myosin binding protein-c: structural and functional complexity

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Editorial Myosin binding protein-C: structural and functional complexity Understanding the potential roles that myosin binding protein-C (MBPC) plays in contractile regulation is impor- tant because in addition to its putative role in stabilizing the structure of thick filaments in solution, interaction between the N-terminus of MBPC and the S2 segment of myosin crossbridges may alter force generation and crossbridge ki- netics during shortening and tension development in striated muscle [1,2]. Thus, MBPC could be an important modulator of crossbridge binding to thin filaments and thus the ability of crossbridges to contribute to cardiac thin filament activation [3]. Additionally, phosphorylation of the unique N-terminus of cardiac MBCP (cMBPC) during b-adrenergic stimulation of PKA and calmodulin dependent kinase may decrease cMBPC interaction with the S2 segment of the myosin cros- sbridges [4,5], raising the possibility that MBPC contributes to the phenotype observed during abnormal protein phospho- rylation seen in failing myocardium [6–8]. Furthermore, about 20% of all cases of familial hypertrophic- cardiomyopathy (FHC) are associated with expression of missense or C-terminal truncation mutants of cardiac MBPC (see Flashman et al. [9] for recent review). MBPC has a complex multi-domain structure and potential interactions with numerous sarcomeric proteins. This complexity and potential functional implications make the article of McClel- lan and colleagues in this issue particularly interesting and informative. cMBPC is composed of a linear sequence of 11 IgI-like and fibronectin domains with domain designation C0–C10; C-terminal domains C6, C7 and C9 are fibronectin domains. C0 is unique to cMBPC and may interact with actin [10,11], the phosphorylation dependent interaction of cMBPC with myosin S2 occurs through a linking sequence between C1 and C2 (the myosin-bindng motif), while domains C8–C10 bind to LMM in the thick filament core, as well as to sar- comeric protein titin [12], as reviewed by Flashman et al. [9]. MBPC is associated with thick filaments in all striated mus- cle types and its location is segregated into a series of 7–9 bands within C-zones which are located adjacent to each side of the thick filament H-zones, occupying about 50% of the thick filament in each half sarcomere. The spacing between bands is 43 nm, close to the 42.9 nm spacing between equivalent myosin crossbridge crowns and equiva- lent to the myosin-binding domain repeat on titin. The stoi- chiometry of MBPC/myosin crossbridges is 1:7–9. This stoi- ciometry implies that MBPC effects on a sub-population of individual crossbridges must be communicated throughout the entire population through interactions within the thick filament core or between crossbridges bound to thin fila- ments during contraction. MBPC is necessary for in vitro formation of thick fila- ments with normal length and ordered myosin crossbridge structure, implying the possibility of a similar role in vivo [13]. However, “knockout” of MBPC expression in cardiac myocytes did not cause significant changes in gross myofi- brillar structure or maximal force generation, but did lead to the FHC phenotype [14]. These differences could be explai- ned by compensatory expression of proteins other than cM- BPC to maintain myofibrillar structure in the knockout mouse model or that the resulting alteration of thick filament structure was too subtle to be observed by electron micros- copy. Alternatively the Harris et al. [14] study could indicate that maintenance of thick filament structure is not the pri- mary function of cMBPC. Functional roles for MBPC are implied by its effect(s) on Ca 2+ -activation of force in cardiac muscle. Knockout of MBPC decreased Ca 2+ -sensivitiy of force in chemically skinned myocytes compared to normal mice [14]. In contrast, when MBPC was extracted from single cardiac myocytes Ca 2+ -sensitivity of force increased, as did unloaded shorte- ning velocity [15]. Interestingly, in spite of different effects on Ca 2+ -sensitivity, F max was unaffected in both prepara- tions. Similarly, in MBPC extracted myocytes and knockout mouse myocytes, crossbridge kinetics and shortening velo- city increased, leading to increased contractile power in the knockout model [2]. The effects of MBPC on contraction seem to imply that MBPC restricts crossbridge interaction with Ca 2+ -activated thin filaments, particularly during shor- tening. Supporting this idea introduction of exogenous myo- sin S2 into transiently permeabilized myocytes increased shortening, unloaded shortening velocity and relaxation rate [16]. Although it was not possible to verify directly, the results seem to imply that exogenous S2 had competed for endogenous MBPC with the S2 segment of endogenous crossbridges, thus relieving the inhibitory effect on shorte- ning. Other studies argue that cMBPC has an important effect on force generation as well as kinetics, because in skinned preparation cMBPC extraction [17] and phosphorylation cMBPC [18] both decrease F max . These seemingly disparate results could result from subtle differences in perturbations used to modify MBPC levels (extraction vs. knockout) and the complexity of cMBPC interactions with thick filaments, titin, crossbridges and thin filaments. Journal of Molecular and Cellular Cardiology 37 (2004) 813–815 www.elsevier.com/locate/yjmcc 0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.07.005

