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Ann Thorac Surg 1999;67:1053-1058
© 1999 The Society of Thoracic Surgeons
a Division of Cardiovascular and Thoracic Surgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
Accepted for publication October 6, 1998.
Address reprint requests to Dr Chen, Department of Surgery, University of California, San Francisco, Box 0470, S343, San Francisco, CA 94131
e-mail: epchen{at}itsa.ucsf.edu
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Abstract |
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Methods. Animals underwent pulmonary artery catheterization to measure pulmonary hemodynamics before and 8 weeks after an injection of monocrotaline pyrrole, 3 mg/kg (n = 8) or placebo (n = 8) (controls). Systolic function was assessed with load-insensitive means (preload-recruitable stroke work). Myocardial biopsy specimens were collected to analyze membrane 
1- and ß-adrenergic receptor density and adenylate cyclase activity.
Results. Eight weeks after injection, significant increases in pulmonary hemodynamic indices were noted in monocrotaline-injected dogs. Significant increases in right ventricular preload-recruitable stroke work were also observed in these animals compared with controls and occurred in association with significant increases in right ventricular 1- and ß-adrenergic receptor density and isoproterenol hydrochloride–stimulated adenylate cyclase activity. No significant differences in basal adenylate cyclase activity in the right ventricle were noted between the two groups.
Conclusions. These data suggest that alterations in right ventricular function in the setting of chronic pulmonary hypertension may partially be due to changes in myocardial adrenergic receptor signaling.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Right ventricular performance in the setting of chronic pulmonary hypertension has been well described, both experimentally [1] and in the clinical setting [2]. Few reports, however, have examined the molecular basis behind this adaptation. Basic investigation of this problem has been limited by lack of an appropriate large-animal model of stable chronic pulmonary hypertension. This study was designed to examine the compensatory mechanisms of the right ventricle, at both the physiologic level and the biochemical level, in the setting of chronic pulmonary hypertension using a canine model of monocrotaline pyrrole–induced chronic pulmonary hypertension [3], load-insensitive means for functional assessment of myocardial performance, and analysis of myocardial adrenergic receptor signaling.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985). The experiments were approved by the Duke University Institutional Animal Care and Use Committee (DUIACUC Assigned Registry No. A621-95-9R1).
Instrumentation and operation
Eight weeks after injection, a median sternotomy and an anterior pericardiotomy were performed in every dog to expose the hearts. An ultrasonic flow probe (T208X; Transonic Systems Inc, Ithaca, NY) was placed around the main pulmonary artery trunk to measure pulmonary blood flow. Micromanometers (MPC-500; Millar Instruments Inc, Houston, TX) were placed into the right and left ventricles, the right and left atria, and the pulmonary artery for continuous recording of intracavitary pressures.
Hemispheric ultrasonic dimension transducers (outer diameter, 1.5 mm) (No. 1-1015-5A; Vernitron, Bedford, OH) were sewn to the epicardial surface of the heart across the base-apex major-axis and anteroposterior minor-axis diameters of the left ventricle. Two additional transducers were placed on the epicardial surfaces of the right and left ventricular free walls, and another was inserted into the interventricular septum to measure the septal–free wall minor-axis diameters of both the right and left ventricles.
Myocardial biopsies
After instrumentation, transmural myocardial biopsy specimens were obtained from both ventricles in every animal in the two groups. A suction drill was used to take a sample of the left ventricular apex, and an excisional biopsy was performed in the right ventricle at the outflow tract. All tissue samples were snap-frozen in liquid chlorodifluoromethane (Laroche Chemicals Inc, Gramercy, LA) and stored in liquid nitrogen until biochemical analysis was performed.
Data acquisition and analysis
Hemodynamic and functional data were obtained from each animal after the myocardial biopsies. Raw data were digitized online, collected, and stored on a microprocessor (PDP 11/23; Digital Equipment Corp, Maynard, MA). All data were analyzed on a Dell XPS P90 personal computer (Dell Computer Corp, Austin, TX) with well-described software [5].
