Roles of One-Carbon Metabolism Enzymes in Oxidative Stress and Epigenetic Regulation in Cardiovascular Diseases

One-carbon metabolism is not merely a matter of folate nutrition, but rather a metabolism-signaling integrated system jointly constituted by the folate cycle, the methionine cycle, and the transsulfuration pathway. On the one hand, this system sustains nucleic acid synthesis and methylation reactions by supplying purines, thymidylate, and methyl donors. On the other hand, it participates in redox homeostasis through homocysteine conversion, cysteine generation, glutathione regeneration, and hydrogen sulfide synthesis. Accordingly, in cardiovascular diseases, the functions of one-carbon metabolism-related enzymes are not limited to substrate conversion, but are deeply embedded throughout oxidative stress amplification, endothelial injury, vascular remodeling, myocardial hypertrophy, and epigenetic reprogramming.

 

Keywords: one-carbon metabolism; folate cycle; methionine cycle; transsulfuration pathway; oxidative stress; epigenetic regulation; homocysteine; S-adenosylmethionine; cardiovascular disease; Nrf2

 

I. Basic Structure and Functional Boundaries of the One-Carbon Metabolism Enzyme Network

1.1 One-carbon metabolism consists of three mutually coupled metabolic modules

(1) The folate cycle is responsible for loading and distributing one-carbon units

The folate cycle uses tetrahydrofolate derivatives as carriers and mediates transfer of one-carbon units across different oxidation states, directly serving purine synthesis, thymidylate synthesis, and methyl donor generation. Its key enzymes include dihydrofolate reductase, serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and the methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase (MTHFD) family.

(2) The methionine cycle determines methyl donor supply and methylation capacity

5-Methyltetrahydrofolate drives remethylation of homocysteine to methionine through methionine synthase, and methionine is subsequently converted to S-adenosylmethionine under the action of methionine adenosyltransferase. SAM is the core methyl donor for DNA, RNA, protein, and lipid methylation, whereas its metabolic byproduct SAH can in turn inhibit methyltransfer reactions. Therefore, the SAM/SAH balance directly determines methylation flux.

(3) The transsulfuration pathway links homocysteine clearance with antioxidant defense

When homocysteine does not enter the remethylation pathway, it can be diverted into the transsulfuration pathway through enzymes such as CBS and CSE, generating cystathionine, cysteine, glutathione precursors, and hydrogen sulfide. This branch simultaneously fulfills three functions: clearance of toxic intermediates, maintenance of reducing capacity, and production of gaseous signaling molecules.

 

1.2 The research focus on one-carbon metabolism enzymes lies in network coupling

(1) Key metabolite levels are controlled by coordinated action of multiple enzymes

The levels of key metabolites such as homocysteine, SAM, SAH, 5-methyltetrahydrofolate, cysteine, and glutathione are not determined by a single enzyme, but are jointly shaped by flux allocation among the folate cycle, the methionine cycle, and the transsulfuration pathway.

(2) Compartmentalization is a prerequisite for understanding functional differences

One-carbon metabolism exhibits clear compartmentalization in the cytosol, mitochondria, and nucleus. Mitochondrial folate metabolism not only contributes to formate export and nucleotide precursor support, but is also tightly linked to local redox status; meanwhile, the nuclear SAM/SAH environment directly affects chromatin methylation reactions.

 

1.3 The remethylation pathway includes both a folate-dependent route and a betaine-dependent route

(1) Methionine synthase constitutes the main folate-dependent remethylation axis

MTR uses 5-methyltetrahydrofolate as the methyl donor to reconvert homocysteine into methionine, representing the key interface coupling the folate cycle and the methionine cycle.

(2) Homocysteine methyltransferase constitutes the betaine-dependent bypass route

Homocysteine methyltransferase can use betaine as a methyl donor to reconvert homocysteine into methionine. This pathway has independent significance in maintaining methyl donor homeostasis, buffering homocysteine accumulation, and supporting SAM regeneration.

 

II. Coupling Mechanisms Between One-Carbon Metabolism Enzymes and Oxidative Stress

2.1 Homocysteine accumulation is the core node linking metabolic imbalance to vascular oxidative injury

(1) Hyperhomocysteinemia can induce endothelial dysfunction

Elevated homocysteine can reduce nitric oxide bioavailability and enhance inflammation, endoplasmic reticulum stress, and procoagulant conditions, thereby promoting endothelial dysfunction and vascular wall injury.

