Additional information on the Winding Filament Hypothesis and titin’s role.


Molecular approach: Exploring the Winding Filament Hypothesis through Transmission Electron Microscopy

Over the last several years, our understanding of titin’s contributions to passive and active muscle properties has exploded. Several models and theories have grown out of this work, most notably the winding filament hypothesis (WFH). This model fills existing muscle theory gaps while building on the sliding filament theory. In the WFHs, the N2A region of titin binds to actin upon Ca2+ influx. The PEVK segment of titin (which lies next to the N2A region) winds on the thin filaments during force development because the cross-bridges not only translate but also rotate the thin filaments. As a muscle shortens or lengthens, it is theorized that the titn’s components interact in such a way that when the muscle is activated, titin acts as a powerful elastic spring.  Titin’s active properties are believed to fill in the gaps unresolved by the Hill muscle model. We are working to demonstrate this model’s feasibility by measuring the passive and active “stretches” of titin’s elastic elements, PEVK and tandon Ig domain regions. The winding filament hypothesis predicts that both the proximal tandem Ig and PEVK domains extend in sarcomeres from passively stretched muscles, but only PEVK will extend in actively stretched muscles due to N2A’s interaction with the thin filament. Previous work on a N2A deficient mouse model (mdm mice) have, thus far, supported the WFH.

The present research will determine the location of the N2A epitope within the I-band and estimate force-length relationships for the proximal tandem Ig and PEVK segments in passive and activated soleus muscles from wild type and mdm mice.  We believe that in mdm muscles, the proximal tandem Ig and PEVK domains should extend in sarcomeres from both passive and activated muscles. Wild-type and mdm muscles will differ in force-extension behavior of the proximal tandem Ig and PEVK domains of titin. Passive and activated muscles will differ in force-extension behavior of the proximal tandem Ig and PEVK domains.

For this work, antibody-labeling studies are critical for understanding contributions of titin to active muscle force. Following in the footsteps of previous work, we will use immunogold labeling with a polyclonal titin N2A antibody to locate the N2A epitope within the I-band and to estimate the force-length relationships of the proximal tandem Ig and PEVK segments.

Prepared muscles, fixed at a range of lengths and forces from wild-type and mdm mice will be embed in a porous plastic resin (LR White), sectioned at 40 nm parallel to the muscle fiber orientation and treated with a polyclonal N2A primary antibody and secondary antibody. This antibody is specific for the primary antibody and has been attached to a colloidal gold particle. Gold particles are visible on transmission electron micrographs, labeling the N2A region. This will allow us to calculate the distances between the antibody label, the edge of the A-band, and the center of the Z-line. From these data, the location of the N2A epitope, and force-extension curves for the proximal tandem Ig and PEVK regions of titin will be obtained.  To date, we have developed and implemented a protocol for passive muscle. Active muscle protocols have been developed and are in their testing stages. The implications of this work, will solidify the WFH into our understanding of the complex properties of muscle contractions.