Tissue analysis
After hemodynamic and functional data were collected, all hearts were excised, and the atria were separated from the ventricles at the atrioventricular groove. The right ventricular free wall was separated from the left ventricle and septum. The right and left ventricles from each animal were weighed, and wall volumes were determined using saline solution displacement. To calculate percent water, all samples were placed in a 120°C oven and dried for 24 hours. The dry weight of each tissue sample, that is, the right ventricle and the left ventricle + septum, was measured, and the weight ratios were compared between the two groups. Percent tissue water content was calculated as follows: percent water = [(wet weight - dry weight) x 100%]/wet weight.
Assessment of biventricular systolic function
Right ventricular function and left ventricular function were assessed with load-insensitive means. The relationship between stroke work and end-diastolic chamber volume, for both the right ventricle and the left ventricle, was fit to a highly linear relationship during vena cava occlusion using least squares linear regression. The slope of these linear regressions is known as the preload-recruitable stroke work and represents a load-independent index of systolic function and myocardial contractility [5]. Dynamic right ventricular volume was measured according to the ellipsoidal shell subtraction method [6].
1-adrenergic and ß-adrenergic receptor ligand-binding assays
Membrane fractions were prepared from hearts and resuspended in binding buffer (150 mmol/L NaCl; 50 mmol/L Tris-[tris(hydroxymethyl)methylamine]-HCl, pH 7.4; 5 mmol/L EDTA [ethylenediaminetetraacetic acid] or 75 mmol/L Tris-HCl, pH 7.4; 12.5 mmol/L MgCl2; 2 mmol/L EDTA). Binding assays were performed on 25 µg of membrane protein using saturated amounts of 2-{ß-(4-hydroxy-3-[125I]iodophenyl)ethyl-aminomethyl}tetralone (300 pmol/L), an 1-adrenergic receptor–specific ligand, or [125I]cyanopindolol (300 pmol/L), a ß-adrenergic receptor–specific ligand. Nonspecific binding was determined in the presence of 50 µmol/L prazosin for -binding and 20 µmol/L alprenolol for ß-binding. Reactions were conducted in either 250 or 500 µL of binding buffer at 37°C for 1 hour and then terminated by suction through glass-fiber filters. All assays were performed in triplicate, and receptor density (femtomoles) was normalized to milligrams of membrane protein.
Adenylate cyclase analysis
Crude myocardial membranes were again prepared from the right and left ventricles of animals in both groups. Membranes (20 to 30 µg of protein) were incubated for 15 minutes at 37°C with [32P] -adenosine triphosphate under basal conditions as well as in the presence of 100 µmol/L isoproterenol hydrochloride and 100 mmol/L sodium fluoride. Cyclic adenosine monophosphate was then quantitated using a well-known method [7] to determine adenylate cyclase activity.
Statistical analysis
Statistical analysis was performed on a Dell personal computer using commercially available software (SigmaStat Version 2.0; Jandel Corp, San Rafael, CA). Data taken before and after injection of monocrotaline pyrrole in the MCTP group were analyzed with standard two-tailed paired Student’s t tests. Unpaired Student’s t tests were used to compare all hemodynamic and functional data between the MCTP and CTL groups. Analysis of biochemical data between the two groups was also performed using unpaired Student’s t tests. The results are expressed as the mean ± the standard error of the mean. A difference was considered significant at a p value of less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Biventricular systolic function in monocrotaline pyrrole–induced chronic pulmonary hypertension
Highly linear relationships (r2 > 0.94) were obtained during vena cava occlusion between the calculated right and left ventricular end-diastolic chamber volumes and stroke work in both groups 8 weeks after injection. No significant differences between the two groups were observed in left ventricular systolic function (92.47 ± 4.95 erg x 103/mL, MCTP group, versus 88.36 ± 7.03 erg x 103/mL), as measured by the preload-recruitable stroke work relationship. However, there were significant increases in the right ventricular preload-recruitable stroke work in the MCTP group (47.95 ± 5.30 erg · 103/mL; p < 0.001) compared with the CTL group (23.65 ± 3.10 erg · 103/mL).
