Fachbereich Veterinärmedizin



    Die Rolle des herz- und skelettmuskelspezifischen Transkriptionsfaktors skNAC bei der Muskeldifferenzierung und bei entzündlichen Kardiomyopathien (2014)

    Breustedt, Theresia (WE 3)
    Berlin: Mensch und Buch Verlag, 2014 — 119 Seiten
    ISBN: 978-3-86387-544-2
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000098183
    Institut für Veterinär-Biochemie

    Oertzenweg 19 b
    14163 Berlin
    +49 30 838 62225

    Abstract / Zusammenfassung

    skNAC is a heart and skeletal muscle-specific protein. To date, little is known about its functions. Evidence exists that skNAC might act as a transcriptional coactivator during myogenesis. At later stages of myogenic differentiation, the protein might act as a molecular chaperone and could as such regulate cytoplasmic processes, especially myofibrillogenesis and sarcomerogenesis. In addition, in recent studies, we could show that in skeletal muscle regeneration and also in inflammatory skeletal muscle diseases, there is a strong induction of skNAC expression. Finally, at the beginning of this study, little was known on skNAC expression in the adult heart.

    So, one goal of this study was to examinate the spatial pattern of skNAC expression in coxsackievirus B3 (CVB-3)-infected hearts. Coxsackieviruses can cause myocarditis in infected hearts. Therefore, paraffin sections of hearts from two mousestrains with different susceptibility for CVB-3 were immunohistochemically analyzed with a skNAC-specific antiserum. However, no specific differences with respect to the staining pattern could be found between the two strains A.BY/SnJ (ABY), which is not able to eliminate the virus, and C57BL/6 (BL6), which is able to eliminate the virus without developing a cardiomyopathy.

    In the second, skeletal muscle-specific part of this study, the function of skNAC in skeletal muscle differentiation was analyzed using an in vitro system. For this purpose, we inhibited skNAC expression in cultured murine C2C12 myoblasts. Specifically, C2C12 cells were transfected with a skNAC-specific siRNA. Subsequently, cell lysates were analyzed for the expression of several myogenic differentiation markers at both the RNA and the protein levels using Northern and Western Blot analysis. We found that inhibition of skNAC-expression has no influence of the expression of Myogenin, Desmin, p21, Caveolin-3, Entactin-1 and α-Actinin. In addition, expression of MyHC was not reduced, by contrast, it appeared even slightly induced at the protein level in some experiments.
    Most likely, posttranscriptional mechanisms are responsible for this effect.
    In addition, we found that the morphology of the transfected cells appeared normal, however, their MyHC staining patterns appeared more diffuse in comparison with the controls. This finding suggests that the processes of myofibrillogenesis and/or sarcomerogenesis might be perturbed after inhibition of the skNAC expression.

    Finally, at the beginning of this study, no in vitro system for the analysis of skNAC functions in cardiomyocytes was available. Thus, we tested if the rat H9c2 cardiomyocyte cell line might be a suitablemodel system. Via Northern Blot analysis, we found that skNAC was indeed expressed in these cells, indicating that the H9c2 line might be a useful model for the analysis of skNAC functions in cardiomyocytes in the future.