Slow Skeletal Muscle Isoform Research Articles

Existence in the actin world of a specialized slow skeletal muscle isoform.

1) Teleosts such as Atlantic salmon Salmo salar and Atlantic herring Clupea harengus , in contrast to other vertebrates such as mammals, synthesize two contextually very different skeletal muscle actins. Each isoform has been studied at the protein level. One isoform accounts for the total actin in the lighter ‘fast’ trunk muscle. Based on closest pairings - two amino acid substitutions (salmon vs. herring) and four substitutions (salmon or herring vs. mammal or bird) - it is classified as ‘fast’ skeletal actin. The other isoform accounts for the total actin in the darker ‘slow’ muscle in the lateral line and represents a specialized slow skeletal muscle actin which is an unrecognized isotype. 2) Atlantic salmon and Atlantic herring also synthesize a cardiac actin that is distinct to the skeletal actins in 1). Intraspecies pairings of fast skeletal actin and cardiac actin have two amino acid substitutions. Conversely, those involving slow skeletal actin have 12 or 13, which is a contextual record. 3) The Salmonidae skeletal actins in 2) differ at residues 2, 3, 103, 155, 165, 278, 281, 310, 329, 358, 360 and 363 (numbering system for mature protein of 375 amino acids). The changes are restricted to subdomains 1 and 3 and have affected the net charge at pH 7, polarity and glycine content. Six can be considered non-conservative: Val103Thr (Val in ‘slow’, Thr in ‘fast’); Ala155Ser; Thr278Ala; Gly281Ser; Gly310Ala and Asp360Gln. The change at residue-360 provides a convenient means of detection via electrophoresis. Biochemical characterization of protein points to an advantageous function by which ‘slow’ actin contributes to the distinct physiology of the dark cruise muscle in the trunk. 4) Slow skeletal muscle actin (no more than two substitutions per pairing) is encoded in the genomes of the following families of Teleostei: Salmonidae, Clupeidae, Anguillidae; Cyprinidae; Esocidae; Ictaluridae; Osteoglossoidei and Serrasalmidae. However, many NCBI entry descriptions are confusing. The ‘slow’ actin group is expected to grow as more genomes are sequenced. 5) Slow skeletal actin genes display variable exon structure in the region encoding amino acids 44–151 (numbering system for 377 amino acids). The other exon-intron boundaries are conserved. In most cases to date, but not all, the ‘slow’ actin gene contains a greater number of exons than the ‘fast’ actin gene from the same species. 6) Slow skeletal actin falls into one of the new actin groups, namely epsilon which has representation in fish, reptile, bird and mammal. To date, only the protein products from Salmonidae and herring have been studied biochemically. • Atlantic salmonidae and herring synthesize slow skeletal, fast skeletal and cardiac muscle isoactins. • The distribution is specific; one isoform per muscle type. • The biochemical properties of slow skeletal actin confer an advantageous function on the dark cruise muscle in the trunk. • Slow skeletal actin is encoded in the genomes of more than a dozen other Teleosts. • Slow skeletal actin falls in the new actin group given the description ACT epsilon 1.

Proteomics analysis as an approach to understand the formation of pale, soft, and exudative (PSE) pork

We investigated ten pale, soft, and exudative (PSE), and ten normal meat samples from pig carcasses. The meat quality at 0, 5, 12, and 24 h post-mortem and the key enzyme activities at 0 and 24 h post-mortem were determined. We selected three PSE and three normal samples for proteomics analysis at 0 h and 24 h post-mortem. No remarkable differences in pyruvate kinase (PK) and lactate dehydrogenase (LDH) activity were observed between samples at 0 h post-mortem; however, creatine kinase (CK) activity was significantly higher in PSE meat. Hexokinase (HK) activity in PSE samples was higher than that in normal samples at 24 h post-mortem. Bioinformatics analysis of the proteome showed that PSE was related to glycolysis, TCA cycle, oxidative phosphorylation, muscle tissue structure, signal transduction, and molecular chaperones. This research found that proteins such as troponin T slow skeletal muscle isoform X, GADPH, L-lactate dehydrogenase A chain, and gamma-enolase isoform X1 might be responsible for PSE.