(2) Its damaging mechanisms are tightly coupled to oxidative stress

Homocysteine can promote reactive oxygen species generation, disturb protein thiol status, enhance endoplasmic reticulum stress, and induce protein homocysteinylation, ultimately resulting in disruption of redox homeostasis in endothelial cells, smooth muscle cells, and cardiomyocytes.

 

2.2 The transsulfuration pathway determines the antioxidant capacity of the one-carbon metabolism network

(1) CBS and CSE support glutathione synthesis through cysteine supply

The transsulfuration pathway converts homocysteine into cysteine, thereby providing substrates for glutathione biosynthesis. Accordingly, its functional state directly influences cellular antioxidant buffering capacity.

(2) CBS and CSE also participate in hydrogen sulfide generation

In the cardiovascular system, CSE is one of the major H2S-generating enzymes, and CBS can also participate in related sulfur-donating reactions. Because H2S has vasodilatory, antioxidant, anti-inflammatory, and mitochondrial-protective effects, the transsulfuration pathway is not merely a degradative branch, but also a source of cardioprotective signaling.

 

2.3 The folate cycle and redox homeostasis are not independent of one another

(1) Folate cycle imbalance can amplify oxidative stress

Abnormal activity of enzymes such as MTHFR, SHMT, and MTHFD can alter one-carbon unit supply, remethylation efficiency, and homocysteine clearance, thereby aggravating oxidative stress and endothelial injury.

(2) Oxidative stress can in turn disrupt the one-carbon metabolism enzyme network

Changes in redox status can affect the expression of key one-carbon metabolism enzymes, cofactor availability, and methyl donor homeostasis, thereby forming a positive-feedback structure of “metabolic imbalance-oxidative stress-further metabolic dysregulation.”

 

Table 1. Key One-Carbon Metabolism Enzymes and Their Functional Positioning in Oxidative Stress and Epigenetic Regulation

 

Enzyme Name	Module	Major Metabolic Function	Relationship to Oxidative Stress	Relationship to Epigenetic Regulation
SHMT1/SHMT2	Folate cycle	Convert serine to glycine and provide one-carbon units	Affect folate flux and coupling to reductive metabolism	Affect generation of nucleotide and methyl donor precursors
MTHFR	Folate cycle	Generate 5-methyltetrahydrofolate	Determines homocysteine clearance efficiency	Affects the methionine cycle and SAM supply
MTR/MTRR	Remethylation	Remethylate homocysteine to methionine	Affect homocysteine burden and redox status	Determine SAM regeneration capacity
Homocysteine methyltransferase	Remethylation bypass	Betaine-dependent remethylation of homocysteine	Reduces homocysteine accumulation and buffers oxidative stress	Maintains methionine and SAM supply
MAT	Methionine cycle	Generate SAM from methionine	Affects antioxidant capacity through the SAM-GSH axis	Directly determines methyl donor formation
AHCY	Methionine cycle	Hydrolyze SAH	Affects methyltransfer reaction flux	Determines the SAM/SAH ratio
CBS	Transsulfuration pathway	Diverts homocysteine into cystathionine generation	Promotes formation of cysteine, GSH, and H2S	Influences methylation by lowering homocysteine and regulating SAM utilization
CSE/CTH	Transsulfuration pathway	Cleave cystathionine to generate cysteine	Supports GSH synthesis and contributes to H2S supply	Indirectly affects redox-epigenetic coupling

 

III. Metabolic Basis of Epigenetic Regulation by One-Carbon Metabolism Enzymes

3.1 The SAM/SAH axis determines the upper limit of methylation capacity

(1) SAM is the direct donor for DNA and histone methylation

DNA methyltransferases and multiple histone methyltransferases all depend on SAM as the methyl donor. Therefore, insufficient SAM supply or restricted utilization can directly reduce methylation capacity.

(2) SAH accumulation inhibits methyltransfer reactions

After donating a methyl group, SAM is converted into SAH, which is an important endogenous inhibitor of methyltransferases. Accordingly, what determines epigenetic output is not merely absolute SAM level, but more critically the SAM/SAH ratio.

