Advertisement

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

  • Andrei B. BorisovEmail author
  • Marina G. Martynova
  • Mark W. Russell
Original Paper

Abstract

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis.

Keywords

Cardiac myocytes Myofibrillogenesis Myosin Obscurin Sarcomere Sarcoplasmic reticulum Z-disks 

Notes

Acknowledgments

We wish to thank Dr. A. Kontrogianni-Konstantopoulos and Dr. Robert Bloch (University of Maryland) for providing the polyclonal antibody to obscurin used in this study. We are thankful to Dr. Bruce Carlson for reading and commenting on the manuscript. The authors gratefully acknowledge support the Department of Pediatrics, the University of Michigan Medical School, from the grants through the Muscular Dystrophy Assocaiation (MDA3803), the NIH (R01 HL 075093-01), and the research funding from the Russian Academy of Sciences. We also thank Pavel Borisov for his help in preparation of the illustrations.

References

  1. Agarkova I, Perriard J-C (2005) The M-band: an elastic web that crosslinks thick filaments in the center of the sarcomere. Trends Cell Biol 15:477–485PubMedCrossRefGoogle Scholar
  2. Agarkova I, Ehler E, Lange S, Schoenauer R, Perriard JC (2003) M-band: a safeguard for sarcomeric stability? J Muscle Res Cell Motil 24:191–203PubMedCrossRefGoogle Scholar
  3. Anversa P, Olivetti G, Bracchi PG, Loud AV (1981) Postnatal development of the M-band in rat cardiac myofibrils. Circ Res 48:561–568PubMedGoogle Scholar
  4. Armani A, Galli S, Giacomello E, Bagnato P, Barone V, Rossi D, Sorrentino V (2006) Molecular interactions with obscurin are involved in the localization of muscle-specific small ankyrin 1 isoforms to subcompartments of the sarcoplasmic reticulum. Exp Cell Res 312:3456–3458CrossRefGoogle Scholar
  5. Arimura T, Matsumoto Y, Okazaki O, Hayashi T, Takahashi M, Inagaki N, Hinohara K, Ashizawa N, Yano K, Kimura A (2007) Structural analysis of obscurin gene in hypertrophic cardiomyopathy. Biochem Biophys Res Commun 362:281–287PubMedCrossRefGoogle Scholar
  6. Bader D, Masaki T, Fischman DA (1982) Immunocytochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol 95:763–770PubMedCrossRefGoogle Scholar
  7. Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V (2003) Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils is striated muscles. J Cell Biol 160:245–253PubMedCrossRefGoogle Scholar
  8. Bang ML, Centner T, Fornoff F, Geach AJ, Gotthardt M, McNabb M, Witt CC, Labeit D, Gregorio CC, Granzier H, Labeit S (2001) The complete gene sequence of titin, expression of an unusual ∼700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ Res 89:1065–1072PubMedCrossRefGoogle Scholar
  9. Borisov AB (1991) Myofibrillogenesis and reversible disassembly of myofibrils as adaptive reactions of cardiac muscle cells. Acta Physiol Scand 142(suppl 599):71–80Google Scholar
  10. Borisov AB, Coro Antich RM, Rumyantsev PP (1985) DNA synthesis in cultures of atrial and ventricular rat cardiomyocytes. Tsitologia 27:990–994Google Scholar
  11. Borisov AB, Goncharova EI, Pinaev GP, Rumyantsev PP (1989) Changes in α-actinin localization and myofibrillogenesis in rat cardiomyocytes in culture. Tsitologia 31:642–646Google Scholar
