Real Ambient Particulate Matter-Induced Myocardial Hypertrophy and Myocardial Lipotoxicity : Roles of PDGFRβ Methylation

Ying Zhang Qingdao University Medical College Xinyun Dun Qingdao University Medical College Benying Li Qingdao University Medical College Hongxu Bao Qingdao University Danchuan Li Sun Yat-Sen University Zijian Xu Qingdao University Medical College Angdong Ji Qingdao University Medical College Zhenzhen Jin Columbia University Jianxun Wang Qingdao University Medical College Rong Zhang Hebei Medical University Rui Chen Capital Medical University Wen Chen Sun Yat-Sen University Yuxin Zheng Qingdao University Medical College Lianhua Cui (  qdlhcui@qdu.edu.cn ) Qingdao University Medical College

(Solarbio, China). Both cells were placed in an incubator containing 95% air and 5% carbon dioxide at 37℃. Cells were seeded in 6-well plates (96-well for CCK8 assay) at the density of 1x106 cells/mL or 1x105 cells/mL. The cells grew adherently need 24 h. Then, cardiomyocytes were treated with PM2.5 samples at a concentration of (25,50,100,200 μg/mL) for 24h. Before exposure to cells, PM2.5 dissolved in DMSO (Solarbio, China) was subjected to ultrasonic treatment for 15min, while DMEM/F12 (1:1) containing the same volume of DMSO was used in the control group. In experiments treated with methylation inhibitors (5AZA), cardiomyocytes were pretreated with 5AZA for 24h before exposure to PM2.5.

Western blotting analysis
The protein expression levels of RAS, RAF, MEK, ERK, pMEK, pERK, PDGFRβ were detected by western blot to analyze impact of PM2.5, hPDGFRβ, 5aza on primary neonatal cardiomyocyte cells. A kit for protein extraction (Solarbio, China) was performed to extract the total protein extraction in myocardial cells and myocardial tissue, and the BCA protein assay (Yeasen, China) was used to measure the concentration of the total protein. The same amounts of lysate proteins (20 mg in primary neonatal cardiomyocyte cells and 50 mg in cardiac tissue) were loaded onto SDS-polyacrylamide gels (10% separation gels). The PVDF membrane then were blocked in phosphate buffer saline (PBS) contained 5% nonfat milk for 2 h, and the PVDF membrane was incubated with the primary antibody (ABclonal, China) at 4℃ overnight, washed with PBST three times for 10 min each time, and incubated with anti-rabbit and anti-mouse Ig G secondary antibody (ABclonal, China) at room temperature for 2 h. Then we washed the PVDF membrane three time (10 min each) with PBST. At last, the antibody-bound proteins were detected by the Omni-ECLTM chemiluminescence reagent (EpiZyme, China). GAPDH was used as loading controls for the total protein content. 7. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) RNA was extracted from the frozen heart tissue samples and harvested cells using TRIzolTM Reagent (EpiZyme, China) according to the manufacturer's protocol. The RNA concentration of each sample was detected by NanoDrop-2000 (Thermo, USA), and the RNA was converted to cDNA through the kit (TargetMol, USA). RT-qPCR reaction was conducted using QuantStudio 7 Flex (Thermo Fisher Scienti c, USA). SYBR Green from TargetMol, USA.The results were expressed as multiples of the increase or decrease of GAPDH. Primer sequences for all tested genes are listed in the table (table1). We conducted the assay in three biological repeats and three duplicated repeats.
After the mouse heart was removed, 4% paraformaldehyde was xed at room temperature for at least 24h. After dehydration and transparency, the hearts were embedded and sliced into slices of 4μm thickness. H&E, Masson's trichrome staining were performed using standard procedures. Tissue oil red 0 staining was performed by freezing sectioning into 10μm slices and following standard procedures as for cell staining (Beyotime, China). For the thickness of the free wall of the right ventricle see Jiang et al [28]. 9. Cell transfection for recombinant plasmid and adenovirus infection.
