ß-arrestin-biased signaling through the ß2-adrenergic receptor promotes cardiomyocyte contraction.

ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.

S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction.

Restoring expression levels of the EF-hand calcium (Ca(2+)) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca(2+) handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca(2+) resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca(2+) leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca(2+)- and ß-adrenergic receptor-triggered proarrhythmogenic SR Ca(2+) leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca(2+) leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca(2+) leak in HF, combining antiarrhythmic potency with chronic inotropic actions.

Ca²âº entry via Trpm2 is essential for cardiac myocyte bioenergetics maintenance.

Ubiquitously expressed Trpm2 channel limits oxidative stress and preserves mitochondrial function. We first demonstrated that intracellular Ca(2+) concentration increase after Trpm2 activation was due to direct Ca(2+) influx and not indirectly via reverse Na(+)/Ca(2+) exchange. To elucidate whether Ca(2+) entry via Trpm2 is required to maintain cellular bioenergetics, we injected adenovirus expressing green fluorescent protein (GFP), wild-type (WT) Trpm2, and loss-of-function (E960D) Trpm2 mutant into left ventricles of global Trpm2 knockout (gKO) or WT hearts. Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O2(.-)) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O2(.-) levels in gKO myocytes and only in the presence of extracellular Ca(2+), indicating sustained Ca(2+) entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (+dP/dt) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower +dP/dt. We conclude 1) cardiac Trpm2-mediated Ca(2+) influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca(2+) influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy.

Extrastriatal binding of [¹²³I]FP-CIT in the thalamus and pons: gender and age dependencies assessed in a European multicentre database of healthy controls.

PURPOSE: Apart from binding to the dopamine transporter (DAT), [(123)I]FP-CIT shows moderate affinity for the serotonin transporter (SERT), allowing imaging of both monoamine transporters in a single imaging session in different brain areas. The aim of this study was to systematically evaluate extrastriatal binding (predominantly due to SERT) and its age and gender dependencies in a large cohort of healthy controls. METHODS: SPECT data from 103 healthy controls with well-defined criteria of normality acquired at 13 different imaging centres were analysed for extrastriatal binding using volumes of interest analysis for the thalamus and the pons. Data were examined for gender and age effects as well as for potential influence of striatal DAT radiotracer binding. RESULTS: Thalamic binding was significantly higher than pons binding. Partial correlations showed an influence of putaminal DAT binding on measured binding in the thalamus but not on the pons. Data showed high interindividual variation in extrastriatal binding. Significant gender effects with 31 % higher binding in women than in men were observed in the thalamus, but not in the pons. An age dependency with a decline per decade (±standard error) of 8.2 ± 1.3 % for the thalamus and 6.8 ± 2.9 % for the pons was shown. CONCLUSION: The potential to evaluate extrastriatal predominant SERT binding in addition to the striatal DAT in a single imaging session was shown using a large database of [(123)I]FP-CIT scans in healthy controls. For both the thalamus and the pons, an age-related decline in radiotracer binding was observed. Gender effects were demonstrated for binding in the thalamus only. As a potential clinical application, the data could be used as a reference to estimate SERT occupancy in addition to nigrostriatal integrity when using [(123)I]FP-CIT for DAT imaging in patients treated with selective serotonin reuptake inhibitors.

Direct evidence of intracrine angiotensin II signaling in neurons.

The existence of a local renin-angiotensin system (RAS) in neurons was first postulated 40 years ago. Further studies indicated intraneuronal generation of ANG II. However, the function and signaling mechanisms of intraneuronal ANG II remained elusive. Since ANG II type 1 receptor (AT1R) is the major type of receptor mediating the effects of ANG II, we used intracellular microinjection and concurrent Ca(2+) and voltage imaging to examine the functionality of intracellular AT1R in neurons. We show that intracellular administration of ANG II produces a dose-dependent elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) in hypothalamic neurons that is sensitive to AT1R antagonism. Endolysosomal, but not Golgi apparatus, disruption prevents the effect of microinjected ANG II on [Ca(2+)]i. Additionally, the ANG II-induced Ca(2+) response is dependent on microautophagy and sensitive to inhibition of PLC or antagonism of inositol 1,4,5-trisphosphate receptors. Furthermore, intracellular application of ANG II produces AT1R-mediated depolarization of hypothalamic neurons, which is dependent on [Ca(2+)]i increase and on cation influx via transient receptor potential canonical channels. In summary, we provide evidence that intracellular ANG II activates endolysosomal AT1Rs in hypothalamic neurons. Our results point to the functionality of a novel intraneuronal angiotensinergic pathway, extending the current understanding of intracrine ANG II signaling.

