Published online before print October 8, 2009, doi: 10.1161/CIRCRESAHA.109.206607
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© 2009 American Heart Association, Inc.
Integrative Physiology |
Metabolic Remodeling Induced by Mitochondrial Aldehyde Stress Stimulates Tolerance to Oxidative Stress in the Heart
From the Department of Regenerative Medicine and Advanced Cardiac Therapeutics (J.E., M. Sano, T.K., S. Morizane, T.M., Y.K., Y.Z., H.I., F.H., K.F.); Cardiology Division (J.E., T.K., T.M., Y.K., S. Ogawa), Department of Internal Medicine; Department of Biochemistry and Integrative Medical Biology (T.H., Y.N., T.A., M. Suematsu); and Division of Geriatric Medicine (K.S.), Keio University School of Medicine, Tokyo, Japan; Precursory Research for Embryonic Science and Technology (PRESTO) (M. Sano), Japan Science and Technology Agency, Saitama, Japan; Department of Biochemistry and Cell Biology (K.N., A.M.W., I.O., S. Ohta), Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, Kawasaki, Japan; Department of Pharmaceutical Sciences (S. Marchitti, V.V.), University of Colorado Health Sciences Center, Denver; Department of Metabolome (H.N., R.T.), University of Tokyo, Japan; and Neuronal Circuit Mechanisms Research Group (Y.H.), Brain Science Institute, RIKEN, Saitama, Japan.
Correspondence to Dr Motoaki Sano, Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582, Japan. E-mail msano{at}sc.itc.keio.ac.jp
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Abstract |
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Rationale: Aldehyde accumulation is regarded as a pathognomonic feature of oxidative stress–associated cardiovascular disease.
Objective: We investigated how the heart compensates for the accelerated accumulation of aldehydes.
Methods and Results: Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2
phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress–resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.)
Conclusions: The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione–redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.
Key Words: cardiac metabolism • oxidative stress • aldehyde • stress response
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Introduction |
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Aldehydes are the major end products of lipid peroxidation. They are highly electrophilic and react with biomolecules, such as proteins and nucleic acids, to generate various adducts.1 Increased levels of aldehyde adducts have been detected in oxidized lipoproteins, atherosclerotic lesions, hearts with coronary artery disease, and Alzheimer brains.2–4 In addition to the pathogenic effect associated with oxidative stress, sublethal levels of aldehydes interact with signaling systems to upregulate gene expression to counteract stressor challenges and to reestablish homeostasis.5–7
In mammalian cells, reactive aldehydes are detoxified by oxidation to carboxylates, a reaction catalyzed by aldehyde dehydrogenases (ALDHs).1 The ALDHs are a superfamily of NAD(P)+-dependent enzymes,8 and, to date, 19 distinct ALDH genes have been identified in the human genome.9 ALDH2 is localized to the mitochondria, a major source of reactive oxygen species and a target of membrane lipid peroxidation. ALDH2 has been implicated in cellular antioxidant processes because ALDH2 deficiency increases oxidative stress.10,11 Furthermore, the cardioprotective effects of moderate alcohol consumption are well documented in animal models and humans,12 and ethanol exposure, followed by sufficient time to metabolize the alcohol before ischemia, induces a delayed form of preconditioning-like cardioprotection.13 The mitochondrial translocation of protein kinase C
and the subsequent activation of ALDH2 have been shown to contribute to the cardioprotective effects of alcohol.14 Indeed, administration of a small-molecule activator of ALDH2 to rats before an ischemic event reduced infarct size by 60%.15
A single nucleotide polymorphism of ALDH2 (ALDH2*2), acts as a dominant-negative gene and is found in Asian populations (Figure I in the Online Data Supplement, available at http://circres.ahajournals.org). ALDH2 acts as a homo- or heterotetramer, and all tetramers that contain at least 1 ALDH2*2 subunit are inactive.16 People homozygous for the ALDH2*2 allele (
