SignificanceThe sympathetic nervous system influences various immune cell functions, in particular via β2-adrenergic receptor (β2AR) signaling. Although immune cell recruitment is critical for cardiac repair following ischemia, the impact of β2AR on this process is unclear. We describe how immune cell-specific β2AR depletion ablates chemokine receptor 2 (CCR2) expression and leukocyte recruitment to the heart postischemia. Reciprocally, β2AR activation increases CCR2 expression and responsiveness in a β-arrestin–dependent manner, and expression of a β-arrestin–biased β2AR in β2AR-depleted immune cells restores CCR2 levels and leukocyte recruitment to the postischemic heart. These results highlight the potential utility of next-generation β-arrestin–biased β2AR ligands to selectively modulate leukocyte responsiveness, and suggest that β-blockers, used commonly in peri/postischemic patients, may impact leukocyte-mediated repair mechanisms.

Background: Immune cell–mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through β-adrenergic receptors (βARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. Methods: Wild-type (WT) mice were irradiated followed by isoform-specific βAR knockout (βARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. Results: β 2 ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. β 2 ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via β-arrestin–dependent β 2 AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to β 2 AR activity, and spleens from human tissue donors treated with β-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of β 2 AR in β 2 ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. Conclusions: Immune cell–expressed β 2 AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

Following myocardial infarction (MI), inflammatory responses are important for initiating and regulating cardiac remodeling and tissue repair. Previous work in our lab has shown the importance of β2‐adrenergic receptors (β2AR) in regulating inflammatory responses through decreasing CCR2 expression and increasing splenic retention of leukocyte populations, resulting in decreased leukocyte infiltration to the damaged heart. Using chimeric mice that lack β2AR expression only on cells of hematopoietic origin we demonstrated that lack of β2AR expression in BM resulted in 100% mortality from cardiac rupture within two weeks of receiving MI, in contrast to their WT counterparts that had ~20% death. Administration of β2AR agonists in vitro and in vivo increased CCR2 expression and BM migration while β2AR antagonists decreased CCR2 expression and increased splenic leukocyte retention in vivo . Use of pepducins as allosteric modulators of β2AR signaling demonstrated the importance of β‐arrestin‐mediated signaling in increasing CCR2 expression and responses, which was confirmed in β‐arrestin KO BM. Furthermore, use of mutant β2AR constructs that are capable of signaling only through Gαs or β‐arrestin dependent pathways confirmed the importance of β‐arrestin signaling in altering CCR2 levels. These results demonstrate the critical role of β2AR‐mediated β‐arrestin signaling in the regulation of CCR2 expression on hematopoietic cells and its importance in mounting an immune response to promote healing following acute cardiac injury. Support or Funding Information This work was supported by NIH grants HL105414 (to D.G.T.), HL091799 (to W.J.K.), and an AHA postdoctoral fellowship (to L.A.G.).

Vasopressin type 1A receptor (V1AR) expression is elevated in chronic human heart failure (HF) and contributes to cardiac dysfunction in animal models, in part via reduced β-adrenergic receptor (βAR) responsiveness. Although cardiac V1AR overexpression and V1AR stimulation are each sufficient to decrease βAR activity, it is unknown whether V1AR inhibition conversely augments βAR responsiveness. Further, although V1AR has been shown to contribute to chronic progression of HF, its impact on cardiac function following acute ischaemic injury has not been reported. Using V1AR knockout (V1AR KO) mice we assessed the impact of V1AR deletion on cardiac contractility at baseline and following ischaemic injury, βAR sensitivity and cardiomyocyte responsiveness to βAR stimulation. Strikingly, baseline cardiac contractility was enhanced in V1AR KO mice and they experienced a greater loss in contractile function than control mice following acute ischaemic injury, although the absolute levels of cardiac dysfunction and survival rates did not differ. Enhanced cardiac contractility in V1AR KO mice was associated with augmented β-blocker sensitivity, suggesting increased basal βAR activity, and indeed levels of left ventricular cAMP, as well as phospholamban (PLB) and cardiac troponin I (cTnI) phosphorylation were elevated compared with control mice. At the cellular level, myocytes isolated from V1AR KO mice demonstrated increased responsiveness to βAR stimulation consistent with the finding that acute pharmacological V1AR inhibition enhanced βAR-mediated contractility in control myocytes. Therefore, although V1AR deletion does not protect the heart from the rapid development of cardiac dysfunction following acute ischaemic injury, its effects on βAR activity suggest that acute V1AR inhibition could be utilized to promote myocyte contractile performance.

