Troponin I Isoforms Research Articles

Myofilament incorporation determines the stoichiometry of troponin I in transgenic expression and the rescue of a null mutation

The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using α-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.

Genetic and biochemical heterogeneity of cardiac troponins: clinical and laboratory implications

The contractile sarcomeric pool of skeletal muscle and heart consists of a highly-ordered arrangement of actin thin filaments, tropomyosin and proteins of the troponin complex: troponin I (TnI), troponin T (TnT) and troponin C (TnC). The cardiac myocyte expresses specific isoforms of both TnI and TnT (cTnI and cTnT, respectively), which can be distinguished from skeletal muscle isoforms by rapid and reliable immunoassays. Due to their superior cardiac specificity, cTnI and cTnT have become the recommended biomarkers for the diagnosis of myocardial injury. The most common applications are for risk stratification and diagnosis of acute coronary syndromes, albeit they can also be used for assessing a variety of other myocardial disorders. However, the excellent diagnostic efficiency of cardiac troponin testing is jeopardized by some pre-analytical issues (unsuitable specimens for testing, analyte stability, handling, transportation and storage of specimens prior to analysis), as well as by analytical concerns related to standardization or harmonization of cTnI immunoassays, imprecision at low concentrations, antibody specificity, immunoreactivity of plasma isoforms released in the blood after myocardial injury, interference from autoantibodies, heterophilic antibodies, rheumatoid factor, and human anti-mouse antibodies. In addition, although the influence of some known mutations in cTnT and cTnI on calcium sensitivity and force generation have been readily demonstrated, their influence on current immunoassays is unknown. Most mutations in these proteins may contribute to the development of certain cardiomyopathies, namely hypertrophic and restrictive. In these situations, the phenotype is characterized by the underlying diseases, and the ability to detect cTnT or cTnI is of relatively little clinical significance. However, the cTnT Arg129Lys polymorphism and those observed in the stable domain of cTnI do not significantly alter the functional properties of the molecule within the myocardium and thus are predictably asymptomatic. While the actual prevalence of these polymorphisms in the general population is still unknown, they might be a source of potential analytical problems; modifying the immunogenicity of the molecule and leading to potential false negative results. The goal of this article is to provide an overview on the potential technical and analytical challenges in the measurement of cardiac troponins, along with the significance of polymorphisms in the genes encoding for cTnI and cTnT in both health and disease.

Isoform‐specific variation in the intrinsic disorder of troponin I

Various intrinsic disorder (ID) prediction algorithms were applied to the three tissue isoforms of troponin I (TnI). The results were interpreted in terms of the known structure and dynamics of troponin. In line with previous results, all isoforms of TnI were predicted to have large stretches of ID. The predictions show that the C-termini of all isoforms are extensively disordered as is the N-terminal extension of the cardiac isoform. Cardiac TnI likely belongs to the group of intrinsically disordered signalling hub proteins. For a given portion of the protein sequence, most ID prediction approaches indicate isoform-dependent variations in the probability of disorder. Comparison of machine learning and physically based approaches suggests the ID variations are only partially attributable to local variations in the ratio of charged to hydrophobic residues. The VSL2B algorithm predicts the largest variations in ID across the isoforms, with the cardiac isoform having the highest probability of structured regions, and the fast-skeletal isoform having no intrinsic structure. The region corresponding to residues 57-95 of the fast-skeletal isoform, known to form a coiled coil substructure with troponin T, was highly variable between isoforms. The isoform-specific ID variations may have mechanistic significance, modulating the extent to which conformational fluctuations in tropomyosin are communicated to the troponin complex. We discuss structural mechanisms for this communication. Overall, the results motivate the development of predictors designed to address relative levels of disorder between highly similar proteins.

