This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca 2 (cid:1) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca 2 (cid:1) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP 2 (cid:2) , the condition conducive to rigor cross-bridge formation, further increased the apparent Ca 2 (cid:1) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca 2 (cid:1) dissociation ( k off ) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity ( i.e. TF (cid:1) S1), an