Amelioration of metabolic disorders in H9C2 cardiomyocytes induced by PM2.5 treated with vitamin C

Abstract Objective Particulate matter with an aerodynamic diameter ≤2.5 μm (PM2.5) is a public health risk. We investigate PM2.5 on metabolites in cardiomyocytes and the influence of vitamin C on PM2.5 toxicity. Materials and methods For 24 hours, H9C2 were exposed to various concentrations of PM2.5 (0, 100, 200, 400, 800 μg/ml), after which the levels of reactive oxygen species (ROS) and cell viability were measured using the cell counting kit-8 (CCK-8) and 2′,7′-dichlorofluoresceindiacetate (DCFH2-DA), respectively. H9C2 were treated with PM2.5 (200 μg/ml) in the presence or absence of vitamin C (40 μmol/L). mRNA levels of interleukin 6(IL-6), caspase-3, fatty acid-binding protein 3 (FABP3), and hemeoxygenase-1 (HO-1) were investigated by quantitative reverse-transcription polymerase chain reaction. Non-targeted metabolomics by LC-MS/MS was applied to evaluate the metabolic profile in the cell. Results Results revealed a concentration-dependent reduction in cell viability, death, ROS, and increased expression of caspase-3, FABP3, and IL-6. In total, 15 metabolites exhibited significant differential expression (FC > 2, p < 0.05) between the control and PM2.5 group. In the PM2.5 group, lysophosphatidylcholines (LysoPC,3/3) were upregulated, whereas amino acids (5/5), amino acid analogues (3/3), and other acids and derivatives (4/4) were downregulated. PM2.5 toxicity was lessened by vitamin C. It reduced PM2.5-induced elevation of LysoPC (16:0), LysoPC (16:1), and LysoPC (18:1). Discussion and conclusions PM2.5 induces metabolic disorders in H9C2 cardiomyocytes that can be ameliorated by treatment with vitamin C.


Introduction
PM 2.5 is one of China's most important air pollutants (Zhang andCao 2015, Geng et al. 2021).Anyang City, located in the most highly populated province (Henan) in the central region of China, has been affected by severe air pollution in recent years due to rapid industrial expansion (Chuai et al. 2019).Epidemiological evidence supports an association between air pollution exposure and health, and delicate particulate matter is reported to be a particular hazard to cardiovascular health (Anderson et al. 2012, Lim et al. 2012, Lu et al. 2018).In China, PM 2.5 increased the risk of cardiorespiratory diseases, such as ischemic heart disease (IHD) (Xing et al. 2016, Song et al. 2017), heart rhythm disturbances, and heart failure (HF) (Zhao et al. 2019b).HF is a heterogeneous clinical syndrome stemming from cardiac overload and injury (Snipelisky et al. 2019).It is the leading cause of mortality, morbidity, and poor quality of life (Tomasoni et al. 2019), affecting more than 23 million patients worldwide (Orso et al. 2017).Acute HF is one of the most common causes of hospital admission (Sinnenberg and Givertz 2020).
In the systemic circulatory system, the heart functions as the main pump.About 70% of the adenosine triphosphate (ATP) produced in the heart comes from the oxidation of fatty acids (FAs) (Li et al. 2018).In patients with HF, FA oxidation is increased (Sakai et al. 1995, Sharma et al. 2004), indicating a role in disrupting cardiac metabolism in this condition.Oxidative stress plays an essential role in the occurrence and development of cardiac metabolic disorders.ROS are produced at low levels and degraded to water by endogenous antioxidants.However, increased ROS production in pathological conditions leads to free radical-mediated reactions that influence lipids (Rubbo et al. 1994) and DNA (Richter et al. 1988) and trigger the cellular apoptotic pathway.Accumulating evidence has helped clarify the mechanisms underlying PM 2.5 toxicity (Feng et al. 2016).Oxidative stress is a critical molecular mechanism of PM 2.5 -mediated lung injury and embryonic toxicity (Xu et al. 2018a, Chen et al. 2019).PM 2.5 exposure leads to increased levels of ROS and circulating inflammatory cytokines, such as tumor necrosis factor-a (TNF-a) and IL-6.These are known to be upregulated by modifiable environmental factors (Delfino et al. 2008, Bo et al. 2016).
Due to the harmful impacts of PM 2.5 , researchers have been working to find solutions to safeguard people's health.Vitamin C (ascorbic acid) has attracted much attention as an antioxidant (Zhang et al. 2018).Vitamin C is a simple, watersoluble, low-molecular-weight carbohydrate (Lykkesfeldt et al. 2014).In reactions, vitamin C is a cofactor catalyzed by Cu þdependent monooxygenases and Fe 2þ -dependent dioxygenases.It is synthesized in vertebrates having this capacity from d-glucuronate (Linster and Van Schaftingen 2007).Vitamin C stimulates the production and activation of immune cells and is relatively safer due to its minimal side effects (van Gorkom et al. 2019).Moreover, vitamin C has therapeutic effects on multiple diseases, including cancer, sepsis, and other diseases, e.g., heart diseases (Fritz et al. 2014, Kuhn et al. 2018, van Gorkom et al. 2019).It is a widely used antioxidant in the clinic, functions as a free radical scavenger inside and outside the cell, and maintains the stability of the redox system ( € Ozkayaet al. 2011).Therefore, we speculated that antioxidants might have an antagonistic effect on the toxicity of PM 2.5 .
Most PM 2.5 -induced oxidative stress and inflammation studies have focused on lung tissues or airway epithelial cells as experimental materials (Zhao et al., 2019b).However, the effects of PM 2.5 exposure on cardiomyocyte metabolism remain to be clarified.LysoPC, as a metabolite, has been postulated to be an essential causal agent in inflammation (Janne et al. 2011).Alterations in the amino acid profile were found to be related to various disease states (Yoheiet al. 2011).However, as metabolites, few studies have been done on amino acid analogs.Finding particular biomarkers might be attainable with the advancement of metabolomics.In order to evaluate the effects of PM 2.5 exposure on metabolites in cardiomyocytes and the impact of vitamin C on PM 2.5 toxicity, we have employed a non-targeted metabolomics technique in the current work.