Molecular prenatal diagnosis for hereditary distal arthrogryposis type 2B

Autosomal dominant distal arthrogryposes (DAs) are a group of muscle diseases characterized by congenital contractures of the limbs. Currently, prenatal diagnosis of DAs depends upon ultrasound examination during late gestation. Recently, five genes encoding fast switch proteins located at 9p13.2, 11p15.5 and 17q13.1 were identified. These included TPM2, TNNI2/TNNT3, and MYH3/MYH8. Last year, we discovered a novel heterozygous mutation c.523_525delAAG (p.K175del) in the TNNI2 gene, which encodes the isoform of troponinI, in a seven-generation Chinese family affected with distal arthrogryposis type 2B (DA2B). Here, we report the molecular prenatal diagnosis of 3 high-risk fetuses of two women in the family by two-point linkage inferential analysis and deletion detection of the TNNI2 gene with chorionic villus sampling (CVS) or amniocentesis. To our knowledge, this is the first description of molecular prenatal diagnosis for DAs.

Developmental changes in passive stiffness and myofilament Ca2+sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth

The giant protein titin, a major contributor to myocardial mechanics, is expressed in two main cardiac isoforms: stiff N2B (3.0 MDa) and more compliant N2BA (>3.2 MDa). Fetal hearts of mice, rats, and pigs express a unique N2BA isoform ( approximately 3.7 MDa) but no N2B. Around birth the fetal N2BA titin is replaced by smaller-size N2BA isoforms and N2B, which predominates in adult hearts, stiffening their sarcomeres. Here we show that perinatal titin-isoform switching and corresponding passive stiffness (STp) changes do not occur in the hearts of guinea pig and sheep. In these species the shift toward "adult" proportions of N2B isoform is almost completed by midgestation. The relative contributions of titin and collagen to STp were estimated in force measurements on skinned cardiac muscle strips by selective titin proteolysis, leaving the collagen matrix unaffected. Titin-based STp contributed between 42% and 58% to total STp in late-fetal and adult sheep/guinea pigs and adult rats. However, only approximately 20% of total STp was titin based in late-fetal rat. Titin-borne passive tension and the proportion of titin-based STp generally scaled with the N2B isoform percentage. The titin isoform transitions were correlated to a switch in troponin-I (TnI) isoform expression. In rats, fetal slow skeletal TnI (ssTnI) was replaced by adult carciac TnI (cTnI) shortly after birth, thereby reducing the Ca2+ sensitivity of force development. In contrast, guinea pig and sheep coexpressed ssTnI and cTnI in fetal hearts, and skinned fibers from guinea pig showed almost no perinatal shift in Ca2+ sensitivity. We conclude that TnI-isoform and titin-isoform switching and corresponding functional changes during heart development are not initiated by birth but are genetically programmed, species-specific regulated events.

Histidine button engineered into cardiac troponin I protects the ischemic and failing heart

The myofilament protein troponin I (TnI) has a key isoform-dependent role in the development of contractile failure during acidosis and ischemia. Here we show that cardiac performance in vitro and in vivo is enhanced when a single histidine residue present in the fetal cardiac TnI isoform is substituted into the adult cardiac TnI isoform at codon 164. The most marked effects are observed under the acute challenges of acidosis, hypoxia, ischemia and ischemia-reperfusion, in chronic heart failure in transgenic mice and in myocytes from failing human hearts. In the isolated heart, histidine-modified TnI improves systolic and diastolic function and mitigates reperfusion-associated ventricular arrhythmias. Cardiac performance is markedly enhanced in transgenic hearts during reperfusion despite a high-energy phosphate content similar to that in nontransgenic hearts, providing evidence for greater energetic economy. This pH-sensitive 'histidine button' engineered in TnI produces a titratable molecular switch that 'senses' changes in the intracellular milieu of the cardiac myocyte and responds by preferentially augmenting acute and long-term function under pathophysiological conditions. Myofilament-based inotropy may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart.

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.