Tissue analysis
Gross inspection of all hearts revealed that the right ventricles were enlarged in the MCTP group compared with the CTL group. Eight weeks after injection, significant increases in right ventricular dry weight (10.29 ± 0.55 g versus 8.40 ± 0.51 g, p < 0.05), right ventricular weight to body weight ratio (0.429 ± 0.024 g/kg versus 0.356 ± 0.021 g/kg, p < 0.05), and right ventricular weight to left ventricular + septal weight ratio (0.446 ± 0.022 versus 0.371 ± 0.010; p < 0.01) were observed in the MCTP group compared with the CTL group. There were no significant differences in left ventricular dry weights (23.32 ± 1.37 g, MCTP group, versus 23.30 ± 1.34 g), right ventricular water content (79.56% ± 0.38%, MCTP group, versus 78.55% ± 0.43%), or left ventricular water content (78.98% ± 0.42%, MCTP group, versus 78.41% ± 0.32%) between the two groups.
ß-adrenergic receptor analysis
Eight weeks after injection, there was no significant difference in ß-adrenergic receptor density in the left ventricle between the two groups. Significant increases were observed, however, in the right ventricular ß-adrenergic receptor density of the MCTP group compared with the CTL group (Fig 1).
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1-adrenergic receptor analysis1-adrenergic receptor density in the left ventricle between the two groups 8 weeks after injection. A significant increase was observed in the right ventricular 1-adrenergic receptor density of the MCTP group compared with the CTL group (Fig 4).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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1-adrenergic and ß-adrenergic systems comprise the two principal receptor components located in myocardial tissue and together play a critical role in regulating overall myocardial performance [8]. Stimulation of these receptors in an intact heart by neurotransmitter (norepinephrine)–or hormone (epinephrine)–mediated chemical signals ultimately affects the actual mechanical events of ventricular contraction [9] and occurs by way of classic guanine nucleotide binding (G) protein–coupled signaling pathways. ß-Adrenergic receptor stimulation constitutes the majority of endogenously generated inotropic support and is the primary mechanism by which cardiac performance is increased in response to physiologic stress [9]. Myocardial 1-adrenergic receptors, on the other hand, are expressed at significantly lower densities than ß-adrenergic receptors, but are still involved in the regulation of both inotropy and chronotropy [10]. Experimental studies [11–13] have demonstrated that 1-adrenergic receptor signaling contributes to both cardiac development and the hypertrophic response of adult myocardium. Alterations in adrenergic receptor signaling have been observed in both human and experimental models of heart failure and cardiac dysfunction. Clinically, a 50% downregulation of myocardial ß-adrenergic receptor density was observed in the setting of congestive heart failure and occurred in association with significant reductions in receptor responsiveness to agonist stimulation [14]. Assessment of adrenergic receptor signaling in the setting of altered left ventricular performance has also been carried out in experimental models of brain death [15], spontaneous systemic hypertension [16], and catecholamine-induced cardiac hypertrophy [17]. Few reports, however, have characterized these signaling pathways during the process of physiologic adaptation before the onset of cardiac failure, particularly with respect to right ventricular mechanics in the setting of elevated pulmonary vascular pressures.
Monocrotaline pyrrole–induced chronic pulmonary hypertension is characterized by a proliferative vasculitis [18]. Pulmonary vascular injury, leading to increased capillary permeability as well as interstitial and alveolar edema, is induced after the initial injection and affects medium- and small-sized pulmonary arterioles. Subsequent vascular remodeling leads to endothelial degeneration and hyperplasia, smooth muscle hypertrophy and medial thickening, and perivascular connective tissue proliferation [18]. The gradual development of these microscopic changes is ultimately responsible for the observed rise in pulmonary vascular pressures.
Admittedly, assessment of right ventricular ß-adrenergic receptor density in the setting of chronically elevated pulmonary vascular pressures has previously been performed in a rat model of monocrotaline pyrrole–induced chronic pulmonary hypertension [19, 20]. In those studies, however, quantification of intrinsic myocardial contractility was not performed in conjunction with examination of ß-adrenergic receptor signaling. The primary disadvantage to using this particular species is the small body size. It would be difficult technically to perform the extremely invasive and previously described instrumentation procedures necessary for accurately measuring dynamic ventricular pressures and volumes; such data are requisite for determining the inotropic state of the heart present in association with a specific level of adrenergic receptor signaling.
