Research Article

Medicine

Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise

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

Dec 27, 2018

https://doi.org/10.7554/eLife.41044

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Version of Record: January 4, 2019
Version of Record: December 27, 2018

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Matthew Hancock

Anne D Hafstad

Adam A Nabeebaccus

Norman Catibog

Angela Logan

Ioannis Smyrnias

Synne S Hansen

Johanna Lanner

Katrin Schröder

Michael P Murphy

Ajay M Shah

Min Zhang

(2018)
Myocardial NADPH oxidase-4 regulates the physiological response to acute exercise

eLife 7:e41044.

https://doi.org/10.7554/eLife.41044
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Additional files

5 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements

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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 …
https://doi.org/10.7554/eLife.41044.002

Figure 1—figure supplement 1

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Full images of Western blots in Figure 1.
https://doi.org/10.7554/eLife.41044.003

Figure 1—figure supplement 2

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Myocardial Nox2 is unchanged during acute exercise.

Protein levels of total Nox2 (A) and p47^phox in membrane fraction (B) in normal mouse hearts after acute exercise (Ex). GAPDH and cadherin were used as cytosolic and membrane protein marker …
https://doi.org/10.7554/eLife.41044.004

Figure 1—figure supplement 3

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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, …
https://doi.org/10.7554/eLife.41044.005

Figure 2 with 1 supplement

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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);…
https://doi.org/10.7554/eLife.41044.006

Figure 2—figure supplement 1

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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 …
https://doi.org/10.7554/eLife.41044.007

Figure 3 with 3 supplements

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Cardiomyocyte-targeted *Nrf2* knockout mice have impaired mitochondrial function and reduced heart performance upon exercise.

(A) Targeting strategy for generation of *Nrf2*^fl/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 …
https://doi.org/10.7554/eLife.41044.008

Figure 3—figure supplement 1

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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, …
https://doi.org/10.7554/eLife.41044.009

Figure 3—figure supplement 2

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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.

https://doi.org/10.7554/eLife.41044.010

Figure 3—figure supplement 3

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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 …
https://doi.org/10.7554/eLife.41044.011

Figure 4 with 3 supplements

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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 …
https://doi.org/10.7554/eLife.41044.012

Figure 4—figure supplement 1

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Full images of Western blots in Figure 4.
https://doi.org/10.7554/eLife.41044.013

Figure 4—figure supplement 2

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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 …
https://doi.org/10.7554/eLife.41044.014

Figure 4—figure supplement 3

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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 …
https://doi.org/10.7554/eLife.41044.015

Figure 5

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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 …
https://doi.org/10.7554/eLife.41044.016

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)	Nrf2^fl/fl	in this paper		Commissioned from Taconic-Artemis (Germany)
Genetic reagent (M. musculus)	Myl2^tm1(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

Transparent reporting form: https://doi.org/10.7554/eLife.41044.017
Download elife-41044-transrepform-v1.docx

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Figures

Nox4 mediates myocardial Nrf2 activation and is essential in the acute response to exercise.

Full images of Western blots in Figure 1.

Myocardial Nox2 is unchanged during acute exercise.

Quantification of Western blots in Figure 1E.

Cardiac Nox4 deficiency impairs heart function and exercise capacity due to lack of Nrf2 activation.

csNox4KO mice exhibit reduced cardiac function at peak exercise.

Cardiomyocyte-targeted Nrf2 knockout mice have impaired mitochondrial function and reduced heart performance upon exercise.

Identification of csNrf2KO mouse.

csNrf2KO mice exhibit normal cardiac morphology and function at baseline.

Respiratory rates in isolated cardiac mitochondria of csNrf2KO mice after exercise.

Cardiomyocyte Nrf2 is required for increase in mitochondrial antioxidant capacity with exercise.

Full images of Western blots in Figure 4.

Nox4 is required for increase in mitochondrial antioxidant capacity with exercise.

Mitochondrial-targeted antioxidant MitoQ improves cardiac response to exercise in csNrf2KO.

Schematic illustrating the role of the cardiomyocyte Nox4-Nrf2 pathway in enhancing mitochondrial and cardiac contractile function during acute physiological exercise.

Tables

Additional files

Transparent reporting form

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Cite this article

Nox4 mediates myocardial Nrf2 activation and is essential in the acute response to exercise.

Full images of Western blots in Figure 1.

Myocardial Nox2 is unchanged during acute exercise.

Quantification of Western blots in Figure 1E.

Cardiac Nox4 deficiency impairs heart function and exercise capacity due to lack of Nrf2 activation.

csNox4KO mice exhibit reduced cardiac function at peak exercise.

Cardiomyocyte-targeted Nrf2 knockout mice have impaired mitochondrial function and reduced heart performance upon exercise.

Identification of csNrf2KO mouse.

csNrf2KO mice exhibit normal cardiac morphology and function at baseline.

Respiratory rates in isolated cardiac mitochondria of csNrf2KO mice after exercise.

Cardiomyocyte Nrf2 is required for increase in mitochondrial antioxidant capacity with exercise.

Full images of Western blots in Figure 4.

Nox4 is required for increase in mitochondrial antioxidant capacity with exercise.

Mitochondrial-targeted antioxidant MitoQ improves cardiac response to exercise in csNrf2KO.

Schematic illustrating the role of the cardiomyocyte Nox4-Nrf2 pathway in enhancing mitochondrial and cardiac contractile function during acute physiological exercise.

Transparent reporting form