G protein-coupled receptors are functionally closely related to ion channels. The G protein-coupled receptors are part of a gene family being identified that binds agonists such as adenosine, catecholamines, acetylcholine, odorants, angiotensin, histamine, opioids, and many others.

There are several interesting structural similarities between ion channels and G protein-coupled receptors. Both are integral membrane proteins with seven-transmembrane domains, which form bundles with a central pocket. In G proteins, the pockets are the binding sites for the receptor ligands, and they are typically located on the extracellular site of the protein; the N-terminal tail is located extracellularly as are three extracellular loops. Three loops connecting the transmembrane domains and the C-terminal domain are located intracellularly (Figs. 15.1 and 15.2). The exact function of the extracellular loops remains largely unknown. It is considered that the transmembrane domains are involved in receptor binding. The intracellular loops, particularly loop III and the C-terminal tail, are important for receptor coupling to the associated G protein. Finally, both loop III and C-terminal tail are important for regulation of receptor function and contain phosphorylation and other posttranslational modification sites .

The G protein receptors interact intimately with G proteins. G proteins are complexes consisting of three subunits—α, β, and χ. There are different classes of G proteins, and attempts have been made to classify them as subtypes. For example, common α-subunits include: (1) α_s (stimulatory) which activates adenylyl cyclase, (2) α_i (inhibitory) which inhibits adenylyl cyclase, (3) α₀ which modulates calcium channels and phospholipase C, and (4) α_z which activates phospholipase C. In general, each α-subunit contains a region that interacts with the receptor, a site that binds guanosine triphosphate (GTP), and a site interacting with the effector system .

The traditional concepts of G protein-coupled receptor function are best illustrated in Fig. 15.1. First, an agonist binds to a receptor molecule, and then the seven-transmembrane-spanning receptor molecule interacts with a specific G protein; this, in turn, modulates a specific effector. Examples of typical agonists, receptors, G proteins, and effectors are provided in Fig. 15.1. While this illustration provides a general summary of the individual components needed for proper G protein-coupled receptor function, a more thorough understanding of the G protein receptor-mediated signaling, as well as its regulation, is best accomplished by the more specific example of the well-characterized beta-adrenergic receptor (β-AR) system. Therefore, we will start our discussion with these physiologically, pathophysiologically, and clinically highly relevant seven-transmembrane-spanning β-ARs .

Table 15.2 summarizes the general features of β-AR subtypes identified to date; note that there are three subtypes [1]. While all of the subtypes can be found in the heart, the predominant subtype in the vasculature is the β2-AR. Importantly, norepinephrine and epinephrine are the endogenous agonists (catecholamines) that bind specifically to all three β-AR subtypes.

Pharmacologically, β1- and β2-receptors are characterized by an equal affinity for the exogenous agonists isoproterenol and epinephrine, while norepinephrine (the neurotransmitter of the sympathetic nervous system) has a ten- to thirty-fold greater affinity for the β1-AR subtype. Also, β1-ARs are more closely located to synaptic nerve termini than β2-ARs, thereby being exposed and activated by higher concentrations of released norepinephrine .

Cardiac β-ARs are likely one of the most important types of receptor systems. β-ARs regulate important cardiovascular functions and are integral to the body’s flight or fight response. For example, during exercise, heart rate and contractility increase, cardiac conduction accelerates, and cardiac relaxation, an active process requiring adenosine triphosphate (ATP), is enhanced. Moreover, vascular relaxation (vasodilation) of many vascular beds can be observed during exercise (e.g., in skeletal muscles). All these effects are at least partially a direct consequence of β-AR activation and involve key elements of G protein receptor signaling: (1) receptor binding, (2) G protein activation, and (3) activation of an effector system. Importantly, most of these specific physiological cardiac effects are mediated via activation of β1-AR, while the vascular effects are mediated through the β2-receptor subtype. However, heart muscle also contains β2-receptors (as well as β3 receptors). Nevertheless, the β1-receptor subtype is the predominant cardiac isoform in healthy humans. More specifically, while there is a substantial population of β2-receptors in the atria, only around 20–30 % of the total β-AR population is β2-ARs in the left ventricle [2]. Not only is the activation of β-ARs and their associated signaling transduction critical in understanding the physiology of the cardiovascular system, but it has been recognized over the past decades that β-AR activations also play key roles in cardiac disease processes such as heart failure [3]. For example, an apparent paradox exists, namely, that β-adrenergic blockers (β-receptor antagonists) are beneficial in patients with heart failure; this will be discussed in more detail later in this chapter.

