Truncation by Glu180 Nonsense Mutation Results in Complete Loss of Slow Skeletal Muscle Troponin T in a Lethal Nemaline Myopathy
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m303469200
ISSN1083-351X
AutoresJian‐Ping Jin, Marco Brotto, M. Moazzem Hossain, Qi-Quan Huang, Leticia Brotto, Thomas M. Nosek, D. Holmes Morton, Thomas O. Crawford,
Tópico(s)Viral Infections and Immunology Research
ResumoA lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene. We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM. The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. Expression of slow and fast isoforms of TnT is fiber-type specific. The lack of slow TnT results in selective atrophy of type 1 fibers. Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays. Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth. The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT. These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM. A lethal form of nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), is linked to a nonsense mutation at codon Glu180 in the slow skeletal muscle troponin T (TnT) gene. We found that neither the intact nor the truncated slow TnT protein was present in the muscle of patients with ANM. The complete loss of slow TnT is consistent with the observed recessive pattern of inheritance of the disease and indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. Expression of slow and fast isoforms of TnT is fiber-type specific. The lack of slow TnT results in selective atrophy of type 1 fibers. Slow TnT confers a higher Ca2+ sensitivity than does fast TnT in single fiber contractility assays. Despite the lack of slow TnT, individuals with ANM have normal muscle power at birth. The postnatal onset and infantile progression of ANM correspond to a down-regulation of cardiac and embryonic splice variants of fast TnT in normal developing human skeletal muscle, suggesting that the fetal TnT isoforms complement slow TnT. These results lay the foundation for understanding the molecular pathophysiology and the potential targeted therapy of ANM. Nemaline myopathies are neuromuscular disorders characterized by muscle weakness and rod-shaped or "nemaline" inclusions in skeletal muscle fibers (1North K.N. Laing N.G. Wallgren-Pettersson C. J. Med. Genet. 1997; 34: 705-713Crossref PubMed Scopus (159) Google Scholar). Recently a new recessively inherited nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), 1The abbreviations used are: ANM, Amish nemaline myopathy; Ca50, Ca2+ concentration producing 50% of maximum force; EDL, extensor digitorum longus; F max, maximum calcium-activated force; mAb, monoclonal antibody; MHC, myosin heavy chain; pCa, log of Ca2+ concentration; RATnT, rabbit polyclonal TnT; TnC, troponin C; TnI, troponin I; TnT, troponin T. was identified among the Old Order Amish in Lancaster County, Pennsylvania. ANM is a severe progressive disorder, with affected children dying of respiratory insufficiency resulting from muscle weakness and stiffness, usually in the second or third year of life. No effective treatment is available. Genetic linkage and DNA sequence analyzes have identified a nonsense mutation within exon 11 of the slow skeletal muscle troponin T (TnT) gene (TNNT1) as a potential genetic cause of ANM (2Johnston J.J. Kelley R.I. Crawford T.O. Morton D.H. Agarwala R. Koch T. Schaffer A.A. Francomano C.A. Biesecker L.G. Am. J. Hum. Genet. 2000; 67: 814-821Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). The mutation converted codon Glu180 into a stop codon that is predicted to truncate the slow TnT polypeptide chain with loss of the COOH-terminal 83 amino acids. Vertebrate skeletal muscle contraction is regulated by the troponin complex and tropomyosin, which are associated with actin thin filament in the sarcomere. With depolarization of the muscle cell membrane, Ca2+ released into the cytoplasm binds to troponin C (TnC), inducing a series of allosteric changes in TnC, troponin I (TnI), TnT, and tropomyosin that activate actomyosin ATPase, powering myofilament sliding and shortening