1602491
The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner
Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0–C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1–C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0–C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0–C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0–C3 in proportion to the total number phosphomimetic replacements. Under load, C0–C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0–C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0–C3 on F:V was well fitted by a model that assumed C0–C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.
6666559
ATPase Activity of Myosin Correlated with Speed of Muscle Shortening
Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca++-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca++-activated myosin ATPase was the same as that of the speed of shortening, Q10 about 2. Higher Q10 values were found for the actin-activated myosin ATPase, especially below 10°C. By using myofibrils instead of reconstituted actomyosin, Q10 values close to 2 could be obtained for the Mg++-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.
45945020
Phosphorylation of the myofibrillar proteins.
In muscle, protein phosphorylation takes place in both the sarcoplasm and the myofibrils. This review deals only with those myofibrillar proteins that can be phosphorylated-i.e. myosin light chain and in a few cases the heavy chain, the inhibitory and tropomyosin-binding subunits of troponin, and tropomyosin. Although research on this topic began as recently as 1972, several interesting results have been accumulated (for reviews, see 86-88, 114, 116).
1336010
In the thick of it: HCM-causing mutations in myosin binding proteins of the thick filament.
In the 20 years since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM), an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins, including the myosin essential and regulatory light chains and cardiac myosin binding protein (cMyBP)-C. However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM-causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin-driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes.
34476398
The location of C-protein in rabbit skeletal muscle
Antibodies to C-protein have been used to label fibres of glycerinated rabbit psoas muscle. The labelling patterns observed in the electron microscope show that C-protein is located on 7 stripes, spaced about 43 nm apart in each half of the A-band. Between two and four C-protein molecules per filament are present at each stripe. The labelling patterns also reveal the existence of two previously unrecognized components of the thick filament present in two further stripes in each half of the A-band. The location of C-protein and these other components accounts for many of the stripes seen in sections of the A-band and in negatively stained A-segments and provides new information on the packing of myosin molecules in the thick filament.
29831768
Interaction of C-protein with myosin, myosin rod and light meromyosin.
C-protein, a component of vertebrate skeletal muscle myofibrils, is known to be located at specific positions along the thick myosin-containingc myofilaments. We have investigated its effects on the assembly of myosin into filaments in vitro and its interaction with low ionic strength aggregates of myosin rod and light meromyosin, the α-helical fragments of myosin. C-protein is not required for the formation of myosin filaments in vitro . Purified myosin, free of C-protein, can form long filaments with a demonstrable 14 nm longitudinal repeat. The presence of C-protein disrupts the regularity of the myosin filament structure, resulting in a reduced and more variable diameter and a loss of longitudinal order. However, C-protein does not appear to have any length-regulating role. Binding measurements at low ionic strength reveal a strong affinity of C-protein for myosin and also for rod and light meromyosin. The limiting stoichiometry of binding is about one mole C-protein per mole for myosin and somewhat less for rod and light meromyosin. The interaction of C-protein with rod and light meromyosin has also been investigated by electron microscopy. In the absence of C-protein, myosin rod forms large sheet-like paracrystals with a 14 nm longitudinal repeat. In the presence of C-protein the formation of these paracrystals is disrupted and in their place we find irregular narrow filaments. Formation of paracrystals of light meromyosin is not disrupted by C-protein. The C-protein forms a series of transverse stripes on the paracrystal with a longitudinal spacing identical to the principal repeat of about 40 nm which characterizes the light meromyosin assembly. We conclude that the C-protein in native thick filaments is probably bound to the shaft of the filament with a periodicity determined by the underlying myosin assembly.
42863174
The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.
Abstract C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.
25841791
Hypercontractile Properties of Cardiac Muscle Fibers in a Knock-in Mouse Model of Cardiac Myosin-binding Protein-C*
Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca2+ sensitivity of force development, particularly at lower Ca2+concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.
3370328
Effects of the N-terminal Domains of Myosin Binding Protein-C in an in Vitro Motility Assay
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 ∼110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.
1301685
Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay.
The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.
30287101
Effects of cardiac myosin binding protein-C on the regulation of interaction of cardiac myosin with thin filament in an in vitro motility assay.
Modulatory role of whole cardiac myosin binding protein-C (сMyBP-C) in regulation of cardiac muscle contractility was studied in the in vitro motility assay with rabbit cardiac myosin as a motor protein. The effects of cMyBP-C on the interaction of cardiac myosin with regulated thin filament were tested in both in vitro motility and ATPase assays. We demonstrate that the addition of cMyBP-C increases calcium regulated Mg-ATPase activity of cardiac myosin at submaximal calcium. The Hill coefficient for 'pCa-velocity' relation in the in vitro motility assay decreased and the calcium sensitivity increased when сMyBP-C was added. Results of our experiments testifies in favor of the hypothesis that сMyBP-C slows down cross-bridge kinetics when binding to actin.