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Page 1: Myosin binding protein-C: structural and functional complexity

Editorial

Myosin binding protein-C: structural and functional complexity

Understanding the potential roles that myosin bindingprotein-C (MBPC) plays in contractile regulation is impor-tant because in addition to its putative role in stabilizing thestructure of thick filaments in solution, interaction betweenthe N-terminus of MBPC and the S2 segment of myosincrossbridges may alter force generation and crossbridge ki-netics during shortening and tension development in striatedmuscle [1,2]. Thus, MBPC could be an important modulatorof crossbridge binding to thin filaments and thus the ability ofcrossbridges to contribute to cardiac thin filament activation[3]. Additionally, phosphorylation of the unique N-terminusof cardiac MBCP (cMBPC) during b-adrenergic stimulationof PKA and calmodulin dependent kinase may decreasecMBPC interaction with the S2 segment of the myosin cros-sbridges [4,5], raising the possibility that MBPC contributesto the phenotype observed during abnormal protein phospho-rylation seen in failing myocardium [6–8]. Furthermore,about 20% of all cases of familial hypertrophic-cardiomyopathy (FHC) are associated with expression ofmissense or C-terminal truncation mutants of cardiac MBPC(see Flashman et al. [9] for recent review). MBPC has acomplex multi-domain structure and potential interactionswith numerous sarcomeric proteins. This complexity andpotential functional implications make the article of McClel-lan and colleagues in this issue particularly interesting andinformative.

cMBPC is composed of a linear sequence of 11 IgI-likeand fibronectin domains with domain designation C0–C10;C-terminal domains C6, C7 and C9 are fibronectin domains.C0 is unique to cMBPC and may interact with actin [10,11],the phosphorylation dependent interaction of cMBPC withmyosin S2 occurs through a linking sequence between C1and C2 (the myosin-bindng motif), while domains C8–C10bind to LMM in the thick filament core, as well as to sar-comeric protein titin [12], as reviewed by Flashman et al. [9].MBPC is associated with thick filaments in all striated mus-cle types and its location is segregated into a series of7–9 bands within C-zones which are located adjacent to eachside of the thick filament H-zones, occupying about 50% ofthe thick filament in each half sarcomere. The spacingbetween bands is 43 nm, close to the 42.9 nm spacingbetween equivalent myosin crossbridge crowns and equiva-lent to the myosin-binding domain repeat on titin. The stoi-chiometry of MBPC/myosin crossbridges is 1:7–9. This stoi-ciometry implies that MBPC effects on a sub-population ofindividual crossbridges must be communicated throughout

the entire population through interactions within the thickfilament core or between crossbridges bound to thin fila-ments during contraction.

MBPC is necessary for in vitro formation of thick fila-ments with normal length and ordered myosin crossbridgestructure, implying the possibility of a similar role in vivo[13]. However, “knockout” of MBPC expression in cardiacmyocytes did not cause significant changes in gross myofi-brillar structure or maximal force generation, but did lead tothe FHC phenotype [14]. These differences could be explai-ned by compensatory expression of proteins other than cM-BPC to maintain myofibrillar structure in the knockoutmouse model or that the resulting alteration of thick filamentstructure was too subtle to be observed by electron micros-copy. Alternatively the Harris et al. [14] study could indicatethat maintenance of thick filament structure is not the pri-mary function of cMBPC.

