Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development
Introduction
The excitation–contraction coupling (E–C coupling) mechanism is a key element in the regulation of the contractile function of muscle cells, where activation of plasma membrane dihydropyridine receptors (DHPRs), localized on T-tubules triggers the opening of the ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) [1], [2]. The organization of the E–C coupling machinery during muscle development requires the coordinate development of both the sarcoplasmic reticulum and T-tubule system [3], [4], [5], [6]. E–C coupling properties differ between embryonic, newborn and adult skeletal muscles [7], [8], [9], [10], [11], [12]. While no Ca2+ influx from extracellular fluids is required to activate contraction of adult skeletal muscle cells, fetal and neonatal E–C coupling show a Ca2+-current-dependent component that progressively decreases to disappear in the adult [8], [10], [13], [14]. Dependence on Ca2+ influx has been proposed to improve contraction in developing muscles, taking into account the small diameter of muscle fibers and the poorly developed T-tubule system in fetal and newborn muscles [15], [16].
During skeletal muscle development, expression of RyR1 and DHPRs is constantly observed from early embryonic stages to adult life [3], [17], [18], [19], [20]. In contrast, RyR3 has been found to be mostly expressed in developing mammalian skeletal muscles, whereas in the adult its expression is down-regulated in most muscles, with the exception of the diaphragm and some muscles of the head and neck region [20], [21], [22], [23]. Previous reports indicated that the RyR3 content in adult rabbit and bovine diaphragm accounts for 0.6% and 5% of total RyRs, respectively [24], [25]. Several evidence suggest that, although RyR1 represents the major component in the E–C coupling machinery of mammalian skeletal muscle cells acting in close association with DHPRs, the RyR3 isoform may be engaged as an additional constituent to improve Ca2+ release from the SR in developing skeletal muscles [20], [26], [27]. Experiments with RyR3 knock out mice showed that RyR3 may contribute to the generation of a calcium-induced calcium release signal in developing, but not in adult muscle cells [20]. In addition, the speed of the Ca2+ signal to diffuse from the membrane to the central region of muscle fibers was reported to be higher in neonatal RyR3 knockout mice compared to control, suggesting that co-expression of RyR3 with RyR1 may contribute to amplify Ca2+ release across the neonatal skeletal muscle fiber [26]. Nevertheless, some questions on the role of RyR3 in the regulation of skeletal muscle contraction during development are still to be answered. At first, a precise estimate of the RyR3 protein content in mouse skeletal muscle during development as well as its relative abundance compared to RyR1 are currently not known. In addition, how the RyR3 protein, that is expected to represent a small fraction of total RyRs even in developing skeletal muscles may account for the large effect on muscle contraction observed in knockout mice, has still not been completely unravelled. In this respect, recent reports by Murayama and Ogawa showed that, at difference with RyR3, the [3H]ryanodine binding activity of the RyR1 isoform is significantly reduced in native sarcoplasmic reticulum membranes, compared to CHAPS solubilized channels, suggesting that these two isoforms may actually differentially contribute to Ca2+ homeostasis in skeletal muscle cells [28], [29], [30].
Here we report experiments aimed to determine the relative levels of expression of RyR1 and RyR3 isoforms in developing mouse hindlimb and diaphragm muscles. In addition, in order to investigate the actual contribution of RyR1 and RyR3 channels to Ca2+ homeostasis of developing and adult muscle cells, the activity of the two channel types has been investigated in native sarcoplasmic reticulum membranes in the presence or in the absence of CHAPS.
Section snippets
Isolation of SR vesicles
Sarcoplasmic reticulum vesicles were prepared from 5- and 60-day-old mice. Muscles were homogenized in 6.7 mM NaOH and a cocktail of protease inhibitors (leupeptin, aprotinin, antipain, chymostatin and pepstatin A at 2 μg/ml each) and centrifuged at 11,000 × g for 10 min at 4 °C. The supernatant was decanted through a cheesecloth and ultracentrifuged at 100,000 × g for 30 min at 4 °C. The microsomal pellet was resuspended in 50 mM NaCl, 10 mM 3-[N-morpholino]-2-hydroxypropanesulfonic acid (MOPSO), pH 6.8
Developing mouse skeletal muscles express non-equivalent levels of RyR1 and RyR3 channels
In order to evaluate the relative content of RyR1 and RyR3 in developing mouse skeletal muscles, we performed [3H]ryanodine binding experiments on sarcoplasmic reticulum vesicles followed by immunoprecipitation with anti-RyR3 antibodies. For a precise determination of maximum binding site values (Bmax), optimal binding conditions included the use of high salt medium containing 1 M NaCl and 1% CHAPS/0.5% lecithin [28]. The specificity of the RyR3 antibodies has been previously described [28], [29]
Discussion
Ca2+ release from the sarcoplasmic reticulum of skeletal muscles is mediated by members of the ryanodine receptor channel family. In most non-mammalian vertebrates, such as chick, frog and fish, two isoforms of RyRs, α and β RyRs (corresponding to mammalian RyR1 and RyR3, respectively) are expressed at equivalent levels [31], [32]. In mammalian skeletal muscles, expression of RyR1 is continuously detected from early skeletal muscle development stages to adult life [17], [19], [33], [34], [35].
