Author + information
- Received January 23, 2001
- Revision received May 1, 2001
- Accepted May 21, 2001
- Published online September 1, 2001.
- Antonio Rapacciuolo, MD∗,
- Giovanni Esposito, MD∗,
- Kathleen Caron, PhD†,
- Lan Mao, MD∗,
- Steven A Thomas, MD, PhD‡ and
- Howard A Rockman, MD∗,* ()
- ↵*Reprint requests and correspondence: Dr. Howard A. Rockman, Department of Medicine, Duke University Medical Center, DUMC 3104, Durham, North Carolina 27710
We sought to define the role of norepinephrine and epinephrine in the development of cardiac hypertrophy and to determine whether the absence of circulating catecholamines alters the activation of downstream myocardial signaling pathways.
Cardiac hypertrophy is associated with elevated plasma catecholamine levels and an increase in cardiac morbidity and mortality. Although considerable evidence suggests that G-protein–coupled receptors are involved in the hypertrophic response, it remains controversial whether catecholamines are required for the development of in vivo cardiac hypertrophy.
We performed transverse aortic constriction (TAC) in dopamine beta-hydroxylase knockout mice (Dbh−/−, genetically altered mice that are completely devoid of endogenous norepinephrine and epinephrine) and littermate control mice. After induction of cardiac hypertrophy, the mitogen-activated protein kinase (MAPK) signaling pathways were measured in pressure-overloaded/wild-type and Dbh−/−hearts.
Compared with the control animals, cardiac hypertrophy was significantly blunted in Dbh−/−mice, which was not associated with altered cardiac function, as assessed by transthoracic echocardiography in conscious mice. The extracellularly regulated kinase (ERK 1/2), c-jun–NH2-terminal kinase (JNK) and p38 MAPK pathways were all activated by two- to threefold after TAC in the control animals. In contrast, induction of the three pathways (ERK 1/2, JNK and p38) was completely abolished in Dbh−/−mice.
These data demonstrate a nearly complete requirement of endogenous norepinephrine and epinephrine for the induction of in vivo pressure-overload cardiac hypertrophy and for the activation of hypertrophic signaling pathways.
Cardiac hypertrophy occurs in response to increased stress on the heart. Although the development of myocardial hypertrophy is believed to be a compensatory mechanism to normalize wall stress and maintain normal cardiac function, epidemiologic studies have shown an association between ventricular hypertrophy and increased cardiac morbidity and mortality (1). Therefore, investigations that lead to a better understanding of the molecular signals that stimulate myocyte growth would have clear therapeutic implications for diseases characterized by cardiac hypertrophy. In this regard, considerable evidence suggests that G-protein–coupled receptors are involved in the hypertrophic response (2); however, the precise ligands and receptors that are most involved are largely unknown. Catecholamines are one possibility, because they work through G-protein–coupled receptors and are clearly relevant to maintain cardiac function in both health and disease. Nonetheless, whether catecholamines are required for the development of in vivo cardiac hypertrophy remains controversial (3).
Studies in both cultured neonatal rat cardiomyocytes (4)and experimental animals (5)have suggested that stimulation by catecholamines can produce a hypertrophic phenotype. In contrast, in vitro (6)and in vivo studies (7)have suggested that catecholamines are not important mediators of the hypertrophic response. To date, studies of gene-targeted mice have not resolved the issue of whether intracellular signals resulting from catecholamine stimulation are important for cardiac growth. For example, transgenic mice with cardiac overexpression of either the wild-type beta1b-adrenergic receptor (AR) or beta2-AR did not result in cardiac hypertrophy (8,9). Likewise, cardiac weight was unaffected in gene-targeted adult mice with disruption of genes encoding the alpha1b-AR, alpha1c-AR or beta1-AR (10–12).