of the sarcomere (3Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1342) Google Scholar). The ANM mutant slow TnT lacks the COOH-terminal T2 domain that binds TnC, TnI, and tropomyosin to form the core of the Ca2+-regulatory system (Fig. 1A) (4Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar, 5Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar, 6Lehrer S.S. Geeves M.A. J. Mol. Biol. 1998; 227: 1081-1089Crossref Scopus (169) Google Scholar, 7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (258) Google Scholar). Three homologous genes have evolved in vertebrates to encode isoforms of TnT, i.e. slow skeletal muscle TnT (TNNT1), fast skeletal muscle TnT (TNNT3), and cardiac TnT (TNNT2) (8Huang Q.-Q. Chen A. Jin J.-P. Gene. 1999; 229: 1-10Crossref PubMed Scopus (46) Google Scholar, 9Barton P.J. Cullen M.E. Townsend P.J. Brand N.J. Mullen A.J. Norman D.A. Bhavsar P.K. Yacoub M.H. Genomics. 1999; 57: 102-109Crossref PubMed Scopus (29) Google Scholar, 10Breitbart R.E. Nadal-Ginard B. J. Mol. Biol. 1986; 188: 313-324Crossref PubMed Scopus (110) Google Scholar, 11Jin J.-P. Huang Q.-Q. Yeh H.-I Lin J.J.-C. J. Mol. Biol. 1992; 227: 1269-1276Crossref PubMed Scopus (101) Google Scholar). Each of these is expressed specifically in differentiated adult slow skeletal, fast skeletal, and cardiac muscles, respectively, with a fiber type-based structural conservation (Fig. 1B). From the pre-mRNA transcripts of these muscle fiber type-specific TnT genes, alternative splicing produces additional isoform variations (8Huang Q.-Q. Chen A. Jin J.-P. Gene. 1999; 229: 1-10Crossref PubMed Scopus (46) Google Scholar, 10Breitbart R.E. Nadal-Ginard B. J. Mol. Biol. 1986; 188: 313-324Crossref PubMed Scopus (110) Google Scholar, 11Jin J.-P. Huang Q.-Q. Yeh H.-I Lin J.J.-C. J. Mol. Biol. 1992; 227: 1269-1276Crossref PubMed Scopus (101) Google Scholar, 12Jin J.-P. Chen A. Huang Q.-Q. Gene. 1998; 214: 121-129Crossref PubMed Scopus (65) Google Scholar). The large number of TnT isoforms with complex variations in structure can be classified into acidic and basic isoforms according to their isoelectric points (pI) (Fig. 1C) (13Wang J. Jin J.-P. Gene. 1997; 193: 105-114Crossref PubMed Scopus (70) Google Scholar). TnT isoform expression is developmentally regulated. The cardiac TnT gene is transiently expressed in embryonic skeletal muscle (14Jin J.-P. Biochem. Biophys. Res. Commun. 1996; 225: 883-889Crossref PubMed Scopus (59) Google Scholar), and alternative RNA splicing generates embryonic to adult isoform transitions of cardiac TnT (15Jin J.-P. Wang J. Zhang J. Gene. 1996; 168: 217-221Crossref PubMed Scopus (47) Google Scholar) and fast skeletal muscle TnT (13Wang J. Jin J.-P. Gene. 1997; 193: 105-114Crossref PubMed Scopus (70) Google Scholar, 16Ogut O. Jin J.-P. J. Biol. Chem. 1998; 273: 27858-27866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Normal adult skeletal muscle expresses both slow skeletal muscle TnT and the alternative RNA splicing-generated adult isoforms of fast skeletal muscle TnT (12Jin J.-P. Chen A. Huang Q.-Q. Gene. 1998; 214: 121-129Crossref PubMed Scopus (65) Google Scholar, 13Wang J. Jin J.-P. Gene. 1997; 193: 105-114Crossref PubMed Scopus (70) Google Scholar). Most vertebrate skeletal muscles are made up of a combination of both fast and slow fibers (muscle cells). The finding that the loss of only one isoform of TnT may cause a lethal myopathy establishes the importance of these functionally differentiated fiber type-specific TnT isoforms. The present study investigates the fate of the truncated slow TnT and the functional significance and developmental regulation of TnT isoforms. The results lay the foundation for understanding the molecular pathology and pathophysiology of ANM and for further studies on a targeted therapy of this devastating disease. Muscle Biopsy Samples from ANM Patients—Diagnostic muscle biopsy samples were obtained from the quadriceps muscle of two 7-week-old ANM patients. This investigation was determined to be exempted research under section IV C criteria by the Johns Hopkins Hospital Institutional Review Board. In addition to clinical diagnosis and family history, genetic analysis of both subjects confirmed a homozygous Glu180 nonsense mutation of TNNT1 (2Johnston J.J. Kelley R.I. Crawford T.O. Morton D.H. Agarwala R. Koch T. Schaffer A.A. Francomano C.A. Biesecker L.G. Am. J. Hum. Genet. 