Conditional Expression of Mutant M-line Titins Results in Cardiomyopathy with Altered Sarcomere Structure

Titin is a giant protein responsible for muscle elasticity and provides a scaffold for several sarcomeric proteins, including the novel titin-binding protein MURF-1, which binds near the titin M-line region. Another unique feature of titin is the presence of a serine/threonine kinase-like domain at the edge of the M-line region of the sarcomere, for which no physiological catalytic function has yet been shown. To investigate the role(s) of the titin M-line segment, we have conditionally deleted the exons MEx1 and MEx2 (encoding the kinase domain plus flanking sequences) at different stages of embryonic development. Our data demonstrate an important role for MEx1 and MEx2 in early cardiac development (embryonic lethality) as well as postnatally when disruption of M-line titin leads to muscle weakness and death at ∼5 weeks of age. Myopathic changes include pale M-lines devoid of MURF-1, and gradual sarcomeric disassembly. The animal model presented here indicates a critical role for the M-line region of titin in maintaining the structural integrity of the sarcomere. Titin is a giant protein responsible for muscle elasticity and provides a scaffold for several sarcomeric proteins, including the novel titin-binding protein MURF-1, which binds near the titin M-line region. Another unique feature of titin is the presence of a serine/threonine kinase-like domain at the edge of the M-line region of the sarcomere, for which no physiological catalytic function has yet been shown. To investigate the role(s) of the titin M-line segment, we have conditionally deleted the exons MEx1 and MEx2 (encoding the kinase domain plus flanking sequences) at different stages of embryonic development. Our data demonstrate an important role for MEx1 and MEx2 in early cardiac development (embryonic lethality) as well as postnatally when disruption of M-line titin leads to muscle weakness and death at ∼5 weeks of age. Myopathic changes include pale M-lines devoid of MURF-1, and gradual sarcomeric disassembly. The animal model presented here indicates a critical role for the M-line region of titin in maintaining the structural integrity of the sarcomere. Titin is the third myofilament of vertebrate striated muscles and spans the half-sarcomere by integrating into the Z-disc and M-line through its amino and carboxyl terminus, respectively (1Gregorio C.C. Granzier H. Sorimachi H. Labeit S. Curr. Opin. Cell. Biol. 1999; 11: 18-25Google Scholar, 2Furst D.O. Osborn M. Nave R. Weber K. J. Cell Biol. 1988; 106: 1563-1572Google Scholar, 3Labeit S. Kolmerer B. Linke W.A. Circ. Res. 1997; 80: 290-294Google Scholar, 4Maruyama K. Rev. Physiol. Biochem. Pharmacol. 1999; 138: 1-18Google Scholar, 5Trinick J. Tskhovrebova L. Trends Cell Biol. 1999; 9: 377-380Google Scholar, 6Wang K. Adv. Biophys. 1996; 33: 123-134Google Scholar). Titin consists of subdomains that perform distinct functions (7Labeit S. Kolmerer B. Science. 1995; 270: 293-296Google Scholar, 8Granzier H. Labeit S. J. Physiol. 2002; 541: 335-342Google Scholar). Close to its carboxyl-terminal M-line region, titin contains a kinase domain that shares homology with the catalytic serine/threonine kinase domain of smooth muscle myosin light chain kinase (MLCK), 1The abbreviations used are: MLCK, myosin light chain kinase; MURF, muscle-specific ring finger; MHC, myosin heavy chain; ES, embryonic stem; FRT, flp recombinase target; KO, knockout; MCK, muscle creatine kinase; CARP, cardiac ankyrin repeat protein; ankrd, ankyrin repeat domain and the invertebrate muscle proteins twitchin, projectin, and stretchin-MLCK and Ce titin (9Ayme-Southgate A. Southgate R. McEliece M.K. Adv. Exp. Med. Biol. 2000; 481: 251-262Google Scholar, 10Olson N.J. Pearson R.B. Needleman D.S. Hurwitz M.Y. Kemp B.E. Means A.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2284-2288Google Scholar, 11Benian G.M. Kiff J.E. Neckelmann N. Moerman D.G. Waterston R.H. Nature. 1989; 342: 45-50Google Scholar, 12Champagne M.B. Edwards K.A. Erickson H.P. Kiehart D.P. J. Mol. Biol. 2000; 300: 759-777Google Scholar, 13Ayme-Southgate A. Vigoreaux J. Benian G. Pardue M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7973-7977Google Scholar, 14Flaherty D. Gernert K. Shmeleva N. Tang X. Mercer K. Borodovsky M. Benian G. J. Mol. Biol. 2002; 323: 533Google Scholar). Although a variety of different functions have been hypothesized for the titin kinase domain, its physiological substrate(s) remain unknown. Based on its crystal structure, Mayanset al. proposed that the titin kinase is under strong autoinhibitory control with a dual activation mechanism that controls kinase activity (15Mayans O. van der Ven P.F. Wilm M. Mues A. Young P. Fnrst D.O. Wilmanns M. Gautel M. Nature. 