 

3.2 Alterations in one-carbon metabolism enzymes can induce epigenetic remodeling

(1) Abnormal folate cycling can affect DNA methylation patterns

Reduced efficiency of the folate cycle and remethylation can result in insufficient methyl donor supply, thereby causing aberrant DNA methylation and reorganization of gene expression programs. This process is highly relevant to gene regulation associated with vascular remodeling, inflammatory amplification, and myocardial hypertrophy.

(2) Oxidative stress and epigenetic changes amplify one another

Oxidative stress can reshape DNA methylation and histone modifications by altering one-carbon metabolic flux, SAM/SAH balance, and related enzyme expression; conversely, epigenetic remodeling can in turn alter expression of antioxidant enzymes, inflammatory mediators, and metabolic enzymes, thereby forming a metabolism-epigenetic positive-feedback loop.

 

3.3 Nuclear and mitochondrial one-carbon metabolism jointly participate in epigenetic regulation

(1) Nuclear one-carbon metabolism more directly supports methylation and nucleotide synthesis

Nuclear or perinuclear one-carbon unit supply can influence local dTMP synthesis and substrate availability for chromatin modification, thereby tightly coupling epigenetic regulation with replication and repair processes.

(2) Mitochondrial one-carbon metabolism indirectly affects epigenetics through energy and redox states

Mitochondrial folate metabolism not only supports formate export and nucleic acid precursor generation, but can also alter the nuclear epigenetic environment by affecting energy metabolism, ROS levels, and metabolic intermediate pools.

 

IV. Roles of One-Carbon Metabolism Enzymes in Major Cardiovascular Pathological Processes

4.1 Atherosclerosis and vascular endothelial injury

(1) Hyperhomocysteinemia promotes endothelial dysfunction

Elevated homocysteine can enhance oxidative stress, inflammation, and procoagulant conditions, thereby disrupting endothelial integrity and promoting atherogenesis.

(2) Epigenetic abnormalities participate in chronic vascular wall remodeling

DNA methylation, histone modifications, and non-coding RNA regulation all participate in phenotypic switching of endothelial cells and smooth muscle cells. One-carbon metabolism enzymes contribute to this process through regulation of methyl donor supply and redox status.

 

4.2 Myocardial hypertrophy and heart failure

(1) The folate-homocysteine axis is associated with myocardial remodeling

Imbalance in one-carbon metabolism can promote cardiomyocyte hypertrophic programs, fibroblast activation, and interstitial fibrosis through homocysteine accumulation, abnormal SAM/SAH balance, and enhanced oxidative stress.

(2) Oxidative stress and methylation remodeling jointly drive myocardial reprogramming

Under conditions of pressure overload, ischemia, and chronic metabolic disturbance, redox imbalance can cooperate with abnormal methylation to promote reprogramming of myocardial energy metabolism and expression of genes related to fibrosis and hypertrophy.

 

4.3 Hypertension, thrombotic tendency, and microvascular pathology

(1) MTHFR dysfunction and homocysteine abnormalities can alter vascular response thresholds

Abnormal MTHFR function and elevated homocysteine can affect vasodilatory capacity, endothelium-dependent responses, and the local proinflammatory environment, thereby contributing to hypertension and thrombotic risk.

(2) One-carbon metabolism imbalance can promote microcirculatory dysfunction

When oxidative stress is enhanced, H2S generation is insufficient, and epigenetic regulation is abnormal, vascular tone regulation, microvascular reactivity, and inflammatory sensitivity can all be shifted.

 

V. Key Pathways, Targets, and Evaluation Metrics in Research and Translation

5.1 Major research pathways

(1) Folate cycle-remethylation pathway

The focus is on how MTHFR, MTR, MTRR, SHMT, MTHFD, and homocysteine methyltransferase influence methyl donor formation and homocysteine clearance.

(2) Transsulfuration-glutathione-hydrogen sulfide pathway

The focus is on how CBS and CSE connect homocysteine diversion, cysteine generation, GSH regeneration, and H2S signaling.

(3) One-carbon metabolism-epigenetic pathway

The focus is on how changes in the SAM/SAH ratio affect DNA methylation, histone methylation, and cardiovascular transcriptional programs.

 

5.2 Key targets

(1) Metabolic node targets

These include MTHFR, MTR, homocysteine methyltransferase, MAT, AHCY, CBS, and CSE, and are used to analyze one-carbon flux allocation and coupling to reducing capacity.

