Transgenic Mice with Cardiac Overexpression of alpha 1B-Adrenergic Receptors
IN VIVO alpha 1-ADRENERGIC RECEPTOR-MEDIATED REGULATION OF beta -ADRENERGIC SIGNALING*

(Received for publication, April 8, 1997, and in revised form, June 3, 1997)

Shahab A. Akhter Dagger , Carmelo A. Milano Dagger , Kyle F. Shotwell Dagger , Myeong-Chan Cho §, Howard A. Rockman §, Robert J. Lefkowitz par ** and Walter J. Koch Dagger Dagger Dagger

From the Departments of Dagger  Surgery and par  Medicine and the ** Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 and the § Department of Medicine, University of California at San Diego, La Jolla, California 92093

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Transgenic mice were generated with cardiac-specific overexpression of the wild-type (WT) alpha 1B-adrenergic receptor (AR) using the murine alpha -myosin heavy chain gene promoter. Previously, we described transgenic mice with alpha -myosin heavy chain-directed expression of a constitutively active mutant alpha 1B-AR that had a phenotype of myocardial hypertrophy (Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109-10113). In animals with >40-fold WT alpha 1-AR overexpression, basal myocardial diacylglycerol content was significantly increased, indicating enhanced alpha 1-adrenergic signaling and phospholipase C activity. In contrast to the mice overexpressing constitutively active mutant alpha 1B-ARs, the hearts of these mice did not develop cardiac hypertrophy despite an 8-fold increase in ventricular mRNA for atrial natriuretic factor. In vivo physiology was studied in anesthetized intact animals and showed left ventricular contractility in response to the beta -agonist isoproterenol to be significantly depressed in animals overexpressing WT alpha 1B-ARs. Membranes purified from the hearts of WT alpha 1BAR-overexpressing mice demonstrated significantly attenuated adenylyl cyclase activity basally and after stimulation with isoproterenol, norepinephrine, or phenylephrine. Interestingly, these in vitro changes in signaling were reversed after treating the mice with pertussis toxin, suggesting that the extraordinarily high levels of WT alpha 1B-ARs can lead to coupling to pertussis toxin-sensitive G proteins. Another potential contributor to the observed decreased myocardial signaling and function could be enhanced beta -AR desensitization as beta -adrenergic receptor kinase (beta ARK1) activity was found to be significantly elevated (>3-fold) in myocardial extracts isolated from WT alpha 1B-AR-overexpressing mice. This type of altered signal transduction may become critical in disease conditions such as heart failure where beta ARK1 levels are elevated and beta -ARs are down-regulated, leading to a higher percentage of cardiac alpha 1-ARs. Thus, these mice serve as a unique experimental model to study the in vivo interactions between alpha - and beta -ARs in the heart.


INTRODUCTION

There have been numerous in vitro studies characterizing the role of alpha 1-adrenergic receptor (AR)1 signaling in cardiac myocytes. Agents that stimulate alpha 1-ARs, leading to the activation of the guanine nucleotide-binding protein Gq, have been shown to induce nuclear transcription factors such as c-myc, c-fos, and c-jun and to mediate morphological changes including increases in myocyte size and volume (1). Signaling through the alpha 1-AR/Gq pathway leads to the activation of the effector enzyme phospholipase C and protein kinase C, both of which may act as biochemical initiators of myocardial hypertrophy (2). In addition to Gq-mediated hypertrophy, recent studies have implicated a p21ras (Ras)-dependent hypertrophic pathway initiated by alpha 1-AR activation (3). Other in vitro alpha 1-AR/Gq-mediated signaling events reported to exist in myocytes include positive inotropy and chronotropy and induction of the egr-1 gene (4). In addition to coupling to Gq, alpha 1-ARs have also been reported to activate pertussis toxin (PTx)-sensitive G proteins, leading, in myocytes, to negative chronotropy, positive inotropy, Na+-K+-ATPase activation, and modulation of intracellular calcium transients and cell shortening (5).