CONGENITAL MYOPATHIES 1 – NEMALINE: P.29 AAV gene therapy for TNNT1-associated Nemaline myopathy

Nemaline myopathy is one of the most common congenital myopathies. The hallmarks of this disorder are early onset muscular weakness, hypotonia, pectus carinatum and rod-like inclusions in muscle cells. The severity spectrum of nemaline myopathy is broad, but early respiratory failure and death are the inevitable outcomes in most of the cases. Several gene mutations have been identified in nemaline myopathy, including a non-sense mutation in exon 11 of TNNT1 gene, encoding for the slow skeletal muscle isoform of troponin T (TnT), which results in selective atrophy of slow-twitch (type I) myofibers and in a unique form of Nemaline myopathy, named Amish Nemaline Myopathy (ANM). Currently there is no treatment for ANM, but the development of an AAV gene therapy is viable and explored in our research. Such an approach requires the restricted expression of TNNT1 to slow-twitch fibers, to avoid potential deleterious effects associated with the ectopic expression of TNNT1 in cardiac and fast-twitch myofibers. We achieved this, in wild-type mice, by using a series of AAV vectors carrying a strong muscle-specific promoter (MHCK7) to express both TNNT1 and suppressive microRNAs (miR208a and miR-133, characteristic of cardiac and fast-twitch myofibers respectively). The next step will be a dose escalation study in Tnnt1−/− mice, to assess TNNT1 expression by western blot and by immunostaining for both TNNT1 and myofiber type specific markers. We will then quantify the changes in skeletal muscle histology (fiber diameter and number of centronucleated fibers) to assess the therapeutic efficacy of our AAV vectors.

The loss of slow skeletal muscle isoform of troponin T in spindle intrafusal fibres explains the pathophysiology of Amish nemaline myopathy.

The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.

Heterozygous variants in MYBPC1 are associated with an expanded neuromuscular phenotype beyond arthrogryposis.

Encoding the slow skeletal muscle isoform of myosin binding protein-C, MYBPC1 is associated with autosomal dominant and recessive forms of arthrogryposis. The authors describe a novel association for MYBPC1 in four patients from three independent families with skeletal muscle weakness, myogenic tremors, and hypotonia with gradual clinical improvement. The patients carried one of two de novo heterozygous variants in MYBPC1, with the p.Leu263Arg variant seen in three individuals and the p.Leu259Pro variant in one individual. Both variants are absent from controls, well conserved across vertebrate species, predicted to be damaging, and located in the M-motif. Protein modeling studies suggested that the p.Leu263Arg variant affects the stability of the M-motif, whereas the p.Leu259Pro variant alters its structure. In vitro biochemical and kinetic studies demonstrated that the p.Leu263Arg variant results in decreased binding of the M-motif to myosin, which likely impairs the formation of actomyosin cross-bridges during muscle contraction. Collectively, our data substantiate that damaging variants in MYBPC1 are associated with a new form of an early-onset myopathy with tremor, which is a defining and consistent characteristic in all affected individuals, with no contractures. Recognition of this expanded myopathic phenotype can enable identification of individuals with MYBPC1 variants without arthrogryposis.

Loss of the Slow Skeletal Muscle Isoform of Troponin T Impairs Motor Coordination in Mice

The loss of the slow skeletal muscle isoform of troponin T (ssTnT) encoded by TNNT1 causes lethal myopathies with symptoms including abnormal neuromuscular reflexes. It has been a challenging question that how the loss of one isoform of TnT results in extremely severe phenotypes while the fast isoform of TnT is present in the patient muscles. We investigated ssTnT knockout (KO) mice for the role of ssTnT in muscle spindles that are a mechanosensors critical to normal neuromuscular function. Immunohistological and spindle protein content studies indicated that unlike extrafusal muscle fibers, adult ssTnT-KO mouse spindle intrafusal fibers express high levels of cardiac TnT, which is significantly increased in intrafusal fibers of ssTnT-KO mice. Impaired motor coordination was observed in ssTnT-KO mice with slower walking during a balance beam performance test as compared with age/sex-matched wildtype (WT) control (p<0.05). An interesting observation from the balance beam performance test is that subcutaneous injection of succinylcholine (SCh), a muscle relaxant believed to alter spindle activities, significantly increased the time for WT mice to walk across the balance beam (p<0.01) whereas SCh treatment produced a trend for decreased time across the beam in ssTnT-KO mice (p=0.10). Taken together, our data suggest that the loss of ssTnT in the KO mice impairs spindle intrafusal fiber contractility, identifying a novel therapeutic target. The attenuated effect of SCh treatment on motor coordination in the ssTnT-KO mice may be due to the potentially adaptive increased expression of cardiac TnT in spindle intrafusal fibers. Further testing is currently underway to assessing the hypothesized adaptation of spindle intrafusal fiber TnT isoform expression and function in ssTnT-KO mice during postnatal development.

