Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C037

Oral Communications

The strong binding states of the myosin head contribute to the activation of the thin filament in human soleus slow-twitch muscle fibers

A. J. Lopez-Davila1, S. Zittrich2, B. Piep1, F. Matinmehr1, A. Málnási-Csizmadia3, A. Rauscher3, J. M. Chalovich4, T. kraft1, B. Brenner1, R. Stehle2

1. Institute for Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany. 2. Institute of Vegetative Physiology, University of Cologne, Cologne, Germany. 3. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, Budapest, Hungary. 4. Department of Biochemistry & Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States.

Force development of striated muscle is triggered by the calcium-induced activation of the troponin complex and the subsequent azimuthal displacement of tropomyosin, which exposes the high affinity myosin binding sites on actin. Several experimental models including proteins in solution, reconstituted thin filaments and isometric contracting muscle fibers have suggested that the thin filament is additionally activated by the strong-binding state of the myosin head although some observations made in isometric contracting muscle fibers have questioned this view. Since these studies have been performed either on cardiac or on fast-twitch muscle preparations, it is unknown whether cycling cross-bridges influence the thin filament activation in slow-twitch skeletal muscle. Human soleus fibers express the same myosin isoform as the human heart and constitute an interesting experimental model for the examination of myosin mutations related to familial hypertrophic cardiomyopathy. To probe the calcium and myosin induced activation of the thin filament in isometric contracting human soleus fibers, the endogenous troponin complex was exchanged for a well characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye IANBD (fsTnIANBD). The effectively exchanged amount of troponin was 70%. To dissect the individual contributions of Ca2+ and cross-bridges on thin filament activation, force-pCa and fsTnIANBD emission-pCa relations were explored in absence and presence of para-amino-blebbistatin (PAB), an inhibitor of the strongly-bound state of myosin. Increasing the calcium concentration from relaxing (pCa 7.5) to saturating levels (pCa 4.5) without PAB induced maximal force development and generated a 32.2 % (± 1.33%, N = 8) decrease in the emission intensity of fsTnIANBD. 50 µM PAB strongly inhibited active force at pCa 4.5 to 6.8 % (± 1%, N = 8) of the active force without PAB while it decreased the Ca2+-induced decrease in emission intensity of fsTnIANBD to only 17.8% (± 2.6%, N = 8). At all calcium concentrations, the calcium induced activation of the thin filament was higher than the myosin induced activation. However, the myosin induced activation increased with the calcium concentration (i.e. myosin contribution to the total activation was about 45%, 35% and 20% at saturating, intermediate and low calcium concentrations, respectively). Importantly, when switching from isometric contraction to isotonic contraction at low load, changes in fluorescence indicating the kinetics of the conformational changes of fsTnIANBD were at least ten times faster than kinetics of force development. Thus our kinetic data indicates that the rate of thin filament switching does not limit the rate of force development.

Where applicable, experiments conform with Society ethical requirements