The preload-recruitable stroke work relationship is a highly sensitive and well-validated model of estimating intrinsic myocardial mechanics and systolic function [5], independent of any changes occurring in ventricular loading conditions. The results of this investigation indicate that monocrotaline pyrrole–induced pulmonary hypertension has a significant impact on right ventricular function and no significant effect on left ventricular function. Eight weeks after drug injection, right ventricular systolic function was significantly elevated in the MCTP group compared with the CTL group. In contrast, no significant differences in left ventricular function were observed between the two groups. Thus, in this model, the right ventricle adapts to the increased afterload without evidence of cardiac failure.
This report also demonstrates that the right ventricular ß-adrenergic receptor system is upregulated in monocrotaline pyrrole–induced chronic pulmonary hypertension, whereas no significant changes occurred in the left ventricular ß-adrenergic receptor system. This upregulation consisted of significantly enhanced right ventricular ß-adrenergic receptor density and significantly increased isoproterenol-stimulated adenylate cyclase activity. The significant upregulation of right ventricular ß-adrenergic signaling occurring in association with the significant increases in right ventricular systolic function suggests that right ventricular adaptation to increased afterload in the setting of chronic pulmonary hypertension may partially be due to increased signaling through the ß-adrenergic receptor pathway.
Tissue dry weight analysis in this study revealed that the right ventricle was significantly enlarged in the MCTP group, whereas no significant change occurred in left ventricular muscle mass. There were also significant elevations in the right ventricular weight to left ventricular + septal weight ratio as well as the ratio of right ventricular weight to body weight in animals with monocrotaline pyrrole–induced chronic pulmonary hypertension in comparison to the CTL group, findings indicating that a significant degree of right ventricular hypertrophy had occurred.
Ventricular hypertrophy is a compensatory mechanism of the heart that first develops in response to increased cardiac loading conditions and commonly occurs in the setting of hypertension as well as valvular heart disease. The majority of cardiac disease states manifest some degree of hypertrophy as an initial adaptive response. Ultimately, such conditions can lead to congestive heart failure with an associated loss of myocardial contractility and function. The biochemical triggers of this compensatory process, however, remain poorly understood.
Experimental evidence has suggested that the 1-adrenergic receptor system may play a significant role in the initiation of myocardial hypertrophy. Administration of 1-adrenergic agonists has been shown to induce a hypertrophic phenotype in cultured neonatal rat ventricular myocytes [11]. In a guinea pig model of pressure overload secondary to aortic banding, development of cardiac hypertrophy was preceded by a significant increase in myocardial 1-adrenergic receptor density and prevented with an 1-adrenergic blocker [12]. In addition, Milano and associates [13] demonstrated that myocardial expression of a constitutively active 1B-adrenergic receptor in transgenic mice leads to cardiac hypertrophy.
In the present study, right ventricular 1-adrenergic receptor density was significantly increased, whereas no significant changes occurred in the 1-adrenergic receptor density of the left ventricle. These increases in 1-adrenergic receptor density were observed in association with significant increases in the right ventricular weight to left ventricular + septal weight ratio. Thus, the significant upregulation in 1-adrenergic receptor density in the setting of monocrotaline pyrrole–induced chronic pulmonary hypertension may be contributing to the hypertrophic response observed in the right ventricle.
Although the comparisons of right ventricular function in this report are, to some extent, validated by the absence of change in left ventricular mechanics, alterations in the systemic adrenergic state at the time of instrumentation and data acquisition could potentially introduce some variation into the assessment of cardiac contractility. Further insight into the adrenergic signaling pathway could be gained if myocardial performance of the experimental animals were assessed during an autonomically blocked state. In addition, it would have been interesting to examine the phenotypic changes occurring in contractile proteins and proteins of the sarcoplasmic reticulum as important adaptations to chronic pressure overload. Examination of such changes will require further investigation. Although there is a possibility that the changes observed in the present study are specific to monocrotaline, previous analysis [21] suggests that monocrotaline itself has no direct toxic effect on cardiac tissue. Nonetheless, the potential exists that increased right ventricular afterload secondary to other causes may produce different findings.
