From the
Department of Physiology and Biophysics,
Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, the
¶Clinic for Special Children, Strasburg,
Pennsylvania 17579, and the ||Departments of
Neurology and Pediatrics, Johns Hopkins University, Baltimore, Maryland
21287
Received for publication, April 3, 2003 , and in revised form, April 29, 2003.
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ABSTRACT |
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INTRODUCTION |
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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 (3). 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) (47).
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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) (811). 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, 1012). 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.
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MATERIALS AND METHODS |
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Specific Anti-TnT AntibodiesA 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 NH2-terminal chymotryptic T1 fragment of TnT (19) (Fig. 2B).
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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).
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Immunohistochemistry and StereomicroscopyThin 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
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
(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
FibersSingle muscle fibers were isolated as described previously
(26). 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
(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 FibersThe
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 µM aprotinin). The sarcomere
length of the mounted muscle fiber was adjusted to 2.6 µm by
monitoring its laser diffraction pattern
(26). 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
(29). Maximum
calcium-activated force (Fmax) was recorded and normalized
to the cross-sectional area of each fiber. The force versus pCa curve
was constructed for each fiber by using Fmax 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 AnalysisDensitometric 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.
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RESULTS |
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Fast and Slow TnT Isoforms Are Expressed in a Fiber-specific MannerWhy 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.
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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. 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.
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Slow TnT Confers Higher Ca2+ Sensitivity and Lower Cooperativity of the Muscle FiberTo 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% Fmax (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 (30). Furthermore, transgenic expression of fast skeletal muscle TnT in mouse cardiac muscle increases cooperativity of the Ca2+-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 Ca2+ sensitivity and cooperativity of muscle contraction.
Although troponin isoforms determine Ca2+ responsiveness, myosin isoform expression determines the Fmax (Fig. 5C). Expression of the slow muscle-specific myosin heavy chain, MHC I, correlates with the lower Fmax 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 MusclesProtein 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).
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DISCUSSION |
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To date, mutations in five genes, nebulin (NEB)
(34), -tropomyosin
(TPM3) (35),
-tropomyosin (TPM2)
(36),
-actin
(ACTA1) (37), and
slow skeletal muscle TnT (TNNT1)
(2), 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 ANMThe 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-(1179) 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-(1179) 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 ClassificationTnT 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 (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 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 (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 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 IsoformsNewborn 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 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 (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.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 216-368-5525; Fax:
216-368-3952; E-mail:
jxj12{at}po.cwru.edu.
1 The abbreviations used are: ANM, Amish nemaline myopathy; Ca50,
Ca2+ concentration producing 50% of maximum force; EDL, extensor
digitorum longus; Fmax, 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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