Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle.

Abstract

In addition to the contractile proteins actin and myosin, contractile filaments of striated muscle contain other proteins that are important for regulating the structure and the interaction of the two force-generating proteins. In the thin filaments, troponin and tropomyosin form a Ca-sensitive trigger that activates normal contraction when intracellular Ca is elevated. In the thick filament, there are several myosinbinding proteins whose functions are unclear. Among these is the myosin-binding protein C (MBP-C). The cardiac isoform contains four phosphorylation sites under the control of cAMP and calmodulin-regulated kinases, whereas the skeletal isoform contains only one such site, suggesting that phosphorylation in cardiac muscle has a specific regulatory function. We isolated natural thick filaments from cardiac muscle and, using electron microscopy and optical diffraction, determined the effect of phosphorylation of MBP-C on cross bridges. The thickness of the filaments that had been treated with protein kinase A was increased where cross bridges were present. No change occurred in the central bare zone that is devoid of cross bridges. The intensity of the reflections along the 43-nm layer line, which is primarily due to the helical array of cross bridges, was increased, and the distance of the first peak reflection from the meridian along the 43-nm layer line was decreased. The results indicate that phosphorylation of MBP-C (i) extends the cross bridges from the backbone of the filament and (ii) increases their degree of order and/or alters their orientation. These changes could alter rate constants for attachment to and detachment from the thin filament and thereby modify force production in activated cardiac muscle.

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

“…Several investigators have proposed that the reduced unloaded shortening velocity in fiber studies is the result of cMyBP-C functioning either as a tether restricting myosin's interactions with actin [15,29,[43][44][45] and/or imparting a viscous load to thick-thin filament sliding [46]. Our data would indicate that neither of these mechanisms can account for the functional effects observed with cMyBP-C in this study.…”

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

“…The C-terminal domains of the protein (C7-C10) bind to the myosin thick filament and play a predominantly structural role, 66 while the first four N-terminal domains (designated C0-C1-m-C2 or C0C2) function by regulating the docking of myosin S1 onto actin thin filaments. 67,68 This regulatory action involves C0C2 interacting with actin and with myosin DS2; the latter being the region of myosin S2 that is proximal to the regulatory and essential light chains of the myosin S1 head. Importantly, the phosphoylation of the m-domain, in response to stimuli, is proposed to ''untethers'' the N-terminus of cMyBP-C from myosin DS2 allowing myosin heads to reposition in a conformation that is more favorable for binding actin, thus affecting the rate and force of heart muscle contraction.…”

Cardiac myosin binding protein c phosphorylation is cardioprotective

“…cMyBP-C is the only thick filament protein that is differentially phosphorylated at multiple sites by the enzymes PKA, PKC, and Ca 2ϩ -calmodulin-activated kinase (13). Reconstitution studies showed that PKA-mediated phosphorylation of cMyBP-C extends the cross-bridges from the backbone of the thick filament, changes their orientation, increases the degree of order of the cross-bridges, and decreases cross-bridge flexibility (11). cMyBP-C phosphorylation can change both filament orientation and contractile mechanics (13), but the in vivo role of these posttranslational modifications on whole-organ function remains obscure.…”

“…Compared with the two skeletal muscle isoforms, the cardiac isoform contains an extra Ig domain at the N terminus (C0), an insertion of 28 residues within the C5 domain, and three potential phosphorylation sites (Ser-273, Ser-282, and Ser-302) that are substrates for cAMP-dependent PKA, Ca 2ϩ -calmodulin-activated kinase, and PKC (8). This region is located between the C1 and C2 domains of the N terminus, which binds to the subfragment 2 (S2) segment of myosin close to the lever arm domain (9)(10)(11), and this interaction may be dynamically regulated by the differential phosphorylation of cMyBP-C (12). cMyBP-C is the only thick filament protein that is differentially phosphorylated at multiple sites by the enzymes PKA, PKC, and Ca 2ϩ -calmodulin-activated kinase (13).…”