(2) Redox and epigenetic interface targets

These include the GSH system, the H2S-generating axis, DNA methyltransferases, histone methyltransferases, and Nrf2-related nodes, and are used to analyze how metabolic changes are translated into gene regulatory changes.

 

5.3 Common evaluation metrics

(1) Metabolic-level indicators

① Homocysteine levels.

② SAM and SAH content and ratio.

③ Levels of folate and related one-carbon intermediates.

④ Readouts related to cysteine, glutathione, and H2S.

(2) Oxidative stress-level indicators

① ROS levels.

② GSH/GSSG ratio.

③ Lipid peroxidation products.

④ Endothelial oxidative injury and mitochondrial function readouts.

(3) Epigenetic-level indicators

① Genome-wide or site-specific DNA methylation.

② Histone methylation modification profiles.

③ Chromatin accessibility and transcription factor binding status.

④ Reprogramming of cardiovascular gene expression.

 

Table 2. Pathways, Targets, and Evaluation Metrics in Research on One-Carbon Metabolism Enzymes

 

Research Direction	Major Pathway	Key Targets	Common Evaluation Metrics
Folate cycle and remethylation	SHMT-MTHFR-MTR axis and the betaine-dependent remethylation bypass	SHMT1/2, MTHFR, MTR, MTRR, homocysteine methyltransferase	5-Methyltetrahydrofolate, Hcy, SAM/SAH
Transsulfuration and antioxidant coupling	CBS-CSE-GSH/H2S axis	CBS, CSE, GSH-related enzymes	Cys, GSH/GSSG, H2S, ROS
Epigenetic donor homeostasis	MAT-AHCY-SAM axis	MAT, AHCY, DNMT-related nodes	SAM, SAH, DNA methylation, histone methylation
Vascular endothelial injury	Hcy-oxidative stress-inflammation axis	MTHFR, CBS, CSE, Nrf2-related nodes	Endothelial function, ROS, inflammatory factors, methylation changes
Myocardial remodeling and heart failure	One-carbon metabolism-epigenetic remodeling axis	MTHFR, MAT, CBS, CSE	Hypertrophy markers, fibrosis indicators, oxidative stress and methylation readouts

 

VI. Commonly Used Products in Related Research

 