Molecular Cloning, Characterization, and Temporal Expression Profile of Troponin I Type 1 (TNNI1) Gene in Skeletal Muscle During Early Development of Gaoyou Duck (Anas Platyrhynchos Domestica)

ABSTRACTTNNI1 encodes the slow skeletal muscle isoform of troponin I. In the present study, the basic characteristic and expressing profile of the TNNI1 gene was first explored in Gaoyou ducks. Full-length TNNI1 cDNA of Gaoyou duck was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA consisted of a 57-base pair (bp) 5′UTR, a 345-bp 3′UTR, and a 564-bp open reading frame. The predicted protein was predicted to be hydrophilic, nonsecretory protein and contained 17 phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted protein was relatively conserved in avian. TNNI1 mRNA could be detected in every tissue analyzed at 70 days of age, and the muscle tissues had relatively high expression level, with the highest level seen in leg muscle. The TNNI1 gene was differentially expressed in the breast muscle and leg muscle during embryonic and posthatching development. Our findings reveal the sequence characterization and expression patterns of the TNNI1 gene, which may provide correlative evidence that TNNI1 gene plays an important role in duck muscle fiber development and meat quality.

Development and Phenotype Studies of a Slow Skeletal Troponin T E180 Nonsense Mutation Knock-In Mouse Line

A nonsense mutation in codon Glu180 of TNNT1 gene encoding the slow skeletal muscle isoform of troponin T (ssTnT) causes a recessively inherited severe nemaline myopathy (Amish nemaline myopathy, ANM). Muscle biopsies of ANM patients showed a total loss of ssTnT. The present study developed and characterized mice with the E180 nonsense mutation knocked-in the Tnnt1 gene (ssTnT-KI). Homozygotes of ssTnT-KI mice survived to adulthood. Histology studies revealed significant increases of centralized nuclei in the soleus muscle of ssTnT-KI when compared with age matched wild type controls. Immunohistochemistry staining showed atrophic slow type 1 fibers in ssTnT-KI mouse soleus muscle with severe inflammatory cells infiltration. Western blots confirmed the absence of full length ssTnT with no ssTnT fragments detectable, mimicking that seen in the muscle of ANM patients. Contractile properties of ssTnT-KI mouse soleus muscle measured in situ demonstrated a decreased fatigue resistance. Significant expression of cardiac TnT is detected in soleus muscle of ssTnT-KI mice, consistent with the presence of active muscle regeneration. Decrease of slow type isoforms and increase of fast type isoforms of other myofilament proteins with a significant hypertrophy of fast fibers in ssTnT-KI mouse soleus muscle indicate a potentially compensatory adaptation to the loss of slow fiber functions. The ssTnT-KI mouse line provides a model of ANM for pathogenesis, pathophysiology and therapeutic studies.

Expanding the MYBPC1 phenotypic spectrum: a novel homozygous mutation causes arthrogryposis multiplex congenita.

Arthrogryposis multiplex congenita (AMC) is characterized by heterogeneous nonprogressive multiple joint contractures appearing at birth. We present a consanguineous Israeli-Druze family with several members presenting with AMC. A variable intra-familial phenotype and pected autosomal recessive inheritance prompted molecular diagnosis by whole-exome sequencing. Variant analysis focused on rare homozygous changes, revealed a missense variant in MYBPC1, NM_002465:c.556G>A (p.E286K), affecting the last nucleotide of Exon 8. This novel variant was not observed in the common variant databases and co-segregated as expected within the extended family. MYBPC1 encodes a slow skeletal muscle isoform, essential for muscle contraction. Heterozygous mutations in this gene are associated with distal arthrogryposis types 1b and 2, whereas a homozygous nonsense mutation is implicated in one family with lethal congenital contractural syndrome 4. We present a novel milder MYBPC1 homozygous phenotype.

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.

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

Structure and expression of the human slow twitch skeletal muscle troponin I gene.

