The impact of C-reactive protein levels on the effectiveness of upadacitinib in patients with rheumatoid arthritis: a 12-month prospective, non-interventional German study.

OBJECTIVES: We investigated whether the effectiveness of upadacitinib in rheumatoid arthritis (RA) treatment is affected by baseline CRP levels in a real-world setting. METHODS: UPwArds was a prospective, non-interventional study. Patients had moderate-to-severe RA and an inadequate response or intolerance to ≥1 disease-modifying anti-rheumatic drug (DMARD). The primary endpoint was clinical remission (Clinical Disease Activity Index [CDAI] ≤2.8) at 6 months. Secondary endpoints at 12 months included clinical remission and low disease activity assessed by CDAI and Simple Disease Activity Index criteria, DAS28-CRP <2.6/≤3.2, and patient-reported outcomes. The impact of baseline CRP levels (normal vs. above the upper limit of normal [ULN]) on primary and secondary endpoints was evaluated. The effect of concomitant MTX and prior inadequate response to biologic or targeted synthetic DMARDs (b/tsDMARD-IR) on the effectiveness of upadacitinib was also assessed. Safety was evaluated through 12 months. RESULTS: 518 patients were included in the effectiveness analyses. At 6 months, 24.4% of patients achieved the primary endpoint (CDAI ≤2.8). At 12 months, similar proportions of patients with normal CRP and CRP above the ULN at baseline achieved CDAI ≤2.8 (27.3% and 29.1%) and other key secondary endpoints. The effectiveness of upadacitinib was comparable with and without concomitant MTX and in b/tsDMARD-naive and b/tsDMARD-IR patients. The safety results were consistent with the known safety profile of upadacitinib; no new safety signals were identified. CONCLUSIONS: Upadacitinib therapy was effective for RA in a real-world setting. Baseline CRP levels had no significant impact on the effectiveness of upadacitinib.

cAMP Imaging at Ryanodine Receptors Reveals ß2-Adrenoceptor Driven Arrhythmias.

[Figure: see text].

Persistent cAMP Signaling by Internalized LH Receptors in Ovarian Follicles.

A crucial event in female reproduction occurs at midcycle, when a LH peak induces the final maturation of ovarian follicles. LH signals via a G protein-coupled receptor selectively expressed in the outermost follicular cell layers. However, how LH signals are relayed inside these cells and finally to the oocyte is incompletely understood. Here, we monitored LH signaling in intact ovarian follicles of transgenic mice expressing a fluorescent cAMP sensor. We found that LH stimulation induces 2 phases of cAMP signaling in all cell layers surrounding the oocyte. Interfering with LH receptor internalization abolished the second, persistent cAMP phase and partially inhibited oocyte meiosis resumption. These data suggest that persistent cAMP signals from internalized LH receptors contribute to transmitting LH effects inside follicle cells and ultimately to the oocyte. Thus, this study indicates that the recently proposed paradigm of cAMP signaling by internalized G protein-coupled receptors is implicated in receptor function and is physiologically relevant.

Transgenic mice for real-time visualization of cGMP in intact adult cardiomyocytes.

RATIONALE: 3',5'-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood. OBJECTIVE: Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes. METHODS AND RESULTS: We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk. CONCLUSIONS: The new sensor model allows visualization of real-time cGMP dynamics and pharmacology in intact adult cardiomyocytes. Förster resonance energy transfer imaging suggests the importance of well-established and potentially novel PDE-dependent mechanisms that regulate cGMP under physiological and pathophysiological conditions.

Enhanced expression of ß3-adrenoceptors in cardiac myocytes attenuates neurohormone-induced hypertrophic remodeling through nitric oxide synthase.

BACKGROUND: ß1-2-adrenergic receptors (AR) are key regulators of cardiac contractility and remodeling in response to catecholamines. ß3-AR expression is enhanced in diseased human myocardium, but its impact on remodeling is unknown. METHODS AND RESULTS: Mice with cardiac myocyte-specific expression of human ß3-AR (ß3-TG) and wild-type (WT) littermates were used to compare myocardial remodeling in response to isoproterenol (Iso) or Angiotensin II (Ang II). ß3-TG and WT had similar morphometric and hemodynamic parameters at baseline. ß3-AR colocalized with caveolin-3, endothelial nitric oxide synthase (NOS) and neuronal NOS in adult transgenic myocytes, which constitutively produced more cyclic GMP, detected with a new transgenic FRET sensor. Iso and Ang II produced hypertrophy and fibrosis in WT mice, but not in ß3-TG mice, which also had less re-expression of fetal genes and transforming growth factor ß1. Protection from Iso-induced hypertrophy was reversed by nonspecific NOS inhibition at low dose Iso, and by preferential neuronal NOS inhibition at high-dose Iso. Adenoviral overexpression of ß3-AR in isolated cardiac myocytes also increased NO production and attenuated hypertrophy to Iso and phenylephrine. Hypertrophy was restored on NOS or protein kinase G inhibition. Mechanistically, ß3-AR overexpression inhibited phenylephrine-induced nuclear factor of activated T-cell activation. CONCLUSIONS: Cardiac-specific overexpression of ß3-AR does not affect cardiac morphology at baseline but inhibits the hypertrophic response to neurohormonal stimulation in vivo and in vitro, through a NOS-mediated mechanism. Activation of the cardiac ß3-AR pathway may provide future therapeutic avenues for the modulation of hypertrophic remodeling.

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and ß-arrestin-2 Overexpression in HEK293 Cells.

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and ß-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and ß-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and ß-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and ß-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/ß-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.

Advances and techniques to measure cGMP in intact cardiomyocytes.

Förster resonance energy transfer (FRET)-based biosensors are powerful tools for real-time monitoring of signaling events in intact cells using fluorescence microscopy. Here, we describe a highly sensitive method which allows FRET-based measurements of the second messenger cGMP in adult mouse ventricular myocytes. Such measurements have been challenging before, primarily due to relatively low cGMP concentrations in cardiomyocytes and limited sensitivity of the available biosensors. With our new technique, one can reliably measure dynamic changes in cGMP upon stimulation of myocytes with natriuretic peptides and other physiological and pharmacological ligands.

FRET microscopy for real-time monitoring of signaling events in live cells using unimolecular biosensors.

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium, cAMP, inositol phosphates and cGMP. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a ß-adrenergic receptor agonist and blocker.