Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C 
(cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the 
Ca(2+) 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 [Ca(2+)] 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 Ca(2+) sensitivity 
with only A8V demonstrating lowered Ca(2+) 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 
Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to 
the TF measured by co-sedimentation. Only D145E increased the Ca(2+) 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 Ca(2+). 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 Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on 
the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain 
the large Ca(2+) 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 the sarcomere lattice disrupting the 
coupling between the cross-bridge and Ca(2+) binding to cTnC.