The Miscommunicative Cardiac Cell: When Good Proteins Go Bad

Troponin (Tn) is made up of three subunits, troponin T (TnT), troponin I (TnI), and troponin C (TnC). In cardiac muscle, TnI can exist as two isoforms, slow skeletal TnI (ssTnI) or cardiac TnI (cTnI), whereas TnT occurs as multiple isoforms. The predominant form of TnI in fetal cardiac muscle is ssTnI, which is derived from a different gene than cTnI. However, the predominant form of cardiac TnT (cTnT) in fetal muscle is cTnT1, which is derived from the same gene that produces the adult cTnT isoform (cTnT3). Fetal cardiac muscle is more sensitive to Ca(2+) than adult muscle and this may be due in part to the fetal cTnT1 and ssTnI isoforms. cTnT1 and/or ssTnI by themselves cause a significant increase in Ca(2+) sensitivity when compared to cTnT3 and/or cTnI. Mutations in the gene for cTnT can cause hypertrophic cardiomyopathy or dilated cardiomyopathy (DCM). Investigation of DCM mutations in the fetal cTnT1 isoform showed that the cTnT isoform is an important determinant of the effect of the mutation. The TnI isoform also affects the physiological function of the cardiac muscle. The presence of both the fetal TnT isoform, containing a DCM mutation, and ssTnI results in larger changes in Ca(2+) sensitivity than the same DCM mutant in the adult TnT isoform and in the presence of cTnI (when compared to their respective wild-type TnT controls). These recent results suggest that some mutations may have different severities in fetal and adult hearts.

Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements.

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

Role of Troponin I Phosphorylation in Protein Kinase C-mediated Enhanced Contractile Performance of Rat Myocytes

Our goal was to define the role of phosphorylated cardiac troponin-I in the adult myocyte contractile performance response to activated protein kinase C. In agreement with earlier work, endothelin enhanced both adult rat myocyte contractile performance and cardiac troponin-I phosphorylation. Protein kinase C participated in both responses. The role of cardiac troponin-I phosphorylation in the contractile function response to protein kinase C was further investigated using gene transfer into myocytes of troponin-I isoforms/mutants lacking one or more phosphorylation sites previously identified in purified cardiac troponin-I. Sarcomeric replacement with slow skeletal troponin-I-abrogated protein kinase C-mediated troponin-I phosphorylation. In functional studies, endothelin slowed relaxation in myocytes expressing slow skeletal troponin-I, while the relaxation rate increased in myocytes expressing cardiac troponin-I. Based on these results, acceleration of myocyte relaxation during protein kinase C activation largely depended on cardiac troponin-I phosphorylation. Experiments with troponin-I isoform chimeras provided evidence that phosphorylation sites in the amino portion of cardiac troponin I-mediated the protein kinase C acceleration of relaxation. The cardiac troponin-I Thr-144 phosphorylation site identified in earlier biochemical studies was not significantly phosphorylated during the acute contractile response. Thus, amino-terminal protein kinase C-dependent phosphorylation sites in cardiac troponin-I are likely responsible for the accelerated relaxation observed in adult myocytes.

Ascidian larva reveals ancient origin of vertebrate-skeletal-muscle troponin I characteristics in chordate locomotory muscle.

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle -like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.

Effects of long-term high-altitude hypoxia on myocardial protein kinase A activity and troponin I isoforms in fetal and nonpregnant sheep

In fetal sheep, we found that the augmentation of cardiac contractility by beta-adrenergic receptor (beta-AR) stimulation was reduced after exposure to long-term hypoxia. However, cyclic adenosine monophosphate (cAMP) production after beta-AR stimulation was higher in long-term hypoxic fetal sheep than in normoxic ones. Therefore, we studied the potential role of changes in myocardial protein kinase A (PKA) activity and troponin I (TnI) isoforms in fetal and nonpregnant sheep exposed to approximately 112 days of hypoxia at high altitude (3820 m). Resting and maximally stimulated (by cAMP) PKA activity was measured by phosphorylation of the artificial peptide, Kemptide. Specificity was confirmed by inhibition with PKI, a specific PKA inhibitor. For TnI isoforms, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve the proteins. We used monoclonal anti-cardiac TnI antibody (clone C5), which also cross-reacted with slow skeletal muscle TnI, to identify TnI isoforms. For the fetal hearts, resting PKA activity was significantly higher in the high-altitude group than the control group, but total PKA activity was not different between the normoxic and hypoxic groups. In the adult hearts, no significant difference was observed in either resting or total PKA activity between normoxic and hypoxic groups. For both the fetal and adult sheep, the predominant TnI was the cardiac isoform, and hypoxic exposure produced no change in the TnI isoform composition. Neither a reduction in PKA activity nor a change in TnI isoforms could explain the reduction in beta-receptor augmentation of cardiac contractility in fetal sheep exposed to long-term hypoxia.

