Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach.

Abstract

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) 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+) 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-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) 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 Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) 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 Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

• Authors: Jose Renato Pinto, Daniel P Reynaldo, Michelle S Parvatiyar, David Dweck, Jingsheng Liang, Michelle A Jones, Martha M Sorenson, James D Potter
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 286 Issue 2 Pg. 1005-13 (Jan 14 2011) ISSN: 1083-351X [Electronic] United States
• PMID: 21056975 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Cross-Linking Reagents
• Myosin Subfragments
• Troponin C
• Calcium

Topics

• Actin Cytoskeleton (physiology)
• Animals
• Calcium (metabolism)
• Cardiomyopathy, Hypertrophic (genetics, metabolism, physiopathology)
• Cross-Linking Reagents (chemistry, metabolism)
• Humans
• Kinetics
• Mutagenesis
• Myocardial Contraction (physiology)
• Myocytes, Cardiac (cytology, physiology)
• Myosin Subfragments (metabolism)
• Protein Conformation
• Rabbits
• Swine
• Troponin C (chemistry, genetics, metabolism)