Functional roles for MBPC are implied by its effect(s) onCa2+-activation of force in cardiac muscle. Knockout ofMBPC decreased Ca2+-sensivitiy of force in chemicallyskinned myocytes compared to normal mice [14]. In contrast,when MBPC was extracted from single cardiac myocytesCa2+-sensitivity of force increased, as did unloaded shorte-ning velocity [15]. Interestingly, in spite of different effectson Ca2+-sensitivity, Fmax was unaffected in both prepara-tions. Similarly, in MBPC extracted myocytes and knockoutmouse myocytes, crossbridge kinetics and shortening velo-city increased, leading to increased contractile power in theknockout model [2]. The effects of MBPC on contractionseem to imply that MBPC restricts crossbridge interactionwith Ca2+-activated thin filaments, particularly during shor-tening. Supporting this idea introduction of exogenous myo-sin S2 into transiently permeabilized myocytes increasedshortening, unloaded shortening velocity and relaxation rate[16]. Although it was not possible to verify directly, theresults seem to imply that exogenous S2 had competed forendogenous MBPC with the S2 segment of endogenouscrossbridges, thus relieving the inhibitory effect on shorte-ning. Other studies argue that cMBPC has an important effecton force generation as well as kinetics, because in skinnedpreparation cMBPC extraction [17] and phosphorylationcMBPC [18] both decrease Fmax. These seemingly disparateresults could result from subtle differences in perturbationsused to modify MBPC levels (extraction vs. knockout) andthe complexity of cMBPC interactions with thick filaments,titin, crossbridges and thin filaments.

Journal of Molecular and Cellular Cardiology 37 (2004) 813–815

www.elsevier.com/locate/yjmcc

0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.yjmcc.2004.07.005

Page 2: Myosin binding protein-C: structural and functional complexity

Recent structural models of MBPC may help explaindifferences in physiologic response between experiments inwhich MBPC expression is altered in transgenic models vs.when MBPC is extracted and reconstituted in skinned car-diac preparations. These models imply that studies invol-ving perturbation of cMBPC structure and interactions withspecific fragments of cMBPC structure, as used by McClel-lan et al. (this issue) may be a fruitful approach. Althoughother arrangements have been proposed [10], Moolman-Smook and colleagues proposed that MBPC exists as aternary complex at each C-band, forming a collar aroundthe thick filaments that stabilizes the thick filament core.The C-terminus of each MBPC molecule interacts with theadjacent monomer in a staggered manner, with the interac-tions being between C5 and C8 domains on the adjacentmolecule [19]. The N-terminal domains of each MBPC donot interact with the thick filament core, but are thought toproject radially from the thick filament surface, enablingpotential interaction with myosin S2 or actin. Implicit in thismodel is the probability that any alteration of interactionbetween the MBPC units making up the MBPC collar willlikely lead to altered thick filament structure with potentialfunctional consequences. In this issue on pages 823-835 [20],McClellan and colleagues probe the function of cMBPCdomain C5 in maintenance of normal thick filament structureand function, and its implications for force generation anddisease. They test for the C5–C8 interaction implicit in theMoolman-Smook model by adding various concentrations ofrecombinant domain C5, or equivalently C2–C5 domains, toskinned cardiac trabeculae. This maneuver presumably ena-bles the exogenous C5 fragment to compete with endogenousC5, thus disrupting the C5–C8 interaction between adjacentMBPCs in the collar structure. As a result they observe areversible concentration dependent decrease in Fmax and theCa2+-sensitivity of force when exogenous C5 (or C2–C5)concentration is below about 15 µM. In contrast when [C5] iselevated above 15 µM Fmax decreases irreversibly, correla-ting with loss of cMBPC, as well as myosin heavy chain,implying severe disruption of thick filament structure. SinceC5 has a high affinity for C8 [19], while not binding to S2 oractin, their observations at low concentrations of C5 implythat maintenance of MBPC structure, mediated by C5–C8interaction, is necessary for normal cardiac contractile acti-vation, as implied by the Moolman-Smook model [19]. Otherinteresting observations that cMBPC is slowly, but conti-nuously, lost from relaxed skinned cardiac preparations andthat this loss is inhibited during force generation emergefrom their data.

