Maternal folic acid over-supplementation impairs cardiac function in mice offspring by inhibiting SOD1 expression.

BACKGROUND: Folic acid (FA) supplementation during pregnancy aims to protect foetal development. However, maternal over-supplementation of FA has been demonstrated to cause metabolic dysfunction and increase the risk of autism, retinoblastoma, and respiratory illness in the offspring. Moreover, FA supplementation reduces the risk of congenital heart disease. However, little is known about its possible adverse effects on cardiac health resulting from maternal over-supplementation. In this study, we assessed the detrimental effects of maternal FA over-supplementation on the cardiac health of the offspring. METHODS AND RESULTS: Eight-week-old C57BL/6J pregnant mice were randomly divided into control and over-supplemented groups. The offspring cardiac function was assessed using echocardiography. Cardiac fibrosis was assessed in the left ventricular myocardium by histological analysis. Proteomic, protein, RNA, and DNA methylation analyses were performed by liquid chromatography-tandem mass spectrometry, western blotting, real-time quantitative PCR, and bisulfite sequencing, respectively. We found that maternal periconceptional FA over-supplementation impaired cardiac function with the decreased left ventricular ejection fraction in the offspring. Biochemical indices and tissue staining further confirmed impaired cardiac function in offspring caused by maternal FA over-supplementation. The combined proteomic, RNA expression, and DNA methylation analyses suggested that key genes involved in cardiac function were inhibited at the transcriptional level possibly due to increased DNA methylation. Among these, superoxide dismutase 1 was downregulated, and reactive oxygen species (ROS) levels increased in the mouse heart. Inhibition of ROS generation using the antioxidant N-acetylcysteine rescued the impaired cardiac function resulting from maternal FA over-supplementation. CONCLUSIONS: Our study revealed that over-supplementation with FA during mouse pregnancy is detrimental to cardiac function with the decreased left ventricular ejection fraction in the offspring and provides insights into the mechanisms underlying the association between maternal FA status and health outcomes in the offspring.

Single-cell mapping of peripheral blood mononuclear cells reveals key transcriptomic changes favoring coronary artery lesion in IVIG-resistant Kawasaki disease.

Background: Intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD) poses a considerable challenge to patients and their families due to its severe complications. Previous researches have highlighted the critical role of immune disorders in its pathogenesis. However, fragmented studies based on isolated cases hinder a comprehensive understanding of this deadly illness. This study aimed to explore the overall landscape of peripheral blood mononuclear cells (PBMCs) in IVIG-resistant KD patients using single-cell RNA sequencing (scRNA-seq). Methods: The scRNA-seq was used to characterize the transcriptomic profiles of IVIG-resistant KD patients, IVIG-responsive KD patients, and healthy controls. Data quality control (QC) and subsequent analysis were conducted using various R packages. These included DoubletFinder and Harmony for QC, Seurat and SingleR for identifying and annotating major cell types, ggpubr for calculating and visualizing the percentages of each cell type, Seurat for characterizing differentially expressed genes (DEGs) between groups, pheatmap for visualizing the DEGs, clusterProfiler for performing Gene Ontology (GO) enrichment analysis of DEGs, scRepertoire for TCR and BCR data analysis, Monocle for assessing cell differentiation trajectories, and CellChat for intercellular interaction evaluation. Results: High-quality single-cell transcriptome data from 12 participants were analyzed, including five with IVIG-resistant KD, four with IVIG-responsive KD, and three healthy controls. We identified 10 major cell types and observed that the differentiation of CD8+ effector T cells was impeded in IVIG-resistant KD patients with coronary artery lesion (CAL) according to cell differentiation trajectory analysis. Subsequent cell communication analysis demonstrated that myeloid cluster with high expression of LCN2, S100P, and LTF played a key role, potentially signaling through MIF-CD74/CXCR4 and MIF-CD74/CD44 ligand-receptor pairs. Conclusion: Complex immunopathological changes occur during the development of CAL in IVIG-resistant KD. Stunted differentiation of CD8+ effector T cells is noted in KD-CAL. Interactions between myeloid cells and T cells activates multiple inflammatory signaling pathways, with ligand-receptor pairs, including MIF-CD74/CXCR4 and MIF-CD74/CD44, potentially playing crucial roles.

