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. 2022 Jun 24;131(1):91-105.
doi: 10.1161/CIRCRESAHA.122.321050. Epub 2022 May 16.

ATF4 Protects the Heart From Failure by Antagonizing Oxidative Stress

Xiaoding Wang  1 , Guangyu Zhang  2 , Subhajit Dasgupta  2 , Erica L Niewold  2 , Chao Li  2 , Qinfeng Li  2 , Xiang Luo  2 , Lin Tan  3 , Anwarul Ferdous  2 , Philip L Lorenzi  3 , Beverly A Rothermel  2   4 , Thomas G Gillette  2 , Christopher M Adams  5 , Philipp E Scherer  6 , Joseph A Hill  2   4 , Zhao V Wang  2
Affiliations

ATF4 Protects the Heart From Failure by Antagonizing Oxidative Stress

Xiaoding Wang et al. Circ Res. .

Abstract

Background: Cellular redox control is maintained by generation of reactive oxygen/nitrogen species balanced by activation of antioxidative pathways. Disruption of redox balance leads to oxidative stress, a central causative event in numerous diseases including heart failure. Redox control in the heart exposed to hemodynamic stress, however, remains to be fully elucidated.

Methods: Pressure overload was triggered by transverse aortic constriction in mice. Transcriptomic and metabolomic regulations were evaluated by RNA-sequencing and metabolomics, respectively. Stable isotope tracer labeling experiments were conducted to determine metabolic flux in vitro. Neonatal rat ventricular myocytes and H9c2 cells were used to examine molecular mechanisms.

Results: We show that production of cardiomyocyte NADPH, a key factor in redox regulation, is decreased in pressure overload-induced heart failure. As a consequence, the level of reduced glutathione is downregulated, a change associated with fibrosis and cardiomyopathy. We report that the pentose phosphate pathway and mitochondrial serine/glycine/folate metabolic signaling, 2 NADPH-generating pathways in the cytosol and mitochondria, respectively, are induced by transverse aortic constriction. We identify ATF4 (activating transcription factor 4) as an upstream transcription factor controlling the expression of multiple enzymes in these 2 pathways. Consistently, joint pathway analysis of transcriptomic and metabolomic data reveal that ATF4 preferably controls oxidative stress and redox-related pathways. Overexpression of ATF4 in neonatal rat ventricular myocytes increases NADPH-producing enzymes' whereas silencing of ATF4 decreases their expression. Further, stable isotope tracer experiments reveal that ATF4 overexpression augments metabolic flux within these 2 pathways. In vivo, cardiomyocyte-specific deletion of ATF4 exacerbates cardiomyopathy in the setting of transverse aortic constriction and accelerates heart failure development, attributable, at least in part, to an inability to increase the expression of NADPH-generating enzymes.

Conclusions: Our findings reveal that ATF4 plays a critical role in the heart under conditions of hemodynamic stress by governing both cytosolic and mitochondrial production of NADPH.

Keywords: cell death; fibrosis; glycine; heart failure; metabolomics.