2000; 67: 814-821Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). The muscle biopsies were rapidly frozen in liquid nitrogen and stored at -80 °C until use. For SDS-PAGE and Western blot analysis, the muscle tissues were thawed on ice and immediately homogenized in SDS-PAGE sample buffer containing 1% SDS. The high concentration of SDS inactivates protease activity and effectively extracts myofilament proteins from the tissue. Human quadriceps muscle biopsy or autopsy samples from control (non-ANM) subjects were prepared by the same procedure. Specific Anti-TnT Antibodies—A monoclonal antibody (mAb) CT3 that recognizes cardiac and slow skeletal muscle TnT, but not fast skeletal muscle TnT, was described previously (17Jin J.-P. Chen A. Ogut O. Huang Q.-Q. Am. J. Physiol. Cell Physiol. 2000; 279: C1067-C1077Crossref PubMed Google Scholar). Using human cardiac TnT expressed from cloned cDNA and purified from Escherichia coli culture as an immunogen, we developed a new mAb, 2C8, that recognizes cardiac, slow, and fast TnTs almost equally in Western blots (Fig. 2A). Mouse hybridomas were produced by previously described methods (18Wang J. Jin J.-P. Biochemistry. 1998; 37: 14519-14528Crossref PubMed Scopus (75) Google Scholar). Western blot analysis using Tris-Tricine SDS-PAGE (17Jin J.-P. Chen A. Ogut O. Huang Q.-Q. Am. J. Physiol. Cell Physiol. 2000; 279: C1067-C1077Crossref PubMed Google Scholar) located the 2C8 mAb epitope in the NH2-terminal chymotryptic T1 fragment of TnT (19Heeley D.H. Golosinska K. Smillie L.B. J. Biol. Chem. 1987; 262: 9971-9978Abstract Full Text PDF PubMed Google Scholar) (Fig. 2B). A mAb T12 raised against rabbit fast TnT (Ref. 20Lin J.J.-C. Feramisco J.R. Blose S.H. Matsumura F. Kennett R.H. Bechtol K.B. McKearn T.J. Monoclonal Antibodies and Functional Cell Lines. Plenum Publishing Corp., New York1984: 119-151Crossref Google Scholar; a gift from Prof. Jim Lin, University of Iowa) and a rabbit polyclonal anti-TnT serum, RATnT (18Wang J. Jin J.-P. Biochemistry. 1998; 37: 14519-14528Crossref PubMed Scopus (75) Google Scholar), were also used in the present study for Western blot analysis. Although mAb T12 binds weakly to cardiac TnT and slow TnT at high concentrations, we have established a Western blot working concentration at which T12 specifically recognizes only fast skeletal muscle TnT (Fig. 5B). Immunohistochemistry and Stereomicroscopy—Thin frozen sections of muscle biopsy samples were fixed in cold acetone. As described (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates, Brooklyn, NY2001: 14.6.1-14.6.13Google Scholar), cross sections were subjected to immunohistochemical staining using the anti-TnT isoform mAbs CT3 and T12 and an anti-cardiac β-myosin heavy chain (β-MHC; which is the same as MHC I in skeletal muscles; Ref. 22Baldwin K.M. Haddad F. J. Appl. Physiol. 2001; 90: 345-357Crossref PubMed Scopus (13) Google Scholar) monoclonal antibody, FA2 (23Jin J.-P. Malik M.L. Lin J.J.-C. Hybridoma. 1990; 9: 597-608Crossref PubMed Scopus (25) Google Scholar), followed by horseradish peroxidase-labeled anti-mouse immunoglobulin second antibody (Sigma) and an H2O2-diaminobenzidine substrate reaction to examine the expression of slow TnT, fast TnT, and MHC I, respectively. Morphometric assessment of type 1 and type 2 muscle fibers was carried out on sections stained by standard histochemical techniques for myosin ATPase at pH 9.4 (24Dubowitz V. Dubowitz V. Muscle Biopsy, A Practical Approach. 2nd Ed. Bailliere Tindall, London1985: 19-40Google Scholar). Measured muscle fibers were selected by unbiased sampling techniques (25Mayhew T.M. J. Neurocytol. 1992; 21: 313-328Crossref PubMed Scopus (311) Google Scholar) from regions of the muscle biopsy slide predetermined to have good cross-sectional orientation. Because anatomic boundaries are not defined in biopsy and autopsy specimens, measurement of fiber number is expressed as a ratio between fiber types. Examination of Myofilament Protein Isoform Content within Single Muscle Fibers—Single muscle fibers were isolated as described previously (26Brotto M.A. Nosek T.M. J. Appl. Physiol. 1996; 81: 731-737Crossref PubMed Scopus (98) Google Scholar). Each fiber was dissolved in 10 μl of SDS-PAGE sample buffer and analyzed by SDS-PAGE as described above. The resulting gels were processed for silver staining as described (27Ogut O. Hossain M.M. Jin J.-P. J. Biol. Chem. 2003; 278: 3089-3097Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). To identify the expression of several specific myofibril protein isoforms in a single muscle fiber, Western blots of duplicate gels were carried out using a mixture of the anti-slow TnT mAb CT3, an anti-TnI mAb, TnI-1 (28Jin J.-P. Yang F.-W. Yu Z.-B. Ruse C.I. Bond M. Chen A. Biochemistry. 2001; 40: 2623-2631Crossref PubMed Scopus (70) Google Scholar), and the anti-cardiac β-MHC/skeletal MHC I mAb, FA2 (23Jin J.-P. Malik M.L. Lin J.J.-C. Hybridoma. 1990; 9: 597-608Crossref PubMed Scopus (25) Google Scholar), as described above. After recording the expression patterns for slow TnT, slow and fast TnI isoforms, and MHC I, the nitrocellulose membranes were reprobed with T12 mAb to examine the expression of fast TnT isoforms. Contractility Analysis on Single Muscle Fibers—The experimental protocol and calculation of solution compositions were similar to those described previously (26Brotto M.A. Nosek T.M. J. Appl. Physiol. 1996; 81: 731-737Crossref PubMed Scopus (98) Google Scholar). Single fibers were skinned by Triton X-100 in the present of protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm leupeptin, 1.0 mm benzamidine, and 10 μm aprotinin). The sarcomere length of the mounted muscle fiber was adjusted to ∼2.6 μm by monitoring its laser diffraction pattern (26Brotto M.A. Nosek T.M. J. Appl. Physiol. 1996; 81: 731-737Crossref PubMed Scopus (98) Google Scholar). Muscle fibers were permitted to relax in pCa 8.5 and then exposed to solutions of varying Ca2+ concentrations to determine the force versus pCa relationship as described (29Brotto M.P. van Leyen S.A. Brotto L.S. Jin J.-P. Nosek C.M. Nosek T.M. Pflugers Arch. 2001; 442: 738-744Crossref PubMed Scopus (38) Google Scholar). Maximum calcium-activated force (F max) was recorded and normalized to the cross-sectional area of each fiber. The force versus pCa curve was constructed for each fiber by using F max at pCa 4.0 as 100%. Sigmaplot 5.0 and Origin 6.0 computer programs (Jandel Scientific) were used to fit the force versus pCa curve for each fiber to the Hill equation. Each fiber used for the contractility assays was examined by Western blotting as described above for troponin and myosin isoform contents to classify its fiber type. Data Analysis—Densitometric analysis of the SDS-PAGE and Western blots used the NIH Image program, version 1.61, on images scanned at 600 dpi. The TnT molecular weight and pI were calculated from amino acid sequences by using programs from DNAStar. Statistical analysis for the protein quantification was done by Student's t test. Contractility data were analyzed by the SigmaStat (Jandel Corp.) program for statistical significance. One-way analysis of variance (ANOVA) was used to test normally distributed data, and the Wilcoxon sign rank test was applied for non-normally distributed data. Neither Intact nor Truncated Slow TnT Was Found in the Muscle of ANM Patients—The fate of the truncated slow TnT in ANM muscle is important for understanding the molecular pathophysiology of the disease. The predicted truncation after Arg179 deletes most of the COOH-terminal T2 domain of TnT that interacts with TnI, TnC, and tropomyosin, but the central tropomyosin-binding site in the NH2-termial T1 region is retained (Fig. 1A). Residual truncated TnT might interact with tropomyosin through the single binding site, disturbing the troponin-tropomyosin complex in a dominant