Multi-disciplinary community-based group intervention for fibromyalgia: a pilot randomized controlled trial.

Fibromyalgia is characterized by widespread pain, fatigue, sleep disturbances, mood disturbances, and cognitive impairment. Most individuals with fibromyalgia experience poorly managed symptoms and increased healthcare service use. Multicomponent therapies, with a focus on nonpharmacological modalities, are increasingly supported in the literature. However, given the limited resources available, implementation in smaller communities remains a challenge. This research tested a community-based multidisciplinary group intervention for individuals diagnosed with FM living in a small urban centre. The primary outcome was perceptions of quality of care and secondary outcomes included disease-related functioning, anxious and depressive symptoms, pain beliefs, and health service utilization. A pilot randomized control trial was conducted in which 60 patients diagnosed with fibromyalgia were randomized into a 10-week community-based multidisciplinary group intervention program or usual care. Treatment components included twice-weekly exercise sessions and weekly education sessions (e.g., pain education, cognitive behavioral strategies for stress, nutrition, peer support). The trial (NCT03270449) was registered September 1 2017. Statistically significant post-intervention improvements were found in the primary outcome, perceived quality of care (Cohen's d = 0.61, 0.66 for follow up care and goal setting, respectively). Secondary outcomes showing statistically significant improvements were disease-related daily functioning (Cohen's d = 0.70), depressive symptoms (Cohen's d = 0.87), and pain beliefs (Cohen's d = 0.61, 0.67, 0.82 for harm, disability and control, respectively). No adverse events were reported. Community-based multidisciplinary group interventions for fibromyalgia show promise for improving satisfaction with quality of care, disease-related functioning, and depression, and fostering more adaptive pain beliefs.

Regulation of GIP and GLP1 receptor cell surface expression by N-glycosylation and receptor heteromerization.

In response to a meal, Glucose-dependent Insulinotropic Polypeptide (GIP) and Glucagon-like Peptide-1 (GLP-1) are released from gut endocrine cells into the circulation and interact with their cognate G-protein coupled receptors (GPCRs). Receptor activation results in tissue-selective pleiotropic responses that include augmentation of glucose-induced insulin secretion from pancreatic beta cells. N-glycosylation and receptor oligomerization are co-translational processes that are thought to regulate the exit of functional GPCRs from the ER and their maintenance at the plasma membrane. Despite the importance of these regulatory processes, their impact on functional expression of GIP and GLP-1 receptors has not been well studied. Like many family B GPCRs, both the GIP and GLP-1 receptors possess a large extracellular N-terminus with multiple consensus sites for Asn-linked (N)-glycosylation. Here, we show that each of these Asn residues is glycosylated when either human receptor is expressed in Chinese hamster ovary cells. N-glycosylation enhances cell surface expression and function in parallel but exerts stronger control over the GIP receptor than the GLP-1 receptor. N-glycosylation mainly lengthens receptor half-life by reducing degradation in the endoplasmic reticulum. N-glycosylation is also required for expression of the GIP receptor at the plasma membrane and efficient GIP potentiation of glucose-induced insulin secretion from the INS-1 pancreatic beta cell line. Functional expression of a GIP receptor mutant lacking N-glycosylation is rescued by co-expressed wild type GLP1 receptor, which, together with data obtained using Bioluminescence Resonance Energy Transfer, suggests formation of a GIP-GLP1 receptor heteromer.

Using Bioluminescence Resonance Energy Transfer to measure ion channel assembly.

Bioluminescence Resonance Energy Transfer (BRET) measures protein interactions within 10 nm of each other. Aside from its ability to probe for interactions at high resolution, this technique operates in live, intact cells, and offers a high throughput method of detection. Thus far, BRET has been widely used in measuring G protein receptor dimerization. In this chapter, we describe the BRET methodology in detail and apply this technique to the measurement of ion channel assembly. In addition, we discuss how BRET can be used to compare the extent of homomeric and heteromeric channel assembly.

Regulation of cell surface expression of functional pacemaker channels by a motif in the B-helix of the cyclic nucleotide-binding domain.

Previous studies have suggested that a portion of the cyclic nucleotide-binding domain (CNBD) of the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) "pacemaker" channel, composed of the A- and B-helices and the interceding beta-barrel, confers two functions: inhibition of channel opening in response to hyperpolarization and promotion of cell surface expression. The sequence determinants required for each of these functions are unknown. In addition, the mechanism underlying plasma membrane targeting by this subdomain has been limitedly explored. Here we identify a four-amino acid motif (EEYP) in the B-helix that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. This motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly.

Hyperpolarization-activated cyclic nucleotide-modulated 'HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cells.

The hyperpolarization-activated cation current (I(f)), and the hyperpolarization-activated cyclic nucleotide-modulated 'HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. I(f) and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing I(f), and the density of I(f) in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The I(f) blocker ZD7288 (0.3 microm) reduced I(f) and the beating rate of embryoid bodies. Taken together, the activation kinetics of I(f) and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of I(f) to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.

HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.