Dynamics of the Glycogen ß-Particle Number in Rat Hepatocytes during Glucose Refeeding.

Glycogen is an easily accessible source of energy for various processes. In hepatocytes, it can be found in the form of individual molecules (ß-particles) and their agglomerates (α-particles). The glycogen content in hepatocytes depends on the physiological state and can vary due to the size and number of the particles. Using biochemical, cytofluorometric, interferometric and morphometric methods, the number of ß-particles in rat hepatocytes was determined after 48 h of fasting at different time intervals after glucose refeeding. It has been shown that after starvation, hepatocytes contain ~1.6 × 108 ß-particles. During refeeding, their number of hepatocytes gradually increases and reaches a maximum (~5.9 × 108) at 45 min after glucose administration, but then quickly decreases. The data obtained suggest that in cells there is a continuous synthesis and degradation of particles, and at different stages of life, one or another process predominates. It has been suggested that in the course of glycogenesis, pre-existing ß-particles are replaced by those formed de novo. The main contribution to the deposition of glycogen is made by an increase in the glucose residue number in its molecules. The average diameter of ß-particles of glycogen during glycogenesis increases from ~11 nm to 21 nm.

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure.

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.

Increased RyR2 activity is exacerbated by calcium leak-induced mitochondrial ROS.

Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under ß-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.

Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease.

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.

Rat Left Ventricular Cardiomyocytes Characterization in the Process of Postinfarction Myocardial Remodeling.

Ischemic lesions of the heart, including myocardial infarction, are the most common pathologies of human cardiovascular system. Despite all the research and achievements of medicine in this field, the mortality from this disease remains heavy. Therefore, studying of processes occurring in the myocardium in the early and late postinfarction periods remains important. Rat left ventricular cardiomyocyte (CMC) ploidy, hypertrophy, hyperplasia, and ultrastructure were investigated in 2, 6, and 26 weeks after experimental myocardial infarction, caused by permanent ligation of left coronary artery. Cytofluorimetric study of CMC ploidy revealed no difference between normal, sham-operated, and infarcted animals for all the tested stages. However, interference microscopy indicated significant changes in cells size. CMC dry mass of infarcted rats in 2 weeks after surgery was 1.5 times lower than in control and sham operated groups. Electron microscopy analysis of CMC revealed disruption of sarcomere structure. However, in 6 weeks after surgery CMC dry mass was 1.6 times higher than in control. In 26 weeks after myocardial infarction CMC dry mass exceeded control only in peri-infarction zone. Cell counting showed that the number of left ventricular CMC, reduced as a result of myocardial infarction, was not restored during myocardial remodeling. © 2019 International Society for Advancement of Cytometry.

Sub-cellular Electrical Heterogeneity Revealed by Loose Patch Recording Reflects Differential Localization of Sarcolemmal Ion Channels in Intact Rat Hearts.

The cardiac action potential (AP) is commonly recoded as an integral signal from isolated myocytes or ensembles of myocytes (with intracellular microelectrodes and extracellular macroelectrodes, respectively). These signals, however, do not provide a direct measure of activity of ion channels and transporters located in two major compartments of a cardiac myocyte: surface sarcolemma and the T-tubule system, which differentially contribute to impulse propagation and excitation-contraction (EC) coupling. In the present study we investigated electrical properties of myocytes within perfused intact rat heart employing loose patch recording with narrow-tip (2 µm diameter) extracellular electrodes. Using this approach, we demonstrated two distinct types of electric signals with distinct waveforms (single peak and multi-peak AP; AP1 and AP2, respectively) during intrinsic pacemaker activity. These two types of waveforms depend on the position of the electrode tip on the myocyte surface. Such heterogeneity of electrical signals was lost when electrodes of larger pipette diameter were used (5 or 10 µm), which indicates that the electric signal was assessed from a region of <5 µm. Importantly, both pharmacological and mathematical simulation based on transverse (T)-tubular distribution suggested that while the AP1 and the initial peak of AP2 are predominantly attributable to the fast, inward Na+ current in myocyte's surface sarcolemma, the late components of AP2 are likely representative of currents associated with L-type Ca2+ channel and Na+/Ca2+ exchanger (NCX) currents which are predominantly located in T-tubules. Thus, loose patch recording with narrow-tip pipette provides a valuable tool for studying cardiac electric activity on the subcellular level in the intact heart.