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Figures

Figure 1.
Figure 1.. Oxidative stress is elevated by pressure overload-induced heart failure.
A. Schematic of experimental design. Wild-type C57BL/6 mice were subjected to sham or transverse aortic constriction (TAC). A series of measurements were made 3, 7, and 21 days after TAC. NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADP+, nicotinamide adenine dinucleotide phosphate; GSSG, glutathione disulfide; GSH, glutathione; ROS, reactive oxygen species. B. TAC caused cardiac dysfunction in mice. Ejection fraction and fractional shortening (%) were determined by echocardiography. Sham, n=5; 3 days after TAC, n=6; 7 days after TAC, n=5; 21 days after TAC, n=8. C. Western blotting showed that cleaved caspase 3 and ANP were upregulated in the heart after TAC. D. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining identified apoptotic cardiac cells 3 weeks after TAC. Scale: 100 μm. Sham, n=8; TAC, n=6. E. The hypertrophied heart at 7 and 21 days after TAC showed an elevated level of 4-HNE (4-hydroxynonenal)-positive signal. Scale: 100 μm. N=4–6. Sham, n=4; 3 days after TAC, n=5; 7 days after TAC, n=6; 21 days after TAC, n=5. F. Both NADPH level and the NADPH/NADP+ ratio were significantly decreased in mice 3, 7, and 21 days after TAC. NADP+ level was significantly increased in mice 3, 7, and 21 days after TAC. Sham, n=5; 3 days after TAC, n=7; 7 days after TAC, n=7; 21 days after TAC, n=8. G. GSSG was increased in the heart 7 and 21 days after TAC. The GSH/GSSG ratio was decreased 7 and 21 days after TAC. Sham, n=5; 3 days after TAC, n=6; 7 days after TAC, n=5; 21 days after TAC, n=4. Kruskal-Wallis test was conducted, followed by Dunnett’s test to determine statistical significance for comparisons of the means of each surgical group (3, 7, and 21 days after TAC) with the mean of the sham group (B, E-G). Unpaired Student’s t test was conducted for D. GSH (F) and GSSG (G) levels from each surgical group (3, 7, and 21 days after TAC) were normalized to the mean of the sham group, which was set to 1. Data are represented as mean±SEM.
Figure 2.
Figure 2.. NADPH production pathways are stimulated in the heart by pressure overload.
A. Schematic of NADPH-generation pathways. B. TAC increased the protein levels of MTFFD2, G6PDX, PHGDH, and PSAT1 in the heart. GAPDH was used as a loading control. C. TAC elevated the mRNA levels of MTHFD2, G6PDX, PHGDH, and PSAT1 in the heart. Sham, n=9; 3 days after TAC, n=10; 7 days after TAC, n=5; 21 days after TAC, n=8. D. Mitochondrial ROS production was measured by oxidation of the fluorescent probe MitoSOX red in NRVMs (neonatal rat ventricular myocytes) after PE treatment for 24 and 48 hours, respectively. N=4 for each group. E. Cytosolic ROS level was determined by H2DCFDA staining and flow cytometry. Vehicle (Veh), n=3; PE, 24 h, n=4; PE, 48 h, n=4. F. NADPH was reduced in NRVMs after PE treatment, whereas the ratio of NADP+/NADPH was increased. Vehicle (Veh), n=6; PE, 24 h, n=7; PE, 48 h, n=6. G. PE treatment in NRVMs stimulated the mRNA expression of MTHFD2 and G6PDX, respectively. Vehicle (Veh), n=3; PE, 24 h, n=4; PE, 48 h, n=4. Kruskal-Wallis test was conducted, followed by Dunn’s test to determine statistical significance for comparisons of the means of each surgical group (3, 7, and 21 days after TAC) with the mean of the sham group (C). Gene expression at the mRNA level from each surgical group (3, 7, and 21 days after TAC) was normalized to the mean of the sham group, which was set to 1 (C). One-way ANOVA was conducted, followed by Dunnett’s test to determine statistical significance for comparisons of the means of each treatment group (PE treatment for various times) with the mean of the vehicle (Veh) group (D-E, G). Mean fluorescent intensity of each PE treatment group was normalized to the mean of the vehicle (Veh) group, which was set to 1 (E). Gene expression at the mRNA level from each PE treatment group was normalized to the mean of the vehicle (Veh) group, which was set to 1 (G). One-way ANOVA was conducted, followed by Tukey’s test to determine statistical significance for comparisons of the means of each treatment group (PE treatment for various times) with the mean of the vehicle (Veh) group (F). NADPH and NADP+ levels from each PE treatment were normalized to the mean of the vehicle (Veh) group, which was set to 1 (F). Data are represented as mean±SEM.
Figure 3.
Figure 3.. ATF4 expression is elevated under cardiomyocyte hypertrophic growth.
A. ATF4 expression was upregulated in the heart 3 and 7 days after TAC. Sham, n=9; 3 days after TAC, n=10; 7 days after TAC, n=5; 21 days after TAC, n=8. B. ATF4 was elevated at the protein level in NRVMs 6, 12, and 24 hours after PE treatment. N=7 for each group. Kruskal-Wallis test was conducted, followed by Dunn’s test to determine statistical significance for comparisons of the means of each surgical group (3, 7, and 21 days after TAC) with the mean of the sham group (A). Gene expression at the mRNA level from each surgical group (3, 7, and 21 days after TAC) was normalized to the mean of the sham group, which was set to 1 (A). One-way ANOVA was conducted, followed by Tukey’s test to determine statistical significance for comparisons of the means of each treatment group (PE treatment for various times) with the mean of the vehicle (Veh) group (B). Gene expression at the protein level from each PE treatment group was normalized to the mean of the vehicle (Veh) group, which was set to 1 (B). Data are represented as mean±SEM.
Figure 4.
Figure 4.. ATF4 controls the one-carbon metabolic pathway and PPP.
A. NRVMs were infected by adenoviruses expressing ATF4 or control GFP. RNA-sequencing was conducted and PCA analysis was performed. N=3 for each group. B. Metabolomics were performed with NRVMs expressing control GFP or ATF4. N=3 for each group. C. Significantly changed genes (RNA-sequencing) and metabolites (metabolomics) were subjected to the joint pathway analysis (JPA). D. Multi-omics analysis using transcriptomic and metabolomic data was performed according to the analytical scheme shown in C. JPA was used to calculate the bubble chart, which is a combination of enrichment P values and gene numbers of individual pathways. E. The top 40 transcripts (left, RNA-sequencing) and 27 metabolites (right, metabolomics) with cutoff threshold of fold change>1.2 or <0.8 and P value adjusted <0.05 are shown. FC, fold change.
Figure 5.
Figure 5.. ATF4 deficiency exacerbates heart failure under pressure overload.
A. After 1 week of sham or TAC, control ATF4F/F (F/F) and ATF4 cardiomyocyte specific conditional knockout (cKO) mice were used for echocardiography measurements. Representative M-mode images are shown. B. ATF4 cKO led to deterioration of cardiac function 1 week after TAC, as revealed by decreases in ejection fraction and fraction shortening. Sham, F/F, n=3; Sham, cKO, n=5; TAC, F/F, n=5; TAC, cKO, n=7. C. ATF4-deficient hearts displayed profound immune cell infiltration after TAC, which was clustered as patches and distributed in myocardium. Fibrosis was increased in cKO hearts compared to controls. Scale: 100 μm. D. TUNEL staining revealed more apoptotic cell death in the ATF4 cKO heart after TAC. Scale: 100 μm. Sham, F/F, n=4; Sham, cKO, n=4; TAC, F/F, n=7; TAC, cKO, n=4. E. ATF4 knockout in the heart caused pathological cardiac remodeling after TAC as revealed by increases of expression of Rcan1.4, caspase 3, cleaved caspase 3, and ANP. F. The mRNA levels of heart failure markers, ANF and BNP, were augmented in cKO hearts. Sham, F/F, n=3; Sham, cKO, n=5; TAC, F/F, n=6; TAC, cKO, n=7. G. Cardiac fibrosis marker genes including TGFβ2, CTGF, and COLa1 were elevated by ATF4 deletion in the heart. Sham, F/F, n=3; Sham, cKO, n=5; TAC, F/F, n=6; TAC, cKO, n=7. Two-way ANOVA was conducted, followed by Tukey’s multiple comparisons test (B, D, F-G). Gene expression at the mRNA level from each group was normalized to the mean of the sham/ATF4F/F group, which was set to 1 (F-G). Data are represented as mean±SEM.
Figure 6.
Figure 6.. ATF4 maintains oxidative stress control.
A. Cardiac specific ATF4 deficiency led to a decrease in the ratio of GSH to GSSG and an increase in GSSG level after TAC. ATF4 cKO mice along with F/F controls were subjected to sham or TAC. After 3 days, cardiac tissues were harvested for various measurements. Sham, F/F, n=4; Sham, cKO, n=5; TAC, F/F, n=4; TAC, cKO, n=4. B. ATF4 cKO hearts showed increased oxidative stress in the heart. Sham, F/F, n=5; Sham, cKO, n=6; TAC, F/F, n=5; TAC, cKO, n=6. C. ATF4 deletion in the heart caused a failure of induction of MTHFD2, G6PDX, PHGDH, and PSAT1 by TAC. Sham, F/F, n=5; Sham, cKO, n=6; TAC, F/F, n=5; TAC, cKO, n=6. D. ATF4 silencing in NRVMs increased mitochondrial ROS as measured by flow cytometry. Two independent siRNA oligos against ATF4 were used. NS, not significant. Vehicle (Veh), Ctrl si, n=4; PE, Ctrl si, n=4; all others, n=3. E. ATF4 knockdown in NRVMs caused an increase in cytosolic ROS as examined by H2DCFDA staining. N=4 for each group. F. ATF4 silencing exacerbated NRVM death after PE treatment, which was significantly rescued by either Tempo or Mito-Tempo treatment. Note that all groups were treated by PE. N=5 for each group. Two-way ANOVA was conducted, followed by Tukey’s multiple comparisons test (A-C). GSH and GSSG levels from each group were normalized to the mean of the sham/ ATF4F/F group, which was set to 1 (A). The 4HNE level from each group was normalized to the mean of the sham/ ATF4F/F group, which was set to 1 (B). Gene expression at the mRNA level from each group was normalized to the mean of the sham/ATF4F/F group, which was set to 1 (C). Kruskal-Wallis test was conducted, followed by Dunn’s test to determine statistical significance for comparisons of the means of each group with the mean of the vehicle (Veh)/Control si (Ctrl si) group (D-F). Mean fluorescent intensity (H2DCFDA) of each treatment group was normalized to the mean of the vehicle (Veh)/Control si (Ctrl si) group, which was set to 1 (E). Data are represented as mean±SEM.
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