© 2000 American Heart Association, Inc.
Editorial |
The Cellular Actions of ß-Adrenergic Receptor Agonists
Looking Beyond cAMP
From the Departments of Pharmacology and Medicine, College of Physicians and Surgeons, Columbia University, New York, NY.
Correspondence to Susan F. Steinberg, MD, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W 168 St, New York, NY 10032. E-mail sfs1{at}columbia.edu
Key Words: ß-adrenergic receptors • cAMP • mitogen-activated protein kinases • phosphatidylinositol 3'-kinase • apoptosis
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Introduction |
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 Top Introduction References |
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General concepts regarding the role of the sympathetic nervous system in the pathogenesis of heart failure (and as a site for therapeutic intervention) have undergone a remarkable transition in the last few years. When ß-adrenergic receptor (ß-AR) blockers were first introduced into clinical practice more than 30 years ago, they were viewed as contraindicated in heart failure. Conventional wisdom held that patients with impaired ventricular function rely on increased sympathetic drive as a mechanism to maintain mechanical performance and would clinically deteriorate if exposed to the negative inotropic actions of ß-AR antagonists. However, clinical practice demonstrated that although positive inotropic agents and vasodilators (agents that directly or indirectly activate neurohormonal pathways) induce short-term hemodynamic improvement, this is offset by long-term adverse effects to accelerate the natural history of heart failure. In contrast, ß-AR–blocking drugs prevent or reverse many of the structural and functional changes that develop during the progression of heart failure and prolong life in experimental animal models.1
The mechanisms whereby long-term ß-AR activation leads to abnormalities in cardiomyocyte growth, energy use, calcium regulation, and a progressively dysfunctional and mechanically inefficient heart have become an important focus of recent research. The cellular actions of catecholamines generally are attributed to the predominant ß1-AR subtype that couples to the stimulatory GTP regulatory protein (Gs), activation of adenylyl cyclase (AC), and accumulation of cAMP. Although cardiomyocytes also express pharmacologically distinct ß2-ARs and these assume increasing importance in heart failure syndromes (where ß1-ARs are downregulated), the traditional teaching holds that the Gs/cAMP pathway also is their primary mode of signaling (ie, ß-AR subtypes are functionally redundant). However, recent studies in transgenic mice challenge this concept. Cardiac-specific overexpression of ß2-ARs at relatively high levels leads to increased basal AC activity and elevated contractile function without obvious cardiotoxicity (unless receptor overexpression is maintained at very high levels or for protracted intervals).2 3 In stark contrast to the relatively wide therapeutic window for ß2-ARs, even low levels of transgenic ß1-AR overexpression lead to rapidly progressive cardiac deterioration with prominent histological evidence of fibrosis and cardiomyocyte apoptosis and hypertrophy.4 The distinct biological consequences of ß1- and ß2-AR overexpression argue for their distinct roles in transmembrane signaling in the heart.
In a study in this issue of Circulation Research, Chesley et al5 use cultured neonatal rat cardiomyocytes to decipher the distinct molecular mechanisms activated by cardiomyocyte ß1- and ß2-ARs. These studies follow on earlier research from Communal et al,6 demonstrating that ß1-ARs promote apoptosis and that the proapoptotic actions of ß1-ARs are countered by ß2-ARs in adult rat ventricular myocytes. In contrast, Chesley et al5 focus on ß2-AR protection from apoptosis induced by hypoxia and H2O2, because the effects of ß-AR subtypes on basal apoptosis were not reproduced in neonatal rat ventricular myocyte cultures.5 The initial attempts from the Colucci laboratory6 to delve into the mechanisms through which chronic ß-AR stimulation alters cardiomyocyte survival focused on cAMP, demonstrating that the proapoptotic actions of ß1-ARs are mediated by a cAMP-dependent mechanism, whereas the opposing effects of ß2-ARs could be attributed to a mechanism activated by a pertussis toxin (PTX)-sensitive G protein. Although an obvious potential target of the cardiomyocyte (PTX-linked) ß2-AR is the AC enzyme, the precise role of cAMP in the antiapoptotic actions of ß2-ARs is uncertain. Two aspects of ß2-AR signaling to AC are predicted to influence this process and must be considered.1 Do ß2-ARs display a generalized action to stimulate AC in all cardiomyocyte preparations?2 Does the ß2-AR/Gi pathway inhibit cAMP accumulation by ß1-ARs?