The cells were cultured into the 6-well plates at a dose of 4 × 105/ mL, high expression PDGFRβ(h PDGFRβ) recombinant enhanced green uorescent protein (EGFP) and empty plasmids (Shanghai Genechem Co.,Ltd ) into human primary cardiomyocytes, when the cell con uence was up to 70-80%. All the steps were conducted according to the instructions of Lipofectamine 2000 (Thermo Fisher Scienti c, USA), respectively. After 24 h, the transfection effect was veri ed by RT-PCR and Western blot. The overexpression vector is provided by the Shanghai Genechem Co.,Ltd . It was injected into the mice through a tail vein and tested 20 days later. 10. Reduced representation bisul te sequencing (RRBS) DNA samples were tested. After quali ed sample detection, a certain proportion of negative control (lambda DNA) was added. First, DNA samples were digested by methylation-insensitive restriction enzyme MspI. The DNA after the enzyme digestion The end of the fragment was repaired, A tail was added, and all cytosine was methylated. DNA fragments with a length of 40-220bp were selected for glue cutting (Meissner,2008). Bisul te was followed after processing (EZ DNA Methylation Gold Kit, Zymo Research), the unmethylated C changed to U (changed to T after PCR ampli cation), while the methylated C remained unchanged, and then PCR ampli cation was performed to obtain the nal DNA library. After the completion of library construction, Qubit2.0 was used for preliminary quantitation, the library was diluted to 1ng/µl, and then Agilent 2100 was used to detect the length of inserted fragments in the library. After meeting the expectation, q-pcr method was used to accurately quantify the effective concentration of the library (effective concentration of the library >2nM) to ensure library quality. After quali ed database inspection, Illumina HiSeq/MiSeq sequencing was performed after pooling of different libraries according to the effective concentration and target depooling data volume requirements.

11.Lipidomics analysis
In this study, liquid mass spectrometry (LC-MS) [29,30] technology was used to conduct lipidomics research. There were 5 samples in each group. The experimental process mainly included sample collection, lipid extraction, LC-MS/MS detection and data analysis, etc.

12.Echocardiography
At a speci ed point in time, rst using hair removal creams to remove chest hair, in mice and by intraperitoneal injection of 80 mg/ml of sodium pentobarbital anesthesia in mice, by tape will be xed on the xed in mice, and the sensor probe was carefully placed on the left side chest between the fourth and sixth frame Then capture m type in papillary muscle level image. Each image loop included 10 to 20cardiac cycles. Data were averaged from at least three cycles per loop. The interventricular septum (IVS), interventricular septal thickness at systolic (IVSs), interventricular septal thickness at diastole (IVSd), left ventricular posterior wall s (LVPW), left ventricular posterior wall of systolic (LVPWs), left ventricular posterior wall of diastolic (LVPWd) were directly measured, while other parameters, such as left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), ejection fraction (EF), stroke Volume (SV), heart rate (HR), were derived automatically by the Vevo 2,100 imaging system (Visual Sonics,Toronto, ON, Canada).

Statistical analysis
The data were analyzed and graphed using GraphPad Prism version 8.0, and all summary data are presented as the mean ± SEM from 2 to 3 independent experiments. The data were analyzed and graphed using GraphPad Prism version 8.0, and all summary data are presented as the mean±SEM from 2 to 3 independent experiments. The sample size required for each experiment is based on the previous research and experimental data. The mice were randomly assigned to different treatment groups. To avoid observation bias, the operator was blindfolded during the selection of mice and samples for echocardiography, histology, simpli ed methylation sequencing, and lipidomics analysis. The Shapiro-Wilk normality test was used for data distribution normality. The data were normally distributed using unpaired, 2-tailed Student t test (for comparing 2 groups) or 1-way ANOVA with Bonferroni post hoc test (for multi-group comparisons). For data that did not conform to normal distribution, 2-tailed Mann-Whitney U test (for comparing two groups) or Kruskal-Wallis test (for multi-group comparisons) was used. There are no cross-test corrections for multiple tests, only in-test corrections. Representative images were selected according to the values closest to the mean of each group. There is a P value in the Fig, and

Results
1. PM induced epigenetic alteration and DNA methylation pro ling identi ed the key gene-PDGFRβ in heart tissue of C57BL/6J mice.
