Functional Differences of Unbound and Weakly Bound Xb States in the Cardiac Myofilament

Abstract

Background: Ca2+ and cross-bridges are involved the activation and deactivation of the cardiac actin filament. The actin filament is decorated with a repeating lattice of Tn and Tm. The affinity of rhodamine-phalloidin for actin is sensitive to the twist of f-actin.Experimental design: (1) Murine cardiac myofibrils were incubated with an excess of dye-labeled cTn under conditions that favor strongly bound (no nucleotide, ADP, ADP + blebbistatin), weakly bound (ADP + Pi, ADP + Pi + blebbistatin), or unbound (ATP, ATP-γ-S) myosin. (2) Rigor and ATP-saturated myofibrils were stained with rhodamine-phalloidin. Samples were immunostained for α-actinin examined by epifluorescence.Results: Strongly-bound and weakly-bound myosin promotes cTn exchange near the z-disc. Unbound myosin promotes cTn exchange near the M-line. The site of cTn exchange correlated with phalloidin binding.Conclusions: The native actin filament exhibits region-dependent stability subject to filament-wide perturbation by strongly-bound and weakly-bound myosin motors.Significance: The native cardiac actin filament has unique functional properties not present in solution studies.