  12. Borisov AB, Dedkov EI, Carlson BM (2001) Interrelations of myogenic response, progressive atrophy of muscle fibers, and cell death in denervated skeletal muscle. Anat Rec 264:203–218PubMedCrossRefGoogle Scholar
  13. Borisov AB, Raeker MO, Kontrogianni-Konstantopoulos A, Yang K, Kurnit DM, Bloch RJ, Russell MW (2003) Rapid response of cardiac obscurin gene cluster to aortic stenosis: differential activation of Rho-GEF and MLCK and involvement in hypertrophic growth. Biochem Biophys Res Commun 310:910–918PubMedCrossRefGoogle Scholar
  14. Borisov AB, Kontrogianni-Konstantopoulos A, Bloch RJ, Westfall MV, Russell MW (2004) Dynamics of obscurin localization during differentiation and remodeling of cardiac myocaytes: obscurin as an integrator of myofibrillar structure. J Histochem Cytochem 52:1117–1127PubMedCrossRefGoogle Scholar
  15. Borisov AB, Sutter SB, Kontrogianni-Konstantopoulos A, Bloch RJ, Westfall MV, Russell MW (2006) Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing. Histochem Cell Biol 125:227–238PubMedCrossRefGoogle Scholar
  16. Burridge K, Wennerberg K (2004) Rho and Rac take center stage. Cell 116:167–179PubMedCrossRefGoogle Scholar
  17. Carlsson E, Kjörell U, Thornell L-E, Lambertsson A, Strehler E (1982) Differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart. Eur J Cell Biol 27:62–73PubMedGoogle Scholar
  18. Dlugosz AA, Antin PB, Nachmias VT, Holtzer H (1984) The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes. J Cell Biol 99:2268–2278PubMedCrossRefGoogle Scholar
  19. Du A, Sanger JM, Linask KK, Sanger JW (2003) Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm. Dev Biol 257:382–394PubMedCrossRefGoogle Scholar
  20. Ehler E, Rothen BM, Haemmerle SP, Komiyama M, Perriard JC (1999) Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments. J Cell Sci 112:1529–1539PubMedGoogle Scholar
  21. Ehler E, Fowler VM, Perriard J-C (2004) Myofibrillogenesis in the developing chicken heart: role of actin isoforms and the pointed end actin capping protein tropomodulin during thin filament assembly. Dev Dyn 229:745–755PubMedCrossRefGoogle Scholar
  22. Epstein ND, Davis JS (2003) Sensing stretch is fundamental. Cell 112:147–150PubMedCrossRefGoogle Scholar
  23. Fridlianskaia II, Goncharova EI, Borisov AB, Krylova TA, Pinaev GP (1989) Monoclonal antibody to the muscle isoform of α-actinin—a marker for studies of differentiation of skeletal and cardiac muscle. Tsitologiia 31:1234–1237PubMedGoogle Scholar
  24. Fukuzawa A, Idowu S, Gautel M (2005) Complete human gene structure of obscurin: implications for isoform generation by differential splicing. J Muscle Res Cell Motil 26:427–434PubMedCrossRefGoogle Scholar
  25. Granzier HL, Labeit S (2005) Titin and its associated proteins: the third myofilament system of the sarcomere. Adv Protein Chem 71:89–119PubMedCrossRefGoogle Scholar
  26. Granzier HL, Labeit S (2006) The giant muscle protein titin is an adjustable molecular spring. Exerc Sport Sci Rev 34:50–53PubMedCrossRefGoogle Scholar
  27. Holtzer H, Hijikata T, Lin ZX, Zhang ZQ, Holzer S, Protasi F, Franzini-Armstrong C, Sweeney HL (1997) Independent assembly of 1.6 micron long bipolar MHC filaments and I-Z-I bodies. Cell Struct Funct 22:83–93PubMedCrossRefGoogle Scholar
  28. Huxley AF (2000) Cross-bridge action: present views, prospects, and unknowns. J Biomech 33:1189–1195PubMedCrossRefGoogle Scholar