To best understand β-AR regulation, it is useful to review the molecular structure of this receptor. Basically, the receptor is composed of three types of domains : (1) the extracellular domain, (2) the transmembrane domains which are involved in the agonist and antagonist binding (ligand binding), and (3) the intracellular or cytoplasmic domains which are important for G protein interactions and interactions with kinases such as β-ARK (β-adrenergic receptor kinase) (Fig. 15.2).

One of the hallmarks of chronic heart failure is adrenergic overdrive leading to β-AR downregulation and desensitization. In particular, β1-ARs have been shown to be downregulated selectively, while the β2-ARs are relatively increased so that, as mentioned above, the population of β1 to β2 shifts from 75/25 to about 50/50. The marked uncoupling of the β-ARs from the G proteins is due to increased levels of β-ARK1, as well as an increased inhibitory G protein (Gi) [14]. This, in turn, results in not only attenuated β-AR signaling but also activation of additional pathways involved in ventricular remodeling.

The shift of the β1-/β2-AR subpopulation to an increased number of β2-receptors is only one of the phenomena associated with chronic heart failure. More specifically, β2-ARs couple to a number of signaling pathways, including a potential coupling to an inhibitory G protein (Gαi). Also, β2-ARs may modulate several G protein-independent pathways, including inhibition of sodium-hydrogen exchanges as well as a nonprotein kinase A-dependent interaction with L-type calcium channels [6, 15]. There are other G protein-independent pathways in which the β2-ARs have been implicated, such as those associated with phosphatidylinositol kinase, phospholipase C/protein kinase C, and/or arachidonic acid signaling. And while many details of these pathways remain poorly understood, it is becoming more clear that some of these pathways play important roles in the molecular pathobiology of heart failure.

One of the widely recognized mechanisms of β-AR desensitization is agonist-dependent β-ARK-mediated phosphorylation of the β-AR itself. Moreover, the beta-gamma subunits of the G proteins can act as signaling molecules themselves, with the potential of activating many downstream targets of pathways such as adenylyl cyclases, PLC, PLA2, PI3K, K⁺ channels, and/or Src-Ras-Raf-MEK-MAPK (mitogen-activated protein kinase) [7].

The potentially toxic effects of long-term β1-AR activation are also illustrated by the fact that selective β1-AR agonist or receptor overexpression leads to increased cardiac apoptosis or programmed cell death [16–18]. The mechanisms by which such apoptosis is activated have been attributed, in part, to initial activation of calcineurin via increased intracellular calcium through L-type calcium channels [19]. Additionally, direct toxic effects of catecholamines on the myocytes have been described as a result of direct β-AR activation [12]. Other effects of chronic β-AR signaling include production of cytotoxicity via calcium overload, increased free radical formation, as well as stimulation of pathologic hypertrophy [7]. For example, the marked uncoupling of β1- and β2-receptors from their physiological signaling pathways not only results in attenuation of the β-AR signaling but also allows for the coupling of the receptors with other possible myopathic signaling pathways involved in ventricular remodeling (e.g., MAPK and PI(3)K cascades). As stated earlier, β-receptor antagonists may be a beneficial therapy in the chronic heart failure patient. Specifically, the β1-specific β-AR blocking drug metoprolol produces reverse remodeling similar to a combined α-β-blocker carvedilol, suggesting that β1-AR signaling is an important determinant of pathologic hypertrophy in human heart failure [20].