1998; 395: 863-869Google Scholar). In vitro phosphorylation experiments using a constitutively active kinase fragment suggested that the titin kinase domain phosphorylates the sarcomeric protein telethonin/T-cap, a Z-line-associated protein. More recently, Centner et al.searched for proteins that specifically interact with the titin kinase region to identify potential titin kinase substrate or regulator molecules (16Centner T. Yano J. Kimura E. McElhinny A.S. Pelin K. Witt C.C. Bang M.L. Trombitas K. Granzier H. Gregorio C.C. Sorimachi H. Labeit S. J. Mol. Biol. 2001; 306: 717-726Google Scholar). This demonstrated that the novel RING finger protein MURF-1 binds to the A168/A169 Fn3/Ig repeats of titin, which locate amino-terminal to the titin kinase domain at the periphery of the M-line lattice. MURF-1 together with its close homologues MURF-2 and MURF-3 form a distinct subgroup of the known RING finger proteins that has been implicated in the control of muscle degeneration, microtubule stabilization, M-line function, and transcriptional regulation (17Spencer J.A. Eliazer S. Ilaria R.L.J. Richardson J.A. Olson E.N. J. Cell Biol. 2000; 150: 771-784Google Scholar,18McElhinny A.S. Kakinuma K. Sorimachi H. Labeit S. Gregorio C.C. J. Cell Biol. 2002; 157: 125-136Google Scholar). Within the MURF family, only MURF-1 interacts with the A168/A169 Fn3/Ig repeats of titin, and it has been suggested that MURF-1 may participate in the regulation of titin kinase activity (16Centner T. Yano J. Kimura E. McElhinny A.S. Pelin K. Witt C.C. Bang M.L. Trombitas K. Granzier H. Gregorio C.C. Sorimachi H. Labeit S. J. Mol. Biol. 2001; 306: 717-726Google Scholar). Recently, titin has been identified as the gene mutated in an autosomal dominant form of dilated cardiomyopathy (19Siu B.L. Niimura H. Osborne J.A. Fatkin D. MacRae C. Solomon S. Benson D.W. Seidman J.G. Seidman C.E. Circulation. 1999; 99: 1022-1026Google Scholar, 20Gerull B. Gramlich M. Atherton J. McNabb M. Trombitas K. Sasse-Klaassen S. Seidman J.G. Seidman C. Granzier H. Labeit S. Frenneaux M. Thierfelder L. Nat. Genet. 2002; 30: 201-204Google Scholar). The mutation results in a truncated titin, and patients that express the truncated protein suffer from heart failure of variable age of onset in the absence of clinically detectable skeletal muscle disease. In contrast, mutations within the carboxyl-terminal region of titin cause the distal type skeletal muscular dystrophy TMD without affecting the heart (21Hackman P. Vihola A. Aravuori H. Archand S. Arpanta J. Eze J. Labeit S. Witt C.C. Peltonen L. Richard I. Udd B. Am. J. Hum. Genet. 2002; (in press)Google Scholar). Because these mutations occur in a constitutively expressed region of titin it is unclear why some of these mutations cause a disease specific to the heart muscle, whereas other mutations cause a skeletal-muscle-specific myopathy. To investigate the physiological function(s) of the titin kinase domain, we have used a conditional knockout approach to selectively delete the M-line exons MEx1 (which encodes the kinase domain) and MEx2 (to maintain the reading frame) in heart and skeletal muscle. When exons MEx1/MEx2 are excised early in embryonic development (using the α-MHC promoter) mice die in utero. In contrast, excision during late embryonic development (using the MCK promoter) allows the mice to survive but causes development of a progressive myopathy, resulting in death at 5 weeks of age. Ultrastructural analysis indicates that sarcomeres incorporate the mutant titins but subsequently disassemble. Gene expression profiling data suggest that a family of molecules involved in myocyte signaling become dysregulated after deletion of MEx1/MEx2, including members of the ankyrin repeat family. This raises the possibility that MEx1/MEx2 are involved in myofibrillar signaling and in maintaining the structural integrity of the contracting sarcomere. A targeting construct was assembled by standard procedures from a mouse genomic BAC clone (bacterial artificial chromosome library MGS1 from mouse ES cells; Genome Systems/Incyte Genomics) spanning the 3′ region of the mouse titin gene (Fig. 1 a). To preserve the open reading frame