Slow Skeletal Muscle Troponin Research Articles

Structural and Functional Impacts of Novel Mutations in Slow Skeletal Muscle Troponin T Found in Non-Amish TNNT1 Nemaline Myopathies

Troponin T (TnT) is the thin filament anchoring subunit of the troponin complex and has two tropomyosin-binding sites for the incorporation of troponin into the sarcomeric structure. A nonsense mutation in exon 11 of the slow skeletal muscle troponin T (ssTnT) gene (TNNT1) truncating the polypeptide chain at Glu180 was found to cause nemaline myopathy (NM) in the Amish, an autosomal recessive disease with severe lethal phenotype. More NM TNNT1 mutations have recently been reported with similar recessive phenotypes. Here we engineered protein constructs representing the mutant ssTnT to investigate their impact on tropomyosin-binding and integration into the thin filament regulatory system. Like the Glu180X mutation, two novel nonsense mutations in exon 9 and exon 11 truncate the ssTnT polypeptide chain at Ser108 and Leu203, respectively, to delete the C-terminal region tropomyosin-binding site 2. A splicing site mutation causes a deletion of a 39 amino acid segment from the middle region tropomyosin-binding site 1. To understand the molecular mechanisms underlying these TNNT1 mutations, we expressed and purified the mutant ssTnT proteins and analyzed their tropomyosin-binding affinity using solid-phase protein binding assays. The results demonstrate that both the Ser108X and exon 8 deletion mutations have similarly decreased tropomyosin-binding as that of Glu180X. We recently showed that the affinity of tropomyosin-binding site 1 is modulated by the isoform-specific N-terminal variable region, with ssTnT having the weakest tropomyosin-binding affinity. Therefore, the N-terminal variable region-based conformational and functional modulation may be a therapeutic target for TNNT1 myopathies. While Leu203X has both tropomyosin-binding sites intact, its recessive phenotype suggests that the incorporation into troponin complex may be required for high affinity binding of TnT to tropomyosin, which may be a protective mechanism against the Leu203X mutation.

Deficiency of slow skeletal muscle troponin T causes atrophy of type I slow fibres and decreases tolerance to fatigue

The total loss of slow skeletal muscle troponin T (ssTnT encoded by TNNT1 gene) due to a nonsense mutation in codon Glu(180) causes a lethal form of recessively inherited nemaline myopathy (Amish nemaline myopathy, ANM). To investigate the pathogenesis and muscle pathophysiology of ANM, we studied the phenotypes of partial and total loss of ssTnT in Tnnt1 gene targeted mice. An insertion of neomycin resistance cassette in intron 10 of Tnnt1 gene caused an approximately 60% decrease in ssTnT protein expression whereas cre-loxP-mediated deletion of exons 11-13 resulted in total loss of ssTnT, as seen in ANM muscles. In diaphragm and soleus muscles of the knockdown and knockout mouse models, we demonstrated that ssTnT deficiency resulted in significantly decreased levels of other slow fibre-specific myofilament proteins whereas fast fibre-specific myofilament proteins were increased correspondingly. Immunohistochemical studies revealed that ssTnT deficiency produced significantly smaller type I slow fibres and compensatory growth of type II fast fibres. Along with the slow fibre atrophy and the changes in myofilament protein isoform contents, ssTnT deficiency significantly reduced the tolerance to fatigue in soleus muscle. ssTnT-deficient soleus muscle also contains significant numbers of small-sized central nuclei type I fibres, indicating active regeneration. The data provide strong support for the essential role of ssTnT in skeletal muscle function and the causal effect of its loss in the pathology of ANM. This observation further supports the hypothesis that the function of slow fibres can be restored in ANM patients if a therapeutic supplement of ssTnT is achieved.

Deficiency of Slow Skeletal Muscle Troponin T Causes Atrophy of Type I Slow Fibers and Decreases Tolerance to Fatigue

Deficiency of Slow Skeletal Muscle Troponin T Causes Atrophy of Type I Slow Fibers and Decreases Tolerance to Fatigue

Human slow troponin T (TNNT1) pre-mRNA alternative splicing is an indicator of skeletal muscle response to resistance exercise in older adults.

Slow skeletal muscle troponin T (TNNT1) pre-messenger RNA alternative splicing (AS) provides transcript diversity and increases the variety of proteins the gene encodes. Here, we identified three major TNNT1 splicing patterns (AS1-3), quantified their expression in the vastus lateralis muscle of older adults, and demonstrated that resistance training modifies their relative abundance; specifically, upregulating AS1 and downregulating AS2 and AS3. In addition, abundance of TNNT1 AS2 correlated negatively with single muscle fiber-specific force after resistance training, while abundance of AS1 correlated negatively with V max. We propose that TNNT1 AS1, AS2 and the AS1/AS2 ratio are potential quantitative biomarkers of skeletal muscle adaptation to resistance training in older adults, and that their profile reflects enhanced single fiber muscle force in the absence of significant increases in fiber cross-sectional area.