To determine whether catecholamines are required for the development of cardiac hypertrophy, we tested whether genetically altered mice that lack endogenous norepinephrine and epinephrine are able to develop cardiac hypertrophy in response to in vivo pressure overload. These mice have been created by targeted deletion of the dopamine beta-hydroxylase gene (Dbh−/−), the essential enzyme in the biosynthetic pathway converting dopamine to norepinephrine (13,14). This model has the advantage of totally eliminating norepinephrine and epinephrine, while preserving the release of co-transmitters and other factors derived from the sympathetic nervous system or adrenal gland, or both. By measuring the response to transverse aortic constriction (TAC) in Dbh−/−and littermate control mice, we are able to definitively assess the role of norepinephrine and epinephrine in the development of cardiac hypertrophy and induction of downstream signaling pathways.
Adult (4 to 5 months old) dopamine beta-hydroxylase knockout mice (Dbh−/−) were used in this study. Age-matched littermate heterozygotes (Dbh+/−) were used as control animals. These Dbh+/−mice have been extensively characterized and shown to have normal levels of norepinephrine and epinephrine (13,14). The animals in this study were handled according to approved protocols and animal welfare regulations by the Institutional Review Board of Duke University Medical Center.
Echocardiography in conscious animals
Transthoracic two-dimensional guided M-mode echocardiography was performed, using an HDI 5000 echocardiograph (ATL, Bothell, Washington), in conscious mice, as previously described (15). In separate conscious Dbh−/−mice, echocardiography was performed before and 5 h after the subcutaneous administration of L-threo-3,4-dihydroxyphenylserine (L-DOPS) (0.4 mg/g) to transiently restore norepinephrine levels. DOPS is a synthetic amino acid that is converted to norepinephrine by aromatic L-amino-acid decarboxylase, which completely restores tissue and circulating norepinephrine levels after dorsal subcutaneous injection (16).
Transverse aortic constriction
After conscious echocardiography, mice were anesthetized and TAC or a sham operation was performed, as previously described (17,18). Seven days after the operation, conscious echocardiography was again performed, and the trans-stenotic gradient systolic pressure was assessed (17,18). The hearts were then excised; the chambers were dissected free and weighed and then frozen in liquid nitrogen. There was no difference in post-TAC mortality between the control and Dbh−/−mice.
In separate anesthetized control and Dbh−/−mice, intravenous infusion of either saline, angiotensin II (0.2 μmol/min) or norepinephrine (0.02 μmol/min) was administered through the jugular vein for 7 to 10 min, while monitoring arterial pressure. The dose of angiotensin II and norepinephrine was chosen to achieve a 50% increase in blood pressure. After infusion, the hearts were harvested and immediately frozen in liquid nitrogen for determination of mitogen-activated protein kinase (MAPK) activity.
Mitogen-activated protein kinase assays
The MAPK assays were performed on myocardial extracts from the left ventricles of the mice, as previously described (19). After immunoprecipitation with antibodies specific for the various MAPKs, reactions were performed in 20 μmol/liter of adenosine triphosphate (ATP), (γ-32P)ATP (20 μCi/ml) and myelin basic protein (0.25 mg/ml) for extracellularly regulated kinase (ERK)/p38 or GST–c-jun(10 μg) for c-jun–NH2-terminal kinase (JNK) and incubated at 30°C for 20 min, then terminated by adding 40 μl of 2× Laemmli buffer. Phosphorylated myelin basic protein and GST–c-junwere resolved by polyacrylamide gel electrophoresis and quantified with a phosphorimager.
Quantitative measurement of kidney renin ribonucleic acid (RNA)
Total kidney RNA was prepared using TRIzol reagent (GibcoBRL, Grand Island, New York), according to manufacturer’s instructions. Amounts of renin RNA per 0.5 mg of total RNA were determined using the ABI 7700 real-time reverse-transcription polymerase chain reaction system (Perkin Elmer Applied Biosystems, Foster, California). Primers and Taqman probes were determined by using Primer Express software (Perkin Elmer Applied Biosystems). The primers and sequences for renin amplification were RenF-5′-ACAGTATCCCAACAGGAGAGACAAG-3′; and RenR-5′-CACCCAGGACCCAGACA-3′. The renin Taqman probe sequence was 5′-FAM-TGGCTCTCCATGCCATGGACATCC-Tamra-3′. Beta-actin was used as the internal standard control agent in each sample. The primers and sequences for beta-actin amplification were beta-actinF-5′-CTGCCTGACGGCCAAGTC-3′; and beta-actinR-5′-CAAGAAGGAAGGCTGGAAAAGA-3′. The beta-actin Taqman probe sequence was 5′-TET-CACTATTGGCAACGAGCGGTTCCG-Tamra-3′. Each sample was measured twice.
