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.
A Novel Mutation in TNNC1-ENCODED Cardiac Troponin C Predisposes to Hypertrophic Cardiomyopathy and Recurrent Episodes of Aborted Sudden Cardiac Death
