Silicon Nitride Cantilevers for Muscle Sarcomere Force Measurements

Titin is a giant structural protein in muscle that spans the half sarcomere from the z-band to the M-line in skeletal muscle. Although much is known about titin’s mechanical properties from tests on isolated molecules [1] or fragments of titin produced recombinantly, there is little information on its behavior within the structural confines of a sarcomere. Since the passive properties of single myofibrils are primarily associated with titin, we tested the hypothesis that titin properties might be reflected well in single myofibrils. To measure the forces produced during our experiments, we fabricated silicon nitride cantilever pairs at the CNF using photolithography and reactive ion etching [2]. Summary of Research: Titin is a giant structural protein in muscle which spans a half sarcomere from the z-band to the M-line (Figure 1) and has been associated with passive force production in cardiac and skeletal muscles. It has spring like properties in its I-band domain dominated by extensible regions associated with the proximal and distal Ig segments and the PEVK region, named so because of its predominance in proline (P), glutamate (E), valine (V) and lysine (K) residues. Since its discovery in the mid 1970s [3], titin has emerged as an important stabilizer of sarcomeres [4], a producer of passive force [5], a regulator of active force [6], and has been associated with a variety of signaling, structural, and mechanical properties [7]. Titin is considered the third sarcomeric protein [8], and knowing its mechanical properties is essential for explaining passive characteristics of muscles. Rabbit psoas myofibrils were used for testing. Myofibrils were harvested from rabbit psoas, chemically and mechanically isolated as described in our previous works [9], and prepared for mechanical testing using silicon nitride cantilevers (stiffness 68 pN/nm) for force measurement at one end of the myofibril (resolution < 0.5 nN), and a glass needle attached to a motor for producing sub-nanometer step sizes at the other end (Figure 2). Testing: Myofibrils (n = 28) were passively stretched from a nominal initial average sarcomere length of 2.5-2.7 μm by 1.0, 2.0, 2.5, and 3.0 μm at a speed of 0.1 μm/s·sarcomere and then returned to the original length at the same speed. A distinct change in stiffness of the force-elongation curves was observed in eight of the tested myofibrils (Figure 3). The smallest sarcomere length where this was observed was 3.5 μm, while the average force at this inflection point was 68 nN (±5 nN) when normalized to 1.0 μm of myofibril crosssectional area. The results of this study suggest that the properties of isolated titin molecules are well reflected in whole myofibril testing. Titin properties appear to be well preserved when titin operates within its structural boundaries of a sarcomere. This result is insofar exciting as passive myofibril testing is rather simple compared to the complex isolation, stabilization and mechanical testing of single titin proteins. Not only is a myofibril approach much easier technically, it also offers the advantage that titin can be studied in its native environment and that titin’s properties can be directly related to sarcomere forces and lengths, and thus can be extrapolated to myofibril, fibre and muscle properties.