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 Ca 2؉ sensitivity of contraction. Mutations in the Ca 2؉ sensor, troponin C (TnC), were generated to increase/ decrease the Ca 2؉ 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 Ca 2؉ 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 Ca 2؉ sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca 2؉ sensitivity, maximal force recovery, and activation of the ATPase at high [Ca 2؉ ]). Steady-state fluorescence was utilized to assess Ca 2؉ affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca 2؉ sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased ␣-helical content correlated with increased Ca 2؉ 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 Ca 2؉ sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca 2؉ sensitivity, maximal force, and ATPase activation of some mutants.

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

“…This is consistent with bona fide HCM cTn mutations, which usually do not alter ATPase inhibition (33). The basal force in skinned fibers was unaffected (data not shown) (35). Taken together, the functional data support the HCM phenotype found in the patient.…”

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy

“…We also used mutant cTnCs because they represent the proximal Ca 2+ binding element of the sarcomere, hence allowing for a more direct modulation of the Ca 2+ -tension relationship without potential confounding effects. The L48Q and I61Q variants used here have been rigorously quantified for their effects on tension and Ca 2+ handling dynamics (Kreutziger et al, 2011; Parvatiyar et al, 2010; Tikunova and Davis, 2004; Wang et al, 2012; Feest et al, 2014). …”

Functional Characterization of TNNC1 Rare Variants Identified in Dilated Cardiomyopathy

“…Mutations Y5H, M103I, D145E, and I148V decrease the ␣-helix content of cTnC, which may be associated with a less coordinated response to Ca 2ϩ binding and decreased Ca 2ϩ sensitivity in skinned fibers (23,28). Interestingly, Y5H is the second cTnC mutation found within the N-helix, known for its ability to modulate the Ca 2ϩ affinity of Site II (40,41).…”