It might be thought that their C5 experiment should befunctionally comparable to the MBPC extraction experi-ments of Hoffman et al. [21]. However, extraction of about70% MBPC in single myocytes, which would be expected todisrupt MBPC collar structure, caused increased Ca2+-sensitivity of force with no effect on Fmax, in contrast to theC5 study of McClellan et al. On the other hand extraction ofcMBPC in another study caused increased Ca2+-sensitivity

of force, with no effect on Fmax for brief (1 h) extractiontimes, while longer times caused further extraction of cM-BPC and decreased Fmax; these effects were reversed byaddition of exogenous cMBPC [17]. The apparent conflict ofresults obtained with C5 disruption of cMBPC function andMBPC extraction imply that the two approaches are notequivalent. The difference could lie in the nature of theintervention. One could speculate that cMBPC might berandomly removed from collar structures by extraction. Inthis case, the removal of one MBPC per collar might leavethick filament relatively unaltered structurally (thus no de-crease in Fmax), but with crossbridge structure altered tomake interaction with thin filaments more probable, thusincreasing sensitivity of contraction to Ca2+. This could bethe case in the study by Kulikovskaya et al. [17] where thepercentage of cMBPC extracted was 25–30%, but not in theHoffman et al. [1] study where extraction was 70% completeand by implication more than one cMBPC lost from eachcollar. Perhaps a better comparison for the results of McClel-lan et al. would be with transgenic models expressingC-terminal truncated FHC MBPC mutants [22,23]. Expres-sion of C-terminal truncated FHC mutant cMBPC, lackingboth titin and LMM myosin binding domains but with C5intact, caused decreased maximal power, consistent with thereversible decrease of Fmax observed by McClellan et al., butwith increased Ca2+-sensitivity of force [22]. On the otherhand the results ofYang et al. [22] are similar to the results inthe present study at higher C5 concentrations. Above 15 µMC5 the irreversibly decreased Fmax and increased Ca2+-sensitvity of force was correlated with both loss of cMBPCand myosin heavy chain, implying extensive disruption ofthick filament structure. The transgenic expression of theC-terminal truncated FHC cMBPC mutant likewise causeddisrupted myofibrillar structure, decreased maximum powerand increased Ca2+-sensitivity of force.

A speculative interpretation of these results might be thatwhen thick filament structure is sufficiently compromisedby loss of myosin and/or cMBPC, crossbridge orientationand ability to interact with Ca2+-activated thin filamentstransiently increases. This idea is consistent with the notionthat crossbridge binding makes a significant contribution tothin filament activation in cardiac muscle. Finally, the appa-rently minor observation in the current study that the loss ofMBPC in the presence of even high C5 concentration wasdiminished or eliminated during force generation seems toimply interaction between strongly bound crossbridges andcMBPC. Prevention of cMBPC loss during contractionsuggests that either cMBPC affinity for myosin S2 in-creases during force generation or that thick filament corestructure is altered in such a way as to increase the bindingof individual cMBPC units to LMM. Of course these arespeculations in need of tests, but they and the interestingresults from many labs are commensurate with the com-plexities of MBPC structure and varied interactions ofMBPC with other contractile proteins in both skeletal andcardiac muscle.

814 Editorial / Journal of Molecular and Cellular Cardiology 37 (2004) 813–815

Page 3: Myosin binding protein-C: structural and functional complexity

References

[1] Hoffman PA, Hartzell HC, Moss RL. Alterations in Ca2+ sensitivetension due to partial extraction of C-protein from rat skinned cardiacmyocytes and rabbit skeletal muscle fibers. J Gen Physiol 1991;97(6):1141–63.

[2] Korte FS, McDonald KS, Hairris SP, Moss RL. Loaded shortening,power output an rate of force redevelopment are increased withknockout of cardiac myosin binding protein-C. Circ Res 2003;93:752–8.

[3] Smith SH, Fuchs F. Length-dependence of cross-bridge mediatedactivation of the cardiac thin filament. J Mol Cell Cardiol 2000;32(5):831–8.

[4] Kunst G, Kress KR, Gruen M, Uttenweiler D, Gautel M, Fink RH.Myosin binding protein C, a phosphorylation-dependent force regula-tor in muscle that controls the attachment of myosin heads by itsinteraction with myosin S2. Circ Res 2000;86(1):51–8.

[5] Gruen M, Prinz H, Gautel M. cAPK-phosphorylation controls theinteraction of the regulatory domain of cardiac myosin binding pro-tein C with myosin-S2 in an on–off fashion. FEBS Lett 1999;453(3):254–9.

