Titin Isoform Size is not Correlated with Thin Filament Length in Rat Skeletal Muscle

Abstract

The striated muscle sarcomere is dependent upon the precise interactions of a variety of myofibrillar proteins for its proper formation. As the largest and third most abundant protein in this milieu, titin plays a number of functions in the sarcomere, including assembly of the thick filaments and preventing overstretch. The titin gene is expressed as multiple splice variants in skeletal muscle, generating a continuum of titin protein sizes. Recently it was reported that thin filament length was related to titin size, and that the latter might be involved in determining thin filament length. We tested this hypothesis using several muscles from wild type rats and from a mutant rat model (Greaser et al J Mol Cellul Cardiol 44:983, 2008) which results in increased titin size. Myofibrils were isolated from skeletal muscles (diaphragm, extensor digitorum longus, gastrocnemius, longissimus dorsi, psoas major, rectus abdominis, and tibialis anterior) using both adult wild type (WT) and homozygous mutant (HM) rats (n=6 each). Thin filament length was estimated using fluorescent dye labeled phalloidin and relaxed sarcomere length was determined by phase contrast microscopy after adding ATP and BDM. No differences in thin filament lengths were found between WT muscles with titin sizes ranging from 3.2 to 3.7 MDa. Similarly the thin filament lengths in the mutant rats did not differ from the paired WT muscles in spite of large differences in titin size with several muscles. However, the relaxed sarcomere length was correlated to titin size in muscles from WT rats, and it was significantly increased relative to WT for within muscle comparisons. The data indicates that, although titin performs many functions, its relationship to thin filament length could not be demonstrated in the rat. Supported by HL77196.