Contribution of the Myosin Binding Protein C Motif to Functional Effects in Permeabilized Rat Trabeculae

Abstract

Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C “motif” or “m-domain” increased Ca2+ sensitivity of tension and increased rates of tension redevelopment (i.e., ktr) at submaximal levels of Ca2+. At concentrations ≥20 μM, recombinant proteins also activated force in the absence of Ca2+ and inhibited maximum Ca2+-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament.

A Gain-of-Function Mutation in the M-domain of Cardiac Myosin-binding Protein-C Increases Binding to Actin

“…In most cases recombinant proteins were omitted from Ca 2ϩ activating solutions because measured forces were not different with or without added protein (not shown), consistent with previous results demonstrating negligible washout of proteins during the short period of Ca 2ϩ activation (23).…”

“…Recombinant Protein Expression and Purification-Recombinant proteins were expressed and purified using murine cDNA sequences as described previously (23,24). For single and paired amino acid substitutions, site-directed mutagenesis of wild-type C1C2 (domains C1-M-C2) was performed using the QuikChange II Site-directed Mutagenesis kit (Stratagene) with primers designed using the Stratagene primer design software.…”

“…Solutions-Relaxing and activating solutions were prepared using a custom software package as previously described (23,27 Mechanical Force Measurements-Force measurements in permeabilized trabeculae were performed as described previously (23). Permeabilized trabeculae were mounted between a force transducer (model 403A, Aurora Scientific Inc.) and a torque motor (model 312-C, Aurora Scientific Inc.) for rapid adjustments of fiber length.…”

Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

“…Previous modeling suggested that the C0 and C1 domains could clash with Tm in its blocked position (25,27,28), whereas experiments in which expressed N-terminal fragments were added to skinned cardiac myocytes implicated the ProAla-rich domain between C0 and C1 in modulating Ca 2+ activation of crossbridge cycling (46). However, in similar skinned fiber experiments, the ProAla-rich domain was found to have no effect, whereas the C1 and M-domains were critical to the Ca 2+ -sensitizing and activating effects of various N-terminal domains (48). This too was the case in the present study, as only the N-terminal fragments containing the C1 and M-domains were able to displace Tm (C0C2) and activate the thin filament (C0C3).…”

Structural Insight into Unique Cardiac Myosin-binding Protein-C Motif

“…Taken together, our structural studies suggest that the C-terminal region of the m-domain may function as an actin-binding protein. Supporting this hypothesis, functional studies showed that the m-domain increased the Ca 2ϩ sensitivity of tension and increased rates of tension redevelopment (53) and that C1-m binds F-actin in a saturable manner (15). Phosphorylation of the m-domain was shown to decrease the actin binding affinity (15).…”