Human cardiac myosin has two isoforms, alpha and beta, sharing significant sequence similarity, but different in kinetics: ADP release from actomyosin is an order of magnitude faster in the alpha myosin isoform. Apparently, small differences in the sequence are responsible for distinct local inter-residue interactions within alpha and beta isoforms, leading to such a dramatic difference in the rate of ADP release. Our analysis of structural kinetics of alpha and beta isoforms using molecular dynamics simulations revealed distinct dynamics of SH1:SH2 helix within the force-generation region of myosin head. The simulations showed that the residue R694 of the helix forms two permanent salt bridges in the beta isoform, which are not present in the alpha isoform. We hypothesized that the isoform-specific electrostatic interactions play a role in the difference of kinetic properties of myosin isoforms. We prepared R694N mutant in the beta isoform background to destabilize electrostatic interactions in the force-generating region of the myosin head. Our experimental data confirm faster ADP release from R694N actomyosin mutant, but is not as dramatic as the difference of kinetics of ADP release in the alpha and beta isoforms. (C) 2019 Elsevier Inc. All rights reserved.