Dynamics of Obscurin Localization During Differentiation and Remodeling of Cardiac Myocytes : Obscurin as an Integrator of Myofibrillar Structure
Departments of Pediatrics and Communicable Diseases (ABB,MWR), Physiology (MVW), and Surgery (MVW), Division of Pediatric Cardiology, University of Michigan Medical School, Ann Arbor, Michigan, and Department of Physiology (AK-K,RJB), School of Medicine, University of Maryland, Baltimore, Maryland
Correspondence to: Andrei B. Borisov, PhD, Room 8200, MSRB III, Div. of Pediatric Cardiology, Dept. of Pediatrics and Communicable Diseases, U. of Michigan Medical School, Ann Arbor, MI 48109. E-mail: aborisov{at}umich.edu
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Summary |
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Key Words: obscurin myofibrillogenesis cardiac myocytes differentiation sarcomere
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Introduction |
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During differentiation, the architecture of the cytoskeleton and the contractile system in cardiac muscle cells undergoes progressive development and structural maturation, which is accompanied by intense formation of new myofibrils and cytoskeletal elements (Rumyantsev 1991). Terminal differentiation of cardiomyocytes has long been considered an irreversible process. However, over the past two decades it has become apparent that adult cardiac myocytes are plastic, able to remodel their structure profoundly both in vivo and in vitro (for literature see Borisov 1991
; Bugaisky 1991
; Claycomb 1991
; Eppenberger et al. 1994
; Swynghedauw 1999
). To better understand the potential functions of obscurin in cardiac muscle, it is important to determine its role during myocyte differentiation and remodeling.
Cardiac cell cultures are an interesting and convenient object for studies of myofibrillar dynamics (for review see Ehler and Perriard 2002). This study was undertaken to elucidate the dynamics of spatial and temporal patterns of obscurin expression during myofibrillogenesis, remodeling, and redifferentiation in primary cultures of neonatal and adult rat cardiomyocytes.
Z-lines are important structures of striated muscle cells that provide the stability and functional integration of sarcomeres and play an important role in myofibril assembly. Because the mechanisms of cardiac myocyte differentiation and remodeling involve significant modifications and changes in the areas of Z-lines of myofibrils (for discussion see Borisov 1991; Sanger and Sanger 2002
), we investigated the localization of obscurin in relation to
-actinin and desmin, two functionally different proteins topographically associated with Z-lines in terminally differentiated striated muscle.
-Actinin, a major structural protein of Z-lines that provides a proper orientation and mechanical fixation of actin filaments within the sarcomeres, is indispensable for normal progression of all stages of myofibrillogenesis. This is one of the first proteins detected in structural precursors of myofibrils. Movement of
-actinin-containing bodies (Z-bodies) along the long axis of nascent myofibrils underlies the formation of Z-lines, apparently through their coalescence and lateral alignment with closely apposed neighboring nascent myofibrils (for literature see Borisov 1991
; Shimada et al. 2002
).
Desmin is a muscle-specific intermediate filament protein that shifts from filamentous to striated pattern of localization at terminal stages of myogenic differentiation, binding myofibrils together at the level of Z-lines and structurally integrating the contractile apparatus during contractionrelaxation cycles (e.g., Saetersdal et al. 1989; Borisov 1991
; Opie 1998
). Taking into account the recent reports concerning the capacity of obscurin to bind titin in solution (Bang et al. 2001
; Young et al. 2001
) and the data concerning the direct involvement of titin in the assembly and function of sarcomeres (reviewed by Shimada et al. 2002
), we also compared the patterns of obscurin and titin localization within nascent and newly formed sarcomeres. Titin is believed to link myosin and, probably, actin filaments to Z-lines and provide the elasticity to sarcomeric structure (for discussion see Sanger and Sanger 2002
; Shimada et al. 2002
). With technical advances of confocal microscopy, it has become feasible to analyze the co-localization of individual proteins in differentiating and dedifferentiating cells at high resolution. Our goal was to analyze the localization of obscurin in relation to
-actinin and desmin during the processes of assembly and disassembly of myofibrils using confocal microscopy and high-resolution digital imaging.