40594948
C‐protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2+ activation.
1. Effects on maximum shortening velocity (Vmax) due to partial extraction of C‐protein were investigated in skinned fibres from rabbit psoas muscles. Up to 80% of endogenous C‐protein was extracted, as assessed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of fibre segments obtained before and after the extraction protocol. Vmax was obtained at 15 degrees C by measuring the times required to take up various amounts of slack imposed at one end of the fibre. 2. During maximal activation with Ca2+, Vmax in control fibres was 4.26 +/‐ 0.16 (mean +/‐ S.E.M., n = 7) muscle lengths per second (ML/s). Following extraction of approximately 40% of endogenous C‐protein, Vmax in these same fibres was 4.41 +/‐ 0.24 ML/s. 3. At sufficiently low levels of submaximal activation, high‐ and low‐velocity phases of unloaded shortening were observed. Partial extraction of C‐protein significantly increased Vmax in the low‐velocity phase but had no effect on the high‐velocity phase. The effect on low‐velocity Vmax was fully reversed by re‐addition of purified C‐protein. 4. At low levels of activation, the amount of shortening to the break‐point between the high‐ and low‐velocity phases was not significantly affected by C‐protein extraction. Under control conditions the average break‐point was 85.6 +/‐ 3.1 nm/half‐sarcomere, while 84.1 +/‐ 3.1 nm/half‐sarcomere was obtained following partial extraction of C‐protein. 5. These results are considered in terms of a model in which an internal load slows Vmax at low levels of activation once a given amount of active shortening has occurred. C‐protein may contribute to this internal load either by binding to actin and myosin or by influencing mechanical properties of myosin cross‐bridges.
10730015
Ablation of Cardiac Myosin-Binding Protein-C Accelerates Stretch Activation in Murine Skinned Myocardium
Cardiac myosin binding protein-C (cMyBP-C) is a thick filament accessory protein that binds tightly to myosin, but despite evidence that mutations in the cMyBP-C gene comprise a frequent cause of hypertrophic cardiomyopathy, relatively little is known about the role(s) of cMyBP-C in myocardium. Based on earlier studies demonstrating the potential importance of stretch activation in cardiac contraction, we examined the effects of cMyBP-C on the stretch activation responses of skinned ventricular preparations from wild-type (WT) and homozygous cMyBP-C knockout mice (cMyBP-C−/−) previously developed in our laboratory. Sudden stretch of skinned myocardium during maximal or submaximal Ca2+ activations resulted in an instantaneous increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (ie, stretch activation) to levels greater than prestretch force. Ablation of cMyBP-C dramatically altered the stretch activation response, ie, the rates of force decay and delayed force transient were accelerated compared with WT myocardium. These results suggest that cMyBP-C normally constrains the spatial position of myosin cross-bridges, which, in turn, limits both the rate and extent of interaction of cross-bridges with actin. We propose that ablation of cMyBP-C removes this constraint, increases the likelihood of cross-bridge binding to actin, and speeds the rate of delayed force development following stretch. Regardless of the specific mechanism, acceleration of cross-bridge cycling in cMyBP-C−/− myocardium could account for the abbreviation of systolic ejection in this mouse as a direct consequence of premature stretch activation of ventricular myocardium.
17486322
Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers
C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3- h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40- 60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension- pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C- protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.
1404682
A role for C‐protein in the regulation of contraction and intracellular Ca2+ in intact rat ventricular myocytes
1 C‐protein is a major component of muscle thick filaments whose function is unknown. We have examined for the first time the role of the regulatory binding domain of C‐protein in modulating contraction and intracellular Ca2+ concentration ([Ca2+]i) in intact cardiac myocytes. 2 Rat ventricular myocytes were reversibly permeabilised with the pore‐forming toxin streptolysin O. Myosin S2 (which binds to the regulatory domain of C‐protein) was introduced into cells during permeabilisation to compete with the endogenous C‐protein‐thick filament interaction. 3 Introduction of S2 into myocytes increased contractility by ∼30%, significantly lengthened the time to peak of the contraction and the time to half‐relaxation, but had no effect on [Ca2+]i transient amplitude. 4 Our data are consistent with increased myofilament Ca2+ sensitivity when there is reduced binding of C‐protein to myosin near the head‐tail junction. 5 We propose that the effects of introducing S2 into intact cardiac cells can be equated with the consequences of selectively phosphorylating C‐protein in vivo, and that the regulation of contraction by C‐protein is mediated by the effects of crossbridge cycling on the Ca2+ affinity of troponin C.
