For the past 60 years, muscle contraction had been thought to be governed exclusively by the contractile filaments, actin, and myosin. This thinking explained most observations for concentric and isometric, but not for eccentric muscle contractions. Just over a decade ago, we discovered that eccentric contractions were associated with a force that could not be assigned to actin and myosin, but was at least in part associated with the filamentous protein titin. Titin was found to bind calcium upon activation, thereby increasing its structural stability, and thus its stiffness and force. Furthermore, there is increasing evidence that the proximal part of titin binds to actin in an activation- and force-dependent manner, thereby shortening its free length, thus increasing its stiffness and force. Therefore, we propose that muscle contraction involves three filaments, actin, myosin and titin, and that titin regulates force by binding calcium and by shortening its spring length by binding to actin.
![Effects of mutations R719W, R723G, and I736T on stiffness of soleus muscle fibers in rigor (absence of nucleotide) and during active contraction. (A) Principle of stiffness measurement in rigor. In rigor fibers generate no force when put in rigor by rapid removal of free Mg2+-ions together with removal of ATP (Brenner et al., 1986). Thus, stiffness was measured by applying a ramp-shaped stretch (see inset for a schematic illustration). Stiffness is the slope when force change is plotted vs. applied change in sarcomere length, measured in nm/half sarcomere (nm/HS). Note that only the dark part of the traces in the inset are included in the force vs. length change plot. Noise of this plot was reduced by signal-averaging responses of several such maneuvers. (B) Fiber stiffness in rigor for the mutations and control fibers. Speed of stretch ~3–5 × 103 (nm/half sarcomere)s−1; T = 5°C; n = 5–10 fibers. Mean values ± SE, normalized control fibers, respectively. *Difference to controls statistically significant, p ≤ 0.05. (C) Principle of stiffness measurements during isometric steady state contraction. When fibers have reached constant isometric force a ramp shaped release is imposed on the fiber length. This results in a drop of isometric force (cf. inset for schematic illustration of length change and force response). Over the initial 2–3 nm of length change the plot of force vs. sarcomere length change is linear. Deviation from the linear response later in the release is due to rapid re-equilibration of myosin heads in their different force-generating states. The slope of the initial linear part is determined by linear regression (red line) and taken as active fiber stiffness. The intercept of this line with the abscissa is the y0-value. This is interpreted as the elastic extension of the actin and myosin filaments plus elastic distortion of the force generating myosin heads (Linari et al., 2007). The plot of force vs. length change is noisier than in (B). This is due to the fact that fewer measurements were signal-averaged than under rigor conditions shown in (A). (D) Fiber stiffness determined during isometric steady state contraction [speed of release ~2–4 × 103 (nm/half-sarcomere)s−1]; T = 10°C to ensure structural integrity of fibers and stability of striation pattern throughout experiments; n = 6–14 fibers. Mean values ± SE, normalized control fibers, respectively. *Difference to controls statistically significant, p ≤ 0.05. (E) Plot of y0, vs. isometric force at different levels of calcium-activation. The y0-values were measured from T-plots (cf. C) at pCa values from 4.5 to 6.6; T = 10°C. Speed of length release 4–5 × 103 (nm/half-sarcomere)s−1. Continuous line obtained by linear regression. Y-axis intercept of solid line is 5.67 nm/HS (HS = half sarcomere) equals elastic extension of force generating heads (cf. double headed arrow). Slope of solid line is filament compliance per half-sarcomere, about 0.025 nm/kPa. Elastic extension of myofilaments at an isometric force of 120 kPa is illustrated by double arrow. Panels (B, D and E) reprinted with modifications from Seebohm et al. (2009) with permission from Elsevier.](https://www.frontiersin.org/_rtmag/_next/image?url=https%3A%2F%2Fwww.frontiersin.org%2Ffiles%2FArticles%2F110620%2Ffphys-05-00392-HTML%2Fimage_m%2Ffphys-05-00392-g002.jpg&w=3840&q=75)
