Myosin binding protein-C (MyBP-C) is a striated muscle, thick filament associated protein [1
]. Its importance is emphasized by the development of familial hypertrophic or dilated cardiomyopathy caused by mutations in the cardiac isoform (cMyBP-C) [2
]. MyBP-C exists in a repeating pattern of 7–9 bands within the C-zones of the sarcomere [5
] with each cMyBP-C containing 11 immunoglobulin or fibronectin-like domains, C0 through C10 () [6
]. Functional roles have been proposed for distinct regions within the protein based on its multiple contractile protein binding partners: myosin [7
], myosin regulatory light chain [8
], titin [9
], and actin [10
]. However, the physiological importance of its in vitro
binding to actin has been called into question with recent evidence that this binding is non-specific and that cMyBP-C phosphorylation, a potential regulator of cMyBP-C function in vivo
, has no effect on such binding [11
]. Here we used single molecule biophysical techniques (i.e. in vitro
motility and laser trap assays) to directly confirm that the N-terminus of cMyBP-C binds actin stereospecifically, as proposed by others [12
], and that this binding is modulated by cMyBP-C phosphorylation.
Figure 1 Schematic of whole cMyBP-C and expressed N-terminal fragments. Immunoglobulin I-like domains are oval, while fibronectin-3 domains are rectangular. cMyBP-C contains several cardiac specific sequences: the C0 domain, and inserts within the MyBP-C motif (more ...)
The impact of MyBP-C on muscle function was derived from early studies in skinned skeletal muscle fibers where MyBP-C had been chemically extracted [14
]. Upon its removal, faster shortening velocities and elevated force production at low calcium levels were observed, which was interpreted as MyBP-C imposing an internal load and limiting myosin head mobility. This effect could be a result of cMyBP-C’s capacity to form a link between the myosin head and thick filament backbone through its N-terminal binding to the myosin S2 region and its C-terminal binding to the myosin LMM [4
]. With the N-terminus of cMyBP-C also capable of binding actin [16
], an equally plausible model is that cMyBP-C forms a tether between the thick and thin filaments. Such a link could modulate actomyosin activity, by competing with myosin for actin-binding sites, by altering muscle activation by modulating tropomyosin movement on the thin filament, or by simply imposing an internal load to muscle shortening.
Phosphorylation of cMyBP-C in response to β-adrenergic stimulation is critically important to the dynamic contractile regulation of the heart [4
]. Phosphorylation occurs within the linker between domains C1 and C2 (i.e. MyBP-C motif) at 4 cardiac isoform-specific serines [18
]. Recent studies suggest that cMyBP-C phosphorylation is reduced in humans with hypertrophic cardiomyopathy or end-stage heart failure [20
]. In fact, transgenic mice expressing non-phosphorylatable alanine substitutions for serines in the cMyBP-C motif in a cMyBP-C null background, exhibited a hypertrophied phenotype [23
], whereas substitution with aspartic acids as phosphomimetics protected the heart from ischemic reperfusion injury [25
]. These data underscore the physiologic importance of cMyBP-C phosphorylation. Phosphorylation by protein kinase A (PKA) in vitro
diminishes the binding affinity of the motif for actin as well as myosin [17
], although the effect of phosphorylation on actin-binding was recently challenged [11
]. Therefore, phosphorylation might eliminate cMyBP-C’s role as a tether and partially account for the enhanced cardiac contractility observed with adrenergic stimulation.
Using a combination of expressed mouse cMyBP-C fragments and single molecule biophysical approaches, we identified potential site(s) within the first 4 N-terminal domains (C0-C3) responsible for actin-binding and the regulatory effect phosphorylation has on this interaction. Through structural deletions and amino acid substitutions, we identified an actin-binding site within the first 17 amino acids of the cMyBP-C motif following the C1 domain that may interact with actin in a stereospecific manner. All N-terminal fragments studied that contain these 17 amino acids inhibit actomyosin motility to the same extent as we previously reported for purified full length cMyBP-C [27
]. Based on direct evidence that the N-terminal fragments transiently bind to a single actin filament in the laser trap assay, we propose that one mode by which cMyBP-C exerts its mechanical effects on actomyosin power generation is by creating a physical link between the thick and thin filaments that imposes an internal viscous load within the sarcomere.