A facile synthesis and microtubule-destabilizing properties of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines.

A series of 4-(1H-benzo[d]imidazol-2-yl)-furazan-3-amines (BIFAs) were prepared in good yields (60-90% for each reaction step) via a novel procedure from aminofurazanyl hydroximoyl chlorides and o-diaminobenzenes. The synthetic sequence was run under mild reaction conditions, it was robust and did not require extensive purification of intermediates or final products. Furthermore, there was no need for protection of reactive moieties allowing for the parallel synthesis of diverse BIFA derivatives. Subsequent biological evaluation of the resulting compounds revealed their anti-proliferative effects in the sea urchin embryo model and in cultured human cancer cell lines. The most active compounds showed 0.2-2 µM activities in both assay systems. The unsubstituted benzene ring of the benzoimidazole template as well as the unsubstituted amino group in the furazan ring were essential prerequisites for the antimitotic activity of BIFAs. Compound 57 bearing the 2-chlorophenyl acetamide substituent at the nitrogen atom of the imidazole ring was the most active molecule in the examined set.

Tailoring single-cycle electromagnetic pulses in the 2-9 THz frequency range using DAST/SiO2 multilayer structures pumped at Ti:sapphire wavelength.

We present a numerical parametric study of single-cycle electromagnetic pulse generation in a DAST/SiO2multilayer structure via collinear optical rectification of 800 nm femtosecond laser pulses. It is shown that modifications of the thicknesses of the DAST and SiO2layers allow tuning of the average frequency of the generated THz pulses in the frequency range from 3 to 6 THz. The laser-to-THz energy conversion efficiency in the proposed structures is compared with that in a bulk DAST crystal and a quasi-phase-matching periodically poled DAST crystal and shows significant enhancement.

DAST/SiO2 multilayer structure for efficient generation of 6 THz quasi-single-cycle electromagnetic pulses.

We propose a DAST/SiO(2) multilayer structure for efficient generation of near-single-cycle THz transients with average frequency around 6 THz via collinear optical rectification of 800 nm femtosecond laser pulses. The use of such a composite material allows compensation for the phase mismatch that accompanies THz generation in bulk DAST crystals. The presented calculations indicate a strong increase in the THz generation efficiency in the DAST/SiO(2) structure in comparison to the case of bulk DAST crystal.

Generation of 30 microJ single-cycle terahertz pulses at 100 Hz repetition rate by optical rectification.

We report the generation of 30 microJ single-cycle terahertz pulses at 100 Hz repetition rate by phase-matched optical rectification in lithium niobate using 28 mJ femtosecond laser pulses. The phase-matching condition is achieved by tilting the laser pulse intensity front. Temporal, spectral, and propagation properties of the generated terahertz pulses are presented. In addition, we discuss possibilities for further increasing the energy of single-cycle terahertz pulses obtained by optical rectification.

Rapid prototyping of optical components for surface plasmon polaritons.

Advanced femtosecond laser technology allows the fabrication of arbitrary 2D and 3D dielectric micro- and nanoscale structures by two-photon polymerization (2PP). In this paper, we present first investigations on excitation of surface plasmon polaritons (SPPs) on dielectric 2D structures fabricated on metal surfaces with this technology. Straight and curved line- and dot- structures built of the negative-tone photoresist ORMOCER (organically modified ceramic) are investigated by plasmon leakage radiation microscopy. Polarization dependent excitation efficiencies and focusing of SPPs are investigated.

Terahertz interferometric and synthetic aperture imaging.

The stand-off imaging properties of a terahertz (THz) interferometric array are examined. For this application, the imaged object is in the near-field region limit of the imaging array. In this region, spherical and circular array architectures can compensate for near-field distortions and increase the field of view and depth of focus. Imaging of THz point sources is emphasized to demonstrate the imaging method and to compare theoretical predictions to experimental performance.