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Supplementary material for Immune Cell Beta2-Adrenergic Receptors Contributes to the Development of Heart Failure by Tanner et al.

The new horizon for cardiac therapy may lie beneath the surface, with the downstream mediators of G protein–coupled receptor (GPCR) activity. Targeted approaches have shown that receptor activation may be biased towardsignaling through G proteins or through GPCR kinases (GRKs) and b-arrestins, with divergent functional outcomes. Inaddition to these canonical roles, numerous noncanonical activities of GRKs and b-arrestins have been demonstratedto modulate GPCR signaling at all levels of receptor activation and regulation. Further, research continues to identifynovel GRK/effector and b-arrestin/effector complexes with distinct impacts on cardiac function in the normal heartand the diseased heart. Coupled with the identification of once orphan receptors and endogenous ligands withbeneficial cardiovascular effects, this expands the repertoire of GPCR targets. Together, this research highlights thepotential for focused therapeutic activation of beneficial pathways, with simultaneous exclusion or inhibition ofdetrimental signaling, and represents a new wave of therapeutic development.

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

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself inducesfurther cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotessurvival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte deathat the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthostericsmall molecules has prevented investigation into whether this pathway relays protection against ischemic injuryin vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed fromthe first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependentmanner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection againstischemia/reperfusion (I/R)-induced injury in vivo.Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice receivedintracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min)followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitorthe impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM)were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrialsuperoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and toinvestigate the associated mechanism(s).Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death andimproved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile functionearly and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxideproduction and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation couldbe detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability ofICL1-9 effects on βarr-dependent β2AR signaling.Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a noveltherapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodelingbenefits.

Objective—To test the hypothesis that loss of IL (interleukin)-19 exacerbates atherosclerosis.Approach and Results—Il19−/− mice were crossed into Ldlr−/− (low-density lipoprotein receptor knock out) mice. Doubleknockout (dKO) mice had increased plaque burden in aortic arch and root compared with Ldlr−/− controls after 14 weeksof high-fat diet (HFD). dKO mice injected with 10 ng/g per day rmIL-19 had significantly less plaque compared withcontrols. qRT-PCR and Western blot analysis revealed dKO mice had increased systemic and intraplaque polarization of Tcells and macrophages to proinflammatory Th1 and M1 phenotypes, and also significantly increased TNF (tumor necrosisfactor)-α expression in spleen and aortic arch compared with Ldlr−/− controls. Bone marrow transplantation suggeststhat immune cells participate in IL-19 protection. Bone marrow-derived macrophages and vascular smooth muscle cellsisolated from dKO mice had a significantly greater expression of inflammatory cytokine mRNA and protein comparedwith controls. Spleen and aortic arch from dKO mice had significantly increased expression of the mRNA stabilityprotein HuR (human antigen R). Bone marrow-derived macrophage and vascular smooth muscle cell isolated from dKOmice also had greater HuR abundance. HuR stabilizes proinflammatory transcripts by binding AU-rich elements in the3′ untranslated region. Cytokine and HuR mRNA stability were increased in dKO bone marrow-derived macrophageand vascular smooth muscle cell, which was rescued by addition of IL-19 to these cells. IL-19–induced expression ofmiR133a, which targets and reduced HuR abundance; miR133a levels were lower in dKO mice compared with controls.Conclusions—These data indicate that IL-19 is an atheroprotective cytokine which decreases the abundance of HuR,leading to reduced inflammatory mRNA stability.

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.