Tropomodulin function and thin filament assembly in cardiac myocytes

tin filament pointed end capping protein, tropomodulin, and its role in regulating thin filament assemblyin cardiac myocytes.

Tropomodulin Function and Thin Filament Assembly in Cardiac Myocytes

Tropomodulin

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Tropomodulin was originally identified as a tropomyosin-binding protein in human erythrocytes, where it has been shown to Carol C. Gregorio and Velia M. Fowler be associatedwith the short actin filaments in the spectrin-actin membrane skeleton [Fowler (1987), Ursitti and The regulation of thin filament length is a fundamental property of all Fowler (1994); for a review, see Fowler (1996)]. It exhibits isoform-specific bindstriated muscles. Tropomodulin is an actin and tropomyosin binding protein that is exclusively associated with the free (pointed) ends of thin ing to the N-terminal end of tropomyosin under certain conditions blocks trofilaments. In vitro and in vivo studies reveal that tropomodulin is an and pomyosin head-to-tail associationsalong actin filament pointed end capping protein, which is required to main- actin filaments in vitro (Fowler 1990,Susstain the final length of thin filaments and is essential for contractile man and Fowler 1992, Sung and Lin activity in embryonic chick cardiac myocytes. Understanding the mech- 1994). In cardiac and skeletalmuscle,troanisms of thin filament assembly, as well as determining the roles of pomodulin is specifically associatedwith the pointed ends of the thin filaments, proteins modulating actin filament dynamics, is important for future where one or two tropomodulin moleconsiderations of the molecular bases for myopathies seen in various cules are thought to bind to both the terminal tropomyosin and actin molecules types of heart disease. (Trends Cardiovasc Med 1996;6:136-141). (Figure 1) (Fowler et al. 1993, Gregorio and Fowler 1995). In vitro, tropomodulin is a potent acThe ability of heart muscle to contract is barbed ends of the thin filaments are tin filament pointed end capping procrucially dependent on the proper as- crosslinked by or-actinin, and anchored tein. It is capable of completely blocking sembly and function of all contractile at the Z disk, whereas the pointed ends the addition and lossof actin monomers proteins. Assembly of contractile proare free and terminate in the A band (G-actin) from the pointed ends of troteins is a complex process that requires overlapping with the bipolar myosin fil- pomyosin-actin filaments in concentracoordinate expression of the constituent aments. “Barbed” and “pointed” ends of tions stoichiometric to that of filament proteins, polymerization of actin and actin filaments refer to the orientation of ends (& 5 1 nM). However, in the abmyosin into thin and thick filaments, arrowheads generated by myosin sub- senceof tropomyosin, tropomodulin is a respectively, and association of the two fragment 1 binding to actin filaments, “leaky” cap, and only partially blocks filament systems into highly organized and are the fast-growing and slow-grow- elongation and depolymerization at the sarcomeres. Newly assembled sarcoing ends of actin filaments, respectively. pointed ends of actin filaments (Kd = meres consist of parallel arrays of ap- Control of actin filament length and dy- 0.1-0.4 pm) (Weber et al. 1994). It is proximately 1.0 pm-long thin filaments namics during myofibril assembly is at- likely that tight capping requires binding to both tropomyosin and actin and rethat interdigitate with laterally aligned tained at several levels. Actin monomer flects the sum of the binding energiesfor 1.6~pm long thick filaments. Rodlike troavailability is likely determined by both molecules (Weber et al. 1994, Babpomyosin molecules are associated with monomer sequestering proteins [for a cock and Fowler 1994). each other head-to-tail along each thin recent review, seeSun et al. (1995)]; the Two functional domains have been filament, forming two polymers, one on rate and site of actin polymerization are identified on the tropomodulin moleeach side of the actin filament. Each likely determined by actin filament capcule: one responsible for tropomyosin tropomyosin molecule binds one tropoping proteins. Considerable information binding and another for actin capping nin complex (composed of troponins T, I, and C); together they regulate the cal- is available about how barbed end cap activity. Quantitative binding studies cium-sensitive interaction of actin and ping proteins control actin filament as- with recombinant fragments of tropomyosin (Figure 1). Thin filaments are sembly and function in many cell types mod&n demonstrate that the N termipolarized in muscle sarcomeres. The [for recent reviews, seeCarlier and Pan- nal half of tropomodulin (amino acids taloni (1994), Shafer and Cooper 6-184) is sufficient for full tropomyosin (IWS)], Carol C. Gregorio and Velia M. Fowler are at the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

