Predicting Cardiomyopathic Phenotypes by Altering Ca2+ Affinity of Cardiac Troponin C

Cardiac diseases associated with mutations in troponin sub-units 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 alpha-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.