Lastly, genetic heterogeneity in expression of the β-AR in the general population has been identified which may be associated with disease susceptibility. For example, a substitution of threonine to isoleucine at amino acid 164 in the β2-AR has been linked with reduced survival and exercise tolerance in patients with heart failure [21, 22].

A summary of the known changes in β-AR signaling is provided in Table 15.3. Lastly, it should be mentioned that investigations using transgenic animal models have greatly helped to elucidate some of the important disease mechanisms associated with adrenergic receptor reception pathways; they have also served as tools in identifying and testing novel or evolving therapeutic targets for the treatment of heart failure [1] .

Two major α-AR subtypes have been identified—α1 and α2. The α1-type receptors have been described to be present in the heart, as well as in the vessels and smooth muscle. The primary response to their activation in cardiac cells is phospholipase C-β which leads to an increase in diacylglycerol and inositol triphosphate (InsP₃) and also activates both protein kinase C and MAPKs. Physiological receptor agonists include norepinephrine and epinephrine, and a Gq protein is the primary associated G protein [23]. In general, the primary physiological importance of α1-receptors is far greater in the vasculature, leading to calcium influx and vasoconstriction via the second messenger InsP₃. In cardiac muscle cells, α1-receptors have been shown to induce a positive inotropic effect; like β-receptors, this response is also associated with formation of InsP₃ [24]. Alpha-2-receptors have been identified on coronary vessels, as well as in the central nervous system (presynaptic); they reduce the activity of AC (and protein kinase A) via an inhibitory G protein.

In summary, the primary physiological role of α1-receptors is for the vasculature regulation of tone via vasoconstriction, which is secondary to calcium influx into the SMCs of the circulatory arterioles.

In general, in heart failure patients, the α1-AR system will elicit an increase in receptor density and is also characterized by changes in inotropic responses [25]. In addition, α1-AR activation has been shown to promote cardiomyocyte hypertrophy via activation of hypertrophic MAPK pathways) .

The muscarinic receptor system in the heart is also an associated G protein-coupled receptor system, one fairly similar to the β-AR. The main cardiac receptor subtype (M2 receptor) is a cholinergic receptor and thus mediates primary parasympathetic (syn. nervus vagus, cholinergic, acetylcholine mediated) nervous system responses. For more details, refer to Chap. 14. Its general function is to simultaneously balance and antagonize the sympathetic effects of β-AR activation, with its most pronounced effects on the controls of the sinoatrial (heart rate, chronotropic effect) and atrioventricular nodes (conduction, dromotropic effect). There exist very few muscarinic M2 receptors in the ventricles, so activated negative inotropic (contractility) effects subsequent to M2 receptor activation are very small. The primary control signal mediated via the vagus nerve is that which leads to a local release of acetylcholine in the sinoatrial and atrioventricular nodes. Acetylcholine then binds to the M2 receptor, activates an inhibitory G protein (Gαi), and essentially decreases the activity of adenylyl cyclase, which directly leads to opening of K⁺ channels. Thus, in the sinoatrial node, vagal stimulation tends to a flattening of the diastolic depolarization, which then induces a slowing of heart rate (bradycardia, negative chronotropic effect) possibly via the effects of reduced cAMP availability on I_f current (hyperpolarization-activated cyclic nucleotide-gated channel) but also via activation of a potassium outward current which in concert decreases the spontaneous firing rate of the sinoatrial node. In the atrioventricular nodal tissue, vagal stimulation also activates an inhibitory G protein, which typically causes a slowing conduction velocity via a decreased calcium influx through L-type calcium channels. Clinically, the effects of vagal stimulation on the atrioventricular node are detected as increased atrioventricular nodal conduction times (e.g., prolonged PR interval).

The M3 muscarinic subtype is primarily found in vascular SMCs. Its activation leads to contraction of vascular smooth muscles via a Gq/11-PLC-InsP3-mediated calcium influx (as well as diacylglycerol/phospholipase C/protein kinase, cAMP elevation) .

