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G-protein coupled receptors (GPCRs) are encoded by nonabundant mRNAs, and it is difficult to detect them reliably with the highly parallel methods that are in general use. Because of this, we developed and validated a sensitive, specific, semi-quantitative method for detecting these transcripts. We have used the method to profile GPCR transcripts in white blood cells (WBCs)–B, CD4, CD8, NK, and dendritic cells; monocytes, and macrophage-like monocytes treated with granulocyte-macrophage colony-stimulating factor–as well as platelets. On average, the white cells studied expressed 160 receptor mRNAs (range, 123–206). Platelets made 69. Some, but far from all, of the receptors we found have been detected earlier. We believe our data should stimulate studies of receptor function and contribute to drug development. [ABSTRACT FROM AUTHOR]

The relationship between duration perception and the feeling of time passing (passage of time) is not yet understood. In the present study, we assessed introspective reaction times (RT) and passage of time judgments in a speeded RT task. Task difficulty was manipulated in a numerical comparison task by numerical distance (distance from the number 45) and notation (digit vs. word). The results showed that both effects were reflected in introspective RTs, replicating previous results. Moreover, passage of time judgments showed a very similar pattern, with slower passage of time for more difficult comparisons. These results suggest that in the millisecond range judgments of duration and passage of time largely mirror each other when participants introspect about their own RT performance. [ABSTRACT FROM AUTHOR]

The role and outcome of the muscarinic M2 acetylcholine receptor (M2R) signaling in healthy and diseased cardiomyocytes is still a matter of debate. Here, we report that the long isoform of the regulator of G protein signaling 3 (RGS3L) functions as a switch in the muscarinic signaling, most likely of the M2R, in primary cardiomyocytes. High levels of RGS3L, as found in heart failure, redirect the Gi-mediated Rac1 activation into a Gi-mediated RhoA/ROCK activation. Functionally, this switch resulted in a reduced production of reactive oxygen species (− 50%) in cardiomyocytes and an inotropic response (+ 18%) in transduced engineered heart tissues. Importantly, we could show that an adeno-associated virus 9-mediated overexpression of RGS3L in rats in vivo, increased the contractility of ventricular strips by maximally about twofold. Mechanistically, we demonstrate that this switch is mediated by a complex formation of RGS3L with the GTPase-activating protein p190RhoGAP, which balances the activity of RhoA and Rac1 by altering its substrate preference in cardiomyocytes. Enhancement of this complex formation could open new possibilities in the regulation of the contractility of the diseased heart. [ABSTRACT FROM AUTHOR]

The assessment of proarrhythmic risks of drugs remains challenging. To evaluate the suitability of rat engineered heart tissue (EHT) for detecting proarrhythmic effects. We monitored drug effects on spontaneous contractile activity and, in selected cases, on action potentials (sharp microelectrode) and Ca2+ transients (Fura-2) and contraction under electrical pacing. The Ito-blocker inhibitor 4-aminopyridine increased action potential duration and T2 and caused aftercontractions, which were abolished by inhibitors of ryanodine receptors (RyR2; JTV-519) or sodium calcium exchanger (NCX; SEA0400). 77 Drugs were then tested at 1-10-1009 free therapeutic plasma concentrations (FTPC): Inhibitors of IKr, IKs, Ito, antiarrhythmics (8), drugs withdrawn from market for torsades des pointes arrhythmias (TdP, 5), drugs with measurable (7) or isolated TdP incidence (13), drugs considered safe (14), 28 new chemical entities (NCE). Inhibitors of IKr or IKs had no effect alone, but substantially prolonged relaxation time (T2) when combined at high concentration. 15/33 drugs associated with TdP and 6/14 drugs considered non-torsadogenic (cibenzoline, diltiazem, ebastine, ketoconazole, moxifloxacin, and phenytoin) induced concentration- dependent T2 prolongations (10-100× FTPC). Bepridil, desipramine, imipramine, thioridazine, and erythromycin induced irregular beating. Three NCE prolonged T2, one reduced force. Drugs inhibiting repolarization prolong relaxation in rat EHTs and cause aftercontractions involving RyR2 and NCX. Insensitivity to IKr inhibitors makes rat EHTs unsuitable as general proarrhythmia screen, but favors detection of effects on Ito, IKs + Ito or IKs + IKr. Screening a large panel of drugs suggests that effects on these currents, in addition to IKr, are more common than anticipated. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM) is a myocardial disease associated with mutations in sarcomeric genes. Three mutations were found in ANKRD1, encoding ankyrin repeat domain 1 (ANKRD1), a transcriptional co-factor located in the sarcomere. In the present study, we investigated whether expression of HCM-associated ANKRD1 mutations affects contraction parameters after gene transfer in engineered heart tissues (EHTs). EHTs were generated from neonatal rat heart cells and were transduced with adeno-associated virus encoding GFP or myc-tagged wild-type (WT) or mutant (P52A, T123M, or I280V) ANKRD1. Contraction parameters were analyzed from day 8 to day 16 of culture, and evaluated in the absence or presence of the proteasome inhibitor epoxomicin for 24 h. Under standard conditions, only WT- and T123M-ANKRD1 were correctly incorporated in the sarcomere. T123M- ANKRD1-transduced EHTs exhibited higher force and velocities of contraction and relaxation than WT- P52A- and I280V-ANKRD1 were highly unstable, not incorporated into the sarcomere, and did not induce contractile alterations. After epoxomicin treatment, P52A and I280V were both stabilized and incorporated into the sarcomere. I280V-transduced EHTs showed prolonged relaxation. These data suggest different impacts of ANKRD1 mutations on cardiomyocyte function: gain-of-function for T123M mutation under all conditions and dominant-negative effect for the I280V mutation which may come into play only when the proteasome is impaired. [ABSTRACT FROM AUTHOR]