Changes in serum fast and slow skeletal troponin I concentration following maximal eccentric contractions

ObjectivesWe tested the hypothesis that fast skeletal muscle troponin I (fsTnI) concentration in serum would increase more than those of slow skeletal muscle troponin I (ssTnI) after eccentric exercise of the elbow flexors using a sensitive blood marker to track fibre specific muscle damage. DesignObservational comparison of response in a single experimental group. MethodsEight young men (26.4±6.2 years) performed 210 (35 sets of 6) eccentric contractions of the elbow flexors on an isokinetic dynamometer with one arm. Changes in serum fsTnI and ssTnI concentrations, serum creatine kinase (CK) activity, and maximal voluntary isometric contraction torque (MVIC) before and 1, 2, 3, 4 and 14 days following exercise were analysed by a Student–Newman–Keuls multiple comparison test. The relationship between serum CK activity and fsTnI or ssTnI concentrations was determined using a Pearson's product moment correlation. ResultsSignificant (P<0.05) decreases in MVIC and increases in serum CK activity and fsTnI were evident after exercise, but ssTnI did not change. The time course of changes in fsTnI was similar to that of CK, peaking at 4 days post-exercise, and the two were highly correlated (r=0.8). ConclusionsIncreases in serum fsTnI concentrations reflect muscle damage, and it seems likely that only fast twitch fibres were damaged by eccentric contractions.

Preliminary proteomic analysis of peripheral skeletal muscle atrophy in chronic obstructive pulmonary disease

To conduct a preliminary proteomic study of chronic obstructive pulmonary disease (COPD) with peripheral skeletal muscle atrophy. A total of 16 COPD patients and 8 aged-matched persons because of bone fractures were recruited in First Hospital Affiliated to Kunming Medical College from February to July in 2010. According to body mass index, fat free mass index, quadriceps femoris perimeter and quadriceps femoris active contraction, they were divided into those with muscle atrophy (group A, n = 8) and those without (group B, n = 8). There were 6 males and 2 females with an average age of (70 ± 8) years in the group A and 5 males and 3 females with an average age of (74 ± 8) years in the group B. And the control group had 6 males and 2 females with an average age of (72 ± 6) years. All samples of total quadriceps protein were separated by two-dimensional gel electrophoresis. The abnormal protein points on electrophoresis were compared by PDQuest image software. And the differential protein expression was detected. Then the corresponding peptide quality fingerprint spectrum was analyzed by mass spectrometer. Finally the differential protein points were partially detected by a search of database. The two-dimensional gel electrophoresis yielded an excellent profile of resolution and repeatability. And 12 proteins likely to cause skeletal muscle atrophy in COPD were identified. Among them, 8 proteins belonged to structural proteins (actin alpha cardiac muscle isoform CRA_c, myosin regulatory light chain 2, ventricular/cardiac muscle isoform, myoglobin isoform myosin heavy polypeptide 7 cardiac muscle beta isoform CRA_c, actin, alpha skeletal muscle, actin alpha cardiac muscle isoform CRA_b, hemoglobin alpha 1 globin chain & myosin light chain 6B) and 4 proteins were of functional proteins (chain A, crystal structure of human enolase; troponin T, slow skeletal muscle isoform alpha, carbonate anhydrase, troponin T & slow skeletal muscle isoform b). COPD patients are often accompanied with obvious peripheral skeletal muscle atrophy. It may be caused by quantitative or qualitative changes of peripheral skeletal structural and functional proteins.

