The ß-arrestin-biased ß-adrenergic receptor blocker carvedilol enhances skeletal muscle contractility.

A decrease in skeletal muscle strength and functional exercise capacity due to aging, frailty, and muscle wasting poses major unmet clinical needs. These conditions are associated with numerous adverse clinical outcomes including falls, fractures, and increased hospitalization. Clenbuterol, a ß2-adrenergic receptor (ß2AR) agonist enhances skeletal muscle strength and hypertrophy; however, its clinical utility is limited by side effects such as cardiac arrhythmias mediated by G protein signaling. We recently reported that clenbuterol-induced increases in contractility and skeletal muscle hypertrophy were lost in ß-arrestin 1 knockout mice, implying that arrestins, multifunctional adapter and signaling proteins, play a vital role in mediating the skeletal muscle effects of ß2AR agonists. Carvedilol, classically defined as a ßAR antagonist, is widely used for the treatment of chronic systolic heart failure and hypertension, and has been demonstrated to function as a ß-arrestin-biased ligand for the ß2AR, stimulating ß-arrestin-dependent but not G protein-dependent signaling. In this study, we investigated whether treatment with carvedilol could enhance skeletal muscle strength via ß-arrestin-dependent pathways. In a murine model, we demonstrate chronic treatment with carvedilol, but not other ß-blockers, indeed enhances contractile force in skeletal muscle and this is mediated by ß-arrestin 1. Interestingly, carvedilol enhanced skeletal muscle contractility despite a lack of effect on skeletal muscle hypertrophy. Our findings suggest a potential unique clinical role of carvedilol to stimulate skeletal muscle contractility while avoiding the adverse effects with ßAR agonists. This distinctive signaling profile could present an innovative approach to treating sarcopenia, frailty, and secondary muscle wasting.

ß-arrestin 1 regulates ß2-adrenergic receptor-mediated skeletal muscle hypertrophy and contractility.

BACKGROUND: ß2-adrenergic receptors (ß2ARs) are the target of catecholamines and play fundamental roles in cardiovascular, pulmonary, and skeletal muscle physiology. An important action of ß2AR stimulation on skeletal muscle is anabolic growth, which has led to the use of agonists such as clenbuterol by athletes to enhance muscle performance. While previous work has demonstrated that ß2ARs can engage distinct signaling and functional cascades mediated by either G proteins or the multifunctional adaptor protein, ß-arrestin, the precise role of ß-arrestin in skeletal muscle physiology is not known. Here, we tested the hypothesis that agonist activation of the ß2AR by clenbuterol would engage ß-arrestin as a key transducer of anabolic skeletal muscle growth. METHODS: The contractile force of isolated extensor digitorum longus muscle (EDL) and calcium signaling in isolated flexor digitorum brevis (FDB) fibers were examined from the wild-type (WT) and ß-arrestin 1 knockout mice (ßarr1KO) followed by chronic administration of clenbuterol (1 mg/kg/d). Hypertrophic responses including fiber composition and fiber size were examined by immunohistochemical imaging. We performed a targeted phosphoproteomic analysis on clenbuterol stimulated primary cultured myoblasts from WT and ßarr1KO mice. Statistical significance was determined by using a two-way analysis with Sidak's or Tukey's multiple comparison test and the Student's t test. RESULTS: Chronic administration of clenbuterol to WT mice enhanced the contractile force of EDL muscle and calcium signaling in isolated FDB fibers. In contrast, when administered to ßarr1KO mice, the effect of clenbuterol on contractile force and calcium influx was blunted. While clenbuterol-induced hypertrophic responses were observed in WT mice, this response was abrogated in mice lacking ß-arrestin 1. In primary cultured myoblasts, clenbuterol-stimulated phosphorylation of multiple pro-hypertrophy proteins required the presence of ß-arrestin 1. CONCLUSIONS: We have identified a previously unappreciated role for ß-arrestin 1 in mediating ß2AR-stimulated skeletal muscle growth and strength. We propose these findings could have important implications in the design of future pharmacologic agents aimed at reversing pathological conditions associated with skeletal muscle wasting.

ß-Arrestin2 mediates progression of murine primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. ß-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, ß-arrestin2 (ßarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between ßarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor ßarr2-knockout (ßarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted ßarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of ßarr2-/- cells. In order to assess the effect of acute loss of ßarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional ßarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic ßarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that ßarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

Conformationally selective RNA aptamers allosterically modulate the ß2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the ß2-adrenoceptor (ß2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

The role of ß-arrestin2-dependent signaling in thoracic aortic aneurysm formation in a murine model of Marfan syndrome.

