Journal of Vascular Medicine & Surgery

Metabolomic Regulation by Oxidative Stress in Cardiac Diseases: An Overview on the State of the Heart

Alan Goncalves Amaral¹ and Aline Marados Santos^{2* 1}Department of Analytical Chemistry, Institute of Chemistry, State University of Campinas, Campinas, Brazil
²Department of Structural and Functional Biology, Institute of Biology, State University of Campinas, Campinas, Brazil
^*Correspondence: Aline Marados Santos, Department of Structural and Functional Biology, Institute of Biology, State University of Campinas, Campinas, Brazil, Email:

Received: 30-May-2022, Manuscript No. JVMS-22-16816; Editor assigned: 01-Jun-2022, Pre QC No. JVMS-22-16816(PQ); Reviewed: 22-Jun-2022, QC No. JVMS-22-16816; Revised: 29-Jun-2022, Manuscript No. JVMS-22-16816(R); Published: 06-Jul-2022, DOI: 10.35248/2329-6925.22.10.456

Introduction

Oxidative stress is a state of imbalance between the production of reactive oxygen species and the endogenous antioxidant defense mechanisms, resulting in excessive ROS production, which is associated with the pathogenesis of multiple cardiovascular diseases [1-6]. ROS are highly reactive compounds containing oxygen, including OH (hydroxyl), O₂ ⁻ (superoxide), ONOO⁻ (peroxynitrite), and non-radicals such as H₂O₂ (hydrogen peroxide). Physiologically, antioxidant defenses are capable of controlling the levels of ROS by the activity of the antioxidant enzymes, including glutathione peroxidase, superoxide dismutase, and catalase. Conversely, an increase in ROS production that overcomes the antioxidant defenses leads to a series of cell damages, including DNA breaks, lipid peroxidation, and protein oxidation [7]. Indeed, the accumulation of oxidative damage is a common factor involved in CVDs progression. This review summarizes current knowledge regarding oxidative stress and its pathological actions on the heart, highlighting cardiomyocyte metabolic alterations and their implications for overall cell metabolism. Specifically, the function of the endogenous antioxidant defense mechanisms and the pathological alterations in the primary cellular source of ROS will be discussed and linked to the development of the most relevant myocardial-related diseases. Then, a discussion of metabolic switches orchestrated by oxidative stress in cardiac diseases will be presented in parallel with results reached by H₂O₂-induced oxidative stress in H₉C₂ cardiomyocytes.

Metabolomic Technologies

The need to investigate different phenomena and improve techniques requires developing new tools. In recent years, we have seen in the area of life sciences the development of genetic engineering with CRISPR [8,9], improvement of bioinformatics [10], new pharmaceutical designs [11-13], and the use of omics [14-16]. The latter is a popular tool lately where large-scale studies are carried out to explore from genes to metabolites [17,18]. For studies on metabolism, we have metabolomics, a term introduced by Oliver Fiehn in 2001 [18]. In general, metabolomics can be defined as the comparison between the metabolome of an object of study in its standard and altered state, being those diseases, the ripening stage of a fruit, or different growth sites of a plant species [18,19]. Its application was facilitated by the development of Mass Spectrometry (MS) techniques, which is the most widely used tool.

Mass spectrometry, developed by J. J. Thomson in 1912 and improved by A. J. Dempster in 1918, is based on the ionization and fragmentation of molecules, generating a spectrum composed of the mass/charge ratio (m/z) of the fragments formed, which, when interpreted, provides information about the structures contained in a given sample [20]. Its use is frequent in metabolomics because of its high selectivity and sensitivity and the variety of commercially available equipment, allowing different couplings (e.g., GC-MS, HPLC-MS, and CEMS), ionization sources (e.g., EI, ESI, MALDI, and DESI), and mass analyzers (e.g., quadrupole, TOF, and Orbitrap) [21-25]. These instruments will generate different information about the samples, which complement each other. Since the type of sample and its preparation influence the choice of instruments, the advantages and limitations of the techniques must be aware [17]. GC-MS is primarily restricted to volatile and semi-volatile compounds, and its main disadvantage is the inclusion of a derivatization procedure in sample preparation [26,27]. On the other hand, it has high robustness, separation efficiency, reproducibility, and credibility in identifying small metabolites [26,27]. The LC-MS allows the analysis of different molecules with different polarities by varying its mobile phase, being employed the Reverse Phase Mode Chromatography (RPLC) for the study of more apolar molecules and the Hydrophilic Interaction Chromatography (HILIC) mode for more polar molecules [28,29]. In CE-MS, we have the separation and analysis of polar ionic compounds present in an electrolyte by an electric field applied to a capillary [30]. Due to its more complex construction than the other two instruments, careful optimization of the electrophoretic parameters is essential [31]. When using samples such as histological sections and cultured cells, imaging techniques such as MALDI (Matrix-Assisted Laser Desorption Ionization), SIMS (Secondary Ion Mass Spectrometry), and DESI (Desorption Electrospray) are typically used. Since the sample is in its native state, i.e., there is barely any sample preparation, there is little or no loss of metabolites during the analysis [24]. These techniques also allow the cells to be studied individually. This sub-area of metabolomics, known as single-cell metabolomics, has been widely pursued due to its great potential [32]. Given all the advantages and possibilities, metabolomic techniques have the potential to improve the understanding of heart diseases, enabling the discovery of new biomarkers and therapeutic targets.

Cellular Metabolism and Redox Balance

Redox reactions are constantly present in aerobic organisms, with their products participating in cellular metabolism. Among cellular components, mitochondria play a crucial role in the redox balance, being the primary source of ROS and antioxidants [33]. As is well known, the mitochondria are responsible for providing energy to the cell via oxidative phosphorylation performed in the mitochondrial electron transport chain. Among the five complexes responsible for this phenomenon, failures can occur in complexes I and III leading to electron leakage that, when reacting with O₂, can produceReactive Oxygen Species (ROS) [34]. Instead of being used toreduce O₂ to H₂O by the enzyme cytochrome coxidase, these electrons migrate to the cytosol and react with O₂, generating the superoxide radical [33,34]. This highly reactive compound derived from O₂ is primarily produced in mitochondria; nevertheless, other sources of O₂^•−, such as NADPH oxidase, monoamine oxidase, and xanthine Oxidoreductase, produce ROS throughout different reactions (Figure 1) [35].

Figure 1: Redox reactions of eukaryotic cells, highlighting (red) Reactive Oxygen Species (ROS).

In response to the physiological production of ROS, eukaryotic organisms developed a range of defense mechanisms known as the antioxidant system. Such mechanisms are based on the reduction of ROS by enzymes, including Super Oxide Dismutase (SOD), catalase, and Thioredoxin (Trx), and by endogenous or non-endogenous biomolecules (e.g., glutathione, vitamins A, C, and E) [36]. Within the metabolic pathways that actively participate in the antioxidant system of organisms, glutathione metabolism plays a fundamental role. Glutathione (GSH) is the most important intracellular antioxidant. Its main action is the removal of HO., ONOO⁻, CO₃ ^•−, and HOCl. In addition, in combination with the enzymes glutathione peroxidase and glutathione reductase, GSH acts in the removal of H₂O₂. The ability to consume cell-damaging species and its regenerative power made the glutathione system a robust antioxidant defense pathway [37]. However, cells undergo oxidative stress when the antioxidant defense is insufficient and the redox balance is disrupted, leading to cellular damage. Several studies highlight the link between oxidative stress and the pathogenesis of several diseases, including CVDs [5].