Sarcomere Thin Filament Regulatory Isoforms: EVIDENCE OF A DOMINANT EFFECT OF SLOW SKELETAL TROPONIN I ON CARDIAC CONTRACTION

Thin filament proteins tropomyosin (Tm), troponin T (TnT), and troponin I (TnI) form an allosteric regulatory complex that is required for normal cardiac contraction. Multiple isoforms of TnT, Tm, and TnI are differentially expressed in both cardiac development and disease, but concurrent TnI, Tm, and TnT isoform switching has hindered assignment of cellular function to these transitions. We systematically incorporated into the adult sarcomere the embryonic/fetal isoforms of Tm, TnT, and TnI by using gene transfer. In separate experiments, greater than 90% of native TnI and 40-50% of native Tm or TnT were specifically replaced. The Ca(2+) sensitivity of tension development was markedly enhanced by TnI replacement but not by TnT or Tm isoform replacement. Titration of TnI replacement from >90% to <30% revealed a dominant functional effect of slow skeletal TnI to modulate regulation. Over this range of isoform replacement, TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction, and this identifies TnI as a potential target to modify contractile performance in normal and diseased myocardium.

Mutations in Genes Encoding Fast-Twitch Contractile Proteins Cause Distal Arthrogryposis Syndromes

The distal arthrogryposes (DAs) are a group of disorders characterized by multiple congenital contractures of the limbs. We previously mapped a locus for DA type 2B (DA2B), the most common of the DAs, to chromosome 11. We now report that DA2B is caused by mutations in TNNI2 that are predicted to disrupt the carboxy-terminal domain of an isoform of troponin I (TnI) specific to the troponin-tropomyosin (Tc-Tm) complex of fast-twitch myofibers. Because the DAs are genetically heterogeneous, we sought additional candidate genes by examining modifiers of mutant Drosophila isoforms of TnI. One of these modifiers, Tm2, encodes tropomyosin, another component of the Tc-Tm complex. A human homologue of Tm2, TPM2, encodes beta-tropomyosin and maps to the critical interval of DA type 1 (DA1). We discovered that DA1 is caused by substitution of a highly conserved amino acid residue in beta-tropomyosin. These findings suggest that DAs, in general, may be caused by mutations in genes encoding proteins of the contractile apparatus specific to fast-twitch myofibers. This provides a new opportunity to directly study the etiology and pathogenesis of multiple-congenital-contracture syndromes.

Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing.

Cyclic AMP-dependent protein kinase (PKA) targets contractile proteins, troponin-I (TnI) and myosin binding protein C (MyBP-C) in the heart and induces a decrease in myofilament Ca2+ sensitivity. Yet, the effect of sarcomere length (SL) change on Ca2+ sensitivity (length-dependent activation: LDA) following PKA-dependent phosphorylation is not clear. To clarify the role of PKA-dependent phosphorylation of TnI and MyBP-C on LDA in the heart, we examined LDA in skinned myocytes from a non-transgenic (NTG) and a transgenic murine model in which the native cardiac isoform (cTnI) was completely replaced by the slow skeletal isoform of TnI (ssTnI-TG) lacking the phosphorylation sites for PKA, while retaining PKA sites on MyBP-C. In NTG myocytes, PKA treatment decreased Ca2+ sensitivity at each SL, but enhanced the impact of SL change on Ca2+ sensitivity. Despite a greater sensitivity to Ca2+ and a reduction in LDA, neither Ca2+ responsiveness nor LDA was affected by PKA treatment in ssTnI-TG myocytes. To determine whether the above observations could be explained by the lateral separation between thick and thin filaments, as suggested by others, we measured interfilament spacing by X-ray diffraction as a function of SL in skinned cardiac trabeculae in the passive state from both NTG and ssTnI-TG models before and following treatment with PKA. Phosphorylation by PKA increased lattice spacing at every SL in NTG trabeculae. However, the relationship between SL and myofilament lattice spacing in ssTnI-TG was markedly shifted downward to an overall decreased myofilament lattice spacing following PKA treatment. We conclude: (1) PKA-dependent phosphorylation enhances length-dependent activation in NTG hearts; (2) replacement of native TnI with ssTnI increases Ca2+ sensitivity of tension but reduces length-dependent activation; (3) MyBP-C phosphorylation by PKA does not alter calcium responsiveness and induces a decrease in myofilament lattice spacing at all sarcomere lengths and (4) length-dependent activation in the heart cannot be entirely explained by alterations in myofilament lattice spacing.