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but

until

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known about the significance of regulating actin filament assembly at the pointed end (Coluccio 1994). This review focuses on the only recognized acScience

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binding

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the tro-

pomyosin binding half of the molecule can be further subdivided into two regions. cDNA deletion analysis and competition solid phasebinding assaysdem-

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Tropmodtdin

Figure 1. the pointed morphological

Molecular

model for the association of tropomodulin with actin and tropomyosin at (slow growing) end of striated muscle thin filaments based on biochemical and analysis. TN-I, troponin I; TN-T, troponin T; and TN-C, troponin C.

onstrate that residues 6-94 contain the binding domain for skeletal muscle tropomyosin and residues 90-184 contain the erythrocyte tropomyosin binding domain [however, it is likely that the two tropomyosin binding domains overlap somewhat (Babcock and Fowler 1994)] (Figure 2). The presence of two different tropomyosin binding domains on tropomodulin can explain why tropomodulin binds with similar affinity to muscle or nonmuscle tropomyosins that contain different N-terminal sequences (Pittenger et al. 1994, Babcock and Fowler 1994). Furthermore, because thin filaments in embryonic muscle contain both nonmuscle and muscle tropomyosin isoforms (Lin et al. 1984), binding of distinct regions of tropomodulin to different tropomyosins may allow for tropomodulin to bind efficiently to all pointed ends and regulate capping activity. The putative actin binding site appears to be on the C terminal end of tropomodulin, because a monoclonal antibody that binds to this site abolishes tropomodulin’s ability to block elongation from the pointed ends of pure actin and actin-tropomyosin filaments (as expected, this antibody has no effect on the binding of tropomodulin to tropomyosin) (Gregorio et al. 1995).

As well as being associated with the pointed ends of actin filaments in erythrocytes and striated muscle, tropomodulin is also a component of the membrane skeleton in several other types of differentiated cells including lens fiber cells and some neuronal cells of the central nervous system (Woo and Fowler 1994, Sussman et al. 1994c). The tropomodulin gene has been mapped to human chromosome 9q22 by fluorescence in situ hybridization (Sung et al. 1991) and to the homologous region of mouse chromosome 4 by Southern blotting and linkage analysis (White et al. 1995). Although Southern blots of mRNA from a variety of tissues demonstrate multiple tropomodulin messages, this appears to be due to utilization of alternative polyadenlylation sites (Sung et al. 1992, Babcock and Fowler 1994, Ito et al. 1995). Furthermore, reverse transcriptase poiymerase chain reaction (RT-PCR) amplification of RNA from a number of mouse tissues, including cerebral cortex, cerebellum, skeletal muscle, heart and spleen has identified only one tropomodulin isoform, which appears to be the product of a single-copy gene (Ito et al. 1995). The derived amino acid sequence of chicken skeletal muscle tropomodulin is

Figure 2. Locations of the skeletal muscle tropomyosin tropomyosin (residues 90-184) binding sites, and the capping activity (residues 184-359) within tropomoclulin.

