Peering into the molecular mechanisms of skeletal muscle

For the last 60 years, skeletal muscular contraction theory has been dominated by the sliding filament theory, which suggests that all muscle force is produced by the interaction of actin and myosin proteins. However, researchers have noted that not all muscle properties, especially history-dependent properties, can be accounted for by the sliding filament theory. Recently, a new muscle model, the winding filament hypothesis (WFH), has emerged as a leading candidate to describe all muscle properties, including those poorly described by the sliding filament model.

N2A of titin bind to Actin during calcium influx  —–The winding filament hypothesis expands on the sliding filament model by incorporating a role for the giant protein titin (Yellow), along with actin (blue) and myosin (green) in muscle force modulation. N2A of titin binds to actin during calcium influx, which causes a stiffer spring to be stretch during active muscle lengthening storing energy and increasing the muscle’s maximum force output. For info on the Winding Filament Hypothesis and titin’s role, see this paper, or this review paper.

Novel to the WFH is that, in addition to actin and myosin, the giant protein titin functions like a molecular spring that can modulate muscle force during muscle length change. The WFH is an original and transformative concept of muscles that has the ability to radically change how researchers, students and clinicians view muscle contractions. My research is focused around two aspects of the WFH. First, I critically study the protein interactions of actin, myosin and titin in passive and active muscles to examine the validity of the WFH claim. Second, I use this information to advance the field of powered bionic prostheses (talked about here!)

AIM I: Critically testing the fundamental mechanism of the WFH

The sarcomere protein titin spans an entire half-sarcomere (functional unit of a muscle), but is generally studied in the I-band (where there is no actin-myosin overlap). In the I-band region, titin is composed of two in-series elastic spring proteins, called the PEVK and Ig regions. These regions flank a non-flexible N2A region. It is known that the Ig region is much more compliant than the PEVK region. Since the PEVK and Ig springs are in series, the compliant Ig spring must fully extend before the stiffer PEVK region extends, which oddly enough seems to rarely occur in the physiological limits of skeletal muscle stretch. However, this observation has only been observed in passive (non-active) muscle stretch because active muscle preps are incredibly hard to prepare. The winding filament hypothesis (WFH), argues that titin functions differently in active muscle. During muscle activation, the N2A region binds to the actin filament, functionally removing the Ig spring from the mechanisms and leaving the PEVK region to be stretched alone during muscle stretch. As the muscle stretches, the stiffer PEVK spring stretches, storing elastic potential energy, adding to the muscle force produced by cross bridges. Computer simulation show that our muscle theory can properly predict all muscular force properties, which has never been done before by a physiologically correct model. Though promising, to date, no active muscle measurements of the Ig and PEVK spring exists to test our claims. My goal in this project is to directly measure the PEVK stretch during active muscle stretch. If the PEVK region stretches during active muscle stretch, than the WFH is supported by direct evidence for the first time.

Using a custom build manipulator (designed by Michael DuVall, Post Doc) that can hold and stretch the functional unit of muscles, myofibrils (1/100 the width of a hair), we stretch passive and active muscles to desired lengths. We then utilize an immunohistochemical (ICH) technique to label the N2A and PEVK regions with antibodies that are visible under a scanning electron microscopy (SEM). With these landmarks in place, the PEVK region extension behavior is directly measured during and after stretch by the SEM at a magnification of 30 thousand times and resolution of 4 nanometers. If the PEVK spring is stretched in active muscles, then the WFH is supported.

Ongoing Work:

Molecular Approach: Exploring the Winding Filament Hypothesis Through Electron MicroscopyA single myofibril. This is the functional unit of muscle. Contractile elements pull the bands closer together, shortening the myofibril. Put a whole bunch of these together, and you have muscle contraction. Credit: Carl Z.

myofibril_4—-A single myofibril. This is the functional unit of muscle and its thickness is 1/100th of a hair. Contractile elements pull the bands closer together, shortening the myofibril. Put a whole bunch of these together in an intense hierarchical structure, and you have muscle contraction. Credit: Carl Zapfe

Myofibril 3 Ph and Flu 200x N2A Myofibril 2 Ph and Flu 200x N2A

—- Above, you can see the desired antibodies (AB) binding to their target. We needed a cheap experiment to make sure our AB’s actually do what we say they do…. In this setup, we used the N2A antibody to bind to the N2A region of titin (this is the space surrounding the black bands in the myofibrils). A secondary AB binds to the N2A AB. This AB is special because it has a florescent tag that allows it to glow under the right conditions. For each picture, you see the general myofibril under regular light (right of each picture), and the same myofibril with the florescent tags glowing. If you are curious, they are all in the right spot! With landmarks like these, we are able to see how titin proteins move during muscle activation and stretch. Photo credit: Michael DuVall

Next steps: Currently, we are starting to stretch passive myofibrils to desired lengths, running them through the AB protocol, and measuring the titin spring lengths. Once we have this data, we will start actively stretch myofibrils to desired lengths, measure the springs and compare them to our passive data for changes.

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