  29. Kaarbo M, Crane DI, Murrell WG (2003) RhoA is highly up-regulated in the process of early heart development of the chick and important for normal embryogenesis. Dev Dyn 227:35–47PubMedCrossRefGoogle Scholar
  30. Kontrogianni-Konstantopoulos A, Jones EM, van Rossum DB, Bloch RJ (2003) Obscurin is a ligand for small ankyrin 1 in skeletal muscle. Mol Biol Cell 14:1138–1148PubMedCrossRefGoogle Scholar
  31. Kontrogianni-Konstantopoulos A, Catino DH, Strong JC, Bloch RJ (2005) De novo myofibrillogenesis in C2C12 cells: evidence for the independent assembly of M-bands and Z-disks. Am J Physiol 290:C626–C637CrossRefGoogle Scholar
  32. Kontrogianni-Konstantopoulos A, Catino DH, Strong JC, Sutter S, Borisov AB, Pumplin DW, Russell MW, Bloch RJ (2006) Obscurin modulates the assembly and organization of sarcomeres and the sarcoplasmic reticulum. FASEB J 20:2102–2111PubMedCrossRefGoogle Scholar
  33. Lange S, Agarkova I, Perriard J-C, Ehler E (2005) The sarcomeric M-band during development and in disease. J Muscle Res Cell Motil 26:375–379PubMedCrossRefGoogle Scholar
  34. Lange S, Ehler E, Gautel M (2006) From A to Z and back? Multicompartment protein in the sarcomere. Trends Cell Biol 16:11–18PubMedCrossRefGoogle Scholar
  35. LeWinter MM, Wu Y, Labeit S, Granzier H (2007) Cardiac titin: structure, functions and role in disease. Clin Chim Acta 375:1–9PubMedCrossRefGoogle Scholar
  36. Miller G, Musa H, Gautel M, Peckham M (2003) A targeted deletion of the C-terminal end of titin, including the titin kinase domain, impairs myofibrillogenesis. J Cell Sci 116:4811–4819PubMedCrossRefGoogle Scholar
  37. Musa H, Meek S, Gautel M, Peddie D, Smith AJH, Peckham M (2006) Targeted homozygous deletion of M-band titin in cardiomyocytes prevents sarcomere formation. J Cell Sci 119:4322–4331PubMedCrossRefGoogle Scholar
  38. Ojima K, Lin ZX, Zhang ZQ, Hijikata T, Holtzer S, Labeit S, Sweeney HL, Holtzer H (1999) Initiation and maturation of I–Z–I bodies in the growth tips of transfected myotubes. J Cell Sci 112:4101–4112PubMedGoogle Scholar
  39. Peng J, Raddatz K, Labeit S, Granzier H, Gotthardt M (2005) Muscle atrophy in titin M-line deficient mice. J Muscle Res Cell Motil 26:381–388PubMedCrossRefGoogle Scholar
  40. Person V, Kostin S, Suzuki K, Labeit S, Schaper J (2000) Antisense oligonucleotide experiments elucidate the essential role of titin in sarcomerogenesis in adult cardiomyocytes in long-term culture. J Cell Sci 113:3851–3859PubMedGoogle Scholar
  41. Porter NC, Resneck WG, O’Neill A, Van Rossum DB, Stone MR, Bloch RJ (2005) Association of small ankyrin 1 with the sarcoplasmic reticulum. Mol Membr Biol 22:421–432PubMedCrossRefGoogle Scholar
  42. Raeker MO, Su F, Geisler SB, Borisov AB, Kontrogianni-Konstantopoulos A, Lyons SE, Russell MW (2006) Obscurin is required for the lateral alignment of striated myofibrils in zebrafish. Dev Dyn 235:2018–1029PubMedCrossRefGoogle Scholar
  43. Rhee D, Sanger JM, Sanger JW (1994) The premyofibril: evidence for its role in myofibrillogenesis. Cell Motil Cytoskeleton 28:1–24PubMedCrossRefGoogle Scholar
  44. Rumyantsev PP (1976) Myofibrillogenesis and its relationships with cell proliferation during development and regeneration of skeletal, cardiac and smooth muscles. Acta Histochem 17(suppl):215–218Google Scholar
