Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Corticotropin-releasing factor receptors: oligomerization and lateral mobility studied by fluorescence spectroscopy

G protein-coupled receptors have been proposed to exist in large signalosomes subject to agonist-driven shifts in the assembly-disassembly equilibrium. Signalosome assembly is also thought to be stabilized by the self-organizing properties of membrane lipids and/or by cortical actin and to result in restricted mobility of receptors. Here I investigated the receptors for corticotropin-releasing factor (CRF), CRFR1 and CRFR2; these are highly homologous and couple to the same G proteins but differ within their hydrophobic core. Fluorescence resonance energy transfer (FRET) microscopy showed that CRFRs existed in oligomeric form on the surface of living cells. CRF enhanced FRET in CRFR2 - but not in CRFR1 - suggesting either a conformational change in or an agonist-induced dissociation of CRFR2 oligomers. Agonist binding accelerated the diffusion of CRFR2 - but not of CRFR1 - in both, HEK293 cells and hippocampal neurons detected by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). However,fluorescence intensity distribution analysis (FIDA) demonstrated that agonist binding did not change the size of CRFR-complexes. Disruption of the actin cytoskeleton or extraction of cholesterol abolished the agonist-dependent increase in CRFR2 mobility. These observations are incompatible with an agonist-induced change in monomer-oligomer equilibrium. However, they are consistent with a model in which agonist occupancy facilitates accommodation of cholesterol by the CRFR2 - but not by CRFR1; this conformational change and/or redistribution of CRF2 into lipid microdomains affords rapid diffusion. The difference between the two receptors is likely to arise from their distinct hydrophobic cores.submitted by Laura Milan-LoboZsfassung in dt. SpracheWien, Med. Univ., Diss., 2008OeBB(VLID)188348

Anti-analgesic effect of the mu/delta opioid receptor heteromer revealed by ligand-biased antagonism.

Delta (DOR) and mu opioid receptors (MOR) can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers may be a novel therapeutic target in the treatment of pain. However, there are currently no ligands selective for DOR/MOR heteromers, and, consequently, their role in nociception remains unknown. In this study, we used a pharmacological opioid cocktail that selectively activates and stabilizes the DOR/MOR heteromer at the cell surface by blocking its endocytosis to assess its role in antinociception. We found that mice treated chronically with this drug cocktail showed a significant right shift in the ED50 for opioid-mediated analgesia, while mice treated with a drug that promotes degradation of the heteromer did not. Furthermore, promoting degradation of the DOR/MOR heteromer after the right shift in the ED50 had occurred, or blocking signal transduction from the stabilized DOR/MOR heteromer, shifted the ED50 for analgesia back to the left. Taken together, these data suggest an anti-analgesic role for the DOR/MOR heteromer in pain. In conclusion, antagonists selective for DOR/MOR heteromer could provide an avenue for alleviating reduced analgesic response during chronic pain treatment

Heteromerization of the μ- and δ-Opioid Receptors Produces Ligand-Biased Antagonism and Alters μ-Receptor TraffickingS⃞

Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and postendocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the μ-opioid receptor (MOR) and δ-opioid receptor (DOR), because the MOR is recycled primarily after endocytosis and the DOR is degraded in the lysosome. Here, we examined the endocytic and postendocytic fate of MORs, DORs, and DOR/MOR heteromers in human embryonic kidney 293 cells stably expressing each receptor alone or coexpressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, whereas MORs in the same cell are significantly more stable. It is noteworthy that we found that the DOR-selective antagonist naltriben mesylate could block both methadone- and [d-Ala2,NMe-Phe4,Gly-ol5]-enkephalin-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows “biased antagonism,” whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer

A single methadone treatment reverses the reduced antinociception produced by the methadone/NTB cocktail.

