Opposing Effects of β1- and β2-Adrenergic Receptors on Cardiac Myocyte Apoptosis
Role of a Pertussis Toxin–Sensitive G Protein
Background—β-Adrenergic receptor (β-AR) stimulation increases apoptosis in adult rat cardiac (ventricular) myocytes (ARVMs) via activation of adenylyl cyclase. β2-ARs may couple to a Gi-mediated signaling pathway that can oppose the actions of adenylyl cyclase.
Methods and Results—In ARVMs, β-AR stimulation for 24 hours increased the number of apoptotic cells as measured by flow cytometry. β-AR–stimulated apoptosis was abolished by the β1-AR–selective antagonist CGP 20712A (P<0.05 versus β-AR stimulation alone) but was potentiated by the β2-AR–selective antagonist ICI 118,551 (P<0.05 versus β-AR stimulation alone). The muscarinic agonist carbachol also prevented β-AR–stimulated apoptosis (P<0.05 versus β-AR stimulation alone), whereas pertussis toxin potentiated the apoptotic action of β-AR stimulation (P<0.05 versus β-AR stimulation alone) and prevented the antiapoptotic action of carbachol.
Conclusions—In ARVMs, stimulation of β1-ARs increases apoptosis via a cAMP-dependent mechanism, whereas stimulation of β2-ARs inhibits apoptosis via a Gi-coupled pathway. These findings have implications for the pathophysiology and treatment of myocardial failure.
We previously demonstrated that exposing adult rat ventricular myocytes (ARVMs) to norepinephrine (NE) for 24 hours increases the number of apoptotic cells.1 This effect is inhibited by the nonselective β-adrenergic receptor (β-AR) antagonist propranolol, mimicked by the adenylyl cyclase stimulator forskolin, and attenuated by an inhibitor of protein kinase A, indicating that NE stimulates apoptosis via a β-AR–mediated increase in cAMP.1 Likewise, Iwai-Kanai et al2 showed that β-AR stimulation increases apoptosis in neonatal rat cardiac myocytes by a mechanism that is dependent on the cAMP–protein kinase A pathway.
Both β1- and β2-ARs are expressed in cardiac myocytes and mediate an increase in contractility via Gs-dependent coupling to adenylyl cyclase.3 However, β2-ARs can also couple to signaling pathways independent of cAMP or Gs and, in particular, to a pertussis toxin (PTX)–sensitive pathway mediated by Gi.4 5 6 We tested the hypotheses that (1) β1- and β2-AR subtypes exert opposing effects on apoptosis in cardiac myocytes and (2) β2-AR activation attenuates β1-AR–stimulated apoptosis via Gi. Accordingly, ARVMs were exposed to NE or isoproterenol in the presence of β1- or β2-AR–selective antagonists. Apoptosis was measured by flow cytometry and confirmed by terminal deoxynucleotidyl transferase (TdT)–mediated nick end-labeling (TUNEL).1 Carbachol and PTX were used to stimulate or inhibit Gi, respectively.
ARVMs were isolated from the hearts of adult (200 to 220 g) male Sprague-Dawley rats,1 plated at a nonconfluent density of 30 to 50 cells/mm2 on 100-mm plastic culture dishes (Fisher) or 40×22-mm glass coverslips (Fisher) precoated with laminin (1 μg/cm2, Becton-Dickinson), and maintained in ACCT medium (DMEM; BSA, 2 mg/mL; l-carnitine, 2 mmol/L; creatine, 5 mmol/L; taurine, 5 mmol/L; penicillin, 100 IU/mL; streptomycin, 100 μg/mL) for 16 hours before drug treatments.
Myocytes were treated with isoproterenol (10 μmol/L) or a combination of NE (10 μmol/L) and prazosin (PZ; 0.1 μmol/L, 30 minutes before NE) for 24 hours. All dishes were supplemented with ascorbic acid (0.1 mmol/L). The β1-AR–selective antagonist CGP 20712A (0.3 μmol/L, RBI) or the β2-AR–selective antagonist ICI 118,551 (0.1 μmol/L, RBI) was added 30 minutes before NE. Carbachol (30 μmol/L, Sigma) or PTX (1 μg/mL, Sigma) was added 30 minutes or 3 hours, respectively, before NE.
