Impact of a real-time diagnostic and antimicrobial stewardship workflow on time to appropriate therapy for infections caused by multidrug-resistant Gram-negative organisms.

INTRODUCTION: Multidrug-resistant (MDR) Gram-negative organisms cause life-threatening infections, and the incidence is rising globally. Timely therapy for these infections has a direct impact on patient survival. This study aimed to determine the impact of a multidisciplinary diagnostic and antimicrobial stewardship (AMS) workflow on time to appropriate therapy (TAP) for these infections using novel beta-lactam/beta-lactamase inhibitors. METHODS: This was a retrospective quasi-experimental study of adult patients with carbapenem-resistant Enterobacterales (CRE) and multidrug-resistant Pseudomonas (MDR PsA) infections at a 1500 bed university hospital. Included patients who received ≥ 72 hours of ceftazidime-avibactam (CZA) or ceftolozane-tazobactam (C/T) from December 2017 to December 2019. During the pre-intervention period (December 2017 to December 2018), additional susceptibilities (including CZA and C/T) were performed only upon providers' request. In 2019, reflex algorithms were implemented for faster identification and testing of all CRE/MDR PsA isolates. Results were communicated in real-time to the AMS team to tailor therapy. RESULTS: A total of 99 patients were included, with no between-group differences at baseline. The median age was 60 years and 56 (56.7%) were in intensive care at the time of culture collection. Identified organisms included 71 (71.7%) MDR PsA and 26 CRE, of which 18 were carbapenemase producers (Klebsiella-producing carbapenemase = 12, New Delhi metallo-ß-lactamase = 4, Verona integron-encoded metallo-ß-lactamase = 2). The most common infections were pneumonia (49.5%) and bacteraemia (30.3%). A decrease was found in median TAP (103 [IQR 76.0-156.0] vs. 75 [IQR 56-100] hours; P < 0.001). Median time from culture collection to final susceptibility results was shorter in the post-intervention group (123 vs. 93 hours; P < 0.001). CONCLUSION: This study identified improvement in TAP in MDR PsA and CRE infections with implementation of a reflex microbiology workflow and multidisciplinary antimicrobial stewardship initiatives.

Correction: Maning et al. Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition. Int. J. Mol. Sci. 2020, 21, 2868.

The authors wish to make the following correction to this paper [...].

A New Delhi metallo-ß-lactamase (NDM)-positive isolate of Klebsiella pneumoniae causing catheter-related bloodstream infection in an ambulatory hemodialysis patient.

The New Delhi metallo-ß-lactamase (NDM) is a mediator of broad antimicrobial resistance among the Enterobacteriaceae and other gram-negative pathogens that cause opportunistic and nosocomial infections. In the decade since its discovery, NDM has spread worldwide and represents an increasing threat to public health. NDM is capable of hydrolyzing nearly all known ß-lactam antibiotics, including the carbapenems, and due to its zinc ion-dependent catalytic mechanism is unaffected by available ß-lactamase inhibitors. We report a case of catheter-related bloodstream infection caused by a pan-resistant, NDM-positive isolate of Klebsiella pneumoniae in an ambulatory end-stage renal disease patient started on hemodialysis approximately 8 weeks prior. The absence of any recent hospitalization indicates that the infection was likely acquired from a hemodialysis center in the United States. This case demonstrates the increasing prevalence of antimicrobial resistance mechanisms in ambulatory as well as inpatient healthcare settings, and highlights the particular risk of the outpatient hemodialysis facility as an optimal environment for colonization with multidrug- and pandrug-resistant pathogens.

Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition.

Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon ß2-adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. ß2AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via ß2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

Sustained GRK2-dependent CREB activation is essential for α2-adrenergic receptor-induced PC12 neuronal differentiation.

