The effect of Mg 2+ on Ca 2+ binding to cardiac troponin C in hypertrophic cardiomyopathy associated TNNC1 variants

Cardiac troponin C (cTnC) is the critical Ca -sensing component of the troponin complex. Binding of Ca to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca binding to site II of cTnC. However, work by several groups including ours suggest that Mg , which is several orders of magnitude more abundant in the cell than Ca , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg may decrease site II Ca -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca and Mg to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca and Mg . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg in modifying the Ca affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg both at baseline and when elevated on modifying the Ca binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction.