contracted twice with isotonic KCl (100 mM); the presence of endothelium was demonstrated by partial relaxation in response to acetylcholine (1 × 10−5M). After washout, a cumulative concentration-response (contraction) curve was determined for noradrenaline, 5-hydroxytryptamine, or KCl as an isotonic solution. Some rings were perfused with 10% buffered neutral formalin, embedded in wax, and 20-μm sections cut and stained with hematoxylin and eosin. Image analysis (Wild-Leitz MD30+ system, Adelaide, Australia) was then used to calculate wall thicknesses.
Characterization of ventricular β-adrenoceptors
Membranes from the left and right ventricular free walls, liver, and lungs were prepared (11); β-adrenoceptors were characterized (12) by using 125I-labeled iodocyanopindolol (ICYP) as ligand. Membranes were incubated with ICYP (∼25,000 dpm/tube) and increasing concentrations (1 × 10−9-1 × 10−4M as nonspecific binding) of the β2-adrenoceptor-selective antagonist ICI 118,551 in a final volume of 300 μl of a buffer containing TRIS-HCl (50 mM) and MgCl2 (1 mM), pH 7.4, for 2 h at 37°C. Bound ICYP was precipitated by adding 0.5 ml bovine γ-globulin (4 mg/ml) and 2 ml polyethylene glycol 6000 (16% solution in water), mixing thoroughly, and centrifuging at 15,000 g for 30 min at 4°C. The counts in the pellet were determined with a γ-counter. Protein concentration was determined by a dye-binding method (13) by using Coomassie blue, read at 595 nm with bovine serum albumin as standard.
All values of change in force of contraction (in mN) or change in rate of contraction (in beats/min) are mean ± SEM. Mean effective concentration (EC50) values were determined from the concentration yielding half-maximal effect from each concentration-response curve. ICYP binding was analyzed by Eadie-Hofstee plots by the LIGAND program (14) to determine receptor densities and Kd values for β1- and β2-adrenoceptors, as in previous studies (5). Dunnett's t test with corrections for multiple comparisons was used for comparison of response curves (15); the unpaired Student's t test was used for comparison of physiological and biochemical parameters. A value of p < 0.05 was considered significant.
Monocrotaline, (−)noradrenaline hydrochloride, and forskolin were purchased from Sigma Chemical (St. Louis, MO, U.S.A.); EMD 57033 was generously provided by Dr. Inge Lues (E. Merck, Darmstadt, Germany). Monocrotaline solution (105 mg/ml) was prepared by dissolving monocrotaline (500 mg) in HCl (1 M; 1.5 ml) and water (1 ml), adjusted to pH 7.0 with NaOH (1 M) and to 4.76 ml with water (9). Noradrenaline was dissolved in water; forskolin and EMD 57033 were dissolved in dimethylformamide. Organ-bath concentrations of dimethylformamide did not exceed 1%, which we have previously shown to be without effect on force or rate of contraction.
Rats given a single injection of monocrotaline (105 mg/kg, s.c.) showed minimal body-weight gain and no significant changes in food and water intake for the first 4 weeks but significant decreases in these parameters in the following 2 weeks (Fig. 1). Four weeks after treatment, rats showed right ventricular and right atrial hypertrophy, pulmonary edema, an increased pulmonary artery wall thickness, and renal hypertrophy (Table 1). These changes were more marked 6 weeks after treatment (Table 1); in addition, these rats showed shallow and rapid respiration, pulmonary effusions, an increased deposition of collagen in the right ventricle, and marked changes to the liver architecture.
Isolated right ventricular papillary muscles from monocrotaline-treated rats showed an increased basal force of contraction, whereas basal force was unchanged in left ventricular muscles (Table 1). Positive inotropic responses to noradrenaline, EMD 57033, and calcium chloride on right and left ventricular papillary muscles are given in Figs. 2 and 3, respectively. Responses in right ventricular papillary muscles to noradrenaline were shifted to higher concentrations after 4 weeks and depressed after 6 weeks; responses to EMD 57033 were depressed, whereas responses to calcium chloride were unaffected. In right ventricular papillary muscles, maximal increases in force after forskolin were decreased from 1.28 ± 0.46 mN (control) to 0.45 ± 0.35 mN (4 weeks) and 0.18 ± 0.35 mN (6 weeks). Papillary muscles from the nonhypertrophied left ventricle showed increased responses to calcium chloride and depressed responses to EMD 57033. Noradrenaline responses decreased, whereas forskolin was more potent after 4 weeks (−log EC50 6.1 ± 0.15, control; 6.50 ± 0.12, 4 weeks; maximal increase, 3.46 ± 0.46 mN, control; 4.04 ± 0.33 mN, 4 weeks); both returned to control values after 6 weeks.