Many aspects of neuronal development, such as neuronal survival and differentiation, are regulated by the transcription factor cAMP-response element-binding protein (CREB). We have previously reported that α2-adrenergic receptors (ARs), members of the G protein-coupled receptor (GPCR) superfamily, induce neuronal differentiation of rat pheochromocytoma (PC)-12 cells in a subtype-specific manner, i.e. α2A<α2B<α2C. Herein, we sought to investigate CREB`s involvement in this α2AR-dependent neurogenic process. We used a combination of gene reporter assays and immunoblotting analysis, coupled with co-immunoprecipitation and morphological analysis, in transfected PC12 cell lines. Chronic α2B- or α2C-AR activation results in sustained CREB activation, which is both necessary and sufficient for neuronal differentiation of PC12 cells expressing these two α2ARs. In contrast, chronic α2A activation only leads to transient CREB activation, insufficient for PC12 neuronal differentiation. However, upon CREB overexpression, α2A-AR triggers neuronal differentiation similarly to α2B- or α2C-ARs. Importantly, NGF (Nerve Growth Factor)`s TrkA receptor transactivation is essential for the sustained activation of CREB by all three α2 subtypes in PC12 cells, whereas protein kinase A (PKA), the prototypic kinase that phosphorylates CREB, is not. Instead, TrkA-activated GPCR-kinase (GRK)-2 mediates the sustained CREB activation during α2AR-induced neuronal differentiation of PC12 cells. In conclusion, catecholaminergic-induced neuronal differentiation of PC12 cells through α2ARs uses a non-canonical pathway involving TrkA transactivation and subsequent GRK2-dependent, sustained phosphorylation/activation of CREB. These findings provide novel insights into catecholaminergic neurogenesis and suggest that α2AR agonists, combined with NGF analogs or GRK2 stimulators, may exert neurogenic/neuroprotective effects.

Deletion of Osteopontin Enhances ß2-Adrenergic Receptor-Dependent Anti-Fibrotic Signaling in Cardiomyocytes.

Cardiac ß2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The ß2AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates the generation of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory cytokine, which also mediates fibrosis in several tissues, including the heart. OPN underlies several cardiovascular pathologies, including atherosclerosis and cardiac adverse remodeling. We found that the cardiotoxic hormone aldosterone transcriptionally upregulates OPN in H9c2 rat cardiac myoblasts-an effect prevented by endogenous ß2AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both ß1AR and ß2AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered ß2AR stimulation capable of completely abrogating transforming growth factor (TGF)-ß-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gαs subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac ß2AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Not all arrestins are created equal: Therapeutic implications of the functional diversity of the ß-arrestins in the heart.

The two ubiquitous, outside the retina, G protein-coupled receptor (GPCR) adapter proteins, ß-arrestin-1 and -2 (also known as arrestin-2 and -3, respectively), have three major functions in cells: GPCR desensitization, i.e., receptor decoupling from G-proteins; GPCR internalization via clathrin-coated pits; and signal transduction independently of or in parallel to G-proteins. Both ß-arrestins are expressed in the heart and regulate a large number of cardiac GPCRs. The latter constitute the single most commonly targeted receptor class by Food and Drug Administration-approved cardiovascular drugs, with about one-third of all currently used in the clinic medications affecting GPCR function. Since ß-arrestin-1 and -2 play important roles in signaling and function of several GPCRs, in particular of adrenergic receptors and angiotensin II type 1 receptors, in cardiac myocytes, they have been a major focus of cardiac biology research in recent years. Perhaps the most significant realization coming out of their studies is that these two GPCR adapter proteins, initially thought of as functionally interchangeable, actually exert diametrically opposite effects in the mammalian myocardium. Specifically, the most abundant of the two ß-arrestin-1 exerts overall detrimental effects on the heart, such as negative inotropy and promotion of adverse remodeling post-myocardial infarction (MI). In contrast, ß-arrestin-2 is overall beneficial for the myocardium, as it has anti-apoptotic and anti-inflammatory effects that result in attenuation of post-MI adverse remodeling, while promoting cardiac contractile function. Thus, design of novel cardiac GPCR ligands that preferentially activate ß-arrestin-2 over ß-arrestin-1 has the potential of generating novel cardiovascular therapeutics for heart failure and other heart diseases.