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation.

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1-/- mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

RATIONALE: Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. OBJECTIVE: To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. METHODS AND RESULTS: Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. CONCLUSIONS: Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the microvascular therapeutic potential of S100A1.

Loudness dependence of auditory evoked potentials in patients with borderline personality disorder--impact of psychopathology.

Alterations of the central serotonergic system are considered to be involved in the pathophysiology of borderline personality disorder (BPD). The loudness dependence of the N1/P2 component of auditory evoked potentials (LD) has been shown to indirectly reflect central serotonergic activity. The aim of this study was to investigate LD in patients with BPD compared to healthy controls, and to evaluate the association between LD and psychopathology such as anxiety, anger or impulsiveness. Female patients with BPD were included and compared to age- and sex-matched healthy subjects. Self-rating instruments, such as the State-Trait Anxiety Inventory (STAI), State-Trait Anger Expression Inventory (STAXI), and the Barratt Impulsiveness Scale (BIS) were used to assess clinical scores of anxiety, anger, and impulsiveness. Evoked potentials were recorded following the application of acoustic stimuli with increasing intensities; the LD was analysed using dipole source analysis. The mean LD was significantly higher in patients with BPD compared to controls. In the entire sample there were significant positive correlations of LD with state anxiety scores and STAXI subscores. The data contribute to the knowledge of neurophysiological alterations in patients with BPD, supporting the hypothesis of serotonergic dysregulation in the pathophysiology of the disorder. The significant clinical correlations suggest monoaminergic modulations of psychopathology on the symptom level.

Reduced cardioprotective action of adiponectin in high-fat diet-induced type II diabetic mice and its underlying mechanisms.

Diabetes exacerbates ischemic heart disease morbidity and mortality via incompletely understood mechanisms. Although adiponectin (APN) reduces myocardial ischemia/reperfusion (MI/R) injury in nondiabetic animals, whether APN's cardioprotective actions are altered in diabetes, a pathologic condition with endogenously reduced APN, has never been investigated. High-fat diet (HD)-induced diabetic mice and normal diet (ND) controls were subjected to MI via coronary artery ligation, and given vehicle or APN globular domain (gAPN, 2 µg/g) 10 min before reperfusion. Compared to ND mice (where gAPN exerted pronounced cardioprotection), HD mice manifested greater MI/R injury, and a tripled gAPN dose was requisite to achieve cardioprotective extent seen in ND mice (i.e., infarct size, apoptosis, and cardiac function). APN reduces MI/R injury via AMP-activated protein kinase (AMPK)-dependent metabolic regulation and AMPK-independent antioxidative/antinitrative pathways. Compared to ND, HD mice manifested significantly blunted gAPN-induced AMPK activation, basally and after MI/R (p<0.05). Although both low- and high-dose gAPN equally attenuated MI/R-induced oxidative stress (i.e., NADPH oxidase expression and superoxide production) and nitrative stress (i.e., inducible nitric oxide synthase expression, nitric oxide production, and peroxynitrite formation) in ND mice, only high-dose gAPN efficaciously did so in HD mice. We demonstrate for the first time that HD-induced diabetes diminished both AMPK-dependent and AMPK-independent APN cardioprotection, suggesting an unreported diabetic heart APN resistance.

[123I] ADAM brainstem binding correlates with the loudness dependence of auditory evoked potentials.

The in vivo assessment of brain serotonergic function might be of clinical relevance in neuropsychiatry. The loudness dependence of auditory evoked potentials (LD) has been proposed as an indirect indicator of cortical serotonergic activity, whereas single photon emission computed tomography (SPECT) and [123I]ADAM allow the selective assessment of brain serotonin transporters (SERT). The aim of this study was to investigate LD and SERT availability as independent variables of the brain serotonergic system in healthy volunteers. Fifteen (six male, nine female) subjects received both neurophysiological and imaging investigations. Evoked potentials were recorded following the application of acoustic stimuli with increasing intensities; the LD was analyzed using dipole source analysis. SPECT was performed four hours after injection of 137 +/- 11.4 MBq [123I]ADAM. As a measure of SERT availability specific ADAM brainstem binding was used. LD correlated significantly with SERT availability (Pearson's correlations: rho = -0.57, p < 0.05). The correlations remained significant after controlling for the effects of age or gender (partial correlations: rho = -0.60, p < 0.05) but were pronounced in the female group (rho = -0.83, p < 0.01). Associations between LD and SERT availability contribute to the understanding of the central serotonergic system and further validate the use of neurophysiological approaches as indirect measures of neurochemical brain activity.