Pathological Implications of Oxidative Stress in Cardiovascular Diseases

General aspects

Cardiovascular diseases represent the leading causes of death worldwide, according to the World Health Organization [38]. In recent decades, with the increase in the incidence of cancer, obesity, and diabetes, in addition to the aging of the population, the risk of CVD has increased, in part due to the accumulation of oxidative damage [39]. Indeed, oxidative stress is a relevant factor in the development of cardiovascular diseases, including myocardial fibrosis and infarction, hypertrophy, and Heart Failure (HF), so significant efforts have been made to elucidate the biomolecular alterations governed by oxidative stress and its consequences CVDs progression [40-45].

The pathophysiological effects of ROS depend on their disponibility and site of production. Radical species with a shorter half-life are more unstable and toxic; conversely, the site of production and diffusion of the radical species throughout the cell and tissue impacts the surrounding molecules directly [46]. In pathological conditions, when ROS production overcomes the antioxidant defenses, ROS are able to cause oxidative modification of cellular macromolecules, such as DNA, lipids, and proteins [7]. Reactive oxygen species are a constant threat to DNA. Its oxidation results in breaks that can generate mutations during the DNA repair, with the risk of disrupting genome function [47]. The genome instability and the mutagenic processes derived from oxidative stress are tightly linked to multiple age-related diseases, such as hypertension and diabetes [48,49]. Otherwise, the oxidation of lipids and proteins induces modifications in the sarcolemma and subcellular organelles, such as mitochondria and sarcoplasmic reticulum, impacting the production of energy and the metabolism of calcium. Such alterations could trigger cardiomyocyte dysfunction and death through apoptosis and necrosis, events linked to contractile dysfunction, impaired cardiac remodeling, hypertrophy, fibrosis, and HF [5,50,51]. Importantly, enzymatic sources for ROS, such as the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase, Xanthine Oxidoreductase, Monoamine Oxidases (MAO), Cytochrome P450 Oxidase, and mitochondria are all considered relevant sources of ROS in CVDs, causing myocardial dysfunction. In the current topic, the most relevant sources of ROS in cardiac tissue will be summarized, and the related knowledge regarding pathological actions of oxidative stress on the heart will be discussed.

NADPH oxidases

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidases (NOXs) are a family of a multisubunit enzymes comprising membrane and cytosolic components responsible for transporting electrons across biological membranes, which leads to the reduction of oxygen into O₂^•−, in a reaction that consumes NADPH as an electron donor [52]. NOX 2 and 4 represent the members that are expressed in cardiomyocytes. While NOX2 is a sarcolemmal enzyme activated by several stimuli such as angiotensin II, growth factors, endothelin-1, TNF-α, and mechanical forces [53,54]; NOX4, found in intracellular membranes, are constitutively active [53-56]. NOX2 inactivation reduces the infarct area and ameliorates the development of heart failure in animal models of myocardial infarction. However, it is unclear whether this is related to vascular NOX or the NOX located in inflammatory cells [57]. NOX2-induced superoxide generation is also involved in angiotensin II-induced cardiomyocyte hypertrophy; thereby, antioxidative therapies have been proposed to ameliorate cardiac hypertrophy by inhibiting the activity of NOX enzymes [57,58].

Furthermore, NOX activity was shown to be upregulated in patients with ischemic or dilated cardiomyopathies, which is associated with increased RAC1 activity [57,58]. NOX4 constitutively produces hydrogen peroxide rather than O₂^•−. In heart tissue, NOX4 seems to be located at the membranes of the endoplasmic reticulum, nuclear envelope, and mitochondria. Several studies have reported a protective role of NOX4 in cardiac hypertrophy and against cardiac remodeling, regulating different transcription factors, such as NRF2, HIF1α, and ATF4. Briefly, the signaling pathways modulated by NOX4-enzyme serve to (a) limit oxidative stress, mitochondrial DNA damage, and cardiomyocyte death; (b) mediate cardiac remodeling and promote angiogenesis to protect the stressed hearts; and (c) activate autophagy in response to energy stress [59].

Xanthine Oxido Reductase (XOR)

Xantina Oxido Redutase (XOR) is also an important source of ROS in human hearts. This enzyme is expressed in the dehydrogenase form, but it is converted to the oxidase form under stress conditions. Both forms oxidate xanthine to uric acid, reducing NAD⁺ to NADH, in the case of the dehydrogenase form, and molecular oxygen to H₂O₂ and O₂^•−, in the case of the oxidase form [60]. In failing hearts, the level of XOR expression is increased when compared with normal myocardium [61]. Recent studies showed that XO inhibition improved myocardial efficiency and reverted left ventricular remodeling in dilated cardiomyopathy and myocardial infarction [61-63]. These results suggest that free radical production by XOR may be a significant cause of myocardial-related diseases.

Mitochondrial oxidative stress

Mitochondria are the predominant source of intracellular ROS, which are produced by the mitochondrial Electron Transfer Chain (ETC) as a byproduct of electron transfer. ROS generation in mitochondria is related to the partial reduction of O₂ to O₂^•− by complexes I and III of the electron transfer chain. O₂^•− generated is rapidly dismutate to H₂O₂ by the Mndependent superoxide dismutase [64]. However, other proteins may also trigger mitochondrial oxidative stress, such as the p66shc, MAOs, and NOX4 [64,65]. Upon stress, p66shc translocates to mitochondria and contributes to mitochondrial ROS production by oxidizing cytochrome C and stimulating hydrogen peroxide production [66].

Excessive ROS production occurs during mitochondrial dysfunction resulting in damage to mitochondrial DNA and defects in the ETC function [64]. The resulting increase in H₂O₂ levels induces cell death and left ventricular dysfunction. Mitochondrial dysfunction has been shown to play an essential role in the development and progression of HF in a murine model of myocardial infarction [45, 53,67]. Targeting catalase to mitochondria prevents HF in response to pressure overload and neurohormonal stimulation [64]. Mitochondrial ROS production is also a critical factor in many diabetes-related cardiovascular diseases [67-69]. Indeed, mice fed with a high-fat high-sucrose diet develop mitochondrial oxidative stress and cardiac hypertrophy [69,70]. An increase in mitochondrial oxidative stress was also described in the atria of diabetic and obese patients [71]. Likewise, in an insulin-dependent diabetes mellitus model, the mitochondrial generation of ROS leads to senescence and apoptosis of cardiac progenitor cells [68]. These data indicate that increased mitochondrial ROS production plays a crucial role in the pathophysiology of cardiac diseases.