Promoting Glutathione Synthesis: A Possibility for Treating Cardiomyopathy Induced by a Maternal Western Diet.

BACKGROUND: The adverse effects of a Western diet on obesity and diabetes among reproductive-aged women pose a significant threat to the cardiovascular health of their offspring. Given the crucial role of glutathione metabolism and glutathione-related antioxidant defense systems in cardiovascular diseases through scavenging ROS and maintaining redox homeostasis, further exploration of their specific influence is imperative to develop therapeutic strategies for cardiomyopathy induced by a maternal Western diet. METHODS: We developed a prenatal maternal Western diet exposure model in C57/B6 mice to investigate cardiac morphology and function through histological analysis and echocardiography. RNA sequencing and analysis were utilized to elucidate the mechanisms underlying the impact of a maternal Western diet and N-acetylcysteine treatment on cardiomyopathy. Additionally, ELISAs, transmission electron microscopy, and flow cytometry were employed to assess the antioxidant defense system and mitochondrial ROS levels in progenitor cardiomyocytes. RESULTS: N-acetylcysteine significantly mitigated cardiomyocyte hypertrophy, myocardial interstitial fibrosis, collagen type I accumulation, and left ventricular remodeling induced by a maternal Western diet, particularly in male offspring. Furthermore, N-acetylcysteine reversed the increase in apoptosis and the increase in the ß/α-MyHC ratio in the myocardium of offspring that results from a maternal Western diet. RNA sequencing and GSEA revealed that the beneficial effects of N-acetylcysteine were linked to its ability to modulate oxidative phosphorylation pathways. Additionally, N-acetylcysteine treatment during pregnancy can markedly elevate glutathione levels, augment glutathione peroxidase (GPx) activity, and mitigate the accumulation of mitochondrial ROS caused by a maternal Western diet. CONCLUSIONS: N-acetylcysteine mitigated cardiomyopathy induced by a maternal Western diet by bolstering glutathione synthesis and enhancing GPx activity, thereby scavenging mitochondrial ROS and modulating oxidative phosphorylation pathways.

Effectiveness of the six-step approach guided online training program to improve knowledge of Mycoplasma pneumoniae pneumonia among pediatricians: a pretest-posttest study.

Background: In 2023, China witnessed an earlier and more widespread outbreak of Mycoplasma pneumoniae pneumonia (MPP). To address this situation, an online training program was designed to enhance the knowledge of MPP among pediatricians in Shanghai, China. Methods: An online training program on the diagnosis and treatment of MPP, guided by Kern's six-step approach, was developed by the Shanghai Pediatric Clinical Quality Control Center. A pre- and post-training survey was conducted using a 20-item self-administered questionnaire to investigate the pediatricians' knowledge of MPP. A linkage mechanism was established to match pretest/posttest questionnaires using personal identifiers. Paired t-tests and McNemar tests were performed to measure the differences, as appropriate, between pre- and post-training groups. A higher survey score indicated better knowledge. Results: There were 289 participants performed pre- and post-tests. The average age of the respondents was 38.7 years (standard deviation: 8.9). Over 80% of the participants were primary (32.5%) and intermediate (47.8%) pediatricians. Those from specialized hospitals accounted for the highest proportion (41.5%). The post-training group achieved significantly higher total scores than the pre-training group (91.3 vs. 67.7, t=22.48, P<0.001), regardless of the professional titles or hospital levels (all P<0.001). The accuracy rates of each question increased significantly in the post-training group (all P<0.001). Conclusions: The online training program effectively enhanced pediatricians' understanding of diagnosing and treating MPP. It is recommended to maintain continuous education and training targeting all healthcare providers.

High paternal homocysteine causes ventricular septal defects in mouse offspring.