Gene transfer of troponin I isoforms, mutants, and chimeras.

Thin filament proteins play an essential role in the regulation of myocardial pressure development. Within the thin filament of the sarcomere, troponin I (TnI) plays a key role in regulating the Ca(2+) sensitivity of force. During myocardial development, there is a transition in TnI isoform expression from the slow skeletal isoform (ssTnI) in embryonic/fetal myocardium to the cardiac isoform (cTnI) expressed in adult hearts. Over a similar developmental time window, the calcium sensitivity of force development also decreases. Gene transfer of ssTnI, and chimeras derived from ssTnI and cTnI, into adult ventricular myocytes have provided insights into the isoform-specific domains of TnI responsible for differentially influencing myofilament Ca(2+) sensitivity. Two separate isoform-specific regions, located in the carboxyl- and amino-portions of the protein, have been identified by comparing Ca(2+)-activated isometric tension in myocytes expressing the TnI isoforms or chimeras. The carboxyl-portion of TnI also contributes to isoform-dependent differences in myofilament sensitivity to acidic pH, which ensues during several myocardial disease states. In contrast, the diminished Ca(2+) sensitivity observed in response to beta-adrenergic-mediated phosphorylation of cardiac TnI requires the amino-portion of the cardiac TnI isoform yet, does not depend on the presence of a specific isoform in the carboxyl-region of TnI. Recent studies with a mutation linked to hypertrophic cardiomyopathy have demonstrated that changes in protein charge also influence the ability of TnI isoforms to regulate myofilament Ca(2+) sensitivity. Information gained from these, and future studies on more localized and specific changes in the amino acid sequence, may one day lead to the use of genetically engineered TnI for therapeutic manipulation of contractile function.

The structural organization of ascidian Halocynthia roretzi troponin I genes.

The organization of troponin I (TnI) genes from the ascidian Halocynthia roretzi have been determined. Halocynthia possesses roughly two types of TnI isoforms. One type is a single-copied adult TnI (adTnI) gene, which contains eight exons and seven introns. adTnI expresses two isoforms, the shorter body wall muscle TnI and the longer cardiac TnI, through alternative splicing. The mRNAs of these TnI isoforms may undergo trans-splicing of the 5'-leader sequences, like the TnI mRNA of another ascidian species, Ciona intestinalis. The other type comprises multi-copied larval TnI (laTnI) genes. Halocynthia has at least three laTnIs (alpha, beta, and gamma), which are composed of five exons and four introns, and two of them (alpha and gamma) are clustered in tandem. All laTnIs have B- and M-regions within their 5'-upstream regions, which have been discovered to be the regulatory elements of Halocynthia larval actin genes. The expression of Halocynthia laTnIs and larval actins may be regulated in the same manner. It is known that Ciona does not possess a larva-specific TnI isoform. The phylogenetic tree of ascidian TnIs suggests that laTnIs might have only been generated within the Pleurogona lineage after Enterogona/Pleurogona divergence, and this scenario well agrees with the absence of laTnIs in Ciona.

