FIGURE 1.

Steady-state and stopped-flow analysis of Ca²⁺ kinetics in human cardiac reconstituted thin filaments (hcRTF). (A) The Ca²⁺ sensitivity [expressed in K _d (μM)] of the wild-type (WT) (n = 8 technical replicates) and I79N hcRTF (n = 6 technical replicates) was determined by steady-state fluorescence spectrometry. The I79N significantly decreased the K _d compared to the WT counterpart by ∼1.7-fold, indicating that these variants increase the Ca²⁺ sensitivity significantly in the hcRTF biochemical system. (B) A stopped-flow instrument was used to determine the Ca²⁺ dissociation rates (expressed as k _off) from the hcRTF. Upon the addition of EGTA, the Ca²⁺ was rapidly removed from troponin C, inducing a conformational change from cTnC within hcRTF in which the fluorescence change was recorded. The I79N hcRTF (n = 11 technical replicates) decreased the k _off by ∼1.2-fold compared to the WT counterpart (n = 9 technical replicates), suggesting that the increase in Ca²⁺ sensitivity observed in the steady state is largely due to a slower Ca²⁺ off-loading rate. Unpaired Student’s t tests were conducted to compare WT versus I79N hcRTF, and a p value <0.05 was considered statistically significant with the following notation: *p < 0.05, **p < 0.01, and ***p < 0.001.