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Materials and Methods |
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Immunocytochemistry and Confocal Microscopy
Cell fixation, processing, and immunostaining were performed as previously described (Borisov et al. 1989). We previously reported the development and characterization of the monoclonal antibody (MAb)EA- 53 to the sarcomeric isoform of
-actinin (Borisov et al. 1989
; Fridlianskaia et al. 1989
) which is now available commercially (Sigma Chemical; St Louis, MO). A polyclonal antibody recognizing the carboxy region of obscurin was prepared in a rabbit host using standard methods, and its specificity has been examined (Kontrogianni-Konstantopoulos et al. 2003
). An anti-titin MAb (clone 9D10) binding to the PEVK segment of titin in the I-bands near the IA interfaces of sarcomeres (Trombitás et al. 1998
) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA). Monoclonal anti-desmin and all secondary antibodies were obtained from Sigma. Immunostained samples of cultured cardiac myocytes were mounted on slides and examined with a Carl Zeiss LSM 510 Meta confocal microscope using the x40 and x63 objectives.
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Results |
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To further examine the possible role of obscurin in binding individual myofibrils into larger functional units, we examined its co-localization with desmin, an intermediate filament protein known to link myofibrils in register at the periphery of Z-lines in terminally differentiated cardiac muscle. We found that obscurin and desmin did not co-localize in differentiating cardiac myocytes (Figures 5A5C) . Instead, the transverse striated pattern of obscurin was already well developed and the fusion and lateral alignment of myofibrils was in progress while desmin was still in diffuse filaments typical of developing muscle. This demonstrates that obscurin is primarily associated with laterally aligning myofibrils before they associate with desmin and that obscurin-mediated primary alignment of the contractile apparatus precedes the maturation of the intermediate filament system.
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Discussion |
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We found that the primary structure of obscurin bears a striking similarity to Unc-89, a protein required for assembly of myofibrils and M-bands in Caenorhabditis elegans (Russell et al. 2002). Given its localization in maturing myofibrils and structural similarity to Unc-89, obscurin may play a similarly vital role in the completion of sarcomere differentiation in mammalian myocytes. Obscurin and its possible embryonic isoforms may perform additional functions at different stages of myofibrillogenesis. Young and co-authors (2001) described the presence of obscurin, which appears to be a fetal isotype, in Z-lines of myofibrils in chick and mouse embryos (<10-somites stage in chick, E9.5 in mouse) and observed noticeable staining of the M-band at later stages. Bang et al. (2001)
found the presence of this protein both in the Z-lines and the areas of M-bands of mature sarcomeres, which agrees with our data. We recently reported elevated expression of obscurin and its intense localization to the new sites of myofibrillogenesis after pharmacological induction of hypertrophy in neonatal rat cardiomyocytes in vitro (Borisov et al. 2003
).
Obscurin binds a small form of ankyrin and co-localizes with it in skeletal muscle cells, suggesting that it may serve as a molecular link between the sarcoplasmic reticulum and the myofibrils (Bagnato et al. 2003; Kontrogianni-Konstantopoulos et al. 2003
). If obscurin plays a similar role in the heart, as seems likely, it may help to align the sarcoplasmic reticulum and, probably, T-system with M- and Z-lines in differentiating cardiomyocytes. The presence of a calmodulin-binding domain in obscurin would be consistent with its involvement in control of calcium homeostasis and calcium-regulated signaling vital for rhythmic contractile function.
Based on our data and previous studies, we propose that obscurin functions as an integrator of myofibrillar structure. Its similarity to Unc-89 supports this model. Like Unc-89, obscurin is a large, muscle-specific protein containing numerous tandem immunoglobulin domains and tandem SH3 and Rho guanine nucleotide exchange factor domains. Mutation of Unc-89 caused marked disorganization of muscle in C. elegans. In particular, the thick (myosin) filaments were not organized into A-bands and there were no M-bands (Waterston et al. 1980). It was suggested that Unc-89, through interactions with other muscle proteins and the sarcolemma, enabled the organization and stabilization of the contractile apparatus (Benian et al. 1996
). As the only known vertebrate orthologue of Unc-89, obscurin may perform similar functions during myogenic differentiation in mammals (Sutter et al. in press
). Obscurin may also play a regulatory, signaling role during the assembly, disassembly and remodeling of myofibrils. The involvement of the Rho-GTPase in myofibril formation and organization (Hoshijima et al. 1998
) supports the possibility that obscurin's Rho-GEF domain is involved in regulating myofibrillar structure.
