Truncation by Glu180 nonsense mutation results in complete loss of slow skeletal muscle troponin T in a lethal nemaline myopathy.

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 (1). Recently a new recessively inherited nemaline myopathy, named "Amish Nemaline Myopathy" (ANM), 1 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 (2). The mutation converted codon Glu 180 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, Ca 2ϩ 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 (3). The ANM mutant slow TnT lacks the COOH-terminal T2 domain that binds TnC, TnI, and tropomyosin to form the core of the Ca 2ϩ -regulatory system ( Fig. 1A) (4 -7).
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) (8 -11). 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 (8, 10 -12). 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) (13). TnT isoform expression is developmentally regulated. The cardiac TnT gene is transiently expressed in embryonic skeletal muscle (14), and alternative RNA splicing generates embryonic to adult isoform transitions of cardiac TnT (15) and fast skeletal muscle TnT (13,16). Normal adult skeletal muscle expresses both slow skeletal muscle TnT and the alternative RNA splicing-generated adult isoforms of fast skeletal muscle TnT (12,13). 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.

MATERIALS AND METHODS
Muscle Biopsy Samples from ANM Patients-Diagnostic muscle biopsy samples were obtained from the quadriceps muscle of two 7-weekold 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 Glu 180 nonsense mutation of TNNT1 (2). The muscle biopsies were rapidly frozen in liquid nitrogen and stored at Ϫ80°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 (17).
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 (18). Western blot analysis using Tris-Tricine SDS-PAGE (17) located the 2C8 mAb epitope in the NH 2 -terminal chymotryptic T1 fragment of TnT (19) (Fig. 2B).
A mAb T12 raised against rabbit fast TnT (Ref. 20; a gift from Prof. Jim Lin, University of Iowa) and a rabbit polyclonal anti-TnT serum, RATnT (18), 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 (21), cross sections were subjected to immunohistochemical staining using the anti-TnT isoform mAbs CT3 and T12 and an anti-cardiac ␤-myosin heavy chain (␤-MHC; which is the same as MHC I in skeletal muscles; Ref. 22) monoclonal antibody, FA2 (23), followed by horseradish peroxidase-labeled anti-mouse immunoglobulin second antibody (Sigma) and an H 2 O 2 -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 (24). Measured muscle fibers were selected by unbiased sampling techniques (25) 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 (26). Each fiber was dissolved in 10 l of SDS-PAGE sample buffer and analyzed by SDS-PAGE as described above. The resulting gels were processed for silver staining as described (27). 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 (28), and the anti-cardiac ␤-MHC/skeletal MHC I mAb, FA2 (23), 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 (26). 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 M aprotinin). The sarcomere length of the mounted muscle fiber was adjusted to ϳ2.6 m by monitoring its laser diffraction pattern (26). Muscle fibers were permitted to relax in pCa 8.5 and then exposed to solutions of varying Ca 2ϩ concentrations to determine the force versus pCa relationship as described (29). 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

FIG. 1. Troponin T isoforms.
A, TnT occupies a central position in the thin filament Ca 2ϩ regulatory system of striated muscle. Location of the functionally characterized chymotrypsin fragments T1 and T2 of TnT and the location of the Glu 180 nonsense mutation found in ANM are indicated. The predicted truncation deletes most of the COOH-terminal T2 domain of TnT that interacts with TnI, TnC, and tropomyosin. B, phylogenetic analysis of TnT isoforms. Amino acid sequences of human, mouse, and chicken cardiac, fast, and slow TnT isoforms were compared by alignment using the MegAlign program (DNAStar) to produce a phylogenetic tree. The result demonstrates that each of the fiber typespecific TnTs is better conserved across species than the three TnT isoforms of one species. C, the pIs of statistically large numbers of cloned mouse TnT isoforms were calculated from the amino acid sequences (12,13,15). The data show that slow, cardiac, and embryonic fast TnTs are all acidic isoforms, whereas the adult fast TnT is basic.

