Clinical and Biophysical Characterization of a Mutation in the N-Helix Region of Cardiac Troponin C: Evidence for an Allosteric Mechanism of Contractile Dysfunction

Abstract

Troponin is a heterotrimeric, Ca2+-binding protein responsible for regulating muscle contraction in the heart. Missense mutations in genes encoding the subunits of the cardiac troponin complex are associated with inherited dilated cardiomyopathy (DCM). However, the underlying molecular mechanisms of disease pathogenesis are not fully understood. Here, we report a clinical case of a 1-year-old female who presented with severe DCM and hypotonia. Whole exome sequencing revealed a previously unreported de novo heterozygous variant in exon 1 of the TNNC1 gene (c.12C>G), resulting in an Ile4Met mutation located in the N-Helix region of cardiac troponin C (cTnC). We utilized various biophysical and biochemical techniques to examine the molecular basis of this pathogenic variant and provide evidence for allosteric communication within the Ca2+-binding subunit of the troponin complex. In vitro extraction of native cTnC and reconstitution with either recombinant WT or I4M cTnC in cardiac muscle preparations revealed decreased Ca2+ sensitivity of isometric force development and slower kinetics of tension redevelopment (ktr) for I4M compared to WT cTnC. Steady-state fluorescence measurements on isolated cTnC using Bis-ANS indicated enhanced Ca2+ binding at the C-terminal domain of I4M compared to WT. Furthermore, cTnC-I4M displayed a smaller magnitude of Ca2+-induced hydrophobic exposure compared to WT. Finally, IANBD fluorescence (singly labeled at cysteine 84) titration studies revealed tighter binding of cardiac TnI to I4M compared to WT. Altogether, these results suggest that perturbed contractile kinetics and altered Ca2+-binding, perhaps by an allosteric mechanism, likely contribute to the contractile dysfunction observed in this proband. Experiments using solution NMR spectroscopy are currently underway to determine how a pathogenic mutation in the N-helix affects the overall structure and dynamics of cTnC. NIH-HL128683.