Can We Reproduce the Latch-State in Vitro at the Molecular Level?

Smooth muscle has a unique property, called the latch-state, during which force is maintained for long periods of time at low energy consumption and low myosin activation (phosphorylation) levels. This property has been observed at the whole muscle level and is mostly associated with tonic rather than phasic smooth muscle. To explain the latch-state, theories have been elaborated that invoke different molecular mechanisms, but have never been verified. One such theory states that, during the cross-bridge cycle, if smooth muscle myosin gets dephosphorylated while attached to actin, it will remain attached, in a load-bearing mode. Other theories involve regulatory proteins, like caldesmon, for force maintenance to occur. In an attempt to reproduce the latch-state at the molecular level, we used the in vitro motility assay to measure the velocity (Vmax) of fluorescently labeled actin filaments when propelled by myosin molecules on a coverslip. A threshold velocity was chosen, above which the filaments were considered to be moving, so that motile fraction (fmot), the percentage of filaments moving, could be calculated. An injection chamber was added to the motility assay so that myosin light chain phosphatase (MLCP) could be injected during the assay to efficiently dephosphorylate myosin without creating bulk flow. We dephosphorylated separately phasic and tonic smooth muscle myosin. Our data suggested that a load was created during dephosphorylation because both Vmax and fmot decreased. However, these data were very noisy because they were obtained at very low levels of activation. Thus, the next protocol involved the injection of MLCP to a mixture of smooth and skeletal muscle myosin. The latter not being regulated by phosphorylation assured that the motility would continue after MLCP injection. The rationale behind this protocol was that if the latch-state occurs, a transient decrease in Vmax and fmot should be observed, due to the load induced by the attached, dephosphorylated SM myosin. The motility would eventually increase back to the level of skeletal muscle myosin after the detachment of the latch bridges. The effect of caldesmon was also tested. We observed a transient decrease in both Vmax and fmot suggesting a load-bearing effect. These findings suggest that the dephosphorylation created latch-bridges. Enhancement of this transient load-bearing phase was observed when adding caldesmon. Unexpectedly, this synergistic transient effect of MLCP and caldesmon did not appear to be muscle specific as the same behavior was observed when the measurements were repeated with skeletal muscle myosin alone. In conclusion, a force-maintenance state was reproduced at the molecular level, which could be the underlying mechanism of the latch-state.%%%%Le muscle lisse a une propriete unique, appelee l'etat-« latch », durant lequel la force est maintenue pour de longues periodes de temps a faible consommation d'energie et a faible niveau d'activation de la myosine (faible niveau de phosphorylation). Cette propriete a ete…