In contrast to these studies, very few reports have investigated in vivo cardiac alpha 1-AR signaling particularly with respect to physiological sequelae and the potential in vivo significance of dual G protein coupling. alpha 1A- and alpha 1B-ARs have been shown to exist in neonatal myocytes (6), whereas in adult human myocardium, the alpha 1A-AR appears to predominate (7). Both the alpha 1A and alpha 1B subtypes have been implicated in myocyte growth and hypertrophy (6). One possible functional role of alpha 1-ARs is as a source of inotropic reserve in pathophysiological conditions where the beta -AR system is down-regulated and uncoupled (8). Thus, an interrelationship may exist in the heart between alpha 1- and beta -ARs. Previous work from our laboratory revealed that cardiac-specific expression of a constitutively active mutant (CAM) of the alpha 1B-AR in transgenic mice leads to myocardial hypertrophy, demonstrating that cardiac alpha 1-adrenergic signaling in vivo can trigger responses similar to myocytes in culture (9).

In this study, we continue our characterization of in vivo myocardial adrenergic signaling in transgenic mice (9-13) by describing animals with cardiac overexpression of the wild-type (WT) alpha 1B-AR. As in our previous studies, cardiac expression was targeted by using the murine alpha -MyHC promoter (9-13). To determine the consequences of WT alpha 1B-AR overexpression, we studied both biochemical signaling and in vivo physiology. We have previously observed for the WT beta 2-AR that when overexpression of these WT receptors reaches extraordinarily high levels, agonist-independent signaling can occur due to a small percentage of spontaneously activated receptors that is significant at high levels of receptor density (10, 14). Both alpha 1- and beta -AR signal transduction was assessed, including measurements of myocardial diacylglycerol (DAG) content, ventricular atrial natriuretic factor (ANF) mRNA levels, adenylyl cyclase activity, and G protein-coupled receptor kinase (GRK) activity. In addition, the presence of myocardial hypertrophy was assessed. Finally, in vivo basal and beta -AR-mediated cardiac function was assessed by catheterization of anesthetized mice. The results from these studies reveal findings that point to potentially important interactions between alpha - and beta -adrenergic signaling in the heart.


EXPERIMENTAL PROCEDURES

Transgene Constructs

A 5.5-kilobase SalI-SacI fragment containing the murine alpha -MyHC promoter (15) was ligated into a previously described plasmid containing the SV40 intron poly(A) signal (9-13) to generate a new plasmid, pGEM-alpha -MyHC-SV40. A 2.0-kilobase SalI-SalI fragment containing the coding sequence for the wild-type hamster alpha 1B-AR was then ligated into pGEM-alpha -MyHC-SV40 to generate pGEM-alpha -MyHC-alpha 1B-AR-SV40. The transgene was then linearized and purified before pronuclear injections done by the Duke Comprehensive Cancer Center Transgenic Facility (9-13). Two lines of mice were established, TG alpha 43 and TG alpha 47. Litter sizes and postnatal development were indistinguishable from nontransgenic littermate controls. Offspring were screened by Southern blot analysis with a probe to the SV40 sequences. Second generation adult animals (2-5 months of age) were used for all studies. Institutional Review Board approval for all mouse experiments was obtained from the University of California at San Diego and from Duke University Medical Center.

Ligand Binding Assays

Membrane fractions were prepared from hearts and resuspended in binding buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, and 5 mM EDTA or 75 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, and 2 mM EDTA). Binding assays were performed on 25 µg of membrane protein using saturating amounts of 125I-HEAT (300 pM), an alpha 1-AR-specific ligand, or 125I-CYP (300 pM), a beta -AR-specific ligand. Nonspecific binding was determined in the presence of 50 µM prazosin for alpha -binding and 20 µM alprenolol for beta -binding. Reactions were conducted in either 250 or 500 µl of binding buffer at 37 °C for 1 h and then terminated by vacuum filtration through glass-fiber filters. All assays were performed in triplicate, and receptor density (fmol) was normalized to mg of membrane protein following the method of Bradford (29).

DAG Quantitation

Lipid fractions were extracted from 50 mg of homogenized myocardial tissue as described (6, 9). Aliquots of lipid and DAG standards were dried under nitrogen, resuspended in detergent micelles, and then completely phosphorylated using Escherichia coli DAG kinase and [gamma -32P]ATP. 32P-Labeled phosphatidic acid (phosphorylated DAG) was isolated by silica gel thin-layer chromatography and quantitated with a PhosphorImager (Molecular Dynamics, Inc.). DAG content was normalized to tissue phospholipid, and the final DAG concentration was expressed as pmol of DAG/nmol of lipid phosphate as described previously (9).