FIG. 2. Anti-TnT mAbs.
A, total protein extracts from representative mammalian and avian cardiac and skeletal muscles were analyzed by Western blotting. The mAb CT3 recognizes cardiac TnT (cTnT) in the heart and slow skeletal muscle TnT (sTnT) in the leg muscles containing mixed fast and slow fibers. The clear size difference between cardiac TnT and slow TnT provides a convenient identification of the two TnT isoforms on Western blots. In contrast, mAb 2C8 recognizes cardiac, slow, and fast skeletal muscle TnT (fTnT) almost equally. B, mAb 2C8 recognizes the NH 2 -terminal T1 fragment of chicken skeletal muscle TnT generated by limited chymotryptic digestion (lane 2) (amino acids 1-178, equivalent to amino acids 1-158 in rabbit fast TnT; Ref. 19). Intact bovine cardiac TnT was included as control (lane 1). Several nonspecific degradation fragments of bovine cardiac TnT were also seen in the 2C8 blots.
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 Arg 179 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 NH 2 -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 NH 2 -terminal T1 fragment (Fig. 2B), showed that multiple fast TnT isoforms were ex-pressed 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 Ϯ 4.3% of total TnI versus 43.2 Ϯ 5.7% in the control muscles, p Ͻ 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 (12). 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. and the anti-all TnT mAb 2C8 and polyclonal antibody RATnT demonstrate that there was no intact slow TnT (sTnT) in the muscles of two ANM patients, in contrast to the control infant muscle. The CT3 blot was re-stained by RATnT without stripping (the CT3 ϩ RATnT blot). No specific truncated slow TnT fragment was detected using the anti-NH 2 -terminal mAb 2C8 and the polyclonal RATnT (arrows point to the region to which the truncated slow TnT-(1-179) would be predicted to migrate). Expression of cardiac TnT (cTnT) in skeletal muscle was down-regulated and only barely detectable in these 7-week-old ANM infants as their myopathy phenotype became evident. Cardiac TnT had ceased expression in the 6-month-old control human skeletal muscle. There was no apparent loss of high molecular weight proteins such as the myosin heavy chain band at 200 kDa, indicating no significant protein degradation in the muscle biopsy samples. Fast TnT (fTnT) isoforms were expressed at significant amounts in both ANM and control muscles. B, normalized to the actin bands on the SDS-gel, densitometric measures of total TnT on 2C8 mAb Western blots in the ANM and control are similar (p Ͼ 0.4). C, Western blotting with mAb TnI-1 that recognizes all TnI isoforms detected both fast and slow TnI in the control and the ANM muscle. However, the ratio of slow to fast TnI was lower in the ANM muscle, also indicating dominant fast thin filament content. The histogram shows densitometry data from multiple Western blots of two ANM and two control muscle samples, demonstrating significant decrease of slow TnI in the patient muscles (p Ͻ 0.001).