Acknowledgements
This work was supported by a grant from Telethon (no. GGP02168), EU grant (HPRN-CT-2002-00331) and MIUR/FIRB 2001 to V.S. and by grant MIUR 2004 to D.R.
References (37)
- et al.
Two centuries of excitation–contraction coupling
Cell Calcium
(2004) - et al.
Sequential docking, molecular differentiation, and positioning of T-tubule/SR junctions in developing mouse skeletal muscle
Dev. Biol.
(2001) - et al.
Development of the excitation–contraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils
Dev. Biol.
(1993) - et al.
Selective expression of the type 3 isoform of ryanodine receptor Ca2+ release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits
Biochem. Biophys. Res. Commun.
(2005) - et al.
Characterization of type 3 ryanodine receptor (RyR3) of sarcoplasmic reticulum from rabbit skeletal muscles
J. Biol. Chem.
(1997) - et al.
Purification and characterization of ryanodine receptor 3 from mammalian tissue
J. Biol. Chem.
(1998) - et al.
RyR3 amplifies RyR1-mediated Ca2+-induced Ca2+ release in neonatal mammalian skeletal muscle
J. Biol. Chem.
(2001) - et al.
Putative roles of type 3 ryanodine receptor isoforms (RyR3)
Trends Cardiovasc. Med.
(2000) - et al.
Selectively suppressed Ca2+-induced Ca2+ release activity of α-ryanodine receptor (α-RyR) in frog skeletal muscle sarcoplasmic reticulum
J. Biol. Chem.
(2001) - et al.
Intracellular calcium release channel expression during embryogenesis
Dev. Biol.
(1999)
Embryonic chicken skeletal muscle cells fail to develop normal excitation–contraction coupling in the absence of the alpha ryanodine receptor. Implications for a two-ryanodine receptor system
J. Biol. Chem.
Involvement of dihydropyridine receptors in excitation–contraction coupling in skeletal muscle
Nature
Differentiation of the sarcoplasmic reticulum and T-tubule system in developing chick skeletal muscle in vitro
J. Biol. Chem.
Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation–contraction coupling in skeletal muscle
Mol. Biol. Cell
mRNA for cardiac calcium channel is expressed during development of skeletal muscle
Dev. Biol.
Progressive predominance of “skeletal” versus “cardiac” type excitation–contraction coupling during in vitro skeletal myogenesis
Pflugers Arch.
Contractile responses in rat extensor digitorum longus muscle at different times of post-natal development
J. Comp. Physiol.
Calcium currents in embryonic and neonatal mammalian skeletal muscle
J. Gen. Physiol.
Cited by (13)
Two ryanodine receptor isoforms in nonmammalian vertebrate skeletal muscle: Possible roles in excitation-contraction coupling and other processes
2011, Progress in Biophysics and Molecular BiologyCitation Excerpt :Immunohistochemical studies revealed that RyR3 is expressed in a subset of fibers (Conti et al., 2005; Flucher et al., 1999). The relative abundance of RyR3 is estimated to be at most ∼10% as compared to RyR1 in mammalian muscles (Rossi et al., 2007). This is much lower than the relative abundance of β-RyR in nonmammalian vertebrate skeletal muscle (∼50%).
RY-3 ryanodine receptor
2009, xPharm: The Comprehensive Pharmacology ReferenceAssociation between statin-associated myopathy and skeletal muscle damage
2009, CMAJ. Canadian Medical Association JournalDetection of Ca<sup>2+</sup>transients near ryanodine receptors by targeting fluorescent Ca<sup>2+</sup>sensors to the triad
2021, Journal of General PhysiologyMolecular determinants of homo- And heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers
2019, Proceedings of the National Academy of Sciences of the United States of America
- 1
The first two authors contributed equally to this work.