TY - JOUR
T1 - Strong cross-bridges potentiate the Ca2+ affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments
T2 - A fast kinetic approach
AU - Pinto, Jose Renato
AU - Reynaldo, Daniel P.
AU - Parvatiyar, Michelle S.
AU - Dweck, David
AU - Liang, Jingsheng
AU - Jones, Michelle A.
AU - Sorenson, Martha M.
AU - Potter, James D.
PY - 2011/1/14
Y1 - 2011/1/14
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M110.168583
DO - 10.1074/jbc.M110.168583
M3 - Article
C2 - 21056975
AN - SCOPUS:78651397869
VL - 286
SP - 1005
EP - 1013
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 2
ER -