Tropomyosin Binding N

Sk. TM

erythrocyte for actin

Actin Filament Capping C

Eryth. TM

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(residues 6-94), and putative region required

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86% identical (91% similar) to that of human erythrocyte tropomodulin (Sung et al. 1992, Babcock and Fowler 1994). This suggests that tropomodulin structure and functions are highly conserved between distantly related species and across tissues. However, more distantly related tropomodulinlike proteins may also exist. For example, the sequence of a human 64 kD auto-antigen associated with Graves’ disease, which is present in many muscle and nonmuscle tissues, contains two regions of similarity with tropomodulin (Dong et al. 199 1, Kendler et al. 1991). Its amino terminal 55 amino acids are 70% homologous to a portion of the tropomyosin binding domain 6f tropomodulin, and an internal region of 182 amino acids is 64% homologous to the carboxy terminal half of tropomodulin (Fowler 1994, C.A. Conley and V.M. Fowler unpublished results). Consequently, tropomodulin or a tropomodulinlike protein may function to cap the pointed ends of tropomyosin-actin filaments in most, if not all, tissues.

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Assembly Myofibrils: Requires

of Tropomodulin Tropomodulin Tropomyosin

Into

Primary cultures of cardiac myocytes are the model of choice to study the role of actin filament end capping proteins during myofibril assembly. Owing to their flat, thin, well-spread shape, these cells are ideally suited for detailed immunolocalization observations of the spatial relationships among assembling myofibrillar proteins. During myofibrillogenesis, the barbed end capping protein capZ assembles at the nascent Z disc early, as expected for a nucleating protein, well before the periodic alignment of actin filaments in the sarcomere (Schafer et al. 1993). In contrast, tropomodulin is incorporated into sarcomeres relatively late as shown by double immunofluorescence and electron microscopy (Gregorio and Fowler 1995). It is not detected in the assemblies of contractile proteins involved in early stages of myofibril assembly (for example, nonstriated myofibrils). Tropomodulin is incorporated into myofibrils only after all the other myofibrillar proteins, such as titin, the thick filament proteins myosin and C-protein, and the thin filament proteins a-actinin, actin, and tropomyosin, are assembled in their characteristic

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Microinjected with Anti-Tmod mAb9 Figure

3. (A) Model for thin filament assembly. (i) Nascent Z-disks containing capZ and a-actinin are assembled with the thin filament proteins, actin, tropomyosin, and the troponins. Pointed ends in nascent myofibrils are uncapped. (ii) Thin filaments that extend across the sarcomere and thus are incorrectly polarized with respect to the myosin cross-bridges are selectively disassembled at their pointed ends and become organized in an antiparallel, periodic fashion in striated myofibrils. (iif) Thin filaments become mature in the presence of a putative “third factor” and their length is subsequently maintained (capped at their pointed ends) by tropomodulin. Copper, Z-line components such as a-actinin; blue, capZ; red, tropomodulin; green, tropomyosin and the troponins; and black, actin filaments. (B) Microinjection of a C terminal domain-specific antibody to tropomodulin disrupts the interaction between actin and tropomodulin and inhibits tropomodulin’s capping activity at the pointed end. This results in an elongation of the actin filaments from their pointed ends across the gap in the middle of the sarcomere (H zone). The microinjected antibody has no effect on the binding of tropomodulin to tropomyosin.

striated patterns (Figure 3A). Thus, both of the known tropomodulin binding proteins, tropomyosin and actin, are organized in their mature periodic pattern before tropomodulin is assembled.The delayed appearance of tropomodulin with respect to the other contractile proteins indicates that this protein is unnecessary in initial stages of myofibril assembly. Tropomodulin is synthesized as a soluble precursor and assemblesinto myofibrils with a t,,z - 7 min as determined from kinetic data obtained from pulsechase [35S]methionine labeling experiments (Gregorio and Fowler 1995). Consistent with this finding, in situ hybridization demonstrates that tropo138

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modulin mRNA is distributed throughout the cytoplasm and is not targeted to thin filament pointed ends by localized translational machinery (Sussman et al. 1994b), unlike some other sarcomeric components that are believed to assemble via cotranslational insertion (Fulton and L’Ecuyer 1993). Interestingly, tropomodulin assemblesinto myofibrils significantly more slowly than does tropomyosin (t,,z I 2 min). In addition, results obtained with a novel saponinpermeabilized cell model for thin filament assembly suggestthat tropomodulin binding to the pointed ends of thin filaments requires the prior association of tropomyosin with actin filaments (Gregorio and Fowler 1995). TropomyoScience