In order to explore the epigenetic changes that occur in mouse hearts after a real-ambient PM exposure via an individual ventilated cage (IVC)-based system, we performed reduced representation bisul te sequencing (RRBS). As can be seen from the distribution map of the number of methylation levels in functional regions, the genes with hypermethylation or hypomethylation were mainly concentrated in intron regions, while the difference between high and low methylation was the largest in repeat region (Fig. 1a). According to the length distribution map of differential methylation region (DMR), the length of DMR is between 0 and 400bp (Fig. 1b). Although the difference in discrete values between the exposed group and the control group was not signi cant, the distribution of DMRS methylation level in the exposed group was relatively concentrated (Fig. 1c). The heat map analysis of the methylation level in the functional region between the samples showed that there was signi cant difference methylation between the exposed group and the control group. Among them, there were 3808 (57.66%) hypermethylated genes and 2796 (42.34%) hypomethylated genes (Fig. 1d). GO and KEGG analysis revealed the GO terms and important KEGG pathways which PM may trigger. According to GO analysis results, in the three aspects of biological process, cell component and molecular function, differentially methylated genes mainly concentrated in the growth and development process of biological process, the development process of anatomical structure, single-multicellular biological process and other categories. In KEGG result analysis, differentially methylated genes mainly regulated the PI3K-Akt signaling pathway, RAS signaling pathway, MAPK signaling pathway and Rap1 signaling pathway. (Fig.   1e and Fig. 1f). Interestingly, the differential methylated gene PDGFRβ in this study was enriched in the MAPK pathway and was associated with multiple pathways. We found that it is closely associated with the downstream MEK/ERK pathway. Overall, we found that PM induced epigenetic changes and RRBS revealed a hypermethylated gene PDGFRβ. Given the established roles of PDGFRβ signaling, we investigated the effects of PDGFRβ and its downstream related pathways in our subsequent experiments.
Exposing mice to 6 weeks of real-ambient PM via the IVC-based system resulted in a signi cantly thickened right ventricular wall in mice ( Fig. 2a, b, c). To verify the methylation status of PDGFRβ, in the following RT-qPCR analysis, compared with the control group, the mRNA expression level of PDGFRβ in the PM exposure group showed a signi cantly decreased trend, which was also consistent with the results of the RRBS analysis (Fig. 2d). Western blot analysis also showed that the protein levels of RAS, RAF, MEK and ERK, which are downstream of PDGFRβ via the MEK/ERK pathway, showed a downward turn in the PM exposure group compared to control (Fig. 2e, f, g, l). Hence, our results indicate that PM directly leads to cardiac hypertrophy and hypermethylation of PDGFRβ in mice, which leads to downregulation of the expression levels of related genes in its downstream MEK/ERK pathway.
MEK/ERK pathway is closely related to cardiac hypertrophy. In order to further understand the mechanism of MEK/ERK pathway induced cardiac hypertrophy after PDGFRβ hypermethylation, primary neonatal rat cardiomyocytes were exposed to different concentrations of PM. CCK8 assay showed a stress-induced increase in cell viability at 25 μg/mL, and then showed a dose-dependent decrease with the increase of PM concentration (Fig. 3a). Finally, we selected three different PM concentrations for further study. The results of RT-qPCR showed that the mRNA expression levels of PDGFRβ and downstream RAS, RAF, MEK and ERK all decreased with increased PM concentrations (Fig. 3b, c, d, e, f). Importantly, these results were consistent with western blot analysis of protein expression (Fig. 3g, h, j, k, l). Based upon these data, we chose 50μg/ mL PM as our experimental concentration for future experiments. However, we also know that PM exposure time has a signi cant effect on experimental results. To assess this, we established PM exposure models at different time points in primary neonatal rat cardiomyocytes to detect the expression of myocardial hypertrophy markers. RT-qPCR results showed that mRNA levels of cardiac hypertrophy related markers in cardiomyocytes showed an upward trend during the rst 0-6 hours of PM exposure. This was followed by a decrease in mRNA expression during the 6-24 hours timepoints (Fig. 3m, n, o). Based upon these results, we conducted the study after 6 hours of PM exposure. We found that when PM concentration was 50μg/ mL and exposure time was 6 hours, the mRNA expression levels of the three DNA methyltransferases followed an upward trend (Fig. 3p, q, r). Thence, we conclude that exposing cardiomyocytes to PM at 50 μg/mL for 6 hours causes changes in DNA methylation levels in cardiomyocytes and may affect cardiac hypertrophy.