Ventricular ANF mRNA

Ventricular tissue was separated from the atria under a dissecting microscope. Total RNA was extracted using RNAzol (Biotecx Laboratories, Houston TX) in a single-step guanidinium-based isolation procedure (16). Total RNA was then fractionated on a 1% formaldehyde-agarose gel and transferred to nitrocellulose as described (9). Blots were prehybridized in a 50% formamide solution for 4 h at 42 °C and then hybridized overnight with a random primer, radiolabeled ANF cDNA probe (9). Blots were washed three times in 0.2 × SSC at 65 °C for 30 min before exposure to x-ray film. All blots were then stripped in water at 95-100 °C for 15 min and reprobed with a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe. The ANF and GAPDH bands were quantitated with the PhosphorImager, and the ANF/GAPDH signal intensity ratio was determined (9).

Adenylyl Cyclase Activity

Crude myocardial membranes were prepared as described above from both transgenic and nontransgenic control hearts. Membranes (20-30 µg of protein) were incubated for 15 min at 37 °C with [alpha -32P]ATP under basal conditions or in the presence of one of the following: 100 µM isoproterenol, 100 µM norepinephrine, 100 µM phenylephrine, or 10 mM NaF. Cyclic AMP was quantitated by standard methods described previously (17).

GRK Activity in Rhodopsin Phosphorylation Assays

Cytosolic extracts were prepared as described previously (11). These were concentrated using a Centricon microconcentrator (Amicon, Inc.). Concentrated cytosolic extracts (300 µg of protein) were incubated with rhodopsin-enriched rod outer segments in 75 µl of lysis buffer (25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 5 mM EGTA, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) with 10 mM MgCl2 and 0.1 mM ATP containing [gamma -32P]ATP. The reactions were incubated in white light for 15 min and quenched with 300 µl of ice-cold lysis buffer and then centrifuged for 15 min at 13,000 × g. Sedimented proteins were resuspended in 25 µl of protein gel loading dye and electrophoresed through SDS-12% polyacrylamide gels. Phosphorylated rhodopsin was visualized by autoradiography of dried polyacrylamide gels and quantified using the PhosphorImager.

Physiological Evaluation

Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg) given intraperitoneally. After endotracheal intubation, mice were connected to a rodent ventilator. Following bilateral vagotomy, the chest was opened, and a 1.8-French high-fidelity micromanometer catheter (Millar Instruments, Inc., Houston, TX) was inserted into the left atrium, advanced through the mitral valve, and secured in the left ventricle (LV). Hemodynamic measurements were recorded at base line and 45-60 s after injection of incremental doses of isoproterenol. Doses of isoproterenol were specifically chosen to maximize the contractile response but to limit the increase in heart rate. Continuous high-fidelity LV pressure and fluid-filled aortic pressure were recorded simultaneously on an eight-channel chart recorder and in digitized form at 2000 Hz for later analysis. Experiments were then terminated with an overdose of pentobarbital. Hearts were rapidly excised, and individual chambers were separated, weighed, and then frozen in liquid N2 for later analysis. Parameters measured were heart rate, LV systolic and end diastolic pressure, and the maximal and minimal first derivative of LV pressure (LV dP/dtmax and LV dP/dtmin). Ten sequential beats were averaged for each measurement.

Statistical Analysis

Data are expressed as mean values ± S.E. Student's t test was used to analyze all biochemical data. Two-way repeated measure analysis of variance was used to evaluate the in vivo hemodynamic measurements under basal conditions and with isoproterenol stimulation. When appropriate, post hoc analysis was performed with a Newman-Keuls test. For all analyses, p < 0.05 was considered significant.


RESULTS AND DISCUSSION

Two transgenic lines were established expressing the WT alpha 1B-AR and were named TG alpha 43 and TG alpha 47. Cardiac-specific transgene expression was documented by Northern analysis of RNAs from different tissues, including heart, lung, diaphragm, quadriceps muscle, kidney, and liver (data not shown). This cardiac-specific expression is consistent with the previously documented pattern of transgene expression achieved with the murine alpha -MyHC promoter (9-13). At 10 weeks of age, transgene expression was quantitated by radioligand binding assays performed on purified myocardial membranes using 125I-HEAT, and the results are shown in Fig. 1. Total cardiac alpha 1-AR density in TG alpha 43 animals was 43-fold greater than in nontransgenic littermate controls (NLC) and 26-fold greater in the TG alpha 47 line compared with NLC. Unless otherwise stated, all subsequent studies were performed on TG alpha 43 animals.