Mono Amine Oxidases (MAO)

Mono Amine Oxidases (MAO) is another source of mitochondrial ROS. These enzymes are localized in the outer mitochondrial membrane and exist as two isoforms, MAO-A and MAO-B [72]. They are members of the flavoenzymes family, responsible for oxidizing biologically active amines in mammals, and are expressed at equivalent levels in the human heart [73]. MAO uses a FAD cofactor to catalyze the oxidative deamination of dietary amines, monoamine neurotransmitters, and catecholamines hormones, including serotonin, dopamine, norepinephrine, and epinephrine. The oxidative deamination generates toxic products (NH₃) and H₂O₂ [74]. In acute or chronic stress situations, MAO-A is a source of deleterious ROS, resulting in cardiolipin peroxidation, cardiomyocyte death, and ventricular dysfunction [75-77]. Likewise, global deletion of MAO-B protects against oxidative stress, left ventricular remodeling, and prevents cardiac failure in a mouse model of congestive heart failure induced by transverse aortic constriction [78]. The cardiac protection of MAO-B depletion was confirmed by the generation of cardiac-specific MAO-B knockout mouse submitted to ischemia/reperfusion injury, suggesting that ROS generation by MAO-B contributes to cardiac injury under stress conditions [79].

Oxidative Stress and Metabolic Switch in Cardiac Diseases

Metabolomics has become a powerful research tool in cardiovascular disease as knowledge of the metabolic bases of CVDs advances. The ability to measure changes in metabolite concentrations and discover new biomarkers of different cardiovascular diseases has provided new insights into diagnoses and the development of new therapeutic approaches for CVDs. We highlight metabolic switches orchestrated by oxidative stress in cardiomyocytes cells, animal models, and human heart biopsies.

Oxidative Stress (OS) has been shown to play an essential role in the pathophysiology of the most severe cardiac diseases. As previously mentioned, a balance between the levels of Reactive Oxygen Species (ROS) and the antioxidant defenses is essential for cell health. Otherwise, excessive production of ROS that overcomes the antioxidant capacity causes damage to macromolecules, including protein, lipids, and DNA, and can trigger cell death [3,80-83]. However, although several signaling pathway studies and functional in vivo experiments have demonstrated the effects of ROS production in CVDs, scarce information is available about measurable changes in the metabolomic profile of cardiomyocytes undergoing oxidative stress.

Recently, in search of a model to explore the effects of oxidative stress, specifically in cardiomyocyte metabolism, Amaral et al. developed an in vitro platform to explore the metabolic switch induced by stressors agents on H₉C₂ cardiomyocytes through LC/MS untargeted metabolomics technique [84]. As a result, the authors depicted metabolic alterations of glucose, lipid, pyrimidine and purine biosynthesis, and glutathione pathways in cardiomyocytes exposed to H₂O₂. Such modulations will be discussed in the context of cardiac diseases.

Glucose metabolism

Glucose metabolism is essential for life maintenance. Carbohydrates, lipids, and proteins are ultimately broken down into glucose, the primary metabolic fuel of mammal cells and the precursor for synthesizing several cellular compounds. Glucose transport into cardiomyocytes is regulated by transmembrane Glucose Transporters (GLUTs) [85]. In the cytoplasm, glucose is converted to glucose-6-phosphate and oxidized to pyruvate, transported into mitochondria, where it will be oxidized by the Tri Carboxylic Acid (TCA) cycle [86].

Several studies have reported that mitochondrial glucose oxidation is defective in the failing heart [87,88]. Ussher et al. demonstrated that in HF, pyruvate dehydrogenase activity is decreased, which reduces pyruvate oxidation by the TCA cycle. As a result, glycolysis is increased, elevating the circulating levels of lactate [89]. Likewise, Amaral et al. showed an upregulation of glycolysis metabolites such as Glucose-6-phosphate (2.01-fold), Glycerol-3-phosphate (0.8-fold), and Lactate (1.59-fold) upon H₂O₂ exposed cardiomyocytes, indicating a shift of energy metabolism to anaerobic glycolysis as an adaptive response to oxidative stress [84]. The authors also identified the accumulation of citrate levels (0.48-fold) and an upregulation of amino acid biosynthesis pathways, indicating dysfunction of the citric acid cycle and a shift toward amino acid biosynthesis [84,90].

Interestingly, Frezza et al. reported the action of HIF-1α factor during oxidative stress to promote the expression of genes involved in shifting the metabolism towards anaerobic glycolysis, impairing the citric acid cycle activity to couple to oxidative stress [91]. Furthermore, in a rat model, glucose oxidation rates were increased during compensated phases of cardiac hypertrophy, while during HF, glucose oxidation was downregulated [92,93]. Another interesting study was published by Ranjbarvaziri et al. [94]. In order to identify the functional components governing Hypertrophic Cardio Myopathy (HCM), the most common heritable cardiovascular disease [95,96], authors performed metabolomics and transcriptomics experiments on human heart samples. The metabolomics approach revealed alterations in carbohydrate metabolism, which significantly decreased in HCM. The levels of glucose, glycolysis intermediates (fructose 6- phosphate and phosphoenolpyruvic acid), pentose phosphate pathway metabolites (ribose 5-phosphate; ribulose 5-phosphate), and TCA cycle intermediates (malate, citrate, succinate) were downregulated, suggesting a global energetic decompensation.

Nowadays, it is accepted that alterations in glucose utilization vary depending on the etiology and severity of heart pathology, which possibly explains the discrepancy among the studies mentioned above [6].

Lipid metabolism

Several abnormalities in lipid metabolism have been identified in cardiovascular diseases. Free fatty acid crosses the sarcolemma and enters the cytoplasm, where it can be converted into Acyl- Coenzyme A (acyl-CoA). Thereafter, acyl-CoA is converted to acylcarnitine to enable its entry into mitochondria to be oxidized in the β-oxidization [86]. Changes in fatty acid oxidation rates or damage in mitochondrial β-oxidation can be reflected in acylcarnitines profiles since these metabolites are derivatives of fatty acyl-CoA [97].

In line with these findings, Amaral et al. recently demonstrated that L-carnitine was upregulated (1.9-fold) in H9C2 cardiomyocytes under oxidative stress [84]. Wang et al. suggested that L-carnitine protects cardiomyocytes against doxorubicininduced oxidative stress and myocardial injury [98]. The upregulation of L-carnitine may be related to a reduction in intra-mitochondrial acetyl-CoA in response to mitochondria oxidative damage and reducing β-oxidation, as previously reported [99]. Hunter et al. demonstrated that the levels of acylcarnitines were increased among HF patients with preserved ejection fraction, and even higher levels were found in those with reduced ejection fraction [100].

Nevertheless, Bedi et al. found reduced levels of these compounds in end-stage HF patients compared to tissue from healthy ones [101]. Ranjbarvaziri et al. also described a decrease in the abundance of acylcarnitines in samples from HCM patients, suggesting a defect in converting free fatty acid to acylcarnitine. Consistently, mitochondrial carnitine Oacetyltransferase, which catalyzes the conversion of acyl-CoA to acylcarnitine, was reduced. Moreover, the expression of genes involved in fatty acid β-oxidation was reduced in relation to normal hearts [94]. Decreased myocardial acylcarnitines might indicate impaired mitochondrial function and reduced β- oxidation [102-104], which is in line with previous findings showing a reduction of fatty acid oxidation during more severe stages of HF [102,105]. This controversial finding among this set of studies may be explained by the inclusion or exclusion of diabetic patients, in which circulating acylcarnitines are often elevated, or by the severity of the cardiac diseases [6,88]. Future metabolomics studies considering aspects of subgroups or in vitro assays with isolated cardiomyocytes will contribute to clarifying these discrepancies. Exclusion of diabetic patients, in which circulating acylcarnitines are often elevated, or by the severity of the cardiac diseases [6,88]. Future metabolomics studies considering aspects of subgroups or in vitro assays with isolated cardiomyocytes will contribute to clarifying these discrepancies.