Inflammatory responses are important for cardiac remodeling and tissue repair after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune responses, in large part through the β2-adrenergic receptor (β2AR), however the influence of β2AR in regulating the inflammatory response following MI is unknown. Thus, to examine the contribution of β2AR on immune cells following MI, wild-type (WT) mice were irradiated and then received β2ARKO or WT control bone marrow (BM) transplants to create immune cell-specific knockout (KO) animals. Lack of β2AR expression in BM resulted in 100% mortality from cardiac rupture within two weeks of receiving MI, in contrast to their WT counterparts that had ∼20% death. Granulocyte populations were sequestered in the spleen of β2ARKO chimeric mice resulting in reductions in post-MI infiltration of monocyte/macrophage, neutrophil and mast cell populations into the heart. Additionally, alterations in chemokine receptor levels, particularly CCR2, on BM resulted in decreased cellular migration, and use of a CCR2 antagonist in vivo recapitulated the β2ARKO chimeric mouse phenotype following MI. Administration of β2AR agonists in vitro and in vivo increased CCR2 expression and BM migration while β2AR antagonists decreased CCR2 expression and increased splenic leukocyte retention in vivo . Use of pepducins as allosteric modulators of β2AR signaling demonstrated the importance of β-arrestin-mediated signaling in increasing CCR2 expression and responses. The impact of β2AR deletion on BM cell CCR2 expression and migration, splenic retention of leukocytes and reciprocal cardiac leukocyte infiltration following MI could be reversed via lentivirus-mediated β2AR rescue in the β2ARKO BM prior to transplantation. These results demonstrate the critical role of β2AR in the regulation of CCR2 expression on hematopoietic cells and its importance in mounting an immune response to promote healing following acute cardiac injury.

Label-free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β-adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label-free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). Catecholamine stimulation of the cells induced a negative DMR deflection resulting in a concentration-dependent pharmacological response that was competitively blocked by βAR blockade and non-competitively blocked by irreversible uncoupling of Gs proteins. Pharmacological profiling of subtype-selective βAR agonists and antagonists revealed a dominant role of β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. Additionally, βAR-mediated cAMP generation was assessed via a fluorescence biosensor, revealing similar kinetics between DMR responses and cAMP generation. As such, βAR-dependent DMR responses were enhanced via inhibition of cAMP degradation, as well as dynamin-mediated receptor internalization. Finally, we assessed G protein-independent βAR signaling through epidermal growth factor receptor (EGFR). While inhibition of EGFR reduced the DMR response to βAR stimulation, our results demonstrate that G protein-dependent signaling produces a majority of the biological response to βAR stimulation in NRCF. Altogether, measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein-dependent and -independent pathways.

β-Adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to relay pro-survival effects via unknown mechanisms. We hypothesized that acute βAR-mediated EGFR transactivation in the heart promotes differential subcellular activation of ERK1/2 and Akt, promoting cell 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 phosphorylation of ERK1/2 (P-ERK1/2) and Akt (P-Akt) in distinct cardiac subcellular fractions. Increased P-ERK1/2 and P-Akt were observed in cytosolic, plasma membrane and nuclear fractions following ISO stimulation. Whereas the P-ERK1/2 response was EGFR-sensitive in all fractions, the P-Akt response was EGFR-sensitive only in the plasma membrane and nucleus, results confirmed in primary rat neonatal cardiomyocytes (RNCM). βAR-mediated EGFR-transactivation also decreased apoptosis in serum-depleted RNCM, as measured via TUNEL as well 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 EGFR transactivation may regulate apoptotic gene transcription. An Apoptosis PCR Array identified tnfsf10 (TRAIL) to be altered by ISO in an EGFR-sensitive manner, results confirmed via RT-PCR and ELISA measurement of both membrane-bound and soluble cardiomyocyte TRAIL levels. βAR-mediated EGFR transactivation induces differential subcellular activation of ERK1/2 and Akt leading to increased cell survival through the modulation of caspase 3 activity and apoptotic gene expression in cardiomyocytes.