Deficiency of Slow Troponin T in Nemaline Myopathy
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 Ca 2ϩ Sensitivity and Lower Cooperativity of the Muscle Fiber-To investigate the relationship between TnT isoform content and muscle fiber contractility, we measured the Ca 2ϩ -activated development of force in Triton X-100-skinned rat single muscle fibers. Fibers were sorted ac-cording 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 (Ca 50 ) at a lower Ca 2ϩ concentration, reflecting higher Ca 2ϩ sensitivity. In contrast, fibers expressing fast TnT show a higher cooperativity during the Ca 2ϩ activation of contraction. Fibers with fast TnT but differing in myosin type are indistinguishable with respect to Ca 50 and cooperativity, indicating a determining role of the thin filament. Previous experiments in chicken skeletal muscle demonstrate that alterations of Ca 2ϩ sensitivity correlate with the TnT isoform but not with the TnI or the TnC isoform (30). Furthermore, transgenic expression of fast skeletal muscle TnT in mouse cardiac muscle increases cooperativity of the Ca 2ϩ -activated contraction (31). Therefore, the TnT isoform appears to be a major determinant of the role of slow and fast troponins in the modulation of Ca 2ϩ sensitivity and cooperativity of muscle contraction.  5. Fiber type-specific expression of TnT isoforms determines contractility. A, comparable amounts of total protein extracts of the predominantly slow soleus and fast EDL muscles of rat were examined by Western blotting. mAb CT3 detected slow TnT (sTnT) expression in soleus but not EDL. The mAb T12 blot detected multiple fast TnT (fTnT) isoforms in both soleus and EDL muscles. B, examples of isolated single fibers of rat soleus and EDL muscles examined for myofilament protein isoform contents. The silver stained SDS-PAGE gel and Western blots using three mAbs against MHC I (FA2), slow TnT (CT3), and TnI (TnI-1) in a mixture or the anti-fast TnT mAb T12 demonstrate matched expression of the slow or fast isoform of TnT (sTnT and fTnt, respectively) and TnI (sTnI and fTnI, respectively) specific to individual muscle fibers. C, Ca 2ϩ -activated contraction was measured on skinned single fibers of rat muscle. The force versus pCa relationship demonstrates a difference in calcium sensitivity between rat EDL and soleus muscle fibers containing the slow or fast isoform of TnT. The inset table summarizes the fact that higher Ca 2ϩ sensitivity but lower cooperativity was found in soleus fibers containing slow TnT compared with EDL and soleus fibers containing fast TnT, whereas myosin isoforms determine the level of absolute maximal force produced. The data are mean Ϯ S.E. F max is the maximum force in kilopascals. Ca 50 is the [Ca 2ϩ ] required to induce 50% of F max in M. *, significantly different (p Ͻ 0.01) from the EDL fast and soleus fast groups. **, significantly different from the soleus groups (p Ͻ 0.01).
Although troponin isoforms determine Ca 2ϩ 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 (22).
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 (14), 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 (13). Slow TnT is also developmentally regulated in normal human muscle, increasing in abundance with maturation (Fig. 6A).

DISCUSSION
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 (32). 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 (33). 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.
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 NH 2 -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 typespecific 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 Ca 2ϩ 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 (19) 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 (38) or decreased stability of the protein fragment. The clear recessive inheritance of ANM (2) 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 (39).
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 FIG. 6. Developmentally regulated expression of TnT isoforms and progression of the ANM phenotype. A, Western blots were carried out on normal 16-week fetus, term newborn, 6-month postnatal, and adult human quadriceps muscle samples by using the anticardiac/slow TnT mAb CT3 and the anti-fast TnT mAb T12. Expression of slow TnT increases with maturation. Both embryonic and adult isoforms of cardiac TnT (the higher and lower molecular weight bands, respectively) are expressed in the embryonic, but not adult, skeletal muscle. Multiple fast TnT isoforms are detected in both embryonic and adult human skeletal muscles, showing a developmental high to low molecular weight switching similar to that observed in mouse muscle (13). B, summary of the developmental regulation of the TnT isoform expression and ANM phenotypes. The time courses show that, although cardiac TnT and embryonic fast skeletal muscle TnT are normally expressed in the fetal skeletal muscle, they are replaced by slow skeletal muscle TnT and adult fast TnT up-regulated in normal adult skeletal muscle. In the absence of slow TnT due to the Glu 180 nonsense mutation, the developmental down-regulation of embryonic fast TnT and cardiac TnT is concurrent with the onset and progression of the ANM phenotype.
in the typing of skeletal muscle fibers (22). 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 (40). 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 (41). 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 Ca 2ϩ 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 (12). 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 Ca 2ϩ activation compared with fast TnT (Fig. 5C) suggests that thin filament responsiveness to Ca 2ϩ is a major factor determining the function of fast and slow fibers. The hypothesis that differential Ca 2ϩ 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 (2). 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 NH 2 -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 (13,16). 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.