Factors determining the subunit composition of tropomyosin in mammalian skeletal muscle.

Adult rat fast-twitch skeletal muscle such as extensor digitorum longus contains alpha- and beta-tropomyosin subunits, as is the case in the corresponding muscles of rabbit. Adult rat soleus muscle contains beta-, gamma- and delta-tropomyosins, but no significant amounts of alpha-tropomyosin. Evidence for the presence of phosphorylated forms of at least three of the four tropomyosin subunit isoforms was obtained, particularly in developing muscle. Immediately after birth alpha- and beta-tropomyosins were the major components of skeletal muscle, in both fast-twitch and slow-twitch muscles. Differentiation into slow-twitch skeletal muscles was accompanied by a fall in the amount of alpha-tropomyosin subunit and its replacement with gamma- and delta-subunits. After denervation and during regeneration after injury, the tropomyosin composition of slow-twitch skeletal muscle changed to that associated with fast-twitch muscle. Thyroidectomy slowed down the changes in tropomyosin composition resulting from the denervation of soleus muscle. The results suggest that the 'ground state' of tropomyosin-gene expression in the skeletal muscle gives rise to alpha- and beta-tropomyosin subunits. Innervation by a 'slow-twitch' nerve is essential for the expression of the genes controlling gamma- and delta-subunits. There appears to be reciprocal relationship between expression of the gene controlling the synthesis of alpha-tropomyosin and those controlling the synthesis of gamma- and delta-tropomyosin subunits.     

Fast and slow isoforms of troponin I and troponin C. Distribution in normal rabbit muscles and effects of chronic stimulation

Polyclonal antibodies were raised against troponin I (TnI) and troponin C (TnC) purified from fast-twitch and slow-twitch rabbit muscles. These antibodies were used to elucidate the distribution of fast and slow isoforms of TnI and TnC in normal and chronically stimulated rabbit hind limb muscles by immunoblots of one-dimensional and two-dimensional electrophoreses. In contrast to the multiplicity of fast and slow troponin T (TnT) isoforms, TnI and TnC were present as unique fast and slow isoforms. Whereas no charge variants were detected for slow TnI, fast TnI was present in at least three charge variants. As judged from the results of alkaline phosphatase digestion, these charge variants represent differently phosphorylated forms. Fast and slow TnC both exist as two charge variants which, however, were unaffected by alkaline phosphatase treatment. Chronic low-frequency stimulation of fast-twitch muscles induced progressive increases in the slow isoforms of TnC and TnI at the expense of their fast isoforms. The extent of the fast-to-slow transition was more pronounced in the case of TnC than in that of TnI. Long-term stimulated muscles with a complete fast-to-slow transition, at the level of the TnT isoforms, still contained fast and slow isoforms of both TnI and TnC. The coexistence of fast and slow isoforms of the three troponin subunits in the transforming muscle was interpreted as indicating the presence of hybrid troponin molecules composed of fast and slow isoforms. Studies at the mRNA level showed changes similar to those at the protein level. However, in long-term stimulated muscles, the fast-to-slow transition of TnI was more pronounced at the mRNA level than at the protein level.     

Transitions from fetal to fast troponin T isoforms are coordinated with changes in tropomyosin and alpha-actinin isoforms in developing rabbit skeletal muscle

In adult fast skeletal muscle, specific combinations of thin filament and Z-line protein isoforms are coexpressed. To determine whether the expression of these sets of proteins, designated the TnT1f, TnT2f, and TnT3f programs, is coordinated during development, we characterized the transitions in troponin T (TnT), tropomyosin (Tm), and alpha-actinin isoforms that occur in developing fetal and neonatal rabbit skeletal muscle. Two coordinated developmental transitions were identified, and a novel pattern of thin filament expression was found in fetal muscle. In fetal muscle, new TnT species--whose protein and immunochemical properties suggest that they are the products of a new TnT gene--are expressed in combination with beta 2 Tm and alpha-actinin1f/s. This pattern, which is found in both back and hindlimb muscles, is specific to fetal and early neonatal muscle. Just prior to birth, there is a transition from the fetal program to the isoforms that define the TnT3f program, TnT3f, and alpha beta Tm. Like the fetal program, expression of the TnT3f program appears to be a general feature of muscle development, because it occurs in a variety of fast muscles as well as in the slow muscle soleus. The transition to adult patterns of thin filament expression begins at the end of the first postnatal week. Based on studies of erector spinae, the isoforms comprising the TnT2f program, TnT2f, alpha 2 Tm, and alpha-actinin2f, appear and increase coordinately at this time. The transitions, first to the TnT3f program, and then to adult patterns of expression indicate that synthesis of the isoforms comprising each program is coordinated during muscle specialization and throughout muscle development. In addition, these observations point to a dual role for the TnT3f program, which is the major thin filament program in some adult muscles, but appears to bridge the transition from developmentally to physiologically regulated patterns of thin filament expression during the late fetal and early neonatal development.     

Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.     

Heterogeneity of contractile proteins. A continuum of troponin-tropomyosin expression in mammalian skeletal muscle

Comparison of the myofibrillar proteins from several adult rabbit skeletal muscles has led to the identification of multiple forms of fast and slow troponin T. In Briggs et al. (Briggs, M. M., Klevit, R., and Schachat, F. H. (1984) J. Biol. Chem. 259, 10369-10375) two species of rabbit fast skeletal muscle troponin T (TnT), TnT1f and TnT2f, were characterized. Here, the distribution of these fast TnT species and the alpha- and beta- tropomyosin (Tm) subunits is characterized in fast muscles and in single muscle fibers. Evidence is also presented for two forms of slow skeletal muscle TnT. The presence of each fast TnT species is associated with the presence of a different Tm dimer: TnT1f with alpha beta-Tm and TnT2f with alpha 2-Tm. Histochemical analysis shows that expression of the fast TnT-Tm combinations is not due to differences in the distribution of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fiber types. The absence of a correlation between histochemical typing and the composition of the thin filament Ca2+-regulatory complex is more apparent in individual fast muscle fibers where both fast TnT-Tm combinations appear to be expressed in a continuum. The implications of these observations for mammalian skeletal muscle fiber diversity are discussed.     

Transitions from fetal to fast troponin T isoforms are coordinated with changes in tropomyosin and α-actinin isoforms in developing rabbit skeletal muscle

In adult fast skeletal muscle, specific combinations of thin filament and Z-line protein isoforms are coexpressed. To determine whether the expression of these sets of proteins, designated the TnT1f, TnT2f, and TnT3f programs, is coordinated during development, we characterized the transitions in troponin T (TnT), tropomyosin (Tm), and α-actinin isoforms that occur in developing fetal and neonatal rabbit skeletal muscle. Two coordinated developmental transitions were identified, and a novel pattern of thin filament expression was found in fetal muscle. In fetal muscle, new TnT species—whose protein and immunochemical properties suggest that they are the products of a new TnT gene—are expressed in combination with β₂ Tm and α-actinin_1f/8. This pattern, which is found in both back and hindlimb muscles, is specific to fetal and early neonatal muscle. Just prior to birth, there is a transition from the fetal program to the isoforms that define the TnT3f program, TnT_3f, and αβ Tm. Like the fetal program, expression of the TnT3f program appears to be a general feature of muscle development, because it occurs in a variety of fast muscles as well as in the slow muscle soleus. The transition to adult patterns of thin filament expression begins at the end of the first postnatal week. Based on studies of erector spinae, the isoforms comprising the TnT2f program, TnT_2f, α₂ Tm, and α-actinin_2f, appear and increase coordinately at this time. The transitions, first to the TnT3f program, and then to adult patterns of expression indicate that synthesis of the isoforms comprising each program is coordinated during muscle specialization and throughout muscle development. In addition, these observations point to a dual role for the TnT3f program, which is the major thin filament program in some adult muscles, but appears to bridge the transition from developmentally to physiologically regulated patterns of thin filament expression during late fetal and early neonatal development.     

Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscle

1. On electrophoresis in dissociating conditions the tropomyosins isolated from skeletal muscles of mammalian, avian and amphibian species migrated as two components. These were comparable with the alpha and beta subunits of tropomyosin present in rabbit skeletal muscle. 2. The alpha and beta components of all skeletal-muscle tropomyosins contained 1 and 2 residues of cysteine per 34000g respectively. 3. The ratio of the amounts of alpha and beta subunit present in skeletal muscle tropomyosins was characteristic for the muscle type. Muscle consisting of slow red fibres contained a greater proportion of beta-tropomyosin than muscles consisting predominantly of white fast fibres. 4. Mammalian and avian cardiac muscle tropomyosins consisted of alpha-tropomyosin only. 5. Mammalian and avian smooth-muscle tropomyosins differed both chemically and immunologically from striated-muscle tropomyosins. 6. Antibody raised against rabbit skeletal alpha-tropomyosin was species non-specific, reacting with all other striated muscle alpha-tropomyosin subunits tested. 7. Antibody raised against rabbit skeletal beta-tropomyosin subunit was species-specific.     

Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle

This study focuses on the effects ofmechanical unloading of rat soleus muscle on the isoform patterns ofthe three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI),and troponin C (TnC). Mechanical unloading was achieved by hindlimbunloading (HU) for time periods of 7, 15, and 28 days. Relativeconcentrations of slow and fast TnT, TnI, and TnC isoforms wereassessed by electrophoretic and immunoblot analyses. HU inducedprofound slow-to-fast isoform transitions of all Tn subunits, althoughto different extents and with different time courses. The effectivenessof the isoform transitions was higher for TnT than for TnI and TnC.Indeed, TnI and TnC encompassed minor partial exchanges of slowisoforms with their fast counterparts, whereas the expression patternof fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in thesame manner by HU. This suggests that the slow and fast counterparts ofthe Tn subunit isoforms are regulated independently in response to HU.The changes in TnTf composition occurred in parallel with previouslydemonstrated transitions within the pattern of the fast myosin heavychains in the same muscles.

     

Adaptation by alternative RNA splicing of slow troponin T isoforms in type 1 but not type 2 Charcot-Marie-Tooth disease

Slow troponin T (TnT) plays an indispensable role in skeletal muscle function. Alternative RNA splicing in the NH₂-terminal region produces high-molecular-weight (HMW) and low-molecular-weight (LMW) isoforms of slow TnT. Normal adult slow muscle fibers express mainly HMW slow TnT. Charcot-Marie-Tooth disease (CMT) is a group of inherited peripheral polyneuropathies caused by various neuronal defects. We found in the present study that LMW slow TnT was significantly upregulated in demyelination form type 1 CMT (CMT1) but not axonal form type 2 CMT (CMT2) muscles. Contractility analysis showed an increased specific force in single fibers isolated from CMT1 but not CMT2 muscles compared with control muscles. However, an in vitro motility assay showed normal velocity of the myosin motor isolated from CMT1 and CMT2 muscle biopsies, consistent with their unchanged myosin isoform contents. Supporting a role of slow TnT isoform regulation in contractility change, LMW and HMW slow TnT isoforms showed differences in the molecular conformation in conserved central and COOH-terminal regions with changed binding affinity for troponin I and tropomyosin. In addition to providing a biochemical marker for the differential diagnosis of CMT, the upregulation of LMW slow TnT isoforms under the distinct pathophysiology of CMT1 demonstrates an adaptation of muscle function to neurological disorders by alternative splicing modification of myofilament proteins. muscle adaptation; demyelination; force and velocity     

Troponin T isoforms alter the tolerance of transgenic mouse cardiac muscle to acidosis

Troponin T (TnT) is an essential protein in the Ca²⁺ regulatory system of striated of muscle. Three fiber type-specific TnT genes have evolved in higher vertebrates to encode cardiac, slow and fast skeletal muscle TnT isoforms. To understand the functional significance of TnT isoforms, we studied the effects of acidosis on the contractility of transgenic mouse cardiac muscle that expresses fast skeletal muscle TnT. Contractility analysis of intact cardiac muscle strips showed that while no differences were detected at physiological pH, the transgenic cardiac muscle had significantly greater decreases in +dF/dt_max at acidic pH than that of the wild-type control. Contractility of skinned cardiac muscles demonstrated that the presence of fast TnT resulted in significantly larger decreases in force and Ca²⁺ sensitivity at acidic pH than that of the wild-type control. The effect of TnT isoforms on the tolerance of muscle to acidosis may explain the higher tolerance of embryonic versus adult cardiac muscles. The results are consistent with the hypothesis that charge differences in TnT isoforms contribute to the contractility of muscle. The data further support a hypothesis that slow TnT is similar to the cardiac, but not fast, and TnT may contribute to the higher tolerance of slow muscles to stress conditions. Therefore, TnT isoform diversity may contribute to the compatibility of muscle thin filaments to cellular environments in different fiber types, during development and functional adaptation.     

The Effect of Passive Movement on Denervated Soleus Highlights a Differential Nerve Control on SERCA and MyHC Isoforms

The sarco-endoplasmic reticulum Ca²⁺ ATP-ase (SERCA) and myosin heavy chain (MyHC) levels were measured in hindlimb-denervated and selectively denervated rat soleus muscles. Selective denervation allowed passive movement of the soleus, whereas hindlimb denervation rendered it to passivity. To minimize chronic effects, we followed the changes only for 2 weeks. Selective denervation resulted in less muscle atrophy, a faster slow-to-fast transition of MyHC isoforms, and less coordinated expressions of the slow vs fast isoforms of MyHC and SERCA. Generally, expression of the slow-twitch type SERCA2a was found to be less dependent, whereas the slow-twitch type MyHC1 was the most dependent on innervation. Our study shows that passive movement is able to ameliorate denervation-induced atrophy of the soleus and that it also accentuates the dyscoordination in the expression of the corresponding slow and fast isoforms of MyHC and SERCA. (J Histochem Cytochem 56:1013–1022, 2008)     

Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading

Weight-bearing skeletal muscles change phenotype in response to unloading. Using the hindlimb suspension rat model, we investigated the regulation of myofilament protein isoforms in correlation to contractility. Four weeks of continuous hindlimb unloading produced progressive atrophy and contractility changes in soleus but not extensor digitorum longus muscle. The unloaded soleus muscle also had decreased fatigue resistance. Along with the decrease of myosin heavy chain isoform I and IIa and increase of IIb and IIx, coordinated regulation of thin filament regulatory protein isoforms were observed: - and -tropomyosin decreased and -tropomyosin increased, resulting in an / ratio similar to that in normal fast twitch skeletal muscle; troponin I and troponin T (TnT) both showed decrease in the slow isoform and increases in the fast isoform. The TnT isoform switching began after 7 days of unloading and TnI isoform showed detectable changes at 14 days while other protein isoform changes were not significant until 28 days of treatment. Correlating to the early changes in contractility, especially the resistance to fatigue, the early response of TnT isoform regulation may play a unique role in the adaptation of skeletal muscle to unloading. When the fast TnT gene expression was upregulated in the unloaded soleus muscle, alternative RNA splicing switched to produce more high molecular weight acidic isoforms, reflecting a potential compensation for the decrease of slow TnT that is critical to skeletal muscle function. The results demonstrate that differential regulation of TnT isoforms is a sensitive mechanism in muscle adaptation to functional demands. troponin T; fatigue resistance; troponin I; tropomyosin; myosin; hindlimb-suspended rat; Western blot protein quantification     

Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle

Fast-twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low-frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+-ATPase isoforms were assessed through the use of an RNase-protection assay. Stimulation-induced increases in slow cardiac Ca2+-ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+-ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow-twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow-twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast-to-slow troponin C isoform transition, previously investigated in the same muscles.     

Loss of fast-twitch isomyosins in skeletal muscles of the diabetic rat.

By means of pyrophosphate electrophoresis the myosin isoenzyme pattern of two fast-twitch skeletal muscles (extensor digitorum longus, gastrocnemius) and one slow-twitch muscle (soleus) was investigated in control rats and was compared with that of rats 4 weeks after induction of diabetes mellitus by streptozotocin injection. In the fast-twitch muscles the isomyosin pattern consisting of FM1 (fast isomyosin 1), FM2 and FM3 was strongly affected by diabetes, resulting in an extensive loss of FM1 and a substantial decrease of FM2. These changes were also apparent when the light chains of the fast isomyosins were analysed by two-dimensional electrophoresis: LC3f (myosin light chain 3f) largely disappeared and LC2f was significantly diminished. In contrast, the isomyosin pattern in soleus muscle, consisting of SM1 (slow isomyosin 1) and SM2, was not affected by the diabetic state, and two-dimensional electrophoresis revealed a normal light-chain pattern of LC1sa, LC1sb and LC2s. These results indicate that the isomyosins of slow-twitch oxidative myofibres are more resistant to the hormonal and metabolic disorders during diabetes mellitus than are the isomyosins of fast-twitch fibres.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.     

Coordinate changes in fast thin filament and Z-line protein expression in the early response to chronic stimulation

The early response to chronic low frequency stimulation is characterized by coordinate changes in fast thin filament and Z-line protein expression prior to the expression of slow contractile proteins. Within the first 3 weeks of intervention there is 1) a transition from expression of the fast troponin (Tn) Ts TnT1f and TnT2f to expression of TnT3f, 2) a parallel change in Z-line protein expression in which alpha-actinin1f/s becomes predominant, and 3) a small but significant increase in the levels of the beta-tropomyosin (Tm) subunit. The timing of these changes coincides with the conversion to thick Z-lines, and the kinetics of changes in fast TnT and alpha-actinin isoforms suggests that the expression of TnT3f with alpha-actinin1f/s and a combination of alpha beta and beta 2-Tm, which we have designated the TnT3f program, is coordinated. Because fast fibers expressing TnT3f with alpha beta and beta 2-Tm, like slow fibers, exhibit a more graded response to calcium (Schachat, F.H., Diamond, M.S., and Brandt, P.W. (1987) J. Mol. Biol., 198, 551-555), this change appears to be an adaptive response, illustrating the contribution of isoform diversity to the physiological plasticity of fast skeletal muscle and indicating that expression of the TnT3f program may be an intermediate phase in the conversion from a fast to a slow molecular phenotype.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

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.     

Effect of different troponin T-tropomyosin combinations on thin filament activation

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.