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. 2019 Apr:129:118-129.
doi: 10.1016/j.yjmcc.2019.02.006. Epub 2019 Feb 13.

The cardiomyocyte "redox rheostat": Redox signalling via the AMPK-mTOR axis and regulation of gene and protein expression balancing survival and death

Daniel N Meijles  1 , Georgia Zoumpoulidou  2 , Thomais Markou  3 , Kerry A Rostron  4 , Rishi Patel  2 , Kenneth Lay  2 , Balvinder S Handa  2 , Bethany Wong  2 , Peter H Sugden  3 , Angela Clerk  3
Affiliations

The cardiomyocyte "redox rheostat": Redox signalling via the AMPK-mTOR axis and regulation of gene and protein expression balancing survival and death

Daniel N Meijles et al. J Mol Cell Cardiol. 2019 Apr.

Abstract

Reactive oxygen species (ROS) play a key role in development of heart failure but, at a cellular level, their effects range from cytoprotection to induction of cell death. Understanding how this is regulated is crucial to develop novel strategies to ameliorate only the detrimental effects. Here, we revisited the fundamental hypothesis that the level of ROS per se is a key factor in the cellular response by applying different concentrations of H2O2 to cardiomyocytes. High concentrations rapidly reduced intracellular ATP and inhibited protein synthesis. This was associated with activation of AMPK which phosphorylated and inhibited Raptor, a crucial component of mTOR complex-1 that regulates protein synthesis. Inhibition of protein synthesis by high concentrations of H2O2 prevents synthesis of immediate early gene products required for downstream gene expression, and such mRNAs (many encoding proteins required to deal with oxidant stress) were only induced by lower concentrations. Lower concentrations of H2O2 promoted mTOR phosphorylation, associated with differential recruitment of some mRNAs to the polysomes for translation. Some of the upregulated genes induced by low H2O2 levels are cytoprotective. We identified p21Cip1/WAF1 as one such protein, and preventing its upregulation enhanced the rate of cardiomyocyte apoptosis. The data support the concept of a "redox rheostat" in which different degrees of ROS influence cell energetics and intracellular signalling pathways to regulate mRNA and protein expression. This sliding scale determines cell fate, modulating survival vs death.

Keywords: Cytoprotection; Immediate early genes; Oxidative stress; Protein synthesis; Raptor; mTOR; p21(Cip1/WAF1).

