Strong Cross-bridges Potentiate the Ca2+ Affinity Changes Produced by Hypertrophic Cardiomyopathy Cardiac Troponin C Mutants in Myofilaments

• Published in: The Journal of biological chemistry Vol. 286; no. 2; pp. 1005 - 1013
• Main Authors: Pinto, Jose Renato, Reynaldo, Daniel P., Parvatiyar, Michelle S., Dweck, David, Liang, Jingsheng, Jones, Michelle A., Sorenson, Martha M., Potter, James D.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01-01-2011
• Subjects: Actin; Biophysics; Calcium; Cardiac Hypertrophy; Cardiac Muscle; Contractile Protein; Fluorescence; Genetic Diseases; Myosin; Troponin; Contractile Protein; Cardiac Muscle; Troponin; Genetic Diseases; Calcium; Actin; Myosin; Fluorescence; Biophysics; Cardiac Hypertrophy
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.168583

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca2+ 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 Ca2+ 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 MgATP2−, the condition conducive to rigor cross-bridge formation, further increased the apparent Ca2+ affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca2+ dissociation (koff) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca2+ affinity and decrease in koff was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca2+ off-rate is most likely to be affected rather than the Ca2+ on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca2+-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower koff from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.