Myocyte apoptosis was assessed primarily by flow cytometry as described and validated.1 Apoptotic cells stained with propidium iodide exhibit a reduced DNA content peak in the hypodiploid region indicative of apoptosis.7
TUNEL staining was performed on cells plated on glass coverslips with a Boehringer Mannheim in situ death detection kit.1 The percentage of TUNEL-positive myocytes (relative to total myocytes) was determined by counting 400 to 500 cells in 20 randomly chosen fields per coverslip on each of 3 coverslips for each experiment.
Cellular cAMP Content
ARVMs (≈2×105) were homogenized at 4°C in buffer (Tris-HCl, 50 mmol/L; MgCl2, 10 mmol/L; EDTA, 1 mmol/L; and PMSF, 5 μmol/L; pH 7.4), and cAMP was measured by radioimmunoassay (Dupont-NEN).
All data are presented as mean±SEM. Differences among multiple conditions were determined by 1-way ANOVA with a post hoc Tukey’s test. Differences were considered significant if the null hypothesis could be rejected at the 0.05 level.
β-AR–Stimulated Apoptosis Is Mediated via β1-ARs
As we previously reported,1 treatment with NE/PZ for 24 hours increased the number of apoptotic myocytes by 1.72±0.08-fold (Figure 1A⇓). Pretreatment with the β1-AR–selective antagonist CGP 20712A5 alone had no effect on the number of apoptotic cells, but it inhibited the apoptotic action of NE/PZ. Conversely, pretreatment with the β2-AR–selective antagonist ICI 118,5515 alone had no effect on the number of apoptotic cells, but it potentiated the apoptotic action of NE/PZ. Likewise, treatment with the nonselective β-AR agonist isoproterenol increased the number of apoptotic myocytes by 1.81±0.08-fold (Figure 1B⇓). As with NE/PZ, this effect was inhibited by CGP 20712A and potentiated by ICI 118,551.
Gi Attenuates β-AR–Stimulated Apoptosis
Pretreatment with PTX had no effect on the number of apoptotic myocytes, but it potentiated the apoptotic action of NE/PZ as measured by flow cytometry (Figure 2A⇓). Likewise, PTX pretreatment increased the number of TUNEL-positive cells after NE/PZ (2.68±0.16-fold) compared with NE alone (1.70±0.14-fold; P<0.05 versus NE/PZ+PTX; n=3).
Carbachol Protects From β-AR–Stimulated Apoptosis via Gi
Pretreatment with carbachol alone had no effect on the number of apoptotic cells, but it abolished the apoptotic action of NE/PZ (Figure 2B⇑). Pretreatment with PTX abolished the protective effect of carbachol for NE-stimulated apoptosis.
Myocyte cAMP Content
cAMP content averaged 12.44±1.24 pmol/mg protein in control cells and increased to 37.4±4.9 pmol/mg protein (P<0.05 versus control) with NE/PZ. Pretreatment with PTX had no effect on basal cAMP (11.1±1.2 pmol/mg protein) or that stimulated by NE/PZ (29.1±3.8 pmol/mg protein).
The major new findings of this study are that (1) β1- and β2-ARs mediate opposing effects of NE on apoptosis in cardiac myocytes (stimulation by β1-ARs, inhibition by β2-ARs) and (2) activation of a PTX-sensitive G protein (presumably Gi) by either β2-ARs or carbachol inhibits β-AR–stimulated apoptosis.
