Anti–β₁-adrenergic receptor antibodies and heart failure: causation, not just correlation

Abstract

Antibodies specific for the β₁-adrenergic receptor are found in patients with chronic heart failure of various etiologies. From work presented in this issue of the JCI, we can now infer that these antibodies actually contribute to the pathogenesis of chronic heart failure. This commentary discusses mechanisms by which these antibodies may engender cardiomyopathy.

Do anti–β-adrenergic receptor (anti–β-AR) antibodies play a role in the pathogenesis of chronic systolic heart failure (CHF)? This question emerged almost 30 years ago (1), when antibodies with β-adrenergic stimulating (agonist) activity were discovered in the serum of patients with Chagas disease, one of the most common causes of CHF worldwide (2). Since that time, IgGs with agonist activity for the β₁-adrenergic receptor (β₁-AR) have been found in sera not only from patients with Chagas disease, but also from patients with idiopathic dilated cardiomyopathy (3) as well as ischemic (4) cardiomyopathy. Whether these antibodies merely correlate with myocardial inflammation that leads to CHF, result from myocardial inflammation, or actually contribute to the pathogenesis of CHF could not be ascertained — until now. In this issue of the JCI, Jahns et al. employed isogenic injections of anti–β₁-AR antiserum in inbred rats to produce a cardiomyopathy that appears to be non-inflammatory (5). In so doing, these authors conclusively demonstrated that agonistic, anti–β₁-AR IgG — by itself — is sufficient to engender the sort of myocardial dysfunction characteristic of CHF. This finding fundamentally advances our understanding of CHF. However, it should not really surprise us, because it represents a logical extension of diverse but congruent investigations conducted over several decades.

To provide historical and mechanistic perspectives for the elegant work of Jahns et al., we address several questions that relate their work to contemporary concepts of β₁-AR pathophysiology: How might IgG activate the β₁-AR, and how could chronic β₁-AR activation result in cardiomyocyte toxicity? What molecular mechanisms regulate the β₁-AR when it is chronically stimulated by IgG or other agonists, and how might these mechanisms affect the pathogenesis of CHF? Lastly, how can these perspectives elucidate the therapeutic efficacy of β-AR antagonists, or “beta blockers,” in CHF?

Activation of cardiac β₁-ARs

The β₁-AR constitutes approximately 80% of the cardiac β-AR complement. Like the β₂-AR (with 54% overall homology), the β₁-AR is a seven-membrane–spanning receptor, with three extracellular polypeptide sequences (“loops”) connecting the transmembrane α helices (Figure 1). With their intracellular domains, the β-ARs couple to the stimulatory heterotrimeric GTP-binding protein (G_s). Activation of the β₁-AR requires a specific receptor conformation — one that is stabilized by agonist (and, apparently the binding of certain IgGs to the second extracellular loop; ref. 5). It also appears that β₁-AR dimerization (6, 7) may be involved in receptor activation (and may underlie the agonist properties of anti-β₁-AR antibodies observed in CHF patients and rats). Stimulation of β-ARs engenders a cascade of consequent activation: first G_s, then adenylyl cyclase (which forms cAMP), then the cAMP-dependent protein kinase (PKA). Activated PKA subsequently phosphorylates molecules critical for regulating sarcoplasmic [Ca²⁺] (8) — thereby increasing cardiomyocyte inotropy, chronotropy, and lusitropy. Activation of G_s can also increase L-type Ca²⁺ channel currents directly (9, 10) (Figure 1). Over the last few years, this time-honored scheme has been modified in ways that illuminate the β₁-AR-specific findings of Jahns et al. (5).

Figure 1

Schema for β₁-AR–mediated cardiomyocyte stimulation and β₁-AR desensitization. The seven-membrane-spanning β₁-AR is stimulated by the physiologic agonist norepinephrine or by IgG specific for the receptor’s second extracellular loop (ECII) (in CHF subjects). The stimulated β₁-AR then activates the heterotrimeric G_s, which dissociates into its Gα_s* and Gβγ substituents. The Gα_s* activates both adenylyl cyclase (AC), which catalyzes cAMP formation, and the L-type calcium channel (L), which then permits Ca²⁺ to enter the cardiomyocyte. This Ca²⁺ can both augment contractility and, on a slower time scale, promote cardiomyocyte apoptosis by activating Ca²⁺/calmodulin kinase II (CaMK II). The cAMP produced by adenylyl cyclase activates PKA, which subsequently phosphorylates (P) numerous substrates important to sarcoplasmic [Ca²⁺] regulation: the L-type Ca²⁺ channel, the ryanodine receptor (RyR), and phospholamban (PLB). The net effect of this activity in the short term is to augment sarcoplasmic [Ca²⁺] and contractility. (In the long term, this activity engenders cardiomyocyte toxicity.) The activated β₁-AR is desensitized when it is phosphorylated by PKA (not shown) and by GRK2, the cellular expression of which increases with chronic β₁-AR stimulation. Translocation of GRK2 from the sarcoplasm to the sarcolemma requires Gβγ subunits, and this translocation is inhibited when GRK2ct is expressed heterologously in cardiomyocytes. Asterisks denote activated proteins.