[6] Dorn II GW, Molkentin JD. Manipulating cardiac contractility in heartfailure. Circulation 2004;109:150–8.

[7] Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, Brasmus-sen R, et al. Stinson, B1 and B2 adrenergic receptor subpopulations innonfailing and failing ventricular myocardium: coupling of bothreceptor subtypes to muscle contraction and elective B1 receptordownregulation in heart failure. Circ Res 1986;59:297–309.

[8] Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS,Lurie K, et al. Decreased catecholamine sensitivity and b-adrenergicreceptor density in failing human hearts. New Engl J Med 1982;307:205–11.

[9] Flashman E, Redwood C, Moolman-Smook J, Watkins H. Cardiacmyosin binding protein C: its role in physiology and disease. Circ Res2004;94:1279–89.

[10] Squire JM, Luther PK, Knupp C. Structural evidence for the interac-tion of C-protein (MBP-C) with actin and sequence identification of apossible actin binding domain. J Mol Biol 2003;331:713–24.

[11] Kulikovskaya I, McClellan G, Flavigny J, Carrier L, Winegrad S.Effect of MyBP-C binding to actin on contractility in heart muscle. JGen Physiol 2003;122:761–74.

[12] Furst DO, Vinkemeier U, Weber K. Mammalian skeletal muscleC-protein: purification from bovine muscle, binding to titin and thecharacterization of a full-length human cDNA. J Cell Sci 1992;102:769–88.

[13] Moos C, Offer G, Starr R, Bennett P. Interaction of C-protein withmyosin, myosin rod and light meromyosin. J Mol Biol 1975;97:1–9.

[14] Harris SP, Bartley CR, Hacker TA, McDonald KS, Douglas PS,Greaser ML, et al. Hypertrophic cardiomyopthay in cardiac myosinbinding protein-C knockout mice. Circ Res 2002;90:594–601.

[15] Hoffman PA, Greaser ML, Moss RL. C-protein limits shorteningvelocity of rabbit skeletal muscle fibres at low levels of Ca2+ activa-tion. J Physiol London 1991;439:701–15.

[16] Calaghan SC, Trinick J, Knight PJ, White E.A role for C-protein in theregulation of contraction and intracellular Ca2+ in intact rat ventricularmyocytes. J Physiol 2000;528(Pt 1):151–6.

[17] Kulikovskaya I, McClellan G, Levine R, Winegrad S. Effect of extrac-tion of myosin binding protein-C on contractility of rat heart. Am JPhysiol 2003;285:H857–H865.

[18] McClellan G, Kulikovskaya I, Winegrad S. Changes in cardiac con-tractility related to calcium-mediated changes in phosphorylation ofmyosin-binding protein C. Biophys J 2001;81:1083–92.

[19] Moolman-Smook J, Flashman E, de Lange W, Li Z, Corfield V,Redwood C, et al. Identification of novel interactions betweendomains of myosin binding protein-C that are modulated by hyper-trophic missense mutations. Circ Res 2002;91:704–11.

[20] Mc Clellan G, Kulikovskaya I, Flavigny J, Carrier L, Winegrad S.Effect of cardiac myosin-binding protein C on stability of the thickfilament. J Mol Cell Cardiol 2004;37:823–35, in this isssue.

[21] Hoffman PA, Hartzell HC, Moss RL. Alterations in Ca2+-sensitivetension due to partial extraction of C-protein from rat skinned cardiacmyocytes and rabbit skeletal fibers. J Gen Physiol 1991;97:1141–63.

[22] Yang Q, Sanbe A, Osinka H, Hewett T, Klevitsky R, Robbins J. Amouse model of myosin binding protein C human familial hyper-trophic cardiomopathy. J Clin Invest 1998;102:1292–300.

[23] Yang Q, Sanbe A, Osinka H, Hewett T, Klevitsky R, Robbins J. In vivomodel of myosin bindng protein C familial hypertrophic cardiomy-opathy. Circ Res 1999;85:841–7.

Donald A. Martyn *Department of Bioengineering, University of Washington,

Box 357962, Seattle, WA 98195, USAE-mail address:

[email protected] (D.A. Martyn).

* Tel.: +1-206-543-4478.

815Editorial / Journal of Molecular and Cellular Cardiology 37 (2004) 813–815