Gi/o signaling and its potential role in cardioprotection

Of the predominant receptor classes – ion channel-linked, enzyme-linked and G-protein-linked receptors (GPCRs) – GPCRs are the largest and most ubiquitously expressed family of receptors, encompassing more than 1000 different members [1] and represent the target of approximately 50–60% of the drugs used in clinical practice [2]. Binding of the GPCR to its ligand facilitates receptor activation and interaction with the G-protein heterotrimer, which consists of an α subunit that binds to and hydrolyzes GTP and a βγ dimer. To convey extracellular events to intracellular signaling moieties, G-proteins undergo an activation and inactivation cycle that actively links GPCRs and effectors. G-proteins can be divided into four families on the basis of the properties of the G-protein α subunit: Gαs, Gαi/o, Gαq/11 and Gα12/13 [3]. In the heart, β-adrenergic receptors (β-ARs) couple to Gαs signaling, resulting in increased cAMP production via adenylyl cyclase, subsequent activation of protein kinase A, and phosphorylation of calcium handling and contractile proteins, with resultant increased cardiac inotropy, chronotropy and lusitropy. Cardiac β-ARs, as well as adenosine and muscarinic receptors, are capable of coupling to the Gαi/o family of G-proteins. Gαi classically inhibits adenylyl cyclase-mediated cAMP production with subsequent decreased cardiac contractility, and Gαo, whose function in cardiac myocytes has until recently been undefined, appears to positively regulate calcium cycling through reduced activity of protein phosphatase 1 [4]. Thus, for β-ARs in the heart, there is a balance between Gs and Gi signaling, which is mediated by distinct β-AR subtypes and this becomes critical in times of cardiac stress.

In the human heart, β-ARs are primarily comprised of β₁-ARs, with approximately 25% β₂-ARs and minimal β₃-ARs. β-AR signal transduction has been demonstrated to be at the center of the pathophysiology of heart failure (HF) at the molecular level, primarily through diminished contractile responsiveness of failing myocardium to circulating catecholamines. Initially, this loss of catecholamine sensitivity through β-ARs, known as desensitization, compensates and protects the heart against elevated sympathetic nervous system activity. Chronically, desensitization leads to β₁-AR-selective downregulation and becomes maladaptive as the heart continues to weaken and subsequently cannot respond to stimulation. The Gαi/o family of G-proteins has been demonstrated to be increased in protein content as well as activity in human HF and, therefore, has been hypothesized to contribute intimately to the dampened and desensitized environment of the failing heart, and is thus an attractive target for therapeutic intervention [5,6]. Increased sympathetic drive in the failing heart is crucial to the proposed mechanism of Gαi/o upregulation in HF, as the Gαi2 promoter contains a cAMP-responsive CCAAT box that increases Gαi2 transcription in an apparent negative-feedback response to excess β-AR signaling [7]. However, normalizing the level of plasma catecholamines through treatment with βHAR blockers, such as metoprolol, or angiotensin-converting enzyme inhibitors, such as captopril, reverses Gi upregulation only partially at best or not at all, respectively [8,9]. The mechanism through which Gi upregulation is maintained in the absence of elevated plasma catecholamines has yet to be formally defined. Furthermore, the functional significance of the upregulation of the Gαi/o family of G-proteins in the failing heart has yet to be definitively established as beneficial or maladaptive.

We recently postulated that targeted inhibition of cardiomyocyte Gi signaling, a key component of the desensitized signaling milieu of the failing heart, might improve cardiac contractility in HF. To address Gi signaling in this regard, we generated a line of transgenic mice that express a Gi-selective inhibitor, termed GiCT, which consists of the carboxy-terminal 63 amino acids of Gαi2, the predominant Gαi/o isoform in the heart, specifically in cardiomyocytes [10]. In fact, in a surgical model of ischemia–reperfusion injury (I–R), class-specific inhibition of Gi signaling in cardiomyocytes led to significantly more cellular injury as assessed by increased infarct size. These data demonstrate that Gi signaling critically contributes to the maintenance of viable myocardium in response to I–R injury through inhibiting myocardial cell death through apoptosis [10]. Furthermore, we demonstrated that constitutive blockade of Gi signaling in cardiomyocytes via expression of GiCT significantly accelerates contractile dysfunction following myocardial infarction (MI), with a rapid transition towards cardiac remodeling and fetal gene re-expression DeGeorge BR Jr, Koch W; Unpublished Data[DeGeorge BR Jr, Koch W; Unpublished Data]. These data provide the first conclusive evidence that the elevated level of Gαi2 in acute post-MI HF is intimately involved in protecting the heart from the pathophysiological transition from the normal to the failing state. Taken together, these studies with mice harboring a Gi-selective inhibitor in cardiomyocytes critically implicates Gi signaling in mediating cardioprotective effects in response to acute myocardial injury through preventing pathologic remodeling and myocardial apoptosis. Thus, Gi signaling is needed to sustain critical myocyte survival and salvage pathways after an acute ischemic episode, a fact that has previously not been demonstrated.