FET PET for the evaluation of untreated gliomas: correlation of FET uptake and uptake kinetics with tumour grading.

PURPOSE: Treatment and prognosis of gliomas depend on their histological tumour grade. The aim of the study was to evaluate the potential of [(18)F]fluoroethyltyrosine (FET) PET for non-invasive tumour grading in untreated patients. METHODS: Dynamic FET PET studies were performed in 54 patients who, based on MRI, were estimated to have low grade (LG; n = 20), intermediate (WHO II-III; n = 4) or high grade (HG; n = 30) tumours. For standard evaluation, tumour SUV(max) and the ratio to background (SUV(max)/BG) were calculated (sum image: 20-40 min). For dynamic evaluation, mean SUV values within a 90% isocontour ROI (SUV90) and the SUV90/BG ratios were determined for each time frame to evaluate the course of FET uptake. Results were correlated with histopathological findings from PET-guided stereotactic biopsies. RESULTS: Histology revealed gliomas in all patients. Using the standard method a statistically significant difference (p = 0.001) was found between LG (n = 20; SUV(max)/BG: 2.16 +/- 0.98) and HG (n = 34; SUV(max)/BG: 3.29 +/- 1.06) gliomas (opt. threshold 2.58: SN71%/SP85%/area under ROC curve [AUC]:0.798), however, with a marked overlap between WHO II to IV tumours. Time activity curves showed slight increase in LG, whereas HG tumours presented with an early peak (10-20 min) followed by a decrease. Dynamic evaluation successfully separated LG from HG gliomas with higher diagnostic accuracy (SN94%/SP100%/AUC:0.967). CONCLUSIONS: Based on the ratio-based method, a statistically significant difference was found between LG and HG gliomas. Due to the interindividual variability, however, no reliable individual grading was possible. In contrast, dynamic evaluation allowed LG and HG gliomas to be differentiated with high diagnostic power and, thus, should supplement the conventional method.

[I-123] ADAM and SPECT in patients with borderline personality disorder and healthy control subjects.

OBJECTIVE: Serotonergic dysfunction is considered to be involved in the pathophysiology of borderline personality disorder (BPD). The aim of this study was to investigate serotonin transporter availability in patients with BPD as a marker of the central serotonergic system. METHODS: Eight unmedicated patients with BPD and 9 healthy control subjects received single photon emission computed tomography (SPECT) 4 hours after injection of 185 MBq [I-123] ADAM (2-([2-([dimethylamino]methyl)phenyl]thio)). As a measure of brain serotonin transporter (SERT) availability, ratios of specific-to-nonspecific [I-123] ADAM binding for the brainstem and hypothalamus were calculated with an occipital reference. Levels of impulsiveness and depressive symptoms were assessed with the Barratt Impulsiveness Scale and the Beck Depression Inventory. RESULTS: Mean specific-to-nonspecific ratios showed a 43% higher brainstem and a 12% higher hypothalamus ADAM binding in patients, compared with control subjects. We found significant correlations of ADAM binding with both age and impulsiveness but not depression. Associations of BIS scores with ADAM binding remained significant after controlling for age and depression (r = 0.69, p < 0.01). CONCLUSION: The study provides evidence of a serotonergic dysfunction in patients with BPD and suggests a serotonergic component in the pathophysiology of the disorder. SERT binding reflected the level of impulsiveness as a common feature in BPD.

Thalamic hypofunction in alcohol hallucinosis: FDG PET findings.

We report findings obtained with positron emission tomography with fluorodeoxyglucose-F18 (FDG PET) as tracer in two patients with acute alcohol hallucinosis. In line with previous findings, both patients had a significant hypometabolism in the left relative to the right thalamus. When the second patient was retested after clinical recovery, FDG PET findings were normalized, suggesting the probable thalamic dysfunction to be a functional rather than a structural abnormality.