Materials and methods
PM 2.5 sampling and preparation PM 2.5 samples were collected from an iron and steel plant in Anyang city (China) in December 2017.The samples were collected onto fiber filters (Art.No.TX40HI20WW, Pall Life Sciences) using a micro-orifice uniform deposit impactor (Art.No.TH-150D, Tianhong Instrument Co. Ltd).The sampling flow rate was 100 L/min for 24 h.The filters were cut into squares (1-2 cm 2 ) and subjected to ultrasonication for 40 min.The PM 2.5 in the samples was removed by agitation in ultrapure water, then frozen, dried, and stored at À20 C.

Cell culture and treatment
The H9C2 embryonic rat heart-derived cell line was obtained from the Shanghai Institute of Life Sciences Cell Resource Center.The cell line was cultured in high glucose Dulbecco's modified Eagle's medium (Cat.No.A4192102, Thermo) supplemented with 10% fetal bovine serum (Cat.No.10100147, Thermo)and 1% antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin; Cat. No.11593027,11860038, respectively, Thermo).The cells were incubated at 37 C in a humidified atmosphere containing 5% CO 2 .The cells were divided into the PM 2.5 group, vitamin C (Cat.No.16468, MedChemExpress) group, PM 2.5 þvitamin C group, and control group.
Cells were exposed to different concentrations (0, 100, 200, 400, and 800 lg/ml) of PM 2.5 for 24 h to determine the optimal concentration for our study.In the PM 2.5 group, cells were cultured for 24 h in a medium containing PM 2.5 (final concentration 200 lg/ml).In the vitamin C group, Cells were cultured for 24 h in a medium containing vitamin C (final concentration 40 lmol/L).Because the physiological range of vitamin C is 40-100 lmol/L.At 40 lmol/L, it had an inhibitory effect on specific genes (Liu et al. 2019, Osipyants et al. 2018, Zhang et al. 2018).In our pre-experiment, the concentration of vitamin C at 40 lmol/L had the best effect on apoptosis induced by PM 2.5 .So I chose this concentration.In the PM 2.5 þvitamin C group, cells were cultured for 24 h in a medium containing PM 2.5 (final concentration 200 lg/ml) and vitamin C (final concentration 40 lmol/L).In the control group, cells were cultured for 24 h in a medium containing an equal volume of PBS used to substitute for the addition of PM 2.5 and vitamin C. All these doses were screened through experiments.

Cell viability determination
Cells were seeded in 96-well plates (Art.No.3599, Corning) at a density of 1 Â 10 4 cells/well.At 80% confluence, cells were exposed to a fresh medium containing different concentrations of PM 2.5 for 24 h.According to the manufacturer's instructions, cell viability was determined using a cell counting kit-8 (Cat.No.G021-1-2, Nanjing Jiancheng Bioengineering Institute).The absorbance of CCK-8 was determined using a microplate reader (Model.No.1681130A, Bio-Rad) at 450 nm.

