Targeting Gαi2 in neutrophils protects from myocardial ischemia reperfusion injury.

Neutrophils are not only involved in immune defense against infection but also contribute to the exacerbation of tissue damage after ischemia and reperfusion. We have previously shown that genetic ablation of regulatory Gαi proteins in mice has both protective and deleterious effects on myocardial ischemia reperfusion injury (mIRI), depending on which isoform is deleted. To deepen and analyze these findings in more detail the contribution of Gαi2 proteins in resident cardiac vs circulating blood cells for mIRI was first studied in bone marrow chimeras. In fact, the absence of Gαi2 in all blood cells reduced the extent of mIRI (22,9% infarct size of area at risk (AAR) Gnai2-/- → wt vs 44.0% wt → wt; p < 0.001) whereas the absence of Gαi2 in non-hematopoietic cells increased the infarct damage (66.5% wt → Gnai2-/- vs 44.0% wt → wt; p < 0.001). Previously we have reported the impact of platelet Gαi2 for mIRI. Here, we show that infarct size was substantially reduced when Gαi2 signaling was either genetically ablated in neutrophils/macrophages using LysM-driven Cre recombinase (AAR: 17.9% Gnai2fl/fl LysM-Cre+/tg vs 42.0% Gnai2fl/fl; p < 0.01) or selectively blocked with specific antibodies directed against Gαi2 (AAR: 19.0% (anti-Gαi2) vs 49.0% (IgG); p < 0.001). In addition, the number of platelet-neutrophil complexes (PNCs) in the infarcted area were reduced in both, genetically modified (PNCs: 18 (Gnai2fl/fl; LysM-Cre+/tg) vs 31 (Gnai2fl/fl); p < 0.001) and in anti-Gαi2 antibody-treated (PNCs: 9 (anti-Gαi2) vs 33 (IgG); p < 0.001) mice. Of note, significant infarct-limiting effects were achieved with a single anti-Gαi2 antibody challenge immediately prior to vessel reperfusion without affecting bleeding time, heart rate or cellular distribution of neutrophils. Finally, anti-Gαi2 antibody treatment also inhibited transendothelial migration of human neutrophils (25,885 (IgG) vs 13,225 (anti-Gαi2) neutrophils; p < 0.001), collectively suggesting that a therapeutic concept of functional Gαi2 inhibition during thrombolysis and reperfusion in patients with myocardial infarction should be further considered.

Non-canonical G protein signaling.

The original paradigm of classical - also referred to as canonical - cellular signal transduction of heterotrimeric G proteins (G protein) is defined by a hierarchical, orthograde interaction of three players: the agonist-activated G protein-coupled receptor (GPCR), which activates the transducing G protein, that in turn regulates its intracellular effectors. This receptor-transducer-effector concept was extended by the identification of regulators and adapters such as the regulators of G protein signaling (RGS), receptor kinases like ßARK, or GPCR-interacting arrestin adapters that are integrated into this canonical signaling process at different levels to enable fine-tuning. Finally, the identification of atypical signaling mechanisms of classical regulators, together with the discovery of novel modulators, added a new and fascinating dimension to the cellular G protein signal transduction. This heterogeneous group of accessory G protein modulators was coined "activators of G protein signaling" (AGS) proteins and plays distinct roles in canonical and non-canonical G protein signaling pathways. AGS proteins contribute to the control of essential cellular functions such as cell development and division, intracellular transport processes, secretion, autophagy or cell movements. As such, they are involved in numerous biological processes that are crucial for diseases, like diabetes mellitus, cancer, and stroke, which represent major health burdens. Although the identification of a large number of non-canonical G protein signaling pathways has broadened the spectrum of this cellular communication system, their underlying mechanisms, functions, and biological effects are poorly understood. In this review, we highlight and discuss atypical G protein-dependent signaling mechanisms with a focus on inhibitory G proteins (Gi) involved in canonical and non-canonical signal transduction, review recent developments and open questions, address the potential of new approaches for targeted pharmacological interventions.

Cerebrovascular Gi Proteins Protect Against Brain Hypoperfusion and Collateral Failure in Cerebral Ischemia.

Cerebral hypoperfusion and vascular dysfunction are closely related to common risk factors for ischemic stroke such as hypertension, dyslipidemia, diabetes, and smoking. The role of inhibitory G protein-dependent receptor (GiPCR) signaling in regulating cerebrovascular functions remains largely elusive. We examined the importance of GiPCR signaling in cerebral blood flow (CBF) and its stability after sudden interruption using various in vivo high-resolution magnetic resonance imaging techniques. To this end, we induced a functional knockout of GiPCR signaling in the brain vasculature by injection of pertussis toxin (PTX). Our results show that PTX induced global brain hypoperfusion and microvascular collapse. When PTX-pretreated animals underwent transient unilateral occlusion of one common carotid artery, CBF was disrupted in the ipsilateral hemisphere resulting in the collapse of the cortically penetrating microvessels. In addition, pronounced stroke features in the affected brain regions appeared in both MRI and histological examination. Our findings suggest an impact of cerebrovascular GiPCR signaling in the maintenance of CBF, which may be useful for novel pharmacotherapeutic approaches to prevent and treat cerebrovascular dysfunction and stroke.