Fig. 1. Myocardial alpha 1-adrenergic receptor density. Sarcolemmal membranes were purified as described under "Experimental Procedures" from NLC (n = 10), TG alpha 47 (n = 5), and TG alpha 43 (n = 15) hearts. The estimated Bmax was determined using 300 pM 125I-HEAT and 25 µg of membrane protein. Data are expressed as mean ± S.E. *, p < 0.001 compared with control (Student's t test).
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Classical alpha 1-AR/Gq coupling leads to stimulation of phospholipase C, generating the second messengers inositol trisphosphate and DAG, which subsequently leads to the activation of protein kinase C (18, 19). To assess the functional coupling of overexpressed WT alpha 1B-ARs, myocardial DAG content was quantitated, and as shown in Fig. 2, base-line DAG content in TG alpha 43 hearts was significantly higher than in control hearts. This indicates that alpha 1B-AR/Gq signaling is enhanced under basal conditions.


Fig. 2. Basal myocardial diacylglycerol content. Lipid extraction was performed from NLC (n = 5) and TG alpha 43 (n = 5) hearts as described under "Experimental Procedures." Diacylglycerol content was quantified using 50 nmol of lipid phosphate as described. Data shown are mean ± S.E. *, p < 0.005 compared with control (Student's t test).
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Since signaling through alpha 1-ARs has been shown to evoke a hypertrophic response in cultured myocytes including activation of fetal gene expression (2, 20), we investigated the levels of ANF mRNA present in the ventricles of these transgenic mice. ANF is a gene normally inactive in the ventricles after maturation and has been shown to be associated with cardiac hypertrophy (21). To examine ANF gene activation present in TG alpha 43 mice, Northern blots of ventricular RNA were generated and probed with a mouse ANF cDNA (Fig. 3A). Control ventricles showed minimal or undetectable ANF signals, which is consistent with the inactivation of this gene in normal adult ventricular myocytes (21). In contrast, there was a strong ANF signal in RNA isolated from TG alpha 43 ventricles, which, when normalized to the control GAPDH mRNA, was ~9-fold higher compared with controls (Fig. 3B). The increase in ventricular ANF mRNA in TG alpha 43 mice was twice that seen in our previously described CAM alpha 1B-AR-overexpressing mice (9). Surprisingly, despite the extraordinarily high ANF mRNA levels, TG alpha 43 animals did not have significantly different LV/body weight ratios or increased LV myocyte cross-sectional areas compared with controls (data not shown). Nonsignificant changes in heart mass were also found in TG alpha 47 animals (data not shown). This is unlike the phenotype in the CAM alpha 1B-AR transgenic animals, which had increased heart mass and increased cross-sectional areas of ventricular myocytes that accompanied the increased ANF signal (9). The lack of a hypertrophic phenotype in these animals is not clearly understood since signaling through alpha 1-ARs is clearly elevated, and ventricular ANF expression is high (Figs. 2 and 3). This suggests that signaling through CAM alpha 1-ARs is somehow different from that through WT receptors.