Data are expressed as the mean value ± SEM. For comparison of the echocardiographic variables before and after TAC or treatment with L-DOPS, twice repeated measures analysis of variance (ANOVA) was used. When appropriate, post hoc analysis was performed using the Scheffé test. Analysis of the slopes of the regression lines for the relationship between cardiac mass and pressure gradient was performed using a test of parallelism by multivariate ANOVA. For heart weight and kinase activity data, unpaired ttests were performed. For all analyses, p < 0.05 was considered significant.
Evaluation of the hypertrophic response seven days after aortic constriction
To evaluate the hypertrophic response after TAC, we measured the increase in the left ventricular weight (LVW) to body weight (BW) ratio and the LVW to tibial length (TL) ratio after seven days of in vivo pressure overload. Seven days after TAC, control mice with normal norepinephrine and epinephrine levels developed significant left ventricular (LV) hypertrophy, with a 60% increase in the LVW/BW ratio and a 62% increase in the LVW/TL ratio, compared with sham-operated control mice. This degree of cardiac hypertrophy was induced with a mean trans-stenotic systolic pressure gradient of 70.7 ± 5.6 mm Hg (Table 1, Fig. 1). In marked contrast, the hypertrophic response after TAC was significantly blunted in the littermate Dbh−/−mice compared with those undergoing a sham operation, as measured by LVW/BW (19.7%) or LVW/TL (14%), even though the trans-stenotic pressure gradient remained elevated at 67.9 ± 5.5 mm Hg (Table 2, Fig. 1). In addition, peak LV systolic pressure, as measured by the carotid artery pressure proximal to the stenosis, was not different in the Dbh−/−mice compared with the control mice (174 ± 8.2 vs. 172 ± 5.6 mm Hg), indicating that the two groups of banded mice had matched hemodynamic loads. The LVW/BW and LVW/TL ratios in the sham-operated animals were not different between the two groups (Tables 1 and 2, Fig. 1).
The impaired hypertrophic response in the Dbh−/−mice occurred over a wide range of pressures, as shown in Figure 2. In the control group, a strong correlation between the index LV mass (LVW/BW) and trans-stenotic systolic pressure gradient was observed (Fig. 2). In contrast, Dbh−/−mice showed no relationship between the pressure load and degree of hypertrophy (Fig. 2).
Evaluation of cardiac function before and after aortic constriction
To eliminate the effect of anesthesia on LV performance, we evaluated LV function by echocardiography in conscious mice. As shown in Table 1, seven days of pressure overload in control mice did not affect the chamber diameters or percent fractional shortening (%FS). Septal wall thickness was increased, consistent with the induction of concentric LV hypertrophy after pressure overload.
Although the hypertrophic response was blunted in the Dbh−/−mice, cardiac function after TAC remained normal. The Dbh−/−mice showed no change in LV end-diastolic dimension after TAC. Furthermore, a small but significant increase in %FS after TAC was found in the Dbh−/−mice, as measured by noninvasive echocardiography (Table 2). Lower basal %FS, heart rate and heart rate–corrected mean velocity of circumferential fiber shortening (Vcfc) in the pre-TAC Dbh−/−mice are also shown in Table 2, compared with the pre-TAC littermate control animals (Table 1).
