Computational Studies of a pH Responsive Histidine-Modified Cardiac Troponin I

Abstract

Cardiac troponin I (cTnI) functions as the molecular switch of the thin filament. Studies have shown that a A164H button engineered into cTnI enhances inotropic function under pathophysiological challenges. In vitro studies of myofilament calcium sensitivity and sarcomere shortening kinetics in intact and permeablized myocytes at baseline (pH 7.4) indicated similar cellular contractile function and myofilament calcium sensitivity between myocytes expressing wildtype cTnI and cTnI A164H while A164R showed a hypercontractile phenotype associated with increased myofilament calcium sensitivity. Under acidic conditions, compared to depressed function in myocytes with wildtype cTnI, myocytes expressing cTnI A164H maintained myofilament calcium sensitivity and contractile performance comparable to the calcium sensitizer cTnI A164R. The role of histidine modified cTnI was assessed by molecular dynamics (MD) simulations and pKa calculations of the wildtype and histidine or arginine-modified cTnI (148-173): cTnC (1-90) complex. The simulations showed similar conformations between the wildtype and the deprotonated cTnI A164H variant. In contrast, simulations of protonated cTnI A164H and cTnI A164R showed diverse conformation changes, both of which included the formation of a cTnI His 164 and cTnC Glu 19 salt bridge. pKa calculations showed no significant pKa shift for all ionizable residues except for cTnI His 164 and cTnC Glu 19 when the salt bridge is formed. The data shed light into the potential mechanism of pH activation of cTnI A164H and the importance of electrostatic interactions in governing the biophysical adjustments in troponin function necessary for nuanced modulation of myofilament function in response to changes in the cytosolic milieu.