Fast Troponin T Research Articles

Early Divergence of the C-Terminal Variable Region of Troponin T Via a Pair of Mutually Exclusive Alternatively Spliced Exons Followed by a Selective Fixation in Vertebrate Heart.

Troponin T (TnT) is the thin filament anchoring subunit of troponin complex and plays an organizer role in the Ca2+-regulation of striated muscle contraction. From an ancestral gene emerged ~ 700 million years ago in Bilateria, three homologous genes have evolved in vertebrates to encode muscle type-specific isoforms of TnT. Alternative splicing variants of TnT are present in vertebrate and invertebrate muscles to add functional diversity. While the C-terminal region of TnT is largely conserved, it contains an alternatively spliced segment emerged early in C. elegans, which has evolved into a pair of mutually exclusive exons in arthropods (10A and 10B of Drosophila TpnT gene) and vertebrates (16 and 17 of fast skeletal muscle Tnnt3 gene). The C-terminal alternatively spliced segment of TnT interfaces with the other two subunits of troponin with functional significance. The vertebrate cardiac TnT gene that emerged from duplication of the fast TnT gene has eliminated this alternative splicing by the fixation of an exon 17-like constitutive exon, indicating a functional value in slower and rhythmic contractions. The vertebrate slow skeletal muscle TnT gene that emerged from duplication of the cardiac TnT gene has the exon 17-like structure conserved, indicating its further function in sustained and fatigue resistant contractions. This functionality-based evolution is consistent with the finding that exon 10B-encoded segment of Drosophila TnT homologous to the exon 17-encoded segment of vertebrate fast TnT is selectively expressed in insect heart and leg muscles. The evolution of the C-terminal variable region of TnT demonstrates a submolecular mechanism in modifying striated muscle contractility and for the treatment of muscle and heart diseases.

First insights about the underlying mechanisms of Martina Franca donkey meat tenderization during aging: A proteomic approach

This study aimed to evaluate the effect of different aging times (1, 6 and 14 days) on the tenderization rate and protein changes in ten Martina Franca donkey striploins using a proteomic approach. During aging, a progressive decrease in share force, hardness, gumminess, and chewiness together with an increase in myofibril fragmentation index were observed. Proteolysis monitored by immunoblotting revealed a progressive degradation of desmin and fast troponin-T over time and an increase of their degradation products up to 14 days aging. Proteomics revealed by means of two-dimensional electrophoresis 37 protein spots corresponding to 15 proteins to change significantly by increasing aging time. These proteins belong to three pathways, these being “muscle contraction, structure, and associated proteins” (9 proteins); ii) “energy metabolism” (5 proteins); and iii) “chaperone” (1 protein). This study is the first to highlight the possible role of interconnected pathways in driving the final quality of donkey meat and predicting its texture.

Role of troponin T and AMP deaminase in the modulation of skeletal muscle contraction

In fast muscle, isoforms of troponin T (TnT) contain an N-terminal hypervariable region that does not bind any protein of the thin filament. The N-terminal domain of TnT is removed by calpain during stress conditions and so could modulate the role of TnT in the regulation of contraction by affecting the TnT-binding affinity for tropomyosin (Tm) depending on the sequence and charge within the domain. During skeletal muscle contraction, the myokinase reaction is displaced by AMP deaminase (AMPD), an allosteric metalloenzyme, toward the formation of ATP. An unrestrained AMPD activity follows the proteolytic cleavage of the enzyme in vivo that releases a 97 aa N-terminal fragment, removing the inhibition exerted by the binding of ATP to a zinc site in the N-terminal region. Rabbit fast TnT or its phosphorylated 50-aa residue N-terminal peptide restores in AMPD the inhibition by ATP, removed in vitro by the release of a 95 aa N-terminal fragment by trypsin. Since the N-terminal region of fast rabbit TnT contains a putative zinc-binding motif, it can be inferred that TnT mimics the regulatory action exerted in native AMPD by the N-terminal domain that holds the enzyme in a less active conformation due to the presence of a zinc ion connecting the N-terminal and C-terminal regions. Together with evidence that AMPD is localized on the myofibril, the data reported in this review on the interactions between AMPD and TnT strongly suggest that these proteins mutually combine to fine-tune the regulation of muscle contraction in fast muscle.

