Cross-talk between cAMP and Ca2+ signalling in cardiac pacemaker cells involves IP3-evoked Ca2+ release and stimulation of adenylyl cyclase 1

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. Here we investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sinoatrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.46 % to 8.17% (P = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.20 %, ST034307 = 16.32 %; P > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transient amplitude (CaT) generated by 10μM PE in the presence and absence of 1μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; P = 0.004), but did not result in an inhibition of CaT amplitude increase in response to PE in atrial cells. The results presented here demonstrate the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Ultra-deep through-skull mouse brain imaging via the combination of skull optical clearing and three-photon microscopy

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and thee-photon fluorescence microscopy (3PM). The Visible-NIR-II compatible Skull Optical Clearing Agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1 mm and neuronal imaging depth of >700 μm through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.

Acute exposure to extracellular BTP2 does not inhibit Ca2+ release during EC coupling in intact skeletal muscle fibers

The inhibitor of store-operated Ca2+ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca2+ leak and electrically evoked Ca2+ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca2+ release during excitation–contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca2+ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca2+ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca2+ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca2+ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca2+ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.

Mapping of individual sensory nerve axons from digits to spinal cord with the Transparent Embedding Solvent System

Understanding the connections and projections of neurons has been a fundamental issue for neuroscience. Although strategies have been developed to map the projection of individual axons within the mouse brain, high resolution mapping of individual peripheral nerve axons in peripheral organs or spinal cord has never been achieved. Here, we designed the Transparent Embedding Solvent System (TESOS) method and developed a technical pipeline for imaging, reconstructing and analyzing large samples containing various tissue types at sub-micron resolution. The mouse whole body was reconstructed at micron-scale resolution. We were able to image, reconstruct and analyze the complete axonal projection of individual sensory neurons within an intact mouse paw or spinal cord at sub-micron resolution. Furtherly, we imaged and reconstructed the entire projection course of individual sensory neurons from adult mouse digits to the spinal cord. The TESOS method provides an efficient tool for micron-scale connectome mapping of the peripheral nervous system.

A Novel Small Molecule Troponin Activator Increases Cardiac Contractile Function Without Negative Impact on Energetics

Background: Current heart failure (HF) therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (i.e. calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents - myotropes - activate the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. Methods: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine (PCr) and ATP concentrations and ATP production were assessed by 31 P NMR spectroscopy on isolated perfused rat hearts. Results: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased rate pressure product (RPP). Dobutamine increased both developed pressure (DevP) and heart rate (HR) accompanied by decreased PCr to ATP ratio and decreased free energy of ATP hydrolysis (ΔG~ ATP ) and elevated left ventricular end-diastolic pressure (LVEDP). In contrast, the TA1 increased DevP without any effect on HR, LVEDP, PCr/ATP ratio or ΔG~ ATP . Conclusions: Novel myotrope, TA1, increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.

Lineage tracing and single-cell analysis reveal proliferative Prom1+ tumour-propagating cells and their dynamic cellular transition during liver cancer progression

ObjectiveHepatocellular carcinoma (HCC) has high intratumoral heterogeneity, which contributes to therapeutic resistance and tumour recurrence. We previously identified Prominin-1 (PROM1)/CD133 as an important liver cancer stem cell (CSC) marker in human HCC. The aim of this study was to investigate the heterogeneity and properties of Prom1+ cells in HCC in intact mouse models.DesignWe established two mouse models representing chronic fibrotic HCC and rapid steatosis-related HCC. We performed lineage tracing post-HCC induction using Prom1C-L/+; Rosa26tdTomato/+ mice, and targeted depletion using Prom1C-L/+; Rosa26DTA/+ mice. Single-cell RNA sequencing (scRNA-seq) was carried out to analyse the transcriptomic profile of traced Prom1+ cells.ResultsProm1 in HCC tumours marks proliferative tumour-propagating cells with CSC-like properties. Lineage tracing demonstrated that these cells display clonal expansion in situ in primary tumours. Labelled Prom1+ cells exhibit increasing tumourigenicity in 3D culture and allotransplantation, as well as potential to form cancers of differential lineages on transplantation. Depletion of Prom1+ cells impedes tumour growth and reduces malignant cancer hallmarks in both HCC models. scRNA-seq analysis highlighted the heterogeneity of Prom1+ HCC cells, which follow a trajectory to the dedifferentiated status with high proliferation and stem cells traits. Conserved gene signature of Prom1 linage predicts poor prognosis in human HCC. The activated oxidant detoxification underlies the protective mechanism of dedifferentiated transition and lineage propagation.ConclusionOur study combines in vivo lineage tracing and scRNA-seq to reveal the heterogeneity and dynamics of Prom1+ HCC cells, providing insights into the mechanistic role of malignant CSC-like cells in HCC progression.

Mechanical mapping of mammalian follicle development using Brillouin microscopy

In early mammalian development, the maturation of follicles containing the immature oocytes is an important biological process as the functional oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. Despite recent work demonstrating the regulatory role of mechanical stress in oocyte growth, quantitative studies of ovarian mechanical properties remain lacking both in vivo and ex vivo. In this work, we quantify the material properties of ooplasm, follicles and connective tissues in intact mouse ovaries at distinct stages of follicle development using Brillouin microscopy, a non-invasive tool to probe mechanics in three-dimensional (3D) tissues. We find that the ovarian cortex and its interior stroma have distinct material properties associated with extracellular matrix deposition, and that intra-follicular mechanical compartments emerge during follicle maturation. Our work provides an alternative approach to study the role of mechanics in follicle morphogenesis and might pave the way for future understanding of mechanotransduction in reproductive biology, with potential implications for infertility diagnosis and treatment.

TTYH family members form tetrameric complexes within the cell membrane

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs, studied following detergent solubilization, shared a dimeric stoichiometry, their spatial organization differed. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrated that mTTYH1 and mTTYH3 form tetramers at the plasma membrane. Blue-native PAGE and fluorescence-detection size-exclusion chromatography analyses revealed that detergent solubilization results in the dissolution of tetramers into dimers, suggesting a dimer-of-dimers assembly mode. As cross-linking analysis of the soluble extracellular domains also showed tetrameric stoichiometry, we explored the effect of membrane solubilization and disulfide bridges integrity and established their contribution to tetramer stability. Future studies of the native tetrameric TTYH characterized here may illuminate their long-sought cellular function.