Maternal hyperhomocysteinemia is widely considered as an independent risk of congenital heart disease (CHD). However, whether high paternal homocysteine causes CHD remains unknown. Here, we showed that increased homocysteine levels of male mice caused decreased sperm count, sperm motility defect and ventricular septal defect of the offspring. Moreover, high levels of paternal homocysteine decrease sperm DNMT3A/3B, accompanied with changes in DNA methylation levels in the promoter regions of CHD-related genes. Folic acid supplement could decrease the occurrence of VSD in high homocysteine male mice. This study reveals that increased paternal homocysteine level increases VSD risk in the offspring, indicating that decreasing paternal homocysteine may be an intervening target of CHD.

Research progress of cardiovascular disease prevention commencing in childhood / 中国学校卫生

Abstract@#Cardiovascular diseases (CVD) have imposed a substantial disease burden in China. Recently, the significance of childhood in CVD prevention has gained a significant attention. Prospective cohort studies have provided conclusive evidence that various risk factors during childhood are associated with the risk of CVD in adulthood. Furthermore, randomized controlled trials have made noteworthy advancements, demonstrating that healthy lifestyles during childhood could significantly promote cardiovascular health. The paper reviews the prevalence characteristics of CVD risk factors in Chinese children, the association with the risk of CVD development in adulthood and the progress of intervention research in childhood, in order to provide a scientific reference for the prevention of CVD from childhood.

Upregulation of miR-21-5p rescues the inhibition of cardiomyocyte proliferation induced by high glucose through negative regulation of Rhob.

Increasing evidence shows that maternal hyperglycemia inhibits cardiomyocyte (CM) proliferation and promotes cell apoptosis during fetal heart development, which leads to cardiac dysplasia. Accumulating evidence suggests that the overexpression of miR-21 in CMs has a protective role in cardiac function. Therefore, we investigated whether miR-21 can rescue CM injury caused by high glucose. First, we performed biological function analysis of miR-21-5p overexpression in H9c2 cells treated with high glucose. We found that the proliferation of H9c2 cells treated with high glucose decreased significantly and was rescued after overexpression of miR-21-5p. CCK-8 and EdU incorporation assays were performed to assess cell proliferation. The cell proliferation of the miR-21-5p mimic transfection group was improved compared with that of the NC mimic group (*p < 0.05, miR-21-5p mimics vs. NC mimics) when the proliferation of H9c2 cells was reduced by high glucose (****p < 0.0001, high glucose (HG) vs. normal glucose (NG)). Then, we verified the targeted and negative regulation of miR-21-5p on Rhob using a dual-luciferase activity assay and RT-qPCR, respectively. We further demonstrated that miR-21-5p regulates Rhob to rescue the inhibition of CM proliferation induced by high glucose. The CCK-8 results showed that the cell proliferation of the siRNA-Rhob group was higher than that of the NC mimic group (***p < 0.001) and that of the cotransfection group with Up-Rhob plasmids and miR-21-5p mimics was lower than that of the miR-21-5p mimic group (*p < 0.05). Conclusion: Overexpression of miR-21-5p rescues the inhibition of high glucose-induced CM proliferation through regulation of Rhob.

Maternal Obesity and Kawasaki Disease-like Vasculitis: A New Perspective on Cardiovascular Injury and Inflammatory Response in Offspring Male Mice.

Maternal obesity affects the risk of cardiovascular disease and inflammatory response in offspring. However, the impact of maternal obesity on offspring with Kawasaki disease (KD), the leading cause of childhood acquired heart disease, is still an understudied area. This study aimed to elucidate the impact of maternal obesity on offspring in KD-like vasculitis and the underlying mechanisms. Offspring of obese female mice and normal diet dams were randomly divided into two subgroups. The pups were injected intraperitoneally with either Candida albicans water-soluble fraction (CAWS) or phosphate buffered saline (PBS) to establish the obesity (OB)-CAWS group, OB group, wild type (WT)-CAWS group, and WT group. Their weight was monitored during the study. After four weeks, echocardiography was applied to obtain the alternation of cardiac structures. Mouse cytokine panel, Hematoxylin-Eosin (HE) staining, western blot, and real-time qPCR were used to study the pathological changes and protein and RNA expression alternations. Based on the study of pathology, serology and molecular biology, maternal obesity lead to more severe vasculitis and induced altered cardiac structure in the offspring mice and promoted the expression of pro-inflammatory cytokines through activating the NF-κB signaling pathway. Maternal obesity aggravated the inflammatory response of offspring mice in KD-like vasculitis.