Ang II type 1a receptor (AT1aR)-mediated activation of MAPKs contributes to thoracic aortic aneurysm (TAA) development in Marfan syndrome (MFS). ß-Arrestin2 (ßarr2) is known to mediate AT1aR-dependent MAPK activation, as well as proproliferative and profibrotic signaling in aortic vascular smooth muscle cells. Therefore, we investigated whether ßarr2-dependent signaling contributes to TAA formation in MFS. We used a murine model of MFS [fibrillin (Fbn)(C1039G/+)] to generate an MFS murine model in combination with genetic ßarr2 deletion (Fbn(C1039G/+)/ßarr2(-/-)). Fbn(C1039G/+)/ßarr2(-/-) mice displayed delayed aortic root dilation compared with Fbn(C1039G/+) mice. The mRNA and protein expression of several mediators of TAA formation, including matrix metalloproteinase (MMP)-2 and -9, was reduced in the aorta of Fbn(C1039G/+)/ßarr2(-/-) mice relative to Fbn(C1039G/+) mice. Activation of ERK1/2 was also decreased in the aortas of Fbn(C1039G/+)/ßarr2(-/-) mice compared with Fbn(C1039G/+) animals. Small interfering RNA targeting ßarr2 inhibited angiotensin-stimulated expression of proaneurysmal signaling mediators in primary aortic root smooth muscle cells. Angiotensin-stimulated expression of the proaneurysmal signaling mediators MMP-2 and -9 was inhibited by blockade of ERK1/2 or the EGF receptor, whereas blockade of the transforming growth factor-ß receptor had no effect. These results suggest that ßarr2 contributes to TAA formation in MFS by regulating ERK1/2-dependent expression of proaneurysmal genes and proteins downstream of the AT1aR. Importantly, this demonstration of the unique signaling mechanism by which ßarr2 contributes to aneurysm formation identifies multiple novel, potential therapeutic targets in MFS.

Antithrombotic therapy: new areas to understand efficacy and bleeding.

INTRODUCTION: Therapeutic options for antithrombotic therapy are limited due to associated adverse bleeding events. Traditionally, the antithrombotic effects of these agents have been closely linked with concomitant risks in bleeding complications. AREAS COVERED: This article will review recent developments in the understanding of the mechanisms underlying physiological hemostasis and pathological thrombosis as well as key findings that may serve to 'uncouple' these two processes. In addition, we highlight the recent work identifying novel therapeutic agents targeting these novel mechanisms of pathological thrombus formation. EXPERT OPINION: Recent research has identified several novel mediators of thrombus formation, including cell-free nucleic acids, histones, histone-DNA complexes and neutrophil extracellular traps that may serve to link inflammation and thrombosis as well as separate physiological hemostasis from pathological thrombosis. Researchers are developing ligands to target these mediators with an achievable goal to identify novel therapies that inhibit thrombus formation without increased bleeding risk.

Recent developments in biased agonism.

The classic paradigm of G protein-coupled receptor (GPCR) activation was based on the understanding that agonist binding to a receptor induces or stabilizes a conformational change to an 'active' conformation. In the past decade, however, it has been appreciated that ligands can induce distinct 'active' receptor conformations with unique downstream functional signaling profiles. Building on the initial recognition of the existence of such 'biased ligands', recent years have witnessed significant developments in several areas of GPCR biology. These include increased understanding of structural and biophysical mechanisms underlying biased agonism, improvements in characterization and quantification of ligand efficacy, as well as clinical development of these novel ligands. Here we review recent major developments in these areas over the past several years.

Emerging paradigms in arterial thrombosis.

A traditional perspective of arterial thrombosis begins with vessel wall injury and exposure of subendothelial proteins, including collagen and tissue factor, to circulating cellular and non-cellular components. Adhesion and activation of platelets, mediated by their interaction with von Willebrand protein and collagen, respectively, coupled with tissue factor-mediated activation of coagulation proteins, results in thrombin generation and fibrin formation. While this time-honored paradigm remains firm and soundly based, emerging evidence suggests that arterial thrombosis is much more complex and dynamic than originally believed. Several novel triggers, templates and facilitators, such as cell-free nucleic acids, histones, DNA-histone complexes, polyphosphates, and microvesicles have recently been identified and require inclusion in the expanding universe of thrombosis as a dominant phenotype of human disease. Because these mediators appear to have modest if any effect on physiologic hemostasis, they likely represent acquired and disease or condition-dependent processes that are highly attractive targets for pharmacologic intervention.

MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound ß(2)-adrenergic receptors.

Lysosomal degradation of ubiquitinated ß(2)-adrenergic receptors (ß(2)ARs) serves as a major mechanism of long-term desensitization in response to prolonged agonist stimulation. Surprisingly, the ßAR antagonist carvedilol also induced ubiquitination and lysosomal trafficking of both endogenously expressed ß(2)ARs in vascular smooth muscle cells (VSMCs) and overexpressed Flag-ß(2)ARs in HEK-293 cells. Carvedilol prevented ß(2)AR recycling, blocked recruitment of Nedd4 E3 ligase, and promoted the dissociation of the deubiquitinases USP20 and USP33. Using proteomics approaches (liquid chromatography-tandem mass spectrometry), we identified that the E3 ligase MARCH2 interacted with carvedilol-bound ß(2)AR. The association of MARCH2 with internalized ß(2)ARs was stabilized by carvedilol and did not involve ß-arrestin. Small interfering RNA-mediated down-regulation of MARCH2 ablated carvedilol-induced ubiquitination, endocytosis, and degradation of endogenous ß(2)ARs in VSMCs. These findings strongly suggest that specific ligands recruit distinct E3 ligase machineries to activated cell surface receptors and direct their intracellular itinerary. In response to ß blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface ß(2)AR expression and, consequently, its signaling.

A guidance pathway for the selection of novel anticoagulants in the treatment of atrial fibrillation.

Oral anticoagulation with vitamin K antagonists has served as the primary treatment for the prevention of stroke and systemic embolization in patients with atrial fibrillation (AF) for decades. Over the past several years, multiple novel oral anticoagulants targeting key mediators of coagulation, including thrombin and factor Xa, have been developed. Specifically, agents targeting thrombin (dabigatran) and factor Xa (apixaban and rivaroxaban) have either reached late stages of clinical development (apixaban) or have received approval (dabigatran, rivaroxaban) by the US Food and Drug Administration for use in patients with nonvalvular AF. The promising results derived from large-scale clinical trials with these agents compared to warfarin expand the available therapeutic options for the prevention of stroke and systemic embolization in this rapidly increasing patient population. Here we present a general guidance pathway for the initiation and selection of oral anticoagulants in patients with AF.

Oral factor Xa inhibitors for the long-term management of ACS.

Despite considerable reductions in cardiovascular events in patients with an acute coronary syndrome (ACS) receiving dual antiplatelet therapy (DAPT), substantial residual risk persists. This unmet need has stimulated the development of anticoagulant drugs that target specific coagulation factors involved in the pathogenesis of thrombosis after atheromatous plaque disruption. Factor Xa is an attractive target for inhibition because of both its integral role in coagulation and its recognized participation in cellular proliferation and inflammation. Several oral, direct factor Xa inhibitors are undergoing investigation and large, phase III clinical trials of two agents, apixaban and rivaroxaban, in patients with an ACS have been completed. On the basis of the known pathobiology of ACS, one might anticipate that drugs in this class of anticoagulant would beneficially reduce ischemic and thrombotic events; however, a strategy of combined anticoagulant therapy and DAPT is likely to increase concomitant bleeding complications. The balance of benefit and risk will ultimately determine uptake in clinical practice. We review the available data on factor Xa inhibitors in the long-term management of patients with an ACS.

Evaluation of dose requirements for prolonged bivalirudin administration in patients with renal insufficiency and suspected heparin-induced thrombocytopenia.

Bivalirudin, a direct thrombin inhibitor, is indicated for patients with suspected heparin-induced thrombocytopenia (HIT) with anticipated percutaneous coronary intervention (PCI). Data is limited on dose selection among patients with renal insufficiency, particularly with prolonged infusion durations. The study cohort comprised 73 patients with renal dysfunction who received bivalirudin for suspected HIT with or without acute coronary syndrome. We reviewed individual pharmacy and medical records for laboratory and bivalirudin dosing information, medical comorbidities, and adverse clinical outcomes during administration. When estimated glomerular filtration rate (eGFR) was calculated by the Cockcroft-Gault (CG; ml/min) formula, the average bivalirudin dose (mg/kg/h) achieving a therapeutic activated partial thromboplastin time (aPTT) was 0.07 ± 0.04, 0.15 ± 0.08, and 0.16 ± 0.07 for patients with eGFR between 15-30, 31-60, and >60, respectively. When eGFR was calculated by the modification of diet in renal disease (MDRD; ml/min/1.73 m(2)) formula, the average bivalirudin dose achieving a therapeutic aPTT was 0.07 ± 0.04, 0.12 ± 0.07, and 0.20 ± 0.07 for patients with eGFR between 15-30, 31-60, >60, respectively. The difference between the dose achieving a therapeutic aPTT for patients with eGFR >60 when calculated by MDRD versus CG was completely abolished when obese patients were excluded from the CG cohort. The results of our series of patients with renal dysfunction receiving prolonged duration of bivalirudin in the setting of acute coronary syndrome (ACS) suggests that dose adjustment is safe and should be considered for patients with moderate to severe renal impairment (eGFR < 60 ml/min/1.73 m(2)).

