Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay

Abstract

The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Cosedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of crossbridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.

Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle

“…While these in vitro studies clearly demonstrated MyBP-C's ability to interact with F-actin and thin filaments, and the consequent functional effects (14,15,18,32,(37)(38)(39), our work demonstrates that this binding is relevant in vivo. The in vitro studies show that both skeletal (14,29,30) and cardiac (15,16,18,31,32) MyBP-C can bind to actin. For our experiments we used frog skeletal muscle, whose excellent order and orientation made it possible to obtain the accurate longitudinal sections needed in this work (20).…”

“…These earlier studies, based on centrifugation assays (14)(15)(16)(30)(31)(32), light and electron microscopy (14,16,29,30,(33)(34)(35), neutron scattering data (36), and motility assays (15,32,37,38), studied binding to F-actin (14-16, 31, 32) and to regulated thin filaments (14,15,(29)(30)(31)(32), and used both tissue-prepared protein (14,29,30,32) and expressed whole MyBP-C or N-terminal fragments (15,16,31). While these in vitro studies clearly demonstrated MyBP-C's ability to interact with F-actin and thin filaments, and the consequent functional effects (14,15,18,32,(37)(38)(39), our work demonstrates that this binding is relevant in vivo. The in vitro studies show that both skeletal (14,29,30) and cardiac (15,16,18,31,…”

Knockdown of fast skeletal myosin-binding protein C in zebrafish results in a severe skeletal myopathy

“…Because the relative content of fast and slow skeletal muscle types was not altered, we exclude an effect caused by a fiber type shift. Previous work using skinned skeletal muscle fibers where MyBPC-2 has been partially extracted (Hofmann et al, 1991b), from MyBPC-3 knockout mice and from in vitro motility assays (Korte et al, 2003;Saber et al, 2008), has suggested that MyBPC slows cross-bridge cycling, possibly via introduction of an internal load or direct effects on the actin-myosin interaction. Our results are consistent with these findings and show that the fast MyBPC-2 slows down cross-bridge cycling in the living fibers, albeit to a comparatively small extent.…”

Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

“…If C0 and C1 preferentially bind to activated thin filaments, then relaxation (after a decline in Ca 2ϩ ) could be slowed. Intriguingly, the position of C0 and C1 on actin is also predicted to overlap with myosin cross-bridge-binding sites, which could explain observations that the cMyBP-C and myosin-S1 compete for actin binding (12,34).…”