Regarding the metabolites involved in the sphingolipid metabolism, Amaral et al. demonstrated an upregulation of sphingosine 1-phosphate (2.65-fold) and phytosphingosine (2.31- fold) in H₉C₂ cardiomyocytes under oxidative stress [84]. In line with these findings, Ranjbarvaziri et al. showed an upregulation of ceramide and sphingomyelin in HCM patients [94]. Ceramides, derived from sphingomyelin metabolism, seem to be involved in the pathogenesis of cardiac diseases, causing lipotoxicity, inflammation, and cell death. Genetic ablation or pharmacological inhibition of ceramide biosynthesis enzymes ameliorates cardiac diseases, such as hypertension and cardiomyopathy [49]. Metabolic profiling studies have further highlighted the potential of such lipids to risk stratification and as biomarkers of heart disease [106].

Pyrimidine and purine biosynthesis

Oxidative phosphorylation, oxygen consumption, and ATP production are reduced during heart failure [107]. Ranjbarvaziri et al. showed evidence of heart decompensation of energy metabolism with a decrease in ATP, ADP, and phosphocreatine in HCM patients [93]. The authors also described a reduction in oxidative phosphorylation capacity through ATP synthase and a lower expression of several other mitochondrial complexes. An upregulation of the Un Coupling Protein 2 (UCP2) was also described. Next, the authors demonstrated that AMPK, the main sensor of cellular energy status [108, 109], was activated in hypertrophic hearts. These data suggest that despite increased metabolic demand and activation of AMPK in HCM hearts, decreased energy supply contributes to energetic deprivation.

Interestingly, Amaral et al. described a downregulation of the products UDP, CDP, and AMP of the pyrimidine and purine metabolism in cardiomyocytes under oxidative stress [83]. The downregulation of AMP levels was shown to maintain AMPK in its inhibited form [109]. When activated by energetic stress, the AMPK inhibits ATP-consuming pathways and triggers ATPproducing pathways, such as fatty acid oxidation, glucose uptake, and glycolysis [110]. Amaral et al. suggest that oxidative stress induces a metabolic adaptation mechanism that allows cell survival and the reactivation of the anabolic pathways in H₉C₂ cardiomyocytes after oxidative stress. Such differences described in the literature may be related to the multifactorial components of cardiac diseases. Future metabolomic experiments using isolated cardiomyocytes urge to clarify the impact of pyrimidine and purine metabolism on the evolution of this class of diseases.

Glutathione metabolism

As mentioned above, glutathione metabolism is the first line of defense against ROS, playing a pivotal role in the cardiovascular system [111]. Several studies have associated CVDs with redox imbalances in the heart, such as hypertension and atherosclerosis, linked to polymorphisms of the enzymes of this pathway [37]. Ranjbarvaziri et al. showed that patients with Hypertrophic Cardiomyopathy present high rates of oxidative stress in hearts, characterized by a high cystine level, low glutathione (GSH) disponibility, and an increased GSSG/GSH ratio (oxidized glutathione/reduced glutathione) [93]. Amaral et al. also reported a significant reduction of glutathione metabolite in cardiomyocytes under oxidative stress [83]. Studies that target oxidative stress as a therapeutic target have shown that the best strategy to improve its efficacy is to enhance the endogenous antioxidant capacity, increasing GSH levels [82,112]. Despite the knowledge concerning the antioxidant action of glutathione metabolism, the functioning of this pathway and its impacts on CVDs needs to be further clarified.

Discussion

Our results showed that a key pathway to be activated under oxidative stress was the alanine, aspartate, and glutamate metabolism. Specifically, we found that the L-aspartate and LAsparagine were upregulated 2.5- and 1.56-fold, respectively, while the N-(L-Arginino) succinate was down regulated 1.76-fold in cardiomyocytes exposed to H₂O₂ over control cells, probably due to increased utilization or decreased synthesis. Although we cannot firmly distinguish between these possibilities, it is likely that N-(L-Arginine) succinate is depleted because of increased utilization. L-aspartate is converted into N-(L-Arginine) succinate, which may be converted into L-arginine, the substrate of Nitric Oxide (NO) synthase. NO is a potent anti-hypertensive agent, produced when L-arginine is converted to citrulline Under oxidative stress, the increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to form the peroxynitrite anion (ONOO−), resulting in both oxidation and nitration of proteins and in lipid peroxidation. This pathway was also up regulated in the cardiomyocytes recovered for 48 h after oxidative stress.

The observed up regulation of the “aspartate metabolism” under OS may also affect related pathways including “glycolysis”, “gluconeogenesis”, “citric acid cycle”, “amino acids biosynthesis”, and “pyrimidine and purine biosynthesis”. Interestingly, upon 48 hours recovery of OS, we observed an up regulation of the glycolysis metabolites such as Glucose-6- phosphate (2.01-fold), Glycerol-3-phosphate (0.8-fold), and Lactate (1.59-fold), which indicated that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in cardiomyocytes H₉C₂. We also identified a modest accumulation in citrate levels (0.48-fold) and an up regulation of amino acid biosynthesis pathways, indicating a dysfunction of the citric acid cycle and a shift towards amino acids biosynthesis Scientific literature have reported the action of the factor HIF-1α during oxidative stress in mediation of gene expression involved in shifting the metabolism towards anaerobic glycolysis without requirement of a hypoxic condition. HIF-1α up regulates the expression of Glut1 and glycolytic enzymes increasing the anaerobic glycolysis pathway. Moreover, HIF-1α has shown to limit the conversion of pyruvic acid to acetyl-CoA in the mitochondria, impairing the citric acid cycle activity as a strategy to couple to oxidative stress.

Conclusion

This review highlights recent advances in metabolomic profile applied to the characterization of the pathophysiology of the most relevant cardiac diseases, focusing on oxidative stress as a damaging agent. The advances in metabolomic platforms, predominantly based on NMR and mass spectrometry, enabled metabolomics to address the molecular mechanisms of cardiac diseases with implications for therapeutic efficacy and the discovery of new biomarkers for ameliorating the diagnostics. Furthermore, such techniques have enabled the description of shifts in the central metabolic pathways of cardiac cells under diverse oxidative stress conditions. We emphasized the discussion of metabolic switches in glucose, lipids, purine and pyrimidines, and glutathione metabolism, orchestrated by oxidative stress in cardiac diseases, and made a parallel with the results reached by H₂O₂-induced oxidative stress in H₉C₂ cardiomyocytes. The advances in knowledge of cardiomyocyte metabolic regulation by oxidative stress will help identify the challenges and opportunities to develop more effective ROSbased therapies.

REFERENCES

1. Hermes-Lima M, Pereira B, Bechara EJ. Are free radicals involved in lead poisoning? Xenobiotica. 1991;21(8):1085-1090.

   [Crossref] [Google Scholar] [Pubmed]

2. Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: What is the clinical significance? Hypertension. 2004;44(3):248-252.

   [Crossref] [Google Scholar] [Pubmed]

3. Rodrigo R, Libuy M, Feliú F, Hasson D. Molecular basis of cardioprotective effect of antioxidant vitamins in myocardial infarction. BioMed Research International. 2013.

