Susceptibility to apoptosis in different murine muscle cell lines MATTHIAS WITTSTOCK 1, *, CHARLOTTE REHFELDT 2 , GERD NU ¨ RNBERG 2 , ULLA RENNE 2 , WOLFGANG BRU ¨ CK 3 , EILHARD MIX 1 and UWE K. ZETTL 1 1 Department of Neurology, University of Rostock, Gehlsheimer Strasse 20, D-18147 Rostock, Germany; 2 Research Institute for the Biology of Farm Animals, Division Muscle Biology and Growth, Genetics and Biometry, Dummerstorf; 3 Department of Neuropathology, Humboldt University, Campus Virchow, Berlin Received 11 December 2002; accepted in revised form 9 April 2003 Abstract Objective of the study was to investigate growth characteristics and susceptibility to apoptosis in different murine muscle cell lines (selected for high body weight, DU-6; randomly mated control, DU-Ks; immortalized myoblast cell line, C 2 C 12 ). Apoptosis was induced by serum deprivation. At days 4, 5, and 6 of cultivation, protein, DNA and the frequency of apoptotic cells were determined. Until day 4, C 2 C 12 accumulated more DNA and protein compared with DU-Ks and DU-6, while exhibiting a lower percentage of apoptotic cells. Serum deprivation impaired the growth of each cell line. C 2 C 12 continued to accumulate DNA and protein after serum deprivation, whereas reductions, indicative of cell death, were apparent in DU-Ks and DU-6. Serum deprivation did not enhance apoptosis in C 2 C 12 . Higher percentages of apoptosis were observed in DU-Ks and DU-6 after 2 days of serum deprivation with greater responsiveness of DU-6 to serum deprivation. The results suggest that cell loss in response to serum deprivation is in part due to induction of apoptosis. C 2 C 12 are less sensitive to sub-optimal culture conditions compared with DU-Ks and DU-6 which are at a closer distance to the in vivo status. Moreover, long- term selection for growth decreases the basic frequency of apoptosis of muscle satellite cells, but increases their susceptibility to apoptosis induction. Introduction Apoptosis is a process of individual cell death regulated by activation of specific genes (Wyllie et al., 1980). Characteristic morphological features of apoptosis in- clude nuclear and cytoplasmatic condensation, frag- mentation of the cell into apoptotic bodies, which become engulfed by phagocytes, and local absence of inflammation. These changes are due to the activation of nuclear endonucleases and cytoplasmatic proteases. The cell death program is modulated by several regulatory genes with pro- and anti-apoptotic function (Vaux and Strasser, 1996). Nuclear changes, which constitute the morphological hallmark of apoptosis, are the result of early high molecular weight DNA fragmentation and late oligonucleosomal DNA fragmentation (Walker et al., 1995). Apoptotic cell death in skeletal muscle occurs under various physiologic and pathologic conditions like reperfusion after ischaemia or diseases such as muscular dystrophies (McArdle and Jackson, 1997). Furthermore, susceptibility to induction of apoptosis appears to be a key property during development of mammalian skel- etal muscle cells under physiological conditions (Wang and Walsh, 1996). The question whether mature, post- mitotic, multinucleated skeletal muscle cells can undergo apoptosis is discussed controversially (Jacobson et al., 1996; Weil et al., 1996). Strong inductors of apoptosis are H 2 O 2 - and NO- dependent oxidative stress (Stangel et al., 1996), the protein kinase inhibitor staurosporine (Jacobson et al., 1996) and serum deprivation (Sandri and Carraro, 1999; Zettl et al., 2000). They are, therefore, suitable for assessment of susceptibility of muscle cells to apoptosis induction in vitro. There are no reports comparing the susceptibility to apoptosis in different muscle cell lines during in vitro myogenesis. Primary cell cultures derived from mice long-term selected for high body weight and cells from respective controls differ in in vitro DNA and protein accumulation and they respond more sensitive to the deprivation of serum as indicated by increased cell loss (Walther et al., 1998; Walther, 1999). It is of impor- tance, whether this higher sensitivity of ‘selected’ cells can be explained by differences in the induction of apoptosis. The aim of the current study was, therefore, to investigate the susceptibility to apoptosis by serum deprivation in three murine muscle cell lines (C 2 C 12 , DU-Ks and DU-6) with different growth characteristics and different degree of selection at two developmental stages representing proliferation and early differentia- tion. * To whom correspondence should be addressed: Tel.: þ49-381- 494-9517; Fax: þ49-381-494-9512; E-mail: matthias.wittstock@ med.uni-rostock.de Journal of Muscle Research and Cell Motility 24: 521–526, 2003. 521 Ó 2003 Kluwer Academic Publishers. Printed in the Netherlands.