Experimental stress in inflammatory rheumatic diseases: a review of psychophysiological stress responses

Introduction  

Stressful events are thought to contribute to the aetiology, maintenance and exacerbation of rheumatic diseases. Given the growing interest in acute stress responses and disease, this review investigates the impact of real-life experimental psychosocial, cognitive, exercise and sensory stressors on autonomic, neuroendocrine and immune function in patients with inflammatory rheumatic diseases.     

PKCα-Specific Phosphorylation of the Troponin Complex in Human Myocardium: A Functional and Proteomics Analysis

Aims

Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.

Methods and Results

Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca²⁺] after exchange. The maximal force (F_max) in the cTn (DD+PKCα) group (17.1±1.9 kN/m²) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m²). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca²⁺-sensitivity of force (pCa₅₀ = 5.59±0.02) compared to cTn (DD) (pCa₅₀ = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa₅₀ to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.

Conclusion

PKCα-mediated phosphorylation of the cTn complex decreases F_max and increases myofilament Ca²⁺-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca²⁺-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.     

Large-scale chromatin de-compaction induced by low light is not accompanied by nucleosomal displacement

Arabidopsis thaliana is widely used as a model to study chromatin compaction dynamics during development and in response to the environment. Signals such as prolonged heat treatment, low light and pathogen infestation are known to induce large-scale de-condensation of nuclear chromatin. Here we demonstrate that the response to different environments varies at the nucleosomal level. Our results show that in contrast to previous reports on heat and biotic infestation, low light intensity signaling does not alter nucleosomal occupancy, despite the marked effects of low light on global chromatin compaction.Key words: Arabidopsis, chromatin, nucleosomes, MNase IThanks to its relatively simple chromatin organization, Arabidopsis thaliana became the model of choice to study dynamics in nuclear chromatin compaction in plants.^1–3 At the microscopic level, highly condensed ‘heterochromatic’ domains (chromocenters), containing compact DNA (mainly repetitive sequences), and less condensed gene-rich ‘euchromatic’ domains can be distinguished upon staining with DAPI (4′,6-diamidino-2-phenylindole). This division however, is not static and compaction changes throughout development (reviewed in ref. ⁴). Chromatin for example de-condensates prior to flowering⁵ and increases with cell differentiation during leaf maturation³ and seedling establishment.⁶ Vice versa, artificially induced cell de-differentiation during protoplastization, results in loosening of compact chromatin.^7,8 Chromatin compaction is also influenced by various environmental signals. These include infestation by pathogenic microorganisms such as Pseudomonas syringae, light and heat signals.^9–11In our recent paper, published in Plant Physiology,¹² we demonstrate that a ∼90% decrease in light intensity (low light) induces a reversible reduction in global chromatin compaction. In addition, also specifically lowering the blue-light wavelengths in the spectrum, or lowering the red-to-far red (R/Fr) ratio induced a significant reduced compaction of the nuclear chromatin. This is interesting from a functional perspective because (1) these are the relevant signals perceived by plants in natural shade conditions occurring in dense-vegetations and (2) because these wavelengths are specifically detected by the light-sensitive photoreceptor proteins. Previously, we demonstrated that the R/Fr-photoreceptor Phytochrome-B (PhyB) is a positive regulator of chromatin compaction in standard light conditions.¹⁰ We now showed that PhyB also controls low light-induced chromatin organization, but that its effect depend on the genetic background of the phyb mutant under study. Likely, PhyB exerts its effects on light-mediated chromatin compaction via stabilization of CRYPTOCHROME 2 (CRY2) protein. This chromatin-associated blue light photoreceptor is a general positive regulator of low light-induced chromatin de-compaction and in addition controls chromatin compaction during floral transition.⁵In addition, we demonstrated that global chromatin de-compaction during floral transition and low light treatment also occurs in euchromatic domains.^5,12 To study possible chromatin changes at the nucleosomal level, we performed Micrococcal Nuclease I (MNase I) analysis. No differences were observed in the nucleosomal occupancy between standard and low light conditions in DNA gels or Southern blots hybridized with different probes for repeated sequences associated to heterochromatin, and dispersed upon low light treatment (Fig. 1). This suggests that the large-scale heterochromatin (de)compaction response observed at the microscopic level under low light conditions is not necessarily accompanied by nucleosomal displacement. These results are in line with the de-condensation conditions induced by protoplastization, where no changes in H3K9Me2 or in DNA methylation (5-mC) levels were found.⁷ However, these results are in contrast to the results of Pecinka and colleagues,¹¹ who demonstrated that prolonged heat stress results in heterochromatin de-condensation and loss of nucleosomes. Moreover, it is in contrast with Pavet and co-workers,⁹ who found reduced 5-mC levels upon infection with P. syringae. Although the results of Pecinka and colleaugues¹¹ were obtained by real-time PCR which may be more sensitive than our Southern blots, we conclude that the response of plants to their environment at the chromatin compaction level may be tailored to the specific signal it is confronted with and that this probably can be dissected at the nucleosomal level.Open in a separate windowFigure 1MNase I analysis of low light treated plants. Southern blots with 3 different probes hybridized to DNA from Col-0 plants cultured under standard (200 µmol m⁻² s⁻¹; control) and low light (15 µmol m⁻² s⁻¹) conditions. For each part, the first two lanes represent control DNA samples (no MNase I), followed by lanes with increasing MNase I concentrations (0.02, 0.1, 0.75 and 3 units MNase I). (A) 5S rDNA probe, (B) 45S rDNA probe, (C) pAl1 probe (180 bp centromeric repeat). M = molecular weight marker.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation

Background  

High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts.     

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO₂peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm⁻²) vs. AB. (41.0 ± 8.1 glands · cm⁻²). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l⁻¹) vs. AB (26.8±11.07 mmol · l⁻¹). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.