Challenges and opportunities in implementing pharmacogenomics testing in the clinics.

The field of pharmacogenomics aims to incorporate individual patient genomic information into treatment selection. This is a rapidly evolving field with significant clinical promise. Implementation into clinical practice has several challenges that must be overcome. Genomics-based information encompasses large databases and requires expert knowledge for interpretation. Existing research suggests there are already several areas where pharmacogenomics-based decision-making is ripe for adoption into clinical practice. Impediments exist that must be overcome prior to large-scale implementation of existing pharmacogenomics-based therapies. There are several institutions and corporations at the forefront of implementation that are leading the development; however, larger systems-based approaches will be necessary. This article will discuss some of the present successes and future challenges that are necessary to overcome in order to implement a more patient-centered healthcare system.

The diversity of wound presentation associated with freon contact frostbite injury.

The authors report two cases of patients presenting with chemical frostbite-like injuries to the hands and wrists after contact exposure to Freon liquid. Although the history and initial physical presentations were quite similar, the severity of these injuries varied widely from superficial bullae to deep tissue injuries, requiring skin grafting and amputation of several digits. Freon is a widely used coolant in refrigerators, air conditioners, freezers, and water coolers, with a boiling point of -41°C. Although several cases of Freon-induced inhalational injury have been reported, few case reports of Freon-associated contact skin injury exist in the literature. The authors detail the broad diversity of injuries resulting from Freon contact as well as the first report of severe Freon injury necessitating skin grafting and amputation of multiple digits.

beta-arrestin-biased agonism at the beta2-adrenergic receptor.

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.

A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling.

For many years, beta-adrenergic receptor antagonists (beta-blockers or betaAR antagonists) have provided significant morbidity and mortality benefits in patients who have sustained acute myocardial infarction. More recently, beta-adrenergic receptor antagonists have been found to provide survival benefits in patients suffering from heart failure, although the efficacy of different beta-blockers varies widely in this condition. One drug, carvedilol, a nonsubtype-selective betaAR antagonist, has proven particularly effective in the treatment of heart failure, although the mechanism(s) responsible for this are controversial. Here, we report that among 16 clinically relevant betaAR antagonists, carvedilol displays a unique profile of in vitro signaling characteristics. We observed that in beta2 adrenergic receptor (beta2AR)-expressing HEK-293 cells, carvedilol has inverse efficacy for stimulating G(s)-dependent adenylyl cyclase but, nonetheless, stimulates (i) phosphorylation of the receptor's cytoplasmic tail on previously documented G protein-coupled receptor kinase sites; (ii) recruitment of beta-arrestin to the beta2AR; (iii) receptor internalization; and (iv) activation of extracellular regulated kinase 1/2 (ERK 1/2), which is maintained in the G protein-uncoupled mutant beta2AR(T68F,Y132G,Y219A) (beta2AR(TYY)) and abolished by beta-arrestin2 siRNA. Taken together, these data indicate that carvedilol is able to stabilize a receptor conformation which, although uncoupled from G(s), is nonetheless able to stimulate beta-arrestin-mediated signaling. We hypothesize that such signaling may contribute to the special efficacy of carvedilol in the treatment of heart failure and may serve as a prototype for a new generation of therapeutic beta2AR ligands.

Domain structure and DNA binding regions of beta protein from bacteriophage lambda.

beta protein from bacteriophage lambda promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. beta protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of beta protein in the presence and absence of DNA using limited proteolysis. Residues 1-130 form an N-terminal "core" domain that is resistant to proteases in the absence of DNA, residues 131-177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178-261 of beta protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of beta protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of beta protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of beta protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1-177 of beta protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA.

Probing the DNA sequence specificity of Escherichia coli RECA protein.

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5'-GCTGGTGG-3'). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a lambda repressor fragment in the presence of RecA, ADP-AlF4 and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.