Functional roles of essential light chains of myosin II

The C-terminus of myosin-S1 forms the alpha-helical lever arm which is associated with an essential and a regulatory light chain. The lever arm is believed to work as an elastic ratchet which moves during force-generation from an up into a down position. The ELC in the heart tethers the tick and thin filaments by binding with its four EF-hand domains to the myosin lever arm, while the antenna-like N-terminal domain binds to a distinct actin binding site. Using cell-penetrating peptides which compete for actin interaction with the N-terminal ELC domain we showed that this interaction regulates contraction velocity of the myosin motor.The interaction of the ELC with the lever arm seems to be more complex. Albeit it is known that the ELC are not essential for myosin ATPase activity, ELC are in particular critical for force generation of a single myosin molecule. Therefore, an increased affinity of ELC should raise the fraction of highforce-generating myosin and force of muscle contraction – without the need of additional rise in activating calcium levels. In addition, increasing the ELC-affinity to myosin could modulate the high-force myosin state and, therefore, force generation. We tested this prediction by investigating the myosin affinity of different cardiac ELC isoforms. We observed that the human ALC-1 has an almost three-fold higher myosin affinity than the human or rat VLC-1 isoform. In a recent study we produced adenoviral vectors with human ALC1 as expression cassette and infected adult rat cardiomyocytes. After two days in culture, we investigated shortening amplitude and intracellular Ca2+ during electrical stimulation of the cardiomyocytes. As predicted, shortening amplitude of ALC-1 producing cells was significantly higher than those cells infected with a β-gal-adenovirus. Please note, that this positive inotropic effect occurred without a secondary increase in activating systolic free calcium levels, as monitored by investigating the Fura-fluorescence. We observed consistent inotropic effects of hALC-1 in cardiac preparations with increasing amounts of expressed ALC-1, namely i) in isolated perfused hearts from transgenic rats overexpressing human ALC-1, ii) skinned human ventricular fibers, and iii) in vivo in patients with heart diseases, who showed an improved ejection fraction with increasing amounts of ALC-1 expressed in the left ventricle. If strong ELC binding to the lever arm → (increased stiffness) → increased myosin force generation, than weak ELC binding to the lever arm → (decreased stiffness) → decreased myosin force generation. We tested this prediction with the help of disease-causing ELC mutations. At the moment, there are seven mutations of the human MYL3 gene coding the VLC-1. They are clustered in exon Session 5. Molecular Mechanisms of Motility