The basal rate of contraction in isolated right atria was unaffected by monocrotaline treatment (Table 1). Chronotropic responses to noradrenaline and forskolin in hypertrophied right atria were unchanged by monocrotaline treatment (Fig. 4; forskolin −log EC50 6.9 ± 0.13, control; 7.0 ± 0.1, 4 weeks; 7.1 ± 0.1, 6 weeks; maximal increase, 200 ± 12 beats/min, control; 158 ± 9 beats/min, 4 weeks; 181 ± 16 beats/min, 6 weeks).
Maximal responses to a depolarizing solution of KCl did not differ between groups (pulmonary arteries: control, 16 ± 2.4 mN; 4 weeks, 15.3 ± 2.3 mN; 6 weeks, 18.8 ± 1.2 mN; thoracic aortic rings: control, 17.3 ± 1.8 mN; 4 weeks, 17.6 ± 3.0 mN; 6 weeks, 18.6 ± 2.3 mN; all n = 6−8). Acetylcholine-mediated (1 × 10−5M) relaxation of KCl-induced contraction of pulmonary artery rings was increased after 6 weeks' treatment (control, 13.9 ± 1.3%; 4 weeks, 17.3 ± 3.8%; 6 weeks, 23.9 ± 4.5%), whereas monocrotaline treatment reduced acetylcholine-mediated relaxation of KCl-contracted thoracic aortic rings (control, 20.8 ± 1.2%; 4 weeks, 9.7 ± 3.2%; 6 weeks, 10.2 ± 3.6%). Vasoconstrictor responses to 5-hydroxytryptamine in isolated pulmonary artery rings were selectively increased after monocrotaline treatment (Fig. 5A). Thoracic aortic responses to noradrenaline, 5-hydroxytryptamine, and potassium chloride were unaltered by monocrotaline treatment (Fig. 5B; noradrenaline −log-EC50, 7.3 ± 0.1, control; 7.4 ± 0.1, 4 weeks; 7.4 ± 0.1, 6 weeks; 5-hydroxytryptamine, −logEC50, 5.4 ± 0.2, control; 5.4 ± 0.2, 4 weeks; 5.5 ± 0.2, 6 weeks).
Right ventricular β1-adrenoceptor density decreased only in 6-week monocrotaline-treated rats; β2-adrenoceptor density was unchanged (Table 1). β1-Adrenoceptor density decreased in the nonhypertrophied left ventricle 4 weeks after monocrotaline. Both β-adrenoceptor subtypes were present in the rat lung and liver; their densities decreased in the lung yet increased in the liver after monocrotaline treatment (Table 1).
In rats, monocrotaline produces pathological changes in the lungs resulting in pulmonary hypertension (16,17). This increase in work load induces selective right ventricular hypertrophy, progressing to heart failure. The molecular mechanisms responsible for the monocrotaline responses remain unclear: the hepatic metabolite, dehydromonocrotaline, may be the reactive intermediate initiating pulmonary damage but has a very short half-life (18), and increased levels of polyamines (19) also have been implicated. Further, inhibition of endogenous NO production combined with increase of endothelin levels may play an important role in the pathogenesis of monocrotaline-induced pulmonary hypertension (20). The usefulness of monocrotaline-treated rats as a model of the pulmonary changes in human pulmonary hypertension has been questioned (17). Thus it is important to characterize the cardiac responses after monocrotaline-induced pulmonary hypertension for comparison with reported changes in cardiac responses in humans with pulmonary hypertension (21).
The development of hypertrophy may change the performance of the heart. Monocrotaline treatment leads to an enhanced right ventricular performance (2), which is consistent with the increased basal force of contraction of right ventricular papillary muscles in this study. However, the responsiveness to positive inotropic compounds may be significantly compromised in the hypertrophied nonfailing ventricle: for example, to noradrenaline in hyperthyroid rats (5), in noradrenaline-induced left ventricular hypertrophy (22), and in renal or spontaneously hypertensive rats (6). The contractile reserve as calcium chloride responses was maintained in muscles from the hypertrophied right ventricle of monocrotaline-treated rats even in heart failure in our study. In contrast, the ability to respond to cAMP generators (noradrenaline and forskolin) and the calcium sensitizer, EMD 57033, was decreased. Heart failure produced decreased responses in right ventricular papillary muscles to all cAMP generators, possibly because a decreased availability of cAMP resulting from a reduced adenylate cyclase activity (3) may be common to all types of heart failure (23,24). As a calcium sensitizer, EMD 57033 binds to the Ca2+/Mg2+ sites of troponin C(25) and decelerates the dissociation of myosin-actin cross-bridges (26). Part of the positive inotropic responses to EMD 57033, especially at higher concentrations, may be caused by inhibition of phosphodiesterase (8,27), although inotropic responses to phosphodiesterase inhibitors such as theophylline are relatively small in the rat heart (10). Thus the reduced inotropic response to EMD 57033 in pulmonary hypertensive hearts may represent a loss of cAMP-mediated responses, as with the cAMP generators, noradrenaline and forskolin. This contrasts with the unchanged responses to EMD 57033 relative to calcium chloride in left ventricular hypertrophy caused by DOCA/salt hypertension or hyperthyroidism (8).
