Calcium Dissociation of HCM Causative R21C Troponin I Mutation

Abstract

Hypertrophic cardiomyopathy (HCM) is a genetic disorder that affects 1/500 globally. A subset of HCM causing mutations resides in the cardiac thin filament (TF), comprised of actin, tropomyosin (Tm), cardiac Troponin I (cTnI), cardiac Troponin T (cTnT), and cardiac troponin C (cTnC). The N-terminal domain of cTnI is structurally close to the N-lobe binding site of cTnC. Glutamate 32 (E32) has been shown to add an extra negatively charged coordinating oxygen that interacts with the positively charged Ca2+. This oxygen contributes to the stabilization of calcium in the binding pocket and influences calcium dissociation kinetics. Mutations in the N-terminus of cTnI and their potential effects on calcium dissociation have largely been unexplored. A recently identified N-terminal cTnI mutation, R21C, causative for HCM with a high prevalence of sudden cardiac death. We hypothesized that R21C would significantly alter the rate of calcium dissociation from Site II of cTnC due to its proximity to E32 in cTnI. Here, we use an atomistic TF computational model and steered molecular dynamics to assess the work required to pull calcium from Site II on cTnC. We found that the average energy barrier created from the Ca2+ and E32 oxygen interaction was decreased in R21C as compared to WT. In addition, the total work to remove calcium from site II was 981.9 kcal/mol and 936.5 kcal/mol for WT and R21C respectively. Root mean squared fluctuation data of the alpha-carbons of cTnI also showed an increased flexibility in the N-terminus. These computational data suggests that the R21C mutation would increase the calcium dissociation rate in vitro (these studies are in progress). We thus conclude that a mutation-specific alteration in the kinetics of Ca2+ dissociation may act as a trigger for downstream Ca2+-driven pathogenic remodeling in cTnI R21C-linked HCM.