Christopher J. Traynham

β-Arrestin1–Biased β1-Adrenergic Receptor Signaling Regulates MicroRNA Processing

Rationale: MicroRNAs (miRs) are small, noncoding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes after activation by a variety of signals such as those stimulated by &bgr;-adrenergic receptors (&bgr;ARs). Initially discovered to desensitize &bgr;AR signaling, &bgr;-arrestins are now appreciated to transduce multiple effector pathways independent of G-protein–mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the &bgr;-arrestin–biased &bgr;AR agonist, carvedilol, activates cellular pathways in the heart. Objective: Here, we tested whether carvedilol could activate &bgr;-arrestin–mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects. Methods and Results: In human cells and mouse hearts, carvedilol upregulates a subset of mature and pre-miRs, but not their pri-miRs, in &bgr;1AR-, G-protein–coupled receptor kinase 5/6–, and &bgr;-arrestin1–dependent manner. Mechanistically, &bgr;-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs. Conclusions: Our findings indicate a novel function for &bgr;1AR-mediated &bgr;-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.

β-arrestin–biased signaling through the β2-adrenergic receptor promotes cardiomyocyte contraction

Significance Commonly prescribed drugs for congestive heart failure (CHF) include β-adrenergic receptor antagonists or β-blockers. These drugs operate by inhibiting deleterious apoptotic signaling and normalizing inotropic signaling from these receptors. As the β-adrenergic receptor (β1AR) (dominant subtype in the heart) is systematically down-regulated during CHF while Gi (a G protein that antagonizes contractile signaling) is up-regulated, the ability to selectively control β2AR signaling becomes an attractive therapeutic approach. It is proposed that biasing receptor interaction with β-arrestins (promoting antiapoptotic signaling and possibly contraction) over G proteins may be therapeutically advantageous for the treatment of CHF. Here, we report a β-arrestin–biased pepducin of the β2AR that is able to induce cardiomyocyte contractility and antiapoptotic signaling to provide a pharmacological template for next-generation cardiovascular pharmaceuticals. β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent β1AR and Gi-dependent β2AR pathways leads to enhanced cardiomyocyte death, reduced β1AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein–dependent signaling, β-arrestin–dependent signaling promotes cardiomyocyte survival. Given that β2AR expression is unaltered in CHF, a β-arrestin–biased agonist that operates through the β2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin–biased agonist that can inhibit basal signaling from βARs and also stimulate cell survival signaling pathways. To understand the relative contribution of β-arrestin bias to the efficacy of select β-blockers, a specific β-arrestin–biased pepducin for the β2AR, intracellular loop (ICL)1–9, was used to decouple β-arrestin–biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1–9 was able to promote β2AR phosphorylation, β-arrestin recruitment, β2AR internalization, and β-arrestin–biased signaling. Interestingly, ICL1–9 was also able to induce β2AR- and β-arrestin–dependent and Ca2+-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1–9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the β2AR and serves as a model for the next generation of cardiovascular drug development.

Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels.

Leukocyte-Expressed β2-Adrenergic Receptors are Essential for Survival Following Acute Myocardial Injury

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 &bgr;-adrenergic receptors (&bgr;ARs); 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 &bgr;AR knockout (&bgr;ARKO) 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: &bgr;2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. &bgr;2ARKO 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 &bgr;-arrestin–dependent &bgr;2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to &bgr;2AR activity, and spleens from human tissue donors treated with &bgr;-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 &bgr;2AR in &bgr;2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. Conclusions: Immune cell–expressed &bgr;2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

Differential Role of G Protein–Coupled Receptor Kinase 5 in Physiological Versus Pathological Cardiac Hypertrophy

RATIONALE G protein-coupled receptor kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is upregulated in heart failure. Although GRK5 is a critical regulator of cardiac G protein-coupled receptor signaling, recent data has uncovered noncanonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early heart failure after transverse aortic constriction (TAC) because of GRK5 nuclear accumulation. OBJECTIVE In this study, we investigated the role of GRK5 in physiological, swimming-induced hypertrophy (SIH). METHODS AND RESULTS Cardiac-specific GRK5 transgenic mice and nontransgenic littermate control mice were subjected to a 21-day high-intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed, including nuclear localization of GRK5, and compared with TAC. Unlike after TAC, swim-trained transgenic GRK5 and nontransgenic littermate control mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC transgenic GRK5 mice was able to preserve cardiac function. CONCLUSIONS These data suggest that although nuclear-localized GRK5 is a pathological mediator after stress, this noncanonical nuclear activity of GRK5 is not induced during physiological hypertrophy.

β2-Adrenergic receptor-dependent chemokine receptor 2 expression regulates leukocyte recruitment to the heart following acute injury

Significance The 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. Following cardiac injury, early immune cell responses are essential for initiating cardiac remodeling and tissue repair. We previously demonstrated the importance of β2-adrenergic receptors (β2ARs) in the regulation of immune cell localization following acute cardiac injury, with deficient leukocyte infiltration into the damaged heart. The purpose of this study was to investigate the mechanism by which immune cell-expressed β2ARs regulate leukocyte recruitment to the heart following acute cardiac injury. Chemokine receptor 2 (CCR2) expression and responsiveness to C-C motif chemokine ligand 2 (CCL2)-mediated migration were abolished in β2AR knockout (KO) bone marrow (BM), both of which were rescued by β2AR reexpression. 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 recruitment to the heart following myocardial infarction (MI) observed in mice with immune cell-specific β2AR deletion. Converse to β2AR ablation, β2AR stimulation increased CCR2 expression and migratory responsiveness to CCL2 in BM. Mechanistically, G protein-dependent β2AR signaling was dispensable for these effects, whereas β-arrestin2–biased β2AR signaling was required for the regulation of CCR2 expression. Additionally, activator protein 1 (AP-1) was shown to be essential in mediating CCR2 expression in response to β2AR stimulation in both murine BM and human monocytes. Finally, reconstitution of β2ARKO BM with rescued expression of a β-arrestin–biased β2AR in vivo restored BM CCR2 expression as well as cardiac leukocyte infiltration following MI. These results demonstrate the critical role of β-arrestin2/AP-1–dependent β2AR signaling in the regulation of CCR2 expression and recruitment of leukocytes to the heart following injury.