Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise
- Matthew Hancock
- Adam A Nabeebaccus
- Norman Catibog
- Angela Logan
- Ioannis Smyrnias
- Synne S Hansen
- Johanna Lanner
- Katrin Schröder
- Ajay M Shah
- Min Zhang
- King's College London British Heart Foundation Centre of Excellence, United Kingdom
- University of Tromsø - The Arctic University of Norway, Norway
- University of Cambridge, United Kingdom
- Karolinska Institutet, Sweden
- Goethe-Universität, Germany
Figures
Figure 1 with 3 supplements
Nox4 mediates myocardial Nrf2 activation and is essential in the acute response to exercise.
(A) Changes in myocardial Nox4 mRNA and protein levels after acute moderate exercise (Ex) compared to sedentary controls (Sed). *p<0.05, **p<0.01, 2-tailed t-test (n = 4–6/group). (B) Exercise capacity of Nox4-null mice (Nox4KO) and littermate wild-types (WT) measured by maximal running distance and running time. **p<0.01, 2-tailed t-test (n = 5–8/group). (C) Ratio of glutathione/glutathione disulfide (GSH/GSSG) in Nox4KO and WT mouse hearts before (Sed) and after exercise (Ex). n = 6–9/group. (D) Protein levels of 4-hydroxynonenal (4-HNE) adducts in the heart. n = 3–4/group. (E and F) Protein and mRNA levels of major Nrf2 targets. n = 3–6/group *p<0.05, **p<0.01 vs respective sedentary controls (Sed); #p<0.05, ##p<0.01 vs WT/Ex, 1-way ANOVA followed by Tukey post hoc analysis. CAT: catalase, GSTA2: glutathione S-transferase A2, GCLC: glutamate cysteine ligase catalytic subunit, TXNRD1: thioredoxin reductase 1.
Figure 1—figure supplement 1
Full images of Western blots in Figure 1.
https://doi.org/10.7554/eLife.41044.003
Figure 1—figure supplement 2
Myocardial Nox2 is unchanged during acute exercise.
Protein levels of total Nox2 (A) and p47phox in membrane fraction (B) in normal mouse hearts after acute exercise (Ex). GAPDH and cadherin were used as cytosolic and membrane protein marker respectively. Sed: sedentary control, T: total heart tissue lysate, Mem: membrane fraction, Cyt: cytosolic protein. n.s: no significance. n = 3/group.
Figure 1—figure supplement 3
Quantification of Western blots in Figure 1E.
Protein levels of Nrf2 and its main targets in the hearts of Nox4KO and WT mice before (Sed) and after exercise (Ex). *p<0.05, **p<0.01 vs respective sedentary control. #p<0.05, ##p<0.01 vs WT/Ex, 1-way ANOVA followed by Tukey post hoc analysis (n = 4–6/group). GSTA2: glutathione S-transferase A2, GCLC: glutamate cysteine ligase catalytic subunit.
Figure 2 with 1 supplement
Cardiac Nox4 deficiency impairs heart function and exercise capacity due to lack of Nrf2 activation.
(A) Expression of Nrf2-related genes in hearts of cardiomyocyte-specific Nox4KO (csNox4KO) and littermate controls after exercise (Ex). n = 3–4/group. **p<0.01 vs respective sedentary controls (Sed); #p<0.05, ##p<0.01 vs Control/Ex. (B) Maximal exercise distance and time. n = 3–5/group. *p<0.05 vs Control/Ex; #p<0.05, ##p<0.01 vs csNox4KO/Ex, 1-way ANOVA followed by Tukey post hoc analysis. (C) Fractional shortening (FS) evaluated by conscious echocardiography in control and csNox4KO mice immediately after the exercise capacity test. Some animals were treated with sulforaphane (SFN) prior to exercise. n = 4–12/group. **p<0.01 vs Sed; ##p<0.01 vs Control/Ex, 2-way ANOVA followed by Tukey post hoc analysis. Cat: catalase, Gsta2: glutathione S-transferase A2, Gclc: glutamate cysteine ligase catalytic subunit, Txnrd1: thioredoxin reductase 1.
Figure 2—figure supplement 1
csNox4KO mice exhibit reduced cardiac function at peak exercise.
Cardiac function was evaluated by awake echocardiography prior to (sedentary, Sed) and after the exercise capacity test (Ex). Some animals were treated with sulforafane (SFN). Mean data of heart rate (HR), left ventricular end-systolic dimension (LVESD) and left ventricular end-diastolic dimension (LVEDD) (A to C). *p<0.05, **p<0.01 vs respective sedentary controls, ##p<0.01 vs Control/Ex, 2-way ANOVA followed by Tukey post hoc analysis (n = 4–12/group).
Figure 3 with 3 supplements
Cardiomyocyte-targeted Nrf2 knockout mice have impaired mitochondrial function and reduced heart performance upon exercise.