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sin does not appear to be sufficient for tropomodulin’s assembly, however, because early in myofibrillogenesis tropomodulin is not associatedwith thin filament pointed ends in some striated myofibrils or any nonstriated myofibrils, all of which contain tropomyosin. This lag in tropomodulin binding to tropomyosin-containing actin filaments early in myofibrillogenesis is not due to delayed tropomodulin synthesisor to insufficient quantities of tropomodulin, as the cells contain a sizable pool of soluble tropomodulin (approximately 35% of the total amount) at all times during myofibrillogenesis (Gregorio and Fowler 1995). It is intriguing to speculate that a pool of monomeric tropomodulin is available to be readily recruited according to the changing requirements of the cell (for example, onset of beating). Taken together, our data suggestthat the incorporation of tropomodulin into myofibrils appears to require an additional factor (or factors): for example, a posttranslational modification or the prior assembly of an additional thin filament component(s) other than actin or tropomyosin (Gregorio and Fowler 1995). In this regard, it is of interest that subcellular fractionation experiments indicate that tropomodulin does bind to a yet unidentified cytoskeletal component(s) in an actin- and tropomyosinindependent binding manner (Fowler 1987).

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Pointed End Regulation Maintains the Length of Thin Filaments in vivo

The temporal order of the assembly of tropomodulin into cardiac myocyte sarcomeressuggestedthat tropomodulin was responsible for maintaining the final length of actin filaments by capping their pointed ends in mature striated myofibrils. This hypothesis was recently demonstrated to be correct by microinjecting into chick cardiac myocytes a C terminal domain-specific, antitropomodulin antibody that inhibits tropomodulin’s capping activity in vitro (Figure 3B) (Gregorio et al. 1995). Microinjection of this antibody inhibits tropomodulin’s in vivo actin filament capping activity by disrupting the interaction between actin and tropomodulin, resulting in a dramatic elongation of the actin filaments from their pointed endsacrossthe gap in the middle

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of the sarcomere (H zone) (Figure 3B). Surprisingly, the microinjected antibody had no effect on the original sarcomeric position of tmpomodulin, presumably because tropomodulin remained bound to tropomyosin. Therefore, the interaction of tmpomodulin with tropomyosin per se is not sufficient to cap the pointed ends of thin filaments, and the interaction of tropomodulin with actin is not required for tropomodulin to bind in its proper sarcomerit location in vivo (Gregorio et al. 1995). Taken together, our data suggest that tropomodulin’s N terminal tropomyosin binding domain and its C terminal actin filament capping domain may be functionally independent (Babcock and Fowler 1994, Gregorio et al. 1995, V.M. Fowler and A. Weber unpublished data). The ability of tropomodulin to function as a pointed end capping protein is also essential for contractile activity: cell beating is abolished in cells that contain elongated actin filaments. The inhibition of beating is likely to be due to the absence of tropomyosin on the newly formed actin filament extensions. Actin filaments without tropomyosin are expected to be unregulated because the interaction of the troponin complex with tropomyosin on the thin filament is required for calcium regulation of contraction in striated muscle. It is possible that the naked actin filament extensions are permanently “on” with respect to their interaction with myosin heads and thus the sarcomeres cannot relax (Weber and Murray 1973). The absence of tropomyosin from the new actin filament extensions is likely to be due to the fact that tropomodulin remains bound to the terminal tropomyosin molecules, thereby preventing additional tropomyosin molecules from binding to the newly formed extensions (Figure 3B). In this role, tropomodulin also acts as a “cap” for the tropomyosin polymers on the thin filaments. This idea is consistent with the observation that under some conditions, tropomodulin blocks head-to-tail associations along actin filaments in vitro (Fowler 1990). It is also worth noting that self-nucleation of new tropomyosin polymers on the newly formed actin filament extensions is unlikely to take place owing to the very low concentrations of free tropomyosin in cardiac myocytes (Wegner 1979, Gregorio and Fowler 1995). Additional support for the in vivo role of TCM Vol. 6, No. 4, 1996