Fig. 3. Ventricular ANF mRNA Levels. A, representative Northern blot of total RNA (15 µg) isolated from the ventricles of NLC and TG alpha 43 hearts (n = 2 each) and probed with a mouse ANF cDNA (top panel). The blots were stripped and reprobed with rat GAPDH cDNA (bottom panel). B, quantitation of the ANF signal. The signals from the ANF blots were counted on a Molecular Dynamics PhosphorImager and normalized to the GAPDH signal as described (9). Data shown are mean ± S.E. for n = 8 in each group. *, p < 0.05 versus control (Student's t test).
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In addition to investigating a possible hypertrophic phenotype in TG alpha 43 mice, the primary aim of this study was to determine the in vivo physiological effects of enhanced signaling through myocardial alpha 1-ARs. In vivo measurements of alpha 1-AR-mediated cardiac hemodynamics using a pharmacological approach is difficult since alpha -agonists are potent vasoconstrictors that can change the loading conditions and secondarily affect cardiac function. In TG alpha 43 mice, we chose to initially study basal cardiac physiological parameters to determine whether the enhanced alpha 1-AR/Gq signaling indicated by increased DAG content can affect basal function. In addition, we examined cardiac responses to the beta -agonist isoproterenol to determine if enhanced alpha 1-AR signaling affects this response, which is the primary mechanism for increasing performance of normal hearts. Following catheterization of anesthetized mice (10-13), we measured several hemodynamic parameters, including heart rate and LV dP/dtmax and LV dP/dtmin, measures of cardiac contractility and relaxation, respectively. The results found in TG alpha 43 animals and NLC mice under basal conditions and in response to isoproterenol are shown in Fig. 4. There was no difference in LV systolic pressure between the two groups (Fig. 4A). Base-line and beta -agonist-stimulated heart rates were significantly depressed in TG alpha 43 mice compared with NLC mice (Fig. 4B). There was no statistically significant difference in basal dP/dtmax or dP/dtmin in TG alpha 43 mice versus NLC mice (Fig. 4, C and D), although the trend was for lower values in TG alpha 43 animals. There was, however, a significant decrease in these parameters in TG alpha 43 animals compared with NLC mice in response to progressive isoproterenol infusion. Thus, beta -AR-mediated LV function is depressed in these animals, suggesting that there is significant cross-talk between the signaling of alpha 1-ARs and beta -ARs in the hearts of these transgenic animals. The increased constitutive alpha 1-AR signaling present in TG alpha 43 mice has a significant effect on cardiac physiological responses elicited by beta -AR stimulation. Although previous studies have demonstrated that alpha 1-AR stimulation can lead to negative chronotropy (5), alpha 1-AR-mediated negative inotropy is a novel finding. To further study alpha 1-AR-mediated effects on cardiac contractility, it would be relevant to study these in vivo parameters in the presence of an alpha -agonist such as phenylephrine. However, the use of phenylephrine presents major fundamental problems due to its predominant peripheral effects on systolic pressure, which would influence myocardial function independent of any myocardial alpha 1-AR signaling. The data presented above could have important clinical significance since, in pathophysiological conditions such as heart failure, there is significant loss of both beta -AR density and functional coupling, which could potentially increase the role of alpha 1-AR signaling in response to endogenous catecholamines.


Fig. 4. In vivo assessment of cardiac function of TG alpha 43 (n = 19) (bullet ) and NLC (n = 12) (open circle ) mice. Cardiac catheterization was performed on intact anesthetized animals. Four measured parameters are shown at base line and after progressive doses of isoproterenol. A, LV systolic pressure; B, heart rate; C, LV dP/dtmax; D, LV dP/dtmin. Data were analyzed with a two-way repeated measure analysis of variance. *, p < 0.0005; #, p < 0.05 (control versus transgenic). A significant between-group main effect in response to isoproterenol was found in B for heart rate (p < 0.001). The pattern of change between groups (interaction) was statistically significant in B for heart rate (p < 0.05), in C for LV dP/dtmax (p < 0.001), and in D for LV dP/dtmin (p < 0.05).
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To investigate possible molecular mechanisms involved in the altered cardiac physiology seen in TG alpha 43 animals, we carried out in vitro biochemical assays on heart extracts to examine the beta -AR system. Signaling through beta -ARs involves the activation of the G protein Gs, which activates adenylyl cyclase, leading to increases in intracellular cAMP and activation of cAMP-dependent protein kinase A (22). Activated protein kinase A phosphorylates several myocardial proteins, leading to positive inotropy as well as chronotropy. The beta -AR system as well as several other G protein-coupled receptors undergo rapid desensitization, which is the loss of response in the continued presence of agonist. This is initiated by targeted receptor phosphorylation via a family of serine/threonine kinases known as the GRKs, of which the beta -AR kinase (beta ARK1) is a prototypic member (23). We first examined beta -AR density in myocardial membranes purified from TG alpha 43 hearts and control nontransgenic myocardial membranes using 125I-CYP and found no significant difference in total beta -AR density (46.5 ± 0.8 fmol/mg of membrane protein in TG alpha 43 mice versus 44.3 ± 1.6 in controls). Therefore, changes in myocardial beta -AR density cannot account for the differences in cardiac contractility seen in TG alpha 43 mice.