Co-IP assays for measuring GPCR-arrestin interactions.

ßarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The ßarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.

Novel Insights into the Crosstalk between Mineralocorticoid Receptor and G Protein-Coupled Receptors in Heart Adverse Remodeling and Disease.

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type⁻specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR's actions, but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter's activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

Biased Agonism/Antagonism of Cardiovascular GPCRs for Heart Failure Therapy.

G protein-coupled receptors (GPCRs) are among the most important drug targets currently used in clinic, including drugs for cardiovascular indications. We now know that, in addition to activating heterotrimeric G protein-dependent signaling pathways, GPCRs can also activate G protein-independent signaling, mainly via the ßarrestins. The major role of ßarrestin1 and -2, also known as arrestin2 or -3, respectively, is to desensitize GPCRs, i.e., uncoupled them from G proteins, and to subsequently internalize the receptor. As the ßarrestin-bound GPCR recycles inside the cell, it serves as a signalosome transducing signals in the cytoplasm. Since both G proteins and ßarrestins can transduce signals from the same receptor independently of each other, any given GPCR agonist might selectively activate either pathway, which would make it a biased agonist for that receptor. Although this selectivity is always relative (never absolute), in cases where the G protein- and ßarrestin-dependent signals emanating from the same GPCR result in different cellular effects, pharmacological exploitation of GPCR-biased agonism might have therapeutic potential. In this chapter, we summarize the GPCR signaling pathways and their biased agonism/antagonism examples discovered so far that can be exploited for heart failure treatment. We also highlight important issues that need to be clarified along the journey of these ligands from bench to the clinic.

ß-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes.

Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins ßarr (ß-arrestin)-1 and ßarr-2 are functionally distinct in the heart. ßarr1 is cardiotoxic, decreasing contractility by opposing ß1AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, ßarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether ßarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that ßarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca2+-ATPase) in vivo and in vitro in a ß1AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. ßarr1 lacks this effect. Moreover, ßarr2 does not desensitize ß1AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to ßarr1. In vivo, post-MI heart failure mice overexpressing cardiac ßarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, ßarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac ßAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac ßarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac ßarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure.

ß1-adrenoceptor Arg389Gly polymorphism confers differential ß-arrestin-binding tropism in cardiac myocytes.

AIM: The ß1-adrenergic receptor (AR) Arg389Gly polymorphism affects efficacy of its procontractile signaling in cardiomyocytes and carriers' responses to ß-blockers. To identify molecular mechanisms underlying functional differences between Arg389 and Gly389 ß1ARs, we examined their binding to ß-arrestins (ßarr-1 and -2), which mediate ß1AR signaling, in neonatal rat ventricular myocytes. METHODS: We tested the ß1AR-ßarr interaction via ß1AR immunoprecipitation followed by ßarr immunoblotting. RESULTS: ßarr1 binds both variants upon isoproterenol, carvedilol or metoprolol treatment in neonatal rat ventricular myocytes. Conversely, the potentially beneficial in the heart ßarr2 only interacts with the Arg389 receptor in response to isoproterenol or carvedilol. CONCLUSION: Arg389 confers unique ßarr2-interacting tropism to the ß1AR in cardiac myocytes, potentially underlying this variant's gain-of-function phenotype and better clinical responses to ß-blockers.

GRK2 Up-Regulation Creates a Positive Feedback Loop for Catecholamine Production in Chromaffin Cells.

