Gurtej K. Dhoot

Polymorphic forms of troponin T and troponin C and their localization in striated muscle cell types.

Abstract 1. 1. Immunochemical studies have shown that the major forms of troponin T present in fast skeletal, slow skeletal and cardiac muscles are different proteins. 2. 2. Similar studies indicate that the major form of troponin C present in fast skeletal muscles differs from troponin C present in slow skeletal and cardiac muscle cells. The forms of troponin C present in slow skeletal and cardiac muscles are immunochemically very similar. 3. 3. The antibodies to the polymorphic forms of troponin T and troponin C are specific for the muscle type, except in the case of the slow skeletal and cardiac muscle forms of troponin C. 4. 4. By the immunoperoxidase technique, it has been shown that the fast skeletal muscle troponin T is localized in type II cells and slow skeletal muscle troponin T in type I cells. 5. 5. Fast skeletal muscle troponin C is present in type II cells and a different troponin C, identified by its reaction with the antibody against cardiac troponin C, is present in type I cells. 6. 6. It is concluded that in normal adult skeletal muscle, fast muscle forms of troponin I, troponin T and troponin C are present together as a homocomplex in type II cells and the slow muscle forms exist as an analagous homocomplex in type I cells.

The localization of the different forms of troponin I in skeletal and cardiac muscle cells

Abstract 1. 1. Antibodies raised against troponin I isolated from human cardiac and rabbit fast and slow skeletal muscles have been shown to be specific for the polymorphic forms of troponin I against which they were raised, i.e. they are tissue specific. 2. 2. These antibodies reacted with the polymorphic forms of troponin I, against which they were raised, that are present in tissues of other species such as the rhesus monkey, hamster and rat, i.e. they were species non-specific. 3. 3. Using the immunoperoxidase staining technique it has been shown that the fast and slow forms of troponin I are located in different cells in virtually all adult normal muscles examined. 4. 4. By comparison of the ATPase staining of skeletal muscle sections at pH 9.4 and 4.2 it is concluded that the fast form of troponin I is located in type II fibres and the slow form in type I fibres. 5. 5. It is suggested that immunoperoxidase staining with the antibodies to the fast and slow forms of troponin I provides an unambiguous new method of muscle fibre typing.

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

SummaryA monoclonal antibody that reacts with all known isoforms of troponin I detected a single isoform of cardiac troponin I in both a trial 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.

The effect of cross-innervation on the tropomyosin composition of rabbit skeletal muscle

Soleus, semitendinosus and crureus muscles of the rabbit were found to contain α‐ and β‐tropomyosin subunits and additional forms that have been provisionally designated γ and δ. Extensor digitorum longus and psoas muscles contained only α and β subunits, the relative proportions of which varied between single fibres of psoas muscle. On cross‐innervation of rabbit soleus and extensor digitorum longus muscles, the fraction of the total tropomyosin present as the β subunit remained constant. The relative proportions of α, γ and δ subunits changed as would be expected from the change in speed that occurred.

The effect of denervation on the distribution of the polymorphic forms of troponin components in fast and slow muscles of the adult rat

SummaryIn the soleus muscle of the normal rat the number of cells containing fast troponin I decreased and those containing slow troponin I increased after birth until less than 10% stained for the fast form in the adult muscle. On denervation of soleus muscle this pattern of change was reversed with the result that the majority of cells stained for fast troponin I. The change was more rapid when denervation was carried out at 12 weeks rather than at 52 weeks of age. Denervation of extensor digitorum longus and tibialis anterior muscles produced little change in the distribution of fast and slow troponin I over a period of 12 weeks. After long periods (>24 weeks) of denervation of these fast muscles, fast troponin I was observed in cells in which originally only slow troponin I could be detected. Similar results to those obtained with troponin I in both fast and slow muscles were obtained using antibodies to the fast and slow forms of troponin C and troponin T.

The isoforms of C protein and their distribution in mammalian skeletal muscle

SummaryA monoclonal antibody that is specific for the slow skeletal muscle isoform of C protein of rabbit muscle has been prepared by immunizing mice with a crude preparation of human myosin. It reacted with the X protein fraction of rabbit skeletal muscle and stained all type I cells in this tissue. It also stained a fraction of the type II cells with varying intensities. The type II cells staining with antibody to slow C protein also stained with a polyclonal antibody prepared against rabbit fast muscle C protein. The type II cells not staining with antibody to slow C protein stained strongly with antibody to fast C protein. In the human skeletal muscle antibody to slow C protein stained all cells whereas antibody to fast C stained only type II cells. It is concluded that the distribution of the isoforms of C protein in adult vertebrate skeletal muscle is more complex than is the case with proteins such as components of the troponin complex.

