| Remote-zone left-ventricular dysfunction (RZLVD) may contribute to the high incidence of pump failure after myocardial infarction. We tested the hypothesis that post-translational modifications to the contractile apparatus are in part responsible for RZLVD. We used cardiac myosin and native thin filaments purified from a mouse model of ischemia-reperfusion (I-R) injury and tested function using the in vitro motility assay. Myosin from the remote-zone showed 50% reduced function due to cysteine oxidation. Also, remote-zone thin filaments were propelled faster than controls. This increase was associated with increased tropomyosin (Tm) phosphorylation. This suggested that Tm phosphorylation might serve as a compensatory mechanism that increases contraction when myosin function is compromised.;alpha-Tm is phosphorylatable near the C-terminus, and Tm polymerizes with adjacent Tm's head to tail. We hypothesized that Tm phosphorylation might influence the strength of Tm polymerization, and thus regulate cooperative activation of the thin filament by myosin binding. We tested Tm phosphorylation effects on the force and velocity of actin-myosin interactions at the level of single actin filaments. Dephosphorylation of Tm did not significantly change actin-Tm binding or sliding velocities. Isometric force measurements showed that thin filaments were cooperatively activated by myosin and increased in force, but only when Tm was phosphorylated. The force increase suggests that Tm phosphorylation may also have single crossbridge effects.;We tested the hypothesis that Tm phosphorylation effects the formation and dissociation of individual actin-myosin bonds. We measured on-rate between single actin-myosin interactions and the bond lifetimes at a range of step loads using a laser trap. Actin-myosin on-rates increased, but only when Tm was phosphorylated. Conversely, bond lifetimes increased only when Tm was phosphorylated and in the open state. Together, these data suggest that Tm adds additional sites of interaction for myosin on the thin filament.;Tm phosphorylation may serve to increase the rate of crossbridge formation during unloaded shortening. In isometric contraction, Tm phosphorylation may serve to increase duty ratio, leading to an increase in time-averaged force production. These effects by Tm phosphorylation may be present in pathologies, when contraction is compromised, or when there is increased contractile demand such as in exercise. |
