A functional and structural study of tropon in C mutations related to hypertrophic cardiomyopathy

Jose Renato Pinto, Michelle S. Parvatiyar, Michelle A. Jones, Jingsheng Liang, Michael John Ackerman, James D. Potter

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Abstract

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca2+ sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca2+] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca2+ sensitivity with only A8V demonstrating lowered Ca2+ sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca2+. None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca2+ affinity of site II measured by 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca2+. Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca2+ sensitivity of the myofilament; 2) the effects of the mutations on the Ca2+ affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca2+ sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along thesarcomerelattice disrupting the coupling between the cross-bridge and Ca2+ binding to cTnC.

Original languageEnglish (US)
Pages (from-to)19090-19100
Number of pages11
JournalJournal of Biological Chemistry
Volume284
Issue number28
DOIs
StatePublished - Jul 10 2009

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Troponin C
Hypertrophic Cardiomyopathy
Troponin
Mutation
Myosins
Adenosine Triphosphatases
Myofibrils
Circular Dichroism
Sedimentation
Assays
Proteins
Fluorescence
Metals
Chemical activation
Fibers

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

A functional and structural study of tropon in C mutations related to hypertrophic cardiomyopathy. / Pinto, Jose Renato; Parvatiyar, Michelle S.; Jones, Michelle A.; Liang, Jingsheng; Ackerman, Michael John; Potter, James D.

In: Journal of Biological Chemistry, Vol. 284, No. 28, 10.07.2009, p. 19090-19100.

Research output: Contribution to journalArticle

Pinto, Jose Renato ; Parvatiyar, Michelle S. ; Jones, Michelle A. ; Liang, Jingsheng ; Ackerman, Michael John ; Potter, James D. / A functional and structural study of tropon in C mutations related to hypertrophic cardiomyopathy. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 28. pp. 19090-19100.
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abstract = "Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca2+ sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca2+] utilizing reconstituted thin filaments (TFs) with 50{\%} mutant and 50{\%} wild type (WT) and 100{\%} mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca2+ sensitivity with only A8V demonstrating lowered Ca2+ sensitization at the 50{\%} ratio when compared with 100{\%}; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca2+. None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca2+ affinity of site II measured by 2-(4′-(2″-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca2+. Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca2+ sensitivity of the myofilament; 2) the effects of the mutations on the Ca2+ affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca2+ sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along thesarcomerelattice disrupting the coupling between the cross-bridge and Ca2+ binding to cTnC.",
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AU - Ackerman, Michael John

AU - Potter, James D.

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