1176127
Contribution of the Myosin Binding Protein C Motif to Functional Effects in Permeabilized Rat Trabeculae
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.
5606984
Effect of Cardiac Myosin Binding Protein-C on Mechanoenergetics in Mouse Myocardium
We examined the effect of cardiac myosin binding protein-C (cMyBP-C) on contractile efficiency in isovolumically contracting left ventricle (LV) and on internal viscosity and oscillatory work production in skinned myocardial strips. A 6-week diet of 0.15% 6-n-propyl-2-thiouracil (PTU) was fed to wild-type (+/+PTU) and homozygous-truncated cMyBP-C (t/tPTU) mice starting at age ≈8 weeks and leading to a myosin heavy chain (MHC) isoform profile of 10% &agr;-MHC and 90% β-MHC in both groups. Western blot analysis confirmed that cMyBP-C was present in the +/+PTU and effectively absent in the t/tPTU. Total LV mechanical energy per beat was quantified as pressure-volume area (PVA). O2 consumption (VO2) per beat was plotted against PVA at varying LV volumes. The reciprocal of the slope of the linear VO2–PVA relation represents the contractile efficiency of converting O2 to mechanical energy. Contractile efficiency was significantly enhanced in t/tPTU (26.1±2.6%) compared with +/+PTU (17.1±1.6%). In skinned myocardial strips, maximum isometric tension was similar in t/tPTU (18.7±2.1 mN · mm−2) and +/+PTU (21.9±4.0 mN · mm−2), but maximum oscillatory work induced by sinusoidal length perturbations occurred at higher frequencies in t/tPTU (7.31±1.17 Hz) compared with +/+PTU (4.48±0.60 Hz) and was significantly more sensitive to phosphate concentration in the t/tPTU. Under rigor conditions, the internal viscous load was significantly lower in the t/tPTU compared with +/+PTU, ie, ≈40% lower at 1 Hz. These results collectively suggest that contractile efficiency is enhanced in the t/tPTU, probably through a reduced loss of mechanical energy by a viscous load normally provided by cMyBP-C and through a gain of phosphate-dependent oscillatory work normally inhibited by cMyBP-C.
23137552
Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain.
Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20 ms) and long (~300 ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.
12066171
Phosphorylation switches specific for the cardiac isoform of myosin binding protein‐C: a modulator of cardiac contraction?
Cardiac myosin binding protein‐C (cardiac MyBP‐C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP‐dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP‐C now reveals regulatory motifs specific for this isoform. Site‐directed mutagenesis identifies a LAGGGRRIS loop in the N‐terminal region of cardiac MyBP‐C as the key substrate site for phosphorylation by both PKA and a calmodulin‐dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform‐specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP‐C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP‐C in cardiac disease. The gene coding for cardiac MyBP‐C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP‐C a candidate gene for chromosome 11‐associated FHC.
5794389
Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.
Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.
11452730
A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function
Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during &bgr;-adrenergic stimulation. Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca2+-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-CSAS), Ala-273-Asp-282-Ala-302 (cMyBP-CADA), or Asp-273-Ala-282-Asp-302 (cMyBP-CDAD), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-CSAS(t/t), cMyBP-CADA(t/t), and cMyBP-CDAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C(t/t) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered &bgr;-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-CSAS(t/t) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of &bgr;-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal &bgr;-adrenergic responsiveness. Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to &bgr;-agonist stimulation.
20414295
Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C.
Ca ions can influence the contraction of cardiac muscle by activating kinases that specifically phosphorylate the myofibrillar proteins myosin-binding protein C (MyBP-C) and the regulatory light chain of myosin (RLC). To investigate the possible role of Ca-regulated phosphorylation of MyBP-C on contraction, isolated quiescent and rhythmically contracting cardiac trabeculae were exposed to different concentrations of extracellular Ca and then chemically skinned to clamp the contractile system. Maximum Ca-activated force (F(max)) was measured in quiescent cells soaking in 1) 2.5 mM Ca for 120 min, 2) 1.25 mM for 120 min, or 3) 1.25 mM for 120 min followed by 10 min in 7.5 mM, and 4) cells rhythmically contracting in 2.5 mM for 20 min. F(max) was, respectively, 21.5, 10.5, 24.7, and 32.6 mN/mm(2). Changes in F(max) were closely associated with changes in the degree of phosphorylation of MyBP-C and occurred at intracellular concentrations of Ca below levels associated with phosphorylation of RLC. Monophosphorylation of MyBP-C by a Ca-regulated kinase is necessary before beta-adrenergic stimulation can produce additional phosphorylation. These results suggest that Ca-dependent phosphorylation of MyBP-C modulates contractility by changing thick filament structure.