   [Crossref] [Google Scholar] [Pubmed]

4. Deidda M, Noto A, Bassareo PP, Cadeddu Dessalvi C, Mercuro G. Metabolomic approach to redox and nitrosative reactions in cardiovascular diseases. Front Physiol. 2018;9:672.

   [Crossref] [Google Scholar] [Pubmed]

5.  Senoner T, Dichtl W. Oxidative stress in cardiovascular diseases: Still a therapeutic target? Nutrients. 2019;11(9):2090.

   [Crossref] [Google Scholar] [Pubmed]

6.   Müller J, Bertsch T, Volke J, Schmid A, Klingbeil R, Metodiev Y, et al. Narrative review of metabolomics in cardiovascular disease. J Thorac Dis. 2021;13(4):2532.

   [Crossref] [Google Scholar] [Pubmed]

7.  Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr Biol. 2014;24(10):453-462.

   [Crossref] [Google Scholar] [Pubmed]

8. Uddin F, Rudin CM, Sen T. CRISPR gene therapy: Applications, limitations, and implications for the future. Front Oncol. 2020;10:1387.

   [Crossref] [Google Scholar] [Pubmed]

9. Westermann L, Neubauer B, Köttgen  M. Nobel Prize 2020 in Chemistry honors CRISPR: A tool for rewriting the code of life. Pflugers Arch. 2021;473(1):1-2.

   [Crossref] [Google Scholar] [Pubmed]

10.  Westermann L, Neubauer B, Köttgen M. Nobel Prize 2020 in Chemistry honors CRISPR: A tool for rewriting the code of life. Methods. 2021;473(1):1-2.

    [Crossref] [Google Scholar] [Pubmed]

11. Fukuda IM, Pinto CF, Moreira CD, Saviano AM, Lourenço FR. Design of Experiments (DoE) applied to pharmaceutical and analytical Quality by Design (QD). Braz J Pharm Sci. 2018;54.

    [Crossref] [Google Scholar]

12. Diab S, McQuade DT, Gupton BF, Gerogiorgis DI. Process design and optimization for the continuous manufacturing of nevirapine, an active pharmaceutical ingredient for HIV treatment. Org. Process Res Dev. 2019;23(3):320-333.

    [Crossref] [Google Scholar]

13.  Hartford A, Thomann M, Chen X, Miller E, Bedding A, Jorgens S, et al. Adaptive designs: Results of 2016 survey on perception and use. Ther Innov Regul Sci. 2020;54(1):42-54.

    [Crossref] [Google Scholar] [Pubmed]

14. Karczewski KJ, Snyder MP. Integrative omics for health and disease. Nat Rev Genet. 2018;19(5):299-310.

    [Crossref] [Google Scholar] [Pubmed]

15. Misra BB, Langefeld C, Olivier M, Cox LA. Integrated omics: Tools, advances and future approaches. J Mol Endocrinol. 2019;62(1):21-45.

    [Crossref] [Google Scholar]

16. Subramanian I, Verma S, Kumar S, Jere A, Anamika K. Multi-omics data integration, interpretation, and its application. Bioinform Biol Insights. 2020;14:1177932219899051.

    [Crossref] [Google Scholar] [Pubmed]

17. Canuto GA, Costa JL, Da Cruz PL, Souza AR, Faccio AT, Klassen A, et al. Metabolômica: Definições, estado-da-arte e aplicações representativas. Quim Nova. 2018;41:75-91.

    [Crossref] [Google Scholar]

18. Fiehn O. Metabolomics the link between genotypes and phenotypes. Plant Mol Biol. 2002:155-171.

    [Crossref] [Google Scholar] [Pubmed]

19. Fiehn O. Combining genomics, metabolome analysis, and biochemical modelling to understand metabolic networks. Comp Funct Genomics. 2001;2(3):155-168.

    [Crossref] [Google Scholar] [Pubmed]

20. Ren JL, Zhang AH, Kong L, Wang XJ. Advances in mass spectrometry-based metabolomics for investigation of metabolites. RSC advances. 2018;8(40):22335-22350.

    [Crossref] [Google Scholar] [Pubmed]

21. Jonsson P, Gullberg J, Nordström A, Kusano M, Kowalczyk M, Sjöström M, et al. A strategy for identifying differences in large series of metabolomic samples analyzed by GC/MS. Anal Chem. 2004;76(6):1738-1745.

    [Crossref] [Google Scholar] [Pubmed]

22. Cubbon S, Antonio C, Wilson J, Thomas‐Oates J. Metabolomic applications of hilic–lc–ms. Mass Spectrom Rev. 2010;29(5):671-684.

    [Crossref] [Google Scholar] [Pubmed]

23. Barbosa CG, Gonçalves NS, Bechara EJ, Assunção NA. Potential diagnostic assay for cystinuria by capillary electrophoresis coupled to mass spectrometry. J Braz Chem Soc. 2013;24(4):534-540.

    [Crossref] [Google Scholar]

24. Feider CL, Krieger A, DeHoog RJ, Eberlin LS. Ambient ionization mass spectrometry: Recent developments and applications. Anal Chem. 2019;91(7):4266-4290.

    [Crossref] [Google Scholar]

25. Theiner S, Schoeberl A, Schweikert A, Keppler BK, Koellensperger G. Mass spectrometry techniques for imaging and detection of metallodrugs. Curr Opin Chem Biol. 2021;61:123-134.

    [Crossref] [Google Scholar] [Pubmed]

26. Coskun O. Separation techniques: Chromatography. North Clin Istanb. 2016;3(2):156.

    [Crossref] [Google Scholar] [Pubmed]

27. Sutherland K. Gas chromatography/mass spectrometry techniques for the characterisation of organic materials in works of art. Phys Sci Rev. 2019;4(6).

    [Crossref] [Google Scholar]

28. Maldaner L, Collins CH, Jardim IC. Fases estacionárias modernas para cromatografia líquida de alta eficiência em fase reversa. Quim Nova. 2010;33(7):1559-1568.

    [Crossref] [Google Scholar]

29. Gama MR, Da Costa Silva RG, Collins CH, Bottoli CB. Hydrophilic interaction chromatography. Trends Anal Chem. 2012;37:48-60.

    [Crossref] [Google Scholar]

30. Assunção NA, Bechara EJ, Simionato AV, Tavares MF, Carrilho E. Eletroforese capilar acoplada à espectrometria de massas (CE-MS): Vinte anos de desenvolvimento. Quim Nova. 2008;31(8):2124-2133.

    [Crossref] [Google Scholar]

31. Viberg P, Nilsson S, Skog K. Nanospray mass spectrometry with indirect conductive graphite coating. Anal Chem. 2004;76(14):4241-4244.

    [Crossref] [Google Scholar] [Pubmed]

32. Ma A, McDermaid A, Xu J, Chang Y, Ma Q. Integrative methods and practical challenges for single-cell multi-omics. Trends Biotechnol. 2020;38(9):1007-1022.

    [Crossref] [Google Scholar] [Pubmed]

33. Bottje WG. Oxidative metabolism and efficiency: The delicate balancing act of mitochondria. Poult Sci. 2019;98(10):4223-4230.

    [Crossref] [Google Scholar] [Pubmed]

34. Kowaltowski AJ, De Souza-Pinto NC, Castilho RF, Vercesi AE. Mitochondria and reactive oxygen species. Free Radic Biol Med. 2009;47(4):333-343.