We then studied myocardial membrane adenylyl cyclase activity. There was significantly lower basal adenylyl cyclase activity in membranes purified from TG alpha 43 hearts compared with nontransgenic myocardial membranes (Table I). This could account for the decreased basal heart rate seen in these animals. As shown in Table I, agonist-stimulated adenylyl cyclase activity in TG alpha 43 membranes compared with control membranes was also significantly depressed following addition of isoproterenol or norepinephrine. Interestingly, the depressed response to norepinephrine, a mixed alpha /beta -agonist, was greater than with the strict beta -agonist isoproterenol. These results indicate that the depressed in vivo cardiac function shown above (Fig. 4) is likely due, at least in part, to the lower adenylyl cyclase activity and an attenuated beta -AR-mediated cAMP response. To examine whether decreased adenylyl cyclase activity in TG alpha 43 membranes was secondary to enhanced alpha 1-AR signaling, we studied adenylyl cyclase activity following addition of an alpha -agonist. Surprisingly, the addition of 100 µM phenylephrine resulted in significant lowering of basal activity in TG alpha 43 membranes, whereas control membranes had no alpha 1AR-mediated cAMP response (Table I). This strongly suggests that further alpha 1-AR stimulation leads to inhibition of membrane adenylyl cyclase activity. This also suggests that the significant decrease in basal adenylyl cyclase activity is due to enhanced basal alpha 1-AR signaling present in TG alpha 43 hearts. Strengthening these conclusions of a receptor-mediated phenomenon, NaF-stimulated adenylyl cyclase activities were similar in membranes from TG alpha 43 animals and nontransgenic controls (Table I).

Table I. Adenylyl cyclase activity in control and TG alpha 43 myocardial membranes

Activity is presented as pmol of cAMP/min/mg of protein. Data are the mean ± S.E. of 7-10 heart samples from experiments performed in triplicate.

Hearts Basal ISOa NE PE NaF

10-4 M 10-4 M 10-4 M 10-2 M
Control 38.53  ± 2.96 68.25  ± 6.55 78.60  ± 4.36 34.70  ± 4.90 269.60  ± 12.78
TG alpha 43 16.87  ± 2.13b 24.55  ± 4.47b 20.02  ± 5.57b 10.21  ± 1.80b,c 245.65  ± 11.83

a ISO, isoproterenol; NE, norepinephrine; PE, phenylephrine.
b p < 0.05 compared with control activity.
c p < 0.05 compared with TG alpha 43 basal activity (Student's t test).

One hypothesis for the decrease in adenylyl cyclase activity in TG alpha 43 myocardial membranes is coupling of WT alpha 1B-ARs to the adenylyl cyclase inhibitory G protein, Gi. As mentioned above, dual coupling of alpha 1-ARs to Gq and Gi has been demonstrated in vitro, but not yet investigated in vivo. To examine the potential involvement of Gi in the alpha 1-AR-mediated cyclase responses, we intraperitoneally injected TG alpha 43 and NLC mice with either 100 µg/kg PTx or 150 µl of saline and sacrificed the animals 24 h later. Myocardial membranes were purified from these animals, and adenylyl cyclase activities were measured. As shown in Table II, PTx treatment reversed the depressed basal cyclase activity in TG alpha 43 membranes, and in fact, the percent increase in TG alpha 43 basal activity was significantly higher compared with PTx-treated controls (Table II), indicating the enhanced Gi coupling of overexpressed WT alpha 1B-ARs. Isoproterenol- and norepinephrine-stimulated adenylyl cyclase activities were also significantly increased in TG alpha 43 membranes following PTx treatment. Additionally and in contrast to findings in saline-treated transgenic animals, phenylephrine did not decrease membrane adenylyl cyclase activity (Table II). PTx treatment also enhanced NaF-stimulated activities compared with saline treatment, as expected, but TG alpha 43 membranes did not differ from controls, indicating that there is no change in the levels or function of myocardial G proteins. To confirm this, we carried out protein immunoblotting of membranes for Galpha s and Galpha i and found no difference in protein levels in TG alpha 43 versus control hearts (data not shown). Overall, PTx treatment converted TG alpha 43 membranes to having the biochemical characteristics of control membranes. Thus, these results indicate that the involvement of PTx-sensitive G proteins is significantly higher in hearts overexpressing the WT alpha 1B-AR, which could lead to the dampened myocardial performance seen in TG alpha 43 animals (Fig. 4).