8% of the Japanese population) do not have any ALDH2 activity, whereas activity in individuals heterozygous for the ALDH2*2 allele (40% of the Japanese population) is as low as 1 in 16 of that in ALDH2*1 (wild-type [Wt]) homozygous individuals.17 Notably, the Aldh2*2 allele has been reported to affect the metabolism of acetaldehyde, as well as other aldehydes, such as benzaldehyde (a metabolite of toluene) and chloroacetaldehyde (generated during the metabolism of vinyl chloride). The ALDH2*2 allele is associated with alcohol flushing syndrome, increased serum lipid peroxide levels,18 and an increased risk for late-onset Alzheimer’s disease.19 Previously, we demonstrated that in stable transfectants of the PC12 neuronal cell line expressing mouse Aldh2*2, mitochondrial, but not cytosolic, Aldh activity was repressed.10,11 The resultant Aldh-deficient transfectants were highly vulnerable to oxidative insult by exogenous 4-hydroxy-2-nonenal (4-HNE) or antimycin A. Furthermore, transgenic (Tg) mice expressing Aldh2*2 in their brains exhibited decreased ability to detoxify 4-HNE in cortical neurons and an accelerated accumulation of 4-HNE in the brain. Consequently, age-associated neurodegeneration accompanied by memory loss appeared in these mice after 1 year of age.20 This phenotype mimics late-onset Alzheimer’s disease in human.
In this study, we developed a Tg loss-of-function model for aldehyde-detoxifying enzymes to exploit the overexpression of Aldh2*2 in the heart, which simulates heterozygotes for the ALDH2*2 allele in individuals already expressing Wt ALDH2*1 in the heart. Despite a significant accumulation of 4-HNE adduct proteins in the mitochondrial matrices, left ventricular systolic and diastolic function were equivalent to that in Wt littermates until at least 2 years of age. Furthermore, the hearts exhibited enhanced tolerance to ischemia/reperfusion (I/R) injury. This is markedly different to the Aldh2*2 Tg brain, where accumulation of 4-HNE in the brain was accompanied by neuronal cell death, indicating that the heart has a superior ability to resist mitochondrial aldehyde stress than the brain and exploits the hormesis-like effect of aldehydes. Thus, we subsequently investigated the molecular basis underlying this aldehyde-induced, hormesis-like stress response in Aldh2*2 Tg hearts.
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Methods |
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Aldh2*2 Tg mice were generated by pronuclear injection of a plasmid carrying Aldh2*2 with a single nucleotide mutation at the same locus as the human Aldh2*2 polymorphism. The mice used for these experiments were more than 10 generations back-crossed to C57BL/6 mice. For fluxome analysis, hearts were perfused with a modified Krebs–Henseleit buffer (120 mmol/L NaCl, 25 mmol/L NaHCO3, 5.9 mmol/L KCl, 1.2 mmol/L MgSO4, 1.75 mmol/L CaCl2, 10 mmol/L glucose, 10 µU/mL insulin, 0.4 mmol/L oleate, and 1% BSA) gassed with 95% O2/5% CO2 at 37°C, according to the Langendorff procedure. After equilibration, the buffer was switched to modified Krebs–Henseleit buffer containing 10 mmol/L 13C-glucose instead of 10 mmol/L glucose.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
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Results |
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Aldh2*2 Tg Mice Represent a Loss-of-Function Model of Aldh Activity
We created a loss-of-function model for aldehyde-detoxifying enzymes by Tg expression of Aldh2*2 under control of the CAG promoter (Figure 1A). Strong expression of the Aldh2*2 protein was observed in the heart and skeletal muscles (Figure 1B). During development to reproductive maturity, Aldh2*2 Tg mice exhibited small body size (Figure 1C and 1D), reduced muscle mass, diminished fat content (Figure 1E), osteopenia, and kyphosis. These phenotypes are not observed in Aldh2-null mice (ie, mice in which normal Aldh2 activity is blocked by knockout of the Aldh2 gene).21 These data indicate that the phenotypes observed in Aldh2*2 Tg mice are not attributable simply to a lack of Aldh2 activity. We suspect that Aldh2*2 inactivates not only Aldh2, but also other Aldh subfamilies, presumably by forming heterotetramers.22,23 Consistent with this, the forced expression of Aldh2*2 impaired Aldh activity against aliphatic aldehydes, including 4-HNE, whereas genomic disruption of Aldh2 did not (Online Table I).
