Laminopathies are a family of monogenic multi-system diseases resulting from mutations in the LMNA gene which include a wide range of neuromuscular disorders. Although lamins are expressed in most types of differentiated cells, LMNA mutations selectively affect only specific tissues by mechanisms that remain largely unknown. We have employed the combination of functional in vitro experiments and transcriptome analysis in order to determine how two LMNA mutations associated with different phenotypes affect skeletal muscle development and metabolism. We used a muscle differentiation model based on C2C12 mouse myoblasts genetically modified with lentivirus constructs bearing wild-type human LMNA (WT-LMNA) or R482L-LMNA/G232E-LMNA mutations, linked to familial partial lipodystrophy of the Dunnigan type and muscular dystrophy phenotype accordingly. We have shown that both G232E/R482L-LMNA mutations cause dysregulation in coordination of pathways that control cell cycle dynamics and muscle differentiation. We have also found that R482/G232E-LMNA mutations induce mitochondrial uncoupling and a decrease in glycolytic activity in differentiated myotubes. Both types of alterations may contribute to mutation-induced muscle tissue pathology.

Heart failure (HF) is associated with skeletal muscle wasting and exercise intolerance. This study aimed to evaluate the exercise-induced clinical response and histological alterations. One hundred and forty-four HF patients were enrolled. The individual training program was determined as a workload at or close to the lactate threshold (LT1); clinical data were collected before and after 12 weeks/6 months of training. The muscle biopsies from eight patients were taken before and after 12 weeks of training: histology analysis was used to evaluate muscle morphology. Most of the patients demonstrated a positive response after 12 weeks of the physical rehabilitation program in one or several parameters tested, and 30% of those showed improvement in all four of the following parameters: oxygen uptake (VO2) peak, left ventricular ejection fraction (LVEF), exercise tolerance (ET), and quality of life (QOL); the walking speed at LT1 after six months of training showed a significant rise. Along with clinical response, the histological analysis detected a small but significant decrease in both fiber and endomysium thickness after the exercise training course indicating the stabilization of muscle mechanotransduction system. Together, our data show that the beneficial effect of personalized exercise therapy in HF patients depends, at least in part, on the improvement in skeletal muscle physiological and biochemical performance.

Aim. Heart failure (HF) is accompanied by skeletal muscle atrophy and exercise intolerance. The aim was to study the molecular mechanisms underlying the therapeutic effect of personalized exercise in patients with HF. Material and methods . RNA sequencing obtained from skeletal muscle biopsies before and after a 12-week exercise course was used to identify changes in gene expression and signaling pathways induced by the physical rehabilitation program for patients with HF. Results. We have shown that personalized exercise program in patients with HF stimulates the activation of molecular pathways regulating the differentiation and functioning of skeletal muscles: commitment of muscle progenitor cells; mechanisms regulating the calcium release and sensitivity of myofibrillar contraction, electrical excitability of the muscle membrane, synaptic vesicle proton gradient creation, maintenance of electrochemical gradients of Na ⁺ /K ⁺ . Also, the analysis of differentially expressed genes revealed an increase in the expression of transcription factors MyoD and MEF2, which are responsible for the differentiation of muscle stem cells, and sarcomeric genes MYOM1 , MYOM2 , MYH7 . Along with this, we observed activation of the CYR61 expression — a potential prognostic biomarker for HF patients. Conclusion. Our data show that the beneficial effect of personalized aerobic exercise in patients with HF depends, at least in part, on an improvement in the physiological and biochemical parameters of skeletal muscle.

Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients’ immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.

ОСОБЕННОСТИ ПРОБОПОДГОТОВКИ В ПРОТОКОЛАХ ИССЛЕДОВАНИЯ РОЛИ ЭПИГЕНЕТИЧЕСКИХ МОДИФИКАЦИЙ ХРОМАТИНА (МЕТИЛИРОВАНИЯ ДНК И МОДИФИКАЦИЙ ГИСТОНОВ) В РЕГУЛЯЦИИ ЭКСПРЕССИИ ГЕНОВ

MicroRNAs in tissues and biological fluids represent a promising class of biomarkers for the molecular diagnostics and therapy of numerous diseases, including oncological diseases. Biomarkers based on easily accessible biological fluids, primarily blood-based biomarkers, are of particular value for diagnostic and prognostic purposes. To explore the potential of microRNAs as prognostic cancer markers and targets for molecular therapy, global microRNA profiling is required, which is provided by next-generation sequencing (NGS). NGS offers high sensitivity, single nucleotide resolution, and the possibility of profiling a large number of samples in parallel. Despite the promising potential of microRNAs as biomarkers and the growing number of works in this area, the literature does not address in sufficient detail the problems associated with sample preparation methods, the specifics of library preparation for microRNA sequencing, and the difficulties of quantitative analysis. Protocols for creating libraries for microRNA sequencing present specific challenges and require selecting conditions for each type of biological sample. Here, we present in detail the preparation of libraries for microRNA sequencing from pituitary adenoma tumor tissue and blood plasma of patients with acromegaly on the Illumina platform. We discuss the difficulties and limitations of the methods and evaluate the effectiveness of sequencing plasma and brain samples. This work can serve as a guide for researchers studying the mechanisms of microRNA regulation in endocrine diseases of the pituitary gland and will also allow for the adaptation of technical procedures for various biological samples in relation to other pathologies.