Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles

The response to caffeine was studied in mouse muscles[diaphragm, soleus, and extensor digitorum longus (EDL)] withdifferent ryanodine receptor isoform (RyR1, RyR3) composition and insingle permeabilized muscle fibers dissected from diaphragm ofwild-type (WT) and RyR3-deficient (RyR3/) mice at 1, 15, 30, and 60 postnatal days (PND). The caffeine response decreased duringdevelopment, and, in adult mice, was greater in diaphragm, lower inEDL, and intermediate in soleus. This suggests a direct relationbetween response to caffeine and RyR3 expression. The lack of RyR3reduced caffeine response in young, but not in adult mice, and did not abolish the age-dependent variation and the intermuscle differences. Indiaphragm single fibers, the response to caffeine increased duringdevelopment and was reduced in fibers lacking RyR3 both at 15 and 60 PND. A population of fibers highly responsive to caffeine was presentin adult WT and disappeared in RyR3/. The results confirm thecontribution of RyR3 to calcium release for contractile response andclarify the contribution of RyR3 to developmental changes andintermuscle differences.

     

Muscle type-specific myosin isoforms in crustacean muscles

Differential expression of multiple myosin heavy chain (MyHC) genes largely determines the diversity of critical physiological, histochemical, and enzymatic properties characteristic of skeletal muscle. Hypotheses to explain myofiber diversity range from intrinsic control of expression based on myoblast lineage to extrinsic control by innervation, hormones, and usage. The unique innervation and specialized function of crayfish (Procambarus clarkii) appendicular and abdominal musculature provide a model to test these hypotheses. The leg opener and superficial abdominal extensor muscles are innervated by tonic excitatory motoneurons. High resolution SDS-PAGE revealed that these two muscles express the same MyHC profile. In contrast, the deep abdominal extensor muscles, innervated by phasic motoneurons, express MyHC profiles different from the tonic profiles. The claw closer muscles are dually innervated by tonic and phasic motoneurons and a mixed phenotype was observed, albeit biased toward the phasic profile seen in the closer muscle. These results indicate that multiple MyHC isoforms are present in the crayfish and that differential expression is associated with diversity of muscle type and function.     

Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties.

To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation.     

Myosin isoforms of skeletal muscles in development

The latest data are reviewed concerning identification of myosin from the skeletal muscle during embryonic and postnatal development in vertebrates. The data are given on the composition of light subunits and specificity of heavy chains of the early isoforms obtained by electrophoresis, peptide mapping, DNA-RNA hybridization, as well as immunological methods with poly-and monoclonals. The substitution of embryonic heavy chains by neonatal and definitive ones is discussed. The following items are also considered: early isoforms of the fast and slow myosin types and, in particular, endogenous program directing the muscle development and protein synthesis towards the "fast phenotype", which is modulated by neurostimulation and other physiological factors inducing slow myosin type. The enzymatic activity of the early isoforms and its physiological importance in embryogenesis are discussed.     

Cloning and characterization of hamster fetal retinoic acid receptor isoforms

Hamsters are routinely employed in toxicology evaluation, particularly for investigating the teratologic potential of chemicals. We have employed Syrian golden hamsters in retinoid-induced teratogenesis, mechanisms of which involve various retinoic acid receptor (RAR) isoforms. The purpose of this study was to clone and characterize different full-length hamster RAR isoforms. A 12-day old fetal hamster cDNA library was constructed and screened for RAR isoforms using human or mouse probes. Three full-length clones representing RARα, β, and γ were isolated, amplified and sequenced, and based on their homology to known mammalian isoforms were termed as hamster RARα variant, RARβ2 and RARγ2, respectively. The respective translated products for these clones were 430, 448 and 406 amino acids long. The clones were homologous to their human or mouse counterparts, although differences, particularly in the N-terminal region, were observed. These differences may represent differential splicing of exons controlled by two promoters for each isoform.     

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).     