Effects of up-regulation of PDGFRβ on MEK/ERK pathway and cardiac hypertrophy in cardiomyocytes.
To investigate the effect of PDGFRβ on MEK/ERK pathway and cardiac hypertrophy. We used a recombinant enhanced green uorescent protein (EGFP) plasmid with high expression of PDGFRβ (hPDGFRβ) and an empty plasmid (PDGFRβ-con) for transient gene transfection in cardiomyocytes after PM exposure. Fluorescence images demonstrate that the transfection was successful (Fig. 4a). Then we performed RT-qPCR and WB to verify that overexpression was achieved. The mRNA level of hPDGFRβ was 28 times higher than that of blank control and PDGFRβ-con (Fig. 4b). The expression level of the protein was also consistent with that of RT-qPCR (Fig. 4c). We found that RT-qPCR results showed that mRNA expression levels of RAS, RAF, MEK and ERK were signi cantly decreased in the PM exposure group compared with the control group, which was consistent with the results of real PM exposure in our previous animal experiments. The mRNA expression levels of RAS, RAF, MEK and ERK in the hPDGFRβ group showed an upward trend (Fig. 4d, e, f, g), and the protein expression levels of MEK and ERK in the WB experiment were also consistent with the results of RT-qPCR ( Fig. 4h, I, j). We further veri ed the effect of hPDGFRβ on cardiac hypertrophy markers by RT-qPCR. We found that the mRNA expression levels of atrial natriuretic factors (ANF), brain natriuretic factor (BNF) and beta-myosin heavy chain (β-MHC) were signi cantly increased in the PM exposure group, which was consistent with the results of our previous experiment. The mRNA expression levels of ANF, BNF and β-MHC in hPDGFRβ group showed a decreasing trend (Fig. 4k, l, m). These results suggest that high expression of PDGFRβ facilitates increased activation of the MEK/ERK pathway, thereby preventing the increase in cardiac hypertrophy markers, suggesting a net effect ofreducing cardiac hypertrophy.

Effect of inhibiting methylation on MEK/ERK pathway and cardiac hypertrophy in cardiomyocytes.
In order to further explore the effects of PDGFRβ methylation on MEK, ERK pathways and cardiac hypertrophy, we established a methylation inhibition model in primary neonatal rat cardiomyocytes. Firstly, CCK8 experiments were conducted to understand the effects of different concentrations of methylation inhibitors (5-Azacytidine: 5AZA) on cell viability (Fig. 5a). According to RT-qPCR results, when the concentration of 5AZA was 10μM, the mRNA expression level of DNA methyltransferase 1(DNMT1) and DNA methyltransferase 3A (DNMT3A) could be decreased (Fig 5b, c), so we chose 10μM as our experimental concentration. We found that the mRNA expression levels of MEK, ERK, RAS and RAF in the methylation inhibitor group showed an upward trend compared with the PM exposure group (Fig. 5d, e, f, g). In the western blot results, the protein expression levels of MEK and ERK were also consistent with the RT-qPCR results (Fig. 5h, I, j). In order to further study the effect of methylation inhibitors on cardiac hypertrophy, we used RT-qPCR to observe the expression levels of markers related to cardiac hypertrophy.