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Graphical abstract
Fig. 1
Fig. 1
H2O2 reduces ATP levels and inhibits protein synthesis in cardiomyocytes. Cardiomyocytes were exposed to the concentrations of H2O2 shown for the times indicated. (A) Concentrations of ATP were measured using a luciferase assay. Results are % of control values and are means ± SEM (n = 4 independent myocyte preparations). (B) Area under curve (AUC) analysis of cardiomyocyte ATP levels from the data in (A). ***p < .001 relative to control (one-way ANOVA with Tukey post-test). (C) Protein synthesis was measured by incorporation of [3H]-Phe. Results are means ± SEM (n = 4 independent myocyte preparations). The IC50 was calculated using the 4-step parameter function in GraphPad Prism 7. ***p < .001, ****p < .0001 relative to 0.1 mM H2O2 (one-way ANOVA with Tukey post-test).
Fig. 2
Fig. 2
H2O2 activates AMPK and promotes AMPK-dependent phosphorylation of Raptor. Cardiomyocytes were exposed to 1 mM H2O2 for the times shown (A), to the concentrations of H2O2 indicated for 5 min (B), or were treated with SU6656 (15 nM) or compound C (60 μM) prior to addition of H2O2 (1 mM; 10 mins) (C). Samples were immunoblotted with antibodies to phosphorylated (P)-AMPKα(Thr172), total AMPKα, P-Raptor(Ser792) or total Raptor as indicated. Representative blots are shown in the left panels, with densitometric analysis and the ratio of Phospho−/Total proteins in the graphs on the right. (A) and (B), Results are means ± SEM (n = 4 independent myocyte preparations). *p < .05, **p < .01, ***p < .001 relative to unstimulated cells (one-way ANOVA with Dunnett's post-test). (C) Results are means ± SEM (n = 3 independent myocyte preparations). ****p < .0001 relative to unstimulated cells, #### p < .0001 relative to H2O2 alone (one-way ANOVA with Bonferroni post-test).
Fig. 3
Fig. 3
H2O2 (0.03–0.3 mM) activates mTOR but concentrations > 1 mM are required for activation of PKB/Akt. Cardiomyocytes were exposed to the concentrations of H2O2 indicated for 15 min (A) and (C), or to 0.2 mM H2O2 for the times shown (B). Samples were immunoblotted with antibodies to phosphorylated (P)-mTOR(Ser2448), P-mTOR(Ser2481), total mTOR, P-PKB/Akt(Ser473), P-PKB/Akt(Thr308) or total PKB/Akt as indicated. Representative blots are shown in the panels on the left, with densitometric analysis and the ratio of Phospho−/Total proteins in the graphs on the right. Results are means ± SEM [n = 4 (A) and (B) or n = 3 (C) independent myocyte preparations]. *p < .05, **p < .01 and ***p < .001 relative to unstimulated cells (one-way ANOVA with Dunnett's post-test).
Fig. 4
Fig. 4
H2O2 upregulates immediate early genes, but not second-phase gene products. (A), (C) and (E) Cardiomyocytes were exposed to 0.2 mM H2O2 for 2 h in the absence or presence of 20 μM cycloheximide (CHX). (B), (D) and (F) Cardiomyocytes were exposed to 0.3 mM or 1 mM H2O2 for the times indicated. mRNA expression was measured by qPCR. Results are means ± SEM (n = 4–6 independent myocyte preparations). (A), (C) and (E) *p < .05, **p < .01 and ***p < .001 relative to unstimulated controls, # p < .05 relative to H2O2 alone (one-way ANOVA with Tukey post-test). (B), (D) and (F) *p < .05, **p < .01 and ***p < .001 relative to unstimulated controls, # p < .05 relative to 0.3 mM H2O2 (one-way ANOVA with Tukey post-test).
Fig. 5
Fig. 5
Recruitment of mRNAs induced by H2O2 to polysomes for translation. Cardiomyocytes were unstimulated (Control) or exposed to 0.2 mM H2O2 (1 h) and total or polysomal RNA prepared for microarray analysis [(A) - (D)] or qPCR (E). (A) Heatmap (Log2 scale) of all probesets with significantly decreased or increased expression (>1.5-Fold change; FDR < 0.05) in total or polysomal RNA fractions. Expression values were normalised to the gene median. (B) Relative fold change of upregulated mRNAs in polysomal vs total RNA pools. Linear regression analysis indicates that most mRNAs are regulated similarly in total and polysomal fractions and are translated efficiently. (C) Overlay plot of mRNAs induced by H2O2 in total and polysomal RNA pools to show differential changes in some genes. (D) Heatmap (Log2 scale) of specific genes relevant to this study is shown on the left, with fold change relative to controls on the right. (E) qPCR validation of microarray data showing similar changes in expression in total or polysomal RNA fractions for Hmox1 and Atf3, enhanced recruitment of Nfil3 and Rasd1 to polysomes for translation and less efficient recruitment of Mdm2 to polysomes. Results are means ± SEM (n = 4 independent myocyte preparations; these are different preparations from those used for microarray analysis). *p < .05, ***p < .001 relative to unstimulated controls, # p < .05 relative to fold-change in total RNA pool (one-way ANOVA with Tukey post-test).
Fig. 6
Fig. 6
H2O2 or doxorubicin increase p21Cip1/WAF1 protein in cardiomyocytes to reduce apoptosis. (A) and (B) Cardiomyocytes were exposed to 0.2 mM H2O2 [(A) and (B)] or to 0.4 mM doxorubicin (E) for the times indicated and total extracts [(A) and (D)] or nuclear and cytosolic extracts (B) immunoblotted with antibodies to p21Cip1/WAF1. Representative immunoblots of at least 4 independent experiments are shown. Densitometric analysis is included to the right panels (A) and (D). Results are means ± SEM (n = 4 independent myocyte preparations). *p < .05, **p < .01 relative to unstimulated cells (one-way ANOVA with Dunnett's post-test). (C) Cardiomyocytes were exposed to the concentrations of H2O2 indicated for 120 min and immunoblotted with antibodies to to p21Cip1/WAF1. Densitometric analysis is included below the panel. Results are means ± SEM (n = 4 independent myocyte preparations). *p < .05, ****p < .0001 relative to unstimulated cells (one-way ANOVA with Dunnett's post-test). (D) and (F), Cardiomyocytes were exposed to 0.2 mM H2O2 (4 h) or 0.4 mM doxorubicin (4 h) alone (Control), following treatment with transfection reagent without oligodeoxynucleotides (no ODN) or following transfection with antisense ODNs (AS-ODNs) for Cdkn1a or scrambled ODNs (Scr-ODNs). Samples were immunoblotted with antibodies to p21Cip1/WAF1, cleaved caspase 3 or the loading control sarcomeric α-actin. Representative immunoblots are shown on the left, with densitometric analysis on the right. Data are means ± SEM (n = 6 independent myocyte preparations). *p < .05, ****p < .0001 relative to vehicle, #p < .01 relative to H2O2 treated cells with no ODNs, @ p < .05 relative to Cdkn1a-AS-ODN (two-way ANOVA with Tukey post-test).
Fig. 7
Fig. 7
Inhibition of mTOR reduces the increase in expression of nuclear p21Cip1/WAF1 in cardiomyocytes induced by H2O2 and promotes apoptosis. (A) Cardiomyocytes were unstimulated (Control) or exposed to 0.2 mM H2O2 (2 h) in the presence of 1 μM KU63794, 10 μM Rapamycin or vehicle only. (B and C) Nuclear or cytosolic samples were immunoblotted with antibodies to p21Cip1/WAF1, [(A) and (B)] or cleaved caspase 3 [(A) and (C)]. Data are means ± SEM (n = 3 independent myocyte preparations); *p < .05, **p < .01, ***p < .001 relative to vehicle control; #p < .05 relative to H2O2 (one-way ANOVA with Tukey post-test). (D) Schematic for regulation of signalling and gene expression under conditions of redox stress vs signalling. In conditions of low H2O2, immediate early genes (IEGs) are upregulated and mTOR is activated to promote protein synthesis, leading to production of antioxidant enzymes and cytoprotective proteins such as p21Cip1/WAF1 to alleviate the stress of increased oxidative burden. Under conditions of high H2O2, ATP levels fall dramatically causing activation of AMPK with phosphorylation of Raptor, inhibition of mTORC1 and decreased protein synthesis. Cells are unable to synthesise cytoprotective proteins such as p21Cip1/WAF1 and undergo apoptosis.
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