Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres.

The effects of amrinone were studied on single skinned fibres isolated from rat hindlimb muscles. In each fibre a force-velocity relation was determined during maximal calcium activation (pCa = 4.45) in control conditions and in the presence of amrinone. The MgATP concentration was 3.93 mM, close to the physiological value. After the experiment the fibre was classified as fast or slow on the basis of its reactivity with anti-myosin monoclonal antibodies. In fast fibres amrinone (3 mM) potentiated isometric tension (Po) by 13.8 +/- 2.9\% (n = 13), reduced maximum shortening velocity (Vmax) by 32.6 +/- 3.2\% and the curvature of the force-velocity relation (a/Po) was increased by 98.9 +/- 46.0\%. All these effects were less pronounced in slow fibres, where Vmax was reduced only by 11.4 +/- 3.6 (n = 16). The effects of amrinone (0.3-6 mM) on the ATPase activity of myofibrils and myosin prepared from fast (tibialis anterior) and slow (soleus) rat skeletal muscles were studied. Amrinone was found to depress Ca-Mg dependent ATPase activity of myofibrillar preparations of the tibialis anterior (up to 16.6 +/- 2\%) and, to a lesser extent, of the soleus (up to 7.2 +/- 1.2\%). On the contrary, Ca-stimulated myosin ATPase activity was significantly increased by amrinone in myosin preparations from the tibialis anterior. Experiments were carried out to test whether amrinone (3 mM) might affect the sensitivity of the contractile system to MgATP concentration ([MgATP]). The results obtained showed that (1) the [MgATP] value at which isometric tension reached its maximum was shifted by amrinone from 0.1 mM to 0.3 mM, (2) the slope of the negative relation between [MgATP] and a/Po was made more steep by amrinone, and (3) the Km of the hyperbolic relation between [MgATP] and Vmax was increased from 0.39 to 1.71 mM by amrinone, thus indicating a reduced affinity of myosin for MgATP. These results are in accordance with the hypothesis that amrinone exerts a direct effect on the contractile mechanism

Stac3 is a component of the excitation–contraction coupling machinery and mutated in Native American myopathy

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca(2+) from internal stores to initiate muscle contraction. Defects in EC coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the EC coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca(2+) imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 as the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies