Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle

Two Ca²⁺ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca²⁺-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twich muscle. Ca²⁺-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca²⁺-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased⁴⁵Ca-binding.These results indicate first that Ca²⁺-ATPase activity in EDL was under neural control, and that because of low Ca²⁺-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.     

Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.     

Cysteine proteinase inhibitors from rabbit skeletal muscle

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.     

Characteristics of sarcoplasmic reticulum from normal and denervated rat skeletal muscle.

Denervation of rat skeletal muscle produces after 14 days a decrease in Ca2+ uptake of a heterogeneous population of sarcoplasmic-reticulum vesicles, when measured in the presence of oxalate. The Mg2+-dependent ATPase (Ca2+-independent) activity increased after the same period and the Ca2+ + Mg2+-dependent ATPase activity decreased. Concomitant with these changes, there was an increase in vesicle size and calcium content. The observations are discussed in terms of changes in altered membrane structure, manifested in the shift of the equilibrium of the ATPase from an enzyme involved in calcium transport to a phosphoenzyme giving rise to an increase in the Mg2+-dependent ATPase activity.     

Purification of morphologically intact triad structures from skeletal muscle

A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.     

Can cells extruded from denervated skeletal muscle become circulating potential myoblasts?

Summary The hypothesis that satellite cells which leave denervated skeletal muscle might become circulating potential myoblasts which could participate in myogenesis in distant sites in the body has been tested.Sixteen mice had one hindlimb denervated and were given 7 daily injections of ³H-thymidine (³H-Tdr). One day later extensor digitorum longus muscle isografts from unlabelled mice were inserted into each hindlimb. As controls, the procedure was repeated in 6 non-denervated labelled mice. Fourteen days after their insertion, isografts in denervated mice contained many labelled myotubes with a labelling index of 55±4% (mean±SEM). In the control isografts in non-denervated mice, 38±4% of myotube nuclei were labelled. The results show that either labelled cells, or ³H-Tdr, had transferred from the host to isografts in both cases. The probability of ³H-Tdr reutilization was demonstrated in regenerating livers of 8 similarly labelled mice, where 34±3% hepatocytes adjacent to crush lesions were labelled after 14 days. This conclusion was reached because only 2–3% of normal hepatocytes incorporate ³H-Tdr under these conditions and this population is inadequate to provide sufficient labelled precursor cells for the large numbers of labelled regenerated hepatocytes. Therefore, it was concluded that ³H-Tdr reutilization is the most likely explanation for labelled myotube nuclei in the muscle isografts (rather than movement of labelled precursor cells), and that additional label for reutilization had been derived from breakdown of labelled cells in denervated muscle. The data do not support the hypothesis of a circulating precursor for skeletal muscle cells.     

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.     

Semaphorin 6C expression in innervated and denervated skeletal muscle

Semaphorins are secreted or transmembrane proteins important for axonal guidance and for the structuring of neuronal systems. Semaphorin 6C, a transmembrane Semaphorin, has growth cone collapsing activity and is expressed in adult skeletal muscle. In the present study the expression of Semaphorin 6C mRNA and immunoreactivity has been compared in innervated and denervated mouse hind-limb and hemidiaphragm muscles. Microscopic localization of immunoreactivity was studied in innervated and denervated rat skeletal muscle. The results show that Semaphorin 6C mRNA expression and immunoreactivity on Western blots are down-regulated following denervation. The mRNA of Semaphorin 6C as well as immunoreactivity determined by Western blots are expressed in extrasynaptic as well as perisynaptic regions of muscle. Immunohistochemical studies, however, show Semaphorin 6C-like immunoreactivity to be concentrated at neuromuscular junctions. The results suggest a role for Semaphorin 6C in neuromuscular communication.     

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.