The contractile protein troponin I is encoded by a multigene family whose members are expressed differentially in various classes of muscle fibers. In vertebrates, the "slow" isoform of troponin I is expressed during early heart and skeletal muscle development but is restricted to slow twitch skeletal muscle in the adult. This diverse expression pattern offers an opportunity to study the regulation of a single gene within different developmental contexts. To initiate such studies, we have cloned the gene encoding the human slow twitch skeletal muscle isoform of troponin I and have identified 5'-flanking sequences required for its expression in skeletal muscle cells. The slow troponin I gene spans 12.5 kilobases and is divided into nine exons. In contrast to many muscle-specific genes, the troponin I promoter does not contain consensus CCAAT or TATA elements. Moreover, the sequence from -9 to +11 resembles an "initiator element" previously shown to direct transcription of some tissue-specific genes lacking TATA boxes (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113; Brand, N. J., Petkovich, M., and Chambon, P. (1990) Nucleic Acids Res. 18, 6799-6806; Weis, L., and Reinberg, D. (1992) FASEB J. 6, 3300-3309). A transcriptional fusion construct, comprising 4.2 kilobases of troponin I 5'-flanking DNA linked to the bacterial chloramphenicol acetyl-transferase gene, exhibited cell type-specific and developmentally regulated expression. A muscle-specific enhancer regulated slow troponin I promoter activity.

Molecular cloning and expression of chicken cardiac troponin C.

We have isolated a full length complementary DNA clone (pCTnC1) from a 19-day embryonic chicken heart library corresponding to cardiac troponin C (TnC). Sequence analysis demonstrated varying homologies with TnC complementary DNA clones isolated from developing chick skeletal muscle. Using pCTnC1 as a hybridization probe, we have determined that cardiac TnC is constitutively expressed in both atria and ventricles of the developing and adult heart. These data along with previous immunochemical studies of TnC expression demonstrate that the slow skeletal and cardiac muscle isoform is the only TnC expressed in the heart. In contrast, expression of slow skeletal and cardiac muscle TnC is developmentally regulated in skeletal muscles of the chicken. The tightly controlled expression of slow skeletal and cardiac muscle TnC in the varying myocyte types of the heart suggests a physiologically significant role of this regulatory protein.

Chromosomal assignment of two myosin alkali light-chain genes encoding the ventricular/slow skeletal muscle isoform and the atrial/fetal muscle isoform (MYL3, MYL4).

In all eukaryotes, myosin plays a major role in the maintenance of cell shape and in cellular movement; in association with actin and other contractile proteins it is also a major structural component of the muscle sarcomere. Several isoforms of myosin alkali light chain have been identified, associated with different muscle types. We have recently localized the gene encoding the fast skeletal muscle alkali light-chain isoforms MLC1F and MLC3F (HGM symbol, MYL1) to human chromosome 2q32.1-qter (Cohen-Haguenauer 1988). We present here the chromosomal assignment of two loci encoding the ventricular muscle isoform MLC1V (equivalent to the slow skeletal muscle isoform MLC1Sb) and the atrial muscle isoform MLC1A (equivalent to the fetal isoform MLC1emb) using a panel of 25 independent man-rodent somatic cell hybrids. The MLC1V gene (HGM symbol, MYL3) was mapped to human chromosome 3 using a human full-length cDNA probe that hybridizes to a single major human TaqI2.8-kb fragment. The MLC1A probe (HGM symbol, MYL4) was a 360-bp mouse cDNA fragment that gave a distinct signal with human DNA using low stringency conditions of hybridization and washings and after presaturation of the Southern blots with rodent DNA. A single PstI 7.8-kb fragment gives an intense signal, and its presence correlates with the presence of chromosome 17 among the hybrids. These data are in keeping with the localizations of the MLC1V gene to mouse chromosome 9, and of the MLC1A gene to mouse chromosome 11, which share some markers in common with human chromosomes 3 and 17 respectively.

Identification and pattern of expression of a developmental isoform of troponin I in chicken and rat cardiac muscle.

A monoclonal antibody that reacts with all known isoforms of troponin I detected a single isoform of cardiac troponin I in both atrial and ventricular chambers of adult chicken and rat hearts in an immunoblotting analysis. Another isoform of troponin I in addition to the adult cardiac form, however, was present in all chambers of the heart during early development in both species. This developmental isoform appeared to have the same electrophoretic mobility on SDS tris glycine polyacrylamide gels as that observed for the adult slow skeletal muscle isoform. In the rat, only the developmental isoform of troponin I was present in the early foetal heart and small amounts of the adult cardiac isoform were not apparent until late in gestation, whereas the developmental and adult isoforms were expressed in approximately equal amounts throughout embryonic development in the chicken. The level of developmental isoform of troponin I in both the chicken and the rat hearts gradually decreased so that only small amounts of this variant were detectable two weeks after birth or post hatching.