We found that compared with PM exposure group, the mRNA expression levels of ANF, BNF and β-MHC in the methylation inhibitor group showed a decreasing trend (Fig. 5k, l, m). These results indicated that the methylation inhibitor group activated the MEK/ERK pathway, reduced the effect of cardiac hypertrophy, and had a protective effect on cardiomyocytes.
5. Effects of up-regulation of PDGFRβ on MEK/ERK pathway and cardiac hypertrophy in heart tissue of C57BL/6J mice.
Since the activation of cardiac-speci c transgenic PDGFRβ has a signi cant effect, we further investigated the regulatory mechanism of high expression of PDGFRβ on cardiotoxicity induced by PM exposure in mice and the potential of PDGFRβ in improving cardiac hypertrophy outcomes. Our adenovirus helper-free system is composed of three plasmids, namely a viral vector, PAAV-RC vector and pHelper vector. We generated AAV9: Ubi by using myocardial cell-speci c ubiquitination protein promoter and triple FIAG marker.3Flag-PDGFR β (hPDGFRβ), as a control, we generated AAV9: Ubi-EGFP (hPDGFRβ-Con), and replaced 3Flag-PDGFR β with EGFP (Fig. 6a). AAV9 was rst injected into mice by tail vein, and PDGFRβ expression was highly speci c in the heart after 20 days. The high expression of PDGFRβ was clearly observed under the light microscope by immunohistochemical experiments, and the green uorescence of AAV9 in the heart tissue was directly observed by frozen sections, which was consistent with the immunohistochemical results ( Fig. 6c). In addition, we also examined the expression level of PDGFRβ protein by western blot assay, and we found that the protein expression level of PDGFRβ group was higher than that of hPDGFRβ-con group (Fig. 6d), so 20 days later, the PDGFRβ high expression model was established. Then we performed tracheal drip, once a week, where the PM of tracheal drip came from IVC-based real-ambient PM exposure system, and the total concentration of tracheal drip was consistent with the total concentration in real-ambient PM exposure system (Fig. 6b). Then we conducted echocardiographic examination of the mice, and it was found that interventricular septum (IVS), interventricular septal thickness at systolic (IVSs), interventricular septal thickness at diastole (IVSd), left ventricular posterior wall (LVPW), left ventricular posterior wall of systolic (LVPWs), left ventricular posterior wall of diastolic (LVPWd) in the PM exposed group showed an increasing trend compared with the control group, indicating that myocardial hypertrophy appeared in the exposed group. Compared with PM exposure group, hPDGFRβ group showed a decreasing trend in all indicators (Fig. 6e, f, g, h, i, j), indicating that hPDGFRβ can reduce cardiac hypertrophy and thus protect the heart. To follow up these data, we further studied this mechanism. We used western blot experiments to study the related genes in the downstream pathway of PDGFRβ. We observed that RAS, RAF, MEK, pMEK, ERK and pERK in the downstream pathway all showed the negative same trend. The protein expression level showed an upward trend (Fig. 6k, l, m, o, p, q), which was consistent with the results of our previous cell experiments.
In conclusion, hPDGFRβ activates downstream MEK and ERK pathways, ameliorates the effects of cardiac hypertrophy and protects the mouse heart.
6. PM -induced myocardial hypertrophy and lipid metabolism disorders.
These data have demonstrated that PM exposure can lead to cardiac hypertrophy in mice. However, whilst is well known that cardiac hypertrophy is closely related to lipid metabolism, we next asked how is PDGFRβ related to lipid metabolism? To further explore the relationship between the two, we rst used HE staining to observe the relationship between the PM exposure group and the control group in AC16 cells (Fig. 7a). We found that compared with the control group, the cardiomyocytes in the exposed group showed a decreased nucleo-plasmic ratio (Fig. 7b) and an increased area ratio (Fig. 7c), combined with our previous experimental results in primary neonatal rat cardiomyocytes, indicating that PM exposure did cause myocardial hypertrophy in cardiomyocytes. Next, oil red O staining was used to stain AC16, and we observed that the PM exposure group had signi cant lipid accumulation (Fig. 7d). Subsequently, oil red O staining was used to observe the heart tissues of the tracheal drip group and the control group, and it was found that the tracheal drip group also had a small amount of lipid accumulation (Fig. 7e).