15863048
Skinned Myocardium Is Eliminated by Ablation of cMyBP-C Mediated Acceleration of the Stretch Activation Response in Murine − Protein Kinase
-Adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C / ) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during -adrenergic stimulation. (Circ Res. 2006;99:884-890.)
13291225
Acceleration of Crossbridge Kinetics by Protein Kinase A Phosphorylation of Cardiac Myosin Binding Protein C Modulates Cardiac Function
Normal cardiac function requires dynamic modulation of contraction. β1-Adrenergic–induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non–PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, krel (s-1), and delayed force development, kdf (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline krel and kdf similar to WT myocardium, but, unlike WT, krel and kdf were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.
12417984
The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner*
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 (Kd) of ∼10 μm and a molar binding ratio (Bmax) 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 Bmax) 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 cross-bridge cycling.
36886575
Myosin Binding Protein C Interaction with Actin
Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and shown that purified cMyBPC binds actin filaments with an affinity of 4.3 ± 1.1 μm and a 1:1 molar ratio with regard to an actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. To determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of the cMyBPC sequence. Recombinant N-terminal fragments such as C0–C1, C0–C4, and C0–C5 cosediment with actin in a linear, nonsaturable manner. At the same time, MyBPC fragments lacking either the C0–C1 or C0–C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via nonspecific electrostatic interactions.
16437293
Cardiac Myosin-Binding Protein C Mutations and Hypertrophic Cardiomyopathy: Haploinsufficiency, Deranged Phosphorylation, and Cardiomyocyte Dysfunction
Background— Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3mut). Methods and Results— Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84±5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the &bgr;-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3mut (20.2±2.7 kN/m2) compared with donor (34.5±1.1 kN/m2). Moreover, Ca2+ sensitivity was higher in MYBPC3mut (pCa50=5.62±0.04) than in donor (pCa50=5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic &bgr;-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca2+ sensitivity between MYBPC3mut (pCa50=5.46±0.03) and donor (pCa50=5.48±0.02). Conclusions— Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca2+ sensitivity in MYBPC3mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.
30202307
Cardiac myosin binding protein c phosphorylation is cardioprotective
Cardiac myosin binding protein C (cMyBP-C) has three phosphorylatable serines at its N terminus (Ser-273, Ser-282, and Ser-302), and the residues' phosphorylation states may alter thick filament structure and function. To examine the effects of cMyBP-C phosphorylation, we generated transgenic mice with cardiac-specific expression of a cMyBP-C in which the three phosphorylation sites were mutated to aspartic acid, mimicking constitutive phosphorylation (cMyBP-CAllP+). The allele was bred into a cMyBP-C null background (cMyBP-C(t/t)) to ensure the absence of endogenous dephosphorylated cMyBP-C. cMyBP-CAllP+ was incorporated normally into the cardiac sarcomere and restored normal cardiac function in the null background. However, subtle changes in sarcomere ultrastructure, characterized by increased distances between the thick filaments, indicated that phosphomimetic cMyBP-C affects thick–thin filament relationships, and yeast two-hybrid data and pull-down studies both showed that charged residues in these positions effectively prevented interaction with the myosin heavy chain. Confirming the physiological relevance of these data, the cMyBP-CAllP+:(t/t) hearts were resistant to ischemia–reperfusion injury. These data demonstrate that cMyBP-C phosphorylation functions in maintaining thick filament spacing and structure and can help protect the myocardium from ischemic injury.
29167740
Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle.
Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40+/-2% of donor, P<0.0001; failing 45+/-3% of donor, P<0.0001). 6 myectomy samples were identified with MYBPC3 mutations, one with MYH7 mutation and two remained unknown, but there was no correlation between MYBPC3 mutation and MyBP-C phosphorylation level. In order to determine the number of phosphorylated sites in human cardiac MyBP-C samples, we phosphorylated the recombinant MyBP-C fragment, C0-C2 (1-453) with PKA using (gamma32)P-ATP up to 3.5 mol Pi/mol C0-C2. This measurement of phosphorylation was used to calibrate measurements of phosphorylation in SDS-PAGE using Pro-Q Diamond stain. The level of phosphorylation in donor heart MyBP-C was calculated to be 4.6+/-0.6 mol Pi/mol and 2.0+/-0.3 mol Pi/mol in myectomy samples. We conclude that MyBP-C is a highly phosphorylated protein in vivo and that diminished MyBP-C phosphorylation is a feature of both end-stage heart failure and hypertrophic cardiomyopathy.