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Mitochondrial Oxidative Stress in Aldh2*2 Tg Hearts
A cell fractionation study revealed that the Aldh2*2 protein is localized in the mitochondria (levels 8-fold higher than those of endogenous Aldh2; Figure 2A). Consistent with this, we found that levels of some 4-HNE adduct proteins were increased in the mitochondrial fraction of Aldh2*2 Tg hearts. However, there was little increase in 4-HNE adduct protein levels in the cytosolic fraction of Aldh2*2 Tg hearts.
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The hearts of Aldh2*2 Tg mice were smaller (in proportion to body size) than those of their Wt littermates (Figure 2B; Online Figure II, A). Light microscopic examination of Aldh2*2 Tg hearts revealed normal alignment of thinner cardiomyocytes. Furthermore, there was no evidence of interstitial fibrosis or cellular degeneration/death (Figure 2C; Online Figure II, C). Ultrastructural analysis by transmission electron microscopy revealed that the sarcomere structure of the myofibrils in Aldh2*2 Tg hearts was intact. Of note, some mitochondria contained electron-dense deposits on a few cristae (Figure 2D). Finally, a decline of mitochondrial function (Figure 3A) and a shift toward the oxidative state were found in mitochondrial matrices of Aldh2*2-expressing cells (Figure 3B).
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Aldh2*2 Tg Hearts Exhibit Greater Tolerance to Oxidative Stress
Echocardiographic examination of 3-month-old Aldh2*2 Tg mice revealed normal left ventricular systolic and diastolic function (Online Figure III), indicating adaptation of the heart to persistent mitochondrial oxidative stress, at least under unstressed conditions.
To determine whether Aldh2*2 Tg hearts are susceptible or resistant to exogenous oxidative stress elicited by I/R injury, isolated hearts were subjected to 30 minutes total global ischemia, followed by 60 minutes aerobic reperfusion. Notably, Aldh2*2 Tg hearts exhibited significantly improved recovery of left ventricular developed pressure and ±dP/dt during reperfusion compared with the control group (n=6; P<0.05; Figure 4A and 4B). Consistent with these findings, total lactate dehydrogenase release into the perfusate during reperfusion was significantly lower in Aldh2*2 Tg compared with control hearts (n=6; P<0.05; Figure 4C). In addition, we confirmed improved handling of I/R injury in in vivo hearts. Reperfusion injury was reduced in hearts from Aldh2*2 Tg mice, as reflected by a reduction in the size of the necrotic zone per area at risk (n=6; P<0.05; Figure 4D).
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Induction of Genes in Aldh2*2 Tg Hearts
Gene chip analysis revealed that the expression of genes for the major antioxidant enzymes was not induced in Aldh2*2 Tg hearts, with the exception of the expression of the glutathione (GSH) S-transferase-a1 and -a2 (Gsta1 and Gsta2) genes (Online Figure IV). Furthermore, there was no change in the expression of genes involved in the production of lipid mediators and reactive oxygen species, including Xdh and components of NADPH oxidase, in Aldh2*2 Tg hearts (data not shown).
