Effect of cMyBP-C ablation on the Ca ²⁺ –force relationship in hiPSC. (A and B) Averaged absolute (A) and normalized (B) twitch force (TF) vs. Ca²⁺ transient (Ca²⁺TR) hysteresis loop traces in day 40–54 cMyBP-C^+/+ (broken traces), cMyBP-C^+/− (grey traces), and cMyBP-C^−/− (black traces) hiPSC-ECTs loaded with Fura2-AM (concurrent TF and Ca²⁺ measurements). (C and D) Box and whisker plots of the time interval from Ca²⁺ transient (Ca²⁺T₁₀₀) peak to twitch force peak (CT₁₀₀; C) and normalized hysteresis areas (D) in hiPSC-ECT. TF and Ca²⁺TR in B and D were normalized by expressing them as TF/TF_max and (F₃₄₀/F₃₈₀)/(F₃₄₀/F₃₈₀)_Max. All experiments were performed at 37°C with pacing at 1.5 Hz. n ≥ 11 biological replicates; *P < 0.05; **P < 0.01; ***P < 0.001 as calculated by one-way ANOVA with Tukey’s multiple comparisons test; see Table S7 for specific P values.