Effect of hypertrophic and dilated cardiomyopathies associated mutations in troponin I on cardiac thin filament dynamics

Troponin I mutations have been linked to genetic hypertrophic and dilated cardiomyopathies. We aimed to understand, at the molecular level, how six HCM mutations (R21C, Q130R, R145G, G203S, and K206Q) and one DCM mutation (A2V) in troponin I affect its structure and function. Circular dichroism, co-sedimentation with actin and ATPase assays demonstrated that these mutations had little or no effect on the folding or the thermal stability of the troponin complex. Isothermal calorimetry, fluorescence spectroscopy, and transient kinetics were used to assess the effect of these mutations on the function of troponin I. We found that: 1) all TnI mutations increased the affinity of the troponin complex for actin in the presence of Ca²+ and increased the Ca²+ affinity of troponin within thin filaments. This suggests an uncoupling between Ca²+ binding and actin binding. 2) The size of the cooperative unit n was not affected by troponin I mutations. 3) A2V, R21C, Q130R, A157V, G203S, and K206Q mutations did not affect the proportion of thin filaments in the blocked state (at low Ca²+). In contrast R145G mutation dramatically reduced the amount of thin filaments switched to the blocked state. This effect was also observed using electron microscopy and helical reconstruction. 4) A2V, R21C, Q130R, R145G, G203S, and K206Q did not affect the observed rate constant of Ca²+ dissociation from troponin and thin filaments. In contrast troponin I A157V showed a decrease in the Ca²+ dissociation rate constant. 5) Finally, we found that calcium alone is sufficient to fully activate the cardiac thin filament while skeletal muscle thin filaments complete activation required both Ca²+ and myosin heads. Overall these results provide insight into the mechanism by which troponin I mutations affect contractility in hypertrophic and dilated cardiomyopathy. These findings could have important clinical consequences.