We found significant upregulation of genes encoding enzymes involved in amino acid biosynthesis and transport in Aldh2*2 Tg hearts (Figure 5A), including genes encoding 3-phosphoglycerate dehydrogenase (Phgdh), phosphoserine aminotransferase 1 (Psat1), and phosphoserine phosphatase (Psph), all of which are involved in the 3-step conversion of 3-phosphoglycerate (a glycolytic intermetabolite) to serine. Serine hydroxymethyltransferase 1/2 (Shmt1/2) catalyzes the conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF, and methylenetetrahydrofolate dehydrogenase 2 catalyzes the interconversion of 5,10-methylene THF and 10-formyl THF. Cystathionase (Cth) is involved in cysteine biosynthesis in the trans-sulfuration pathway. These metabolic pathways eventually converge on GSH biosynthesis (for a metabolic map, see Figure 5B). Accordingly, despite the significant oxidative stress in the mitochondrial matrix, the intracellular concentration of GSH was increased 1.37-fold in Aldh2*2 Tg compared with Wt hearts (n=8; P<0.05; Figure 6A). The synthesis of GSH from its constituent amino acids, namely L-glutamate, L-cysteine, and L-glycine, involves 2 enzymatic steps catalyzed by 
-glutamyl-cysteine ligase (Gclm, Gclc) and GSH synthetase (Gss).24 However, induction of GSH biosynthetic enzymes was not particularly evident in Aldh2*2 Tg hearts (Figure 5 and Online Figure V). The solute carrier (SLC) family of amino acid transporters, including members involved in cysteine biosynthesis or transport (Slc1A4, Slc3A2), was also upregulated in Aldh2*2 Tg hearts. Notably, transcripts for the ATF/CREB (cyclic AMP response element–binding protein) (Atf4, Atf5) families of transcription factors, master regulators of amino acid metabolism,25–28 were increased in Aldh2*2 Tg hearts.
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Glucose Biotransformation Is Shifted Toward the Pentose Phosphate Pathway
To further delineate changes in cardiac metabolism, intracellular concentrations of metabolites were measured by high-throughput metabolomics using capillary electrophoresis–mass spectrometry (CE-MS). The Aldh2*2 Tg hearts exhibited characteristic changes in levels of metabolic intermediates of the glycolysis pathway (Online Figure VI). Specifically, levels of upstream glycolytic metabolites tended to increase, whereas those of downstream glycolytic metabolites tended to decrease in Aldh2*2 Tg hearts. Notably, Aldh2*2 Tg hearts contained higher levels of intermediate metabolites of the pentose phosphate pathway, including 6-phosphogluconate (1.42-fold increase) and ribose 5-phosphate (1.57-fold increase; n=6; P<0.05 vs Wt littermates). The myocardial NADPH/NADP+ ratio was elevated in Aldh2*2 Tg mice (n=6; P<0.05 versus Wt littermates; Figure 6B through 6D). There were no differences in glycogen content between Wt and Aldh2*2 Tg hearts (0.016±0.002 and 0.018±0.003 µg/mg tissue, respectively).
Consistent with the upregulation of genes involved in amino acid metabolism and GSH biosynthesis, intracellular levels of glycine, homocysteine, and cystathionine were significantly higher in Aldh2*2 Tg than Wt hearts (n=6; P<0.05; Figure 6E through 6G).
To verify the changes in glucose metabolism, we performed in vivo pulse-chase analysis of 13C-labeled glucose (fluxome analysis) in Langendorff-perfused hearts (Figure 7). 13C-Labeled metabolites were quantified by CE-MS 5 and 20 minutes after administration of 13C-labeled glucose. 13C-Labeled intermediate metabolites of the glycolytic and pentose phosphate pathways were higher in Aldh2*2 Tg versus Wt hearts at both time points (n=6; P<0.05).
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Phgdh, a rate-limiting enzyme for serine biosynthesis from intermediate metabolites of the glycolytic pathway (3-phosphoglycerate), was highly induced in Aldh2*2 Tg hearts (Figure 8A). Accordingly, 13C-labeled serine was markedly induced in ex vivo (Langendorff-perfused) and in vivo hearts (Online Figure VII).