Description of the medical services provided to children in Shanghai: a cross-sectional study of the characteristics and disparities of hospitals of different levels and types.

Background: The development of the pediatric care system is uneven in China. Limited research has been conducted on pediatric care in Shanghai, which is a well-developed region in China, in which the National Children's Medical Centers are located. Methods: In November 2021, under the commission of the Shanghai Center for Medical Quality Control, a city-wide questionnaire designed to examine the provision of medical services to children in Shanghai in 2020 was conducted at 86 hospitals providing pediatric care. The overall characteristics and disparities between the general hospitals and children's hospitals and suggestions for future developments were explored. Results: In 2020, there were 86 hospitals providing pediatric care, covering all 16 municipal districts of Shanghai, with an average distribution of 1.4 hospitals per 100 km2. The hospitals were mainly public (94.2%) and general (96.5%) hospitals. With a response rate of 90.7%, the questionnaire results revealed that there were 2,683 in-service pediatricians in Shanghai, with an average of 1.1 pediatrician per 1,000 children aged 0-14 years in Shanghai. The pediatricians were mainly women (71.8%), aged 40 years or younger (60.6%), who held a bachelor's degree or higher (99.5%). The total number of pediatric outpatient and emergency visits was approximately 8 million, with an average of 2,973 visits per pediatrician in 2020. There were >370,000 visits to fever clinics. The number of pediatric inpatient visits exceeded 160,000, with an average hospital stay length of 5.8 days. The uneven development between the children's hospitals and general hospitals represents a major challenge facing Shanghai's pediatric care system, and the close links between the 2 types of hospitals need to be further strengthened. Conclusions: Shanghai provides an overall superior medical service to children in China. The close link between the children's hospitals and general hospitals should be further strengthened to optimize the distribution of high-quality resources and greatly improve the overall provision of pediatric medical services.

Direct costs of community-acquired pneumonia for hospitalized children in Shanghai, China from 2018 to 2020: a cross-sectional analysis.

Background: Community-acquired pneumonia (CAP) is an acute respiratory infection with a high clinical and economic burden. Clarifying the burden is important for health policy making. However, there is inadequate data on the economic burden of childhood CAP in China. In this study, the direct disease burden of CAP in children was analyzed using city-level data. Methods: A cross-sectional study of the direct costs of CAP for hospitalized children aged 28 days to 18 years old in Shanghai from January 2018 to December 2020 was performed. Information, including the hospitalization costs from the first page of the children's hospitalized medical records, was obtained. The direct costs included medical services, diagnostics, medications, and medical supplies. The continuous variables with non-normal distributions are expressed as the median (interquartile range). Comparisons between groups were performed using the Kruskal-Wallis H test. The enumeration data are expressed as the number (percentage), and comparisons between groups were performed using the χ2 test. Results: A total of 59 hospitals and 63,614 hospitalized CAP patients were included in this study. Significantly fewer patients were discharged in 2020 than 2018 and 2019 (6,662, 27,943, and 29,009, respectively, P<0.001). Among the patients, 27,741 patients (43.6%) were covered by social medical insurance, 13,509 (21.2%) by commercial health insurance, and 22,364 (35.2%) were self-paying. The annual total direct costs for 2018, 2019, and 2020 were 118.553, 140.865, and 40.064 million Chinese Yuan (CNY), respectively. The average direct costs per hospital stay due to pediatric CAP in Shanghai was 4,707.83 CNY in 2018, a sum that accounted for 7.3% and 16.7% of the per capita disposable income in Shanghai and China in 2018, respectively. The total costs of the group aged <1 year were significantly higher than those of the other age groups (6,271.1 vs. 3,244.3~4,610.7 CNY, P<0.001). The total costs of severe cases were significantly higher than those of non-severe cases (5,200.6 vs. 3,170.4 CNY, P<0.001). The median duration of hospital stay was 6.0 days (5.0, 8.0). Conclusions: CAP hospitalization continues to represent a high clinical and economic burden in Shanghai, China. Specialized hospitals, severe cases, and the length of hospital stay were positively correlated with inpatient costs.

