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(Circulation. 2004;110:713-717.)
© 2004 American Heart Association, Inc.

Original Articles

Neuregulins Regulate Cardiac Parasympathetic Activity

Muscarinic Modulation of ß-Adrenergic Activity in Myocytes From Mice With Neuregulin-1 Gene Deletion

Katashi Okoshi, MD; Masaharu Nakayama, MD; Xinhua Yan, MD; Marina P. Okoshi, MD; Adam J.T. Schuldt, BA; Mark A. Marchionni, PhD; Beverly H. Lorell, MD

From the Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass (K.O., M.N., X.Y., M.P.O., A.J.T.S., B.H.L.), and NRG Biotech, Arlington, Mass (M.A.M.).

Correspondence to Beverly H. Lorell, MD, Cardiovascular Division–East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Room RW453, Boston, MA 02215. E-mail blorell{at}bidmc.harvard.edu

Received October 22, 2003; de novo received February 17, 2004; accepted April 26, 2004.

	Abstract

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Background— Neuregulins are required for maintenance of acetylcholine receptor–inducing activity of nicotinic receptors in neurons and skeletal muscle, but effects of neuregulins on muscarinic receptors are not known. In the normal heart, parasympathetic activation counterbalances ß-adrenergic activation. To test the hypothesis that neuregulins modify parasympathetic function in the heart, we studied cardiomyocytes from mice heterozygous for neuregulin-1 gene deletion (NRG-1^+/–) and examined the effects of ß-adrenergic stimulation on contractility in the presence and absence of the muscarinic agonist carbachol.

Methods and Results— We evaluated contraction and intracellular Ca²⁺ transients ([Ca²⁺]_i) in left ventricular (LV) myocytes loaded with Fluo-3 from NRG-1^+/– and wild-type (WT) mice. Under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca²⁺]_o, 25°C), characteristics of myocyte contraction/relengthening and systolic/diastolic [Ca²⁺]_i were not different between WT and NRG-1^+/– mice. The steady-state increases in fractional shortening (FS) and peak-systolic [Ca²⁺]_i in response to isoproterenol were similar in both groups. In WT myocytes stimulated with isoproterenol, carbachol decreased FS, peak-systolic [Ca²⁺]_i, and cAMP levels. In NRG-1^+/– myocytes, carbachol did not attenuate either FS or peak-systolic [Ca²⁺]_i, associated with the failure to decrease cAMP levels. Investigation of muscarinic receptor signaling showed no difference of LV protein levels of muscarinic M₂ receptors or G protein G_i1,2, G_i3, and G_o subunits.

Conclusions— Cardiomyocytes deficient in neuregulin signaling are unable to adequately counterbalance ß-adrenergic activation by inhibitory parasympathetic activity. This mechanism may contribute to the known increased risk of heart failure in injured human hearts when neuregulin signaling is suppressed.

Key Words: acetylcholine • neuregulins • calcium • contractility • myocytes

	Introduction

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Neuregulins are growth factors that interact with a subclass of tyrosine kinase receptors within the epidermal growth factor family, including erbB2 and erbB4 receptors in the heart.^1–3 Neuregulins promote cell growth, differentiation, and survival during normal development and healing during injury^4,5 and are essential for embryonic cardiac development.⁶ Interference with the oncogenic properties of neuregulins has been exploited in cancer chemotherapy with the use of trastuzumab, a monoclonal antibody against the erbB2 receptor.

Cancer patients treated with trastuzumab in addition to doxorubicin have a higher risk of developing severe heart failure than those treated with doxorubicin alone.^7,8 We demonstrated that left ventricular (LV) erbB2 and erbB4 protein levels are decreased in experimental heart failure⁹ and that mice with conditional erbB2 mutation develop cardiomyopathy.^10,11 These data support the concept that neuregulin-erbB signaling is cardioprotective, but the responsible mechanisms are incompletely understood.

One major biological effect of neuregulin signaling in the nervous system is the maintenance of nicotinic acetylcholine receptors (acetylcholine receptor–inducing activity [ARIA]).^12,13 In cardiac tissue, it is not known whether neuregulins also regulate parasympathetic muscarinic receptors or their activation. The inability to activate muscarinic receptors might interfere with the normal cardioprotective parasympathetic modulation of excess ß-adrenergic stimulation.