Distribution of isoforms of the myofibrillar proteins in myoid cells of thymus.

SummaryMyoid cells of calf and rat thymus have been identified by staining with a monoclonal antibody to the heavy chain of myosin that is not isoform specific. Heterogeneity in the protein composition of myoid cells has been demonstrated by staining with antibodies to the skeletal muscle isoforms of the myosin heavy chain, C-protein and components of the troponin complex. The immunochemical studies suggest that the myoid cells contain proteins closely resembling if not identical with those present in the myofibrils of skeletal muscle. The slow and fast skeletal muscle isoforms of the myofibrillar proteins are present in a large proportion of the myoid cells. A fraction of the myoid cells contains only the fast isoforms of the myofibrillar proteins but there is no sharp compartmentalization of the isoforms as occurs in type 1 and type 2 fibres of skeletal muscle. In general the pattern of gene expression is similar to that of developing skeletal muscle.

Identification and distribution of some developmental isoforms of myosin heavy chains in avian muscle fibres

SummaryTwo monoclonal antibodies that react with all the slow skeletal myosin heavy chains in the mammalian skeletal muscles appeared to react with only SM1 myosin heavy chain in the post-hatch muscles of chicken. Further studies on the developing chicken showed one of these two antibodies to react with an additional myosin heavy chain in the early embryonic skeletal muscle as well as with the cardiac muscle. It is concluded that this antibody identified a slow muscle-like embryonic isoform of myosin heavy chain during earlier stages of development. While this embryonic isoform was more abundant during early development, the synthesis of SM1 myosin heavy chain was restricted to only presumptive slow muscle cells.

Identification and distribution of the fast class of troponin T in the adult and developing avian skeletal muscle

SummaryIn the adult chicken, two groups of fast troponin T, the higher molecular weight (B1 and B2) present only in the pectoral muscle and the lower molecular weight (L1–L5) the only group present in the leg muscle, were identified by the immunoblotting procedure using monoclonal antibodies against fast skeletal troponin T. The presence of significant amounts of three major variants of leg muscle type troponin T (L3–L5), however, could also be detected in the adult chicken pectoral muscle. Although none of the antibodies cross-reacted with slow troponin T itself, the proportions of both leg and pectoral type troponin T variants belonging to the fast class varied in fast muscles that contained slow muscle fibres or fast muscles devoid of slow muscle cells.The troponin T present in the early embryonic skeletal muscles did not react with any of the antibodies raised against adult fast isoforms. The gradual expression of some of the adult isoforms of troponin T was detected at about day 13in ovo. However, the adult isoforms did not all appear simultaneously and their full complement was not achieved until after hatching. In addition to the increased number of bands in the leg type troponin T region, the presence of two other protein bands (neonatal forms) with slower electrophoretic mobility than the other fast isoforms of troponin T, was detected in post-hatch pectoral muscle tested at 1–12 weeks of age. These neonatal forms (N1 and N2) in the pectoral muscle were undetectable at eight months of age. The presence of breast type troponin T in the leg muscle was not detected with these antibodies at any stage of development.

Effect of thyroidectomy on the distribution of the fast and slow forms of troponin I in rat soleus muscle

The effects of thyroid hormones on the properties of muscle in both experimental and clinical situations are well-documented [1-9]. These studies indicate that the speeds of contraction and relaxation in skeletal muscle are related to the thyroid hormone status and that the changes in physiological properties of the muscles observed when this status is altered are associated with appropriate changes in the proportion of type I and type II fibres [6,7,9,10]. The modifications that occur in the myosin light chain composition after thyroidectomy correlate well with the changes in fibre types and physiological properties [8,10]. As similar changes in myosin light chain composition can be induced by cross innervation and by chronic electrical stimulation, some workers have concluded that the thyroid effect on skeletal muscle may be neurally mediated [8]. On the other hand, the results in [11] suggest that thyroid hormones may have a direct effect on gene expression at least so far as mitochondrial enzymes are concerned. Many of the studies on thyroid effects have been carried out on rat soleus muscle without making allowance for the pronounced changes in the proportion of type I and type II fibres which in the normal animal take place for at least up to 24 weeks after birth [ 12,13]. We have, therefore, re-examined the effect of thyroidectomy on fibre type changes in the rat soleus using the immunoperoxidase procedure with antibodies to the fast and slow forms of troponin I to identify cell types and paying particular attention to the continuing maturation process that occurs in this muscle. Our studies suggest that thyroid hormones