Table II. Adenylyl cyclase activity in nontransgenic (control) and TG alpha 43 myocardial membranes after treatment with PTx

Activity is presented as pmol of cAMP/min/mg of protein. Data shown are the mean ± S.E. of three hearts from saline-treated animals and 7-10 hearts from the Ptx-treated group. All assays were performed in triplicate.

Hearts Basal ISOa NE PE NaF

10-4 M 10-4 M 10-4 M 10-2 M
Control
  Saline 37.91  ± 3.41 70.08  ± 6.88 74.52  ± 7.06 32.40  ± 4.78 250.16  ± 15.12
  PTx 46.03  ± 6.36 83.63  ± 13.89 94.27  ± 16.02 43.59  ± 6.30 338.01  ± 25.91b
TG alpha 43
  Saline 20.07  ± 4.61 28.10  ± 5.43 23.85  ± 4.97 13.17  ± 2.22c 241.91  ± 20.36
  PTx 50.67  ± 7.21d 90.53  ± 10.86d 83.74  ± 16.45d 46.98  ± 9.78d 365.92  ± 27.06d,e

a ISO, isoproterenol; NE, norepinephrine; PE, phenylephrine.
b p < 0.05 compared with control activity (saline).
c p < 0.05 compared with control activity (saline) and p < 0.05 compared with TG alpha 43 basal activity (saline).
d p < 0.05 compared with TG alpha 43 (saline) and p = not significant compared with control (PTx) activities.
e p < 0.05 compared with TG alpha 43 (saline) (Student's t test).

Potentially, there could be additional contributors to the decreased beta -AR-mediated myocardial signaling and function seen in TG alpha 43 animals. Since beta -AR density is unaltered in TG alpha 43 hearts, desensitization and functional uncoupling may be enhanced. In fact, the attenuated adenylyl cyclase activities and in vivo beta -AR cardiac responses are similar to the phenotype we have previously described for transgenic mice with cardiac overexpression of either the beta ARK1 (11) or GRK5 (12). These two members of the GRK family are expressed in the heart and can produce desensitization and functional uncoupling of myocardial beta -ARs (11, 12). Interestingly, it has recently been reported that beta ARK1 can be regulated by protein kinase C (24, 25). This regulation involves the enhancement of beta ARK activity following the phosphorylation of beta ARK1 by protein kinase C (24, 25). This is of significance in the present study since protein kinase C activity is apparently increased in the hearts of TG alpha 43 animals as indicated by the measured increase in myocardial DAG content (Fig. 2). To investigate any in vivo regulation of beta ARK in the hearts of these transgenic mice, we carried out in vitro phosphorylation assays using myocardial extracts and the G protein-coupled receptor substrate rhodopsin (11, 12). Fig. 5 contains our findings using soluble myocardial fractions that represent GRK activity primarily attributable to beta ARK1. As shown, GRK activity was increased 3-fold in soluble extracts from the hearts of TG alpha 43 animals compared with NLC heart extracts. Thus, this enhanced GRK activity seen in TG alpha 43 hearts could contribute to the attenuated beta -AR signaling observed in these animals, and interestingly, the increase in beta ARK activity is in the same range as that in transgenic mice overexpressing beta ARK1 (11) and following the development of pressure overload cardiac hypertrophy (26). The increased soluble GRK activity appears not to involve beta ARK1 up-regulation as protein immunoblots revealed no changes in the levels of beta ARK1 in the hearts of TG alpha 43 animals compared with NLC (data not shown). Thus, these data are the first to suggest possible enhancement of beta ARK activity as an in vivo consequence of increases in the alpha 1-AR/Gq/protein kinase C cascade. Thus, previous in vitro findings of protein kinase C regulation of beta ARK1 (24, 25) may have important in vivo implications. In additional studies, we examined the protein content of GRK5, which is found exclusively in the membrane fraction of extracts (27), and found that the levels of this GRK are unaltered in TG alpha 43 hearts (data not shown).