The Cardiac Troponin T Mutant Missing the N-Terminal Extension Causes Dose-Dependent Effects on Cardiac Function and Remodeling in Transgenic Mice

The N-terminal extension (NTE; residues 42-73) of mouse cardiac troponin T (TnT) desensitizes cardiac myofilaments to Ca2+ by stabilizing thin filaments in the blocked-state. We arrived at this conclusion using detergent-skinned muscle from transgenic (TG) mouse hearts that expressed 54% of chimeric TnT (residues 1-73 of mouse cardiac TnT were replaced by residues 1-41 of mouse fast skeletal TnT). Here, we extended our investigation to include higher dose effects of the modified TnT on cardiac myofilament function/phenotype using detergent-skinned fiber studies and echocardiography measurements in two different TG mouse lines (TG-55 and TG-64 that expressed 55% and 64% of chimeric TnT, respectively). Both TG-55 and TG-64 mice showed a similar increase in myofilament Ca2+ sensitivity at sarcomere lengths (SL) of 1.9 and 2.3 μm. However, Ca2+-activated maximal tension increased significantly only in TG-64 mice at either SL. There was a progressive decrease in the overall heart size and heart-to-body weight ratios in both TG-55 and TG-64 mice. Left ventricular diastolic functional parameters (isovolumic relaxation time and E-wave deceleration time) showed a graded increase in TG-55 and TG-64 mice; however, such effects were only significant in TG-64 mice, suggesting impaired relaxation. Systolic functional parameters (stroke volume, ejection fraction and fractional shortening) were unaffected in TG-55 mice, but significantly decreased in TG-64 mice. Thus, higher levels of chimeric TnT (64%) depressed both diastolic and systolic function significantly in TG-64 mice. We will discuss the link between the effects of the modified N-terminus of TnT on cardiac myofilament function and the resultant pathological remodeling of the heart. Our findings have pathological relevance because a growing number of disease-related mutations are found both in and near the NTE of cardiac TnT.

Complex tropomyosin and troponin T isoform expression patterns in orbital and global fibers of adult dog and rat extraocular muscles

We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and β-Tm) and slow (γ-Tm and β-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, β and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.

Comparative transcriptional and biochemical studies in muscle of myotonic dystrophies (DM1 and DM2)

Myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (proximal muscular myopaty/DM2) are caused by similar dynamic mutations at two distinct genetic loci. The two diseases also lead to similar phenotypes but different clinical severity. Dysregulation of alternative splicing has been suggested as the common pathogenic mechanism. Here, we investigate the molecular differences between DM1 and DM2 using reverse transcriptase-polymerase chain reaction of troponin T (TnT) and the insulin receptor (IR), as well as immunoblotting of TnT in muscle biopsies from DM1 and DM2 patients. We found that: (a) slow TnT was encoded by two different transcripts in significantly different ratios in DM1 and DM2 muscles; (b) DM2 muscles exhibited a higher degree of alternative splicing dysregulation for fast TnT transcripts when compared to DM1 muscles; (c) the distribution of TnT proteins was significantly skewed towards higher molecular weight species in both diseases; (d) the RNA for the insulin-independent IR-A isoform was significantly increased and appeared related to the fibre-type composition in the majority of the cases examined. On the whole, these data should give a better insight on pathogenesis of DM1 and DM2.

Decreased Fatigue Tolerance In Diaphragm Muscle Of Slow Troponin T Knockdown Mice

The loss of slow skeletal muscle troponin T (TnT) results in a severe type of nemaline myopathy in the Amish (ANM). The genes encoding TnT and troponin I (TnI) are closely linked in pairs in which the 5′-enhancer region of the slow TnT gene overlaps with the cardiac TnI gene. In a mouse line with the entire cardiac TnI gene deleted, a partial destruction of the slow TnT gene promoter produces a knockdown effect. By crossing with transgenic mouse lines that over-express a core structure of cardiac TnI (cTnI-ND) under the control of cloned alpha-MHC promoter, we rescued the postnatal lethality of the cardiac TnI gene-deleted mice with no detrimental cardiac phenotypes or leaking expression in non-cardiac tissues. The double transgenic mice exhibited decreased expression of slow TnT mRNA and protein in adult diaphragm muscle. Functional analysis of isolated muscle strips showed that the slow TnT deficient (sTnT-KD) diaphragm had significantly decreased fatigue tolerance evident by the faster decrease in force and slower rate of recovery as compared with that in wild type controls. As a consequence of slow TnT deficiency, the sTnT-KD diaphragm muscle contained a higher proportion of fast TnT, decreased slow TnI with increased fast TnI, and decreased type I myosin with increased type II myosin. Consistent with the switch toward fast myofilament contents, the sTnT-KD diaphragm muscle produced higher specific tension in twitch and tetanic contractions as well as shorter time to develop peak tension in twitch contractions. The decreased fatigue tolerance of sTnT-KD diaphragm muscle explains the terminal respiratory failure seen in virtually all ANM patients and this double transgenic mouse model provides a useful experimental system to study the pathogenesis and treatment of ANM.