205810981
Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.
A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.
27203316
Slip sliding away: load-dependence of velocity generated by skeletal muscle myosin molecules in the laser trap.
Skeletal muscle's ability to shorten and lengthen against a load is a fundamental property, presumably reflecting the inherent load-dependence of the myosin molecular motor. Here we report the velocity of a single actin filament translocated by a mini-ensemble of skeletal myosin approximately 8 heads under constant loads up to 15 pN in a laser trap assay. Actin filament velocity decreased with increasing load hyberbolically, with unloaded velocity and stall force differing by a factor of 2 with [ATP] (30 vs. 100 muM). Analysis of actin filament movement revealed that forward motion was punctuated with rapid backward 60-nm slips, with the slip frequency increasing with resistive load. At stall force, myosin-generated forward movement was balanced by backward slips, whereas at loads greater than stall, myosin could no longer sustain forward motion, resulting in negative velocities as in eccentric contractions of whole muscle. Thus, the force-velocity relationship of muscle reflects both the inherent load-dependence of the actomyosin interaction and the balance between forward and reverse motion observed at the molecular level.
9858239
Smooth muscle myosin cross-bridge interactions modulate actin filament sliding velocity in vitro
Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.
22046195
R403Q and L908V mutant β-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level
Familial hypertrophic cardiomyopathy (FHC) is a disease of the sarcomere. In the β-myosin heavy chain gene, which codes for the mechanical enzyme myosin, greater than 40 point mutations have been found that are causal for this disease. We have studied the effect of two mutations, the R403Q and L908V, on myosin molecular mechanics. In the in vitro motility assay, the mutant myosins produced a 30% greater velocity of actin filament movement (νactin). At the single molecule level, νactin ∼d/ton, where d is the myosin unitary step displacement and ton is the step duration. Laser trap studies were performed at 10 μM MgATP to estimate d and ton for the normal and mutant myosin molecules. The increase in νactin can be explained by a significant decrease in the average ton's in both the R403Q and L908V mutants (∼30 ms) compared to controls (∼40 ms), while d was not different for all myosins tested (∼7 nm). Thus the mutations affect the kinetics of the cross-bridge cycle without any effect on myosin's inherent motion and force generating capacity. Based on these studies, the primary signal for the hypertrophic response appears to be an apparent gain in function of the individual mutant myosin molecules.
26269509
Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay.
Point mutations in cardiac myosin, the heart's molecular motor, produce distinct clinical phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathy. Do mutations alter myosin's molecular mechanics in a manner that is predictive of the clinical outcome? We have directly characterized the maximal force-generating capacity (F(max)) of two HCM (R403Q, R453C) and two DCM (S532P, F764L) mutant myosins isolated from homozygous mouse models using a novel load-clamped laser trap assay. F(max) was 50% (R403Q) and 80% (R453C) greater for the HCM mutants compared with the wild type, whereas F(max) was severely depressed for one of the DCM mutants (65% S532P). Although F(max) was normal for the F764L DCM mutant, its actin-activated ATPase activity and actin filament velocity (V(actin)) in a motility assay were significantly reduced (Schmitt JP, Debold EP, Ahmad F, Armstrong A, Frederico A, Conner DA, Mende U, Lohse MJ, Warshaw D, Seidman CE, Seidman JG. Proc Natl Acad Sci USA 103: 14525-14530, 2006.). These F(max) data combined with previous V(actin) measurements suggest that HCM and DCM result from alterations to one or more of myosin's fundamental mechanical properties, with HCM-causing mutations leading to enhanced but DCM-causing mutations leading to depressed function. These mutation-specific changes in mechanical properties must initiate distinct signaling cascades that ultimately lead to the disparate phenotypic responses observed in HCM and DCM.
42561693
Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.
Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.
24339735
Adaptive control of cardiac contraction to changes in loading: from theory of sarcomere dynamics to whole-heart function
The heart accommodates to rapid changes in demands. This review elucidates the adaptive control of cardiac function by loading conditions, and integrates the sarcomeric control of contraction (SCC) with isolated trabeculae and in vivo whole-heart studies. The SCC includes two feedback mechanisms: (1) cooperativity that regulates cross-bridge (XB) recruitment and the force–length relationship, and (2) mechanical feedback, whereby the filament-sliding velocity determines the XB-weakening rate and the force–velocity relationship. An isolated rat trabeculae study tested the suggested mechanisms during sarcomeric lengthening. The observations indicate that lengthening decreases the XB-weakening rate in a velocity-dependent manner, congruent with the suggested hypothesis and in contrast to alternative theories. A whole-heart level study in sheep reveals the existence of a preload-independent linear relationship between the external work (EW) and pressure–time integral during transient vena cava occlusions, for any given afterload, and not just at isovolumic contractions. The slope of this relationship decreases as the afterload increases. These findings highlight the mechanisms underlying the pressure (Frank’s phenomenon) and EW (Starling’s phenomenon) generation and the roles that the preload and afterload play. The theoretical, isolated fibers and whole-heart studies provide complementary information that strengthens our understanding of cardiac function from the top-down and bottom-up.