In summary, PM exposure can lead to lipid metabolism disorder in the mouse heart. Since PPARα and PPARγ are closely related to lipid metabolism, we rst used western blot analysis to investigate changes in PPARα and PPARγ protein expression in the heart tissue of real-ambient PM exposure system. We found that the protein expression levels of PPARα and PPARγ in the PM exposure group were increased compared with the control group ( Fig. 7f, g, h). Further, PM exposure is closely related to cardiac lipid metabolism. And how does this relate to PDGFRβ? As a result, we established a model of PDGFRβ overexpression in AC16 cells. Using western blot experiments, we found that PPARα protein expression was increased in the PM-exposed group compared with the control group, consistent with previous results, but PPARα protein expression was lower in the hPDGFRβ group than in the exposed group (Fig. 7i,  j). Similarly, we also obtained consistent results in the high expression model of PDGFRβ after tracheal drip, with PPARα protein expression increased in the PM exposure group compared with the control group, but decreased in the hPDGFRβ group compared with the exposure group (Fig. 7k, l). In conclusion, hPDGFRβ protects the mouse heart by alleviating PM-induced lipid metabolism disturbances in cardiomyocytes and the mouse heart.
In order to further study the PM induced changes in lipid metabolism, we performed lipidomics analysis  (Fig. 8b). We found that the PC subclass was highest in both positive and negative ion modes. Further analysis of the different lipid compounds revealed that a total of 1226 differential lipid compounds were detected in positive ion mode, among which 34 which were signi cantly different, 30 which were signi cantly up-regulated and 4 which were signi cantly downregulated. In the negative ion mode, a total of 582 different lipid compounds were detected, among which 38 were signi cantly different, 29 were signi cantly up-regulated and 9 were signi cantly down-regulated.
Then, we observed the overall distribution of different lipid compounds from the volcano diagram. It could be seen intuitively that no matter in the positive ion mode or the negative ion mode, more lipid compounds were up-regulated than down-regulated, and the expression multiple of lipid compounds in different groups changed signi cantly (Fig. 8c, d). So, we then looked at the fold change (FC) analysis of differential lipid compounds and found that in the positive ion mode, Creatine had the largest differential multiple, followed by canrenone. In the negative ion mode, carnosine had the largest differential multiple, followed by L-Histidine (Fig. 8e, f). In order to compare the differences of metabolic expression patterns between two groups and within the same comparison group, hierarchical cluster analysis was performed on the obtained metabolites. We found signi cant differences between the control group and the exposed group (Fig. 8g, h). In order to check the consistency of lipid compounds and lipid compounds, Pearson correlation coe cient between all lipid compounds was calculated to analyze the correlation between each lipid compound. We found that as the linear relationship between the two lipid compounds increased, the positive correlation tended to 1, and the negative correlation tended to -1 (Fig. 8i, j). In general, these data suggest that PM exposure can lead to changes in cardiac lipid metabolism in mice.

Discussion
One of the most important ndings of this study is that we demonstrate for the rst time that PM-induced PDGFRβ methylation induces cardiac hypertrophy and that this PM-induced PDGFRβ methylation is also closely associated with myocardial lipid toxicity. Interestingly, high expression of PDGFRβ mitigated this hypertrophy and myocardial lipid toxicity. This activity was demonstrated in PDGFRβ -overexpressed mouse heart and neonatal mouse primary cardiomyocytes, as well as in human cardiomyocytes AC16.The effects of PDGFRβ methylation on myocardial hypertrophy and myocardial lipid toxicity de ne the important role of PDGFRβ in maintaining cardiac stability.
