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Abstract Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β1-adrenoceptors, arrestin-biased signalling via β2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol’s cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system.

Abstract Background Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3′,5′-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. Results To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. Conclusions We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs.

PurposeWe previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective.MethodsWild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET).ResultsSimultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility.ConclusionsTogether, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.

The problem of interference is one of the most complex issues related to language interaction, so it is especially important to investigate its workings on the example of the language of Russian Germans in the Kirov region. The article realises the historical and linguocultural approaches to the study of the interrelationship between folk-colloquial speech and the traditional culture of Russian Germans, residing on the territory of the Kirov region. The authors present the results of an in-depth analysis of interference features in the Russian speech of German bilinguals under the influence of the German language and its dialects, namely, the phonetic, lexical and grammatical features that occur under the influence of interference with the German language. The Russian speech of German bilinguals is heterogeneous and varies from "virtually without an accent" to "unnatural" for Russian monolingual hearing. The interaction of the Russian and German languages in the speech of German bilinguals resulted in the increased invasion of the norms of one language system into the framework of another language. This leads to the so-called levelling of the interacting languages. In other words, we see the emergence of a third–intermediate system that does not coincide either with the German or Russian languages and performs in the bilingual consciousness an adaptive function to the environment language. This study contributes to German dialectology, enriching both the theory and typology of island dialects, which retain archaic features and the theory and practice of scientifically grounded language policy and language preservation.