Analysis of force-related kinetic parameters of myofibrils from demembranated replated hESC-CMs. (A) Example of a force trace indicating the protocol (on top) of sarcomeric activation (+Ca²⁺) and relaxation (−Ca²⁺) and the force-related parameters: rate constant of Ca²⁺-induced force development (k_act), amplitude of the isometric force generated by cycling crossbridges (F_act), rate constant of mechanically induced force redevelopment of Ca²⁺-activated myofibrils (k_tr), rate constant of the linear force decay during the first, slow phase of relaxation (k_lin), and the rate constant of the monoexponential force decay during the second, fast phase of relaxation (k_rel). (B) Force traces during force redevelopment and relaxation for myofibrils of hESC-CMs replated for 2 (grey) or 9 (black) days. Small horizontal arrows indicate the duration of the first relaxation phase (t_lin) on days 2 and 9. (C and D)k_tr, reflecting crossbridge cycling kinetics (C), and k_lin (D), reflecting crossbridges leaving force-generating states in replated hESC-CMs on indicated days, and in 35 d old non-replated hESC-CMs (data imported from Iorga et al. [2018]). Mean ± SD; n = 2–20 myofibrillar bundles obtained from 12 coverslips derived from one differentiation. Insets: Diagrams representing the two states of cycling cross-bridges. f_app: rate constant or probability of cross-bridges entering force-generating states); g_app: rate constant or probability of cross-bridges leaving force-generating states.