Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms.

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.

Ligand-based virtual screening identifies a family of selective cannabinoid receptor 2 agonists.

The cannabinoid receptor 2 (CB2R) has been linked with the regulation of inflammation, and selective receptor activation has been proposed as a target for the treatment of a range of inflammatory diseases such as atherosclerosis and arthritis. In order to identify selective CB2R agonists with appropriate physicochemical and ADME properties for future evaluation in vivo, we first performed a ligand-based virtual screen. Subsequent medicinal chemistry optimisation studies led to the identification of a new class of selective CB2R agonists. Several examples showed high levels of activity (EC50<200 nM) and binding affinity (Ki<200 nM) for the CB2R, and no detectable activity at the CB1R. The most promising example, DIAS2, also showed favourable in vitro metabolic stability and absorption properties along with a clean selectivity profile when evaluated against a panel of GPCRs and kinases.

Neural alterations associated with anxiety symptoms in obstructive sleep apnea syndrome.

BACKGROUND: Neuropsychological comorbidities, including anxiety symptoms, accompany obstructive sleep apnea (OSA); structural and functional brain alterations also occur in the syndrome. The objective was to determine whether OSA patients expressing anxiety symptoms show injury in specific brain sites. METHODS: Magnetic resonance T2-relaxometry was performed in 46 OSA and 66 control subjects. Anxiety symptoms were evaluated using the Beck Anxiety Inventory (BAI); subjects with BAI scores >9 were classified anxious. Whole brain T2-relaxation maps were compared between anxious and nonanxious groups using analysis of covariance (covariates, age and gender). RESULTS: Sixteen OSA and seven control subjects showed anxiety symptoms, and 30 OSA and 59 controls were nonanxious. Significantly higher T2-relaxation values, indicating tissue injury, appeared in anxious OSA versus nonanxious OSA subjects in subgenu, anterior, and mid-cingulate, ventral medial prefrontal and bilateral insular cortices, hippocampus extending to amygdala and temporal, and bilateral parietal cortices. Brain injury emerged in anxious OSA versus nonanxious controls in bilateral insular cortices, caudate nuclei, anterior fornix, anterior thalamus, internal capsule, mid-hippocampus, dorsotemporal, dorsofrontal, ventral medial prefrontal, and parietal cortices. CONCLUSIONS: Anxious OSA subjects showed injury in brain areas regulating emotion, with several regions lying outside structures affected by OSA alone, suggesting additional injurious processes in anxious OSA subjects.

Neural alterations and depressive symptoms in obstructive sleep apnea patients.

STUDY OBJECTIVES: Depressive symptoms are common in obstructive sleep apnea (OSA) patients, and brain injury occurs with both OSA and depression independently. The objective was to determine whether brain alterations in OSA bear relationships to depressive symptoms. DESIGN: Cross-sectional study. SETTING: University-based medical center. PARTICIPANTS: 40 treatment-naive OSA subjects and 61 control subjects without diagnosed psychopathology. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Whole-brain maps of T2 relaxation time, a measure sensitive to injury, were calculated from magnetic resonance images, transformed to common space, and smoothed. Control and OSA groups were classified by Beck Depression Inventory (BDI)-II scores (> or =12 symptomatic, <10 asymptomatic for depressive symptoms). The OSA group separated into 13 symptomatic (mean +/- SD: BDI-II 21 +/- 8; age 47.6 +/- 11; apnea hypopnea index [AHI] 28.3 +/- 17), and 27 asymptomatic (4 +/- 3; 47.5 +/- 8; 31.5 +/- 16) subjects. The control group included 56 asymptomatic (BDI-II 2.5 +/- 2.6; age 47.3 +/- 9) subjects. Asymptomatic OSA subjects exhibited higher AHI. T2 maps were compared between groups (ANCOVA), with age and gender as covariates. Injury appeared in symptomatic vs asymptomatic OSA subjects in the mid- and anterior cingulate, anterior insular, medial pre-frontal, parietal, and left ventrolateral temporal cortices, left caudate nucleus, and internal capsule. Relative to asymptomatic controls, symptomatic OSA patients showed damage in the bilateral hippocampus and caudate nuclei, anterior corpus callosum, right anterior thalamus, and medial pons. CONCLUSIONS: Neural injury differed between OSA patients with and without depressive symptoms. Depressive symptoms may exacerbate injury accompanying OSA, or introduce additional damage in affective, cognitive, respiratory, and autonomic control regions.

Spontaneous contractions of the pig urinary bladder: the effect of ATP-sensitive potassium channels and the role of the mucosa.

OBJECTIVE: To investigate the influence of the mucosa on the inhibitory effects of the ATP-sensitive potassium channel (K(ATP) channel) opener, cromakalim, on the spontaneous contractions of pig bladder strips from the bladder dome and trigone. Little is known about the influence of the mucosa on spontaneous contractions and whether the nature of these contractions differs between the bladder dome and trigone. MATERIALS AND METHODS: Paired longitudinal strips of female pig bladders were isolated from the dome and trigone. The mucosa was removed from one strip per pair and tissues were set up in organ baths. Spontaneous activity was allowed to develop and recorded, and then cumulative concentration-response curves to cromakalim were obtained. The time needed for spontaneous contractions to develop, the frequency and amplitude of spontaneous contractions, and the effect of cromakalim were analysed. The strips of mucosa removed from the dome to produce denuded strips were also analysed by immunofluorescence using antibodies specific for vimentin and alpha-smooth muscle actin (alpha-SMA). RESULTS: In the dome removal of the mucosa delayed the development of spontaneous contractions compared with mucosa-intact strips, whilst the trigone strips developed spontaneous contractions soon after set up in the organ baths irrespective of the presence or absence of mucosa. In the dome, cromakalim was more potent in suppressing spontaneous contractions when the mucosa was absent; whilst in the trigone the effects of cromakalim were similar in mucosa-intact and denuded strips. Upon examination of the strips of mucosa by immunofluorescence these strips were shown to contain cells positive for alpha-SMA or vimentin and cells positive for both, suggesting the presence of not only urothelium but also suburothelium and some detrusor smooth muscle bundles. CONCLUSION: In the dome, the urothelium and suburothelium reduce the inhibitory effect of cromakalim on spontaneous contractions, whilst in the trigone these structures appear to have little influence. The mechanism for generating spontaneous contractions in the intact strips seems to be linked to the urothelium and suburothelium in the dome but not in the trigone.