To test the hypothesis that neuregulins modify parasympathetic function in the heart, we studied cardiomyocytes from mice heterozygous for neuregulin-1 gene deletion (NRG-1^+/–) and examined the effects of ß-adrenergic stimulation on contractility in the presence and absence of the muscarinic agonist carbachol. We also investigated the possible mechanisms involved downstream of the muscarinic receptor.

	Methods

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Measurement of Myocyte Contraction and Intracellular Ca²⁺
We studied LV myocytes from 10- to 14-week-old NRG-1^+/– and wild-type (WT) mice used as controls (n=9 animals per group). This transgenic mouse was created by Meyer and Birchmeier⁶ by deletion of exons 7, 8, and 9 of the EGF domain of the neuregulin-1 gene and used to generate mice heterozygous for neuregulin-1 gene deletion. These NRG-1^+/– animals exhibit normal postnatal viability and cardiac development in the absence of a superimposed injury.⁶ In the present experiment, LV myocytes were dissociated and loaded with the Ca²⁺-sensitive fluorescence indicator Fluo-3. Cell contraction and [Ca²⁺]_i transients were then measured simultaneously with a video and fluorescence microscopy system following methods described in detail from our laboratory.¹⁴ Myocytes were paced with field stimulation at 0.5 Hz with 1.5 mmol/L [Ca²⁺]_o at 25°C. First, after at least 5 minutes of stabilization, myocytes were stimulated with 1 µmol/L isoproterenol for 3 minutes (WT, n=33 myocyte experiments from 9 hearts; NRG-1^+/–, n=41 experiments from 9 hearts). Next, the myocytes were randomized to be stimulated with isoproterenol for an additional 6 minutes (WT, n=16 myocyte experiments from 9 hearts; NRG-1^+/–, n=20 experiments from 9 hearts) or with stepped changes in carbachol (10 µmol/L followed by 100 µmol/L for 3 minutes at each concentration) in the presence of isoproterenol (WT, n=17 myocyte experiments from 9 hearts; NRG-1^+/–, n=21 experiments from 9 hearts). In additional experiments, the effects of carbachol in the absence of isoproterenol were studied (n=20 myocyte experiments from 8 hearts). All of the experimental procedures were approved and conducted in conformity with guidelines of the Institutional Animal Care and Use Committee.

cAMP Measurements
cAMP levels were determined in freshly isolated myocytes stimulated with 1 µmol/L isoproterenol alone (10 minutes at 37°C) or plus 100 µmol/L carbachol (15 minutes before isoproterenol at 37°C), with the use of a commercially available enzyme immunoassay system (RPN225-Amersham Biosciences) and according to the protocol provided by the company. Myocytes isolated from each LV were divided in 6 wells (control: average of 2 wells; isoproterenol alone: average of 2 wells; isoproterenol+carbachol: average of 2 wells) (WT, n=6 experiments from 6 hearts; NRG-1^+/–, n=5 experiments from 5 hearts).

Western Blotting
LV sarcolemmal plasma membranes and LV and brain lysates were prepared, and Western blotting was performed.^9,15 The following antibodies were used to measure levels of LV proteins: anti–muscarinic AChR-M₂ (Santa Cruz Biotechnology); anti–G protein subunits G_i1,2, G_i3, and G_o (Calbiochem); and anti–endothelial NO synthase (eNOS) and anti–inducible NO synthase (iNOS) (BD Biosciences). Protein levels of the nicotinic receptor AChR-ß₁ (Santa Cruz Biotechnology) were investigated in brain lysates. The Western Blot Lightning-Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences) was used to detect bound antibodies. Protein levels were normalized to those of GAPDH (n=5 mice per group).

Statistical Analysis
Values are expressed as mean±SEM. Comparisons among the groups were analyzed by ANOVA followed by a post hoc test. Two-way ANOVA with repeated measures was used to compare the response of myocytes to isoproterenol with and without carbachol. Student t test was used to compare 2 groups. Statistical significance was accepted at the level of P<0.05.

	Results

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The body weight (24.2±0.64 versus 27.7±1.99 g; P=NS) and LV weight/body weight ratio (6.83±0.15 versus 6.80±0.33 mg/g; P=NS) were similar in NRG-1^+/– and WT mice. The total number of myocyte experiments and baseline characteristics are shown in Table 1. Myocyte diastolic length and area and parameters of cell contraction/relengthening and systolic/diastolic [Ca²⁺]_i transients were similar between the groups. There was no effect of carbachol alone on any parameter. Isoproterenol (1 µmol/L) alone caused similar augmentation of fractional shortening (FS), dL/dt, and peak-systolic [Ca²⁺]_i in both groups (Table 2).