Increased afterload results in 'pathological' cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2 weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4 % under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6 %), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRs in myocardial disease processes. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure. [ABSTRACT FROM AUTHOR]

Patients with chronic inflammatory rheumatic diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and especially primary Sjögren's syndrome (SS), are at higher risk than the general population of developing B-cell non-Hodgkin lymphoma (NHL). Analyses of the association between various lymphoma subtypes and specific disease entities suggest that this association might be mediated by disease-specific mechanisms, as well as by mechanisms unique to lymphoma subtype. These specific associations can provide important information about abnormal B-cell stimulation in these conditions. Patients with primary SS, SLE and RA are at high risk of developing diffuse large B-cell lymphomas, a group of high-grade NHLs with remarkable heterogeneity. Patients with primary SS are at particularly high risk of developing marginal-zone B-cell lymphomas. The risk factors of lymphoma development in primary SS seem to be closely related to the underlying mechanisms of abnormal stimulation and/or impaired censoring mechanisms of B cells. In patients with RA and SLE, more intense disease activity and/or long-lasting disease might be indications of a higher risk of lymphoma development. This Review will focus on the risk of lymphoma, common and disease-specific mechanisms of B-cell lymphoma development, and on the clinical consequences of lymphoma in patients with inflammatory rheumatic diseases. [ABSTRACT FROM AUTHOR]

Reverse transcription-polymerase chain reaction (RT-PCR) of individual B-lymphocytes has been shown to be a powerful tool for the simultaneous analysis of different mRNA specificities in both malignant and non-malignant B cell subpopulations. However, especially for longitudinal studies, this may also require analyses of cryopreserved cells. Therefore, the current study assessed whether cryopreserved (liquid nitrogen, dimethyl sulfoxide [DMSO] – stored) viable B cells are an alternative source for single cell RT-PCR analysis. Fresh (non-frozen) and post-thawed human peripheral blood B cells were analyzed by fluorescence-activated cell sorting (FACS). As a result, different B cell subpopulations could be reliably stained and separated from both fresh and post-thawed cells by four-color flow cytometry, although slightly diminished fluorescence intensities of some subpopulation markers were observed when analyzing cryopreserved cells. Subsequently, viable individual CD19+CD27+ memory B cells were sorted into single wells and analyzed for the expression of mRNA transcripts of the ‘house-keeping gene’ glyceraldehyde phosphate dehydrogenase (GAPD), the constitutive B cell homing receptor CXCR4, and immunoglobulin heavy chain variable region (IgVH) genes by nested RT-PCR protocols. Comparing both B cell sources, RT-PCR analysis revealed comparable yields of cells expressing transcripts for the three mRNA specificities tested (GAPD, CXCR4, IgVH) indicating the integrity of the respective mRNAs in cryopreserved B␣cells. In conclusion, these data indicate that optimally cryopreserved B cells may be an alternative source for single-cell RT-PCR analysis, especially in longitudinal B cell studies. However, the settings for both FACS analysis and RT-PCR should be re-evaluated for each distinct subpopulation and target mRNA of interest when analyzing post-thawed cells. [ABSTRACT FROM AUTHOR]

