R482L Mutation of the LMNA Gene Affects Dynamics of C2C12 Myogenic Differentiation and Stimulates Formation of Intramuscular Lipid Droplets.

Mutations in the LMNA gene resulting in the substitution of the highly conserved arginine 482 residue in the globular C-terminal domain of lamin A/C are associated with the Dunnigan-type familial partial lipodystrophy (FPLD2) often accompanied by impairments in the muscle tissue development. The mechanisms underlying these impairments remain unknown. The purpose of our work was to investigate the effects of the LMNA gene mutation R482L on the muscle differentiation and intramuscular fat accumulation using C2C12 mouse myoblasts transduced with the lentiviral constructs carrying the wild-type human LMNA gene (LMNA-WT) or the LMNA-R482L mutant gene. After stimulation of myogenesis and adipogenesis in the transduced cell, expression of muscle and adipose tissue differentiation markers, morphology of differentiated myotubes, and formation of intramuscular lipid droplets were analyzed. C2C12 cells carrying the LMNA-R482L construct exhibited upregulated desmin expression at all stages of muscle differentiation and transformed into hypertrophied myotubules (in comparison with C2C12 myoblasts transduced with LMNA-WT). Reduced expression levels of the myogenic transcription factor Myf6 in the cells with the LMNA-R482L mutant indicated delayed maturation of muscle fibers. These cells more actively accumulated fat in response to the stimulation of adipose differentiation than myoblasts modified with the wild-type LMNA; they also expressed the markers of lipid droplets, such as FABP4 (fatty acid-binding protein 4), ATGL (adipose triglyceride lipase), and PLIN2 (perilipin 2). Therefore, the R482L mutation of the LMNA gene affects the dynamics of C2C12 myoblast differentiation into myotubules and stimulates formation of fat deposits in the myoblasts and myotubules in a tissue-specific manner.

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.