    [Crossref] [Google Scholar] [Pubmed]

35. Panth N, Paudel KR, Parajuli K. Reactive oxygen species: A key hallmark of cardiovascular disease. Adv Med Sci. 2016;2016.

    [Crossref] [Google Scholar] [Pubmed]

36. Kayama Y, Raaz U, Jagger A, Adam M, Schellinger IN, Sakamoto M, et al. Diabetic cardiovascular disease induced by oxidative stress. Int J Mol Sci. 2015;16(10):25234-25263.

    [Crossref] [Google Scholar] [Pubmed]

37. Matuz-Mares D, Riveros-Rosas H, Vilchis-Landeros MM, Vázquez-Meza H. Glutathione Participation in the Prevention of Cardiovascular Diseases. Antioxidants. 2021;10(8):1220.

    [Crossref] [Google Scholar] [Pubmed]

38. Ezzati M, Obermeyer Z, Tzoulaki I, Mayosi BM, Elliott P, Leon DA. Contributions of risk factors and medical care to cardiovascular mortality trends. Nat Rev Cardiol. 2015;12(9):508-530.

    [Crossref] [Google Scholar] [Pubmed ]

39. Tzoulaki I, Elliott P, Kontis V, Ezzati M. Worldwide exposures to cardiovascular risk factors and associated health effects: Current knowledge and data gaps. Circulation. 2016;133(23):2314-2333.

    [Crossref] [Google Scholar] [Pubmed]

40. Hermida N, Michel L, Esfahani H, Dubois-Deruy E, Hammond J, Bouzin C, et al. Cardiac myocyte β3-adrenergic receptors prevent myocardial fibrosis by modulating oxidant stress-dependent paracrine signaling. Eur Heart J. 2018;39(10):888-898.

    [Crossref] [Google Scholar] [Pubmed]

41. Dubois-Deruy E, Cuvelliez M, Fiedler J, Charrier H, Mulder P, Hebbar E, et al. MicroRNAs regulating superoxide dismutase 2 are new circulating biomarkers of heart failure. Sci Rep. 2017;7(1):1-10.

    [Crossref] [Google Scholar] [Pubmed]

42. Li JM, Gall NP, Grieve DJ, Chen M, Shah AM. Activation of NADPH oxidase during progression of cardiac hypertrophy to failure. Hypertension. 2002;40(4):477-484.

    [Crossref] [Google Scholar] [Pubmed]

43. Dai DF, Johnson SC, Villarin JJ, Chin MT, Nieves-Cintrón M, Chen T, et al. Mitochondrial oxidative stress mediates angiotensin II–induced cardiac hypertrophy and Gαq overexpression–induced heart failure. Circ Res. 2011;108(7):837-846.

    [Crossref] [Google Scholar] [Pubmed]

44. Dai DF, Hsieh EJ, Liu Y, Chen T, Beyer RP, Chin MT, et al. Mitochondrial proteome remodelling in pressure overload-induced heart failure: The role of mitochondrial oxidative stress. Cardiovasc Res. 2012;93(1):79-88.

    [Crossref] [Google Scholar] [Pubmed]

45. Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura KI, et al. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ Res. 2001 Mar 16;88(5):529-535.

    [Crossref] [Google Scholar] [Pubmed]

46. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev. 2014;94(3):909-950

    [Crossref] [Google Scholar] [Pubmed]

47. Poetsch AR. The genomics of oxidative DNA damage, repair, and resulting mutagenesis. Comput Struct Biotechnol J. 2020;18:207-219.

    [Crossref] [Google Scholar] [Pubmed]

48. Tubbs A, Nussenzweig A. Endogenous DNA damage as a source of genomic instability in cancer. Cell. 2017;168(4):644-656.

    [Crossref] [Google Scholar] [Pubmed]

49. Iliou A, Mikros E, Karaman I, Elliott F, Griffin JL, Tzoulaki I, et al. Metabolic phenotyping and cardiovascular disease: An overview of evidence from epidemiological settings. Heart. 2021 ;107(14):1123-1129.

    [Crossref] [Google Scholar] [Pubmed]

50. Lancel S, Qin F, Lennon SL, Zhang J, Tong X, Mazzini MJ, et al. Oxidative posttranslational modifications mediate decreased SERCA activity and myocyte dysfunction in Gαq-overexpressing mice. Circ Rec. 2010;107(2):228-232.

    [Crossref] [Google Scholar] [Pubmed]

51. Steinberg SF. Oxidative stress and sarcomeric proteins. Circ. Res. 2013;112(2): 393-405.

    [Crossref] [Google Scholar] [Pubmed]

52. Panday A, Sahoo MK, Osorio D, Batra S. NADPH oxidases: An overview from structure to innate immunity-associated pathologies. Cell Mol. Immunol. 2015;12(1):5-23.

    [Crossref] [Google Scholar] [Pubmed]

53. Moris D, Spartalis M, Tzatzaki E, Spartalis E, Karachaliou GS, Triantafyllis AS,et al. The role of reactive oxygen species in myocardial redox signaling and regulation. Ann. Transl. Med. 2017;5(16):324.

    [Crossref] [Google Scholar] [Pubmed]

54. Sag CM, Santos CX, Shah AM. Redox regulation of cardiac hypertrophy. J. Mol. Cell Cardiol. 2014;73:103-111.

    [Crossref] [Google Scholar] [Pubmed]

55. Santos CX, Nabeebaccus AA, Shah AM, Camargo LL, Filho SV, Lopes LR. Endoplasmic reticulum stress and Nox-mediated reactive oxygen species signaling in the peripheral vasculature: Potential role in hypertension. Antioxid. Redox Signal. 2014;20(1):121-34.

    [Crossref] [Google Scholar] [Pubmed]

56. Münzel T, Camici GG, Maack C, Bonetti NR, Fuster V, Kovacic JC. Impact of oxidative stress on the heart and vasculature: part 2 of a 3-part series. J Am. Coll Cardiol.;70(2):212-29

    [Crossref] [Google Scholar] [Pubmed]

57. Gang C, Qiang C, Xiangli C, Shifen P, Chong S, Lihong L. Puerarin suppresses angiotensin II-induced cardiac hypertrophy by inhibiting NADPH oxidase activation and oxidative stress-triggered AP-1 signaling pathways. Pharm. Pharm. Sci. 2015;18(2):235-248.

    [Crossref] [Google Scholar] [Pubmed]

58. Zhang Q, Tan Y, Zhang N, Yao F. Polydatin prevents angiotensin II-induced cardiac hypertrophy and myocardial superoxide generation. Exp. Biol. Med. 2015;240(10):1352-1361 

    [Crossref] [Google Scholar] [Pubmed]

59. Sciarretta S, Zhai P, Shao D, Zablocki D, Nagarajan N, Terada LS, et al. Activation of NADPH oxidase 4 in the endoplasmic reticulum promotes cardiomyocyte autophagy and survival during energy stress through the protein kinase RNA-activated-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 pathway. Circ. Res. 2013;113(11):1253-1264.

    [Crossref] [Google Scholar] [Pubmed]

60. Nishino T, Okamoto K, Eger BT, Pai EF, Nishino T. Mammalian xanthine oxidoreductase–mechanism of transition from xanthine dehydrogenase to xanthine oxidase. FEBS J. 2008;275(13):3278-3289.

