Desmin mutations impact the autophagy flux in C2C12 cell in mutation-specific manner.

Desmin is the main intermediate filament of striated and smooth muscle cells and plays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway by knocking down Bag3 can promote desmin aggregate formation.

Extracellular MicroRNAs and Mitochondrial DNA as Potential Biomarkers of Arrhythmogenic Cardiomyopathy.

Differential diagnosis of arrhythmogenic cardiomyopathy (ACM) during the disease latent phase is a challenging clinical problem that requires identification of early ACM biomarkers. Because extracellular nucleic acids are stable, specific, and can be easily detected, they can be used as reliable biomarkers of various diseases. In this study, we analyzed the levels of extracellular microRNAs and mitochondrial DNA in the conditioned medium collected from cardiomyocytes differentiated from induced pluripotent stem cells of ACM patients and healthy donor. Several microRNAs were expressed differently by the affected and healthy cardiomyocytes; therefore, they could be considered as potential ACM biomarkers.

THE ROLE OF LMNA MUTATIONS IN MYOGENIC DIFFERENTIATION OF PRIMARY SATELLITE CELLS AND C2C12 CELLS.

Nuclear lamins form nuclear lamina localized under the inner nuclear membrane. It was previously considered that the nuclear lamina predominantly plays a structural role, however, its involvement have been recently described in the regulatory processes such as chromatin organization and gene transcription. It is known that mutations in the LMNA gene lead to the development of a large number of diseases, laminopathies, which mainly affect mesenchymal tissue. Nowadays, the mechanisms by which the lamina can regulate cell differentiation remain incompletely understood. In the present work, we have studied the effect of LMNA gene mutations on the process of muscle differentiation of primary satellite cells and Ñ2Ñ12 cell line. The genome of satellite cells and Ñ2Ñ12 cell line was modified by the introduction of lentiviral constructs encoding LMNA G232E associated with the development of muscular dystrophy Emery­Dreyfus and LMNA R571S associated with the development of dilated cardiomyopathy. The morphology of the cells was estimated using immunofluorescence, the expression level of myogenic genes were analyzed by qPCR. We have shown that the analyzed mutations reduce the ability of cells to differentiate, to fuse and to form myotubes. We have suggested that it is due to enhanced expression of markers at the early stages and to reduced expression markers at the late stages of myogenesis. Therefore, mutations in nuclear lamins can influence the process of muscle differentiation.

[Nuclear lamins regulate osteogenic differentiation of mesenchymal stem cells].

Nuclear lamins are the major proteins of nuclear envelope and provide the strength of nuclear membrane as well as the interaction of extra-nuclear structures with components of cell nucleus. Recently, it became clear that lamins not only play a structural role in the cell, but could also regulate cell fate, for example lamins could influence cell differentiation via interaction with components of the Notch signaling pathway. Human mutations in LMNA, encoding lamin A/C lead to diseases commonly referred to as laminopathies. Different mutations cause tissue specific phenotypes that affect predominantly a tissue of mesenchymal origin. The nature of this phenomenon, as well as the mechanisms by which lamins regulate cell differentiation remain poorly understood. The aim of this study was to investigate the effect of different mutations of the LMNA on human mesenchymal stem cell (MSC) osteogenic differentiation, and to explore a possible interaction of lamins and Notch signaling pathway. We modified human MSC with mutant LMNA bearing known mutations with tissue specific phenotype associated with different laminopathies. We have shown that mutations associated with different diseases have different effects on the efficiency of MSC osteogenic differentiation and on the expression of specific osteogenic markers SPP1, IBSP and BGLAP. We have also shown that one of the mechanisms involved in the regulation of MSC differentiation may be an interaction of lamins A/C with components of Notch signaling.

[Comparative assessment of different approaches for obtaining terminally differentiated muscle cells].

Relevant cell model is essential to study pathogenesis of muscle disorders. However, in the field of muscle research there is no ultimate cell line considered as a standard for studying muscular and neuromuscular diseases. Standard cell line claimed to be well differentiated in muscle lineage, be morphological and physiological similar to mature muscle cells and be easily genetically modified. Therefore, the goal of our study was to pick up available and fruitful cell model of muscle differentiation, that could be further applied for examination of muscular disorder pathogenesis in vitro. We characterized human mesenchymal stem cells (MSC), mature murine muscle fibers and primary murine satellite cells. It has been shown that MSC have very small capacity to myogenic differentiation; moreover, they were able to differentiate only in presence of C2C12 cells. Lentiviral transduction exhibited rather high toxic effect on primary myofibers, and positively transduced cells were not able to response to electrical stimulation, i. e. were functionally inactive. Satellite cells turned out to be the most fruitful cell model since they were easily transduced via lentiviruses and rapidly formed myotubes in differentiation media. Functional analysis of obtained myotubes has confirmed their ability to react to electrical and chemical stimulations; besides, potassium and calcium channels availability has been also demonstrated via patch-clump technique. Taken together, these results imply that satellite cells are the most promising cell line for further experiments aimed at exploring the molecular pathways of muscle pathologies.

[Lamin A/C mutations change differentiation potential of mesenchymal stem cells].

Mutations in lamin A/C gene (LMNA) lead to development of severe disorders--laminopathies. Unlike most other types of intermediate filaments, where the pathological effect of mutations is tightly linked to alteration of mechanical and integrative functions, the detailed mechanism of lamin mutations is still unclear and possibly involves the alteration of nuclear signaling and transcriptional processes. Since the mesenchymal lineage tissues such as myocardium, skeletal muscle, adipose and bone tissues are mostly affected in laminopathies, the role of lamin A/C in differentiation process of mesenchymal stem cells has been assumed. The aim of the study was to estimate the effect of LMNA mutations of differentiation of mesenchymal stem cells into adipose lineages. In vitro mitagenesis was performed on wild type LMNA gene incorporated in a lentiviral vector. Several previously described mutations in LMNA were used, each associated with a certain phenotype. Adipose-derived mesenchymal stem cells from healthy donors were transduced with lentiviruses bearing either wild-type or mutant LMNA. Cells were then induced to adipose differentiation. We show that mutant LMNA/C promotes differentiation capacity of mesenchymal stem cells as seen by morphological changes and by expression of specific adipose markers.

Do the results of adrenalectomy in symptomatic arterial hypertension depend on morphological alterations in adrenal glands?

The article deals with surgical treatment of patients presenting with resistant arterial hypertension and detected pathology in the adrenal glands. The authors analysed the outcomes of surgical management of 97 patients with resistant arterial hypertension, also determining the factors contributing to prediction of the antihypertensive effect of adrenalectomy depending on morphological damage to the adrenal glands.

[Molecular characteristics of cyclodextrin molecules, obtained in studies of the properties of their solutions].

Molecules of three cyclodextrins have been studied by molecular hydrodynamics methods in water, dimethylformamide, and dimethylsulfoxide. The molecular masses have been estimated, and the values correlate well with the calculated values. The hydrodynamic radii of cyclodextrin molecules have been also determined. The experimental data have been compared with theoretical calculations for toroidal molecules. The experimental data may be reconciled with the theoretical on the assumption that solvent molecules are adsorbed on the external surface of cyclodextrin molecules.