Monocrotaline treatment produced hypertrophy of the pulmonary artery and increased responses to 5-HT, as also reported by others (9,28,29). In addition, administration of a selective 5-HT2 antagonist attenuates monocrotaline-induced changes (29). Possible explanations include a decreased endothelial 5-HT1-receptor-mediated vasorelaxant response or an increased vascular smooth-muscle 5-HT2-receptor-mediated vasoconstrictor response. The latter may be more consistent with the increased response to acetylcholine in pulmonary arteries. Increased responses to noradrenaline and KCl were reported in endothelium-denuded pulmonary artery rings (9), unlike our results in rings with an intact endothelium. Contraction responses in the nonhypertrophied thoracic aortic rings were unchanged in our study.
The progression from hypertrophy at 4 weeks to heart failure at 6 weeks allows the assessment of the time course of changes in responsiveness. The most pronounced change is the further increase in responsiveness to 5-HT in the pulmonary arteries; 5-HT from platelets may contribute to both the initiation and progression of hypertension in this model (29). Positive inotropic responses to the cAMP generators, noradrenaline and forskolin, were reduced in muscles from the hypertrophied right ventricle before heart failure occurred; heart failure led to further decreases in responses. Responses in the nonhypertrophied left ventricle were either unchanged or increased in the failing heart, possibly as a partial compensation for the right ventricular failure.
Significantly, inotropic responses to calcium were not reduced in the hypertrophied ventricle, showing that the contractile reserve of the muscles is not changed, even in severe heart failure. Decreases in inotropic responses are thus the result of decreases in access to the contractile reserve, a situation similar to that described in the human heart (30). Mechanisms include decreased cAMP levels (23), partly resulting from decreased adenylate cyclase activity (3), possibly caused by increased Gia-protein levels (31), shifts from α- to β-myosin in hypertension-induced hypertrophy (4,24), or changes in ventricular ion channels (32). These mechanisms seem not to apply to the hypertrophied right atria because chronotropic responses were unaffected.
Increased proportions of types III and V collagen have been shown in the hypertrophied and failing right ventricle of monocrotaline-treated rats, with structural changes in collagen fibrillar sheaths accompanied by replacement fibrosis, especially in the failing heart (33). Our results showed a significant increase in total collagen content in the hypertrophied and failing right ventricle only. An increase in collagens indicates an increased secretion of locally produced or circulating hormones such as endothelin, angiotensin II, or aldosterone, which are known to increase cardiac collagen synthesis, causing myocardial fibrosis (34).
Heart failure resulting from primary pulmonary hypertension in humans produces marked decreases in cardiac β1-adrenoceptor density, positive inotropic responses, cardiac stores of noradrenaline, and activity of the catalytic subunit of adenylate cyclase in the failing right ventricle (21). Similar changes were observed in the right ventricle of monocrotaline-treated rats (3; this study). Both rats and humans show a similar functional response as decreases in responses to cAMP generators without a corresponding decrease in the ability to produce force, measured as calcium-induced contractile responses (21). Vascular hyperresponsiveness to 5-HT has been reported in isolated pulmonary arteries from patients with primary pulmonary hypertension (35), as in this study with monocrotaline-treated rats. Further, these patients had an increased plasma 5-HT concentration and decreased platelet 5-HT storage (36), suggesting that impaired 5-HT handling is associated with human pulmonary hypertension.
In contrast to patients with pulmonary hypertension (21), β-adrenoceptor densities decreased in the nonhypertrophied left ventricle of the monocrotaline-treated rat, suggesting that this may be the result of circulating factors rather than the development of hypertrophy per se. Monocrotaline treatment also produces pathological changes in both the lungs and the liver. The decrease in the lung β-adrenoceptor density correlates with increasing respiratory difficulties. However, the functional correlate to the increased liver β-adrenoceptor density is not obvious, although monocrotaline disturbs sulfur metabolism and the bile composition (37).
In summary, monocrotaline-treated rats showed marked right ventricular and pulmonary artery hypertrophy 4 weeks after treatment, progressing to severe heart failure within a further 2 weeks. The major change at 4 weeks was a marked increase in constrictor responses to 5-HT of the isolated pulmonary artery. Positive inotropic responses to cAMP generators and a calcium sensitizer were reduced after 4 weeks but more markedly reduced after 6 weeks. These disease-induced changes in function seem to be the result of local changes, as neither the nonhypertrophied thoracic aorta nor the left ventricle showed similar changes. Further, monocrotaline-induced right ventricular hypertrophy in rats mirrors the functional changes in humans and thus may be a useful model by which to define therapeutic strategies in humans (38).
Acknowledgment: This study was partially supported by the National Health and Medical Research Council of Australia (C.S.) and The University of Queensland.
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