we flanked exon MEx1, which contains the kinase domain, and the neighboring downstream exon MEx2 with loxP sites. Briefly, a PCR-based strategy was used to introduce a neomycin expression cassette flanked by loxP- and FRT-sites into the intron 3′ of exon MEx2 (for nomenclature of M-line titin exons see Ref. 22Kolmerer B. Olivieri N. Witt C.C. Herrmann B.G. Labeit S. J. Mol. Biol. 1996; 256: 556-563Google Scholar). An additional loxP site was placed in the intron 5′ of MEx1. The targeting vector was verified by sequencing. Homologous recombination with this targeting vector occurred in 10% of all G418-resistant ES cell colonies. ∼30% of the clones also contained the loxP site 5′ to MEx1. These clones were used to derive chimeric animals as described previously (23Willnow T.E. Herz J. Methods Cell Biol. 1994; 43: 305-334Google Scholar). The intronic neomycin cassette has the potential to affect the phenotype of knockout animals (24Kaul A. Koster M. Neuhaus H. Braun T. Cell. 2000; 102: 17-19Google Scholar). Therefore, we mated heterozygous animals that contained the altered titin locus to transgenic mice that expressed the Flp recombinase in their germline (25Dymecki S.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6191-6196Google Scholar). Offspring from this mating in which the neomycin cassette had been removed by Flp-mediated excision was used to generate a colony of homozygous mice that only contained the loxP sequences in the two introns flanking MEx1 and MEx2 and one residual FRT site in the 3′ intron (Fig. 1 a). Genomic DNA from embryonic and postnatal mouse tails or yolk sacs was genotyped as described (Fig. 1 and Refs. 26Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar, 27Herz J. Gotthardt M. Willnow T.E. Curr. Opin. Lipidol. 2000; 11: 161-166Google Scholar). Primers were designed to detect homologous recombination, presence of the 5′ loxP site, and Flp- and Cre-mediated recombination (Fig. 1 a). For Southern genotyping, genomic DNA was digested with HindIII and probed with a SmaI fragment 3′ of Exon MEx2 (Fig. 1, aand b) according to standard procedures (26Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). Timed matings and harvesting of embryos were performed as follows: female mice were superovulated using 10 IU intraperitoneal pregnant mare's serum (Sigma), followed 48 h later by 10 IU intraperitoneal human chorionic gonadotropin (Sigma) and mating to corresponding males. The morning of detection of a vaginal plug was regarded as day 0.5 postconception. Embryos and resorbtion bodies were harvested at E15. Transgenic mice were generously provided by Susan Dymecki (Flp deleter) (25Dymecki S.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6191-6196Google Scholar), Jun-Ichi Miyazaki (CAGcre) (28Araki K. Araki M. Miyazaki J. Vassalli P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 160-164Google Scholar), Michael Schneider (MHCcre) (29Agah R. Frenkel P.A. French B.A. Michael L.H. Overbeek P.A. Schneider M.D. J. Clin. Invest. 1997; 100: 169-179Google Scholar), and Ronald Kahn (MCKcre) (30Bruning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Google Scholar). ROSA-26 β-galactosidase reporter mice were obtained from the Jackson Laboratories (www.jax.org). All animal experiments were carried out under protocols approved by the University of Texas Southwestern Institutional Review Committee for animal use and followed the National Institutes of Health guidelines, “Using Animals in Intramural Research.” Cryosections (25 of were with and with using the for according to the to generate in were from genomic DNA and the kinase region and the exon 3′ of the kinase domain, were using the with HindIII or and with using the in vitro Embryos and were in and were used for in as described previously M. M. J. Richardson J.A. J. Herz J. J. Biol. 2000; Scholar). heart muscle were with an the titin M-line region Ref. T. Yano J. Kimura E. McElhinny A.S. Pelin K. Witt C.C. Bang M.L. Trombitas K. Granzier H. Gregorio C.C. Sorimachi H. Labeit S. J. Mol. Biol. 2001; 306: 717-726Google or the region of titin T. A. Witt C. Granzier H. Trombitas K. Gregorio C.C. Labeit S. Adv. Exp. Med. Biol. 2000; 481: Scholar), and for as described previously K. M. Labeit S. M. M. Granzier H. J. Cell Biol. 1998; Scholar). and for protein were and with of 10% at Biophys. J. 1995; Scholar). Titin protein and were performed and as described M. Trombitas K. Biophys. J. 1996; using specific to T. A. Witt C. Granzier H. Trombitas K. Gregorio