(A) Targeting strategy for generation of Nrf2fl/fl mice (Flox). The endogenous Nrf2 locus is shown at the top and the targeting vector at the bottom. Exons 2–5 were flanked by LoxP sites which are represented by blue triangles; FRT (Neo) and F3 (Puro) sites are shown by double red triangles. Cre-mediated recombination deletes a 5.5 kb fragment including most of the open-reading frame and the 3’UTR. (B) Southern blot analysis of genomic DNA from selected ES cell clones, performed after excision at HincII sites, showing correct 3’ homologous recombination in all clones. WT indicates wild-type. (C) Nrf2 mRNA levels (n = 6/group) and (D) protein levels (n = 3/group) in the hearts of csNrf2KO and Flox control mice. **p<0.01, 2-tailed t-test. (E) Increase in Nox4 mRNA levels in csNrf2KO and control mouse hearts after acute moderate exercise. **p<0.01 vs respective sedentary controls, 2-tailed t-test (n = 6/group). (F) Fractional shortening (FS) by echocardiography immediately after the exercise capacity test. *p<0.05 vs sedentary (Sed) Flox control mice, 1-way ANOVA followed by Tukey post hoc analysis (n = 6–7/group). (G) Respiration rates in isolated cardiac mitochondria of csNrf2KO and control mice after exercise. Both ADP-limited oxidative phosphorylation (OXPHOSLim) and maximal oxidative capacity (OXPHOSMax) were measured (see Materials and methods for details). *p<0.05 vs csNrf2KO/Sed, # p<0.05 Flox/Ex, 1-way ANOVA followed by Tukey post hoc analysis (n = 4–6/group). (H) Citrate synthase (CS) activity in the heart. n = 4–6/group.
Figure 3—figure supplement 1
Identification of csNrf2KO mouse.
(A) Full images of Western blot of Nrf2 using whole heart lysates from csNrf2KO mice and their littermate Flox controls. (B) Nrf2 mRNA levels by real-time PCR in different tissues including lung, liver and skeletal muscle (Skel Mus). n.s: no significance. n = 5/group.
Figure 3—figure supplement 2
csNrf2KO mice exhibit normal cardiac morphology and function at baseline.
(A) Heart weight/body weight ratio (HW/BW), and (B) Ejection fraction (EF). n.s: no significance. n = 5–6/group.
Figure 3—figure supplement 3
Respiratory rates in isolated cardiac mitochondria of csNrf2KO mice after exercise.
(A) The relationship between molecules of ADP phosphorylated and oxygen consumed (P/O ratio). (B) Respiratory coupling ratio (RCR). (C) Cytochrome C was added after oxidative capacity measurement to investigate mitochondrial membrane integrity (OXPHOSCyt). (D) Cardiac mitochondrial function evaluated by a substrate-uncoupler-inhibitor-titration (SUIT) protocol. Mitochondrial proton leak using malate and palmityol carnitine as substrates (LeakFA), maximal oxidative phosphorylation with a saturated amount of ADP (OXPHOXFA), following the addition of complex I substrates, pyruvate and glutamate (OXPHOXFA+CI), and following the addition of complex II substrate, succinate (OXPHOXFA+CI+CII). Maximal electron transport chain (ETC) capacity was measured following the addition of carbonyl cyanide m-chlorophenyl hydrazone (ETCFA+CI+CII), and rotenone was added to measure ETC capacity using complex II substrates (ETCCII). *p<0.05 vs respective sedentary controls, # p<0.05 vs Flox/Ex, 1-way ANOVA followed by Tukey post hoc analysis (n = 4–6/group).
Figure 4 with 3 supplements
Cardiomyocyte Nrf2 is required for increase in mitochondrial antioxidant capacity with exercise.
(A) Western blots for peroxiredoxin III (PRXIII), thioredoxin reductase-2 (TRXR2) and superoxide dismutase-2 (SOD2) in heart of csNrf2KO and Flox controls after acute exercise (Ex). Mean data from n = 4–6/group are shown on the right. *p<0.05 vs respective sedentary controls (Sed); #p<0.05, ##p<0.01 vs Flox/Ex, 1-way ANOVA followed by Tukey post hoc analysis. (B) In vivo mitochondrial H2O2 levels assessed by the MitoP/MitoB ratio in hearts of csNrf2KO mice and controls after acute exercise. **p<0.01 vs Flox/Sed, 1-way ANOVA followed by Tukey post hoc analysis (n = 5–6/group). (C) MitoQ treatment improved cardiac contractile performance at peak exercise in csNrf2KO mice. n = 6–7/group. (D) and (E) The effect of MitoQ treatment on maximal running distance and time. n = 7–11/group. *p<0.05, **p<0.01 vs Flox/Ex, #p<0.05, ##p<0.01 vs csNrf2KO/Ex, 2-way ANOVA followed by Tukey post hoc analysis.