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To examine whether glucose uptake is actually augmented in Aldh2*2 Tg hearts, Langendorff-perfused hearts were incubated with 2-deoxy-D-glucose (2-DG) for 5 minutes. When 2-DG is taken up into cells by glucose transporters, it is phosphorylated to 2-deoxy-d-glucose-6-phosphate (2-DGP). Because it cannot undergo further glycolysis, 2-DGP can be used as a measure of glucose uptake. As expected, intramyocardial accumulation of 2-DGP was 1.69-fold higher in Aldh2*2 Tg compared with Wt hearts (n=6; P<0.05).
ATF4 Is a Key Transcriptional Regulator in Response to Aldehydes in the Heart
It is known that phosphorylation of the subunit of eukaryotic translation initiation factor 2 (eIF2) and subsequent translational activation of ATF4 result in the induction of additional members of the ATF/CREB family of transcription factors, which, together, upregulate a coordinately expressed set of genes involved in amino acid biosynthesis in mammalian cells.25–28 Consistent with this, Western blot analysis demonstrated increased Ser51 phosphorylation on eIF2 and increased protein expression of Atf4 and Phgdh (Figure 8A) in Aldh2*2 Tg versus Wt hearts.
To clarify the role of Atf4 in aldehyde-induced activation of amino acid metabolism and resistance to oxidative stress, Aldh2 Tg mice were mated with Atf4-knockout mice.29 Homozygous Atf4 knockout was lethal, whereas Atf4 heterozygous knockout (Atf4+/–) mice, having half the Wt levels of Atf4 expression (Figure 8B), were viable with no cardiac anomalies under unstressed conditions. The increased expression of cardiac genes involved in amino acid metabolism in Aldh2 Tg mice was significantly attenuated in Aldh2 Tg mice carrying heterozygous alleles of Atf4 (Figure 8C). Western blot analysis demonstrated that the increased levels of Phgdh protein in Aldh2 Tg mice were reduced by half in Aldh2 Tg mice carrying heterozygous alleles of Atf4. In addition, the increased intracellular GSH levels in Aldh2 Tg mice were significantly attenuated in mice carrying the heterozygous alleles of Atf4 (Figure 8D). Accordingly, heterozygous knockout of Atf4 attenuated the oxidative stress–resistant phenotype observed in Aldh2*2 Tg hearts (Figure 8E).
Activation of ATF4 Is Crucial for the Oxidative Stress–Resistant Phenotype
Adenovirus-mediated expression of Aldh2*2 in cultured cardiomyocytes increased immunoreactivity for 4-HNE adducts (Online Figure VIII, A), induced expression of Atf4 and Phgdh (Online Figure VIII, B), and increased intracellular levels of reduced GSH (Online Figure VIII, C). The short interference (si)RNA-mediated knockdown of Atf4 abolished the induction of Phgdh and the increase in intracellular GSH in Aldh2*2-expressing cardiomyocytes.
Consistent with the oxidative stress–tolerant phenotype observed for Aldh2*2 Tg hearts, Aldh2*2-expressing cardiomyocytes exhibited enhanced tolerance to oxidative stress induced by glucose-free anoxia–reoxygenation or high-dose 4-HNE or antimycin A (Online Figure VIII, D; data not shown). The siRNA-mediated knockdown of Atf4 rendered the LacZ-expressing control cardiomyocytes vulnerable to oxidative stress and abrogated the oxidative stress–resistant phenotype of the Aldh2*2-expressing cardiomyocytes.
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Discussion |
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The present study has demonstrated, for the first time, that the heart has an intrinsic capacity to change the metabolic pathways to counteract life-long mitochondrial oxidative damage. Mitochondrial aldehyde stress triggers phosphorylation of eIF2
and the combined transcriptional and translational activation of Atf4 upregulates the gene expression of enzymes involved in amino acid biosynthesis and transport that ultimately provide precursor amino acids for GSH biosynthesis, thereby increasing intracellular GSH levels. Surprisingly, hearts with chronic mitochondrial aldehyde stress exhibited improved tolerance to I/R injury despite the presence of substantial mitochondrial oxidative damages. Heterozygous knockout of Atf4 blunted the increase in intracellular GSH levels in mitochondrial Aldh–deficient hearts and attenuated the oxidative stress–resistant phenotype. These findings indicate that Atf4-dependent activation of amino acid metabolism and GSH biosynthesis are causally involved in the oxidative stress–resistant phenotype observed in mitochondrial Aldh–deficient hearts. Enhanced supply of NADPH via the pentose phosphate pathway concomitantly helps in the recycling of oxidized GSH (GSH disulfide [GSSG]).
