Myosin light chain phosphorylation during contraction of chicken fast and slow skeletal muscles

A modified automatic freezing apparatus (K. M. Kretzschmar and D. R. Wilkie, 1962, J. Physiol. (London), 202, 66–67) was used for studying light chain phosphorylation during the early phase of contraction of the fast, posterior latissimus dorsi, and slow, anterior latissimus dorsi, muscles of chicken at 37 °C. The frozen muscles were worked up under conditions which avoid artifacts in quantitating the level of light chain phosphorylation in contracting and resting muscles. The posterior latissimus dorsi muscle reached 80% of its maximal isometric tension at 0.1 s of tetanic stimulation. At the same time, light chain phosphorylation increased by 60% of its maximal extent. The peak tension of the posterior muscle at 0.2 s of stimulation was accompanied by maximal light chain phosphorylation. In case of the slow anterior latissimus dorsi muscle, maximal tetanic tension was developed in 2.5 – 5 s and light chain phosphorylation also proceeded at a much slower rate than in the fast posterior muscle. When contralateral posterior latissimus dorsi muscles were stimulated for 0.2 s and one muscle was frozen at the height of tetanus while the other muscle was allowed to relax and frozen 0.4 s after terminating the stimulation, both contracted and relaxed muscles exhibited maximal light chain phosphorylation. However, when the muscle was allowed to relax for 0.8 s before freezing, half of the phosphorylated light chain became dephosphorylated. The resting level of phosphate content of the light chain was restored in both the posterior and anterior muscles during a longer time after relaxation.     

Developmental changes in the structure and kinetic properties of myosin adenosinetriphosphatase of rabbit skeletal fast muscle.

Structural and functional changes in myosin of fast muscles during early post-natal development were studied to seek correlations with well-known physiological changes in the contraction rate. The findings were as follows: 1. It is known that fetal fast muscle myosin contains three kinds of light chains. It was confirmed that their molecular weights were the same as those of adult fast muscle myosin, but different from those of adult slow muscle myosin. The amount of the smallest light chain, g3, was confirmed to increase markedly during the postnatal period. 2.The ATPase [EC3.6.1.3] activity of fetal fast muscle myosin (-1 day) was found to be about 50% of that of adult myosin. The pH-activity curve of fetal myosin ATPase was confirmed to be similar to that of adult myosin. 3. The rate of formation of the reactive myosin-phosphate-ADP complex, MADPP, was found not to change during post-natal development. 4. It was found that the rate of decomposition of MADPP in the presence of F-actin increased markedly during the post-natal period, and that the rate of decomposition of the complex of fetal mysoin was only 1/6 to 1/4 of that of adult myosin. The change in the actomyosin ATPase activity was found to be closely correlated with the increase in the g3 content during development.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.     

Enzymatic activities and ATP-induced fluorescence enhancement of myosin from fast and slow skeletal and cardiac muscles.

The maximal ATP-induced enhancement of fluorescence and the dependence of this enhancement on ATP concentration were determined for myosins from fast and slow skeletal and cardiac muscle of the rabbit. With myosins from slow and cardiac muscle modifications in the preparative procedure and chromatography on DEAE-Sephadex were required to obtain preprations which were free of actin, which exhibited the maximal fluorescence enhancement and which bound two moles of ATP per mole of myosin. Since the fluorescence enhancement of cardiac and slow muscle myosins is labile at slightly alkaline pH, it was also necessary to minimize incubation at pH greater than 7 in order to attain the maximal enhancement. With fast muscle myosin the changes in preparative procedure, together with chromatography, led to a 50 to 100% increase in the steady-state rate of ATP hydrolysis and fluorescence enhancement, without changing the maximal binding of ATP. From a comparison of the rate of steady-state hydrolysis of ATP with the rate of decay of the enhanced fluorescence, it appears that for all three myosins, both ATP binding sites have the same enzymatic activity, the steady-state rate per site being slower for cardiac and slow muscle myosins than for fast muscle myosin.     

Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression

Work induced hypertrophy of the slow postural soleus and the fast phasic plantaris muscles was produced by tenotomy of the synergistic gastrocnemius muscle. Increases in weight of both muscles were associated with proportionately even larger increases in total RNA and mRNA levels. Alterations in levels of specific myosin heavy chain (MHC) isoform mRNAs were measured using the slot blot procedure with radioactively labelled oligonucleotides as probes. Type 1 MHC gene expression was unaffected in both muscles by work overload, whereas type 2a was deinduced in the soleus and type 2b was deinduced in the plantaris. The neonatal MHC gene was transiently reinduced in the plantaris.     

Roles of troponin isoforms in pH dependence of contraction in rabbit fast and slow skeletal and cardiac muscles.

Skinned fibers prepared from rabbit fast and slow skeletal and cardiac muscles showed acidotic depression of the Ca2+ sensitivity of force generation, in which the magnitude depends on muscle type in the order of cardiac>fast skeletal>slow skeletal. Using a method that displaces whole troponin-complex in myofibrils with excess troponin T, the roles of Tn subunits in the differential pH dependence of the Ca2+ sensitivity of striated muscle were investigated by exchanging endogenous troponin I and troponin C in rabbit skinned cardiac muscle fibres with all possible combinations of the corresponding isoforms expressed in rabbit fast and slow skeletal and cardiac muscles. In fibers exchanged with fast skeletal or cardiac troponin I, cardiac troponin C confers a higher sensitivity to acidic pH on the Ca2+ sensitive force generation than fast skeletal troponin C independently of the isoform of troponin I present. On the other hand, fibres exchanged with slow skeletal troponin I exhibit the highest resistance to acidic pH in combination with either isoform of troponin C. These results indicate that troponin C is a determinant of the differential pH sensitivity of fast skeletal and cardiac muscles, while troponin I is a determinant of the pH sensitivity of slow skeletal muscle.     

Regulation of glucose uptake in rat slow and fast skeletal muscles

1. Regulation of glucose uptake was compared between extensor digitorum longus (EDL) and soleus (Sol) muscles in rats. 2. Insulin stimulated glucose uptake more in EDL than in Sol. 3. Under high concentrations of insulin, the glucose uptake was higher in EDL than Sol. 4. Inhibition of oxidative phosphorylation by anoxia or an uncoupler stimulated glucose uptake more in EDL than in Sol. 5. Anoxia abolished the effect of insulin on glucose uptake in both EDL and Sol. 6. The blocker to glucose transport system reduced glucose uptake more in Sol than in EDL.     

Relation of fast and slow skeletal, and atrial and ventricular myosin in various mammals

Myosin was isolated from adult mouse, rat, rabbit and cat atrial and ventricular myocardium and fast and slow skeletal muscles and examined by measuring Ca2+-ATPase activity and by electrophoretic fractionation of chymotryptic peptides and MLCs. The myosin from mouse atrial and ventricular myocardium were very similar. The properties of cat soleus muscle myosin and ventricular myocardium were also very similar (ATPase activity and electrophoretic pattern of chymotryptic peptides of myosin). The electrophoretic pattern of MLCs, however, was distinct when comparing mouse and feline muscles. These observations are consistent with the idea that atrial and ventricular alpha MHCs are closely related and that beta MHCs from ventricular myocardium and slow skeletal muscle fibres are also closely related.     

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.     

Effect of hypothyroidism on actin subdomain-1 movement induced by myosin subfragment-1 binding in fast and slow rat skeletal muscles

The orientation and mobility of an N-(iodoacetyl)-(1-naphtyl-5-sulpho-ethylenediamine) fluorescent probe (1.5-IAEDANS) specifically bound to Cys-374 of actin in ghost muscle fibers isolated from fast and slow rat muscles were studied by polarized fluorimetry in the absence and presence of a myosin subfragment-1 (S1) in intact rats and in animals with a gradual (2–5 weeks) reduction in the level of thyroid hormones (development of hypothyroidism). The binding of S1 to F-actin of ghost muscle fibers was shown to induce changes in the orientation of dipoles of the 1.5-IAEDANS fluorescent probe and in the relative amount of the randomly oriented fluorophores that indicates changes in actin subdomain-1 orientation and mobility resulting from formation of its strong binding with S1. This effect is markedly inhibited by the development of hypothyroidism. The maximal effect of hypothyroidism is observed after 34 days of the development of the disease. It is suggested that the change in the thyroid status in muscle inhibits the ability of F-actin to form strong binding with myosin, which is essential for the generation of force.     