Gene methylation is a common form of modi cation in eukaryotic cells and a major epigenetic form of gene expression regulation in mammals, which may provide a clearer understanding of the molecular mechanism of cardiac hypertrophy by PM [1-3, 7, 10, 31]. Myocardial hypertrophy affects the structure and function of the heart in a variety of ways, resulting in myocardial lipotoxicity [17,32]. However, the relationship between PM-induced hypertrophy and myocardial lipotoxicity has not been reported. Given its unique role in these processes, PM-induced gene methylation is an important target for studying myocardial hypertrophy and myocardial lipotoxicity.
The PM component is very complex. PM exposure not only increases the risk of cardiovascular disease, particles with small particle size (such as PM 2.5 and PM 0.1 ) can directly enter alveolar deposition and enter the body through blood circulation, causing serious damage to human organs. Studies have shown that a thickening of the right ventricular wall, which is typical after lung injury [33]. Since the right ventricle is usually associated with cardiopulmonary effects, the changes we observed suggest that PM exposure may be related to the pulmonary system and the right ventricle. It's worth noting that, our previous study has analyzed the composition and exposure concentration of PM [11], but it is not yet possible to detect all components of PM in blood. In addition, we also performed ICP mass spectrometry analysis of metal content in mouse hearts, and found deposition of Na, K, Se and Fe in PM exposure group [12]. Deposition of some metals is known to alter DNA methylation patterns in genes, leading to the development of a range of diseases [34,35].
Therefore, RRBS was performed on C57BL/6J mice in the individual ventilated cage (IVC)-based realambient PM exposure system. In our model of real-ambient PM exposure system, reduced representation bisul te sequencing (RRBS) identi ed one of the key genes, PDGFRβ, that is involved in the regulation of multiple pathways, especially the MEK/ERK pathway in the MAPK pathway. Activation of PDGFRβ, a platelet-derived growth factor receptor, regulates myocardial infarction healing and angiogenesis.
PDGFRβ is also associated with myocardial cell proliferation and myocardial regeneration [36,37]. The MEK/ERK pathway is one of the most studied signaling pathways because it controls many important cellular mechanisms [38]. The MEK/ERK pathway may occupy a central regulatory position in the signaling layer of cardiomyocytes because of its unique ability to respond to almost all of the characteristic hypertrophy agonists and stress stimuli examined to date, based on its ability to promote cardiomyocyte growth in vitro [39]. However, the role of MEK/ERK in regulating cardiac hypertrophy is currently an area of ongoing debate [38]. Studies have shown that ERKs is one of the necessary conditions for inducing cardiac hypertrophy [40,41]. Other studies have shown that activation of ERKs is associated with the prevention of cardiomyocyte hypertrophy [42]. In the real-ambient PM exposure system, we found that the mRNA and protein expression levels of MEK and ERK in the downstream PDGFRβ pathway decreased in the PM exposure group, suggesting that PM-induced PDGFRβ methylation leads to the down-regulation of PDGFRβ expression and inhibits the expression of genes related to the downstream pathway, leading to myocardial hypertrophy.
Most studies on PM toxicity have been validated in the cardiovascular system and endothelial cells. However, studies on PM-induced cardiomyocyte related toxicity are limited [43]. It is well known that different concentrations of PM have a bipolar effect on cell growth. Low concentrations of PM can promote cell proliferation, while high concentrations can inhibit cell proliferation [44,45]. Although numerous studies have shown that the cytotoxicity of PM depends on its concentration [46], the effects of PM exposure time on cells are different [47,48]. Some studies have shown that long-term exposure increases the risk of cardiovascular death more than short-term exposure [49]. Other studies have found that short-term PM exposure leads to intense vascular remodeling and exacerbates the transition from left ventricular failure to right ventricular hypertrophy [50,51]. So, in order to meet the relevant requirements of myocardial hypertrophy and methylation, we nally selected concentration of PM exposure was 50 μg/mL and PM exposure time of 6 hours in primary neonatal rat cardiomyocytes. We know that DNA methyltransferase catalyzes DNA methylation [52,53]. At the same time, the above experimental conditions also satis ed the expression of three DNA methyltransferases.
Our study is the rst to show that real-ambient PM exposure leads to changes in PDGFRβ methylation levels. The role of PDGFRβ signaling in angiogenesis and early hematopoiesis has also been established.
