A new in vitro motility assay technique to evaluate calcium sensitivity of the cardiac contractile proteins

Abstract

We attempted to introduce calcium regulation into in vitro motility assay. Cardiac thin filament was reconstituted from actin and tropomyosin-troponin complex purified from rat myocardium separately. Double staining of the filaments showed tropomyosin-troponin complex was integrated along actin filaments homogeneously. The reconstituted thin filaments were made to slide on cardiac myosin fixed on a glass coverslip in the presence of MgATP while varying free Ca²⁺ concentration of the medium ([Ca²⁺]). Filaments showed only Brownian motion when [Ca²⁺] was below 10^−6.4 M. However, filaments slid at a constant velocity when [Ca²⁺] exceeded 10^−6.4 M, showing that the sliding was regulated in an on-off manner. The threshold [Ca²⁺] increased to 10^−5.0 M under acidic conditions, indicating a decrease in Ca²⁺ sensitivity of the contractile proteins. Simple actin filaments slid at a constant velocity independently of [Ca²⁺], demonstrating that the regulatory proteins were responsible for this on-off manner regulation. This new assay technique may be a powerful tool to directly evaluate the Ca²⁺ sensitivity of the contractile apparatus and to investigate how cardiac contraction is regulated by Ca²⁺.