Acute RyR1 Ca2+ leak enhances NADH-linked mitochondrial respiratory capacity

Abstract

Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.

Conflict of interest statement

U.D.M. and J.N.F. are employees of Nestlé Research, which is part of the Société des Produits Nestlé SA. A.R.M. is chair of the SAB and holds stock in ARMGO Pharma Inc., a biotech company developing drugs targeting RyR channel leak. The remaining authors declare no competing interests.

© 2021. The Author(s).

Figures

Fig. 1. One session of SIT induces RyR1 post-translational modifications, calstabin1 dissociation from the RyR1 complex and leads to increased OXPHOS protein expression in human muscle.

a Models of MICT (a1) and SIT (a2) in humans. b, c Assessment of b knee extensor maximal voluntary contraction (MVC) force; n = 8 participants per group and c the ratio of electrically evoked forces at 10 and 100 Hz; n = 7 and 8 participants for MICT and SIT, respectively. Two-way ANOVA followed by Sidak’s multiple comparisons test. d Representative immunoblots (IB) of immunoprecipitated (IP) RyR1, RyR1 post-translational modifications and calstabin1 dissociation. DNP (2,4-dinitrophenylhydrazone): RyR1 oxidation. P RyR1 Ser2844: RyR1 phosphorylation at serine 2844. Cys NO: RyR1 nitrosylation. SK SR: skeletal muscle sarcoplasmic reticulum vesicle. SK SR treated with 200 µM H2O2, 250 µM NOC-12 and 5 units PKA per reaction: positive control for RyR1 oxidation and nitrosylation and calstabin1 dissociation. No antibody in IP: negative control. The whole gel and an additional control are shown in Supplementary Fig. 1f. e–h Quantification of immunoblots in (d); n = 8 participants per group. Two-way ANOVA followed by Sidak’s multiple comparisons test. i Representative immunoblots of PGC-1α. j Representative immunoblots of mitochondrial OXPHOS proteins. All the cropped parts of OXPHOS proteins are part of the same blot that is shown in Supplementary Fig. 5. k Quantification of PGC-1α proteins in (i) related to GAPDH protein and expressed as % of Pre-MICT; n = 8 participants per group. Two-way ANOVA. l–p Quantification of OXPHOS proteins in (j) related to GAPDH protein and expressed as % of Pre-MICT; n = 8 participants per group. Two-way ANOVA, followed by Sidak’s multiple comparisons test (l, m). Data are mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and $main effect of time. Source data are provided as a Source Data file.

Fig. 2. S-SIT in C2C12 myotubes induces RyR1 post-translational modifications, calstabin1 dissociation and decreased SR Ca2+ content.

a S-MICT (a1) and S-SIT (a2) models in C2C12 myotubes. b Metabolomic analysis immediately after stimulation showing changes expressed in % of CTRL values; n = 2 independent biological experiments of six technical replicates each. c–f Representative immunoblots (c) and quantifications (d–f) of RyR1 immunoprecipitation and assessment of RyR1 post-translational modifications immediately after stimulation. DNP (2,4-dinitrophenylhydrazone): RyR1 oxidation. Cys NO: RyR1 nitrosylation. SK SR: skeletal muscle sarcoplasmic reticulum vesicle. SK SR treated with 200 µM H2O2 and 250 µM NOC-12: positive control for RyR1 oxidation, nitrosylation and calstabin1 dissociation. No antibody in IP: negative control; n = 4 independent biological experiments per group. One-way ANOVA followed by Tukey’s multiple comparisons test and Kruskal–Wallis ANOVA, followed by Dunn’s multiple comparison test (e). The whole gel and an additional control are shown in Supplementary Fig. 2g. g–h Original recording (g) and quantifications (h) of normalized Fluo-4 fluorescence in response to 2.5 mM caffeine immediately after S-MICT or S-SIT; n = 7 independent biological experiments per group. One-way ANOVA followed by Tukey’s multiple comparisons test. i, j Fluorescence resonance energy-based assessment of SR Ca2+ content (i) and quantifications of D1ER ratio (j) immediately after S-MICT and S-SIT (i). (i1) D1ER plasmid distribution recorded at 535 nm. (i2) Detail of the reticular pattern of the D1ER signal. (i3) Ca2+ distribution pattern is rendered in pseudocolor scale (from dark blue, low Ca2+ level to red, high Ca2+ level, see arrow); n = 3 independent biological experiments per group. One-way ANOVA followed by Tukey’s multiple comparisons test. k–l Representative immunoblots (k) and quantification (l) of STIM1 after S-MICT and S-SIT; n = 3 independent biological experiments per group. One-way ANOVA. m–o Characterization of store-operated Ca2+ entry (SOCE) in S-MICT and S-SIT myotubes immediately after stimulation. Original recording of SOCE (m); SR Ca2+ store quantifications (n) and SOCE quantifications (o); n = 3 independent biological experiments per group. One-way ANOVA. Data are mean ± SD. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. Source data are provided as a Source Data file.