View this table: [in this window] [in a new window]   TABLE 1. Baseline Characteristics of Myocyte Contraction and [Ca2+]i Transients

View this table: [in this window] [in a new window]   TABLE 2. Effects of Isoproterenol on Myocyte Contraction and [Ca2+]i Transients

Figure 1 shows the effects of the addition of carbachol in the presence of isoproterenol stimulation in comparison with isoproterenol stimulation alone in WT and NRG-1^+/– myocytes. In WT myocytes stimulated with isoproterenol, carbachol caused a concentration-dependent depression of both FS (P<0.001) and peak-systolic [Ca²⁺]_i (P=0.001) in comparison with WT myocytes stimulated continuously with isoproterenol alone. In contrast, in NRG-1^+/– myocytes stimulated with isoproterenol, carbachol failed to attenuate either FS (P=NS) or peak-systolic [Ca²⁺]_i (P=NS) in comparison with isoproterenol alone (Figure 1). As shown, in WT myocytes, isoproterenol-stimulated FS (13.5±0.81% versus 15.6±0.85%) and peak-systolic [Ca²⁺]_i (542±34 versus 691±52 nmol/L) were depressed in presence versus absence of 100 µmol/L carbachol. As shown, in NRG-1^+/– myocytes, isoproterenol-stimulated FS (16.7±0.66% versus 15.9±0.84%) and peak-systolic [Ca²⁺]_i (640±43 versus 650±56 nmol/L) were similar in presence versus absence of 100 µmol/L carbachol.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of carbachol (CARB) on FS and [Ca2+]i transients in the presence of isoproterenol (ISO) in myocytes from NRG-1+/– and WT mice. In each panel, left circles represent basal stimulation with isoproterenol alone for 3 minutes. Black circles represent myocytes stimulated with isoproterenol (1 µmol/L) alone for 3, 6, and 9 minutes. Cells stimulated with isoproterenol plus carbachol are represented with white circles. In WT myocytes stimulated with isoproterenol, carbachol caused significant reduction in both FS (A; representative tracings are shown in E) and peak-systolic [Ca2+]i (B; representative tracings are shown in F). In contrast, carbachol failed to reduce contractility during ß-adrenergic stimulation in myocytes from NRG-1+/– myocytes (C and D; representative tracings are shown in G and H, respectively).

In WT and NRG-1^+/– myocytes, basal control levels of cAMP were similar (WT: 1680±137 fmol/3000 cells; NRG-1^+/–: 1604±39 fmol/3000 cells; P=NS). In WT myocytes, carbachol attenuated isoproterenol-stimulated cAMP levels (isoproterenol: 238±15% control; isoproterenol+carbachol: 170±15% control; P<0.005). In contrast, in NRG-1^+/– myocytes, carbachol failed to abrogate isoproterenol-stimulated levels (isoproterenol: 244±7% control; isoproterenol+carbachol: 246±14% control; P=NS).

Consistent with the reported reduction of neuronal nicotinic receptors in NRG-1^+/– mice,^12,13 we observed a decreased level of nicotinic receptor protein in brain lysate of NRG-1^+/– compared with WT mice (Figure 2A). However, we did not identify any difference in protein levels of LV muscarinic M₂ receptors (Figure 2B), levels of G-protein subunits G_i1,2, G_i3, G_o, or levels of eNOS and iNOS (data not shown) in NRG-1^+/– and WT mice.

View larger version (13K): [in this window] [in a new window]   Figure 2. Acetylcholine nicotinic ß1 and muscarinic M2 receptors protein levels. A, In brain lysate, the protein level of nicotinic ß1 receptor was lower in NRG-1+/– mice than in WT animals. B, In LV membranes, the muscarinic M2 receptor protein level was not different between NRG-1+/– and WT mice. n=5 experiments per group.

	Discussion

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This study demonstrates that muscarinic receptor stimulation fails to depress contractility during ß-adrenergic stimulation in cardiomyocytes from mice heterozygous for neuregulin-1 gene deletion (NRG-1^+/–). This observation supports a role for neuregulins in maintaining normal parasympathetic modulation of excess ß-adrenergic stimulation of the heart.