大鼠骨骼肌快肌肌钙蛋白T的同工型

骨骼肌快肌的收缩主要是由钙离子通过肌钙蛋白所调节控制。这些肌钙蛋白位于肌纤维之中。肌蛋白包括肌钙蛋白T、肌钙蛋白C、肌钙蛋白I。采用双向聚丙烯酰胺凝胶电泳和免疫学技术,对大鼠胚胎、新生大鼠和成年大鼠的骨能肌快肌肌钙蛋白T的同工型进行了研究。在成年大鼠的骨能肌快肌中,发现了10种肌钙蛋白T同工型。在大鼠胚胎和新生大鼠的骨能肌中,发现了7种肌钙蛋白T同工型。作为不同动物、不同发育阶段和不同组织发育的特殊标记,这些肌钙蛋白T同工型具有重要意义[动物学报49(3)：362—369,2003]。     

Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: possible roles in excitation-contraction coupling and other processes

The ryanodine receptor (RyR) is a Ca²⁺ release channel in the sarcoplasmic reticulum in vertebrate skeletal muscle and plays an important role in excitation–contraction (E–C) coupling. Whereas mammalian skeletal muscle predominantly expresses a single RyR isoform, RyR1, skeletal muscle of many nonmammalian vertebrates expresses equal amounts of two distinct isoforms, α-RyR and β-RyR, which are homologues of mammalian RyR1 and RyR3, respectively. In this review we describe our current understanding of the functions of these two RyR isoforms in nonmammalian vertebrate skeletal muscle. The Ca²⁺ release via the RyR channel can be gated by two distinct modes: depolarization-induced Ca²⁺ release (DICR) and Ca²⁺-induced Ca²⁺ release (CICR). In frog muscle, α-RyR acts as the DICR channel, whereas β-RyR as the CICR channel. However, several lines of evidence suggest that CICR by β-RyR may make only a minor contribution to Ca²⁺ release during E–C coupling. Comparison of frog and mammalian RyR isoforms highlights the marked differences in the patterns of Ca²⁺ release mediated by RyR1 and RyR3 homologues. Interestingly, common features in the Ca²⁺ release patterns are noticed between β-RyR and RyR1. We will discuss possible roles and significance of the two RyR isoforms in E–C coupling and other processes in nonmammalian vertebrate skeletal muscle.     

Ca2+对骨骼肌钙释放通道的调节

钙释放通道（calcium release channel）又称Ryanodine受体（RyR）,是细胞内质网膜上介导细胞内钙信号转导的离子通道。RyR1在骨骼肌细胞的兴奋-收缩偶联过程中起重要作用,是肌质网快速释放Ca^2＋的通道。许多调节因素,如一些内源性蛋白（FK结合蛋白、钙调素、钙结合蛋白）和一些离子（Ca^2＋、Mg^2＋）,通过不同的作用位点与RyR1结合,调控RyR1的结构与功能。研究表明,Ca^2＋是众多调节RyR1因素中的核心成分和前提条件,其对RyR1的结构与功能有重要的调控作用。     

Expression of the ryanodine receptor isoforms in immune cells.

Ryanodine receptor (RYR) is a Ca(2+) channel that mediates Ca(2+) release from intracellular stores. We have used RT-PCR analysis and examined its expression in primary peripheral mononuclear cells (PBMCs) and in 164 hemopoietic cell lines. In PBMCs, type 1 RYR (RYR1) was expressed in CD19(+) B lymphocytes, but less frequently in CD3(+) T lymphocytes and in CD14(+) monocytes. Type 2 RYR (RYR2) was mainly detected in CD3(+) T cells. Induction of RYR1 and/or RYR2 mRNA was found after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha (MIP1alpha) or TGF-beta. Type 3 RYR (RYR3) was not detected in PBMCs. Many hemopoietic cell lines expressed not only RYR1 or RYR2 but also RYR3. The expression of the isoforms was not associated with specific cell lineage. We showed that the RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced Ca(2+) release and thereby confirmed functional expression of the RYR in the cell lines expressing RYR mRNA. Moreover, concordant induction of RYR mRNA with Ca(2+) channel function was found in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM) had no effect on Ca(2+) release, whereas 4CmC (<400 microM) caused Ca(2+) release after the induction of RYR2 and RYR3 that occurred after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha, or TGF-beta. Our results demonstrate expression of all three isoforms of RYR mRNA in hemopoietic cells. Induction of RYRs in response to chemokines and TGF-beta suggests roles in regulating Ca(2+)-mediated cellular responses during the immune response.     

Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle

Adult skeletal muscle undergoes adaptation in response to endurance exercise, including fast-to-slow fiber type transformation and enhanced angiogenesis. The purpose of this study was to determine the temporal and spatial changes in fiber type composition and capillary density in a mouse model of endurance training. Long-term voluntary running (4 wk) in C57BL/6 mice resulted in an approximately twofold increase in capillary density and capillary-to-fiber ratio in plantaris muscle as measured by indirect immunofluorescence with an antibody against the endothelial cell marker CD31 (466 ± 16 capillaries/mm² and 0.95 ± 0.04 capillaries/fiber in sedentary control mice vs. 909 ± 55 capillaries/mm² and 1.70 ± 0.04 capillaries/fiber in trained mice, respectively; P < 0.001). A significant increase in capillary-to-fiber ratio was present at day 7 with increased concentration of vascular endothelial growth factor (VEGF) in the muscle, before a significant increase in percentage of type IIa myofibers, suggesting that exercise-induced angiogenesis occurs first, followed by fiber type transformation. Further analysis with simultaneous staining of endothelial cells and isoforms of myosin heavy chains (MHCs) showed that the increase in capillary contact manifested transiently in type IIb + IId/x fibers at the time (day 7) of significant increase in total capillary density. These findings suggest that endurance training induces angiogenesis in a subpopulation of type IIb + IId/x fibers before switching to type IIa fibers. adaptation; capillary density; endothelial cells; fiber type transformation; vascular endothelial growth factor     

Distinct mechanisms for dysfunctions of mutated ryanodine receptor isoforms

Ryanodine receptor (RyR) is the Ca²⁺-induced Ca²⁺ release channel in cells. RyR1 and RyR2 are its isoforms expressed in the skeletal and cardiac muscles, respectively. Their missense mutations, which are clustered in three regions that correspond to each other, cause hereditary disorders such as malignant hyperthermia and central core disease in skeletal muscle and catecholaminergic polymorphic ventricular tachycardia in cardiac muscle. Their pathogeneses, however, are not well understood. The following hypotheses are favorably discussed in this article: phenotypes with RyR1 and RyR2 mutations are mainly caused by dysregulations of their functions through the interdomain interaction and luminal Ca²⁺, respectively.     

Composition of titin isoforms of skeletal and cardiac muscles in pathologies

An electophoretic study of changes in composition of titin isoforms in human and rat skeletal and cardiac muscles is carried out. A more considerable decrease in the content of intact titin isoforms was observed than in the content of N2A-titin in the dorsal muscle of patients with the “stiff-person syndrome” and in m. soleus of humans and rats during development of “muscle hypogravity syndrome” and than in the content of N2BA- and N2B-titins in hypertrophic heart of spontaneously hypertensive rats. The relation between reduction of titin content in m. soleus and the increase of time the rats were in conditions of simulated microgravity is revealed. On electrophoregrams of left ventricle myocardium of patients with terminal stage of dilated cardiomyopathy the intact titin and N2BA-titin were absent and a considerable decrease in the content of N2B-titin was observed. This could be the consequence of the terminal stage of pathology. It follows that development of the diseases is accompanied by a greater destruction of intact titin than of its other forms which may be important for diagnostics of pathological processes.     

Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the rev-erbAalpha orphan receptor gene

The rev-erbAalpha orphan protein belongs to the steroid nuclear receptor superfamily. No ligand has been identified for this protein, and little is known of its function in development or physiology. In this study, we focus on 1) the distribution of the rev-erbAalpha protein in adult fast- and slow-twitch skeletal muscles and muscle fibers and 2) how the rev-erbAalpha protein influences myosin heavy chain (MyHC) isoform expression in mice heterozygous (+/-) and homozygous (-/-) for a rev-erbAalpha protein null allele. In the fast-twitch extensor digitorum longus muscle, rev-erbAalpha protein expression was linked to muscle fiber type; however, MyHC isoform expression did not differ between wild-type, +/-, or -/- mice. In the slow-twitch soleus muscle, the link between rev-erbAalpha protein and MyHC isoform expression was more complex than in the extensor digitorum longus. Here, a significantly higher relative amount of the beta/slow (type I) MyHC isoform was observed in both rev-erbAalpha -/- and +/- mice vs. that shown in wild-type controls. A role for the ratio of thyroid hormone receptor proteins alpha1 to alpha2 in modulating MyHC isoform expression can be ruled out because no differences were seen in MyHC isoform expression between thyroid hormone receptor alpha2-deficient mice (heterozygous and homozygous) and wild-type mice. Therefore, our data are compatible with the rev-erbAalpha protein playing an important role in the regulation of skeletal muscle MyHC isoform expression.     

Myosin isoforms of the developing skeletal muscles in the loach

Changes in the myosin isozyme spectrum were studied in the loach developing skeletal muscle. It was shown using disk-electrophoresis in polyacrylamide gel and peptide mapping that light and heavy myosin chains from the larval muscles, as well as from the red and white muscle of adult fish differ from each other. Forms of myosin light and heavy chains were found which were characteristic of the larval muscle only.     

A skeletal muscle ryanodine receptor interaction domain in triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.     

Properties of ryanodine receptor in rat muscles submitted to unloaded conditions

Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype.