Orientation of the Calcium Sensitizing Agent dfbp-o, when Bound to Troponin in a Muscle Fiber as Determined by Solid-State NMR Spectroscopy

Abstract

Heart failure is characterized by the inability of the heart to supply the body with oxygenated blood. There is a large range of treatment strategies employed by physicians; however, many of these options are limited in their ability to improve mortality during acute heart failure. A more recently identified therapeutic option involves a class of pharmaceuticals termed “calcium sensitizers”. These molecules enhance the contractile apparatus's response to calcium, instead of increasing the concentration of cytosolic calcium. Levosimendan is the most widely used calcium sensitizer, but since it is unstable, its exact mode of action has yet to be identified. Recently, we have determined that the stable structural analog of levosimendan, dfbp-o, functions as a calcium sensitizer in a similar manner as levosimendan [Robertson et al., J Mol Cell Cardiol. 2010 Aug 27. [Epub ahead of print]]. Two fluorine atoms are present on dfbp-o which can be used to elucidate useful structural information via 19F-NMR spectroscopy. 19F is a particularly attractive nucleus for study by NMR spectroscopy because like 1H it is ∼100% naturally abundant, has a spin of 1/2, and possesses a large gyromagnetic ratio. We have used 19F solid-state NMR spectroscopy to study the orientation of dfbp-o in demembranated rabbit psoas muscle fibers. Upon incubating the muscle fiber in relaxing solution containing dfbp-o, the length of the fiber decreased approximately three fold, presumably due to the calcium sensitizing nature of dfbp-o. This approach will allow us to establish the orientation of the dfbp-o:troponin complex with respect to the thin filament axis. In addition, the results give us further evidence that dfbp-o binds muscle fibers to elicit its calcium sensitization effect.