Chronic stimulation of β-adrenergic receptors (βAR) can promote survival signaling via transactivation of epidermal growth factor receptor (EGFR) but ultimately alters cardiac structure and contractility over time, in part via enhanced cytokine signaling. We hypothesized that chronic catecholamine signaling will have a temporal impact on cardiac transcript expression in vivo, in particular cytokines, and that EGFR transactivation plays a role in this process. C57BL/6 mice underwent infusion with vehicle or isoproterenol (Iso) ± gefitinib (Gef) for 1 or 2 wk. Cardiac contractility decreased following 2 wk of Iso treatment, while cardiac hypertrophy, fibrosis, and apoptosis were enhanced at both timepoints. Inclusion of Gef preserved contractility, blocked Iso-induced apoptosis, and prevented hypertrophy at the 2-wk timepoint, but caused fibrosis on its own. RNAseq analysis revealed hundreds of cardiac transcripts altered by Iso at each timepoint with subsequent RT-quantitative PCR validation confirming distinct temporal patterns of transcript regulation, including those involved in cardiac remodeling and survival signaling, as well as numerous cytokines. Although Gef infusion alone did not significantly alter cytokine expression, it abrogated the Iso-mediated changes in a majority of the βAR-sensitive cytokines, including CCL2 and TNF-α. Additionally, the impact of βAR-dependent EGFR transactivation on the acute regulation of cytokine transcript expression was assessed in isolated cardiomyocytes and in cardiac fibroblasts, where the majority of Iso-dependent, and EGFR-sensitive, changes in cytokines occurred. Overall, coincident with changes in cardiac structure and contractility, βAR stimulation dynamically alters cardiac transcript expression over time, including numerous cytokines that are regulated via EGFR-dependent signaling.

β-adrenergic receptor (βAR)-mediated transactivation of epidermal growth factor receptor (EGFR) has been shown to promote cardioprotection in a mouse model of heart failure and we recently showed that this mechanism leads to enhanced cell survival in part via regulation of apoptotic transcript expression in isolated primary rat neonatal cardiomyocytes. Thus, we hypothesized that this process could regulate cardiac transcript expression in vivo. To comprehensively assess cardiac transcript alterations in response to acute βAR-dependent EGFR transactivation, we performed whole transcriptome analysis of hearts from C57BL/6 mice given i.p. injections of the βAR agonist isoproterenol in the presence or absence of the EGFR antagonist gefitinib for 1 hour. Total cardiac RNA from each treatment group underwent transcriptome analysis, revealing a substantial number of transcripts regulated by each treatment. Gefitinib alone significantly altered the expression of 405 transcripts, while isoproterenol either alone or in conjunction with gefitinib significantly altered 493 and 698 distinct transcripts, respectively. Further statistical analysis was performed, confirming 473 transcripts whose regulation by isoproterenol were significantly altered by gefitinib (isoproterenol-induced up/downregulation antagonized/promoted by gefinitib), including several known to be involved in the regulation of numerous processes including cell death and survival. Thus, βAR-dependent regulation of cardiac transcript expression in vivo can be modulated by the EGFR antagonist gefitinib.

