Molecular Mechanisms of A164H cTnI

Abstract

Troponin I (TnI) has a central, isoform-dependent role in ischemic contractile failure. The fetal heart expresses the slow skeletal TnI (ssTnI) isoform which confers protection from ischemia-mediated contractile failure relative to the adult expressed cardiac TnI (cTnI) isoform. A single codon substitution in cTnI, A164H, reverts significantly to an ssTnI phenotype, in specifics, conferring protection from ischemia-mediated contractile failure. Importantly, unlike ssTnI, cTnI A164H does not alter contractile performance under baseline conditions. The molecular mechanisms for the marked enhancement in myocyte function under pathophysiological conditions with no detected effects under baseline conditions have yet to be determined. Isoform specific residues in helix 4 of TnI were investigated through structure/function analysis to gain insight into this mechanism. Molecular dynamics simulation for cTnI, Q157R/A164H/E166V/H173N cTnI (QAEH), A164H cTnI, and ssTnI in complex with cTnC, showed that substitution of cTnI with the ssTnI residues alters the intermolecular interactions between TnI and cTnC. These findings suggest that these residues are important for the conformation of helix 4 in regards to cTnC. To investigate if these substitutions alter function, adult cardiac myocytes were transduced with adenovirus expressing QAEH cTnI and sarcomere dynamics were analyzed. QAEH cTnI showed a similar phenotype to ssTnI, increasing sarcomere shortening and slowing relaxation at pH 7.4 while maintaining normal contractile function under acidic conditions. To elucidate this further, double and triple mutants were analyzed to determine the importance of each of the three residues to baseline contractility. The data show that H173 and Q157 are necessary for the reduced contractility at baseline while E166 exerts the opposite effect. Taken together, these new findings suggests that the conformation of helix 4 in TnI is an important determinate of contractility in ischemia.