Association between High-Fat Diet during Pregnancy and Heart Weight of the Offspring: A Multivariate and Mediation Analysis.

Maternal nutrition and health status in the peri-pregnancy period are closely related to offspring health. Currently, population studies are unable to provide quantitative relationships and effective measures of peri-pregnancy high-fat diet and offspring myocardial remodeling due to the difficulty of obtaining human samples. This study aimed to establish the mouse models of maternal obesity and high-fat diet supplementation and deprivation during pregnancy. The effects of obesity, periconceptional high-fat diet window, fetal weight, sex, and placental weight on myocardial remodeling in the offspring were measured by single-factor and multiple-factor regression analyses. Moreover, the relationship between perinatal high-fat diet/fetal weight and offspring myocardial remodeling was explored using the mediation analysis model. The multivariate analysis showed that the heart weight to body weight (HW/BW) ratio of the offspring decreased by -1.6525 mg/g for every 1-g increase in fetal weight. The offspring HW/BW increased by 1.1967 mg/g if pregnant women were exposed to a high-fat diet throughout pregnancy. The mediation analysis model of a perinatal high-fat diet for the myocardial remodeling of offspring revealed that fetal weight had a suppression effect on the myocardial weight of offspring, accounting for 60.70%; also, it had a mediating effect on the HW/BW of offspring, accounting for 17.10%. Moreover, subgroup analysis showed an interaction between offspring sex and HW/BW in a maternal high-fat diet during pregnancy. Additionally, a quantitative real-time polymerase chain reaction experiment further proved that a perinatal high-fat diet could change the important indicators of myocardial remodeling in offspring. In conclusion, this study found that a high-fat diet in the periconceptional period influenced factors in offspring myocardial remodeling. Moreover, maternal high-fat diet deprivation attenuated the changes in offspring myocardial remodeling. In addition, the role of fetal weight in mediating maternal high-fat diet-mediated offspring myocardial remodeling was quantified. Our study showed that a sensible and healthy diet during the perinatal period, especially during pregnancy, played a positive role in the health of the offspring.

Size-segregated particle number concentrations and outpatient-department visits for pediatric respiratory diseases in Shanghai, China.

BACKGROUND: Few studies have simultaneously explored which size of particles has the greatest impact on the risk for pediatric asthma, bronchitis and upper respiratory tract infections (URTIs). OBJECTIVES: To investigate the short-term association between size-segregated particle number concentrations (PNCs) and outpatient-department visits (ODVs) for major pediatric respiratory diseases. METHODS: Daily counts of pediatric ODVs for asthma, bronchitis and URTIs were obtained from 66 hospitals in Shanghai, China, from 2016 to 2018. Pollutant effects were estimated using Poisson generalized additive models combined with polynomial distributed lag models. We also fitted co-pollutant cumulative effects models included six criteria air pollutants and conducted stratifying analyses by gender, age, season and geographic distances. RESULTS: We identified a total of 430,103 patients with asthma, 1,547,013 patients with bronchitis, and 2,155,738 patients with URTIs from the hospitals. Effect estimates increased with decreasing particle size. Ultrafine particle (UFP) and PNCs of 0.10-0.40 µm particles (PNC0.10-0.40) were associated with increased ODVs for asthma, bronchitis and URTIs at cumulative lags up to 3d. Associations tended to appear stable after adjusting for criteria air pollutants. At the cumulative lag 0-2d, each interquartile range increase in UFP was associated with increased ODVs due to asthma (relative risk 1.21, 95% CI: 1.07, 1.38), bronchitis (1.20, 95% CI: 1.07, 1.34) and URTI (1.17, 95% CI: 1.06, 1.30), whereas the associations for PNC0.10-0.40 remained significant but attenuated in magnitude. CONCLUSIONS: UFP may be a leading contributor to the adverse respiratory effects of particulate air pollution and the effects increased with decreasing particle size.

