Elsevier

Cell Calcium

Volume 41, Issue 6, June 2007, Pages 573-580
Cell Calcium

Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development

https://doi.org/10.1016/j.ceca.2006.10.007Get rights and content

Abstract

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.

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)

  • A. Ivanenko et al.

    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.

    (1995)
  • E. Rios et al.

    Involvement of dihydropyridine receptors in excitation–contraction coupling in skeletal muscle

    Nature

    (1987)
  • E.B. Ezerman et al.

    Differentiation of the sarcoplasmic reticulum and T-tubule system in developing chick skeletal muscle in vitro

    J. Biol. Chem.

    (1967)
  • B.E. Flucher et al.

    Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation–contraction coupling in skeletal muscle

    Mol. Biol. Cell

    (1994)
  • N. Chaudahari et al.

    mRNA for cardiac calcium channel is expressed during development of skeletal muscle

    Dev. Biol.

    (1993)
  • C. Cognard et al.

    Progressive predominance of “skeletal” versus “cardiac” type excitation–contraction coupling during in vitro skeletal myogenesis

    Pflugers Arch.

    (1992)
  • Y. Pereon et al.

    Contractile responses in rat extensor digitorum longus muscle at different times of post-natal development

    J. Comp. Physiol.

    (1993)
  • K.G. Beam et al.

    Calcium currents in embryonic and neonatal mammalian skeletal muscle

    J. Gen. Physiol.

    (1988)
  • 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 Biology
      Citation 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 Reference
    • Molecular 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
    View all citing articles on Scopus
    1

    The first two authors contributed equally to this work.

    View full text