14667699
Loaded Shortening, Power Output, and Rate of Force Redevelopment Are Increased With Knockout of Cardiac Myosin Binding Protein-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−/−) 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 Ca2+ activations. Isometric force was not different between the two groups with force being 17.0±7.2 and 20.5±3.1 kN/m2 in wild-type and MyBP-C−/− myocytes, respectively. Peak normalized power output was significantly increased by 26% in MyBP-C−/− myocytes (0.15±0.01 versus 0.19±0.03 P/Po · ML/sec) during maximal Ca2+ activations. Interestingly, peak power output in MyBP-C−/− myocytes was increased to an even greater extent (46%, 0.09±0.03 versus 0.14±0.02 P/Po · ML/sec) during half-maximal Ca2+ activations. There was also an effect on the rate constant of force redevelopment (ktr) during half-maximal Ca2+ activations, with ktr being significantly greater in MyBP-C−/− myocytes (WT=5.8±0.9 s−1 versus MyBP-C−/−=7.7±1.7 s−1). These results suggest that cMyBP-C is an important regulator of myocardial work capacity whereby MyBP-C acts to limit power output.
33050691
Cardiac Myosin Binding Protein-C Phosphorylation in a &bgr;-Myosin Heavy Chain Background
Background— Cardiac myosin binding protein-C (cMyBP-C) phosphorylation modulates cardiac contractility. When expressed in cMyBP-C-null (cMyBP-C(t/t)) hearts, a cMyBP-C phosphomimetic (cMyBP-CAllP+) rescued cardiac dysfunction and protected the hearts from ischemia/reperfusion injury. However, cMyBP-C function may be dependent on the myosin isoform type. Because these replacements were performed in the mouse heart, which contains predominantly &agr;-myosin heavy chain (&agr;-MyHC), the applicability of the data to humans, whose cardiomyocytes contain predominantly &bgr;-MyHC, is unclear. We determined the effect(s) of cMyBP-C phosphorylation in a &bgr;-MyHC transgenic mouse heart in which >80% of the &agr;-MyHC was replaced by &bgr;-MyHC, which is the predominant myosin isoform in human cardiac muscle. Methods and Results— To determine the effects of cMyBP-C phosphorylation in a &bgr;-MyHC background, transgenic mice expressing normal cMyBP-C (cMyBP-CWT), nonphosphorylatable cMyBP-C (cMyBP-CAllP−), or cMyBP-CAllP+ were bred into the &bgr;-MyHC background (&bgr;). These mice were then crossed into the cMyBP-C(t/t) background to ensure the absence of endogenous cMyBP-C. cMyBP-C(t/t)/&bgr; and cMyBP-CAllP−:(t/t)/&bgr; mice died prematurely because of heart failure, confirming that cMyBP-C phosphorylation is essential in the &bgr;-MyHC background. cMyBP-CAllP+:(t/t)/&bgr; and cMyBP-CWT:(t/t)/&bgr; hearts showed no morbidity and mortality, and cMyBP-CAllP+:(t/t)/&bgr; hearts were significantly cardioprotected from ischemia/reperfusion injury. Conclusions— cMyBP-C phosphorylation is necessary for basal myocardial function in the &bgr;-MyHC background and can preserve function after ischemia/reperfusion injury. Our studies justify exploration of cMyBP-C phosphorylation as a therapeutic target in the human heart.
