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Familial hypertrophic cardiomyopathy-related cardiac troponin C mutation L29Q affects Ca²⁺ binding and myofilament contractility

¹ Cardiac Membrane Research Laboratory, Kinesiology, Simon Fraser University, Burnaby
² Cardiovascular Sciences, Child & Family Research Institute, Vancouver, British Columbia
³ Integrative Biology, University of Guelph, Guelph, Ontario, Canada
⁴ Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio

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 McTnC^F27W and NIQD McTnC^F27W was 1.3- and 1.9-fold higher, respectively, than that of McTnC^F27W. The Ca²⁺ 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 Ca²⁺ binding to site II was higher in L29Q McTnC^F27W and NIQD McTnC^F27W 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.

cardiac thin filament; myocardial contractility