Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4

Abstract

Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O(2) sensor in skeletal muscle, and O(2)-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O(2)-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.

Fig. 1.

pO₂-dependent regulation of RyR1 by endogenous ROS. (A and B) In SR vesicles isolated from rabbit hind-limb skeletal muscle, RyR1 activity assessed by [³H]-ryanodine binding (A) and ROS production assessed by DHE fluorescence (B) were enhanced progressively as pO₂ increased from 1% to 5–20% O₂. Single and double asterisks indicate significant difference versus 20% O₂ and 5% O₂, respectively (P < 0.01; n = 3–5). (C) PEG-catalase largely eliminated the enhancement of RyR1 activity at high versus low pO₂ (n = 3).

Fig. 2.

Characterization of the ROS-generating activity of SR. In SR vesicles isolated from skeletal muscle of rabbit, (A) ROS production (DHE fluorescence) and (B) RyR1 activity ([³H]-ryanodine binding) were enhanced at high versus low pO₂. At high pO₂, addition of NADPH (1 mM) enhanced production of O₂⁻, and this enhancement was eliminated by DPI. (n = 4–6).

Fig. 3.

Colocalization of Nox4 and RyR1 in skeletal muscle. (A) Subcellular fractionation of rat hind-limb muscle showed that Nox4 and RyR1 coenriched and were most abundant in the SR-enriched fraction isolated from the microsomal fraction by densit- gradient centrifugation. (B) Samples taken progressively from the top to the bottom of the SR-enriched gradient segment, which are progressively enriched in junctional SR (heavy SR) (45, 51), were increasingly enriched in both RyR1 and Nox4, whereas the dihydropyridine receptor/Ca²⁺ channel (DHPR), a constituent of transverse tubule membranes, exhibited a disparate pattern of enrichment. (C) Nox4 coimmunoprecipitates with RyR1 from solubilized SR vesicles; results of two separate experiments are shown.

Fig. 4.

ROS generated by Nox4 mediate pO₂-dependent regulation of RyR1 in C2C12 cells. RyR activity in the microsomal fraction from differentiated C2C12 cells (assessed by [³H]-ryanodine binding) is enhanced at high versus low pO₂ and by the addition of NADPH (1 mM), and enhancement is abrogated by DPI (20 μM) and by siRNA-mediated knockdown of Nox4. For samples at 20% O₂, asterisks indicate significant difference versus control (*P < 0.05 re control at 20% O₂; n = 4–6).

Fig. 5.

Nox4 regulates stimulus-induced Ca²⁺ release through RyR1. (A) In primary skeletal muscle myocytes, electrically induced Ca²⁺ transients are greater at high than at low pO₂, and this difference is largely eliminated by PEG-catalase (n = 3–4; asterisk indicates P < 0.05). (B) In C2C12 cells, maximal cytoplasmic Ca²⁺ levels following depolarization by KCl (50 mM) are greater at high than at low pO₂, and this difference is eliminated by Nox4 knockdown with Nox4-specific siRNA. Nox4 knockdown does not affect Ca²⁺ levels at low pO₂ or the time to peak amplitude at high pO₂. Each data point was obtained by integrating fluorescence emission over three to five entire myofibers within a single microscopic field of view during a single experiment (myofibers were depolarized only once); n = 6–9. Asterisks indicate significant differences (P < 0.05). (C) For each condition, maximal depolarization-induced Fluo 3 fluorescence was measured by integrating within 12 subfields distributed within three to five fibers, in each of six to nine experiments. As indicated by an asterisk, ANOVA indicated a significant difference (P ≤ 0.016) between the magnitude of Ca²⁺ release at high pO₂ in control siRNA-treated samples and all other conditions; there were no significant differences (P ≥ 0.465) between any other pair of conditions (excluding the effects of ryanodine). Note that ryanodine eliminated K⁺-induced Ca²⁺ release (n = 3), which therefore may be ascribed to RyR activity, and that elimination of H₂O₂ with PEG-catalase also eliminated the enhancement of Ca²⁺ release at high pO₂ (n = 5).

Fig. 6.

pO₂-dependent regulation of muscle contractility by endogenous ROS. (A) Contractile force evoked by tetanic stimulation (40–250 Hz) of isolated, intact mouse EDL muscles at 1% O₂, 5% O₂, or 20% O₂ and (B) the effects of PEG-catalase (500 U/mL). (A) Plots of force generation versus stimulus frequency reveal a leftward (facilitatory) shift as a function of increasing pO₂. *P < 0.05, 1% and 20% O₂; **P < 0.05, 1% versus 5% O₂ (two-way ANOVA; n = 3–4). (B) Force generation is reduced by PEG-catalase at 20% and at 5% O₂ but not at 1% O₂. *P < 0.05, control values versus values in the presence of PEG-catalase (two-way ANOVA; n = 3–4). (C) Contractile force induced by maximal tetanic stimulation (250 Hz) of isolated, intact mouse EDL muscle decreases in proportion to the degree of shRNA-mediated Nox4 knockdown. (D) A histogram illustrates the decrease in tetanic force generation for muscles in which knockdown of Nox4 was ≥60% (*P < 0.05 re control; n = 5).