A Multi-Tool Mouse Model to Study the Elasticity of Native Titin

Abstract

The elasticity of muscle is principally determined by the I-band region of titin, consisting of 40-100 immunoglobulin-like (Ig) domains and two unstructured segments. Traditional force spectroscopy of titin can study only eight tandem Ig domains at a time, and the dominant entropic effect of the massive native titin polypeptide is lost. Here, we have developed a knock-in mouse model to study titin in its entirety. We genetically encoded a HaloTag enzyme, flanked with two protease sites, in the constitutively expressed region of the I-band. This multi-tool mouse model permits: covalent protein immobilization for force spectroscopy, fluorescent labeling for sarcomere length measurements, and titin specific proteolysis for muscle mechanics experiments. The HaloTag, engineered close to the I-A junction of titin, allows titin immobilization on glass coverslips functionalized with chloroalkane chemistry. Using T12 antibody coated paramagnetic beads, the N-terminus of titin can be picked up and stretched with magnetic tweezers. For the first time, this has enabled the study of the mechanical properties of the entire intact titin I-band. For different muscle groups, the results show unfolding and refolding of individual Ig domains at forces in the physiological range, below 10 pN. Moreover, accumulated step size histograms show two populations in the unfolding and refolding transitions, suggesting the presence of reduced and oxidized disulfide bonds in several Ig domains. Additionally, fluorescent labeling of the HaloTag with Alexa488 conjugated chloroalkane permits accurate sarcomere length measurements of intact myofibrils. We propose muscle mechanics experiments in which stretched myofibrils are treated with TEV protease to cleave titin at the I-A junction so that titin's contribution to passive elasticity, and perhaps active contraction, can be measured. This versatile mouse model allows for a diverse set of biophysical experiments to elucidate the role of the giant protein titin in muscle function.