Figure 4—figure supplement 1
Full images of Western blots in Figure 4.
https://doi.org/10.7554/eLife.41044.013
Figure 4—figure supplement 2
Nox4 is required for increase in mitochondrial antioxidant capacity with exercise.
Peroxiredoxin III (PRXIII), thioredoxin reductase-2 (TRXR2) and superoxide dismutase-2 (SOD2) in the hearts of Nox4-null and littermate wild-type control mice (WT) after acute exercise (Ex). Mean data from n = 4–6/group are shown on the right. *p<0.05, **p<0.01 vs respective sedentary controls (Sed); #p<0.05, ##p<0.01 vs WT/Ex, 1-way ANOVA followed by Tukey post hoc analysis.
Figure 4—figure supplement 3
Mitochondrial-targeted antioxidant MitoQ improves cardiac response to exercise in csNrf2KO.
Cardiac function was evaluated by conscious echocardiography prior to (sedentary, Sed) and after exercise (Ex) with or without MitoQ treatment. Mean data for heart rate (HR), left ventricular end-systolic dimension (LVESD) and left ventricular end-diastolic dimension (LVEDD) (A to C). *p<0.05, **p<0.01 vs respective sedentary controls, ##p<0.01 vs Flox/Ex, 2-way ANOVA followed by Tukey post hoc analysis (N = 6–7/group).
Figure 5
Schematic illustrating the role of the cardiomyocyte Nox4-Nrf2 pathway in enhancing mitochondrial and cardiac contractile function during acute physiological exercise.
Cardiac Nox4 levels increase in response to acute exercise and induce an activation of Nrf2. The Nrf2-dependent gene program includes endogenous antioxidants that are critical in maintaining mitochondrial redox balance during peak exercise. The maintenance of mitochondrial redox balance is essential to achieve maximal mitochondrial respiration and cardiac contractile function at peak exercise. The sites of action of the Nrf2 activator sulforaphane and the mitochondria-targeted antioxidant MitoQ are shown.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Nox4-null | PMID: 20921387 | Dr. Schröder (Institut für Kardiovaskuläre Physiologien, Goethe-Universität) | |
| Genetic reagent (M. musculus) | cardiomyocyte-specific Nox4 knockout | PMID: 29040462 | Dr. Shah (King's College London British Heart Foundation Centre of Excellence) | |
| Genetic reagent (M. musculus) | Nrf2fl/fl | in this paper | Commissioned from Taconic-Artemis (Germany) | |
| Genetic reagent (M. musculus) | Myl2tm1(cre)Krc | The Jackson Laboratory (Stock No: 029465) | RRID:IMSR_JAX:029465 | |
| Antibody | Anti-GSTA2 (rabbit polyclonal) | Sigma-Aldrich Cat# SAB1401163 | RRID:AB_10609834 | |
| Antibody | Mouse Anti-GAPDH Monoclonal Antibody | Sigma-Aldrich Cat# G8795 | RRID:AB_1078991 | |
| Antibody | Anti-4 Hydroxynonenal Rabbit Polyclonal Antibody | Abcam Cat# ab46545 | RRID:AB_722490 | |
| Antibody | Anti-MnSOD Rabbit Polyclonal Antibody | Abcam Cat# ab13533 | RRID:AB_300434 | |
| Antibody | Anti-GCLC Rabbit Polyclonal Antibody | Novus Cat# NBP1-49762 | RRID:AB_10011848 | |
| Antibody | Anti-Catalase Rabbit Polyclonal Antibody | Millipore Cat#219010 | RRID:AB_211660 | |
| Antibody | TrxR2 Goat Polyclonal Antibody | Santa Cruz Biotechnology Cat# sc-46278 | RRID:AB_2210408 | |
| Antibody | PRX III (C-14) Goat Polyclonal Antibody | Santa Cruz Biotechnology Cat# sc-23973 | RRID:AB_2237431 | |
| Antibody | NFE2L2-Rabbit Polyclonal Antibody | Santa Cruz Biotechnology Cat# sc-13032 | RRID:AB_2263168 | |
| Antibody | NFE2L2-Rabbit Polyclonal Antibody | Santa Cruz Biotechnology Cat# sc-722, | RRID:AB_2108502 | |
| Antibody | VE-cadherin (F-8) monoclonal antibody | Santa Cruz Biotechnology Cat# sc-9989 | RRID:AB_2077957 | |
| Antibody | Anti-p47-phox (mouse) antibody | Millipore Cat# 07–500 | RRID:AB_310668 | |
| Antibody | gp91phox mouse monoclonal antibody | BD Biosciences Cat# 611414 | RRID:AB_398936 | |
| Antibody | Nox4 rabbit polyclonal antibody | PMID: 18467643 | Anilkumar et al. (2008) |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.41044.017
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Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise
eLife 7:e41044.
https://doi.org/10.7554/eLife.41044
