Ontogenic appearance of Na+ channels characterized as high affinity binding sites for tetrodotoxin during development of the rat nervous and skeletal muscle systems

The appearance of the voltage-dependent Na+ channel during the fetal and post-natal development of rat brain, cerebellum and skeletal muscle has been followed using a highly radiolabelled derivative of tetrodotoxin. The number of Na+ channels is low at the fetal stage and increases drastically during post-natal development. The time-course of this increase is different in brain, cerebellum and skeletal muscle. Changes in affinity of the Na+ channel for tetrodotoxin occur during brain and cerebellum development. The results are discussed in relation with the maturation of the three types of excitable tissues.     

Two types of ryanodine receptors in mouse brain: skeletal muscle type exclusively in Purkinje cells and cardiac muscle type in various neurons.

Two types of ryanodine receptors, channels for Ca2+ release from intracellular stores, are known. We detected the skeletal muscle type only in cerebellum by immunoblot analysis of microsomes and partially purified proteins. The cardiac muscle type was found in all parts of the mouse brain. Immunohistochemical study showed that the cardiac muscle type was localized mainly at the somata of most neurons. Analysis of mutant cerebella suggested that the skeletal muscle type was present exclusively in Purkinje cells. These results suggest that Ca(2+)-induced Ca2+ release, probably mediated by the cardiac muscle receptor, functions generally in various neurons, whereas depolarization-induced Ca2+ release, probably mediated by the skeletal muscle receptor, functions specifically in Purkinje cells.     

Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles.

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post-natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3(-/- )mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation-contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).     

Comparative aspects and temperature dependence of [3H]1,4-dihydropyridine Ca2+ channel antagonist and activator binding to neuronal and muscle membranes

Binding of [3H]nitrendipine, [3H]nimodipine, and (+)[3H]PN 200-110 to microsomal preparations of guinea pig smooth and cardiac muscle and brain synaptosomes revealed high affinity interaction with KD values in the sequence, (+)PN 200-110 greater than nitrendipine greater than nimodipine. Bmax values for a particular tissue were independent of the 1,4-dihydropyridine employed in radioligand binding at 25 degrees C. The temperature dependence of [3H]nitrendipine binding in cardiac and smooth muscle microsomal preparations and brain synaptosomes was measured from 0 degrees to 37 degrees C and for skeletal muscle preparations from 0 degrees to 30 degrees C. Bmax values increased with temperature for cardiac membranes, but did not vary in other tissues. van't Hoff plots were nonlinear in all tissues, enthalpy and entropy changes becoming increasingly negative with increasing temperature. Competition binding of the activator-antagonist enantiomeric 1,4-dihydropyridine pairs of Bay k 8644 and PN 202-791 for [3H]nitrendipine in smooth muscle did not reveal significant thermodynamic differences between activator and antagonist molecules.     

Biochemical and pharmacologic properties of the Ca2+ channel of skeletal muscle: appearance during myogenesis and the relation between its structure and function

Two distinct and interdependent binding sites for inhibitors of voltage-dependent Ca2+ channels have been identified. They include one site for molecules of the 1,4-dihydropyridine serie such as nitrendipine, nifedipine or PN200-110 and one site for a chemically heterogenous group of compounds comprising verapamil, D600 and desmethoxyverapamil, bepridil and diltiazem. Ca2+ binds to its own coordination site which is distinct from the receptor site for organic Ca2+ channel inhibitors. The molecular size of the native [3H] nitrendipine receptor of transverse tubule membrane, brain and heart, have been determined using the radiation inactivation technique. The [3H] nitrendipine receptor is found to have a Mr of 210,000 +/- 20,000. CHAPS solubilization and purification indicate that the dihydropyridine receptor contains polypeptides of apparent molecular weights of 142,000, 32,000 and 33,000 which copurifie with (+) [3H] PN200-110 binding activity. Two stages in which there is an increased binding of [3H]nitrendipine have been observed during chick myogenesis. The first one occurs during embryonic life and has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors. This increase is accompanied by a decrease in the affinity of nitrendipine for its receptor by a factor of 4 to 10. The second stage of development is partly under innervation control and its expression is modulated by the intracellular cyclic AMP content. The two dihydropyridines Bay K8644 and CGP 28932 work preferentially on polarized membranes. 45Ca2+ flux experiments yielded results which are in good agreement with electrophysiological, contraction and binding data obtained with rat cardiac cells and skeletal muscle cells.     

