Beyond Calcium: Cross-bridge Binding Regulates Titin's Contribution to Muscle Force

Since the 1950s, muscle contraction has been explained by the sliding filament and cross-bridge theories involving actin and myosin. However, these theories do not account for certain muscle properties, such as residual force enhancement (rFE). The sarcomeric protein titin has been proposed to contribute to active force and rFE, but its role remains unclear. A leading hypothesis suggests that titin binds to actin, thereby shortening its spring-like segment, with calcium regulating this interaction. We investigated the roles of calcium and cross-bridge formation in titin mechanics by measuring the length of titin's PEVK region in sarcomeres during (i) passive stretch, (ii) active stretch with cross-bridge inhibition (via BDM), and (iii) active isometric contraction. PEVK lengths were similar for passive and cross-bridge inhibited conditions but were longer for active contractions. Our results suggest that cross-bridge engagement, not calcium alone, modulates titin extensibility and passive force under physiological conditions.