Fig. 3. S-SIT in C2C12 myotubes induces higher mitochondrial protein content, supercomplexes levels and respiratory capacity as compared to MICT, which are blunted by S107-induced RyR1 stabilization.

a–d Representative immunoblots (a) and quantifications (b–d) of immunoprecipitated RyR1 post-translational modifications 3 h post stimulation without or with S107 treatment for 3 h; n = 4 (CTRL) and 3 (S-MICT, S-SIT) independent biological experiments. One-way ANOVA, followed by Tukey’s multiple comparisons test (c, d). e Quantification of Fluo-4/AM fluorescence ratio 3 h post stimulation without or with S107; n = 4 (CTRL, S-SIT) and 3 (S-SIT S107) independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparisons test. f–k Representative immunoblots (f) and quantifications (g–k) of mitochondrial OXPHOS proteins 72 h post stimulation without or with S107 treatment for 72 h. Whole OXPHOS blot shown in Supplementary Fig. 6; n = 7 (CTRL), 6 (S-MICT, S-SIT) and 3 (S-MICT S107, S-SIT S107) independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparisons test. l–p Immunoblots (l) and quantification (m–p) of supercomplexes after stimulations; n = 3 independent biological experiments per group. One-way ANOVA followed by Tukey’s multiple comparisons test (m–o). q–r MitoTracker red fluorescence (q) and quantification of mitochondrial area (r) 72 h post stimulation without or with S107 treatment; n = 6 independent biological experiments per group. One-way ANOVA with Sidak’s multiple comparisons test. s–w O2 flux per mass (pmol/s/mg of protein) 72 h after stimulation and S107 treatment. s MPL: malate pyruvate leak state. t NP: N-linked OXPHOS state with ADP-stimulated. u NSP: N- and S-OXPHOS pathways. v NSE: ET state, noncoupled and w SE, S-pathway; n = 5 (CTRL, S-SIT), 4 (S-MICT) and 3 (S-SIT S107) independent biological experiments. One-way ANOVA with Sidak’s multiple comparisons test. x–z NADH (x), NAD (y) concentration and NADH/NAD ratio (z) in S-SIT myotubes without or with S107 treatment for 72 h; n = 6 independent biological experiments per group. Unpaired t test. For S107 groups, 10 µM of S107 treatment was applied immediately after stimulation for the indicated time. Data are mean ± SD. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. Source data are provided as a Source Data file.

Fig. 4. Proteomic analysis showing the global mapping of RyR1 Ca2+ leak on muscle adaptations to S-SIT.

a–c Proteomic analysis of protein groups related to Gene Ontology Biological Processes (GoBP), Molecular Function (GoMF) and Cellular Component (GoCC) that are significantly decreased S-SIT after 10 µM S107 treatment for 72 h (the treatment was applied immediately after stimulation). Protein groups exceeding 400 proteins were excluded. The median values of S-SIT − S-SIT S107 difference were calculated, and a score affected to the amplitude of the difference. The positive scores display the pathways significantly inhibited by S107 treatment. n = 5 per group. Benjamini–Hochberg corrected t test. Source data are provided as a Source Data file.