Co-expression of skeletal and cardiac troponin T decreases mouse cardiac function

In contrast to skeletal muscles that simultaneously express multiple troponin T (TnT) isoforms, normal adult human cardiac muscle contains a single isoform of cardiac TnT. To understand the significance of myocardial TnT homogeneity, we examined the effect of TnT heterogeneity on heart function. Transgenic mouse hearts overexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT was investigated in vivo and ex vivo as an experimental system of concurrent presence of two classes of TnT in the adult cardiac muscle. This model of myocardial TnT heterogeneity produced pathogenic phenotypes: echocardiograph imaging detected age-progressive reductions of cardiac function; in vivo left ventricular pressure analysis showed decreased myocardial contractility; ex vivo analysis of isolated working heart preparations confirmed an intrinsic decrease of cardiac function in the absence of neurohumoral influence. The transgenic mice also showed chronic myocardial hypertrophy and degeneration. The dominantly negative effects of introducing a fast TnT into the cardiac thin filaments to produce two classes of Ca(2+) regulatory units in the adult myocardium suggest that TnT heterogeneity decreases contractile function by disrupting the synchronized action during ventricular contraction that is normally activated as an electrophysiological syncytium.

Troponin T isoform expression is modulated during Atlantic halibut metamorphosis.

BackgroundFlatfish metamorphosis is a thyroid hormone (TH) driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT), a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied.ResultsIn the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT) gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT) expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature.ConclusionMuscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

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: gamma- and beta-tropomyosin decreased and alpha-tropomyosin increased, resulting in an alpha/beta 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.

Calcium Sensitivity of Human Single Muscle Fibers following Plyometric Training

To study the effect of plyometric training on Ca2+ sensitivity and the influence of troponin T (TnT) isoforms on Ca2+ -activation properties in skinned human muscle fibers. Biopsies were obtained from the vastus lateralis of eight men before and after the training period. Chemically skinned fibers were evaluated regarding their Ca2+ -activation properties and were classified according to their myosin heavy chain (MHC) contents and analyzed regarding their slow and fast TnT isoforms. After training, significant improvements (P < 0.05) were found for static jump, countermovement jump, 6 x 5-m shuttle-run test, and leg-press performances. An 8% increase in the proportion of type IIa fibers (P < 0.05) was observed. Single-fiber diameters increased by 11% in type I (P < 0.01), 10% in type IIa (P < 0.001), and 15% in type IIa/IIx fibers (P < 0.001). Peak fiber force increased by 35% in type I (P < 0.001), 25% in type IIa (P < 0.001), and 57% in type IIa/IIx fibers (P < 0.01). The Ca2+ -activation threshold was not altered by training, but the Ca2+ concentration required to elicit half-maximal activation showed a decreasing trend, with significant changes in type I fibers (P < 0.001). Cooperativity at low Ca2+ concentrations was increased in type I and type IIa/IIx fibers (P < 0.05). Type I fibers exclusively expressed slow TnT isoforms, and type II fibers were always associated with fast TnT isoforms, independent of training status. Therefore, changes in Ca2+ sensitivity after training could not be explained by differential fast or slow TnT isoform expression. Plyometric training increased single-fiber Ca2+ sensitivity, especially in type I fibers. These changes could not be explained by a modified TnT isoform expression pattern.

Troponin T Modulates Sarcomere Length-Dependent Recruitment of Cross-Bridges in Cardiac Muscle

The heterogenic nature of troponin T (TnT) isoforms in fast skeletal and cardiac muscle suggests important functional differences. Dynamic features of rat cardiac TnT (cTnT) and rat fast skeletal TnT (fsTnT) reconstituted cardiac muscle preparations were captured by fitting the force response of small amplitude (0.5%) muscle length changes to the recruitment-distortion model. The recruitment of force-bearing cross-bridges (XBs) by increases in muscle length was favored by cTnT. The recruitment magnitude was ∼1.5 times greater for cTnT- than for fsTnT-reconstituted muscle fibers. The speed of length-mediated XB recruitment ( b) in cTnT-reconstituted muscle fiber was 0.50–0.57 times as fast as fsTnT-reconstituted muscle fibers (3.05 vs. 5.32 s −1 at sarcomere length, SL, of 1.9 μm and 4.16 vs. 8.36 s −1 at SL of 2.2 μm). Due to slowing of b in cTnT-reconstituted muscle fibers, the frequency of minimum stiffness ( f min) was shifted to lower frequencies of muscle length changes (at SL of 1.9 μm, 0.64 Hz, and 1.16 Hz for cTnT- and fsTnT-reconstituted muscle fibers, respectively; at SL of 2.2 μm, 0.79 Hz, and 1.11 Hz for cTnT- and fsTnT-reconstituted muscle fibers, respectively). Our model simulation of the data implicates TnT as a participant in the process by which SL- and XB-regulatory unit cooperative interactions activate thin filaments. Our data suggest that the amino-acid sequence differences in cTnT may confer a heart-specific regulatory role. cTnT may participate in tuning the heart muscle by decreasing the speed of XB recruitment so that the heart beats at a rate commensurate with f min.