To determine whether the reduced basal cardiac function in the Dbh−/−mice reflects a developmental defect caused by norepinephrine deficiency, or one that results from a physiologic deficit of norepinephrine, we quickly restored norepinephrine levels by administering L-DOPS to separate out Dbh−/−mice, and then we measured cardiac function. DOPS is a synthetic amino acid that is converted to norepinephrine by aromatic L-amino-acid decarboxylase and restores tissue and circulating norepinephrine levels in the mouse 5 h after subcutaneous injection (16). As shown in Table 3, LV end-systolic dimension, heart rate, mean Vcfc and %FS significantly increased in the Dbh−/−mice 5 h after the early administration of DOPS. These results indicate that differences in basal function between control and Dbh−/−mice are the result of the physiologic loss of norepinephrine, rather than being secondary to a fixed developmental abnormality.
Role of norepinephrine and epinephrine in pressure-overload–induced MAPK activation
In hearts from six sham and six TAC control animals, the activity of ERK 1/2, JNK, p38 and p38-beta was evaluated seven days after the operation. As shown in Figure 3, a significant increase in activity of the three known MAPK pathways was found after TAC in hypertrophied control mice. In contrast, despite a comparable and persistent pressure load in the Dbh−/−mice, the induction of ERK 1/2, p38, p38-beta and JNK activation was completely abolished (Fig. 3). In a separate set of animals, the basal MAPK activity was not found to be different between the control group and the Dbh−/−group (n = 3 each).
To test whether the MAPK pathways in the hearts of Dbh−/−mice could respond to the exogenous administration of neurohormones, MAPK activity was assessed after a 7- to 10-min infusion of either norepinephrine or angiotensin II in separate Dbh−/−and control mice. As shown in Figure 4, a robust increase in ERK and JNK MAPK activity was seen in the Dbh−/−mice, which was equal to or even greater than that observed in the control mice. Neither p38 nor p38-beta was activated by short infusion of norepinephrine or angiotensin II in either control or Dbh−/−mice. These data demonstrate that the hearts of Dbh−/−mice are able to respond to extracellular signals that activate MAPK pathways and support our hypothesis that endogenous norepinephrine and epinephrine are required for the development of cardiac hypertrophy in response to in vivo pressure overload.
Activation of the renin-angiotensin system after aortic constriction
The sympathetic nervous system is a very potent stimulatory factor for the control of renin secretion, which is mediated primarily by stimulation of beta1-ARs on juxtaglomerular cells (20). To determine whether the sympathetic nervous system plays a prominent role in activation of the renin-angiotensin system during development of in vivo pressure-overload cardiac hypertrophy, renal renin messenger RNA (mRNA) levels were measured by real-time reverse-transcription polymerase chain reaction. It has been shown that renal renin mRNA levels accurately reflect homeostatic changes in the renin-angiotensin system (21). As shown in Figure 4, the renin level was significantly increased (2.5-fold) in the banded control mice compared with the sham-operated animals, indicating a robust activation of the renin-angiotensin system after TAC. In contrast, no increase in kidney renin levels was found after aortic banding in the Dbh−/−animals (Fig. 5).
The main purpose of this study was to define the role of norepinephrine and epinephrine in the development of cardiac hypertrophy and to determine whether the absence of circulating norepinephrine and epinephrine alters the activation of downstream signaling pathways. The first finding is that endogenous norepinephrine and epinephrine are required, to a large extent, for the development of cardiac hypertrophy in response to the physiologic stress of pressure overload. Most likely, this is the result of both a direct effect of norepinephrine and epinephrine on cardiac adrenergic receptors and an indirect effect through stimulation of renal renin release. Although autonomic regulation of cardiac contractile function is important, sympathetic nervous system hyperactivity can also initiate or accelerate cardiovascular pathology. The detrimental effects of catecholamines are likely due, in part, to the induction of myocyte apoptosis by chronic beta-AR stimulation (22).
Role of norepinephrine and epinephrine in development of cardiac hypertrophy
Early studies by Simpson (4)showed that cultured neonatal rat cardiomyocytes respond to norepinephrine stimulation, with an increase in myocyte size. Furthermore, long-term infusion of catecholamines in experimental animals can induce cardiac hypertrophy (5). In contrast, in experiments using loaded and unloaded papillary muscles in a cat model of ventricular overload, Cooper et al. (7)showed that catecholamines may not be important mediators of the hypertrophic response. Other studies (6)have also supported the conclusion that mechanical factors predominate in the development of cardiac hypertrophy, and, while catecholamines may play a role, their importance is not certain (3). The data from the present study clearly establish the important role of norepinephrine and epinephrine in the induction of cardiac hypertrophy and suggests that blocking the action of norepinephrine and epinephrine would not only be favorable with regard to cell survival, but it would also inhibit cardiac hypertrophy.
