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The Effects of Force Inhibition by Sodium Vanadate on Cross-Bridge Binding, Force Redevelopment, and Ca²⁺ Activation in Cardiac Muscle

D. A. Martyn ^*, L. Smith, K. L. Kreutziger ^*, S. Xu , L. C. Yu  and M. Regnier ^*

^* Department of Bioengineering, University of Washington, Seattle, Washington; and National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland

Correspondence: Address reprint requests to Donald A. Martyn, PhD, Dept. of Bioengineering, Box 355061, University of Washington, Seattle, WA 98195. Tel.: 206-543-4478; Fax: 206-685-3300; E-mail: dmartyn{at}u.washington.edu.

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 K_i 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 (k_TR) 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 k_TR was unaffected by Vi. Interestingly, when force was inhibited by Vi k_TR increased at submaximal levels of Ca²⁺-activated force. Additionally, k_TR is faster at saturating Ca²⁺ at [Vi] that inhibit force by >70%. The effects of Vi on k_TR imply that k_TR is determined not only by the intrinsic properties of the cross-bridge cycle, but also by cross-bridge contribution to thin filament activation.

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