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 EFFECT OF PLAKOPHILIN-2 GENE MUTATIONS ON ACTIVITY OF THE CANONICAL Wnt SIGNALING PATHWAY].

Plakophilin-2 is a desmosomal protein encoded by PKP2 gene. Desmosomal proteins are usually considered as structural proteins with the main function of maintaining intercellular interactions. Genetic studies revealed that mutations in desmosomal genes could lead to arrhythmogenic right ventricular cardiomyopathy, heart disease characterized by substitution of cardiomyocytes by adipose and fibrotic tissue predominantly in right ventricle. Wnt signaling pathway is one of the signal transduction pathways which could be involved in the formation of the pathology. The purpose of this study was to investigate Wnt activity changes caused by PKP2 mutations during adipogenic and cardiomyogenic differentiation. We used multipotent mesenchymal stromal cells and iPS cells generated from patient carrying PKP2 gene mutation. We show that Wnt activity is lower in the cells with mutant PKP2. This data indicate a possible signaling role of plakophilin-2 by regulating Wnt activity.

[Comparative characteristics of the stem cells isolated from subcutaneous and subepicardial adipose tissue].

BACKGROUND: Stem cells (SCs) considerably vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. The comparative analysis of their biological properties is essential for the optimal choice of SCs for regenerative therapies. METHODS: Using immunocytochemistry, flow cytometry, histochemistry and real-time RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissue and cultured under similar culture conditions without any differentiation-promoting factors. RESULTS: The cultures were similar in the high proportion of proliferating cell nuclear antigen (PCNA)-positive cells. In both cultures, immunophenotyping has revealed high expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105, low expression of CD31, CD34 and CD45, and wide variability in CD117, CD146 and CD309 expression. The only distinction in CD marker profile was significantly lower expression of CD90 in SCs from SEC-AT. Histochemical analysis has shown the lack of Oil Red O-positive cells in both cultures and about ten-fold higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In the both cultures, immunocytochemistry has detected similar low expression of slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Gap junctional protein Connexin-43 expression was markedly higher in SCs from SC-AT, and epithelial cell marker Cytokeratin-19 expression was detected only in these cells. By RT-PCR, GATA4 mRNA was found to be highly expressed only in SCs from SEC-AT. CONCLUSIONS: Our results suggest that SC-AT, as compared with SEC-AT, is richer in epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs, and can be considered as an alternative to SC-AT as a source of SCs for cell cardiotherapy.