Role of norepinephrine and epinephrine on induction of MAPK activity
The second finding is that induction of a hypertrophic response is associated with activation of the three major MAPK pathways, and these MAPK signaling pathways may be activated both directly and indirectly by norepinephrine and epinephrine. G-protein–coupled receptors and G proteins are involved in regulation of the hypertrophic response through activation of MAPK pathways (19,23). Among the G proteins, Gq has been shown to be associated with the development of in vivo cardiac hypertrophy (17,24)and induction of MAPK activity, particularly ERK and JNK (19). A variety of ligands can activate Gq-coupled receptors, such as the alpha1-AR (norepinephrine and phenylephrine), the angiotensin II type-1a receptor (AT1a) and endothelin-1 receptor, which have all been shown to trigger cellular hypertrophy in cultured neonatal rat ventricular myocytes (4,25,26). The role of beta-ARs in the development of cardiac hypertrophy is more controversial. Early in vitro studies did not show that beta-AR stimulation contributes to myocyte hypertrophy (4), whereas more recent studies suggest that beta-AR stimulation can lead to both cellular hypertrophy and MAPK activation (27,28). In this regard, recent evidence suggests that the phosphorylated beta-ARs can activate the ERK pathway by switching coupling to the G protein called Gi, a process that involves the beta-gamma subunits (29).
Activation of the renin-angiotensin system by norepinephrine and epinephrine
The small residual hypertrophy observed in the pressure overloaded Dbh−/−mice could be due to autocrine or paracrine signaling mediated by fibroblast growth factor-2 (30)and/or other peptide growth factors, such as angiotensin II and endothelin I. Although it is interesting to note that the AT1aknockout mice still develop robust hypertrophy in response to pressure overload (31,32), supporting our data of an important role for catecholamines, it is possible that signaling through either AT1b(33)or AT2(34)receptors is sufficient for the development of hypertrophy. However, it is unlikely that the circulating renin-angiotensin system is responsible for the low residual hypertrophy in the Dbh−/−mice, on the basis of our third finding in this study. That is, endogenous norepinephrine and epinephrine are necessary for the activation of the renin-angiotensin system after imposition of mechanical pressure overload. Previous work has shown that release of renin from juxtaglomerular cells is under the control of beta1-adrenergic stimulation (20). In this study, we show that renin release, in response to hemodynamic stress, is dependent on activation of the sympathetic nervous system. It is interesting that the basal renin levels were normal in the Dbh−/−mice, suggesting that under normal homeostatic conditions, regulation of the renin-angiotensin system does not require endogenous norepinephrine or epinephrine.
It has been previously demonstrated that LV mass in normotensive and hypertensive patients is closely coupled to an increased cardiac sympathetic activity (35). In the same study, arterial blood pressure did not correlate with LV mass (35). Data from our study suggest that normalization of cardiac sympathetic tone is extremely important to reduce or prevent LV hypertrophy. Finally, this model will be valuable to determine whether preventing cardiac hypertrophy has adverse long-term consequences on cardiac function.
We thank Dr. Robert J. Lefkowitz for his critical review of the manuscript and Debbie Colpitts for her expert secretarial assistance.
☆ This work was supported in part by Grant HL-61558 (to Dr. Rockman) from the National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland. Dr. Rockman is a recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.
- adrenergic receptor
- body weight
- dopamine beta-hydroxylase knockout mice
- extracellularly regulated kinase
- percent fractional shortening
- c-jun–NH2-terminal kinase
- left ventricular
- left ventricular weight
- mitogen-activated protein kinase
- transverse aortic constriction
- tibial length
- Received January 23, 2001.
- Revision received May 1, 2001.
- Accepted May 21, 2001.
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