%0 Generic
%A Krsmanovic, Pavle
%D 2013
%F heidok:14851
%K myoblasts differentiation, myofibrillar myopathies, desminopathy, cardiomyopathy
%R 10.11588/heidok.00014851
%T Analyses of Desmin Disease Mutants in vivo with Emphasis on their Effects on Myoblast Differentiation
%U https://archiv.ub.uni-heidelberg.de/volltextserver/14851/
%X Mutations in genes of various intermediate filament (IF) proteins are the causes of a wide range of diseases. For example, a premature aging disorder, Hutchinson-Gilbert progeria syndrome, is caused by mutation in the nuclear lamins. Mutations in a muscle-specific IF protein desmin cause a wide range of myofibrillar myopathies, and in severe cases cardiomyopathies. The effects of the desmin mutations vary with respect to the severity of their impact on its filament formation capacity. One of the observations on the tissue sections of the patients includes the occurrence of both apparently normal association of desmin filaments with Z-discs as well as the occurrence of desmin accumulations in the muscle cells of patients. However, the exact mechanisms leading to such phenotype remain unknown.  The purpose of this PhD thesis was to analyse the effects of some of the known desmin mutations on cell differentiation. In addition, considering what appears to be concurrent occurrence of desmin aggregation and IFs formation as indicated above we aimed to determine to what extent are normal and mutant desmin proteins segregated in these cells. For that purpose, we have employed C2C12 murine myoblast cell line for analyses of the impacts of desmin mutantions. As these cells can be induced to differentiate they are commonly used as a model in studies on muscle cells. Therefore, we have transfected genes of the normal human desmin (hDesWT) and some known desmin mutants into these cells. In order to detect the transfected protein antibodies specific for hDes were generated.  Two desmin gene mutations were used for the analyses, namely hDesE245D and hDesR350P. Whereas the former desmin mutant was found to be incorporated into the IF-system of fibroblasts, the later segregates from it and accumulates in small aggregates through the cell. The analyses performed included cell fractionation and immuno-cytochemistry. hDesWT and hDesE245D mutant were predominantly found to incorporate into endogenous desmin IFs, whereas the extent of hDesR350P mutant incorporation into the filaments varied in the cell population. By subsequent cell passaging the number of hDesR350P-expressing cells was reduced, whereas both hDesWT and hDesE245D were still expressed to a similar extent.  Subsequently transfected C2C12 cells were induced to differentiate. Similarly to the observations in cell passaging, the amount of hDesR350P but not hDesWT or hDesE245D in cells was found to be reduced upon differentiation. Expression of a myosin heavy chain, a C2C12 differentiation marker, was higher in cells expressing either of the mutants than in untransfected or the cells transfected with the hDesWT desmin. Finally, the transfected cells were subjected to immuno-cytochemical analyses with respect to the coexpression of myosin heavy chain and of the various transfected desmins. Whereas hDesWT and hDesE245D were both present in approximately 70% of myosin heavy chain positive cells, about 10% of differentiating cells contained hDesR350P.  Additional studies on C2C12 cells included the analyses of distribution of the A-type lamins in undifferentiated and differentiated cells. Whereas in the undifferentiated C2C12 cells some soluble A-type lamins could have been extracted, the differentiated cells appeared to contain very little of such lamins. Furthermore, the A-type lamin distribution in cells transfected with hDesR350P, but not hDesWT or hDesE245D, and subjected to differentiation was the same as in myoblasts, indicating the cells likely fail to undergo differentiation. These results demonstrate a very close connection between cytosolic and nuclear IFs. Finally, they indicate that the late onset of desminopathies could be correlated to the steady hDesR350P clearance from the cultured myoblasts leading to their failure of cells to differentiate.