C.C. Labeit S. Adv. Exp. Med. Biol. 2000; 481: Scholar), CARP, and M.L. McElhinny A.S. Trombitas K. R. Sorimachi H. Granzier Gregorio C.C. Labeit S. J. Cell Biol. 2001; Scholar, N. J. G. 2000; Scholar). To conditional excision of the titin kinase region we have used homologous recombination in ES to introduce loxP and FRT recombination into introns flanking the kinase (Fig. 1 a). To maintain an open reading exon MEx1, which encodes the kinase domain, and the neighboring downstream exon MEx2 were to of loxP into the gene with and had no in mice that were homozygous for the The targeting was with of the targeting including the site 3′ of MEx1 mice from ES were mated to Flp transgenic mice to the intronic neomycin expression cassette and to potential physiological (24Kaul A. Koster M. Neuhaus H. Braun T. Cell. 2000; 102: 17-19Google Scholar). Offspring in which the neomycin cassette had been deleted was used to derive a colony of homozygous mice that only contained the loxP sequences in the two introns flanking MEx1 and MEx2 and one residual FRT site in the 3′ intron (Fig. 1 a). These animals no or and were from type and analysis of and recombination was performed as in recombination with the recombinase as well as with the recombinase (Fig. 1 The of recombination in the germline through the recombination has been to (25Dymecki S.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6191-6196Google Scholar). In animals of were as from germline of all titin were the of the site with titin gene function heterozygous matings to at the and heterozygous and homozygous animals with and without the neomycin cassette were To the titin kinase domain from striated muscle, we obtained transgenic animals that recombination to heart and skeletal muscle (29Agah R. Frenkel P.A. French B.A. Michael L.H. Overbeek P.A. Schneider M.D. J. Clin. Invest. 1997; 100: 169-179Google Scholar, J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Google Scholar). of the recombinase under control of the heavy chain (MHCcre) in recombination of the reporter construct in heart and of the (Fig. expression under control of the muscle creatine kinase (MCKcre) recombination in all striated muscle including the heart and in smooth muscle in (Fig. The in (Fig. a of of the kinase domain from cardiac titin during development the to early embryonic bodies from knockout animals were at embryonic day (Fig. a). homozygous was a critical role for titin in heart development. In to the animals the recombinase from the early stages during the skeletal muscle-specific of mice that expressed the recombinase and that were homozygous for the at the These in which deletion of the titin kinase domain in skeletal muscle was a by progressive muscle which their and and of the at to was at weeks of with of the phenotype death at ∼5 weeks of age (Fig. the heart of titin animals but in when with for or a). To that this phenotype was to the of the titin M-line and by of the which introduce additional in the carboxyl-terminal of the we by in that exons 3′ of the deleted kinase region were at (Fig. the of MEx1/MEx2 and of the open reading frame when to In demonstrated that by weeks of titin was in only a (Fig. whereas mutant titin protein had titin in heart muscle (Fig. with and and by demonstrated that of the expressed titin was in its kinase region. The presence of the carboxyl in the titin protein was by with the kinase domain or at the and carboxyl of titin (Fig. a and of control animals sarcomeres with well M-line in of animals contained with sarcomeres with of the of the as well as with sarcomeres in stages of of animals had and pale M-lines that were devoid of M-line in and When sarcomeres were with M-line were obtained that were in control animals but in the animals this had (Fig. and the to with the of of the with with also used an MURF-1 and M-line in control animals (Fig. but absence of M-line in animals (Fig. Because in vitro have that MURF-1 binds to the that are by MEx1 (16Centner T. Yano J. Kimura E. McElhinny A.S. Pelin K. Witt C.C. Bang M.L. Trombitas K. Granzier H. Gregorio C.C. Sorimachi H. Labeit S. J. Mol. Biol. 2001; 306: 717-726Google Scholar), absence of MURF-1 the that MEx1 has been excised from sarcomeres in whereas with indicates that the titin carboxyl is titin function by no other gene for a protein of and is Therefore, we that mice after gene targeting of the titin locus die during early embryonic of the role of titin in muscle. we an deletion in a specific region of titin using a conditional that the deletion of the MEx1 exon into structural and of titin MEx1 for the M-line of titin, which is and contains a serine/threonine kinase domain and a site for the RING finger protein the of the titin kinase in the heart and in skeletal muscle at different stages of embryonic and postnatal development by using transgenic animals that express recombinase under control of the well muscle-specific α-MHC and These transgenic animals used to derive the have been described by (29Agah R. Frenkel P.A. French B.A. Michael L.H. Overbeek P.A. Schneider M.D. J. Clin. Invest. 