24492361
Structural Insight into Unique Cardiac Myosin-binding Protein-C Motif
Background: Cardiac myosin-binding protein-C is a sarcomeric assembly protein necessary for the regulation of sarcomere structure and function. Results: The cMyBP-C motif is composed of two subdomains, a largely disordered N-terminal portion and a more ordered C-terminal subdomain. Conclusion: The C-terminal subdomain is capable of forming a three-helix bundle. Significance: The three-helix bundle may provide a platform for actin binding. The structural role of the unique myosin-binding motif (m-domain) of cardiac myosin-binding protein-C remains unclear. Functionally, the m-domain is thought to directly interact with myosin, whereas phosphorylation of the m-domain has been shown to modulate interactions between myosin and actin. Here we utilized NMR to analyze the structure and dynamics of the m-domain in solution. Our studies reveal that the m-domain is composed of two subdomains, a largely disordered N-terminal portion containing three known phosphorylation sites and a more ordered and folded C-terminal portion. Chemical shift analyses, dNN(i, i + 1) NOEs, and 15N{1H} heteronuclear NOE values show that the C-terminal subdomain (residues 315–351) is structured with three well defined helices spanning residues 317–322, 327–335, and 341–348. The tertiary structure was calculated with CS-Rosetta using complete 13Cα, 13Cβ, 13C′, 15N, 1Hα, and 1HN chemical shifts. An ensemble of 20 acceptable structures was selected to represent the C-terminal subdomain that exhibits a novel three-helix bundle fold. The solvent-exposed face of the third helix was found to contain the basic actin-binding motif LK(R/K)XK. In contrast, 15N{1H} heteronuclear NOE values for the N-terminal subdomain are consistent with a more conformationally flexible region. Secondary structure propensity scores indicate two transient helices spanning residues 265–268 and 293–295. The presence of both transient helices is supported by weak sequential dNN(i, i + 1) NOEs. Thus, the m-domain consists of an N-terminal subdomain that is flexible and largely disordered and a C-terminal subdomain having a three-helix bundle fold, potentially providing an actin-binding platform.
1965427
Binding of Myosin Binding Protein-C to Myosin Subfragment S2 Affects Contractility Independent of a Tether Mechanism
Mutations in the cardiac myosin binding protein-C gene (cMyBP-C) are among the most prevalent causes of inherited hypertrophic cardiomyopathy. Although most cMyBP-C mutations cause reading frameshifts that are predicted to encode truncated peptides, it is not known if or how expression of these peptides causes disease. One possibility is that because the N-terminus contains a unique binding site for the S2 subfragment of myosin, shortened cMyBP-C peptides could directly affect myosin contraction by binding to S2. To test this hypothesis, we compared the effects of a C1C2 protein containing the myosin S2 binding site on contractile properties in permeablized myocytes from wild-type and cMyBP-C knockout mice. In wild-type myocytes, the C1C2 protein reversibly increased myofilament Ca 2+ sensitivity of tension, but had no effect on resting tension. Identical results were observed in cMyBP-C knockout myocytes where C1C2 increased Ca 2+ sensitivity of tension with the half-maximal response elicited at ≈5 &mgr;mol/L C1C2. Maximum force was not affected by C1C2. However, phosphorylation of C1C2 by cAMP-dependent protein kinase reduced its ability to increase Ca 2+ sensitivity. These results demonstrate that binding of the C1C2 peptide to S2 alone is sufficient to affect myosin contractile function and suggest that regulated binding of cMyBP-C to myosin S2 by phosphorylation directly influences myofilament Ca 2+ sensitivity.
206209556
Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C).
Myosin-binding protein C (MyBP-C) is an ∼130-kDa rod-shaped protein of the thick (myosin containing) filaments of vertebrate striated muscle. It is composed of 10 or 11 globular 10-kDa domains from the immunoglobulin and fibronectin type III families and an additional MyBP-C-specific motif. The cardiac isoform cMyBP-C plays a key role in the phosphorylation-dependent enhancement of cardiac function that occurs upon β-adrenergic stimulation, and mutations in MyBP-C cause skeletal muscle and heart diseases. In addition to binding to myosin, MyBP-C can also bind to actin via its N-terminal end, potentially modulating contraction in a novel way via this thick-thin filament bridge. To understand the structural basis of actin binding, we have used negative stain electron microscopy and three-dimensional reconstruction to study the structure of F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions varying from 25 to 180 mM KCl concentration. Three-dimensional helical reconstructions, carried out at the 180-mM KCl level to minimize nonspecific binding, showed MyBP-C density over a broad portion of the periphery of subdomain 1 of actin and extending tangentially from its surface in the direction of actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it could modulate tropomyosin position and would interfere with myosin head binding to actin.
5269759
The C0C1 fragment of human cardiac myosin binding protein C has common binding determinants for both actin and myosin.
