Background. We used the “Mitochondrial Membrane Potential Kit” (Sigma-Aldrich) to detected MMP but encountered difficulties by applied this kit because in manual there not JC-10 concentration and it not allowing for account cell type, size, density, differences in incubation time for different cell cultures. Objective. Adaptation of the method for determining MMP (mitochondrial membrane potential) in C2C12 cells using in microplate reader and electron microscope. Design and methods. MMP in C2C12 cells was measured by two way, using fluorescence microscopy (Zeiss, Zen program) and using a plate fluorimeter (CLARIOstar (BMG LABTECH). JC-10 and TMRE dyes (Sigma-Aldrich) were used as fluorescent probes. Results. Optimal conditions for detection changes in mitochondrial membrane potential in C2C12 cells were selected. 100- fold dilution of the dye JC-10 (Dye Loading Solution) and replacement of the manufactured buffer to PBS led to repeatability and reproducibility results. Conclusion. When using ready-made kits for measuring MMP, the method proposed by the manufacturer may not be suitable for the selected cell line. In our study to mouse myoblasts of the C2C12 line, a dilution of the dye for loading was required 100 times compared to that recommended by the manufacturer.

Filamin С is a key an actin-binding protein of muscle cells playing a critical role in maintaining structural integrity and sarcomere organization. FLNC mutations contribute to various types of cardiomyopathies and myopathies through potentially different molecular mechanisms. Here, we described the impact of two clinically distinct FLNC variants (R1267Q associated with arrhythmogenic cardiomyopathy and V2264M associated with restrictive cardiomyopathy) on calcium homeostasis, electrophysiology, and gene expression profile of iPSC-derived patient-specific cardiomyocytes. We demonstrated that R1267Q FLNC variant leads to greater disturbances in calcium dynamics, Nav1.5 kinetics and action potentials compared to V2264M variant. These functional characteristics were accompanied by transcriptome changes in genes linked to action potential and sodium transport as well as structural cardiomyocyte genes. We suggest distinct molecular effects of two FLNC variants linked to different types of cardiomyopathies in terms of myofilament structure, electrophysiology, ion channel function and intracellular calcium homeostasis providing the molecular the bases for their different clinical phenotypes.

Technology of generation of induced pluripotent stem cells followed by differentiation into relevant cell type allows obtaining cell models for studying various inherited human diseases. In the study, an induced pluripotent stem cell (iPSC) line (ICGi046-A) was generated as a result of reprogramming of mononuclear cells of a RASopathy patient carrying pathogenic c.775T>A (p.Ser259Thr) variant in RAF1 to the pluripotent state. The ICGi046-A line demonstrated morphology characteristic of human pluripotent cells, normal karyotype (46,XY), expression of pluripotency markers (OCT4, NANOG, SOX2, TRA-1-60), and capacity to give rise to derivatives of three germ layers during spontaneous differentiation. The iPSC line can be used for studying molecular mechanisms of RASopathies.

Introduction. The development of heart failure is closely associated with the appearance of life threatening arrhythmias, which are often a terminal event for these patients. An analysis of randomized clinical trials of inhibitors of sodium-glucose cotransporter type 2 indicates the clinically significant potential of these drugs as agents with antiarrhythmic properties. However, at the moment the full mechanism by which this effect can be realized is still not fully understood.Aim. To evaluate the effect of empagliflozin on the transmembrane calcium currents and the intracellular calcium transients on isolated ventricular cardiomyocytes of mice under conditions of normoglycemia.Materials and methods. In the experiment, ventricular cardiomyocytes were isolated from 12 outbred male mice. 2 groups were formed: group № 1 – control ventricular cardiomyocytes; group № 2 – ventricular cardiomyocytes after two hours incubation with 5 µmol/L empagliflozin solution. Transmembrane calcium currents were recorded and intracellular calcium transients were assessed.Results and discussion. Incubation of ventricular cardiomyocytes with empagliflozin significantly increased ICa current density and accelerated Ca2+ temporal dynamics. The amplitude of the Ca2+ wave and the rate of rise and decay were increased and the duration of the Ca2+ wave was shortened.Conclusion. The result of the experiment indicates that empagliflozin is able to modulate Ca2+-dependent mechanism of the excitation-contraction-coupling, enhancing and accelerating Ca2+ release into cytoplasm and reuptake. This presumably can optimize, namely reduce the time of systole and enhance it, which may be one of the important elements in the manifestation of empagliflozin antiarrhythmic properties.

