NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression
Alexis Boulinguiez, Christian Duhem, Alicia Mayeuf-Louchart, Benoit Pourcet, Yasmine Sebti, Kateryna Kondratska, Valérie Montel, Stéphane Delhaye, Quentin Thorel, Justine Beauchamp, Aurore Hebras, Marion Gimenez, Marie Couvelaere, Mathilde Zecchin, Lise Ferri, Natalia Prevarskaya, Anne Forand, Christel Gentil, Jessica Ohana, France Piétri-Rouxel, Bruno Bastide, Bart Staels, Helene Duez, Steve Lancel
Alexis Boulinguiez, Christian Duhem, Alicia Mayeuf-Louchart, Benoit Pourcet, Yasmine Sebti, Kateryna Kondratska, Valérie Montel, Stéphane Delhaye, Quentin Thorel, Justine Beauchamp, Aurore Hebras, Marion Gimenez, Marie Couvelaere, Mathilde Zecchin, Lise Ferri, Natalia Prevarskaya, Anne Forand, Christel Gentil, Jessica Ohana, France Piétri-Rouxel, Bruno Bastide, Bart Staels, Helene Duez, Steve Lancel
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Research Article Cell biology Muscle biology

NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression

Abstract

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase–dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/– mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.

Authors

Alexis Boulinguiez, Christian Duhem, Alicia Mayeuf-Louchart, Benoit Pourcet, Yasmine Sebti, Kateryna Kondratska, Valérie Montel, Stéphane Delhaye, Quentin Thorel, Justine Beauchamp, Aurore Hebras, Marion Gimenez, Marie Couvelaere, Mathilde Zecchin, Lise Ferri, Natalia Prevarskaya, Anne Forand, Christel Gentil, Jessica Ohana, France Piétri-Rouxel, Bruno Bastide, Bart Staels, Helene Duez, Steve Lancel

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Figure 2

NR1D1 represses myoregulin expression through direct binding to its promoter.

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NR1D1 represses myoregulin expression through direct binding to its prom...
(A–L) RyR1, Serca1, and Serca2 gene expression in (A–C) muscle from Nr1d1+/+ and Nr1d1–/– mice (n = 6) and (D–F) pBabe- and NR1D1-overexpressing differentiated C2C12 (n = 3). Myoregulin (Mln) expression in (G) muscle from Nr1d1+/+ and Nr1d1–/– mice (**P = 0.0025 vs. Nr1d1+/+; n = 9), (H) muscle from SR9009-treated wild-type animals (**P = 0.0069 vs. vehicle; n = 5), (I) NR1D1-overexpressing C2C12 (**P = 0.0023 vs. pBabe; n = 5), (J) C2C12 treated with 10 μM of NR1D1 agonist SR9009 (*P = 0.0155 vs. DMSO; n = 3), (K) C2C12 transfected with siNr1d1 (***P < 0.001 vs. siCTRL; n = 3), and (L) C2C12 treated with 10 μM of NR1D1 antagonist SR8278 (***P < 0.0001 vs. DMSO; n = 6). Unpaired t test. (M) Schematic representation of Mln promoter, indicating 3 putative Rev-erbα response elements (RevRE), located approximately 1.4 kb, 5.4 kb, and 6.7 kb upstream of the transcription initiation site. (N) ChIP analysis using an anti-NR1D1 antibody or control IgG. –6.7 kb, **P = 0.0017; –5.4 kb, ***P < 0.0001; –1.4 kb, ***P = 0.001 vs. IgG; n = 6–8 mice. (O) Mln expression in mice with muscle-specific expression of a mutated isoform of NR1D1 lacking the DNA-binding domain (Nr1d1 DBDmutfl/fl, MCKCre/+) and control Nr1d1 DBDmutfl/fl mice. *P = 0.0139 vs. Nr1d1 DBDmutfl/fl, unpaired t test; n = 3–5. (P) TG-induced SR Ca2+ release in pBabe- and Mln-overexpressing differentiated C2C12 expressed as Delta F/F0 ratio. n = 7. (Q) Peak fluorescence intensity of TG-induced SR Ca2+ release in pBabe- and Mln-overexpressing differentiated C2C12, normalized to pBabe. **P = 0.024 vs. pBabe, unpaired t test; n = 7. (R) TG-induced SR Ca2+ release in pBabe- and NR1D1- and NR1D1/Mln-overexpressing differentiated C2C12 expressed as Delta F/F0 ratio; n = 6. (S) Peak fluorescence intensity of TG-induced SR Ca2+ release in pBabe- and NR1D1- and NR1D1/Mln-overexpressing differentiated C2C12, normalized to pBabe. *P < 0.026 vs. pBabe, $P < 0.0293 vs. NR1D1, 1-way ANOVA, Tukey’s multiple comparison test; n = 6. Data are shown as the mean ± SEM.