Of special interest are the changes in obscurin localization after induction of disassembly of myofibrils in terminally differentiated heart cells with fetal bovine serum. Early decrease of immunoreactivity for obscurin after induction of myofibril disassembly demonstrates that this protein leaves sarcomeres earlier than the contractile system disintegrates during reversible dedifferentiation. Its early dissociation from myofibrils supports the view that at least some domains of this protein may be located on the external surfaces of the M- and Z-lines as demonstrated recently in skeletal muscle (Kontrogianni-Konstantopoulos et al. 2003).
An important role of obscurin may be linked to its structural and functional interactions with another giant muscle protein, titin, that extends through several sarcomeric regions. One of the major functions of titin in mature sarcomeres is to link the thick (myosin) filaments to the Z-lines (Sanger and Sanger 2002; Shimada et al. 2002
). Titin is believed to function as an elastic spring-like element during muscle contraction and relaxation, and is postulated to define the spatial positions of other contractile proteins during myofibril assembly (Freiburg et al. 2000
; Hein et al. 2002
). In developing muscle, titin plays a role as a molecular blueprint for the structural layout of sarcomeric elements (Gregorio et al. 1999
; Trinick and Tskhovrebova 1999
; Tskhovrebova and Trinick 2003
). Early in myofibrillogenesis, titin co-localizes with
-actinin at Z-lines (Tokuyasu and Maher 1987
; Komiyama et al. 1993
), and may integrate myosin filaments with IZI complexes of nascent myofibrils (Barral and Epstein 1999
; Shimada et al. 2002
). The comparison of the localization of obscurin and titin in nascent and mature myofibrils suggests that these two proteins may function together to coordinate sarcomeric assembly.
A similar linkage is unlikely to exist between obscurin and desmin, one of the earliest muscle-specific proteins expressed during development of skeletal muscle and the heart. Earlier, we did not find co-localization of desmin and obscurin after induction of cardiac cell hypertrophy (Borisov et al. 2003). In terminally differentiated cardiac myocytes, the major muscle-specific intermediate filament protein, desmin, binds bundles of myofibrils at the level of their Z-lines, thus helping to align them into striations (for discussion and illustrations see Saetersdal et al. 1989
; Borisov 1991
; Opie 1998
). Desmin is also believed to play a role in stabilizing the myofibrillar apparatus during muscle contraction (Capetanaki and Milner 1998
). Because obscurin assembles into striations and associates with aligning myofibrils before desmin, it may play a more important role than desmin at early stages of myofibrillar integration in the developing muscle. This may provide a partial explanation for the surprising fact that the desmin null (/) mice generated through homologous recombination by Capetanaki and co-workers (1997)
are viable and fertile. Because cardiac muscle starts intense contractile activity early in embryonic development, the presence of an elastic support system reversibly uniting myofibrils during contractions provides an important functional advantage that permits easy spatial remodeling during heart growth under conditions of increasing hemodynamic load. Indeed, its size (
720 kD), distribution, and multidomain structure suggest that obscurin may interact with several sarcomeric elements. Together with titin, it may serve to integrate and stabilize the contractile apparatus at the Z-lines and the A- and M-bands. Therefore, giant molecules of obscurin and titin may work as elastic staples or springs, integrating and stabilizing different structural elements of the sarcomeres, the sarcoplasmic reticulum and, possibly, the T-system during the cycles of contraction and relaxation in cardiac muscle.
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Acknowledgments |
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We wish to thank Chris Edwards and Bruce Donohoe, the members of Micropscopy and Image Analysis Laboratory, for support of this work. We also thank Pavel Borisov for help with preparation of the manuscript.
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Footnotes |
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