Protective Effect of Creatine Elevation against Ischaemia Reperfusion Injury Is Retained in the Presence of Co-Morbidities and during Cardioplegia.

AIMS: Ischaemic heart disease is most prevalent in the ageing population and often exists with other comorbidities; however the majority of laboratory research uses young, healthy animal models. Several recent workshops and focus meetings have highlighted the importance of using clinically relevant models to help aid translation to realistic patient populations. We have previously shown that mice over-expressing the creatine transporter (CrT-OE) have elevated intracellular creatine levels and are protected against ischaemia-reperfusion injury. Here we test whether elevating intracellular creatine levels retains a cardioprotective effect in the presence of common comorbidities and whether it is additive to protection afforded by hypothermic cardioplegia. METHODS AND RESULTS: CrT-OE mice and wild-type controls were subjected to transverse aortic constriction for two weeks to induce compensated left ventricular hypertrophy (LVH). Hearts were retrogradely perfused in Langendorff mode for 15 minutes, followed by 20 minutes ischaemia and 30 minutes reperfusion. CrT-OE hearts exhibited significantly improved functional recovery (Rate pressure product) during reperfusion compared to WT littermates (76% of baseline vs. 59%, respectively, P = 0.02). Aged CrT-OE mouse hearts (78±5 weeks) also had enhanced recovery following 15 minutes ischaemia (104% of baseline vs. 67%, P = 0.0007). The cardioprotective effect of hypothermic high K+ cardioplegic arrest, as used during cardiac surgery and donor heart transplant, was further enhanced in prolonged ischaemia (90 minutes) in CrT-OE Langendorff perfused mouse hearts (76% of baseline vs. 55% of baseline as seen in WT hearts, P = 0.02). CONCLUSIONS: These observations in clinically relevant models further support the development of modulators of intracellular creatine content as a translatable strategy for cardiac protection against ischaemia-reperfusion injury.

Relationship between obstructive sleep apnea severity and sleep, depression and anxiety symptoms in newly-diagnosed patients.

Obstructive sleep apnea (OSA) occurs in at least 10% of the population, and leads to higher morbidity and mortality; however, relationships between OSA severity and sleep or psychological symptoms are unclear. Existing studies include samples with wide-ranging comorbidities, so we assessed relationships between severity of OSA and common sleep and psychological disturbances in recently diagnosed OSA patients with minimal co-morbidities. We studied 49 newly diagnosed, untreated OSA patients without major co-morbidities such as mental illness, cardiovascular disease, or stroke; subjects were not using psychoactive medications or tobacco (mean +/- std age: 46.8+/-9.1 years; apnea/hyponea index [AHI]: 32.1+/-20.5 events/hour; female/male: 12/37; weight <125 kg). We evaluated relationships between the AHI and daytime sleepiness (Epworth Sleepiness Scale; ESS), sleep quality (Pittsburg Sleep Quality Index; PSQI), depressive symptoms (Beck Depression Inventory-II; BDI), and anxiety symptoms (Beck Anxiety Inventory; BAI), as well as sex and body mass index (BMI). AHI was similar in females and males. Mean levels of all symptoms were above normal thresholds, but AHI was not correlated with age, ESS, PSQI, BDI, or BAI; only BMI was correlated with OSA severity. No differences in mean AHI appeared when subjects were grouped by normal versus elevated values of ESS, PSQI, BDI, or BAI. Consistent with other studies, a strong link between OSA severity and psychological symptoms did not appear in these newly diagnosed patients, suggesting that mechanisms additional to the number and frequency of hypoxic events and arousals occurring with apneas contribute to adverse health effects in OSA. OSA patients presenting with mild or moderate severity, and no major co-morbidities will not necessarily have low levels of sleep or psychological disturbances.

Recent advances in basic science for overactive bladder.

PURPOSE OF REVIEW: Detrusor overactivity is a relatively common yet embarrassing symptom complex with significant impact on quality of life. The mainstay of current pharmacological treatment involves the use of muscarinic receptor antagonists, but their therapeutic effectiveness is limited by a combination of limited efficacy and troublesome side effects and has recently been challenged by Herbison et al. Recognition of the limitations of existing therapy has started the search for pharmacotherapeutic agents acting on alternative pathways underlying detrusor overactivity with the intention of improving storage symptoms of urgency, frequency and urge incontinence. RECENT FINDINGS: Recent research has suggested that several transmitters may modulate bladder storage. However, no agents currently available, acting via mechanisms other than muscarinic receptors have entered clinical practice so far. It is clear that far from being a passive container for urine, the urothelium is a crucial area within the bladder wall and its functions are complex and only now beginning to be appreciated. The release of several neurotransmitters from urothelium in response to distension and its action on receptors on sensory neurons is being increasingly recognized. The role for this afferent stimulation on the micturition reflex is gradually gaining importance in the pathophysiology of detrusor overactivity. SUMMARY: In this article, the recent developments in basic science related to the pathogenesis and pharmacological basis for future drug targets for effective management of overactive bladder are discussed.