The N-terminal domains of cardiac myosin binding protein C (MyBP-C) play a regulatory role in modulating interactions between myosin and actin during heart muscle contraction. Using NMR spectroscopy and small-angle neutron scattering, we have determined specific details of the interaction between the two-module human C0C1 cMyBP-C fragment and F-actin. The small-angle neutron scattering data show that C0C1 spontaneously polymerizes monomeric actin (G-actin) to form regular assemblies composed of filamentous actin (F-actin) cores decorated by C0C1, similar to what was reported in our earlier four-module mouse cMyBP-C actin study. In addition, NMR titration analyses show large intensity changes for a subset of C0C1 peaks upon addition of G-actin, indicating that human C0C1 interacts specifically with actin and promotes its assembly into filaments. During the NMR titration, peaks corresponding to cardiac-specific C0 domain are the first to be affected, followed by those from the C1 domain. No peak intensity or position changes were detected for peaks arising from the disordered proline/alanine-rich (P/A) linker connecting C0 with C1, despite previous suggestions of its involvement in binding actin. Of considerable interest is the observation that the actin-interaction "hot-spots" within the C0 and C1 domains, revealed in our NMR study, overlap with regions previously identified as binding to the regulatory light chain of myosin and to myosin ΔS2. Our results suggest that C0 and C1 interact with myosin and actin using a common set of binding determinants and therefore support a cMyBP-C switching mechanism between myosin and actin.
5021301
Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle
Myosin-binding protein C (MyBP-C) is a thick filament protein playing an essential role in muscle contraction, and MyBP-C mutations cause heart and skeletal muscle disease in millions worldwide. Despite its discovery 40 y ago, the mechanism of MyBP-C function remains unknown. In vitro studies suggest that MyBP-C could regulate contraction in a unique way—by bridging thick and thin filaments—but there has been no evidence for this in vivo. Here we use electron tomography of exceptionally well preserved muscle to demonstrate that MyBP-C does indeed bind to actin in intact muscle. This binding implies a physical mechanism for communicating the relative sliding between thick and thin filaments that does not involve myosin and which could modulate the contractile process.
2793452
Smooth Muscle Heavy Meromyosin Phosphorylated on One of Its Two Heads Supports Force and Motion*
Smooth muscle myosin is activated by regulatory light chain (RLC) phosphorylation. In the unphosphorylated state the activity of both heads is suppressed due to an asymmetric, intramolecular interaction between the heads. The properties of myosin with only one of its two RLCs phosphorylated, a state likely to be present both during the activation and the relaxation phase of smooth muscle, is less certain despite much investigation. Here we further characterize the mechanical properties of an expressed heavy meromyosin (HMM) construct with only one of its RLCs phosphorylated (HMM-1P). This construct was previously shown to have more than 50% of the ATPase activity of fully phosphorylated myosin (HMM-2P) and to move actin at the same speed in a motility assay as HMM-2P (Rovner, A. S., Fagnant, P. M., and Trybus, K. M. (2006) Biochemistry 45, 5280–5289). Here we show that the unitary step size and attachment time to actin of HMM-1P is indistinguishable from that of HMM-2P. Force-velocity measurements on small ensembles show that HMM-1P can generate approximately half the force of HMM-2P, which may relate to the observed duty ratio of HMM-1P being approximately half that of HMM-2P. Therefore, single-phosphorylated smooth muscle HMM molecules are active species, and the head associated with the unphosphorylated RLC is mechanically competent, allowing it to make a substantial contribution to both motion and force generation during smooth muscle contraction.
22548926
The molecular basis of frictional loads in the in vitro motility assay with applications to the study of the loaded mechanochemistry of molecular motors
Molecular motors convert chemical energy into mechanical movement, generating forces necessary to accomplish an array of cellular functions. Since molecular motors generate force, they typically work under loaded conditions where the motor mechanochemistry is altered by the presence of a load. Several biophysical techniques have been developed to study the loaded behavior and force generating capabilities of molecular motors yet most of these techniques require specialized equipment. The frictional loading assay is a modification to the in vitro motility assay that can be performed on a standard epifluorescence microscope, permitting the high‐throughput measurement of the loaded mechanochemistry of molecular motors. Here, we describe a model for the molecular basis of the frictional loading assay by modeling the load as a series of either elastic or viscoelastic elements. The model, which calculates the frictional loads imposed by different binding proteins, permits the measurement of isotonic kinetics, force‐velocity relationships, and power curves in the motility assay. We show computationally and experimentally that the frictional load imposed by alpha‐actinin, the most widely employed actin binding protein in frictional loading experiments, behaves as a viscoelastic rather than purely elastic load. As a test of the model, we examined the frictional loading behavior of rabbit skeletal muscle myosin under normal and fatigue‐like conditions using alpha‐actinin as a load. We found that, consistent with fiber studies, fatigue‐like conditions cause reductions in myosin isometric force, unloaded sliding velocity, maximal power output, and shift the load at which peak power output occurs. © 2010 Wiley‐Liss, Inc.