Fig. 5. Assessment of soluble myocardial GRK activity. A, representative autoradiogram of a dried gel showing phosphorylated rhodopsin (Rho; arrow) in which 300 µg of protein from soluble extracts of TG alpha 43 and NLC hearts were assayed (see "Experimental Procedures"); B, histogram of composite data of GRK activity. Data are the mean ± S.E. of experiments done on eight hearts in each group, where phosphorylated rhodopsin was counted using a Molecular Dynamics PhosphorImager. *, p < 0.05 (Student's t test).
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In summary, we have demonstrated that alpha -MyHC-directed cardiac overexpression of the WT alpha 1B-AR can have profound effects on adrenergic signaling and in vivo cardiac physiology. This is evident even under basal conditions, suggesting that WT alpha 1-ARs can signal spontaneously at this level of overexpression (>40-fold), much like the findings in mice overexpressing the WT beta 2-AR (10, 14). In TG alpha 43 animals, Gq signaling was enhanced as assessed by myocardial DAG content and ventricular ANF mRNA expression, but in contrast to our previously described transgenic mice overexpressing a CAM alpha 1B-AR, myocardial hypertrophy was not associated with this enhanced Gq signaling. In vivo hemodynamic evaluation of TG alpha 43 mice revealed significantly dampened left ventricular function in response to beta -agonist stimulation compared with nontransgenic controls. Probing possible mechanisms for altered myocardial function revealed that dual coupling of the alpha 1B-ARs exists as PTx-sensitive G protein-mediated attenuation of adenylyl cyclase was seen in myocardial membranes purified from TG alpha 43 animals. The surprising lack of hypertrophy in TG alpha 43 and TG alpha 47 animals suggests that the agonist-independent signaling of the CAM alpha 1B-AR differs from the enhanced signaling through WT alpha 1B-ARs. One possible explanation for this phenomenon is the significant dual G protein coupling seen in animals overexpressing the WT alpha 1B-AR, which greatly alters beta -AR signaling in these mice. This alpha 1-AR regulation of beta -AR signal transduction is not evident in the CAM alpha 1B-AR transgenic mice,2 which suggests a true difference in the biochemical phenotype of these mice. Interestingly, we have recently observed significant coupling of overexpressed WT beta 2-ARs to Gi in the hearts of transgenic animals.3 We also observed enhanced GRK activity, presumably beta ARK1, in the hearts of TG alpha 43 mice, which also could contribute to the observed phenotype. Enhanced beta ARK activity is probably due to elevated protein kinase C activity as a result of enhanced alpha 1-AR/Gq signaling, in as much as protein kinase C activation of beta ARK1 has been observed in vitro (24, 25). In support of the notion that this is a Gq-mediated phenomenon, CAM alpha 1B-AR animals have a similar increase in soluble GRK activity.2

These results demonstrate that myocardial alpha 1-AR signaling can significantly alter the signaling through myocardial beta -ARs via two distinct receptor-mediated mechanisms. First, alpha 1-AR coupling to PTx-sensitive G proteins can occur in vivo, and second, enhanced alpha 1-AR signaling can lead to the enhanced activity of beta ARK1, which can cause functional uncoupling of beta -ARs. Such cross-talk between different adrenergic signaling pathways could have important implications in organs like the heart that contain alpha - and beta -ARs and might become critical in pathological conditions where additional signaling alterations take place. For example, beta ARK1 expression and activity have been shown to be increased in end-stage human congestive heart failure (28). Thus, the TG alpha 43 animals represent a unique model to study specific in vivo interactions between alpha 1- and beta -AR signaling as well as other pathways that can be regulated by GRK activity.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant HL16037 (to R. J. L.), National Research Service Award HL09436 (to S. A. A.), and a grant from the North Carolina affiliate of the American Heart Association (to W. J. K.).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.
   Present address: University of North Carolina at Chapel Hill, Dept. of Medicine, Chapel Hill, NC 27599.
Dagger Dagger    To whom correspondence should be addressed: Dept. of Surgery, Duke University Medical Center, P. O. Box 2606, Durham, NC 27710. Tel.: 919-684-3007; Fax: 919-681-5262.
1   The abbreviations used are: AR, adrenergic receptor; PTx, pertussis toxin; CAM, constitutively active mutant; WT, wild-type; alpha -MyHC, alpha -myosin heavy chain; DAG, diacylglycerol; ANF, atrial natriuretic factor; GRK, G protein-coupled receptor kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NLC, nontransgenic littermate control(s); beta ARK1, beta -adrenergic receptor kinase; 125I-HEAT, 2-(beta -(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl)tetralone; 125I-CYP, 125I-cyanopindolol.
2   S. A. Akhter, C. A. Milano, and W. J. Koch, unpublished observations.
3   R. P. Xiao, S. A. Akhter, and W. J. Koch, unpublished observations.

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