    [Crossref] [GoogleScholar] [Pubmed]

61. Cappola TP, Kass DA, Nelson GS, Berger RD, Rosas GO, Kobeissi ZA, et al. Allopurinol improves myocardial efficiency in patients with idiopathic dilated cardiomyopathy. Circulation. 2001;104(20):2407-2411.

    [Crossref] [Google Scholar] [Pubmed]

62. Lee BE, Toledo AH, Anaya-Prado R, Roach RR, Toledo-Pereyra LH. Allopurinol, xanthine oxidase, and cardiac ischemia. J. Investig. Med. 2009;57(8):902-909.

    [Crossref] [Google Scholar] [Pubmed]

63. Minhas KM, Saraiva RM, Schuleri KH, Lehrke S, Zheng M, Saliaris AP, et al. Xanthine oxidoreductase inhibition causes reverse remodeling in rats with dilated cardiomyopathy. Circ Res. 2006;98(2):271-279.

    [Crossref] [Google Scholar] [Pubmed]

64. Peoples JN, Saraf A, Ghazal N, Pham TT, Kwong JQ. Mitochondrial dysfunction and oxidative stress in heart disease. Exp Mol Med. 2019;51(12):1-3.

    [Crossref] [Google Scholar] [Pubmed]

65.  Giorgio M, Migliaccio E, Orsini F, Paolucci D, Moroni M, Contursi C, et al. Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell. 2005;122(2):221-33.

    [Crossref] [Google Scholar] [Pubmed]

66. Orsini F, Migliaccio E, Moroni M, Contursi C, Raker VA, Piccini D, et al. The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential. J. Biol. Chem. 2004;279(24):25689-25695.

    [Crossref] [Google Scholar] [Pubmed]

67. D’Oria R, Schipani R, Leonardini A, Natalicchio A, Perrini S, Cignarelli A, et al. The role of oxidative stress in cardiac disease: from physiological response to injury factor. Oxid. Med. Cell Longev. 2020; 5732956.

    [Crossref] [Google Scholar] [Pubmed]

68. Rota M, LeCapitaine N, Hosoda T, Boni A, De Angelis A, Padin-Iruegas ME, et al. Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene. Circ. Res. 2006;99(1):42-52.

    [Crossref] [Google Scholar] [Pubmed]

69. Sverdlov AL, Elezaby A, Qin F, Behring JB, Luptak I, Calamaras TD, et al. Mitochondrial reactive oxygen species mediate cardiac structural, functional, and mitochondrial consequences of diet‐induced metabolic heart disease. J Am Heart Assoc. 2016;5(1):e002555.

    [Crossref] [Google Scholar] [Pubmed]

70. Jeong EM, Chung J, Liu H, Go Y, Gladstein S, Farzaneh‐Far A, et al. Role of mitochondrial oxidative stress in glucose tolerance, insulin resistance, and cardiac diastolic dysfunction. J. Am Heart Assoc. 2016;5(5): e003046.

    [Crossref] [Google Scholar] [Pubmed]

71. Niemann B, Chen Y, Teschner M, Li L, Silber RE, Rohrbach S. Obesity induces signs of premature cardiac aging in younger patients: The role of mitochondria. J Am Coll Cardiol. 2011;57(5):577-585.

    [Crossref] [Google Scholar] [Pubmed]

72. Edmondson DE, Binda C, Wang J, Upadhyay AK, Mattevi A. Molecular and mechanistic properties of the membrane-bound mitochondrial monoamine oxidases. Biochemistry. 2009;48(20):4220-4230.

    [Crossref] [Google Scholar] [Pubmed]

73. Maggiorani D, Manzella N, Edmondson DE, Mattevi A, Parini A, Binda C, et al. Monoamine oxidases, oxidative stress, and altered mitochondrial dynamics in cardiac ageing. Oxid Med Cell Longev. 2017.

    [Crossref] [Google Scholar] [Pubmed]

74. Machado-Vieira R, Mallinger AG. Abnormal function of monoamine oxidase-A in comorbid major depressive disorder and cardiovascular disease: Pathophysiological and therapeutic implications. Mol Med Rep. 2012;6(5):915-922.

    [Crossref] [Google Scholar] [Pubmed]

75. Manzella N, Santin Y, Maggiorani D, Martini H, Douin‐Echinard V, Passos JF, et al. Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy. Aging cell. 2018;17(5):e12811.

    [Crossref] [Google Scholar] [Pubmed]

76. Santin Y, Sicard P, Vigneron F, Guilbeau-Frugier C, Dutaur M, Lairez O, et al. Oxidative Stress by Monoamine Oxidase-A Impairs Transcription Factor EB Activation and Autophagosome Clearance, Leading to Cardiomyocyte Necrosis and Heart Failure. Antioxid. Redox Signal. 2016;25(1):10-27.

    [Crossref] [Google Scholar] [Pubmed]

77.  Mialet-Perez J, Parini A. Cardiac monoamine oxidases: At the heart of mitochondrial dysfunction. Cell Death Dis. 2020; 11(1):1-3.

    [Crossref] [Google Scholar] [Pubmed]

78. Kalaudercic N, Carpi A, Nagayama T, Sivakumaran V, Zhu G, Lai EW, et al. Monoamine oxidase B prompts mitochondrial and cardiac dysfunction in pressure overloaded hearts. Antioxid Redox Signal. 2014;20(2):267-280.

    [Crossref] [Google Scholar] [Pubmed]

79. Heger J, Hirschhäuser C, Bornbaum J, Sydykov A, Dempfle A, Schneider A, et al. Cardiomyocytes-specific deletion of monoamine oxidase B reduced irreversible myocardial ischemia/reperfusion injury. Free Radic Biol Med. 2021;165:14-23.

    [Crossref] [Google Scholar] [Pubmed]

80. Meneghini R. Iron Homeotasis, Oxidative Stress, and DNA Damage. Free Radic Biol Med. 1997; 23(5):783-792.

    [Crossref] [Google Scholar] [Pubmed]

81. Alkadi H. A review on free radicals and antioxidants. Infect Disord. Drug Targets 2020;20(1):16-26.

    [Crossref] [Google Scholar] [Pubmed]

82. Poljsak B, Šuput D, Milisav I. Achieving the balance between ROS and antioxidants: When to use the synthetic antioxidants. Oxid Med Cell Longev. 2013;956792.

    [Crossref] [Google Scholar] [Pubmed]

83. Van Der Pol A, Van Gilst WH, Voors AA, Van Der Meer P. Treating oxidative stress in heart failure: Past, present and future. Eur J Heart Fail. 2019;21(4):425-435.

    [Crossref] [Google Scholar]

84. Amaral AG, Moretto IA, Zandonadi FS, Zamora-Obando HR, Rocha I. Sussulini A. Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of in vitro Samples. Front Chem. 2022;10:836478.

    [Crossref] [Google Scholar] [Pubmed]

85. Robinson R, Robinson LJ, James DE, Lawrence JC. Glucose transport in L6 myoblasts overexpressing GLUT1 and GLUT4. J Biol Chem. 1993;268(29):22119-22126.

    [Crossref] [Google Scholar] [Pubmed]

86. Schwenk RW, Luiken JJFP, Bonen A, Glatz JFC. Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease. Cardiovasc. Res. 2008;79(2):249-258.

