MOLECULAR DYNAMICS-BASED PREDICTIONS OF THE STRUCTURAL AND FUNCTIONAL EFFECTS OF CARDIAC TROPONIN MUTATIONS ASSOCIATED WITH FAMILIAL HYPERTROPHIC CARDIOMYOPATHY

Abstract

Familial Hypertrophic cardiomyopathy (FHC) affects one in every 500 people. Many FHC cases occur without overt symptoms and patients may be unaware of their condition prior to sudden cardiac death. FHC is caused by a growing list of largely sarcomeric mutations, including 69 in the cardiac troponin (cTn) complex. Understanding these mutations is crucial to the accurate diagnosis and development of novel treatments for FHC. The cTn complex contains three proteins: cTnC, a Ca2+ sensor protein; cTnI, an inhibitory protein; and cTnT, a tether to the thin filament. To date six FHC-linked mutations have been found in cTnC, a highly conserved protein that initiates cardiac contraction in response to the cytosolic level of Ca2+. Upon Ca2+ binding, a hydrophobic patch on cTnC is exposed, which causes the inhibitory peptide of cTnI to withdraw from the actin filament, initiating muscle contraction. Mutations that alter the ability of cTnC to bind Ca2+ are hypothesized to induce either hypertrophic or dilated cardiomyopathies depending on whether Ca2+ affinity is increased or decreased, respectively. The FHC-associated mutations A8V, L29Q and C84Y in the N-terminal domain were studied. The structural effects of these mutations were modeled through molecular dynamics and their functional impact assessed using free energy calculations of the cTnC-Ca2+ interaction. In each mutant, the equilibrated structures were very similar to those of wild-type (WT), the site II Ca2+ coordination, size of cTnC-Ca2+ interface, and the stability of the structures were consistent. Changes were noted in the angle between helices A and B, indicative of the exposure of the hydrophobic patch. Additionally, a change in the free energy of Ca2+ binding was calculated between the mutant and WT structures. The change in free energy due to the cTnC-Ca2+ interaction was the highest for the A8V mutation, followed by the WT protein, C84Y and finally, L29Q. Only the A8V mutation caused a free energy change in Ca2+ binding that was greater than WT. This is contrary to the hypothesis that higher affinity for Ca2+ is associated with FHC. Interestingly, the degree to which the hydrophobic patch on cTnC was exposed was greater than WT in each of the mutants. Taken together, these results suggest that while the Ca2+ affinity does not change, the energy barrier to transduction of the Ca2+-binding signal has been lowered in each of the mutated proteins.