negative manner. Western blots obtained with mAb 2C8, recognizing both fast and slow TnT (Fig. 2A) and the TnT NH2-terminal T1 fragment (Fig. 2B), showed that multiple fast TnT isoforms were expressed in ANM muscle; but, in comparison to control muscle, no additional low molecular weight TnT bands were detected (Fig. 3A). The absence of truncated slow TnT was confirmed by Western blotting with rabbit polyclonal anti-TnT antibody, RATnT, that recognizes multiple epitopes (Fig. 3A). Fast and Slow TnT Isoforms Are Expressed in a Fiber-specific Manner—Why fast TnT does not compensate for the loss of slow TnT in ANM muscle is unknown. Quantitative densitometry analysis of Western blots of control and the two ANM muscle samples in multiple loadings, using the anti-all TnT mAb 2C8 (normalized by densitometry of the actin band on parallel SDS gels), detected no difference in the stoichiometry of total TnT of ANM relative to control (Fig. 3B). Combined with the selective atrophy but normal number of muscle fibers expressing slow myosin (Fig. 4) and a diminished abundance of slow TnI (28.2 ± 4.3% of total TnI versus 43.2 ± 5.7% in the control muscles, p < 0.001, Fig. 3C), the unchanged TnT to actin ratio suggests that, in ANM, there is selective loss of slow thin filaments. We have shown previously that most skeletal muscles of large mammals contain both slow and fast isoforms of TnT (12Jin J.-P. Chen A. Huang Q.-Q. Gene. 1998; 214: 121-129Crossref PubMed Scopus (65) Google Scholar). Fast TnTs are found in ANM, normal human infant and adult quadriceps muscle (Figs. 3A and 6A), and the predominantly slow fiber adult rat soleus muscle (Fig. 5A). To investigate whether TnT isoform expression is evenly mixed in all fibers of the muscle or, instead, is specific to individual fibers, we examined the expression of TnT isoforms at the single fiber level. Expression of slow isoforms of myosin (MHC I) and troponin subunits is highly fiber type-specific (Fig. 5B). In a typical fast muscle, e.g. the rat extensor digitorum longus, EDL, all fibers express only fast TnT, fast TnI, and no MHC I. In contrast, all of the rat soleus fibers examined express MHC I. 50% of the soleus fibers studied express slow TnT, 26.5% express fast TnT, and only 23.5% express a mixture of slow and fast isoforms of TnT. Slow and fast TnI isoforms that are distinguished by mobility in SDS-PAGE are co-expressed with slow and fast TnT, respectively. The results demonstrate that regulation of troponin isoforms is specific to the type of individual muscle fiber. These data suggest that fast TnT in slow muscles is unable to compensate for the loss of slow TnT, because it is only expressed in a small fraction of the fibers. The unchanged ratio of total TnT to actin in ANM muscle further supports the hypothesis that slow thin filaments are lost selectively. Slow TnT Confers Higher Ca2 + Sensitivity and Lower Cooperativity of the Muscle Fiber—To investigate the relationship between TnT isoform content and muscle fiber contractility, we measured the Ca2+-activated development of force in Triton X-100-skinned rat single muscle fibers. Fibers were sorted according to myosin and TnT isoform content into one of three groups (Fig. 5) as follows: (a) EDL fibers containing only fast myosin (MHC I-negative) and fast TnT; (b) soleus fibers (MHC I-positive) containing slow TnT; and (c) soleus fibers (MHC I-positive) containing fast TnT. Muscle fibers expressing slow or fast TnT differ in calcium sensitivity without respect to myosin type (Fig. 5C). Slow TnT-containing fibers produce 50% F max (Ca50) at a lower Ca2+ concentration, reflecting higher Ca2+ sensitivity. In contrast, fibers expressing fast TnT show a higher cooperativity during the Ca2+ activation of contraction. Fibers with fast TnT but differing in myosin type are indistinguishable with respect to Ca50 and cooperativity, indicating a determining role of the thin filament. Previous experiments in chicken skeletal muscle demonstrate that alterations of Ca2+ sensitivity correlate with the TnT isoform but not with the TnI or the TnC isoform (30Ogut O. Granzier H. Jin J.