The notion that cAMP is an obligate downstream effector of ß2-ARs in all cardiomyocyte preparations remains the focus of lingering controversy. Xiao et al8 have put forth the model that ß1- and ß2-ARs both activate AC but in different cellular compartments. According to this model, ß1-ARs (acting through Gs proteins) generate a cAMP signal that is broadcast throughout the cell. In contrast, ß2-ARs promote cAMP accumulation, but the actions of cAMP are confined to effectors at the sarcolemma as a result of simultaneous ß2-AR activation of a PTX-sensitive G protein with opposing function. This model is based upon experiments demonstrating that PTX functionally enhances ß2-AR (but not ß1-AR) signaling (with the target of the ß2-AR/Gi pathway identified as an intracellular phosphatase that counters the stimulatory effects of protein kinase A [PKA] at intracellular sites such as phospholamban). Although inhibitory regulation of AC is a more traditional target for PTX-sensitive G proteins, the consensus of several recent studies is that the ß2-AR/Gi pathway does not inhibit AC.6 9 Because other results establish the integrity of the Gi-dependent pathway for muscarinic cholinergic receptor (mAChR) inhibition of AC in the same cells, these studies argue for specificity in Gi-protein signaling. It suggests that ß2-ARs and mAChRs couple to distinct species or pools of Gi proteins and that the use of mAChR agonists as a strategy to obtain independent confirmation of the Gi-dependent actions of ß2-ARs (an approach adopted by Chesley et al5 in the present study) may not be optimal.6 9 10
Other laboratories identify an alternative mechanism for the distinct signaling properties of ß1- and ß2-ARs. Here, experimental results demonstrate that ß2-ARs elevate cAMP levels in cultured neonatal rat cardiomyocytes but not in adult rat and embryonic mouse cardiomyocytes (where control experiments identify pronounced elevations of cAMP by ß1-ARs).11 12 13 These age- and species-dependent differences in ß2-AR linkage to cAMP provide a plausible explanation for the differential functional effects of ß1- and ß2-ARs (including on cell survival). For example, ß2-AR coupling to the proapoptotic cAMP signal in neonatal, but not adult, cells could (at least in part) explain the failure of Chesley et al5 to reproduce the reciprocal actions of ß-AR subtypes on apoptosis in neonatal rat cardiomyocyte cultures. To date, ß2-AR–dependent protection from ß1-AR–induced apoptosis has been reported only in adult rat cardiomyocytes.6 Traditional random collision-coupling models for receptor action do not provide an obvious mechanism for selective activation of AC by cell surface ß1-ARs but not by ß2-ARs (coexpressed on the cell surface at levels sufficient to provide functional inotropic support11 ). Rather, compartmentation of components of the receptor complex to membrane subdomains (caveolae), with distinct submembrane distributions for ß1- and ß2-ARs, allows for specificity in ß-AR–subtype activation of AC.14
Chesley et al5
build on recent efforts to identify the cAMP-independent pathways
recruited by agonist-activated ß-AR subtypes. Nonconventional
pathways for ß-ARs initially came under scrutiny in the context of
efforts to identify catecholamine-dependent hypertrophic signaling
mechanisms. Here, bifurcating pathways via
Gs/cAMP/PKA and Gi
protein ß
dimers/Src/Ras/Raf/extracellular signal–regulated
kinase (ERK) were implicated in the anabolic response to
ß-ARs.15 Two studies place
ERK activation downstream from ß1- and
ß2-AR subtypes; there is agreement that ERK
activation by ß2-AR is the quantitatively more
robust response, but the data regarding the role of PTX-sensitive G
proteins are less
consistent.5 12
The ERK cascade generally is credited with conferring protection from
