Strong cross-bridges potentiate the Ca2+ affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: A fast kinetic approach

Jose Renato Pinto, Daniel P. Reynaldo, Michelle S. Parvatiyar, David Dweck, Jingsheng Liang, Michelle A. Jones, Martha M. Sorenson, James D. Potter

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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 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 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.

Original languageEnglish (US)
Pages (from-to)1005-1013
Number of pages9
JournalJournal of Biological Chemistry
Volume286
Issue number2
DOIs
StatePublished - Jan 14 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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