Although structurally different, the general principles of receptor signaling as discussed for the β-AR apply also for nitric oxide/guanylyl cyclase signaling. Specifically, the agonist nitric oxide (NO) is considered as a universal signaling molecule present in all tissues, and the role of NO signaling is similar to other signal transduction systems, not only important in normal cardiac function but also in disease processes. Physiologically, nitric oxide is produced by several isoforms of the enzyme nitric oxide synthase (NOS). To date, three isoforms have been described to function within the cardiovascular system—eNOS, iNOS, and nNOS. Nitric oxide is a small, lipid-soluble, gas molecule that readily crosses cell membranes. Inside the cell, it typically binds to a receptor, the soluble (cytoplasmic) guanylyl cyclase (GC, an enzyme which converts GTP to cyclic 3′-5′ GMP). Cyclic GMP is the second messenger in this signaling cascade and, in the cardiovascular system, can activate two major effector systems—cGMP-regulated phosphodiesterases and cGMP-dependent protein kinases (cGKs).

In the vascular SMCs, NO inhibits vascular smooth muscle constriction (causes vasodilation) via inhibition of cytoplasmic calcium release. However, the underlying mechanisms for the important vasodilatory effect of NO are complicated and minimally involve a cGMP-dependent protein kinase (cGKI); these pathways have been reviewed in detail elsewhere [31]. Nitric oxide has also been shown to reduce vascular smooth muscle proliferation (clinically important in in-stent restenosis) and migration. It is well established that NO release may reduce platelet adhesion and activation, as well as vascular inflammation [32]. Importantly, NO insufficiencies are considered to be an important factor in endothelial dysfunction, leading to the increased susceptibility for arterial thrombosis formations [33].

Nevertheless, the relevance of NO signaling is not limited to vascular biology or pathobiology; it has been demonstrated that NO has an important pathophysiological role in heart failure as well, i.e., in the modulation of cardiac function, cardiovascular protection, and/or in the regulation of apoptosis [34–36]) .

To date, at least seven membrane-bound enzymes synthesizing cyclic GMP (cGMP) have been identified. All seven of these membrane guanylyl cyclases (GCs) have a common structure (Fig. 15.5). For the purpose of this discussion, guanylyl cyclase-A has the greatest importance relative to the heart and will be discussed in detail .

Currently, three natriuretic peptides have been identified—ANP, BNP, and CNP. Both ANP and BNP bind to GC-A and are released in the heart; they are considered as cardiac hormones. In general, CNP is mainly produced by the vascular endothelium and may be a regulator of vascular tone and cell growth through GC-B activation. This receptor consists of an extracellular domain, a kinase homology domain, an α-helical amphipathic region (hinge region), and a C-terminal guanylyl cyclase (catalytic) domain (Fig. 15.5). This receptor is considered to exist in dimers (two molecules coupled together).

The major effects of GC-A are via the formation of, and the activation by, cGMP. Since, in general, the agonists are circulating hormones (peptides), a variety of organ systems can be affected simultaneously following ANP/BNP release. ANP is mainly produced in the atrium, while BNP is produced primarily in the ventricles. The primary triggering factors for ANP/BNP release are wall stretch and/or pressure increases, but their release may also involve neurohumoral factors (glucocorticoids, catecholamines, angiotensin II, etc.). The primary effect of ANP/BNP release is modulation of blood pressure/volume; the half-lives of ANP/BNP are around 2–5 min. Interestingly, although both ANP and BNP bind to the same receptors, it has been shown that targeted genetic disruption of ANP or BNP in mice induces a unique phenotype; ANP-deficient mice show arterial hypertension, hypertrophy, and cardiac death. In contrast, the BNP-deficient mice phenotype is characterized by mostly cardiac fibrosis. More specifically, BNP has a lower affinity to GC-A than ANP and may act mostly as a local paracrine (antifibrotic) factor. Also, under physiological conditions, the ANP levels are much higher than BNP levels in the circulating blood, and the potency for induced vasorelaxation is also less for BNP vs. ANP [37].