proapoptotic stimuli. However, Chesley et
al5 provide evidence that ERK
activation is not required for ß2-AR
protection from hypoxia-induced apoptosis. Rather, these investigators
place the phosphoinositide 3'-kinase (PI3K)/Akt pathway (a
survival signal previously implicated in ß-AR–dependent induction of
atrial natriuretic factor
expression16 ) downstream
from the PTX-sensitive ß2-AR subtype and
demonstrate that this pathway figures critically in
ß2-AR protection from apoptosis induced by
hypoxia or
H2O2.5
Noticeably absent from the study by Chesley et
al5 is any consideration of
p38–mitogen-activated protein kinase (MAPK), another MAPK family
member that variably has been placed downstream from
PI3K17 18 and has
been the focus of considerable attention (and confusion) as an
intermediate in signaling pathways leading to cardiac hypertrophy and
apoptosis. Recent studies by Sabri et
al12 indicate that p38-MAPK
is activated largely by the ß1-AR subtype (and
a PTX-insensitive pathway) in embryonic mouse cardiomyocytes and (in
the absence of ERK activation) is not sufficient to induce
cardiomyocyte hypertrophy. Other studies from Communal et
al10 identify p38-MAPK
activation by ß1- and
ß2-AR and argue that p38-MAPK figures
importantly in the antiapoptotic Gi-dependent
pathway for ß2-ARs in adult rat ventricular
myocytes. However, these conclusions regarding the role of p38-MAPK in
antiapoptotic signaling by ß2-ARs are entirely
on the basis of results of experiments with high concentrations of the
inhibitor compound SB203580 and may be open to question. Recent studies
indicate that micromolar SB203580 blocks Akt phosphorylation by
phosphoinositide-dependent protein kinase
1.19 Hence, the most
parsimonious interpretation of available literature is that PI3K/AKT is
the prosurvival signal activated by
ß2-ARs.
With present enthusiasm for ß-AR antagonists as therapeutic agents for heart failure, studies to decipher the signaling properties of individual cardiomyocyte ß-AR subtypes and distinguish their roles on cardiac muscle biology become critical. Key unresolved issues include the following.
What is the structural basis for the distinct signaling properties of ß1- and ß2-ARs (to cAMP and nontraditional signaling pathways)? Recent literature identifies considerable heretofore-unrecognized complexity for ß-AR–subtype signaling. Differences in ß-AR subtype/G-protein linkage, ß-AR association with scaffolding proteins that assemble second messenger–regulated signaling enzymes, and compartmentalization to membrane subdomains are among the mechanisms that can impart diversity in signaling that require additional study.
How do ß1-ARs promote apoptosis?
Studies in cardiomyocytes implicate a
Gs/cAMP/PKA pathway and calcium entry via
voltage-dependent calcium
channels.20 A recent study
identifies the calcium-dependent target of the proapoptotic ß-AR as
calcineurin (possibly acting through the dephosphorylation of the
protein Bad21 ) and comes as
a surprise; calcineurin has attracted considerable attention as a
mediator of cardiac hypertrophy and prevented apoptosis in a previous
study.22 However, given the
broad range of targets for calcineurin in the physiological context
(including to effectors that suppress and induce apoptosis), its
influence on the decision to hypertrophy versus commit to the apoptosis
program is likely to be defined by the identity and magnitude of
associated receptor-activated signals. In this context, recent studies
identify Src family tyrosine kinases as alternate effectors for the
G
s pathway leading to apoptosis in
thymocytes.23 Because
signaling pathways frequently are very context-dependent, direct
examination of this process in cardiomyocytes is warranted in future
studies.