Severalprocedures for inactivating viruses are used presently in the context of bonetissue transplants. Common methods used are gamma irradiation (25kGy), treatment with moist heat (82.5°C/15min., lobator-sd2-system) as well as chemical sterilisation usingperacetic acid-ethanol treatment (PES, 2% peracetic acid, 96% ethanol, Aqua[2:1:1], 200 mbar, agitation, 4 hours). Based on national andinternational guidelines, we tested the antivirucidal effectiveness of thesemethods in human bone transplants. Three enveloped viruses: humanimmunodeficiency virus type 2 (HIV-2), pseudorabies virus (PRV), bovine virusdiarrhoea virus (BVDV), and three non–enveloped viruses were used:hepatitis A virus (HAV), poliovirus (PV-1), porcine/bovine parvovirus (PPV,BPV). Defatted spongiosa cuboids served as model in chemical treatmentexperiments, while cortical diaphyses were used in gamma irradiationexperiments, and the effects of thermal treatment were tested in preparedfemoral heads. The log10 reduction was measured by cytopathogeniceffects after virus titration (TCID50/mL). A dose of at least 33.9kGy (bone model) at −30 ± 5°C wasnecessary to achieve a sufficient reduction (4 log10 steps) of BPV,the most resistant one of all viruses investigated. Thermal treatment as wellasPES treatment led to a reduction of virus titres by more than 4log10. Only HAV showed a reduction below 4 log10 (2.87)with PES. After validation of the defatting step included for HAV-infectedcells, a HAV-reduction of over 7 log10 was found. All threesterilisation methods tested are recommended for bone transplant sterilisation,but only provided that additional safety measures (anamnestic informations,infectious serology, PCR in case of multiorgan donors) are taken. [ABSTRACT FROM AUTHOR]

A lysyl-proline derivate (LP) known to stimulate angiogenesis and formation of granulation tissue was tested as a local additive to allogeneic demineralized bone matrix (DBM) using a rat craniotomy model. Peracetic-acid sterilized DBM (10 mg/defect) was implanted into three groups of 45 animals each with 0, 6 and 20 μg LP. Subsequent evaluation was done by descriptive histology, histomorphometry, and determination of the calcium content of the explants 7, 14, 28, 42 and 84 days post-implantation. Grafting with DBM alone resulted in defect bridging by newly formed bone with incorporated DBM residues on day 84. Addition of LP to the implants caused an enhanced capillarization on day 14 and 28 as well as an enhanced mineralization on day 14, 28, 42 and 84. Both effects were dose-dependent. These data suggest that the local application of a synthetic angiogenic factor significantly improve bone regeneration in DBM-grafted trephine defects in rats. Thereby, they reinforce the opinion that early angiogenesis is crucial for a number of subsequent events in the bone regeneration process. [ABSTRACT FROM AUTHOR]

G protein–coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 μg of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines. [ABSTRACT FROM AUTHOR]

Human iPSC-derived engineered heart tissue (hEHT) has been used to remuscularize injured hearts in a guinea pig infarction model. While beneficial effects on cardiac remodeling have been demonstrated, the arrhythmogenic potential of hEHTs is a major concern. We investigated whether hiPSC-derived hEHTs increase the incidence of ventricular arrhythmias. HEHTs were created from human iPSC-derived cardiomyocytes and endothelial cells. Left-ventricular cryo-injury was induced in guinea pigs (n = 37) and telemetry sensors for continuous ECG monitoring were implanted. 7 days following the cryo-injury, hEHTs or cell-free constructs were transplanted into the surviving animals (n = 15 and n = 9). ECGs were recorded over the following 28 days. 10 hEHT animals and 8 control animals survived the observation period and were included in the final analysis. After implantation of hEHTs or cell-free constructs, ventricular arrhythmias (premature ventricular contractions, couplets, triplets and non-sustained ventricular tachycardia) were observed in animals of both groups. The fraction of animals with the respective arrhythmias as well as the rate of arrhythmic events did not differ between groups. Following hEHT implantation, no clinically relevant sustained ventricular tachycardia or ventricular fibrillation was detected. Our telemetric data provides first evidence for the electrical safety of human iPSC-derived EHTs in this experimental model, thereby supporting further development of this approach. [ABSTRACT FROM AUTHOR]