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 Ca2+
affinity of site II, the regulatory site of mammalian cardiac
troponin C (McTnC). Here we report on the Ca2+ binding
characteristics of L29Q McTnC and D2N/V28I/L29Q/G30D McTnC (NIQD)
utilizing the Phe27 Trp (F27W) substitution, allowing one to monitor
Ca2+ binding and release. We also studied the effect of
these mutants on Ca2+ activation of force generation in
single mouse cardiac myocytes using cTnC replacement, together with
sarcomere length (SL) dependence. The Ca2+-binding
affinity of site II of L29Q McTnCF27W and NIQD
McTnCF27W was 1.3- and 1.9-fold higher, respectively,
than that of McTnCF27W. The Ca2+ disassociation
rate from site II of L29Q McTnCF27W and NIQD
McTnCF27W was not significantly different than that of
control (McTnCF27W). However, the rate of Ca2+
binding to site II was higher in L29Q McTnCF27W and NIQD
McTnCF27W relative to control (1.5-fold and 2.0-fold
respectively). The Ca2+ sensitivity of force generation
was significantly higher in myocytes reconstituted with L29Q
McTnC (1.4-fold) and NIQD
McTnC (2-fold) compared with those
reconstituted with McTnC. Interestingly, the change in
Ca2+ sensitivity of force generation in response to an SL
change (1.9, 2.1, and 2.3 µm) was significantly reduced in myocytes
containing L29Q McTnC or NIQD McTnC. These results demonstrate that
the L29Q mutation enhances the Ca2+-binding
characteristics of cTnC and that when incorporated into cardiac
myocytes, this mutant alters myocyte contractility.