ROS determination
The DCFH2-DA (Cat.No.4091990, MedChemExpress) fluorescent probe was used to detect the accumulation of intracellular ROS by fluorescence microscopy.After PM 2.5 treatment, cells were plated in 6-well plates and incubated with 10 mM DCFA-DA for 30 min at 37 C. Fluorescent images were captured by an inverted fluorescence microscope (TE2000U, Nikon) and analyzed using Image Pro (Version 5.0, Media Cybernetic).

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated using the Total RNA Kit (Cat.No.R6834-01, Omega).The total RNA (100 ng) was reverse transcribed using an M-MLV First Strand Kit (Cat. No.11752050, Invitrogen), and the cDNA thus obtained was used as a template for the subsequent reaction.The qRT-PCR was carried out using the ABI 7500 Fast PCR System (ABI, USA), and the reaction mixture consisted of 5 lL SYBR Green PCR Master Mix (Cat. No.4364344,Thermo), 2 lL cDNA, 2.6 lL nucleasefree water; and 0.4 lL primers (0.2 lL forward primer, 0.2 lL reverse primer).The sequences of the primers used were as shown in Supplementary Table S1.The housekeeping gene 18S was used as a normalization control.The thermal cycling program was as follows: 10 s denaturation at 95 C, followed by 40 cycles of 5 s denaturation at 95 C, 30 s annealing at 65 C, and 20 s extension at 72 C (Li et al. 2018).Relative gene expression was calculated using the 2 ÀDDCT method.

LC-MS/MS analysis
Cell samples were applied to the extraction procedure and extracted with methanol.Samples were ultrasonicated (40 kHz) for 30 min and then stored at À20 C for 1 h.After centrifugation for 15 min at 12 000 rpm and 4 C, 200 lL of supernatant was transferred to a vial for LC-MS analysis.
The samples were then analyzed using a mass spectrometer equipped with an electrospray ionization source at the resolution and in positive/negative ion mode.The sample scanning ranges were 50 to 1,000 m/z, with a scan time of 0.03 s and an inter-scan time of 0.02 s.

Statistical analysis
The data were extracted and preprocessed with the XCMS R package.Data were then normalized and edited into a twodimensional data matrix (retention time (RT), mass-to-charge ratio (MZ), observations (samples), and peak intensity) with Excel 2010 software.Multivariate analysis (MVA), "principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA)" was performed using SIMCA-P software (Umetrics AB, Umea, Sweden).Pathway analysis was performed using the Pathway Analysis features in MetaboAnalyst 3.0 (Impact > 0).
All other data were expressed as mean ± standard deviation (SD).The student's t-test was used to assess the significance of differences between data groups, and P < 0.05 indicated statistical significance.
PM 2.5 exposures increased the expression of genes involved in fatty acid transport, inflammation, and apoptosis H9C2 cardiomyocytes were treated with PM 2.5 (final concentration 200 lg/ml) in the presence or absence of vitamin C (final concentration 40 l mol/L).After treating H9C2 cardiomyocytes with PM 2.5 for 24 h, the mRNA expression of HO-1, caspase-3, FABP3, and IL-6 increased (FABP3, P < 0.01; others P < 0.05, Figure 2).This indicates that oxidative stress, cell apoptosis, fatty acid transport, and inflammation were increased.
PM 2.5 -induced changes in H9C2 cell metabolism LC-MS/MS analysis revealed 811 endogenous metabolites in H9C2 cardiomyocytes in the control and PM 2.5 groups.Among these, 15 metabolites exhibited significant differential expression (P < 0.05) (Figure 3(A) and Table 1).PCA (Figure 3(B)) and PLS-DA (Figure 3(C)) analyses revealed good separation between the data for the two groups, indicating marked differences in the metabolic characteristics between the control and PM 2.5 groups.In addition, these results showed good reproducibility of the data obtained in this experiment.The 15 differentially expressed metabolites detected in the PM 2.5 group included LysoPC, amino acids and analogs, and other acids and derivatives.
Compared to the control group, all three differentially expressed LysoPCs were significantly elevated in the PM 2.5 group (FC ¼ 3.7, 2.4, and 2.7, respectively; all P < 0.05).Further, five metabolites of the amino acids (b-alanine, L-proline, L-glutamic acid, L-phenylalanine, and L-tryptophan) and three differentially expressed analogs of the amino acid were identified (all downregulated) in the group treated with PM 2.5 (FC ¼ 0.43, 0.38, 0.27, 0.27, 0.35, 0.22, 0.33, and 0.41, respectively, all P < 0.05).In addition, when compared to the control group, all four differentially expressed metabolites classified as other acids (citric acid and pyruvic acid), and derivatives (4-pyridoxic acid and pyridoxamine) were significantly downregulated (FC ¼ 0.24, 0.38, 0.27, and 0.28, respectively, all P P < 0.05) in the group that received PM 2.5.
Pathway analysis in the group treated with PM 2.5 With an impact value threshold >0, the pathway analysis showed that the key metabolites were involved in six metabolic pathways (Table 2).The three top-ranking impacted canonical pathways were alanine, aspartate, and glutamate metabolism; cysteine and methionine metabolism; arginine and proline metabolism.

