PEVK EXTENSION OF HUMAN SOLEUS MUSCLE TITIN REVEALED BY IMMUNOLABELING WITH THE ANTI-TITIN ANTIBODY 9D10

Titin is a giant protein that spans half of the striated muscle sarcom ere, The I-band portion of titin extends as the sarcomere is stretched , developing what is known as passive force. This portion of the molec ule is composed mainly of tandem immunoglobulin (Ig) segments, consist ing of serially linked Ig-like domains, and a recently discovered uniq ue sequence termed the PEVK segment. The tandem Ig and PEVK segments h ave been suggested to extend sequentially when sarcomeres are stretche d, with PEVK extension dominating at moderate to high degrees of sarco mere stretch (M. Gautel and D. Goulding, 1996, FEES Lett. 385, 11-14; W. Linke et al., 1996, J, Mol. Biol, 261, 62-71; K. Trombitas et al., 1998). Previously we observed that the anti-titin antibody 9D10 labels a region in the I-band that increases in width as sarcomeres are stre tched. Here we tested whether 9D10 labels the PEVK segment. The 9D10-l abeled region of human soleus fibers was followed by immunoelectron mi croscopy as sarcomeres were stretched. It was found that 9D10 labeled a region in the I-band that was similar to 100 nm wide at a sarcomere length of 2.4 mu m and similar to 550 nm wide at a sarcomere length of 4.0 mu m. Results were compared with those obtained with sequence-spe cific antibodies that were used to mark the boundaries of the PEVK seg ment. Findings indicate that 9D10 labels the PEVK segment from close t o its N-terminal end to its C-terminal end. Consistent with this concl usion are the results on cardiac myocytes that express a much shorter PEVK segment than skeletal muscle and where 9D10 labels a region that is much less wide than in skeletal muscle. The anti-titin antibody 9D1 0 is a useful tool for investigating the extensible behavior of the PE VK segment in both skeletal and cardiac muscles.