Is cardiac protection mediated by a specific PTX-sensitive Gi protein? Present research implicating Gi proteins in ß2-AR signaling comes from studies with PTX, which cannot distinguish individual Gi protein family members (and could be confounded by direct cellular actions of the PTX cell surface–binding B-oligomer24 ). Ultimately, molecular (rather than pharmacological) strategies to ablate Gi proteins are required to validate these conclusions and identify the pertinent Gi proteins. On the basis of their distinct actions to inhibit AC, this approach also is predicted to distinguish Gi-dependent pathways for mAChR and ß2-ARs.
What is the biological significance of signals recruited by
the minor ß2-AR subtype typically only at
high-agonist concentrations? One of the more unnoticed features of
ß-AR signaling to growth regulatory pathways is the
concentration-response relationship for agonist activation. ß-AR
activation of AC is maximal at 0.1 µmol/L, but studies by Chesley et
al5 and
others12 typically rely on
100-fold higher agonist concentrations to optimally engage
cAMP-independent growth regulatory pathways. Recent experiments with
ß2-AR G-subunit fusion proteins demonstrate
that the pharmacologic profile of the ß2-AR
can be influenced by the identity of the G-protein subunit to which
it binds.25 Because
ß2-ARs adopt a conformation that displays
higher affinity for ligand when coupled with Gs
than with Gi, these observations are compatible
with a Gs-independent pathway for growth
regulation by ß2-ARs.
Which is the optimal system to study the cellular actions of ß-ARs in cardiomyocytes? The discrepancy between the studies describing the actions of ß-ARs on basal apoptosis in adult and neonatal cardiomyocytes serves to emphasize the uncertainties regarding the optimal model for investigations of catecholamine action in the heart. Because experience maintaining adult ventricular myocytes cultures that retain a highly differentiated phenotype has become more widespread, there has been a growing temptation to dismiss research in neonatal rat ventricle cultures as irrelevant. However, such categorical conclusions may be premature, because the preferred assay system may differ depending on the nature of the stimulus, response, or cardiomyocyte (normal or diseased) under study. For example, certain components of growth regulatory pathways are more abundant in neonatal than in normal adult cardiomyocytes. This could undermine the validity of extrapolating results obtained in neonatal cardiomyocytes to the normal adult ventricle. However, several examples of disease-associated functional increases in regulatory kinases in adult cardiomyocytes might suggest that neonatal cardiomyocytes are a valid model for the diseased adult heart. The cell type that provides the best surrogate for ß2-AR signaling in human cardiomyocytes also must be taken into consideration, given the evidence for distinct modes for ß2-AR coupling to cAMP accumulation and activation of nontraditional cAMP-independent growth regulatory pathways between neonatal and adult cardiomyocytes. Although knowledge of ß-AR signaling in human cardiac tissue is still limited, the preponderance of available evidence identifies a robust cAMP-dependent pathway for ß2-ARs in human ventricular myocardium.7 This pathway is more similar to the mode for ß2-AR signaling in neonatal (rather than adult) cardiomyocytes and suggests that neonatal cardiomyocytes also may be the preferred cell type for studies of ß2-AR actions.
Does the polymorphic variation of the ß2-AR with impaired Gs coupling and AC activation confer protection from apoptosis? Recent studies identify polymorphisms of both ß1- and ß2-ARs, with patients harboring a hypofunctional ß2-AR variant (using coupling to Gs as the endpoint) at increased risk for heart failure progression.26 27 The signaling properties of structurally distinct ß-ARs to cAMP-independent pathways have not been examined (and are not necessarily predictable). This line of study could reveal additional mechanisms whereby genetic variations contribute to interindividual difference in heart failure progression and sympathetic responsiveness.
Ultimately, studies directed at these and other issues will refine prevailing models for ß-AR action in the heart and provide a framework to develop newer strategies targeted to the sympathetic nervous system to optimize heart failure management.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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