Myocardial adaptation in response to chronic pressure overload may undergo a spectrum of changes ranging from an initial hypertrophic response to eventual heart failure, and is highly dependent on the duration and severity of the physiologic stress to which the heart is subjected. In this investigation, the molecular and functional alterations occurring in the right ventricle in the setting of chronic pulmonary hypertension were assessed only at a single time after initial drug injection. On the basis of the data presented, it is difficult to determine when the described mechanisms of adaptation were first initiated, or whether further significant alterations would be observed at intervals longer than 8 weeks after monocrotaline pyrrole injection. In addition, the issue of whether the increased receptor density caused the ventricular hypertrophy and functional adaptations or whether the myocardial adaptations led to increases in receptor density remains unclear. Such conclusions are not possible with our results, but future studies could assess the changes occurring in intrinsic cardiac function as well as myocardial adrenergic receptor signaling at regular time intervals after creation of chronic pulmonary hypertension.
In summary, a canine model of monocrotaline pyrrole–induced chronic pulmonary hypertension provides a useful means to assess the molecular and functional mechanisms of right ventricular adaptation in chronic pulmonary hypertension. In this setting of monocrotaline pyrrole–induced chronic pulmonary hypertension, right ventricular function adapts to the increased afterload without evidence of cardiac failure. The significant increase in right ventricular preload-recruitable stroke work occurring in association with the significant increases in right ventricular ß-adrenergic receptor density and isoproterenol-stimulated adenylate cyclase activity suggests that this compensation is at least partially due to increased signaling through the ß-adrenergic receptor pathway, and that the significant upregulation in 1-adrenergic receptor density may contribute to the hypertrophic response observed in the right ventricle. These molecular changes in the right ventricle may provide additional insights into the biochemical mechanisms of right ventricular adaptation to increased afterload in the setting of chronic pulmonary hypertension.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy. J Biol Chem 1993;268:15374-15380.1-adrenergic activity in progression of cardiac hypertrophy in guinea pig hearts with pressure overload. Cardiovasc Res 1989;23:315-322.[Medline]
1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci USA 1994;91:10109-10113.- and ß-adrenoceptors in catecholamine-induced cardiac hypertrophy. Circ Res 1989;65:1417-1425.This article has been cited by other articles:
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  P. Pokreisz, G. Marsboom, and S. Janssens Pressure overload-induced right ventricular dysfunction and remodelling in experimental pulmonary hypertension: the right heart revisited Eur. Heart J. Suppl., December 1, 2007; 9(suppl_H): H75 - H84. [Abstract] [Full Text] [PDF]
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 M. Kret and R. Arora Pathophysiological Basis of Right Ventricular Remodeling Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2007; 12(1): 5 - 14. [Abstract] [PDF]
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 S. L. Gaynor, H. S. Maniar, J. B. Bloch, P. Steendijk, and M. R. Moon Right Atrial and Ventricular Adaptation to Chronic Right Ventricular Pressure Overload Circulation, August 30, 2005; 112(9_suppl): I-212 - I-218. [Abstract] [Full Text] [PDF]
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 H. K. Eltzschig, T. Mihaljevic, J. G. Byrne, R. Ehlers, B. Smith, and S. K. Shernan Echocardiographic evidence of right ventricular remodeling after transplantation Ann. Thorac. Surg., August 1, 2002; 74(2): 584 - 586. [Abstract] [Full Text] [PDF]
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 L. Chen, X. T. Gan, J. V. Haist, Q. Feng, X. Lu, S. Chakrabarti, and M. Karmazyn Attenuation of Compensatory Right Ventricular Hypertrophy and Heart Failure following Monocrotaline-Induced Pulmonary Vascular Injury by the Na+-H+ Exchange Inhibitor Cariporide J. Pharmacol. Exp. Ther., August 1, 2001; 298(2): 469 - 476. [Abstract] [Full Text] [PDF]
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 A. Rajek, T. Pernerstorfer, J. Kastner, P. Mares, M. Grabenwoger, D. I. Sessler, G. Grubhofer, and M. Hiesmayr Inhaled Nitric Oxide Reduces Pulmonary Vascular Resistance More Than Prostaglandin E1 During Heart Transplantation Anesth. Analg., March 1, 2000; 90(3): 523 - 530. [Abstract] [Full Text] [PDF]
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