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tropomodulin as an actin filament capping protein comes from a preliminary study in which antisense approaches were used to reduce the intracellular levels of tropomodulin in rat cardiac myocytes. This approach led to the replacement of myofibrils, which when stained for actin, appeared striated with myofibxils that appared nonstriated (Sussman et al. 1994a). In summary, the delayed assembly of tropomodulin into samomeres, as well as its critical role in regulating the length of actin filaments in cardiac myocytes, indicates that pointed end capping by tropomodulin is essential for maintaining actin filament length and that this is required for contractile function.

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Model for Thin Filament Assembly: Tropomodulin Maintains Thin Filament

Length

Based on the known properties of tropomodulin and other sarcomeric components, it is possible to envision the following mechanism for thin filament assembly [Gregorio and Fowler (1995), Epstein and Fischman (1991) and references therein] (Figure 3A): 1. Nascent Z-disks containing capZ and a-actinin are assembled with the thin filament proteins actin, tropomyosin, and troponins. At this stage, the barbed end capping protein capZ is likely to nucleate the process of actin filament assembly, thus specifying the polarity of the actin filaments and organizing actin filaments within the sarcomere (Schafer et al. 1995). In the absence of tropomodulin during these early stages in the assembly of sarcomeres, the pointed ends are expected to be uncapped and the absolute length of individual polarized thin filaments in stress fiberlike structures to be less well defined than the length of thin filaments found in mature myofibrils (Figure 3‘4). 2. Next, we surmise that portions of thin filaments that extend across the sarcomere and thus are incorrectly polarized with respect to the myosin cross-bridges are selectively disassembled at their pointed ends and become organized in an antiparallel, periodic distribution in striated myofibrils by a yet unknown mech-

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anism. However, it is not clear how actin and tropomyosin can assume their mature striated organization in the absence of tropomodulin (assembly intermediates: Figure 3Aii), especially considering tropomodulin’s essential role as a pointed end capping protein in mature myofibrils in vivo [see earlier here and Gregorio et al. (1995)]. It is possible that a new, currently undetectable, isoform of tropomodulin or a more distantly related pointed end capping protein might be associated with the pointed ends of thin filaments in these nascent striated myofibrils. The idea that thin filaments are initially polymerized as long filaments of varying lengths is supported by ultrastructural observations of myofibril assembly, which show that some thin filaments initially extend all the way across the sarcomere. Restriction of filament length and separation of thin filaments into two half sarcomeres (as evidenced by defined H zones and M lines) occurs late in myofibril assembly, after interdigitation of thick and thin filaments in both cardiac and skeletal muscle (Peng et al. 1981, Shimada and Obinata 1977, Legato 1972, Markwald 1973, Brooks et al. 1983). Alternatively, another model proposes that premyofibrils consist of minisarcomeres, composed of short thin fila2 ments that grow in length during the maturation process (Rhee et al. 1994). The absence of tropomodulin (the pointed ends are free) from nonstriated myofibrils or premyofibrils, is consistent with either of these models. In other words, the acquisition of mature thin filament length may he contingent upon the availability of uncapped pointed ends in nascent myofibrils. 3. Finally, thin filaments become mature in the presence of a putative “third factor” and the actin and tropomyosin polymers are subsequently stabilized (capped) at their pointed ends by tropomodulin (Figure 3Aiii). In this capacity, the assembly of tropomodulin into sarcomeres is required to maintain the final length of thin filaments, as well as perhaps being the rate-limiting

