Effects of the N-terminal Domains of Myosin Binding Protein-C in an in Vitro Motility Assay

Abstract

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of ϳ110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where fila- Myosin binding protein-C (MyBP-C)2 is a sarcomeric protein associated with the thick filaments of vertebrate striated muscle (1). Although the precise function of MyBP-C within the sarcomere is not well understood, evidence from MyBP-C knock-out mice that lack cardiac MyBP-C (2) indicate cMyBP-C slows cross-bridge cycling and rates of force development, especially at submaximal [Ca 2ϩ ] (3-5). The idea that MyBP-C limits cross-bridge kinetics was initially proposed by Hofmann et al. (6) who suggested that MyBP-C acts as an internal load within the sarcomere based on their observations that partial extraction of MyBP-C from skeletal fibers reversibly accelerated a low velocity phase of shortening at submaximal Ca 2ϩ activation (7). Although the exact structural arrangement of MyBP-C within the sarcomere is not known, MyBP-C could contribute to an internal load by tethering myosin heads to the thick filament and thereby limiting the extension of attached myosin heads as shortening proceeds (6). Consistent with this idea, Calaghan et al. (8) proposed that simultaneous binding of MyBP-C to two positions on myosin, i.e. to myosin S2 (near the S1/S2 junction) and to the light meromyosin segment of myosin rod, could restrict the extension of myosin heads away from the thick filament. The net effect might be to limit myosin interactions with actin. However, a recombinant MyBP-C protein containing only the C1C2 domains and thus a single S2 binding site increased Ca 2ϩ sensitivity of force in myocytes from cMyBP-C knock-out mice (9). Because effects of C1C2 did not depend on a second myosin binding site, the results implied that the C1C2 domains could affect actomyosin interactions independent of tethering myosin heads to thick filaments.The current experiments were performed to investigate mechanisms by which N-terminal domains of MyBP-C influence myosin contractile properties and whether these effects depend on organization of myosin into thick filaments. Results from in vitro motility assays demonstrate that organized thick filaments are not required for recombinant proteins containing N-terminal domains of MyBP-C to affect mechanical properties of myosin and further suggest that effects of MyBP-C to slow cross-bridge kinetics may be due to slow...

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

“…However, a reduced number of cross-bridges interacting with actin cannot solely account for the cMyBP-C induced reduction in velocity, as long actin filaments, which interact with greater number of cross-bridges on the motility surface, do not exhibit faster velocities (see Results; Figure 6). Thus, cMyBP-C, presumably through its interaction with the thin filament, modulates either the inherent mechanics and/or kinetics of the myosin cross-bridge [22]. Specifically, cMyBP-C could reduce the displacement generated by the myosin motor and/or the rate at which the cross-bridge detaches from actin following the powerstroke, both of which govern actin filament velocity at the molecular level [47].…”

“…In contrast, using an expressed C5 domain peptide resulted in a rightward shift, whereas the C0-C1 peptide had no effect on tension [21]. Recent studies in an in vitro motility assay have shown that the cMyBP-C domain fragments may activate the thin filament at low calcium levels but depress thin filament sliding speed at high calcium levels [22]. Thus individual domains appear to have contrasting effects on contractile function making interpretation of the overall function of the intact cMyBP-C challenging.…”

“…These data indicate that the binding of cMyBP-C to LMM or myosin S2 is not required for whole cMyBP-C to exert its inhibitory effect on thin filament motility. Harris and colleagues demonstrated a reduction in thin filament sliding velocity with the C1-C2 domain fragment for both HMM and myosin S1 in the motility assay [22,37]. In these experiments a ten-fold higher concentration of C1-C2 was used to see this effect suggesting that either the binding affinity of this domain fragment is less than intact cMyBP-C protein and/or that whole cMyBP-C affects contractile function in a somewhat different manner than the C1-C2 peptide.…”

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

“…18). In the in vitro motility assay, actin filament sliding over myosin is slowed by N-terminal fragments of cMyBP-C to the same extent as whole cMyBP-C (19), possibly by slowing the myosin detachment rate from actin (19) or tethering the thick to the thin filament (16,19). In an assay closer to the in vivo situation, the sliding of F-actin over native cardiac thick filaments was slowed specifically in the C-zone, and this slowing was ablated by removal of C0C1 and the first 17 amino acids of the M-domain [known as C0C1f (16); Fig.…”

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

“…The C terminus of cMyBP-C binds to the thick filament backbone via interactions with myosin and titin primarily through C7-C10 (2,7). The C1-C2 region of MyBP-C also has been shown to bind to myosin S2 segment (8) and appears to interact with actin thin filament (9).…”