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

This study focuses on the effects of mechanical unloading of rat soleus muscle on the isoform patterns of the three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). Mechanical unloading was achieved by hindlimb unloading (HU) for time periods of 7, 15, and 28 days. Relative concentrations of slow and fast TnT, TnI, and TnC isoforms were assessed by electrophoretic and immunoblot analyses. HU induced profound slow-to-fast isoform transitions of all Tn subunits, although to different extents and with different time courses. The effectiveness of the isoform transitions was higher for TnT than for TnI and TnC. Indeed, TnI and TnC encompassed minor partial exchanges of slow isoforms with their fast counterparts, whereas the expression pattern of fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in the same manner by HU. This suggests that the slow and fast counterparts of the Tn subunit isoforms are regulated independently in response to HU. The changes in TnTf composition occurred in parallel with previously demonstrated transitions within the pattern of the fast myosin heavy chains in the same muscles.

Myofibrillar protein isoform expression is correlated with synaptic efficacy in slow fibres of the claw and leg opener muscles of crayfish and lobster.

In the crayfish and lobster opener neuromuscular preparations of the walking legs and claws, there are regional differences in synaptic transmission even though the entire muscle is innervated by a single excitatory tonic motor neuron. The innervation of the proximal fibres produced larger excitatory postsynaptic potentials (EPSPs) than those of the central fibres. The amplitudes of the EPSPs in the distal fibres were intermediate between those of the proximal and central regions. These differences in EPSP amplitudes were correlated with differences in short-term facilitation between the three regions. When given a 10- or 20-pulse train of stimuli, the proximal fibres showed greater short-term facilitation initially, often followed by a maximization of short-term facilitation towards the end of a train. In contrast, the central fibres showed a linear increase in short-term facilitation throughout a stimulus train. The distal fibres showed intermediate short-term facilitation compared with the other two regions. Analysis of myofibrillar isoforms showed that levels of troponin-T(1) (TnT(1)), a 55 kDa isoform expressed in slow-tonic (S(2)) fibres, were correlated with synaptic properties. Proximal fibres had the highest levels of TnT(1), with lower levels in distal fibres; central fibres lacked TnT(1), which is characteristic of slow-twitch (S(1)) fibres. In addition, differences in troponin-I isoforms correlated with TnT(1) levels between the proximal, central and distal regions. The correlation between slow fibre phenotype and strength of innervation suggests a relationship between synaptic structure and expression of troponin isoforms.

Localization of Regions of Troponin I Important in Deactivation of Cardiac Myofilaments by Acidic pH

G. Li, A. F. Martin and R. J. Solaro. Localization of Regions of Troponin I Important in Deactivation of Cardiac Myofilaments by Acidic pH. Journal of Molecular and Cellular Cardiology (2001) 33, 0000–0000. Ca2+-activation of cardiac muscle myofilaments is more sensitive to depression by acidic pH than is the case with skeletal myofilaments. We tested the hypothesis that this difference is related to specific regions of the TnI (troponin I) isoforms in these muscles. We exchanged native Tn complex in detergent-extracted fiber bundles from mouse ventricles with Tn containing various combinations of fast (fsTnI) or slow skeletal (ssTnI) complexed with either cardiac TnC (cTnC) or fsTnC, and with cTnC complexed with the following chimeras: (1) fsTnI N-terminal region (fN) plus cTnI inhibitory peptide (cIp) and cTnI C-terminal region (cC); and (2) cTnI N-terminal region (cN)-cIp-fsTnI C-terminal region (fC). We determined the change in half maximal Ca2+(ΔEC50) for tension activation at pH 7.0 and pH 6.5. Similar ΔEC50values were obtained for unextracted controls (5.53±0.30 μ m), for preparations containing cTnI-cTnC (5.74±0.40μ m), and preparations exchanged with cTnI-fsTnC (5.63±0.40 μ m). However, replacement of cTnI with fsTnI significantly decreased ΔEC50to 3.95±0.17 μ m. Replacement of cTnI with ssTnI also significantly depressed ΔEC50to 2.07±0.15 μ m. Results of studies using the chimeras demonstrated that the C-terminal domains of cTnI and fsTnI are responsible for these differences. This conclusion also fits with data from experiments in which we measured Ca2+-binding to the regulatory site of cTnC in binary complexes containing cTnC with cTnI, fsTnI, or the chimeras. Our results localize a region of TnI important in effects of acidosis on cardiac myofilaments and extend our earlier data indicating that C-terminal regions of cTnI outside the Ip are critical for activation by Ca.2+