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.

Keywords: Calcium; Cell Biology; Muscle; Muscle Biology.

Figure 1. NR1D1 regulates SR Ca²⁺ homeostasis in skeletal muscle.

(A) In situ measurement of gastrocnemius-developed force upon an electrical stimulus in wild-type (Nr1d1^+/+) and Nr1d1^–/– mice. ***P = 0.0004 vs. Nr1d1^+/+, unpaired t test; n = 9–10. (B) Representative curves of SERCA-inhibitable Ca²⁺ uptake in microsomal fractions prepared from muscle from Nr1d1^+/+ and Nr1d1^–/– mice. Decrease in fluorescence indicates Ca²⁺ uptake by microsomes. Arrows indicate Ca²⁺ or thapsigargin (TG) injections. The red rectangle was used for (C) slope calculation of fluorescence decrease, indicative of specific SERCA Ca²⁺ uptake. *P = 0.0203 vs. Nr1d1^+/+, unpaired t test; n = 6. (D) Representative curves of SERCA-inhibitable Ca²⁺ uptake in microsomes prepared from muscle from vehicle- or SR9009-treated wild-type mice. (E) Slopes of the decreasing fluorescence over time. *P = 0.0408 vs. vehicle, unpaired t test; n = 4. (F) TG-induced SR Ca²⁺ release in control pBabe- and NR1D1-overexpressing C2C12 myotubes. Cells were loaded with Fluo4-AM to detect cytosolic Ca²⁺. SR Ca²⁺ content depletion was induced by TG (1 μM) (n = 10). (G) Delta F/F0 ratio normalized to pBabe values, obtained 5 minutes after TG-induced Ca²⁺ release. **P = 0.007 vs. pBabe, unpaired t test; n = 10. (H) Representative Fura-2/AM experiments (ratio F340/F380) in pBabe- and NR1D1-overexpressing cells. (I) Normalized SR calcium concentration released upon TG treatment in pBabe- and NR1D1-overexpressing cells. (J) Normalized basal cytosolic calcium concentration (mean of the 100 first seconds) in pBabe- and NR1D1-overexpressing cells. (I and J) Box-and-whisker plots show all points, with minimums and maximums (n > 300 in each group). ***P < 0.0001 vs. pBabe, unpaired t test. (K) TG-induced SR Ca²⁺ release in C2C12 cells transfected by control (siCTRL) or Nr1d1 siRNA (siNr1d1). Results are shown as Delta F/F0 ratio (n = 17). (L) Delta F/F0 ratio normalized to siCTRL values obtained 5 minutes after TG-induced Ca²⁺ release. **P = 0.0044 vs. siCTRL, unpaired t test; n = 17. Data are shown as the mean ± SEM.

Figure 2. NR1D1 represses myoregulin expression through direct binding to its promoter.

(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 DBD_mut^fl/fl, MCK^Cre/+) and control Nr1d1 DBD_mut^fl/fl mice. *P = 0.0139 vs. Nr1d1 DBD_mut^fl/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.

Figure 3. NR1D1 activation alleviates Duchenne muscular dystrophy features both in mice and human myoblasts.

(A) NR1D1 expression in muscle biopsies from controls (n = 14) and patients with Duchenne muscular dystrophy (DMD, n = 23). Data are from GEO data set GSE6011. **P = 0.0092, unpaired t test. (B) NR1D1 expression in control or DMD primary myotubes. *P = 0.0404 vs. control cells, unpaired t test; n = 5–7. (C) Representative curves and (D) peak fluorescence intensity of thapsigargin-induced (TG-induced) sarcoplasmic reticulum (SR) Ca²⁺ release in myoblasts from controls or patients with DMD treated with SR9009 (10 μM) or vehicle. Cells were loaded with Fluo4-AM and SR Ca²⁺ release was induced by the addition of 1 μM TG. Results are expressed as (mean ± SEM) the Delta F/F0 ratio. **P = 0.0049 vs. control cells, unpaired t test; n = 3 controls, n = 7 in both DMD groups. (E) H&E and Sirius red staining of tibialis anterior muscles obtained from vehicle- and SR9009-injected mdx/Utr^+/– mice. Scale bars: 100 μm. (F) Myofiber cross-sectional area distribution. *P < 0.05 vs. vehicle-treated mdx/Utr^+/– mice by 2-way ANOVA; n = 7–9. (G) Circulating creatine phosphokinase (CPK) activity. *P = 0.0329 vs. vehicle-treated mdx/Utr^+/– animals; n = 8–9. (H) Muscular hydroxyproline. *P = 0.0172; n = 8–10. (I) Col1a2 (*P = 0.0314), (J) Pdgfra (*P = 0.0164), and (K) Mln (*P = 0.0402) gene expression; n = 8–12. (L) SERCA activity (n = 8–10) in muscular microsomes from mdx/Utr^+/– mice treated for 20 days with SR9009 (100 mg/kg) or vehicle. *P = 0.0266. (M) In situ measurement of gastrocnemius-developed force. *P = 0.0301; n = 4–9. (G–M) Unpaired t test.