β2-Adrenergic Receptor Signaling in the Cardiac Myocyte is Modulated by Interactions With CXCR4

Department of Medicine, Division of Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY 10029, USA.
Journal of cardiovascular pharmacology (Impact Factor: 2.14). 11/2010; 56(5):548-59. DOI: 10.1097/FJC.0b013e3181f713fe
Source: PubMed


Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective β-adrenergic receptor (βAR) agonist, isoproterenol. As a result of the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR-dependent. We demonstrated a physical interaction between CXCR4 and β2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-β2AR complex could represent a hitherto unknown target for therapeutic intervention.

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    • "In addition, we also examined possible heterodimerization between CXCR4 and a non-chemokine GPCR, β2 adrenergic receptor (β2AR). Although not widely studied, activated CXCR4 has been shown to physically interact with β2AR and regulate its downstream signaling pathway in rat cardiac myocytes [16]. This interaction has been shown previously using co-immunoprecipitation, confocal microscopy, and BRET and we have confirmed this interaction using our proximity biotinylation assay (Figure S4). "
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    ABSTRACT: Dimerization of G protein-coupled receptors (GPCRs) represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET)-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP) in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "These results are at variance with the negative inotropic effect of CXCR4 signaling demonstrated on rat papillary muscle and isolated cardiomyocytes [19]. In addition, a recent report showed that SDF-1α/CXCR4 signaling decreases β-adrenergic receptor-induced protein kinase A activity as assessed by cAMP accumulation and phosphorylation of phospholamban [20]. Both studies were performed on isolated adult rat cardiomyocytes in which excitation-contraction coupling is ryanodine rather than IP3-gated [9] while, in our model, gene expression was higher for IP3Rs than for RyRs and the two pathways contributed to mobilization of intracellular calcium. "
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    ABSTRACT: Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency. Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP3R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP3R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax. These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP3-gated fashion leading to a positive chronotropic and inotropic effect.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "These results describe that the cardioprotection elicited by CXCR4 may employ multiple mechanisms including regulation of LTCC function, calcium homeostasis and VEGF regulation. Previously we reported that CXCR4 prevents isoproterenol induced diastolic Ca 2+ overload and influences downstream β-AR signaling in isolated cardiac myocytes [5]. Here we have shown that CXCR4 may Table 1 Echocardiography: two-dimensional images and M-mode tracings were recorded on the short-axis at the level of the papillary muscle to determine percent fractional shortening and ventricular dimension. "
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    ABSTRACT: Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adrenoceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest that AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure.
    Full-text · Article · Jun 2012 · Journal of Molecular and Cellular Cardiology
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