The effects of force inhibition by sodium vanadate on cross-bridge binding, force redevelopment, and Ca2+ activation in cardiac muscle.

Strongly bound, force-generating myosin cross-bridges play an important role as allosteric activators of cardiac thin filaments. Sodium vanadate (Vi) is a phosphate analog that inhibits force by preventing cross-bridge transition into force-producing states. This study characterizes the mechanical state of cross-bridges with bound Vi as a tool to examine the contribution of cross-bridges to cardiac contractile activation. The Ki of force inhibition by Vi was ∼40 μM. Sinusoidal stiffness was inhibited with Vi, although to a lesser extent than force. We used chord stiffness measurements to monitor Vi-induced changes in cross-bridge attachment/detachment kinetics at saturating [Ca2+]. Vi decreased chord stiffness at the fastest rates of stretch, whereas at slow rates chord stiffness actually increased. This suggests a shift in cross-bridge population toward low force states with very slow attachment/detachment kinetics. Low angle x-ray diffraction measurements indicate that with Vi cross-bridge mass shifted away from thin filaments, implying decreased cross-bridge/thin filament interaction. The combined x-ray and mechanical data suggest at least two cross-bridge populations with Vi; one characteristic of normal cycling cross-bridges, and a population of weak-binding cross-bridges with bound Vi and slow attachment/detachment kinetics. The Ca2+ sensitivity of force (pCa50) and force redevelopment kinetics (kTR) were measured to study the effects of Vi on contractile activation. When maximal force was inhibited by 40% with Vi pCa50 decreased, but greater force inhibition at higher [Vi] did not further alter pCa50. In contrast, the Ca2+ sensitivity of kTR was unaffected by Vi. Interestingly, when force was inhibited by Vi kTR increased at submaximal levels of Ca2+-activated force. Additionally, kTR is faster at saturating Ca2+ at [Vi] that inhibit force by >∼70%. The effects of Vi on kTR imply that kTR is determined not only by the intrinsic properties of the cross-bridge cycle, but also by cross-bridge contribution to thin filament activation.