Fig. 5. (S-)SIT-induced leaky RyR1 increases mitochondrial Ca2+ uptake and decreases PDH phosphorylation levels in muscle cells.

a–e Normalized Rhod-2 fluorescence imaging in C2C12 myotubes. A 300 s time-lapse confocal recording of a bout of slightly modified S-SIT and S-MICT protocols: b S-MICT and c S-SIT. d, e Mitochondrial Ca2+ uptake in S-SIT myotubes pre-treated with 10 µM S107 for 1 h (d) or 20 µM mitoxantrone (MTX) (e). Horizontal black lines indicate the periods of stimulation; vertical red arrows indicate the amplitude of mitochondrial Ca2+ at the end of the recordings. f–g Maximal amplitude of normalized Rhod-2 fluorescence during stimulations (f) and 80 s after the end of S-MICT and S-SIT stimulations (g) in (a–e); n = 5 (CTRL, S-SIT and S-SIT S107) and 3 (S-MICT, S-SIT MTX) independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparisons test. h–j Normalized Rhod-2 fluorescence imaging in mouse FDB intact single muscle fibres. Same protocols as described for (a–c), except that the voltage was 40 V. k–l Maximal amplitude of normalized Rhod-2 fluorescence in FDB muscle fibres in (h–j) during (k) and 80 s after the end (l) of S-MICT and S-SIT stimulations; n = 3 mice per group. One-way ANOVA followed by Tukey’s multiple comparisons test. m–n Representative immunoblots (m) and quantification (n) of phosphorylated PDH E1α at serine 293 related to total PDH E1α in myotubes 1 h after stimulation; 10 µM S107 or 20 µM MTX were applied after the stimulation for 1 h when indicated; n = 7 (CTRL, S-MICT, S-SIT and S-SIT S107) and 3 (S-SIT MTX) independent biological experiments. One-way ANOVA followed by Sidak’s multiple comparisons test. o, p Representative immunoblots (o) and quantification (p) of phosphorylated PDH E1α at serine 293 related to total PDH E1α in human muscles; n = 8 participants per group. Two-way ANOVA followed by Sidak’s multiple comparisons test. q Proposed schematic of RyR1 Ca2+ leak-activated mitochondrial PDH E1α dephosphorylation in response to S-SIT. Data are mean ± SD. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. Source data are provided as a Source Data file.

Fig. 6. Mitochondrial Ca2+ uptake contributes to mitochondrial adaptations to S-SIT.

a Immunoblots of MCU, RyR1, SERCA1, GAPDH and calstabin1 in si-MCU (siRNAs directed against MCU) compared to si-CTRL (negative control siRNAs) myotubes. b Original recordings of normalized Rhod-2 fluorescence imaging in si-MCU compared to si-CTRL myotubes at 72 h post transfection. c Representative immunoblots of mitochondrial OXPHOS and MCU proteins expression in S-SIT si-MCU myotubes at 72 h post stimulation (cells were transfected immediately after the stimulation with the siRNAs against MCU) compared to S-SIT si-CTRL myotubes (cells were transfected immediately after the stimulation with the negative control siRNAs) and si-CTRL (non-stimulated myotubes transfected with the negative control siRNAs). All the cropped parts of OXPHOS proteins are part of the same blot that is shown in Supplementary Fig. 6. d–i Quantifications of the immunoblots in (c) related to total protein and expressed as % of si-CTRL; n = 6 independent biological experiments per group. One-way ANOVA followed by Tukey’s multiple comparisons test (d, e, h). Data are mean ± SD. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. Source data are provided as a Source Data file.