Muscarinic Modulation of ß-Adrenergic Activation
Classic physiology studies have shown that stimulation of muscarinic receptors by carbachol causes little or no direct negative inotropic effect on ventricular myocardium;¹⁶ however, it induces the reduction of contractility in myocardium stimulated by ß-adrenergic agonists.^16–18 Thus, in the normal heart, parasympathetic muscarinic receptor activation plays a cardioprotective role by counterbalancing excess ß-adrenergic activation. A large body of evidence suggests that excess ß-adrenergic activation in association with diminished parasympathetic activity is detrimental in heart failure. Studies in dogs showed autonomic imbalance in the early stage of experimental heart failure,^19,20 and other studies suggest that attenuation of parasympathetic tone precedes sympathetic activation during the development of heart failure.²¹ Clinical studies have shown that both low heart rate variability^22,23 and increased heart rate,²⁴ indicating increased sympathetic tone and/or decreased parasympathetic tone, are associated with increased risk of sudden death in heart failure. Multiple clinical trials in patients have provided evidence of the beneficial results of ß-adrenergic blockers on mortality in heart failure.²⁵ Although therapy aimed at enhancing parasympathetic activation has not been tested in clinical trials, animal studies have shown that vagal stimulation protected against lethal arrhythmias in conscious dogs with healed infarction²⁶ and improved survival and cardiac remodeling in a rodent model of postinfarction heart failure.²⁷

In cancer patients treated with the cardiotoxin doxorubicin, pharmacological suppression of neuregulin signaling increases the development of heart failure.^7,8 The contribution of abnormal neuregulin signaling is not understood and is attributed in part to effects on cardiomyocyte viability and survival.²⁸ The present study supports the possibility that inhibition of neuregulin signaling may exacerbate heart failure via loss of cardioprotective parasympathetic modulation of the excessive ß-adrenergic activation that is characteristic of heart failure.

Role of cAMP
The anti–ß-adrenergic effect of parasympathetic stimulation is mediated via coupling of the muscarinic M₂ receptor, the predominant myocardial receptor subtype, with G protein subunits G_i and G_o.²⁹ Using G_i2-null mice and G_i3-null mice, Nagata et al¹⁸ reported that G_i2, but not G_i3, is necessary for anti–ß-adrenergic effect in cardiomyocytes. In addition, Valenzuela et al³⁰ showed that muscarinic inhibition of L-type Ca²⁺ channels requires muscarinic receptor coupling to G_o. Muscarinic receptor stimulation causes inhibition of adenylyl cyclase via G_i protein and consequently reduction of intracellular cAMP, which leads to a reduction of L-type Ca²⁺ current.^31,32 In the present study, we demonstrated that myocytes of NRG-1^+/– mice fail to decrease levels of ß-adrenergic–stimulated cAMP in response to carbachol. Because protein levels of the muscarinic M₂ receptor as well as G_i1,2, G_i3, and G_o appear to be similar in WT and NRG-1^+/– mice, further studies will be necessary to elucidate the defect in coupling of the M₂ receptor to cAMP in this model.

Recently, it has been suggested that the attenuation of ß-adrenergic signaling by muscarinic receptor agonists requires eNOS and NO activation of cGMP, stimulation of type II phosphodiesterase, and subsequent decrease of cAMP.¹⁷ However, others found no alteration in the response to muscarinic stimulation in mice lacking eNOS.^33,34 Therefore, the importance of NO on muscarinic attenuation of ß-adrenergic stimulation remains controversial. In the present study, we found no difference in protein levels of eNOS and iNOS between NRG-1^+/– and WT mice, but we did not measure NO production.

Study Limitations
Although the M₂ receptor is the predominant receptor subtype expressed in myocardium, M₁, M₃, M₄, and M₅ subtypes have also been identified.^35,36 M₁, M₃, and M₅ receptors couple preferentially with G_q/11 protein and activate the phospholipase C–diacylglycerol-inositolphosphate system.³⁷ Additionally, stimulation of M₂ receptor may result in activation of G protein -subunit and subsequent release of the ß-complex, which is able to activate the phospholipase C pathway.³⁸ Further studies will be needed to elucidate the relationship between neuregulins and these signaling pathways in normal and NRG-1^+/– mice and to explore the contribution of neuregulins to cardiac parasympathetic activation in heart failure.

	Acknowledgments

 
This study was supported by a postdoctoral fellowship from CNPq (200767/01-1) and UNESP, Brazil, to Dr K. Okoshi. Drs Lorell, Yan, and Nakayama were supported by research grants from the National Heart, Lung, and Blood Institute and the National Space Biologic Research Institute. Dr M.P. Okoshi received a postdoctoral fellowship from FAPESP and UNESP, Brazil.

	References

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