Genetic and pharmacologic approaches have demonstrated that the β₁-AR plays the predominant role in mediating cardiac inotropic and chronotropic responses to catecholamines (8). Cardiac preparations from β₁-AR knockout mice fail to augment contractility or their rate of contraction in response to isoproterenol, which stimulates both β₁- and β₂-ARs (11). Remarkably, β₁-AR knockout hearts have almost the same β₂-AR density as cognate wild-type controls, and can demonstrate contractile responses equivalent to controls — when contractility is augmented directly via adenylyl cyclase rather than via β-ARs (11). Even isoproterenol-induced adenylyl cyclase activity in cardiac homogenates is mediated overwhelmingly via the β₁-AR, in a manner disproportionate to the relative densities of β₁- and β₂-ARs (11). The failure of cardiac β₂-ARs to promote cAMP production to a degree commensurate with their expression level may be attributable to a signaling property that the cardiac β₂- and β₁-AR do not share: the ability to activate both the inhibitory heterotrimeric G protein (G_i) and G_s (12, 13). (A β₁-AR subtype-specific intracellular binding protein appears to prevent β₁-AR/G_i coupling [ref. 14].) In human subjects, β₂-AR–selective agonists have been used to augment left ventricular inotropy (15). However, a large fraction of this inotropic effect is mediated by β₁-ARs, and the role of β₂-ARs in this process may be largely to augment norepinephrine release from cardiac sympathetic neurons (15).

Excessive β₁-AR activation yields cardiomyocyte toxicity

Excessive isoproterenol stimulation has long been known to produce cardiomyocyte toxicity, myocardial scarring, and CHF (16, 17). More recently, chronic administration of submaximal isoproterenol doses has also been shown to produce cardiomyopathy, independent of myocardial scarring (18). That this isoproterenol-induced cardiomyopathy results primarily from β₁-AR activation can be inferred from the β₁-AR knockout mouse studies discussed above (11), as well as from a host of in vitro studies with rodent cardiomyocytes (13, 19). Further evidence that chronic β₁-AR hyperstimulation causes cardiomyocyte toxicity has emerged from studies with transgenic mice displaying modest (∼15-fold), cardiac-specific overexpression of the β₁-AR: these mice not only possessed enhanced β₁-AR activity, but also developed cardiomyopathy by age 4–9 months (20). In contrast, transgenic mice overexpressing the β₂-AR at higher absolute levels failed to develop any cardiomyopathy by this age (21).

The particular signaling pathways responsible for β₁-AR–induced cardiomyocyte toxicity remain somewhat enigmatic. Increasing cardiomyocyte PKA activity by as little as 2.4-fold can engender CHF (22), and cardiomyocyte overexpression of Gα_s engenders CHF (23). However, overexpression of adenylyl cyclase type VI (which also augments cardiomyocyte cAMP levels) not only avoids CHF but also can alleviate CHF in the Gα_q-overexpressing mouse (24). In addition, β₁-AR–promoted cardiomyocyte apoptosis can result from Ca²⁺/calmodulin-dependent protein kinase activity, independently from the PKA pathway (19). The diverse studies delineating molecular mechanisms responsible for β₁-AR–promoted cardiomyocyte toxicity have been reviewed recently (8). In light of these data, it is intriguing that levels of plasma norepinephrine (which activates the β₁-AR, like the anti–β₁-AR IgG of Jahns et al. [ref. 5]) have been directly and independently associated with CHF mortality in human subjects (25).

Regulatory mechanisms for subduing the β₁-AR signaling system

In the face of persistent β₁-AR hyperstimulation (either in CHF or experimental systems), both receptor-based and non-receptor counter-regulatory mechanisms are engaged in the cardiomyocyte — perhaps to bridle cellular toxicity. These mechanisms result in approximately 50% downregulation of the β₁-AR itself (26) (through mechanisms that appear to involve PI3K [ref. 27]), a decrease in adenylyl cyclase activity, and upregulation of the multifunctional G_i (which can inhibit adenylyl cyclase) (23). In addition, myocardium from CHF patients demonstrates a two- to threefold upregulation of G protein–coupled receptor kinase-2 (GRK2) (26), which phosphorylates and desensitizes the β₁-AR (28, 29). This upregulation of GRK2 even precedes the onset of left ventricular failure in mice with transgenic myocardial overexpression of calsequestrin (30). Although these “desensitizing” mechanisms are insufficient to prevent CHF, some of them may, paradoxically, contribute to CHF pathophysiology. Perhaps the most thoroughly studied of these cases is the role of GRK2.