-P. Am. J. Physiol. Cell Physiol. 1999; 276: C1162-C1170Crossref PubMed Google Scholar). Furthermore, transgenic expression of fast skeletal muscle TnT in mouse cardiac muscle increases cooperativity of the Ca2+-activated contraction (31Huang Q.-Q. Brozovich F.V. Jin J.-P. J. Physiol. (Lond.). 1999; 520: 231-242Crossref Scopus (40) Google Scholar). Therefore, the TnT isoform appears to be a major determinant of the role of slow and fast troponins in the modulation of Ca2+ sensitivity and cooperativity of muscle contraction. Although troponin isoforms determine Ca2+ responsiveness, myosin isoform expression determines the F max (Fig. 5C). Expression of the slow muscle-specific myosin heavy chain, MHC I, correlates with the lower F max without respect to the TnT isoform. These results are consistent with the fact that slow myosin has a lower ATPase activity than that of the fast myosin isoenzyme (22Baldwin K.M. Haddad F. J. Appl. Physiol. 2001; 90: 345-357Crossref PubMed Scopus (13) Google Scholar). Developmental Switching of TnT Isoform Expression in Human Skeletal Muscles—Protein extracts from normal human quadriceps muscle at 16 weeks of gestation, term, 6 months, and adult were evaluated by SDS-PAGE and Western blots using the anti-cardiac/slow TnT mAb CT3 and the anti-fast TnT mAb T12. As observed in other vertebrates (14Jin J.-P. Biochem. Biophys. Res. Commun. 1996; 225: 883-889Crossref PubMed Scopus (59) Google Scholar), cardiac TnT is expressed in fetal skeletal muscle with minimal expression by term. In comparison to adult muscle, fetal skeletal muscle expresses embryonic isoforms of fast TnT with higher molecular weight than the adult isoforms, which in agreement with previous observations in mouse (13Wang J. Jin J.-P. Gene. 1997; 193: 105-114Crossref PubMed Scopus (70) Google Scholar). Slow TnT is also developmentally regulated in normal human muscle, increasing in abundance with maturation (Fig. 6A). Slow TnT and Myopathy—A number of codon deletion, splice site, and missense dominant mutations of TNNT2 (cardiac TnT) have been found in hypertrophic cardiomyopathy (32Knollmann B.C. Potter J.D. Trends Cardiovasc. Med. 2001; 11: 206-212Crossref PubMed Scopus (54) Google Scholar). The TNNT1 ANM mutation represents the first recessive TnT mutation found in human diseases. Truncation and null recessive mutations in the Caenorhabditis elegans TnT gene (mup-2) produce abnormal body wall muscle twitching and hypercontraction (33Myers C.D. Goh P.Y. Allen T.S. Bucher E.A. Bogaert T. J. Cell Biol. 1996; 132: 1061-1077Crossref PubMed Scopus (86) Google Scholar). This loss of slow TnT produces a characteristic phenotype in ANM and provides novel evidence for the critical function of TnT in the regulation of muscle contraction and the importance of the muscle fiber type-specific TnT isoforms. To date, mutations in five genes, nebulin (NEB) (34Pelin K. Hilpelä P. Donner K. Sewry C. Akkari P.A. Wilton S.D. Wattanasirichaigoon D. Bang M.L. Centner T. Hanefeld F. Odent S. Fardeau M. Urtizberea J.A. Muntoni F. Dubowitz V. Beggs A.H. Laing N.G. Labeit S. de la Chapelle A. Wallgren-Pettersson C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2305-2310Crossref PubMed Scopus (283) Google Scholar), α-tropomyosin (TPM3) (35Laing N.G. Wilton S.D. Akkari P.A. Dorosz S. Boundy K. Kneebone C. Blumbergs P. White S. Watkins H. Love D.R. Haan E. Nat. Genet. 1995; 9: 75-79Crossref PubMed Scopus (280) Google Scholar), β-tropomyosin (TPM2) (36Donner K. Ollikainen M. Ridanpaa M. Christen H.J. Goebel H.H. de Visser M. Pelin K. Wallgren-Pettersson C. Neuromuscul. Disord. 2002; 12: 151-158Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), α-actin (ACTA1) (37Nowak K.J. Wattanasirichaigoon D. Goebel H.H. Wilce M. Pelin K. Donner K. Jacob R.L. Hubner C. Oexle K. Anderson J.R. Verity C.M. North K.N. Iannaccone S.T. Muller C.R. Nurnberg P. Muntoni F. Sewry C. Hughes I. Sutphen R. Lacson A.G. Swoboda K.J. Vigneron J. Wallgren-Pettersson C. Beggs A.H. Laing N.G. Nat. Genet. 