Opposing Effects of β1-ARs and β2-ARs on Apoptosis
Both β1- and β2-ARs can stimulate adenylyl cyclase by coupling to Gs. Recently, it has been appreciated that β2- but not β1-ARs can also couple to non–adenylyl cyclase pathways via Gi.4 The Gi-mediated action may oppose the effect mediated by Gs and in some cases may be the predominant β2-AR effect. Xiao and Lakatta3 showed that inhibition of Gi potentiated the ability of β2-AR stimulation to increase contractility, intracellular calcium, and calcium current in ARVMs. The coupling of β2-ARs to Gi may be regulated by a Gs-mediated, protein kinase A–dependent phosphorylation of the β2-ARs that favors coupling to Gi.4
Although NE and isoproterenol stimulate both β1- and β2-ARs in ARVMs, the net effect is to increase apoptosis. Because β1-ARs account for ≈80% of β-ARs in ARVMs,8 the predominance of the β1-AR effect on apoptosis may reflect the stoichiometry of the subtypes. β1-AR–stimulated cAMP might also inhibit non–cAMP-dependent pathways coupled to β2-ARs.6 9
Role of Gi
PTX, which inhibits Gi, increased the magnitude of β-AR–stimulated apoptosis and thus mimicked the effect of β2-AR blockade. Conversely, pretreatment with carbachol, which stimulates Gi in ARVMs via activation of M2 muscarinic receptors, inhibited β-AR–mediated apoptosis. The antiapoptotic action of carbachol was abolished by PTX, supporting the role of Gi. Gi might oppose the actions of Gs by inhibiting the activation of adenylyl cyclase, which appears to be central to β-AR–stimulated apoptosis in cardiac myocytes.1 2 However, PTX did not inhibit the β-AR–stimulated increase in cAMP, suggesting that Gi might inhibit β-AR–stimulated apoptosis via a cAMP-independent mechanism.4 10
These findings support the thesis that increased sympathetic activity to the myocardium contributes to myocardial failure via β1-AR–stimulated apoptosis of cardiac myocytes. This premise is supported by the demonstration that mice overexpressing Gs developed dilated cardiomyopathy associated with myocyte apoptosis,11 and mice overexpressing β1-ARs developed dilated cardiomyopathy.12 13 In contrast, mice overexpressing β2-ARs did not develop myocardial dysfunction at ages up to 4 months.14 Our findings further demonstrate that activation of Gi by a muscarinic agonist or β2-AR stimulation opposes the apoptotic action of β1-AR stimulation and thus raises the possibility that strategies to activate Gi or to increase the coupling of Gi to β2-ARs may be of therapeutic value.
This study was supported in part by NIH grants HL-52320, HL-42539, and HL-61639 (Dr Colucci), HL-057947 (Dr Singh), and HL-03878 (Dr Sawyer); a Grant-in-Aid from the AHA, Massachusetts Affiliate (Drs Singh and Sawyer); a Merit grant from the Department of Veterans Affairs (Dr Singh); and a fellowship from the AHA, Massachusetts Affiliate (Dr Communal).
- Received August 26, 1999.
- Revision received October 5, 1999.
- Accepted October 8, 1999.
- Copyright © 1999 by American Heart Association
Communal C, Singh K, Pimentel DR, Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the β-adrenergic pathway. Circulation. 1998;98:1329–1334.
Iwai-Kanai E, Hasegawa K, Araki M, Kakita T, Morimoto T, Sasayama S. α- and β-Adrenergic pathways differentially regulate cell type–specific apoptosis in rat cardiac myocytes. Circulation. 1999;100:305–311.
Xiao RP, Lakatta EG. β1-Adrenoreceptor stimulation and β2-adrenoreceptor stimulation differ in their effects on contraction, cytosolic calcium, and calcium current in single rat ventricular cells. Circ Res. 1993;73:286–300.
Xiao RP, Ji X, Lakatta EG. Functional coupling of the β2-adrenoreceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol. 1995;47:322–329.
Xiao RP, Avdonin P, Zhou Y, Cheng H, Akhter SA, Eschenhagen T, Lefkowitz RJ, Koch WJ, Lakatta EG. Coupling of β2-adrenoreceptor to Gi protein and its physiological relevance to murine cardiac myocytes. Circ Res. 1999;84:43–52.
Kuznetsov V, Pak E, Robinson RB, Steinberg SF. β2-Adrenergic receptor actions in neonatal and adult rat ventricular myocytes. Circ Res. 1995;76:40–52.
Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3′,5′-monophosphate. Science. 1993;262:1065–1069.
Luttrell LM, Ferguson SSG, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F-T, Kawakatsu H, Owada K, Luttrell DK, Caron MG, Lefkowitz RJ. β-Arrestin-dependent formation of β2 adrenergic receptor-Src protein kinase complexes. Science. 1999;283:655–661.
Geng Y, Ishikawa Y, Vatner DE, Wagner TE, Bishop SP, Vatner SF, Homcy CJ. Apoptosis of cardiac myocytes in Gs alpha transgenic mice. Circ Res. 1999;84:34–42.
Port JD, Weinberger HD, Bisognano JD, Knudson OA, Bohlmeyer TJ, Pende A, Bristow MR. Echocardiographic and histopathological characterization of young and old transgenic mice over-expressing the human β1-adrenergic receptor. J Am Coll Cardiol. 1998;31:177A. Abstract.
Engelhardt S, Hein L, Wiesmann F, Lohse MJ. Progressive hypertrophy and heart failure in β1-adenergic receptor transgenic mice. Proc Natl Acad Sci U S A. 1999;96:7059–7064.