The cardiac troponin C (cTnC) mutation, L29Q, has been found in a patient with 
familial hypertrophic cardiomyopathy. We previously showed that L29, together 
with neighboring residues, Asp2, Val28, and Gly30, plays an important role in 
determining the Ca(2+) affinity of site II, the regulatory site of mammalian 
cardiac troponin C (McTnC). Here we report on the Ca(2+) binding characteristics 
of L29Q McTnC and D2N/V28I/L29Q/G30D McTnC (NIQD) utilizing the Phe(27) --> Trp 
(F27W) substitution, allowing one to monitor Ca(2+) binding and release. We also 
studied the effect of these mutants on Ca(2+) activation of force generation in 
single mouse cardiac myocytes using cTnC replacement, together with sarcomere 
length (SL) dependence. The Ca(2+)-binding affinity of site II of L29Q 
McTnC(F27W) and NIQD McTnC(F27W) was approximately 1.3- and approximately 
1.9-fold higher, respectively, than that of McTnC(F27W). The Ca(2+) 
disassociation rate from site II of L29Q McTnC(F27W) and NIQD McTnC(F27W) was 
not significantly different than that of control (McTnC(F27W)). However, the 
rate of Ca(2+) binding to site II was higher in L29Q McTnC(F27W) and NIQD 
McTnC(F27W) relative to control (approximately 1.5-fold and approximately 
2.0-fold respectively). The Ca(2+) sensitivity of force generation was 
significantly higher in myocytes reconstituted with L29Q McTnC (approximately 
1.4-fold) and NIQD McTnC (approximately 2-fold) compared with those 
reconstituted with McTnC. Interestingly, the change in Ca(2+) sensitivity of 
force generation in response to an SL change (1.9, 2.1, and 2.3 mum) was 
significantly reduced in myocytes containing L29Q McTnC or NIQD McTnC. These 
results demonstrate that the L29Q mutation enhances the Ca(2+)-binding 
characteristics of cTnC and that when incorporated into cardiac myocytes, this 
mutant alters myocyte contractility.