Here, we showed that the forced expression of Aldh2*2 in the heart impairs Aldh activity against aliphatic aldehydes, including 4-HNE. Several lines of evidence suggest that ALDH2*2 inactivates not only ALDH2 but also other ALDH subfamilies by forming heterotetramers.22,23 For example, alignment of ALDH2 and ALDH1B1 amino acid sequences reveals a high degree of conservation between the regions required for dimer formation (ie, the -helix G residues 247 to 259, β-sheet 18 residues 450 to 453, β-sheet 19 residues 486 to 495, and the "oligomerization domain" residues 140 to 158 and 486 to 495). Notably, there is 100% conservation of residues involved in tetramer formation (ie, β-sheet 5 residues 141 to 144). These findings suggest that ALDH1B1 is one of the targets of ALDH2*2. We are currently investigating other enzymes that are inactivated by Aldh2*2.
However, kinetic assays may not address overall metabolic function or changes in other metabolic pathways, and increases in Gsta1/2 levels seen in the Aldh2*2 Tg hearts may compensate for defects in mitochondrial Aldh activity. A cell fractionation study revealed Aldh2*2 protein in the mitochondria. Consistent with this, we found that some 4-HNE adduct proteins were also increased in the mitochondrial fraction of Aldh2*2 Tg hearts. Furthermore, 4-HNE immunoreactivity was increased following adenovirus-mediated overexpression of Aldh2*2 in cultured cardiomyocytes. Together, these results strongly suggested that aldehyde metabolism in mitochondria is indeed affected in the Tg hearts.
Myocardial GSH content influences susceptibility to I/R injury.30 The rate of GSH synthesis is determined primarily by -glutamyl-cysteine ligase activity and the availability of precursor amino acids, especially cysteine. In the present study, there was only marginal induction of the GSH biosynthetic enzyme -glutamyl-cysteine ligase in Aldh2*2 Tg hearts. In contrast, significant upregulation was observed in Aldh2*2 Tg hearts of genes encoding enzymes involved in serine biosynthesis (Phgdh, Psat1, and Psph), the conversion of serine to glycine (Shmt1/2), the trans-sulfuration pathway (Cth), and the transportation of cysteine and cystine (Slc1A4, Slc3A2).31 Because -glutamyl-cysteine ligase activity is subject to feedback inhibition by increased levels of GSH,32 we speculate that activation of the biosynthesis and transport of precursor amino acids plays a key role in the maintenance of higher levels of GSH biosynthesis for cardioprotection.