Short term and long term effects of beta-adrenergic effectors and cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle

The effects of short term stimulation of beta-adrenergic receptors and elevations in intracellular cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle cells in vitro has been studied using both the 45Ca2+ flux technique and [3H] nitrendipine-binding experiments. Isoproterenol increased the nitrendipine-sensitive 45Ca2+ influx under depolarizing conditions. The effects of isoproterenol were additive to those of depolarization and were antagonized by alprenolol. Half-maximal inhibition of 45Ca2+ influx induced both by depolarization and by isoproterenol occurred at a nitrendipine concentration of 1 nM. Treatments that resulted in an increased level of intracellular cyclic AMP, such as treatment with 1-methyl-3-isobutylxanthine, theophylline, dibutyryl cyclic AMP, or 8-bromocyclic AMP also resulted in an increased rate of 45Ca2+ entry via nitrendipine-sensitive Ca2+ channel. In contrast, long term treatment of myotubes in culture with isoproterenol and other compounds that increased intracellular cyclic AMP led to a large increase in the number of nitrendipine receptors. This increase was accompanied by a 4-10-fold decrease in the affinity of the receptors for nitrendipine. Alprenolol inhibited the long term effects of isoproterenol. In vivo treatment of 7-day-old chicks with reserpine and alprenolol produced a decrease in the number of skeletal muscle nitrendipine receptors. This decrease in receptor number was accompanied by an increase in the affinity of nitrendipine for its receptor by a factor of 4 to 5. These effects on the nitrendipine receptor were prevented by simultaneous injection of isoproterenol. The results are discussed in relation to the role of beta-adrenergic receptors and intracellular cyclic AMP in the regulation of skeletal muscle Ca2+ channels.     

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.     

Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.     

Levels of carnitine and glycogen in rabbit tissues during development

This study attempts to add further light on the development of metabolic pathways in mammalians from fetal to post-natal life, by examining ongoing modifications of carnitine (in terms of total acid soluble, short chain esterified and free carnitine) and glycogen levels in the liver, heart, muscle and brain of rabbit during development.     

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)     

The nitrendipine-sensitive Ca2+ channel in chick muscle cells and its appearance during myogenesis in vitro and in vivo

The nitrendipine-sensitive Ca2+ channel of chick skeletal myotubes in culture has been studied using both the 45Ca2+ flux technique and [3H]nitrendipine binding experiments. Ca2+ uptake is insensitive to nitrendipine when chick myotubes in culture are polarized. Whereas depolarization reveals a new component of 45Ca2+ influx which is inhibited by nitrendipine. Half-maximal inhibition occurs at a nitrendipine concentration of 0.7 nM. This value is similar to the dissociation constant Kd = 0.4 nM found in [3H]nitrendipine binding experiments. During myogenesis in vitro the nitrendipine receptor is absent in myoblasts and appears in parallel with the fusion process. Two stages of increased binding have been observed in vivo. The first one, which occurs during embryonic life, has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors. This increase is accompanied by a decrease of affinity of nitrendipine for its receptor by a factor of 4 to 10. Chronic denervation produces a further increase in the number of nitrendipine receptors which reaches a factor of about 2 at 15 days of denervation. Results are discussed in relation to the particular localization of these channels in transverse tubules and with the innervation.     

Voltage-dependent K+ channel beta subunits in muscle: differential regulation during postnatal development and myogenesis

Voltage-dependent potassium channels contribute to the electrical properties of nerve and muscle by affecting action potential shape and duration. The complexity of the currents generated is further enhanced by the presence of accessory beta subunits. Here we report that while all Kvbeta mRNA isoforms are present in rat brain, muscle tissues express only Kvbeta1 (Kvbeta1.1-Kvbeta1.3) and Kvbeta2, but not Kvbeta3. Kvbeta subunits were close regulated through post-natal development in brain and striated muscle, as well as during myogenesis in the rat skeletal muscle cell line L6E9. While the alternatively spliced Kvbeta mRNA products from Kvbeta1 gene were differentially expressed, Kvbeta2.1 was associated with myogenesis. These results show that Kvbeta genes are strongly regulated in muscle and suggest a physiological role for voltage-gated K(+) channels during development and myotube formation.     

A novel fetal insulin-like growth factor (IGF) I receptor. Mechanism for increased IGF I- and insulin-stimulated tyrosine kinase activity in fetal muscle

