Abstract
The mechanisms controlling thin filament length (TFL) in muscle remain controversial. It was recently reported that TFL was related to titin size, and that the latter might be involved in TFL determination. Titin plays several crucial roles in the sarcomere, but its function as it pertains to the thin filament has not been explored. We tested this relationship using several muscles from wild type rats and from a mutant rat model (Greaser et al., 2008) which results in increased titin size. Myofibrils were isolated from skeletal muscles [extensor digitorum longus (EDL), external oblique (EO), gastrocnemius (GAS), longissimus dorsi (LD), psoas major (PM), and tibialis anterior(TA)] using both adult wild type (WT) and homozygous mutant (HM) rats (n = 6 each). Phalloidin and antibodies against tropomodulin-4 (Tmod-4) and nebulin's N-terminus were used to determine TFL. The WT rats studied express skeletal muscle titin sizes ranging from 3.2 to 3.7 MDa, while the HM rats express a giant titin isoform sized at 3.8 MDa. No differences in phalloidin based TFL, nebulin distance, or Tmod distance were observed across genotypes. However, the HM rats demonstrated a significantly increased (p < 0.01) rest sarcomere length relative to the WT phenotype. It appears that the increased titin size, predominantly observed in HM rats' middle Ig domain, allows for increased extensibility. The data indicates that, although titin performs many sarcomeric functions, its correlation with TFL and structure could not be demonstrated in the rat.
Highlights
One of the hallmarks of the striated muscle sarcomere is its precise periodicity, composed of a number of proteins held in tight alignment
It was previously reported that this mutation alters the normal titin splicing pattern in the heart, and the mutant titin molecules do not decrease in size with age (Greaser et al, 2008)
Our data supports previous studies showing that Tmod appeared furthest from the Z-disk, and that the N-terminus of nebulin is shorter than that of the thin filament determined by Tmod staining (Castillo et al, 2009)
Summary
One of the hallmarks of the striated muscle sarcomere is its precise periodicity, composed of a number of proteins held in tight alignment. The sarcomeric filament systems (thick, thin, titin, and nebulin) are optimized for specific functionalities in the muscle, and their relationship is key for contraction (Figure 1A). Because of the precise control necessary for contractile function, a multitude of protein-protein interactions must be maintained. In contrast to other F-actin environments, the actin of striated muscle primarily exhibits shrinkage and elongation from its pointed end, in the central region of the sarcomere (Littlefield et al, 2001; MardahlDumesnil and Fowler, 2001). While the actin thin filament length (TFL) can vary across and within muscles of a species, its length within a sarcomere changes very little (Fowler, 1996)
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