Using fluxome analysis, we confirmed the increased flux toward the glycolytic and pentose phosphate pathways in Aldh2*2 Tg hearts. Enhanced flux toward the pentose phosphate pathway should be linked with the amount of NADP+ provided by GSH reductase, which converts GSSG to GSH. Because the NADPH/NADP+ ratio is increased in Aldh2*2 Tg hearts, there may be an active mechanism to shift glucose biotransformation from glycolysis toward the pentose phosphate pathway. The precise mechanism underlying the redirection of metabolic flux from the glycolytic to the pentose phosphate pathway under conditions of oxidative stress remains unknown.33,34
The present work has extended the concept of cardioprotection by preconditioning. According to the present understanding of cardioprotection by preconditioning,35 mitochondria are targets for protection from cell death. During sustained ischemia and reperfusion, the opening of the mitochondrial permeability transition pore induces mitochondrial swelling, depolarization, and ultimately cell death. Cardioprotection by preconditioning induced by brief nonlethal episodes of I/R activates a variety of signaling cascades, all of which culminate in the inhibition of mitochondrial permeability transition pore opening. Here, we showed that mitochondrial retrograde signals (signals originating from mitochondria)36 play a key role in cardioprotection. A shift toward the oxidative state in mitochondrial matrices signals to the nucleus to change nuclear gene expression, enabling cells to adapt and thus compensate for mitochondrial oxidative stress. Moreover, these signals favor tolerance to acute I/R injury. We also demonstrated that the eIF2-Atf4 pathway provides the key mitochondrial retrograde signals in response to mitochondrial aldehyde stress. Interestingly, characteristic transcriptional changes in Aldh2*2 Tg hearts were observed in human hybrid cells that harbor MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episode) and NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) mitochondrial DNA mutations37 and in frataxin-deficient murine hearts (Friedreich’s ataxia model),38 which are characterized by mitochondrial dysfunction and oxidative stress.
This study was limited by the uncertainty as to whether the cardioprotection observed for the Aldh2*2 Tg hearts actually exists in individuals carrying the ALDH2*2 allele. More generally, it would be intriguing to know whether a mitochondrial retrograde response transduced via the eIF2-ATF4 pathway is involved in cardioprotection during human aging and age-related diseases. Aging is accompanied by increased reactive oxygen species production and increased oxidative damage to mitochondrial DNA, proteins, and lipids.39 In addition, loss of cardioprotection in aged hearts40 is a likely consequence of the age-associated reduction in retrograde-response signaling. Notably, a paradoxical decline in the relative levels of eIF2 phosphorylation and ATF4 expression were demonstrated in aged rat tissues including heart,41 suggesting that the eIF2-ATF4-mediated retrograde response to mitochondrial dysfunction would also operate less efficiently in aged heart. How this retrograde regulation is affected by aging and age-related diseases represents an important area of future research.
The present study provides insight into the clinical significance of the hormesis-like effects of aldehydes. Hormesis is generally defined as a biphasic dose–response curve to treatments that are beneficial at low levels but noxious at higher levels.42 However, for practical reasons, most researchers in the fields of aging and molecular biology use a limited number of doses in the optimal or hormetic zone to study adaptive mechanisms. Thus, these researchers report hormetic effects without having to confirm a biphasic dose–response curve. This is certainly true for many examples of preconditioning.43,44 We extend the concept to hormetic mimetics: the fact that exposure occurs throughout life is not a problem, because the hormetic effects can be induced within the setting of chronic exposure that persists throughout the lifetime of mice.
Mitochondrial aldehyde stress produces different tissue-specific outcomes. It is essential to determine how aldehydes switch between being a hormetic signal and cytotoxicity and any alternative functions of aldehydes. Given the central role of aldehydes in the stress-induced signaling required to establish hormetic conditions, mimetic triggers of hormesis may be a promising approach for the prevention of the onset of age-associated diseases and senescence itself without the risk of overwhelming damage associated with the use of aldehydes themselves.
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Acknowledgments |
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We thank M. Nishijima, EJ. Calabrese, Y. Oike, N. Mizushima, and Y. Suzuki for critical discussions and K. Kaneki, M. Abe, M. Doi, Y. Miyake, and Y. Shiozawa for technical assistance. M. Sano, T.A., and M. Suematsu are core members of the Global Center of Excellence (GCOE) for Human Metabolomics Systems Biology, Ministry of Education, Culture, Sports, Science and Technology.
Sources of Funding
This work was supported by a PRESTO (Metabolism and Cellular Function) grant from the Japanese Science and Technology Agency (to M. Sano) and also by NIH grants EY11490 and EY AA016875 (to V.V.).
Disclosures
None.
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Footnotes |
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*Authors contributed equally to this work.
Original received June 19, 2009; resubmission received August 3, 2009; revised resubmission received September 25, 2009; accepted September 29, 2009.
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