Relieving excessive β₁-AR desensitization

Inhibition of cardiomyocyte GRK2 activity has been shown to ameliorate CHF in several mouse models, including deficiency of muscle LIM protein (31) and myocardial calsequestrin overexpression (32). GRK2 inhibition in these studies was achieved by transgenic myocardial overexpression of a polypeptide that comprises the carboxyl-terminal third of GRK2. This molecule, termed GRK2ct, inhibits GRK2 activity on receptors by binding to the heterotrimeric G protein βγ subunits required for GRK2 recruitment to the receptors (33) (Figure 1). Because “GRK2ct therapy” presumably does not alter the fundamental cardiomyocyte problems leading to myocardial dysfunction in these mouse models, its success points to the possibility that CHF-related enhancement of cardiomyocyte GRK2 activity may itself be maladaptive, and contribute to the pathogenesis of CHF. Remarkably, GRK2ct-expressing myocardium demonstrates attenuation of CHF-related GRK2 upregulation (31), β₁-AR downregulation (31), and β₁-AR/adenylyl cyclase desensitization (31, 32). In interpreting these data, however, it is important to note that GRK2ct binds a large variety of Gβγ subunits as well as phosphatidylinositol 3,5-bisphosphate (33). These binding activities could, beyond inhibiting GRK2, also contribute to the effects observed with GRK2ct (27, 34).

β-AR antagonists: possible mechanisms underlying therapeutic efficacy

Improvements in myocardial contractility, exercise tolerance, and mortality have been observed in CHF patients treated chronically with the β-AR antagonists metoprolol, bisoprolol, and carvedilol (35). This clinical improvement cannot depend upon reversal of β₁-AR downregulation, since carvedilol does not promote such a reversal (35). Moreover, this clinical improvement seems unlikely to be mediated through β₁-ARs at all, since it can be observed under conditions precluding agonist stimulation of β₁-ARs (36). However, probably because it diminishes toxic hyperstimulation of the β₁-AR by elevated sympathetic tone, prolonged β-AR antagonist therapy reduces cardiomyocyte apoptosis in CHF (37) and effects a partial recovery of CHF-associated derangements in gene expression (38, 39) and PKA-mediated hyperphosphorylation of proteins (40, 41), including those constituting the signaling and Ca²⁺-handling machinery downstream of the β₁-AR. These “receptor-distal” mechanisms can enhance myocardial performance despite ongoing β-AR antagonist occupancy (36).

As a specific example of how mechanisms distal to the receptors can affect cardiomyocyte function, let us again consider the role of GRK2 in CHF. Bisoprolol (42) and carvedilol (43) have been used in animals to reduce GRK expression. (We should note that bisoprolol was used by Jahns et al. to abolish the adenylyl cyclase response to anti–β₁-AR IgG [ref. 5].) Because GRK2 can desensitize a multitude of heptahelical receptors (33), reduced GRK2 levels could enhance signal transduction, and thus inotropy, evoked by heptahelical receptors other than the (antagonist-occupied) β₁-AR, such as endothelin receptors (44). From the perspective of this hypothesis, it is intriguing that metoprolol and GRK2ct reduced calsequestrin-associated cardiomyopathy in a synergistic manner (32).

Perspectives

From decades of CHF investigations, we should indeed have expected that agonistic, anti–β₁-AR antibodies would promote the development of heart failure. Now that Jahns et al. have provided an important proof of principle (5), we have another excellent reason to increase the clinical use of β-AR antagonist therapy in CHF of all causes. Whether immunoadsorption of anti–β₁-AR antibodies (45) will provide CHF patients with benefits beyond those obtainable from β-AR antagonists alone, however, remains to be determined.

Acknowledgments

R.J. Lefkowitz is an Investigator of the Howard Hughes Medical Institute. This work was also supported in part by NIH grants HL-64744 (N.J. Freedman), HL-16037, and HL-70631 (R.J. Lefkowitz).

Footnotes

See the related article beginning on page 1419.

Nonstandard abbreviations used: β-adrenergic receptor (β-AR); β₁-adrenergic receptor (β₁-AR); chronic systolic heart failure (CHF); cyclic AMP–dependent protein kinase (PKA); G protein–coupled receptor kinase-2 (GRK2); GRK2 carboxyl-terminal polypeptide (GRK2ct); inhibitory heterotrimeric G protein (G_i); stimulatory heterotrimeric GTP-binding protein (G_s).

Conflict of interest: R.J. Lefkowitz is cofounder of Norak, a company developing an inhibitor to GRK2.

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