1999; 23: 208-212Crossref PubMed Scopus (342) Google Scholar), and slow skeletal muscle TnT (TNNT1) (2Johnston J.J. Kelley R.I. Crawford T.O. Morton D.H. Agarwala R. Koch T. Schaffer A.A. Francomano C.A. Biesecker L.G. Am. J. Hum. Genet. 2000; 67: 814-821Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), have been found in different forms of hereditary nemaline myopathy. All five nemaline myopathy-related genes encode thin filament-associated proteins that relate to the Z disk of the sarcomere, from which nemaline bodies appear to be derived. Thus, the genetic forms of nemaline myopathy likely represent a class of sarcomeric thin filament diseases. Complete Loss of Slow TnT as the Molecular Basis of ANM—The absence of detectable truncated slow TnT is consistent with the observed recessive inheritance of the disease. This result provides the first direct evidence that the loss of slow skeletal muscle TnT is the molecular cause of ANM and establishes an important foundation for understanding that the molecular pathology of the disease is caused by loss of the TnT protein rather than by a dominant negative effect caused by a TnT NH2-terminal fragment. This finding shifts the focus of pathophysiologic inquiry to the functional role of slow TnT in slow muscle fibers. The significant atrophy of slow fibers in ANM muscle indicates that the lack of slow TnT results in either decreased formation or decreased stability of myofibrils. Amish nemaline myopathy thus highlights a critical role for fiber type-specific TnT isoforms in skeletal muscle function. The slow TnT defect-based loss of myofibrils in ANM muscle indicates that TnT is not only required for the Ca2+ regulation of contraction but is also critical for muscle development and growth. Although the slow TnT-(1–179) fragment retains one tropomyosin-binding site, deletion of the COOH-terminal T2 region should abolish the binding to TnI and TnC (Fig. 1A). The complete loss of slow TnT in ANM muscle indicates a critical role of the COOH-terminal T2 domain in the integration of TnT into myofibrils. The results suggest that the two sites binding to tropomyosin (19Heeley D.H. Golosinska K. Smillie L.B. J. Biol. Chem. 1987; 262: 9971-9978Abstract Full Text PDF PubMed Google Scholar) and/or the formation of troponin complex is essential for incorporation of TnT into the muscle thin filament. The mechanism for the absence of the truncated slow TnT-(1–179) protein fragment remains to be investigated. It may result from either accelerated nonsense-mediated decay of the mutant mRNA (38Maquat L.E. RNA. 1995; 1: 453-465PubMed Google Scholar) or decreased stability of the protein fragment. The clear recessive inheritance of ANM (2Johnston J.J. Kelley R.I. Crawford T.O. Morton D.H. Agarwala R. Koch T. Schaffer A.A. Francomano C.A. Biesecker L.G. Am. J. Hum. Genet. 2000; 67: 814-821Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) suggests that truncated TnT is not incorporated into the troponin-tropomyosin complex and, therefore, is not accumulated. Otherwise, truncated TnT expressed in the muscle of ANM heterozygotes would likely result in a phenotype. Precedent for this phenomenon is provided by the dominantly inherited cardiomyopathy caused by a truncated cardiac TnT due to a splice-site mutation in intron 16 of TNNT2 (39Watkins H. Seidman C.E. Seidman J.G. Feng H.S. Sweeney H.L. J. Clin. Invest. 1996; 98: 2456-2461Crossref PubMed Scopus (112) Google Scholar). Troponin Isoforms as Novel Markers for Skeletal Muscle Fiber Classification—TnT isoform expression in post-natal muscle is specific to the muscle fiber type and influences contractile properties of the fiber. Myosin isoforms have been widely used in the typing of skeletal muscle fibers (22Baldwin K.M. Haddad F. J. Appl. Physiol. 