Muscle RANK is a key regulator of Ca2+ storage, SERCA activity, and function of fast-twitch skeletal muscles

Abstract

Receptor-activator of nuclear factor-κB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscles preventing the loss of maximum specific force while promoting muscle atrophy, fatigability, and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 content, a Ca(2+)sensor, and altered activity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) modulating Ca(2+)storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus muscles. These data identify a novel role for RANK as a key regulator of Ca(2+)storage and SERCA activity, ultimately affecting denervated skeletal muscle function.

Keywords: receptor-activator of nuclear factor-κB; sarco(endo)plasmic reticulum Ca2+-ATPase.

Fig. 1.

Receptor-activator of nuclear factor-κB (RANK) expression in skeletal muscles and myotubes. A: Western blot showing that fully differentiated C₂C₁₂ myotubes but not myoblasts express RANK protein. GAPDH is shown as a loading control. B: confocal images showing colocalization of the intracellular face of the cytoplasmic membrane/sarcolemma dystrophin (green) and RANK (red) in RANK^f/f and the absence of RANK in RANK^mko extensor digitorum longus (EDL) muscles. Thymus sections were used as positive controls for RANK immunofluorescence. Omission of primary antibody was used as a negative control. Because nonmuscle cells in skeletal muscles can also express RANK, confocal images, rather than Western blots, were required to confirm the absence of muscle RANK in RANK^mko mice. Bar = 100 μm. C: PCR analysis of genomic DNA isolated from soleus (Sol) muscle, EDL muscle, heart, liver, spleen, and kidney showing efficient Cre-mediated recombination of loxP sites in skeletal muscles from RANK^mko mice.

Fig. 2.

Impact of RANK deletion on muscle contractility, fatigue, and phenotype. A: muscle atrophy was significantly more pronounced in denervated RANK^mko relative to RANK^f/f EDL muscles. B: however, ex vivo force measurements show that RANK ablation preserves the specific force tension of denervated (Den) EDL muscles but not in slow-twitch Sol muscles. C: RANK deletion increases the ratio of twitch tension (P_t) to absolute force production (P₀) in Sol and EDL muscles following denervation. D: muscle glycolytic fatigue protocol was induced by a train of stimulations (200 ms on-800 ms off, 50 Hz) until Sol muscle force reaches 70% of its initial force. Fatigue time is decreased in denervated muscle but similar between RANK^f/f and RANK^mko muscles. E: muscle glycolytic fatigue protocol was induced by a train of stimulations (200 ms on-800 ms off, 50 Hz) until EDL muscle force reached 50% of its initial force. Sham and denervated RANK^mko EDL muscles exhibit increased fatigability compared with sham and denervated RANK^f/f EDL muscles. F: fiber typing of EDL muscle analysis showed that the slow-twitch fibers were nearly absent in sham RANK^mko EDL muscles, whereas the proportion of fast-twitch fibers (IIA + IIB + IIX) was significantly increased in denervated RANK^mko EDL muscles compared with denervated RANK^f/f EDL muscles. The levels of significance were set at *P < 0.05, **P < 0.01, and ***P < 0.001 for genotype (RANK^f/f vs. RANK^mko) or #P < 0.05 for treatment (Sham vs. Den). Data are presented as means ± SE, n = 3–7 experiments.

Fig. 3.

Percentage of type I and II fibers in Sol muscles and proportion of each fiber type expressing sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)-1a and SERCA-2a in Sol and EDL muscles. A: as opposed to fast-twitch EDL muscles, no changes in phenotype were observed in sham or denervated RANK^f/f and RANK^mko in Sol muscles. B: sham and denervated RANK^mko muscles exhibited a lower proportion of fast-twitch fibers expressing SERCA-1a and a higher proportion of fast-twitch fibers expressing SERCA-2a. The levels of significance were set at *P < 0.05 for genotype (RANK^f/f vs. RANK^mko) or #P < 0.05 for treatment (Sham vs. Den). Data are presented as means ± SEM, n = 6–8.

Fig. 4.

Total Ca²⁺ ([CaT]) content, SERCA activity, and Ca²⁺ protein contents in RANK^f/f and RANK^mko EDL muscles. A–C: [CaT] was decreased in sham RANK^mko EDL muscles relative to sham RANK^f/f EDL muscles but increased sharply in denervated RANK^mko EDL muscles. D: Western blots show that SERCA-1a content is reduced, whereas SERCA-2a content is increased, following denervation. No changes in calsequestrin content were observed, but stromal interaction molecule 1 (Stim1) content increased markedly in denervated RANK^mko EDL muscles. E: SERCA Ca²⁺-dependent ATPase activity was assessed in muscle homogenates from sham and denervated RANK^f/f and RANK^mko EDL muscles over Ca²⁺ concentrations ranging from pCa 7.4-5.0. F and G: maximal ATPase activity (V_max, F) and pCa value required to elicit 50% of maximal activity (pCa₅₀, G). The levels of significance were set at *P < 0.05, **P < 0.01, and ***P < 0.001 for genotype (RANK^f/f vs. RANK^mko) or #P < 0.05 for treatment (Sham vs. Den). Data are presented as means ± SE, n = 3–7.

Fig. 5.

SERCA Ca²⁺-dependent ATPase parameters from sham and denervated RANK^f/f and RANK^mko in Sol muscles. Denervation induced a significant reduction of SERCA Ca²⁺-dependent ATPase activity (A), V_max (B), and pCa₅₀ (C) that was similar between control and experimental Sol muscles. The level of significance treatment (Sham/Den) was set at #P < 0.05. Data are presented as means ± SE, n = 3–7.

Fig. 6.

Schematic representation of the RANK/RANKL/OPG pathway as a common regulator of bone and muscle cells. In osteoclast, RANK/RANKL interaction regulates osteoclastogenesis and/or cell apoptosis through modulation of SERCA activity, Ca²⁺ oscillation, and Ca²⁺-calcineurin-nuclear factor of activated T cells (NFAT) and NF-κB pathways. Muscle cells also express RANK, and RANK/RANKL interaction is an important regulator of SERCA activity and Ca²⁺ storage in fast-twitch EDL muscles. These results highlight the importance of a common signaling pathway opening potentially new treatment for both skeletal muscle and bone.