Effects of vitamin C on cell viability and ROS generation in H9C2 treated with PM 2.5
Compared with the Control group, PM 2.5 treatment significantly reduced H9C2 cell viability (P < 0.05).While vitamin C treatment alone did not affect H9C2 cell viability, co-treatment with vitamin C and PM 2.5 for 24 h significantly reduced the damage induced by PM 2.5 (P < 0.05) (Figure 4(A)).Nonetheless, analysis of ROS levels (Figure 4(B,C)) showed that ROS accumulation was significantly decreased in the group of PM 2.5 þvitamin C (P < 0.05).

Discussion
In RAW264.7 macrophage cells, PM 2.5 was reported to upregulate HO-1 (Xu et al. 2018b) via ROS accumulation (Deng      Total, the total number of compounds in the pathway.Hits, the number of compounds that match with our experimental data.Raw P, original p values calculated from the enrichment analysis.Impact, pathway impact value calculated from pathway topology analysis.The upregulation of HO-1, IL-6, and caspase-3 mRNA expression after 24 hours of exposure to PM 2.5 in H9C2 cardiomyocytes suggests that PM 2.5 exposure increases H9C2 cell damage due to increased oxidative stress, inflammation, and apoptosis. Studies revealed that PM 2.5 exacerbates lipid accumulation in macrophage foam cells (Liu et al. 2019a) and adversely affects lipid metabolism in ApoE knockout mice (Chen et al. 2013).Such as, PM 2.5 -induced total cholesterol (TC) and lowdensity lipoprotein (LDL) were increased significantly in ApoE knockout mice.(Qiang et al. 2014, Qiang et al. 2020).However, PM 2.5 on myocardial cell metabolites was not explored.Our study found that exposure of myocardial cells to PM 2.5 for 24 h upregulated mRNA expression of FABP3, which transports fatty acids (Glatz et al. 1994) to the mitochondria for b-oxidation (Furuhashi and Hotamisligil 2008).We detected significant upregulation of three LysoPCs in H9C2 cardiomyocytes exposed to PM 2.5 .LysoPC, a phospholipid metabolite, effectively induces oxidative stress (Treede et al. 2007) and increases LysoPC levels, indicating abnormal lipid metabolism and an imbalance in the oxidative environment (Long et al. 2015).In addition, LysoPC (16:0) has been shown to upregulate IL-6 expression in human umbilical vein endothelial cells (Kim et al. 2014) and peripheral blood mononuclear cells (Shi et al. 2007).We also found that an upregulated IL-6 expression in the H9C2 cardiomyocytes accompanied the PM 2.5 -induced increase in LysoPCs.Thus, it can be speculated that the PM 2.5 -induced increase in LysoPC may, in turn, cause increased IL-6 expression (Tseng et al. 2018).
Previous studies have shown that few amino acids are used preferentially in the heart as regulators of energy metabolism and not as substrates for direct energy production (Burns and Reddy 1978, Martin 1981, Aquilani et al. 2017).However, the reliance of cardiomyocytes on amino acids for the production of energy increases during HF (Neubauer et al. 1997).Thus, an inadequate supply of amino acids may exacerbate the cellular energy deficit by altering the mitochondrial tricarboxylic acid (TCA) cycle (Kalantar-Zadeh et al. 2004, Taegtmeyer et al. 2008).Research suggests a correlation between amino acid-associated left ventricular energy content, contractility force, and left ventricular function in heart disease patients.This indicates the parallelism between reduced amino acids and progressive heart dysfunction (Aquilani et al. 2017).Downregulated cysteine levels may decrease the anti-oxidative capacity of myocardial cells and increase oxidative stress (Boudina et al. 2009, Akhmedov et al. 2015).
Furthermore, the expression of glutathione synthetase, which catalyzes glutathione production from cysteine, is upregulated in HF (Schisler et al. 2015, Aquilani et al. 2017).Following these reports, we found that cysteine levels were significantly decreased, and oxidative stress was increased in H9C2 cardiomyocytes exposed to PM 2.5 for 24 h.The essential amino acid tryptophan and its metabolites are associated with inflammatory responses (Le Floc'h et al. 2011).Serum tryptophan levels were significantly decreased inPM 2.5 -treated mice (Zhao et al. 2019a), and tryptophan deficiency is associated with inflammation (Waclawikov a and El Aidy 2018, Zhang et al. 2018a, Zhang et al. 2018b).Under these observations, our findings suggest that downregulated amino acids contribute to the elevated oxidative stress and inflammation brought on by PM 2.5 exposure in H9C2 cardiomyocytes.
It has been shown previously that vitamin C protects against PM 2.5 -induced cell damage and ROS generation in human bronchial epithelial cells (Jin et al. 2016, Baccarelli et al. 2008).This study showed the detrimental effects of PM 2.5 exposure on H9C2 cell viability, and ROS levels were ameliorated by co-treatment with vitamin C. Furthermore, vitamin C protected lipid and amino acid metabolism in cells exposed to PM 2.5 .Vitamin C may protect H9C2 cardiomyocytes by lowering ROS levels or decreasing amino acid loss.However, the mechanism of the protective effects of vitamin C under these conditions remains to be elucidated.This is also the subject of our future in-depth studies.
In this study, a non-targeted metabolomics approach was made to investigate PM 2.5 on metabolites in cardiomyocytes.The results revealed that three LysoPCs in H9C2 cardiomyocytes were upregulated.However, the downregulation of citric acid, pyruvic acid, 4-pyridoxic acid, amino acids, amino acid analogs, and pyridoxamine suggests PM 2.5 causes metabolic problems in H9C2 cardiomyocytes.Correspondingly, the influence of vitamin C on PM 2.5 toxicity was explored.It was discovered that vitamin C relieved the reduction of L-phenylalanine, L-tryptophan, L-glutamic acid, and L-cystathionine and lowered the rise of three LysoPCs, indicating that vitamin C can treat metabolic abnormalities in cardiomyocytes brought on by PM 2.5 .
This work was supported by the Science and technology key project in the science and technology bureau of Anyang: A studyon the effect and mechanism of PM2.5 in Anyang city on rat myocardial cell H9C2 and a Study on the mechanism of FABP3 on myocardial cells under high glucose [grant number 2021C01SF038].The funders played no role in this study's design, conduct, or reporting.