Radioactive labeling and location of specific thiol groups in myosin from fast, slow and cardiac muscles.

1. Based on incorporation of radioactively labeled N-ethylmaleimide, the readily reactive thiol groups of isolated myosin (EC 3.6.1.3) from fast, slow and cardiac muscles could be classified into 3 types. All 3 myosins contain 2 thiol-1, 2 thiol-2 and a variable number of thiol-3 groups per molecule. Both thiol-1 and thiol-2 groups which are essential for functioning of the K+-stimulated ATPase, are located in the heavy chains in all 3 myosin types. 2. The variation in the incorporation pattern of N-ethylmaleimide over the 3 thiol group classes under steady-state conditions of Mg(2+) - ATP hydrolysis allowed different conformations of some reaction intermediates to be characterized. In all 3 types of myosin the hydrolytic cycle of Mg(2+) - ATP was found to be controlled by the same step at 25 degrees C. In all three cases, this rate-limiting step is changed in the same way by lowereing temperature. 3. Using the chemically determined molecular weights for myosin light chains, their stoichiometry was found on the basis of sodium dodecyl sulfate electrophoresis to be 1.2 : 2.1 : 0.8 for light chain-1: light chain-2:light chain-3 per molecule of fast myosin, 2.0 : 1.9 for light chain-1:light chain-2 per molecule of slow myosin and 1.9 : 1.9 for light chain-1:light chain-2 per molecule of cardiac myosin. This qualitative difference in light subunit composition between the fast and the two types of slow myosin is not reflected in the small variations of the characteristics exhibited by the isolated myosins, but rather seems to be connected with their respective myofibrillar ATPase activities.     

Transformation of individual contractile responses during tetanus in rat fast and slow skeletal muscles

Using a computer graphics approach, the last contractile responses (LCR_N, where N is a number of elementary contractile responses in tetanus) were separated from integral tetanic responses of rat fast muscles, m. Eхtensor digitorum longus (m. EDL), and slow muscles, m. Soleus, evoked by trains of 5, 10 and 50 stimuli. In m. Soleus, at a stimulation frequency of 20 Hz, the LCR₅ average amplitude decreased to 64 ± 9% compared to the single contraction amplitude. As N increased, LCR_N recovered and then rose to the values exceeding almost twofold initial elementary contractile responses (up to 211 ± 10% for LCR₅₀). Simultaneously, against the background of rising elementary contractile responses, a significant shortening of their half-decay time (～by 50%) and the formation of a stationary plateau within LCR_N was observed. In m. EDL, at a stimulation frequency of 50 Hz, there was only a single-phase LCR_N rise (up to 165 ± 18% for LCR₅₀) without changes in half-decay time and plateau formation. In skeletal muscles of both types, the prolonged (up to 30 s) ‘hyper-relaxation effect’ was found to develop after the end of tetanic responses manifested as a reduction of muscle tension followed by its recovery to the initial level. Possible mechanisms of these events are discussed. It is hypothesized that transformation of elementary contractile responses in skeletal muscles can be fulfilled due to the existence of specialized microdomains in muscle fibers which regulate accumulation and extrusion of Ca²⁺ ions during tetanic activity. The possibility that the basic, depolarization-induced, Ca²⁺ release (DICR) is complemented by an additional, Ca²⁺-induced, Ca²⁺release (CIRC) is analyzed.     

Evaluation of RNA polymerase activities in nuclei isolated from slow and fast skeletal muscles

The optimal incubation conditions were determined for the assay of the α-amanitin-resistant, DNA-dependent RNA polymerase A and the α-amanitin-sensitive, DNA-dependent RNA polymerase B in nuclei isolated from rat skeletal muscles. Significantly higher levels of activity of RNA polymerase B were found in the nuclei isolated from the slow-twitch soleus compared with nuclei from the fast-twitch extensor digitorum longus.