Методом газово-жидкостной хроматографии с масс-спектрометрией в эксудате рылец лилии (Lilium longiflorum L., сорт Белое небо) и табака (Nicotiana tabacum L., сорт. Маленькая Гаванна SR1) был обнаружен сквален (2,6,10,15,19,23-гексаметилтетракоза-2,6,10,14,18,22-гексаен). Данное вещество идентифицировано в эксудате цветковых впервые. Концентрацию сквалена в эксудате можно выразить на среднюю массу рыльца (у табака она составляет в среднем 2,7 мг, для лилии около 60 мг), которую определяли гравиметрическим методом в ходе эксперимента. Таким образом. на 1 мг массы рыльца табака приходится 0,18 нг сквалена, а для лилии 0,29 нг.

The effects of tonoplast and rafts isolated from vacuolar membranes on the import of DNA into mitochondria of potato tubers (Solanum tuberosum) were studied. It was shown that the addition of rafts isolated from the tonoplast to mitochondria increased 8-fold the activity of the DNA import into mitochondria as compared to the import in the untreated mitochondria.

Natural competence of mitochondria for DNA uptake has been known for the last 20 years. Until the present time, all studies of this process have been conducted exclusively in isolated mitochondria, as no system for investigation of the DNA transport into the mitochondria in intact cells has been available. The objective of this work was to improve and standardize the existing approaches for investigating DNA import into plant mitochondria in an in organello system. A method for detecting the import of fluorescently labeled DNA substrates has been developed. Based on the features of DNA import into the mitochondria, we suggested an efficient method for the evaluation of the DNA import efficiency by quantitative PCR. We also developed and characterized the in vivo system that allows to detect DNA transport from the cytoplasm to the mitochondrial matrix in Arabidopsis thaliana protoplasts. A combination of the proposed techniques for studying the DNA uptake by plant mitochondria might be useful for elucidating whether the properties of the mitochondrial DNA import established in the in organello system are preserved in vivo.

Аннотация.Изолированные митохондрии растений обладают способностью импортировать молекулы ДНК.В данной работе импорт ДНК проводили в реконструированной системе, включающей изолированные митохондрии клубней картофеля и

In recent decades, it has become evident that the condition for normal functioning of mitochondria in higher eukaryotes is the presence of membrane transport systems of macromolecules (proteins and nucleic acids). Natural competence of the mitochondria in plants, animals, and yeasts to actively uptake DNA may be directly related to horizontal gene transfer into these organelles occurring at much higher rate compared to the nuclear and chloroplast genomes. However, in contrast with import of proteins and tRNAs, little is known about the biological role and molecular mechanism underlying import of DNA into eukaryotic mitochondria. In this review, we discuss current state of investigations in this area, particularly specificity of DNA import into mitochondria and its features in plants, animals, and yeasts; a tentative mechanism of DNA import across the mitochondrial outer and inner membranes; experimental data evidencing several existing, but not yet fully understood mechanisms of DNA transfer into mitochondria. Currently available data regarding transport of informational macromolecules (DNA, RNA, and proteins) into the mitochondria do not rule out that the mechanism of protein and tRNA import as well as tRNA and DNA import into the mitochondria may partially overlap.

Aim. Investigation of different features of DNA import into plant and human mitochondria, for a better understanding of mitochondrial genetics and generation of biotechnological tools.Methods.DNA up-take experiments with isolated plant mitochondria, using as substrates various sequences associated or not with the specific terminal inverted repeats (TIRs) present at each end of the plant mitochondrial linear plasmids.Results.It was established that the DNA import efficiency has a non-linear dependence on DNA size.It was shown that import into plant mitochondria of DNA molecules of «medium» sizes, i. e. between 4 and 7 kb, barely has any sequence specificity: neither TIRs from the 11.6 kb Brassica plasmid, nor TIRs from the Zea mays S-plasmids influenced DNA import into Solanum tuberosum mitochondria.Conclusions.The data obtained support the hypothesis about species-specific import mechanism operating under the mitochondrial linear plasmids transfer into plant mitochondria.

We investigated import of DNA of various length (109 bp, 269 bp, 717 bp, 1540 bp, 2732 bp, 9000 bp and 11600 bp) into the isolated mitochondria from potato tubers (Solanum tuberosum). The results imply existence of multiple pathways of DNA transfer into plant mitochondria. It was established that the transport of short length DNA (100-300 bp) is apparently carried out with the participation of several protein carriers which nature is currently unknown. We identified factors that influence the process of DNA transfer into mitochondria. These factors include the length of nucleic acid molecule and the presence of terminal inverted repeats that are specific to linear mitochondrial plasmids of plants.