CaMKII inhibition in heart failure, beneficial, harmful, or both

Calmodulin-dependent protein kinase II (CaMKII) has been proposed to be a therapeutic target for heart failure (HF). However, the cardiac effect of chronic CaMKII inhibition in HF has not been well understood. We have tested alterations of Ca(2+) handling, excitation-contraction coupling, and in vivo β-adrenergic regulation in pressure-overload HF mice with CaMKIIδ knockout (KO). HF was produced in wild-type (WT) and KO mice 1 wk after severe thoracic aortic banding (sTAB) with a continuous left ventricle (LV) dilation and reduction of ejection fraction for up to 3 wk postbanding. Cardiac hypertrophy was similar between WT HF and KO HF mice. However, KO HF mice manifested exacerbation of diastolic function and reduction in cardiac reserve to β-adrenergic stimulation. Compared with WT HF, L-type calcium channel current (I(Ca)) density in KO HF LV was decreased without changes in I(Ca) activation and inactivation kinetics, whereas I(Ca) recovery from inactivation was accelerated and Ca(2+)-dependent I(Ca) facilitation, a positive staircase blunted in WT HF, was recovered. However, I(Ca) response to isoproterenol was reduced. KO HF myocytes manifested dramatic decrease in sarcoplasmic reticulum (SR) Ca(2+) leak and slowed cytostolic Ca(2+) concentration decline. Sarcomere shortening was increased, but relaxation was slowed. In addition, an increase in myofilament sensitivity to Ca(2+) and the slow skeletal muscle troponin I-to-cardiac troponin I ratio and interstitial fibrosis and a decrease in Na/Ca exchange function and myocyte apoptosis were observed in KO HF LV. CaMKIIδ KO cannot suppress severe pressure-overload-induced HF. Although cellular contractility is improved, it reduces in vivo cardiac reserve to β-adrenergic regulation and deteriorates diastolic function. Our findings challenge the strategy of CaMKII inhibition in HF.

Expression of slow skeletal TnI in adult mouse hearts confers metabolic protection to ischemia

Changes in metabolic and myofilament phenotypes coincide in developing hearts. Posttranslational modification of sarcomere proteins influences contractility, affecting the energetic cost of contraction. However, metabolic adaptations to sarcomeric phenotypes are not well understood, particularly during pathophysiological stress. This study explored metabolic adaptations to expression of the fetal, slow skeletal muscle troponin I (ssTnI). Hearts expressing ssTnI exhibited no significant ATP loss during 5min of global ischemia, while non-transgenic littermates (NTG) showed continual ATP loss. At 7min ischemia TG-ssTnI hearts retained 80±12% of ATP versus 49±6% in NTG (P<0.05). Hearts expressing ssTnI also had increased AMPK phosphorylation. The mechanism of ATP preservation was augmented glycolysis. Glycolytic end products (lactate and alanine) were 38% higher in TG-ssTnI than NTG at 2min and 27% higher at 5min. This additional glycolysis was supported exclusively by exogenous glucose, and not glycogen. Thus, expression of a fetal myofilament protein in adult mouse hearts induced elevated anaerobic ATP production during ischemia via metabolic adaptations consistent with the resistance to hypoxia of fetal hearts. The general findings hold important relevance to both our current understanding of the association between metabolic and contractile phenotypes and the potential for invoking cardioprotective mechanisms against ischemic stress. This article is part of a Special Issue entitled "Possible Editorial".

Proteomics Comparison of Longissimus Muscle between Hanwoo and Holstein Cattle

This study was conducted to compare proteins expressed in M. longissimus from Hanwoo and Holstein steers immediatelyafter slaughter. Two-dimensional electrophoresis (2DE)/LC-MS/MS analysis revealed that the total number of detectableprotein spots from longissimus muscle tissues was slightly higher in Hanwoo (575 ± 65) than Holstein (534 ± 13) steers, butthat these numbers were not statistically significant due to large variation between replicates. A total of twelve protein spot sdid not match between sample groups, eight of which were expressed in the Hanwoo sample and four that were expressedin the Holstein sample. The protein spots detected in the Hanwoo sample included smooth muscle and non-muscle myosinalkali light chain 6B isomers, αB crystallin isomers, hemoglobin β-A chains, slow myosin heavy chains, and slow skeletalmuscle troponin T chains. Collectively, these proteins are a class of slow-twitch muscle fiber and mirror that Hanwoo mus-cle tissue sampled for the current study contained more slow-twitch muscle fibers than Holstein one. Conversely, proteinsdetected from the Holstein sample included ankyrin repeat domain 2 and creatin kinase isomers. Given that creatin kinaseisomers are related to the fast-twitch muscle, these results likely indicate that Holstein muscle tissue sampled for the curren tstudy contained more fast-twitch muscle fibers than Hanwoo beef.Key words: Hanwoo, Holstein, muscle proteome, meat quality, slow-twitch muscle

Abstract 3984: Effect of zeranol on beef skeletal muscle growth by differential image gel electrophoresis (DIGE)