1997; 100: 169-179Google Scholar, J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Google but have been by their (Fig. with muscular expression of the MCK In expression of MCK at embryonic day to of activity by and at day 10 of Biophys. 1990; Scholar). hypothesized that the activity of the may homozygous mice to survive through early postnatal whereas expression by the α-MHC may to early recombination with potential embryonic and T. Schneider M.D. Circ. Res. 2001; 88: Scholar). These were by the β-galactosidase in from animals (Fig. a and and by the early embryonic of the The mutant titin were by in which the exons for in the periphery of the and exon for were at the protein a for of the M-line of titin was including the site for the RING finger protein MURF-1 and the kinase domain of with when the myofibrillar proteins on protein titin from the mutant mice an on The of myofibrillar titin in the mutant and mice were (Fig. 5 Therefore, the mutant titin from the exon gene locus is and into The strategy here for the of the titin exons is for the deletion of other of in titin, the of this giant The phenotype of mice is their progressive muscle to a for by of (Fig. to and at weakness is at weeks of the mutant mice become at weeks of age. This that the muscle weakness it a of cardiac The sarcomeres mutant titin to Within the region of the sarcomeric from sarcomeres with pale M-lines devoid of (Fig. to sarcomeres (Fig. the two in the of the by in the control but to in the animals (Fig. The in control sarcomeres is with a model in which titins from by within M-lines Gautel M. van der Ven P.F. Weber K. D.O. J. Cell Biol. 1996; Scholar). The of mutant titin that titin molecules but of the of the of titin from the of the a region of the under is to the of the sarcomere, and this may to sarcomeric disassembly. Our mouse model on the physiological role(s) of the titin kinase domain and its the potential for the titin and MURF-1 (15Mayans O. van der Ven P.F. Wilm M. Mues A. Young P. Fnrst D.O. Wilmanns M. Gautel M. Nature. 1998; 395: 863-869Google Scholar, T. Yano J. Kimura E. McElhinny A.S. Pelin K. Witt C.C. Bang M.L. Trombitas K. Granzier H. Gregorio C.C. Sorimachi H. Labeit S. J. Mol. Biol. 2001; 306: 717-726Google Scholar), were in animal model as verified by and analysis In contrast, for and its were and with specific protein in the titin mice (Fig. 5 have been to with titin and during myofibrillar which that titin, CARP, and form an signaling M.L. McElhinny A.S. Trombitas K. R. Sorimachi H. Granzier Gregorio C.C. Labeit S. J. Cell Biol. 2001; Scholar). in has been to in expression myofibrillar N. J. G. 2000; Scholar). Our that and are in titin mice raises the possibility that and signaling is to to myofibrillar H. Labeit S. J. Physiol. 2002; 541: 335-342Google and signaling to muscle gene expression S. J. 1997; Scholar), we that the signaling with the regulation of muscle gene In we used a conditional gene targeting strategy as a novel approach of the titin M-line region of from cardiac titin during development the leads to absence of homozygous This indicates a critical role for in heart development. This is with the by et al. patients with heterozygous titin but no homozygous in of cardiomyopathy B. Gramlich M. Atherton J. McNabb M. Trombitas K. Sasse-Klaassen S. Seidman J.G. Seidman C. Granzier H. Labeit S. Frenneaux M. Thierfelder L. Nat. Genet. 2002; 30: 201-204Google Scholar). In the titin M-line region is to a critical role in maintaining the structural integrity of the and myofibrillar signaling by the when skeletal muscle are and the heart is the M-line region of the sarcomere. The conditional knockout used in this to well for of the giant titin during development and in muscle This approach also to of titin that cause in the of and and the of critical by M. G. is to Michael for the and during the early of this and to for critical reading of the and