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β-Adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has beenshown to relay pro-survival effects via unknown mechanisms. We hypothesized that acute βAR-mediatedEGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promotingcell survival through modulation of apoptosis. C57BL/6 mice underwent acute i.p. injection with isoproterenol(ISO)±AG 1478 (EGFR antagonist) to assess the impact of βAR-mediated EGFR transactivation on the phosphorylationof ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 andP-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereasthe P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in theplasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). βARmediatedEGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL aswell as caspase 3 activity/cleavage, which were sensitive to the inhibition of either ERK1/2 (PD184352) or Akt(LY-294002) signaling. Caspase 3 activity/cleavage was also sensitive to the inhibition of transcription, which,with an increase in nuclear P-ERK1/2 and P-Akt in response to ISO, suggested that βAR-mediated EGFRtransactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) tobe altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of bothmembrane-bound and soluble cardiomyocyte TRAIL levels. βAR-mediated EGFR transactivation induces differentialsubcellular activation of ERK1/2 and Akt leading to increased cell survival through themodulation of caspase3 activity and apoptotic gene expression in cardiomyocytes.

Label-free systems for the agnostic assessment of cellular responses to receptorstimulation have been shown to provide a sensitive method to dissect receptorsignaling.b-adenergic receptors (bAR) are important regulators of normal andpathologic cardiac function and are expressed in cardiomyocytes as well as car-diac fibroblasts, where relatively fewer studies have explored their signalingresponses. Using label-free whole cell dynamic mass redistribution (DMR)assays we investigated the response patterns to stimulation of endogenousbARin primary neonatal rat cardiac fibroblasts (NRCF). The EPIC-BT by Corningwas used to measure DMR responses in primary isolated NRCF treated withvariousbAR and EGFR ligands. Additional molecular assays for cAMP genera-tion and receptor internalization responses were used to correlate the DMRfindings with establishedbAR signaling pathways. Catecholamine stimulation ofNRCF induced a concentration-dependent negative DMR deflection that wascompetitively blocked bybAR blockade and non-competitively blocked by irre-versible uncoupling of Gs proteins. Subtype-selectivebAR ligand profilingrevealed a dominant role forb2AR in mediating the DMR responses, consistentwith the relative expression levels ofb2AR andb1AR in NRCF.bAR-mediatedcAMP generation profiles revealed similar kinetics to DMR responses, each ofwhich were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, G protein-independentbAR signalingthrough epidermal growth factor receptor (EGFR) was assessed, revealing asmaller but significant contribution of this pathway to the DMR response tobAR stimulation. Measurement of DMR responses in primary cardiac fibro-blasts provides a sensitive readout for investigating endogenousbAR signalingvia both G protein-dependent and–independent pathways

Following cardiac injury, early immune cell responses are essentialfor initiating cardiac remodeling and tissue repair. We previouslydemonstrated the importance of β2-adrenergic receptors (β2ARs)in the regulation of immune cell localization following acute cardiacinjury, with deficient leukocyte infiltration into the damagedheart. The purpose of this study was to investigate the mechanismby which immune cell-expressed β2ARs regulate leukocyte recruitmentto the heart following acute cardiac injury. Chemokine receptor2 (CCR2) expression and responsiveness to C-C motif chemokineligand 2 (CCL2)-mediated migration were abolished in β2AR knockout(KO) bone marrow (BM), both of which were rescued by β2ARreexpression. Chimeric mice lacking immune cell-specific CCR2 expression,as well as wild-type mice administered a CCR2 antagonist,recapitulated the loss of monocyte/macrophage and neutrophil recruitmentto the heart following myocardial infarction (MI) observedin mice with immune cell-specific β2AR deletion. Converseto β2AR ablation, β2AR stimulation increased CCR2 expression andmigratory responsiveness to CCL2 in BM. Mechanistically, G proteindependentβ2AR signaling was dispensable for these effects,whereas β-arrestin2–biased β2AR signaling was required for theregulation of CCR2 expression. Additionally, activator protein 1(AP-1) was shown to be essential in mediating CCR2 expressionin response to β2AR stimulation in both murine BM and humanmonocytes. Finally, reconstitution of β2ARKO BM with rescued expressionof a β-arrestin–biased β2AR in vivo restored BM CCR2 expressionas well as cardiac leukocyte infiltration following MI. Theseresults demonstrate the critical role of β-arrestin2/AP-1–dependentβ2AR signaling in the regulation of CCR2 expression and recruitmentof leukocytes to the heart following injury.

Chronic stimulationof -adrenergic receptors ( AR) can promote survival signalingvia transactivation of epidermal growth factor receptor (EGFR)but ultimately alters cardiac structure and contractility over time, inpart via enhanced cytokine signaling. We hypothesized that chroniccatecholamine signaling will have a temporal impact on cardiactranscript expression in vivo, in particular cytokines, and that EGFRtransactivation plays a role in this process. C57BL/6 mice underwentinfusion with vehicle or isoproterenol (Iso) gefitinib (Gef) for 1 or2 wk. Cardiac contractility decreased following 2 wk of Iso treatment,while cardiac hypertrophy, fibrosis, and apoptosis were enhanced atboth timepoints. Inclusion of Gef preserved contractility, blockedIso-induced apoptosis, and prevented hypertrophy at the 2-wk timepoint,but caused fibrosis on its own. RNAseq analysis revealedhundreds of cardiac transcripts altered by Iso at each timepoint withsubsequent RT-quantitative PCR validation confirming distinct temporalpatterns of transcript regulation, including those involved incardiac remodeling and survival signaling, as well as numerouscytokines. Although Gef infusion alone did not significantly altercytokine expression, it abrogated the Iso-mediated changes in amajority of the AR-sensitive cytokines, including CCL2 and TNF- .Additionally, the impact of AR-dependent EGFR transactivation onthe acute regulation of cytokine transcript expression was assessed inisolated cardiomyocytes and in cardiac fibroblasts, where the majorityof Iso-dependent, and EGFR-sensitive, changes in cytokines occurred.Overall, coincident with changes in cardiac structure and contractility, AR stimulation dynamically alters cardiac transcript expression overtime, including numerous cytokines that are regulated via EGFRdependentsignaling.