Difference in postmortem degradation pattern among troponin T isoforms expressed in bovine longissimus, diaphragm, and masseter muscles

The postmortem degradation of troponin T (TnT) in bovine longissimus (LT), diaphragm (DP), and masseter (MS) was analyzed. A 28.3 kDa (conventional 30 kDa) fragment of fast-type TnT isoforms showed the highest content in both LT and DP, where a 35.4 kDa isoform had the highest expression among the other fast isoforms. Meanwhile, a 26.0 kDa fragment was found to be the most highly produced among the fast TnT fragments in MS, where the expression of 36.5 and 32.8 kDa isoforms was higher than that of 35.4 and 34.8 kDa isoforms. Thus, the compositions of both the intact TnT isoform proteins and the postmortem fragments differed among the muscles examined, indicating that each TnT isoform degrades into a specific fragment in each muscle. Among the muscles, the LT muscle showed a high extent of TnT degradation and the highest expression of fast TnT isoforms containing a taste-related peptide sequence.

Role of the Fetal and α/β Exons in the Function of Fast Skeletal Troponin T Isoforms: Correlation with Altered Ca 2+ Regulation Associated with Development

In mammalian fast skeletal muscle, constitutive and alternative splicing from a single troponin T (TnT) gene produce multiple developmentally regulated and tissue specific TnT isoforms. Two exons, alpha (exon 16) and beta (exon 17), located near the 3' end of the gene and coding for two different 14 amino acid residue peptides are spliced in a mutually exclusive manner giving rise to the adult TnTalpha and the fetal TnTbeta isoforms. In addition, an acidic peptide coded by a fetal (f) exon located between exons 8 and 9 near the 5' end of the gene, is specifically present in TnTbeta and absent in the adult isoforms. To define the functional role of the f and alpha/beta exons, we constructed combinations of TnT cDNAs from a single human fetal fast skeletal TnTbeta cDNA clone in order to circumvent the problem of N-terminal sequence heterogeneity present in wild-type TnT isoforms, irrespective of the stage of development. Nucleotide sequences of these constructs, viz. TnTalpha, TnTalpha + f, TnTbeta - f and TnTbeta are identical, except for the presence or absence of the alpha or beta and f exons. Our results, using the recombinant TnT isoforms in different functional in vitro assays, show that the presence of the f peptide in the N-terminal T1 region of TnT, has a strong inhibitory effect on binary interactions between TnT and other thin filament proteins, TnI, TnC and Tm. The presence of the f peptide led to reduced Ca2+-dependent ATPase activity in a reconstituted thin filament, whereas the contribution of the alpha and beta peptides in the biological activity of TnT was primarily modulatory. These results indicate that the f peptide confers an inhibitory effect on the biological function of fast skeletal TnT and this can be correlated with changes in the Ca2+ regulation associated with development in fast skeletal muscle.

Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 microM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa-tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release-restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.

Mechanism of Production of Troponin T Fragments during Postmortem Aging of Porcine Muscle

Troponin T (TnT) is one of the myofibrillar proteins that is easily degraded during postmortem aging of pork. In this study, we determined the N-terminal amino acid sequences of TnT degradation fragments produced during postmortem aging and by m-calpain hydrolysis. The N-terminal amino acid sequences of TnT fragments produced during postmortem aging were EVHEPEEKPRPKLTAP, EKPRPKLTAPKIPEG, and APKIPEGEKVDF. On the other hand, the N-terminal amino acid sequences of TnT fragments produced by the action of m-calpain were APPPPAEV, EVHEPEEK, and APK. These sequences of degradation fragments could be mapped on fast type TnT isoform 2. The peptide bonds of His37-Glu38 and Thr51-Ala52 in fTnT2 were cleaved during postmortem aging as well as by the calpain hydrolysis; therefore, calpain was concluded to have an important role in TnT degradation during postmortem aging. It was also found that the sourness-suppressing peptide APPPPAEVHEVHEEVH (Okumura et al. Biosci. Biotechnol. Biochem. 2004, 68, 1657-1662) derived from TnT degradation could be produced by the action of calpains on Glu21-Ala22 and His37-Glu38 sites.

Cellular Fate of Truncated Slow Skeletal Muscle Troponin T Produced by Glu180 Nonsense Mutation in Amish Nemaline Myopathy

A nonsense mutation at codon Glu180 in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C2C12 myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1-179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosin-binding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

Identification of a Region of Fast Skeletal Troponin T Required for Stabilization of the Coiled-coil Formation with Troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.

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

Troponin T (TnT) is an essential protein in the Ca 2+ 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 +d F/d t 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 Ca 2+ 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.