Name	CAS No.	Experimental Step	Key Use	Notes for Use
Homocysteine methyltransferase	9012-40-2	In vitro remethylation system	Used to construct homocysteine remethylation reaction systems and evaluate methyl transfer efficiency and methionine generation capacity	Suitable for use together with betaine, homocysteine, and methionine generation assays
Betaine	107-43-7	Remethylation bypass studies	Used as the methyl donor in betaine-dependent remethylation reactions to evaluate bypass compensation capacity	Suitable for use together with homocysteine methyltransferase or BHMT-related systems
L-Homocysteine	6027-13-0	Hyperhomocysteinemia model construction	Used to establish endothelial injury, oxidative stress, and proinflammatory response models	Commonly used in endothelial cell, smooth muscle cell, and cardiomyocyte systems; dose and exposure time should be optimized
Folic acid	59-30-3	Folate cycle intervention	Used to evaluate the effects of folate supplementation on homocysteine clearance, methyl donor homeostasis, and oxidative stress	Suitable for joint analysis with MTHFR, MTR, and methylation indicators
5-Methyltetrahydrofolate	31690-09-2	Remethylation validation	Used directly to evaluate folate-dependent remethylation capacity and methyl donor limitation effects	Better suited for mechanistic validation and should not be interpreted only through a nutritional supplementation framework
Calcium folinate	1492-18-8	Folate cycle compensation studies	Used to evaluate the effects of active folate donors on one-carbon flux and epigenetic readouts	Suitable for parallel comparison with folic acid and 5-MTHF to distinguish different supplementation forms
S-Adenosyl-L-methionine iodide	29908-03-0	Methyl donor supplementation	Used to simulate a methyl donor-sufficient state and analyze changes in DNA and histone methylation	Suitable for combined use with SAM/SAH ratio, methylation level, and transcriptional readouts
S-Adenosyl-L-homocysteine	979-92-0	Construction of methylation-inhibited conditions	Used to simulate suppression of methyltransfer reactions and evaluate the effects of SAM/SAH axis imbalance	Suitable for paired use with SAM to observe restricted methylation flux
L-Methionine	63-68-3	Methionine cycle intervention	Used to evaluate the effects of methyl donor precursor supplementation on SAM generation and methylation output	Suitable for combined use with MAT activity, SAM levels, and methylation readouts
L-Cysteine	52-90-4	Transsulfuration and GSH synthesis studies	Used as a glutathione precursor to evaluate downstream sulfur supply capacity of transsulfuration	Attention should be paid to auto-oxidation and culture system stability
Reduced glutathione (GSH)	70-18-8	Antioxidant buffering validation	Used to evaluate the buffering capacity of the transsulfuration pathway and antioxidant systems against oxidative injury in cardiovascular cells	Suitable for combined use with ROS, GSH/GSSG ratio, and lipid peroxidation indicators
Oxidized glutathione (GSSG)	27025-41-8	Redox state analysis	Used to construct or assess redox imbalance states	Commonly paired with GSH to assess overall cellular reducing capacity
N-Acetyl-L-cysteine (NAC)	616-91-1	Transsulfuration and antioxidant intervention	Used as a cysteine precursor and reducing intervention agent to evaluate the effects of GSH supplementation on pathological phenotypes	Suitable for validating reversibility under conditions of transsulfuration insufficiency or elevated oxidative stress
Morpholin-4-ium 4-methoxyphenyl morpholino phosphinodithioate (GYY4137)	1202757-89-8	Sustained-release H2S studies	Used as a slow-releasing H2S donor to simulate a sustained sulfur-supplying environment	Suitable for long-term treatment experiments and vascular homeostasis models
DL-Propargylglycine (PAG)	1078-19-9	CSE-related inhibition studies	Used to inhibit CSE activity and analyze H2S generation and downstream sulfur supply through transsulfuration	Suitable for combined use with H2S, GSH, and vascular reactivity indicators
5-Azacytidine	320-67-2	Epigenetic intervention	Used to intervene in DNA methylation status and observe transcriptional responses under altered one-carbon metabolism	Suitable for combined use with DNA methylation, inflammation, and vascular remodeling gene detection
Decitabine	2353-33-5	Epigenetic intervention	Used as a tool for DNA demethylation studies to analyze cardiovascular epigenetic remodeling	Better suited for actively replicating cell systems and should be used with careful control of cytotoxicity and dose window
BIX-01294	935693-62-2	Histone methylation intervention	Used to inhibit G9a-related H3K9 methylation and analyze coupling between one-carbon metabolism and histone modifications	Suitable for combined analysis with SAM donor status and chromatin readouts
Nicotinamide	98-92-0	Crosstalk between methylation and redox regulation	Used to study the relationship among NAD+ metabolism, methyl donor homeostasis, and oxidative stress buffering	Suitable for parallel analysis with Sirtuins, ROS, and methylation indicators
Reduced nicotinamide adenine dinucleotide phosphate (NADPH)	2646-71-1	Redox enzyme system analysis	Used to evaluate activity of glutathione- and thioredoxin-related reducing systems	Suitable for combined use with GSH, Trx, and oxidative stress enzyme activity readouts
S-Adenosyl-L-methionine p-toluenesulfonate	97540-22-2	Methyl donor supplementation	Used for enhancement of methyl donor availability and restoration of methylation reactions	Can be used as an alternative donor form to SAM iodide
5-Methylcytosine	554-01-8	DNA methylation detection	Used for DNA methylation quantification or methodological standardization	Suitable for use together with global or site-specific methylation methods

 

The oxidative stress and epigenetic regulation mediated by one-carbon metabolism enzymes are not two parallel processes, but rather a continuous metabolism-signaling coupling system formed through homocysteine clearance, SAM/SAH balance, glutathione generation, and H2S supply. In cardiovascular diseases, this system determines both the tolerance of vascular and myocardial cells to oxidative stress and the direction and intensity of epigenetic remodeling. Therefore, its functions should be understood within the unified framework of “folate cycle-methionine cycle-transsulfuration pathway-redox homeostasis-epigenetic output.”

 

For more related articles, please see below:

[1] Epigenetic Modifiers

[2] Methods for Establishing Animal Models of Cardiovascular Diseases