    [Crossref] [Google Scholar] [Pubmed]

87. Stanley WC, Recchia FA, Lopaschuk GD. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev. 2005;85(3):1093-1129.

    [Crossref] [Google Scholar] [Pubmed]

88. Gupte AA, Hamilton DJ, Cordero-Reyes AM, Youker KA, Yin Z, Estep JD. et al. Mechanical unloading promotes myocardial energy recovery in human heart failure. Circ Cardiovasc Genet. 2014;7(3):266-276.

    [Crossref] [Google Scholar] [Pubmed]

89. Ussher J R, Elmariah S, Gerszten RE, Dyck JR. The emerging role of metabolomics in the diagnosis and prognosis of cardiovascular disease. J Am Coll Cardiol. 2016;68(25):2850-2870.

    [Crossref] [Google Scholar] [Pubmed]

90. Mullarky E, Cantley LC, Nakao K, Minato N, Uemoto S. Diverting glycolysis to combat oxidative stress in: Innovative medicine: Basic research and development. Springer;2015.

    [Crossref] [Google Scholar] [Pubmed]

91. Frezza C, Zheng L, Tennant DA, Papkovsky DB, Hedley BA, Kalna G, et al. Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival. PLoS One. 2011;6(9):e24411.

    [Crossref] [Google Scholar] [Pubmed]

92.  Degens H, Brouwer KFJ, Gilde AJ, Lindhout M, Willemsen HM, Janssen BJ, et al. Cardiac fatty acid metabolism is preserved in the compensated hypertrophic rat heart. Basic Res Cardiol. 2006; 101(1):17-26.

    [Crossref] [Google Scholar] [Pubmed]

93. Doenst T, Pytel G, Schrepper A, Amorim P, Färber G, Shingu Y, et al. Decreased rates of substrate oxidation ex vivo predict the onset of heart failure and contractile dysfunction in rats with pressure overload. Cardiovasc Res. 2010; 86(3):461-470.

    [Crossref] [Google Scholar] [Pubmed]

94. Ranjbarvaziri S, Kooiker KB, Ellenberger M, Fajardo G, Zhao M, Roest ASV, et al. Altered cardiac energetics and mitochondrial dysfunction in hypertrophic cardiomyopathy. Circulation. 2021;144(21): 1714-1731.

    [Crossref] [Google Scholar] [Pubmed]

95. Maron BJ, Maron MS. Hypertrophic cardiomyopathy. Lancet. 2013;381(9862):242-255.

    [Crossref] [Google Scholar] [Pubmed]

96. Semsarian C, Ingles J, Maron MS, Maron BJ. New perspectives on the prevalence of hypertrophic cardiomyopathy. J Am Coll Cardiol. 2015;65(12):1249-1254.  

    [Crossref] [Google Scholar] [Pubmed]

97. Bain JR, Stevens RD, Wenner BR, Ilkayeva O, Muoio DM, Newgard CB. Metabolomics applied to diabetes research: Moving from information to knowledge. Diabetes. 2009;58(11):2429-2443.

    [Crossref] [Google Scholar] [Pubmed]

98. Wang ZY, Liu YY, Liu GH, Lu HB, Mao CY. L-Carnitine and heart disease. Life Sci. 2018; 194: 88-97.

    [Crossref] [Google Scholar] [Pubmed]

99. Mate A, Miguel-Carrasco JL, Vázquez CM. The therapeutic prospects of using L-carnitine to manage hypertension-related organ damage. Drug Discov. 2010;15(11-12):484-492.

    [Crossref] [Google Scholar] [Pubmed]

100. Hunter WG., Kelly JP, McGarrahill RW, Khouri, MG, Craig D, Haynes C, et al. Metabolomic Profiling Identifies Novel Circulating Biomarkers of Mitochondrial Dysfunction Differentially Elevated in Heart Failure With Preserved Versus Reduced Ejection Fraction: Evidence for Shared Metabolic Impairments in Clinical Heart Failure. J Am Heart Assoc. 2016;5(8), e003190.

     [Crossref] [Google Scholar] [Pubmed]

101. Bedi Jr KC, Snyder NW, Brandimarto J, Aziz M, Mesaros C, Worth AJ, et al. Evidence for intramyocardial disruption of lipid metabolism and increased myocardial ketone utilization in advanced human heart failure. Circulation. 2016;133(8):706-716.

     [Crossref] [Google Scholar] [Pubmed]

102. Lopaschuk GD, Ussher JR, Folmes CD, Jaswal JS, Stanley WC. Myocardial fatty acid metabolism in health and disease. Physiol Rev. 2010;90(1):207-258.

     [Crossref] [Google Scholar] [Pubmed]

103. Rosenblatt-Velin N, Montessuit C, Papageorgiou I, Terrand J, Lerch R. Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism. Cardiovasc Res. 2001;52(3):407-416.

     [Crossref] [Google Scholar] [Pubmed]

104. Sack MN, Rader TA, Park S, Bastin J, McCune SA, Kelly DP. Fatty acid oxidation enzyme gene expression is downregulated in the failing heart. Circulation. 1996;94(11):2837-2842.

     [Crossref] [Google Scholar] [Pubmed]

105.  Taegtmeyer H, Young ME, Lopaschuk GD, Abel ED, Brunengraber H, Darley-Usmar V, et al. Assessing cardiac metabolism: A scientific statement from the American Heart Association. Circ Res. 2016;118(10):1659-1701.

     [Crossref] [Google Scholar] [Pubmed]

106. Chen H, Wang Z, Qin M, Zhang B, Lin L, Ma Q, et al. Comprehensive Metabolomics Identified the Prominent Role of Glycerophospholipid Metabolism in Coronary Artery Disease Progression. Front Mol Biosci. 2021;8.

     [Crossref] [Google Scholar] [Pubmed]

107. Neubauer S. The failing heart: An engine out of fuel. N Engl J Med. 2007 ;356(11):1140-1151.

     [Crossref] [Google Scholar] [Pubmed]

108. Garcia D, Shaw RJ. AMPK:  Mechanisms of cellular energy sensing and restoration of metabolic balance. Mol Cell. 2017;66(6):789-800.

     [Crossref] [Google Scholar] [Pubmed]

109. Lin SC, Hardie DG, AMPK: Sensing glucose as well as cellular energy status. Cell Metab. 2018;27(2):299-313.

     [Crossref] [Google Scholar] [Pubmed]

110. Lipovka Y, Konhilas JP. AMP-activated protein kinase signalling in cancer and cardiac hypertrophy. Cardiovasc Pharm. 2015;4(3).

     [Crossref] [Google Scholar] [Pubmed]

111. Rochette L, Lorin J, Zeller M, Guilland JC, Lorgis L, Cottin Y, et al. Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: Possible therapeutic targets? Pharmacol Ther. 2013;140(3):239-257.

     [Crossref] [Google Scholar] [Pubmed]

112. Van Der Pol A, Gil A, Silljé HH, Tromp J, Ovchinnikova ES, Vreeswijk-Baudoin I, et al. Accumulation of 5-oxoproline in myocardial dysfunction and the protective effects of OPLAH. Sci Transl Med. 2017;9(415):8574.

     [Crossref] [Google Scholar] [Pubmed]