Abstract-Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (ϩ/Ϫ) and homozygous (Ϫ/Ϫ) for the knockout allele. Wild-type (ϩ/ϩ), cMyBP-C ϩ/Ϫ , and cMyBP-C Ϫ/Ϫ mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C ϩ/Ϫ mice were indistinguishable from wild-type littermates, cMyBP-C Ϫ/Ϫ mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C Ϫ/Ϫ mice. Ca 2ϩ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C Ϫ/Ϫ but not cMyBP-C ϩ/Ϫ mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function. Key Words: myosin binding protein-C Ⅲ heart Ⅲ myocardium Ⅲ gene knockout Ⅲ sarcomeric proteins M yosin binding protein-C (MyBP-C), also known as C-protein, 1 is a thick filament accessory protein that is present in nearly all vertebrate striated muscles but whose function is unknown. Nonetheless, there is compelling evidence to suggest that MyBP-C is a significant determinant of muscle contractile properties. In particular, cardiac MyBP-C (cMyBP-C) is a target for phosphorylation in response to various inotropic stimuli, including sympathetic stimuli that effect trisphosphorylation of cMyBP-C via cAMP-dependent protein kinase (PKA). 2 In addition, mutations of the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy (FHC), 3 an inherited disorder linked to mutations in cardiac contractile proteins (for review, see Bonne et al 4 and Seidman and Seidman 5 ).However, despite clues suggesting the importance of cMyBP-C to cardiac health, the function of cMyBP-C has remained enigmatic. For instance, although numerous studies have investigated effects of PKA on cardiac contractility (eg, Strang et al 6 and Patel et al 7 ), the role, if any, of cMyBP-C in mediating contractile responses to PKA has been difficult to discern. 8 -10 Similarly, the mechanisms by which cMyBP-C mutations affect cardiac function are not well understo...

Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K d ) of ϳ10 M and a molar binding ratio (B max ) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced B max ) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase crossbridge cycling. Cardiac myosin-binding protein C (cMyBP-C)2 is a thick filament accessory protein that performs both structural and regulatory functions within vertebrate sarcomeres. Both roles are likely to be essential in deciphering how a growing number of mutations found in the cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure in a substantial number of the world's population (1, 2).Considerable progress has recently been made in determining the regulatory functions of cMyBP-C and it is now apparent that cMyBP-C normally limits cross-bridge cycling kinetics and is critical for cardiac function (3-5). Phosphorylation of cMyBP-C is essential for its regulatory effects because elimination of phosphorylation sites (serine to alanine substitutions) abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge cycling kinetics and blunts cardiac responses to inotropic stimuli (6). The substitutions further impair cardiac function, reduce contractile reserve, and cause cardiac hypertrophy in transgenic mice (6, 7). By contrast, substitution of aspartic acids at these sites to mimic constitutive phosphorylation is benign or cardioprotective (8).Although a role for cMyBP-C in modulating cross-bridge kinetics is supported by several transgenic and knock-out mouse models (6, 7, 9, 10), the precise mechanisms by which cMyBP-C exerts these effects are not completely understood. For instance, the unique regulatory motif or "m-domain" of cMyBP-C binds to the S2 subfragment of myosin in vitro (11) and binding is abolished by PKA-mediated phosphorylation of the m-domain (12). These observations have led to the idea that (un)binding of the m-domain from myosin S2 mediates PKA-induced increases in cross-bridge cycling kinetics. Consistent with this idea, Calaghan and colleagues (13) showed that S2 added to transiently permeabilized myocytes increa...

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...

Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58 ± 0.01 μm. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra “forbidden” meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.

Abstract-Myosin binding protein-C (MyBP-C) is localized to the thick filaments of striated muscle where it appears to have both structural and regulatory functions. Importantly, mutations in the cardiac MyBP-C gene are associated with familial hypertrophic cardiomyopathy. The purpose of this study was to examine the role that MyBP-C plays in regulating force, power output, and force development rates in cardiac myocytes. Skinned cardiac myocytes from wild-type (WT) and MyBP-C knockout (MyBP-C Ϫ/Ϫ ) mice were attached between a force transducer and position motor. Force, loaded shortening velocities, and rates of force redevelopment were measured during both maximal and half-maximal Ca 2ϩ activations. ). These results suggest that cMyBP-C is an important regulator of myocardial work capacity whereby MyBP-C acts to limit power output.