PDGFRβ is involved in a variety of well-de ned signaling pathways, such as MRK/ERK-MAPK, PI3K, and PLC-γ, and is involved in a variety of cellular and developmental responses [26]. However, the mechanism described here of PDGFRβ methylation induced cardiotoxicity by PM exposure is novel. To further investigate this, we overexpressed PDGFRβ in primary cardiomyocytes and utilized methylation inhibition.
We found that the expression of MEK, ERK was signi cantly increased in the hPDGFRβ group, and the expression of myocardial hypertrophic markers was signi cantly decreased in the hPDGFRβ group, suggesting that MEK/ERK pathway was activated in the hPDGFRβ group and the effect of myocardial hypertrophy was inhibited. At this point, it is not clear that PDGFRβ methylation affects the downstream pathway, so the methylation inhibitor 5-Azacytidine(5AZA) was used to inhibit the methylation of cells. 5-Azacytidine is a kind of deoxycytidine analogue, usually used for demethylation through promoters [54,55]. We found that the methylation inhibitor group also signi cantly increased the expression levels of MEK and ERK, while signi cantly reduced the expression levels of markers of cardiac hypertrophy. These results indicated that methylation inhibitor group activated MEK/ERK pathway and inhibited myocardial hypertrophy effect, which was consistent with PDGFRβ high expression group. In summary, PM exposure leads to methylation of PDGFRβ in primary cardiomyocytes, resulting in decreased gene expression in the downstream MEK/ERK pathway and ultimately cardiac hypertrophy. However, high expression of PDGFRβ and methylation inhibition improved myocardial hypertrophy, thereby protecting myocardial cells. This is also the rst time that we found the protective effect of high expression of PDGFRβ on the heart under PM exposure.
Adeno-associated virus (AAV) is known to be an unenveloped virus that can be engineered to deliver DNA to target cells. Recombinant AAV particles of DNA sequences for a variety of therapeutic applications have proven to be one of the safest strategies for gene therapy to date [56]. In laboratory studies, PM of tracheal drip are often seen to negatively affect cardiovascular activities such as heart function, blood pressure and cardiomyopathy [57]. Similarly, in the mouse heart, the continuous activation of PDGFR-β signal mediated by AAV9 vector can also improve myocardial hypertrophy caused by tracheal drip, providing gene therapy strategies for reducing myocardial hypertrophy.
Related studies have shown that cardiac hypertrophy is closely related to myocardial metabolism, and lipid overload can cause cardiac hypertrophy and cardiac dysfunction [58-60]. PM has been found to cause cardiac hypertrophy [31], however, no studies related to cardiac lipid metabolism caused by PM have been reported so far. Upfront we found PM group exposed mice displayed right ventricular free wall thickening [10,11]. In cardiomyocytes, we found a decreased nucleo-plasmic ratio in PM exposed cardiomyocytes [61], area ratio increases [62,63]. All three markers of cardiac hypertrophy were elevated in the PM exposure group and signi cantly decreased in the PDGFRβ group. Importantly, we also found lipid deposition in both the mouse heart and myocardial cells in the PM exposed group.  Compliance with ethical standards Con ict of interest.
The authors declare that they have no con ict of interest. Tables   Table 1. PCR primer design  Reduced representation bisul te sequencing was performed on C57BL/6J mice. Mice were exposed to        hPDGFRβ ameliorates the effects of PM exposure on myocardial hypertrophy and myocardial lipid metabolism disorders in AC16 cells. a AC16 cells were stained with hematoxylin and eosin in the control and PM exposed groups. (Magni cation, 40x); b Nucleo-plasmic ratio was expressed in AC16 cells in the control and PM exposed groups; c the area ratio of AC16 cells in the control group and PM exposed group was relative; d Oil red O staining of AC16 cells in control group and PM exposed group. The lower panels Page 38/40 are high magni cation (40×) images corresponding to the upper panels (5×); e The heart tissue of C57BL/6J mice was stained with oil red O in the control group and PM exposed group.

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