  45. Rumyantsev PP (1977) Interrelations of the proliferation and differentiation processes during cardiac myogenesis and regeneration. Int Rev Cytol 51:187–273Google Scholar
  46. Rumyantsev PP (1991) Growth and hyperplasia of cardiac muscle cells. Harwood Academic Publishers, New YorkGoogle Scholar
  47. Russell MW, Raeker MO, Korytkowski KA, Sonneman KJ (2002) Identification, tissue expression and chromosomal localization of human obscurin-MLCK, a member of the titin and Dbl families of myosin light chain kinases. Gene 282:237–246PubMedCrossRefGoogle Scholar
  48. Sanger JW, Kang S, Siebrands CC, Freeman N, Du A, Wang J, Stout AL, Sanger JM (2005) How to build a myofibril. J Muscle Res Cell Motil 26:343–354PubMedCrossRefGoogle Scholar
  49. Schoenauer R, Bertoncini P, Machaidze G, Aebi U, Perriard JC, Hegner M, Agarkova I (2005) Myomesin as a molecular spring with adaptable elasticity. J Mol Biol 349:367–379PubMedCrossRefGoogle Scholar
  50. Schultheiss T, Lin ZX, Lu MH, Murray J, Fischman DA, Weber K, Masaki T, Imamura M, Holtzer H (1990) Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils. J Cell Biol 110:1159–1172PubMedCrossRefGoogle Scholar
  51. Seeley M, Huang W, Chen Z, Wolff WO, Lin X, Xu X (2007) Depletion of zebrafish titin reduces cardiac contractility by disrupting the assembly of Z-discs and A-bands. Circ Res 100:238–245PubMedCrossRefGoogle Scholar
  52. Tokuyasu KT, Maher PA (1987) Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryo. II. Generation of alpha-actinin dots within titin spots at the time of the first myofibril formation. J Cell Biol 105:2795–2801PubMedCrossRefGoogle Scholar
  53. Trinick J, Tskhovrebova L (1999) Titin: a molecular control freak. Trends Cell Biol 9:377–380PubMedCrossRefGoogle Scholar
  54. Trombitás K, Greaser M, French G, Granzier H (1998) PEVK extension of human soleus muscle titin revealed by immunolabeling with the anti-titin antibody 9D10. J Struct Biol 4:679–689Google Scholar
  55. Tskhovrebova L, Trinick J (2003) Titin: properties and family relationship. Nat Rev Mol Cell Biol 4:679–689PubMedCrossRefGoogle Scholar
  56. Wei L, Imanaka-Yoshida K, Wang L, Zhan S, Schneider MD (2002) Inhibition of Rho family GTPases by Rho GDP dissociation inhibitor disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation. Development 129:1705–1714PubMedGoogle Scholar
  57. Weinert S, Bergmann N, Luo X, Erdmann B, Gotthargt M (2006) M line-deficient titin causes cardiac lethality through impaired maturation of the sarcomere. J Cell Biol 173:559–570PubMedCrossRefGoogle Scholar
  58. Witt CC, Burkart C, Labeit D, McNabb M, Wu Y, Granzier H, Labeit S (2006) Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo. EMBO J 25:3843–3855PubMedCrossRefGoogle Scholar
  59. Young P, Ehler E, Gautel M (2001) Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly. J Cell Biol 154:123–136PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Andrei B. Borisov
    • 1
    Email author
  • Marina G. Martynova
    • 2
  • Mark W. Russell
    • 1
  1. 1.Division of Pediatric Cardiology, Congenital Heart Center, Department of Pediatrics and Communicable DiseasesUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Cellular Cardiology Research Group, Laboratory of Cell MorphologyInstitute of Cytology RASSt PetersburgRussia

Personalised recommendations