Insulin and insulin-like growth factor (IGF) I receptors from fetal and adult rat skeletal muscle were compared in order to gain insight into the evolving functions of the hormones during development. Basal, insulin-stimulated, and IGF I-stimulated receptor phosphorylation and tyrosine kinase activity are severalfold higher in partially purified receptor preparations from fetal muscle in comparison with equal numbers of receptors from adult muscle. There are distinct insulin and IGF I receptors with Mr 95,000 beta subunits in adult muscle, as evidenced by hormone dose-response curves, immunoprecipitation with specific antibodies, binding to insulin and IGF I affinity columns, and analysis of tryptic phosphopeptides. In addition to these two receptor species, fetal muscle contains a receptor with a Mr 105,000 beta subunit. The fetal receptor is structurally more closely related to the IGF-I receptor than the insulin receptor on the basis of its precipitation with specific antibodies, binding to an IGF I affinity column, and tryptic phosphopeptide map. The fetal receptor does not appear to bind insulin but, unlike the IGF-I receptor, its phosphorylation is stimulated by low physiological concentrations of both insulin and IGF I. This could be explained by the cross-phosphorylation of fetal receptors by activated insulin receptors. Expression of the fetal receptor is highest in the fetus and decreases markedly during the first 2 weeks of postnatal life. The fetal receptor appears to account for the high tyrosine kinase activity of fetal muscle and may be an important mediator of responses to both insulin and IGF I early in development.     

Differentiation of receptor sites for [3H]nitrendipine in chick hearts and physiological relation to the slow Ca2+ channel and to excitation-contraction coupling

The properties of interaction of the Ca2+ channel antagonist [3H]nitrendipine have been investigated in chick hearts at various stages of in ovo and post-natal development and in cultured cells. The dissociation constant of the [3H]nitrendipine-receptor complex is between 0.4 nM and 0.5 nM for intact ventricle and cultured cells. [3H]Nitrendipine binding is antagonized by nitrendipine analogs. The order of efficacy of the different dihydropyridine molecules is nitrendipine greater than nimodipine greater than nifedipine greater than nisoldipine with Kd values ranging from 0.5 to 4 nM. Inhibition of [3H]nitrendipine binding by other antiarrhythmic molecules like amiodarone, F13004 and bepridil was observed. Half-maximum inhibitions (K0.5) were found for verapamil and D600 at concentrations between 0.23 and 0.26 microM. The potency of organic Ca2+ blockers to depress by 50% the maximum amplitude of spontaneous beating of heart cells is closely related to K0.5 values obtained from [3H]nitrendipine binding experiments. Electrophysiological results indicate that the slow channel is insensitive to nitrendipine at the younger stage of development (3-day-old) whereas, in adult like cells, nitrendipine (50 nM) abolished both slow action potential due to the slow Ca2+ channel and contraction. The maximum binding capacity for [3H]nitrendipine is found to increase during development of the embryonic heart from 40 fmol/mg protein at day 3 to 100 fmol/mg protein at day 14, to stay relatively stable until day 18. Then the number of sites increases rapidly to reach a second plateau at 210 fmol/mg protein on day 4 after hatching. Treatment with 6-hydroxydopamine results in 35% increase in [3H]nitrendipine binding, whereas reserpine treatment is without effect. Developmental properties of nitrendipine-sensitive Ca2+ channels have been compared with those of tetrodotoxin-sensitive Na+ channels and muscarinic receptors. These results indicate that nitrendipine receptors exist at the early stage of development (3-day-old-hearts) but that they do not correspond to functional slow Ca2+ channels, that in ovo development corresponds both to an increase of the number of [3H]nitrendipine receptors and to the transformation of silent Ca2+ channels into functional Ca2+ channels, and that there is a regulation of the level nitrendipine-sensitive Ca2+ channels by innervation.     

Myostatin shows a specific expression pattern in pig skeletal and extraocular muscles during pre- and post-natal growth

Abstract Myogenesis is driven by an extraordinary array of cellular signals that follow a common expression pattern among different animal phyla. Myostatin (mstn) is a secreted growth factor that plays a pivotal role in skeletal muscle mass regulation. The aim of the present study was to investigate mstn expression in a large mammal (the pig) in order to ascertain whether distinct expression changes of this factor might be linked to the fiber-type composition of the muscle examined and/or to specific developmental stages. To assess the expression pattern of mstn in relation to myogenic proliferative (Pax7 and MyoD) and differentiative (myogenin) markers, we evaluated muscles with different myosin heavy-chain compositions sampled during pre- and post-natal development and on myogenic cells isolated from the same muscles. Skeletal muscles showed higher levels of mRNA for mstn and all other genes examined during fetal development than after birth. The wide distribution of mstn was also confirmed by immunohistochemistry experiments supporting evidence for cytoplasmic staining in early fetal periods as well as the localization in type 1 fibers at the end of the gestation period. Extraocular muscles, in contrast, did not exhibit decreasing mRNA levels for mstn or other genes even in adult samples and expressed higher levels of both mstn mRNA and protein compared with skeletal muscles. Experiments carried out on myogenic cells showed that mstn mRNA levels decreased when myoblasts entered the differentiation program and that cells isolated at early post-natal stages maintained a high level of Pax7 expression. Our results showed that mstn had a specific expression pattern whose variations depended on the muscle type examined, thus supporting the hypothesis that at birth, porcine myogenic cells continue to be influenced by hyperplastic/proliferative mechanisms.