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muscle

Specification of Thin Filament Length

Determination of thin filament length in striated muscle is likely to require multiple interacting components.Tropomodulin alonecannot specify the length of thin filaments, as actin and other thin filament proteins assembleinto a striated samomeric pattern before tropomodulin assembles at the pointed end. One possibility is that thin filament length is determined by copolymerization of actin and tropomyosin alongsidea template molecule of the appropriate length. For example, in skeletal muscle myofibrils, the giant protein nebulin is coextensive with the thin filaments and is proposed to function as a template based on its repeating domain structure, close associationwith thin filaments, and inextensible nature during contraction and stretching of myofibrils. Also consistent with the hypothesis that nebulin acts as a template is the observation that the molecular massof nebulin correspondsto variations in length of thin filaments found in different skeletalmuscles[for recent reviews, seeFowler (1996) Trinick (1994), Keller (1995)]. However, an enigma is that nebulin is not detected in its mature full length staining pattern, extending from the barbed ends at the 2 line to the pointed ends in the H zone, until rather late in myofibxil assembly, well after the actin staining has already become periodic (Furst et al. 1989, Komiyama et al. 1992). Moreover, a large nebulinlike molecule has not been identified in cardiac muscle (Itoh et al. 1988). Recently, however, a petite (107 kD) nebulin-like protein, referred to as nebulette, hasbeenidentified in this tissue:immunofluorescence localization reveals that nebulette is localized to the I-Z-I complex of myofibrils. The possiblerelationship of nebulette to cardiac muscle thin filament length is at present unclear, as the small size of nebulette indicates that it could maximally span about 25% of the thin filament length (Moncman and Wang 1995). Another idea proposed many years ago is that tropomyosin might function to specify thin filament length in striated muscle through a vernierlike mechanism based on the difference between the length of tmpomyosin moleculesand the 140

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helical pitch of the actin filament (Spudich et al. 1972). In this model, precise alignment of the individual helical repeats of the actin and tropomyosin copolymers can only occur at a particular length. This alignment could perhaps specify the location for a hypothetical “terminator” molecule (tropomodulin?) to bind tightly to the terminal actin and tmpomyosin molecules at the pointed end of the thin filament (Ishiwata and Funatsu 1985). If so, this may partly explain why tropomodulin is not found associatedwith stressfiberlike structures (pmmyofibrils) that contain actin filaments of various lengths. On the other hand, a problem with this hypothesis is that although there is a extremely narrow length distribution of thin filaments in striated muscle (Page and Huxley 1963), thin filaments in cardiac musclehave a length distribution varying over about a twofold range (Robinson and Winegmd 1979).For a more extensive discussion of this hypothesis, see Fowler (1996).In conclusion, it appearslikely that tropomodulin, tropomyosin, and at least one additional component are likely to function together at the pointed ends to determine, as well as to maintain, thin filament lengths.

l

summary

Cytoskeletal .proteins are essential for muscle contraction, as well as for the proper functioning of many motile processesin nonmuscle cells. Mutations in many of the abundant cardiac contractile proteins (for example, or-tropomyosin, troponin T, C-protein, or l3-myosin heavy chain) have been implicated in causing both inherited and secondary forms of heart disease[for a recent review, see Vikstrom and Leinwand (1996)]. Here, we describe the properties of a stoichiometrically minor, yet essential, sarcomeric component, tropomodulin. Based on tropomodulin’s essential role in regulating myofibril architecture and contractility of cultured cardiac myocytes, it is tempting to speculate that in the future, defects in the expression of tropomodulin might be linked to a specific cardiomyopathy. Our work and that of others on striated muscle contractile proteins continue to demonstrate that the sarcomere is an intricately balanced unit depending on all components for its proper functioning. Science

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Acknowledgments

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The authors thank Annemarie Weber (University of Pennsylvania) for insightful comments on this review and Peggy Meyer for preparation of the figures. This work was supported by grants from the National Institutes of Health (NIH) to V.M.F. C.C.G. is a recipient of an American Heart Association California Affiliate postdoctoral fellowship award.

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