Abstract Zeranol is a resorcylic acid lactone produced by fungi of the genus Fusarium that grows on corn, wheat, barley, oats and sorghum. As such Zeranol is a virtually unavoidable contaminant of crops used to feed animals that are consumed by humans. Zeranol has been shown to have a positive impact on muscle growth during the finishing phase of beef cattle production, was first approved and licensed for use as a growth promotant in cattle and sheep in the USA by FDA in 1969. However, the mechanism of this interaction is unknown but is likely related to changes in muscle specific proteins observable during the increased growth phase during finishing. To determine the affect of Zeranol on beef cattle muscle growth, we performed four DIGE experiments. We compared protein expression level changes between beef cattle treated with Zeranol and untreated beef cattle. On day zero, 10 cattle were implanted with 72 mg of Zeranol pellets, the other half (n = 10) were implanted with vehicle. The beef cattle were raised at the Ohio State University Department of Animal Sciences Beef Barn in accordance with industry standards for 120 days. At 60 days, half the implanted beef cattle and half of the control beef cattle were harvested and the remaining beef cattle were harvested at day 120. Muscle samples were analyzed by DIGE and bioinformatics Seven proteins associated with energy metabolism (creatine kinase, glycogen phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucomutase I, aconitase 2, enolase I, and triosephophate isomerase), 5 proteins with regulation of muscle contraction (slow skeletal muscle troponin T, calsequestrin I, myosin light chains 2 and 3, tropomyosin 3), 3 structural proteins (alpha actin, myosin binding protein C and kelch repeat and BTB domain), a protein associated with myotube formation, dihydrolipoamide dehydrogenase, and HSP70, a protein associated with meat quality, and albumin were identified. The differential regulation of these proteins indicates that muscle growth in cattle implanted with Z is due to fast skeletal muscle. (Supported by NIH grant R01 ES 015212). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3984.

Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle

The loss of slow skeletal muscle troponin T (TnT) results in a recessive nemaline myopathy in the Amish featured with lethal respiratory failure. The genes encoding slow TnT and cardiac troponin I (TnI) are closely linked. Ex vivo promoter analysis suggested that the 5'-enhancer region of the slow TnT gene overlaps with the structure of the upstream cardiac TnI gene. Using transgenic expression of exogenous cardiac TnI to rescue the postnatal lethality of a mouse line in which the entire cardiac TnI gene was deleted, we investigated the effect of enhancer deletion on slow TnT gene expression in vivo and functional consequences. The levels of slow TnT mRNA and protein were significantly reduced in the diaphragm muscle of adult double transgenic mice. The slow TnT-deficient (ssTnT-KD) diaphragm muscle exhibited atrophy and decreased ratios of slow versus fast isoforms of TnT, TnI, and myosin. Consistent with the changes toward more fast myofilament contents, ssTnT-KD diaphragm muscle required stimulation at higher frequency for optimal tetanic force production. The ssTnT-KD diaphragm muscle also exhibited significantly reduced fatigue tolerance, showing faster and more declines of force with slower and less recovery from fatigue as compared with the wild type controls. The natural switch to more slow fiber contents during aging was partially blunted in the ssTnT-KD skeletal muscle. The data demonstrated a critical role of slow TnT in diaphragm function and in the pathogenesis and pathophysiology of Amish nemaline myopathy.

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.

Thin filament proteins mutations associated with skeletal myopathies: Defective regulation of muscle contraction

In humans, more than 140 different mutations within seven genes (ACTA1, TPM2, TPM3, TNNI2, TNNT1, TNNT3, and NEB) that encode thin filament proteins (skeletal alpha-actin, beta-tropomyosin, gamma-tropomyosin, fast skeletal muscle troponin I, slow skeletal muscle troponin T, fast skeletal muscle troponin T, and nebulin, respectively) have been identified. These mutations have been linked to muscle weakness and various congenital skeletal myopathies including nemaline myopathy, distal arthrogryposis, cap disease, actin myopathy, congenital fiber type disproportion, rod-core myopathy, intranuclear rod myopathy, and distal myopathy, with a dramatic negative impact on the quality of life. In this review, we discuss studies that use various approaches such as patient biopsy specimen samples, tissue culture systems or transgenic animal models, and that demonstrate how thin filament proteins mutations alter muscle structure and contractile function. With an enhanced understanding of the cellular and molecular mechanisms underlying muscle weakness in patients carrying such mutations, better therapy strategies can be developed to improve the quality of life.

