Predicting cardiomyopathic phenotypes by altering Ca2+ affinity of cardiac troponin C

Abstract

Cardiac diseases associated with mutations in troponin subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca2+ sensitivity of contraction. Mutations in the Ca2+ sensor, troponin C (TnC), were generated to increase/decrease the Ca2+ sensitivity of cardiac skinned fibers to create the characteristic effects of DCM, HCM, and RCM. We also used a reconstituted assay to determine the mutation effects on ATPase activation and inhibition. One mutant (A23Q) was found with HCM-like properties (increased Ca2+ sensitivity of force and normal levels of ATPase inhibition). Three mutants (S37G, V44Q, and L48Q) were identified with RCM-like properties (a large increase in Ca2+ sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca2+ sensitivity, maximal force recovery, and activation of the ATPase at high [Ca2+]). Steady-state fluorescence was utilized to assess Ca2+ affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca2+ sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased α-helical content correlated with increased Ca2+ sensitivity in skinned fibers and vice versa. The main findings from this study were as follows: 1) cTnC mutants demonstrated distinct functional phenotypes reminiscent of bona fide HCM, RCM, and DCM mutations; 2) a region in cTnC associated with increased Ca2+ sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca2+ sensitivity, maximal force, and ATPase activation of some mutants. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.