β-adrenergic receptors (βAR) are critical regulators of cardiac function normally and during (HF). The importance of βAR on cardiomyocyte contractility and survival is well defined however, following myocardial infarction (MI), inflammatory responses occur, which are critical for healing and scar formation. Catecholamines acting through βAR, particularly the β2AR subtype, are known to modulate immune responses, however, the influence of β2AR in regulating the inflammatory response following MI is unknown. To investigate the contribution of β2AR on immune cells following myocardial infarction (MI), wild-type (WT) mice were irradiated and then received β2ARKO or WT control BM transplants to create immune cell specific knockout (KO) animals. Following bone marrow reconstitution, mice were subjected to MI and cardiac function and survival were monitored. Cardiac function, as assessed by echocardiography, did not differ between WT and β2ARKO chimeric mice. However, mice lacking β2ARKO in their BM resulted in 100% mortality from cardiac rupture within two weeks of receiving MI in contrast to their WT counterparts that had ~20% death. Masson trichrome staining demonstrated infarct expansion in β2ARKO chimeric mice occurred more rapidly than their WT counterparts. Flow cytometric analysis showed decreased mobilization of granulocytes from bone marrow in β2ARKO mice. Additionally, β2ARKO chimeric mice reductions in infiltrating monocyte/macrophage, neutrophil and mast cell populations in the heart with no change in total cell infiltration suggesting a disruption in the ratio of infiltrating immune cells. Alterations in chemokine receptor levels, particularly CCR2, on BM resulted in decreased cellular migration. These results demonstrate the critical role of β2AR in mounting an immune response and promoting healing following MI.

Chronic catecholamine stimulation of β-adrenergic receptors (βAR) is ultimately deleterious during heart failure (HF). While alterations in cytokines contribute to HF pathogenesis and βAR have been demonstrated to regulate cytokines in different models of HF, a comprehensive understanding of this relationship is lacking. Thus, we sought to characterize the impact of chronic βAR signaling on cardiac cytokine expression in vivo. C57BL/6 mice underwent infusion with vehicle or isoproterenol (Iso; 3 mg/kg/day) via minipumps for 1 or 2 weeks and cardiac function was monitored via echocardiography. At study termination, hearts were excised and assessed for changes in hypertrophy, fibrosis and apoptosis, each of which were enhanced by Iso. Expression of cardiac transcripts were assessed via whole transcriptome analysis, where 780 and 689 transcripts were significantly altered at 1 and 2 weeks of Iso, respectively, with only 115 transcripts regulated similarly between the two cohorts. Significant changes in cytokine transcript expression was observed in response to chronic Iso and Ingenuity Pathway Analysis (IPA) predicted the involvement of additional upstream cytokine regulators potentially regulated by Iso. Transcriptome results and IPA predictions were confirmed via qRT-PCR. A cytokine array also confirmed temporally-distinct alterations in the expression of 42 cytokines at the protein level. Differential alterations in cytokine expression resulting from 1 versus 2 weeks of Iso infusion suggest that cytokine-directed therapies may have distinct temporally-dependent consequences on cardiac function and survival under conditions of chronic catecholamine stress.

β‐adrenergic receptors (βAR) are critical regulators of cardiac function normally and during heart failure (HF). While alterations in cytokines contribute to HF pathogenesis, and βAR have been demonstrated to regulate cytokines in different models of HF, a comprehensive understanding of this relationship is lacking. Thus, we sought to characterize the impact of chronic βAR signaling on cardiac cytokine expression in vivo. C57BL/6 mice underwent infusion with isoproterenol (Iso; 3 mg/kg/day) via minipumps for 1 or 2 weeks, versus vehicle. Cardiac function was monitored by echocardiography and hearts were excised at study termination for biochemistry and immunohistology, which showed Iso‐induced hypertrophy, fibrosis and apoptosis. Expression of cardiac transcripts were assessed via whole transcriptome analysis, where 780 and 689 transcripts were significantly altered at 1 and 2 weeks of Iso, respectively, with only 115 transcripts regulated similarly between the two cohorts. Protein expression of 53 cytokines were assessed, revealing significant alterations in 42, including distinct changes in expression at the 1 versus 2 week timepoints. Differential alteration in cytokine expression following 1 versus 2 weeks of chronic Iso stimulation suggests distinct consequences of chronic βAR signaling on cytokine‐mediated processes at different stages in the development of HF.