2001; 90: 345-357Crossref PubMed Scopus (13) Google Scholar). The relationship between myosin isoform and muscle fiber type is complex, i.e. MHC I and MHC IIa are associated with slow fibers, whereas MHC IIb and IIx are specific to fast fibers at various relative amounts. In contrast, most muscle fibers express only one isoform of TnT and TnI. The well characterized fast fiber EDL muscle demonstrated exclusive expression of fast TnT and fast TnI and no MHC I. Although both slow and fast TnT are detected in the homogenate of whole soleus muscle (Fig. 5A), most soleus fibers express either slow TnT and TnI or fast TnT and TnI (Fig. 5). The matched expression of TnT and TnI isoforms in fast and slow muscle fibers is in agreement with their closely related function and co-evolutionary relationship (40Huang Q.-Q. Jin J.-P. J. Mol. Evol. 1999; 49: 780-788Crossref PubMed Scopus (26) Google Scholar). Thus, the troponin isoform provides a novel and, possibly, a more specific marker for the functional classification of skeletal muscle fiber type. Functional Difference between Slow and Fast TnT Isoforms— Slow fibers are important in the sustained contraction of muscle (41Fitts R.H. Riley D.R. Widrick J.J. J. Exp. Biol. 2001; 204: 3201-3208PubMed Google Scholar). The presence of fast TnT in a limited number of MHC I-positive fibers (Fig. 4B) does not compensate for the absence of slow TnT in ANM. Therefore, the Ca2+ regulatory functions of the slow thin filament rather than the distinctive contractile force determined by myosin type determines the function of slow fibers that is critical to the molecular pathology of ANM. The primary structure of slow TnT is better conserved across species than those of fast and cardiac TnTs (12Jin J.-P. Chen A. Huang Q.-Q. Gene. 1998; 214: 121-129Crossref PubMed Scopus (65) Google Scholar). Slow TnT may thus play a more fundamental role in vertebrate muscle function. Our finding that slow TnT confers a higher sensitivity but lower cooperativity to Ca2+ activation compared with fast TnT (Fig. 5C) suggests that thin filament responsiveness to Ca2+ is a major factor determining the function of fast and slow fibers. The hypothesis that differential Ca2+ sensitivity and cooperativity of slow versus fast fibers has a critical role in the normal function of skeletal muscle deserves further investigation. Significance of the Developmental Regulation of TnT Isoforms—Newborn babies with ANM have normal muscle power but quickly develop tremors, followed by progressive weakness with muscle rigidity or contracture (2Johnston J.J. Kelley R.I. Crawford T.O. Morton D.H. Agarwala R. Koch T. Schaffer A.A. Francomano C.A. Biesecker L.G. Am. J. Hum. Genet. 2000; 67: 814-821Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). This postnatal onset and infantile progression of the ANM phenotype corresponds to the time course of developmental down-regulation of cardiac TnT and the alternative splicing-generated embryonic isoforms of fast TnT in skeletal muscle (Fig. 6). Slow, fast, and cardiac TnTs are conserved in their COOH-terminal and central regions, reflecting a conserved core function among the three muscle type-specific TnTs. The highly variable NH2-terminal region is responsible for the distinct overall charge of TnT isoforms. Cardiac and embryonic fast, and slow TnTs are all acidic isoforms, whereas only the adult fast TnT is basic (Fig. 1C). Charge characteristics are likely a major functional determinant of TnT isoforms (13Wang J. Jin J.-P. Gene. 1997; 193: 105-114Crossref PubMed Scopus (70) Google Scholar, 16Ogut O. Jin J.-P. J. Biol. Chem. 1998; 273: 27858-27866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The normal developmental coupling of decreased expression of cardiac and embryonic fast TnT to increased expression of slow TnT suggests that these acidic isoforms complement one another in slow muscle fibers. The cardiac TnT and embryonic fast TnT expressed in fetal skeletal muscles may compensate sufficiently for the loss of slow TnT to produce the normal muscle function of ANM neonates (Fig. 6B). Their postnatal down-regulation removes this compensation and corresponds to the progression of myopathy phenotype. This observation suggests a potential specific therapy for ANM directed toward increasing the slow fiber expression of these embryonic TnT isoforms. We thank Dr. Jim J.-C. Lin for providing the T12 mAb.
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