Figure 3 .
Figure 3. Metabolites in H9C2 cardiomyocytes treated with PM 2.5 .(A) Heat map showing the most significantly abundant metabolites in H9C2 cardiomyocytes.Each sample (5 control samples shown in left, 5 PM 2.5 samples shown in right) is represented by a single column.Each column represents a different metabolite.Deep color indicates higher abundance, while light color indicates lower abundance.PCA (B) and PLS-DA (C) plots of the H9C2 metabolome.Control samples are shown in star and PM 2.5 samples are shown in triangular.
et al. 2013), induce inflammatory responses(Chenxu et al. 2018), and increase the expression of pro-inflammatory cytokines, such as IL-6 and TNF-a(Zhao et al. 2016).Besides, PM 2.5 has been shown to induce oxidative stress, inflammation, and apoptosis(Guan et al. 2019, Jin et al. 2019).This study showed that PM 2.5 decreased the viability of the H9C2 embryonic rat heart-derived cell line and increased ROS accumulation in a dose-dependent manner.

Figure 4 .
Figure 4. Effects of vitamin C on cell viability and ROS generation in H9C2 cardiomyocytes exposed to PM 2.5 .(A) Cell viability was evaluated with the CCK-8 assay.(B) ROS levels were assessed by fluorescence microscopic analysis of DCFH-DA staining intensity.(C) DCFH-DA staining of H9C2 cardiomyocytes.Data represent the means ± SD ( Ã p < 0.05 or ÃÃ p < 0.01 vs. Control, # p < 0.05 vs. PM 2.5 ).

Table 1 .
Significant differentially expressed metabolites in H9C2 cardiomyocytes exposed to PM 2.5 .
FC, fold change: CON, control; VIP value, variable importance in the projection; p Values, from Student's t-test and vs. PM 2.5 group;Metabolites, displaying significant changes between control and PM 2.5 group (P < 0.05 and VIP > 1).

Table 2 .
Pathway analysis of key metabolites.