The role of Gi signaling following the acute infarct period and in established HF, remains unresolved by these studies, and is of great consequence both at a clinical and fundamental scientific level. Although blockade of Gi signaling prior to MI may not be the appropriate time-point for therapeutic intervention, this does not per se rule out the validity of Gi as a pharmacologic target in more advanced HF. Importantly, other gene delivery and pharmacologic approaches that restore β-AR signaling have demonstrated efficacy in restoring cardiac performance in HF models [9,11,12]. The apparent cardioprotective role of Gi signaling in the acute infarct development period may become detrimental in the milieu of chronic HF. Our current viewpoint is that Gi signaling is a double-edged sword temporally, in that acutely enhancing Gi signaling is therapeutic; however, in chronic HF, blocking Gi signaling should facilitate increased cardiac contractile performance. The latter half of this hypothesis has yet to be thoroughly tested or studied.

Importantly, chronic stimulation via β₁- and β₂-ARs both provide the predominant means to modulate cardiac contractility and relaxation. Furthermore, both subtypes presumably also participate in maladaptive cardiac hypertrophy, remodeling, apoptosis and necrosis. Recently, it has been established that subtype-specific b-AR signaling results in activation of markedly different signaling within the cardiomyocyte [13]. Specifically, cAMP generated via β₁-AR signaling is distributed throughout the cardiomyocyte, with chronic stimulation activating hypertrophic and apoptotic signaling pathways. Conversely, signaling through the β₂-AR results in localized accumulation of cAMP, which appears to activate prosurvival pathways through Gi, resulting in activation of PI3K and Akt. Blockade of prosurvival β₂-AR signaling in vitro has been demonstrated to increase the susceptibility of cardiomyocytes to apoptosis in response to ischemia [14], and we have identified that in vivo blockade of this pathway is critical to apoptotic cell death in response to ischemic stress using transgenic mice harboring the Gi-selective inhibitor, GiCT [10].

Modulation of β-adrenergic pathways through Gi signaling has only recently been discovered; however, now Gαo as well as Gai2 appears to be capable of physically coupling to β₂-ARs [15]. The potential therapeutic value of Go signaling was suggested in a recent work by Zhu and colleagues, which demonstrated that transgenic mice expressing a constitutively active form of Gαo, specifically in cardiomyocytes, possessed enhanced calcium cycling and contractile function [4]. Future studies will be required to assess the respective contributions of Gi and Go to the prosurvival component of β₂-AR signaling. Interestingly, the phenotype of mice overexpressing Gαo is in direct opposition to mice overexpressing Gαi2, which demonstrate decreased cardiac inotropy and chronotropy [16]. Importantly, these members of the same G-protein family demonstrate marked phenotypic differences when exogenously overexpressed in cardiac myocytes; however, both appear to be elevated in failing myocardium. Nevertheless, it is presently unclear whether targeting Go signaling in myocardium will significantly alter survival or cardiac function in HF.

We propose that a gene therapy-based approach combining novel gene silencing technology, in addition to selective Gi/o signaling inhibitors, will allow investigators the opportunity to convincingly establish the potential cardioprotective properties of Gαi/o signaling in the myocardium. The use of adeno-associated virus-based gene delivery will permit temporal investigation into both the acute and chronic ramifications of Gαi/o signaling, and gene silencing technology will make it possible to discriminate the functional properties of Gαi/o isoforms in the context of HF. Gene therapy with Gαi2 may prove to be beneficial in the acute ischemic period, as Gi signaling during this time-frame appears to be critical to preventing myocyte dropout through apoptosis; initial studies indicate that Go gene delivery may facilitate inotropic support, via enhanced calcium cycling, in failing myocardium. Overall, it is clear that Gi/o signaling in the heart, and especially in compromised myocardium, should continue to be explored and not forgotten when molecular mechanisms of cardiac injury and potential repair or protection are discussed.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.