Identification and analysis of teleost slow muscle troponin T (sTnT) and intronless TnT genes

In the present study cDNA clones representing two slow skeletal muscle troponin T genes ( sTnT1sb and sTnT2sb) in the sea bream ( Sparus auratus), an important aquaculture species, were isolated and characterised. A third, intronless, TnT gene ( iTnTsb), which is an apparent orthologue of a previously described zebrafish TnT, was also isolated. In adult sea bream sTnT expression was restricted to red muscle and, using northern blotting, a single low abundance transcript was identified for sTnT1sb (1260 nucleotides) and a single high abundance transcript was identified for sTnT2sb (1000 nucleotides). In contrast, iTnTsb is predominantly expressed in adult fast muscle. All three TnT genes are also expressed during larval development. Phylogenetic analysis of sea bream sTnT proteins to identify maximum parsimony showed that iTnTsb, sTnT1sb and sTnT2sb each cluster in independent groups. sTnT1sb clustered with other vertebrate sTnTs, while sTnT2 clustered with a group of fish specific sequences (from Fugu rubripes, Oryzia latipes and Salmo trutta). The teleost sTnT2 and iTnT each constitute new, apparently teleost specific, TnT groups. Analysis of the corresponding Fugu scaffold indicates that sTnT2sb is encoded by a gene with twelve exons. The two sTnT cDNAs isolated in sea bream probably arose by duplication of an ancestral gene, and iTnT by reverse transcription. It remains to be established if the encoded proteins have different structural and mechanistic roles in fish muscle.

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.

The slow skeletal muscle troponin T gene is expressed in developing and diseased human heart.

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease. (Mol Cell Biochem 263: 91-97, 2004).

Genomic structure of the chicken slow skeletal muscle troponin T gene

Troponin T (TnT) is a key protein for Ca 2+-sensitive molecular switching of muscle contraction. In vertebrates, three TnT genes have been identified, which produce isoforms characteristic of cardiac, fast skeletal, and slow skeletal muscles through alternative splicing in a tissue-specific and developmentally regulated manner. The diversification of myofibers into forms with specific metabolic and contractile characteristics is thought to be closely associated with the differential expression of these TnT isoforms. Herein, we determined the nucleotide sequence of the chicken slow skeletal muscle TnT gene and its upstream region. The gene was simpler in structure than the two other chicken genes. The transcription initiation site was positioned 183 bp upstream of the 3′ end of exon 1. Alternative splicing of exon 5 using an internal acceptor site generated two distinct slow skeletal muscle troponin T (sTnT) transcripts. We identified possible regulatory elements, M-CAT-like, CACC-box, and E-box (E-box1 to E-box3) motifs in the upstream region and an E-box motif (E-box4) in exon 1.

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.

Mutations in the β-tropomyosin ( TPM2) gene – a rare cause of nemaline myopathy

Nemaline myopathy is a clinically and genetically heterogeneous muscle disorder. In the nebulin gene we have detected a number of autosomal recessive mutations. Both autosomal dominant and recessive mutations have been detected in the genes for α-actin and α-tropomyosin 3. A recessive mutation causing nemaline myopathy among the Old Order Amish has recently been identified in the gene for slow skeletal muscle troponin T. As linkage studies had shown that at least one further gene exists for nemaline myopathy, we investigated another tropomyosin gene expressed in skeletal muscle, the β-tropomyosin 2 gene. Screening 66 unrelated patients, using single strand conformation polymorphism analysis and sequencing, we found four polymorphisms and two heterozygous missense mutations. Both mutations affect conserved amino acids, and in both cases, the mutant allele is expressed. We speculate that the observed mutations affect the formation of the tropomyosin dimer and its actin-binding properties.

A pH-Sensitive Interaction of Troponin I with Troponin C Coupled with Strongly Binding Cross-Bridges in Cardiac Myofilament Activation

Slow skeletal muscle troponin I (ssTnI) expressed predominantly in perinatal heart confers a marked resistance to acidic pH on Ca2+ regulation of cardiac muscle contraction. To explore the molecular mechanism underlying this phenomenon, we investigated the roles of TnI isoforms (ssTnI and cardiac TnI (cTnI)) in the thin filament activation by strongly binding cross-bridges, by exchanging troponin subunits in cardiac permeabilized muscle fibers. Fetal cardiac muscle showed a marked resistance to acidic pH in activation of the thin filament by strongly binding cross-bridges compared to adult muscle. Exchanging ssTnI into adult fibers altered the pH sensitivity from adult to fetal type, indicating that ssTnI also confers a marked resistance to acidic pH on the cross-bridge-induced thin filament activation. However, the adult fibers containing ssTnI or cTnI but lacking TnC showed no pH sensitivity. These findings provide the first evidence for the coupling between strongly binding cross-bridges and a pH-sensitive interaction of TnI with TnC in cardiac muscle contraction, as a molecular basis of the mechanism conferring the differential pH sensitivity on Ca2+ regulation.