<p>A) Proposed model of reduced analgesia produced by the MOR/DOR heteromer in mice treated with methadone/NTB cocktail, where DOR/MORs are anti-analgesic and contribute to poor antinociception. B) Proposed model of a single methadone exposure on the trafficking of MOR and DOR/MOR heteromers after induction of poor antinociception. MOR on day 6 will be activated, internalized and recycled back to the plasma membrane in response to the methadone treatment. Normal cycling will keep the MOR ready for further activation. DOR/MOR on day 6 will be activated, internalized and degraded as a consequence of the methadone treatment. C) Antinociception to escalating doses of methadone on day 7, in the same mice previously shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058362#pone-0058362-g002" target="_blank">Figure 2C & D</a> (closed squares for methadone group: ED₅₀ and 95% confidence intervals: 4.5 (3.4–6.7) mg/Kg, and closed triangles for cocktail group: ED₅₀ and 95% confidence intervals: 3.8 (3.5–4.2) mg/Kg). Open circles show antinociception to methadone on day 6 in the cocktail group: ED₅₀ and 95% confidence intervals: 8.6 (5.4–12.4) mg/Kg. Data represents mean ± SEM; n = 20 mice per group.</p

Development of reduced antinociception after chronic treatment with a cocktail of methadone and NTB.

<p>A & B) Proposed model of the trafficking of MOR and DOR/MOR in response to methadone (A) or to methadone/NTB cocktail treatment (B); MOR will be activated, internalized and recycled back to the plasma membrane in response to methadone. Normal cycling will keep the MOR ready for further activation. DOR/MOR will be activated, internalized and degraded in response to methadone. In the presence of the DOR antagonist NTB, activation and trafficking of MOR in response to methadone will remain unaffected, whereas DOR/MOR heteromers will be occupied by NTB and methadone resulting in the activation of the receptor complex without subsequent endocytosis and degradation. C–E) Antinociception to escalating doses of methadone was measured in naïve wild type mice on day 1 (closed squares). ED₅₀ values calculated via linear regression analysis and 95% confidence intervals are as follows: Day1, MD treatment: 3 (1.9–3.8) mg/Kg and MD+NTB treatment: 3.2 (2.3–4.2) mg/Kg. On days 2, 3, 4 and 5, mice were injected s.c. once daily with the ED₅₀ dose of methadone (3 mg/Kg) (C) or a cocktail of methadone (3 mg/Kg) combined with NTB (0.01 mg/Kg) (D). On day 6 (open circles), antinociception to methadone was measured again in mice treated with only methadone (C) or the cocktail (D); ED₅₀ values and 95% confidence intervals are as follows: Day 6, MD treatment: 4.3 (3.6–5.3) mg/Kg and MD+NTB treatment: 8.6 (5.4–12.4) mg/Kg. E) Shows an additional dose range of methadone on day 6 for the group of mice receiving injections of methadone/NTB cocktail. Data represents mean ± SEM; n = 20 mice per group.</p

Peptide-based interactions with calnexin target misassembled membrane proteins into endoplasmic reticulum-derived multilamellar bodies.

Oligomeric assembly of neurotransmitter transporters is a prerequisite for their export from the endoplasmic reticulum (ER) and their subsequent delivery to the neuronal synapse. We previously identified mutations, e.g., in the gamma-aminobutyric acid (GABA) transporter-1 (GAT1), which disrupted assembly and caused retention of the transporter in the ER. Using one representative mutant, GAT1-E101D, we showed here that ER retention was due to association of the transporter with the ER chaperone calnexin: interaction with calnexin led to accumulation of GAT1 in concentric bodies corresponding to previously described multilamellar ER-derived structures. The transmembrane domain of calnexin was necessary and sufficient to direct the protein into these concentric bodies. Both yellow fluorescent protein-tagged versions of wild-type GAT1 and of the GAT1-E101D mutant remained in disperse (i.e., non-aggregated) form in these concentric bodies, because fluorescence recovered rapidly (t(1/2) approximately 500 ms) upon photobleaching. Fluorescence energy resonance transfer microscopy was employed to visualize a tight interaction of GAT1-E101D with calnexin. Recognition by calnexin occurred largely in a glycan-independent manner and, at least in part, at the level of the transmembrane domain. Our findings are consistent with a model in which the transmembrane segment of calnexin participates in chaperoning the inter- and intramolecular arrangement of hydrophobic segment in oligomeric proteins

doi: 10.1111/j.1600-0854.2008.00761.x Membrane Localization is Critical for Activation of the PICK1 BAR Domain

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative a-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including th