Platelets and the Cybernetic Regulation of Ischemic Inflammatory Responses through PNC Formation Regulated by Extracellular Nucleotide Metabolism and Signaling.

Ischemic events are associated with severe inflammation and are here referred to as ischemic inflammatory response (IIR). Recent studies identified the formation of platelet-neutrophil complexes (PNC) as key players in IIR. We investigated the role of extracellular platelet nucleotide signaling in the context of IIR and defined a cybernetic circle, including description of feedback loops. Cybernetic circles seek to integrate different levels of information to understand how biological systems function. Our study specifies the components of the cybernetic system of platelets in IIR and describes the theoretical progression of IIR passing the cybernetic cycle with positive and negative feedback loops based on nucleotide-dependent signaling and functional regulation. The cybernetic components and feedback loops were explored by cytometry, immunohistological staining, functional blocking antibodies, and ADP/ATP measurements. Using several ex vivo and in vivo approaches we confirmed cybernetic parameters, such as controller, sensor, and effector (VASP phosphorylation, P2Y12, ADORAs and GPIIb/IIIa activity), as well as set points (ADP, adenosine) and interfering control and disturbance variables (ischemia). We demonstrate the impact of the regulated platelet-neutrophil complex (PNC) formation in blood and the resulting damage to the affected inflamed tissue. Taken together, extracellular nucleotide signaling, PNC formation, and tissue damage in IIR can be integrated in a controlled cybernetic circle of platelet function, as introduced through this study.

Heterotrimeric G-protein subunit Gαi2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

Gαi2 , a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of αIIb ß3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gαi2 and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 µg/mL or 5 µg/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and αIIb ß3-integrin activation. These molecular alterations were significantly less pronounced in Gαi2 -deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gαi2 signaling in governing platelet activation and apoptosis.

Lack of Gαi2 leads to dilative cardiomyopathy and increased mortality in ß1-adrenoceptor overexpressing mice.

AIMS: Inhibitory G (Gi) proteins have been proposed to be cardioprotective. We investigated effects of Gαi2 knockout on cardiac function and survival in a murine heart failure model of cardiac ß1-adrenoceptor overexpression. METHODS AND RESULTS: ß1-transgenic mice lacking Gαi2 (ß1-tg/Gαi2 (-/-)) were compared with wild-type mice and littermates either overexpressing cardiac ß1-adrenoceptors (ß1-tg) or lacking Gαi2 (Gαi2 (-/-)). At 300 days, mortality of mice only lacking Gαi2 was already higher compared with wild-type or ß1-tg, but similar to ß1-tg/Gαi2 (-/-), mice. Beyond 300 days, mortality of ß1-tg/Gαi2 (-/-) mice was enhanced compared with all other genotypes (mean survival time: 363 ± 21 days). At 300 days of age, echocardiography revealed similar cardiac function of wild-type, ß1-tg, and Gαi2 (-/-) mice, but significant impairment for ß1-tg/Gαi2 (-/-) mice (e.g. ejection fraction 14 ± 2 vs. 40 ± 4% in wild-type mice). Significantly increased ventricle-to-body weight ratio (0.71 ± 0.06 vs. 0.48 ± 0.02% in wild-type mice), left ventricular size (length 0.82 ± 0.04 vs. 0.66 ± 0.03 cm in wild types), and atrial natriuretic peptide and brain natriuretic peptide expression (mRNA: 2819 and 495% of wild-type mice, respectively) indicated hypertrophy. Gαi3 was significantly up-regulated in Gαi2 knockout mice (protein compared with wild type: 340 ± 90% in Gαi2 (-/-) and 394 ± 80% in ß1-tg/Gαi2 (-/-), respectively). CONCLUSIONS: Gαi2 deficiency combined with cardiac ß1-adrenoceptor overexpression strongly impaired survival and cardiac function. At 300 days of age, ß1-adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction while there was overt cardiomyopathy in mice additionally lacking Gαi2. We propose an enhanced effect of increased ß1-adrenergic drive by the lack of protection via Gαi2. Gαi3 up-regulation was not sufficient to compensate for Gαi2 deficiency, suggesting an isoform-specific or a concentration-dependent mechanism.