Elevated sympathetic nervous system (SNS) activity aggravates several diseases, including heart failure. The molecular cause(s) underlying this SNS hyperactivity are not known. We have previously uncovered a neurohormonal mechanism, operating in adrenomedullary chromaffin cells, by which circulating catecholamine (CA) levels increase in heart failure: severe dysfunction of the adrenal α2-adrenergic receptors (ARs) due to the up-regulation of G protein-coupled receptor-kinase (GRK)-2, the kinase that desensitizes them. Herein we looked at the potential signaling mechanisms that bring about this GRK2 elevation in chromaffin cells. We found that chronic CA treatment of either PC12 or rat primary chromaffin cells can in itself result in GRK2 transcriptional up-regulation through α2ARs-Gi/o proteins-Src-ERK1/2. The resultant GRK2 increase severely enhances the α2AR desensitization/down-regulation elevating not only CA release but also CA biosynthesis, as evidenced by tyrosine hydroxylase up-regulation. Finally, GRK2 knockdown leads to enhanced apoptosis of PC12 cells, indicating an essential role for GRK2 in chromaffin cell homeostasis/survival. In conclusion, chromaffin cell GRK2 mediates a positive feedback loop that feeds into CA secretion, thereby enabling the adrenomedullary component of the SNS to turn itself on.

GPCRs of adrenal chromaffin cells & catecholamines: The plot thickens.

The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as "presynaptic autoreceptors" in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α2ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and ß-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and ß-arrestins in these processes and particular points of focus for future research will be highlighted, as well.

Impact of CYP2D6 Genetic Variation on the Response of the Cardiovascular Patient to Carvedilol and Metoprolol.

Carvedilol and metoprolol are two of the most commonly prescribed ß-blockers in cardiovascular medicine and primarily used in the treatment of hypertension and heart failure. Cytochrome P450 2D6 (CYP2D6) is the predominant metabolizing enzyme of these two drugs. Since the first description of a CYP2D6 sparteinedebrisoquine polymorphism in the mid-seventies, substantial genetic heterogeneity has been reported in the human CYP2D6 gene, with ~100 different polymorphisms identified to date. Some of these polymorphisms render the enzyme completely inactive while others do not modify its activity. Based on all the identified variants, four metabolizer phenotypes are nowadays used to characterize drug metabolism via CYP2D6 in humans: ultra-rapid metabolizer (UM); extensive metabolizer (EM); intermediate metabolizer (IM); and poor metabolizer (PM) phenotypes. As a consequence of these CYP2D6 metabolizer phenotypes, pharmacokinetics and bioavailability of carvedilol and metoprolol can range from therapeutically ineffective levels (in the UM patients) to excessive (overdose) and potentially toxic concentrations (in PM patients). This, in turn, can result in elevated risks for either treatment failure (in terms of blood pressure reduction of hypertensive patients and of improving survival and cardiovascular function of heart failure patients) or for adverse effects (e.g. hypotension and bradycardia). The present review will discuss the impact of these CYP2D6 genetic polymorphisms on the therapeutic responses of cardiovascular patients treated with either of these two ß-blockers. In addition, the potential advantages and disadvantages of implementing CYP2D6 genetic testing in the clinic to guide/personalize therapy with these two drugs will be discussed.

Adrenal G protein-coupled receptor kinase-2 in regulation of sympathetic nervous system activity in heart failure.

Heart failure (HF), the number one cause of death in the western world, is caused by the insufficient performance of the heart leading to tissue underperfusion in response to an injury or insult. It comprises complex interactions between important neurohormonal mechanisms that try but ultimately fail to sustain cardiac output. The most prominent such mechanism is the sympathetic (adrenergic) nervous system (SNS), whose activity and outflow are greatly elevated in HF. SNS hyperactivity confers significant toxicity to the failing heart and markedly increases HF morbidity and mortality via excessive activation of adrenergic receptors, which are G protein-coupled receptors. Thus, ligand binding induces their coupling to heterotrimeric G proteins that transduce intracellular signals. G protein signaling is turned-off by the agonist-bound receptor phosphorylation courtesy of G protein-coupled receptor kinases (GRKs), followed by ßarrestin binding, which prevents the GRK-phosphorylated receptor from further interaction with the G proteins and simultaneously leads it inside the cell (receptor sequestration). Recent evidence indicates that adrenal GRK2 and ßarrestins can regulate adrenal catecholamine secretion, thereby modulating SNS activity in HF. The present review gives an account of all these studies on adrenal GRKs and ßarrestins in HF and discusses the exciting new therapeutic possibilities for chronic HF offered by targeting these proteins pharmacologically.