The functional verification and analysis of Fugu promoter of cardiac gene tnni1a in zebrafish.

Troponin I type 1b (Tnni1b) is thought to be a novel isoform that is expressed only in the zebrafish heart. Knocking down of tnni1b can lead to cardiac defects in zebrafish. Although both the zebrafish tnni1b and human troponin I1 (TNNI1) genes are thought to be closely associated with fatal cardiac development, the regulatory molecular mechanisms of these genes are poorly understood. Analyzing the functionally conserved sequence, especially in the noncoding regulatory region involved in gene expression, clarified these mechanisms. In this study, we isolated a 3 kb fragment upstream of Fugu tnni1a that can regulate green fluorescence protein (GFP) expression in a heart-specific manner, similar to the pattern of zebrafish homologue expression. Three evolutionarily conserved regions (ECRs) in the 5'-flanking sequence of Fugu tnni1a were identified by sequence alignment. Deletion analysis led to the identification of ECR2 as a core sequence that affects the heart-specific expression function of the Fugu tnni1a promoter. Interestingly, both the Fugu tnni1a promoter and ECR2 sequence were functionally conserved in zebrafish, although they shared no sequence similarity. Together, the findings of our study provided further evidence for the important role of tnni1a homologous in cardiac development and demonstrated that two functionally conserved sequences in the zebrafish and Fugu genomes may be ECRs, despite their lack of similarity.

Melatonin alleviates vascular endothelial cell damage by regulating an autophagy-apoptosis axis in Kawasaki disease.

OBJECTIVES: Melatonin has been reported to be an appropriate candidate for mitigating various cardiovascular injuries, owing to its versatility. This study aimed to explore the role of melatonin in Kawasaki disease (KD)-associated vasculitis and its underlying mechanisms. MATERIAL AND METHODS: The role of melatonin was evaluated in human coronary artery endothelial cells (HCAECs), peripheral blood mononuclear cells from KD patients, human THP1 cell line in vitro, and a Candida albicans water-soluble fraction (CAWS)-induced KD mouse model in vivo. Cell proliferation assay, cell apoptosis assay, cell co-culture, RNA extraction, RNA sequencing, reverse transcription quantitative PCR, enzyme-linked immunosorbent assay (ELISA), transwell assay, western blot, dual-luciferase reporter assay, and autophagic flux assay were performed to investigate the function and regulatory mechanisms of melatonin in vitro, while haematoxylin and eosin staining, Verhoeff's van Gieson staining, ELISA, and immunohistochemical analysis were performed to detect the effect of melatonin in vivo. RESULTS: Melatonin suppressed cell apoptosis directly reduced the expression of endothelial cell damage markers in HCAECs, and alleviated vasculitis in the CAWS-induced KD mouse model. Mechanistically, melatonin promoted autophagy by activating the melatonin/ melatonin receptor (MT)/cAMP-response element binding protein (CREB) pathway and upregulating the expression of autophagy-related gene-3, thereby suppressing cell apoptosis in an autophagy-dependent manner. Additionally, melatonin decreased the production of pro-inflammatory cytokines in macrophages and indirectly reduced the immunopathological damage of HCAECs. CONCLUSIONS: This study revealed that melatonin protects vascular endothelial cells in KD, by suppressing cell apoptosis in an autophagy-dependent manner and reducing the immunopathological damage mediated by macrophages.

Nicotinamide Mononucleotide Alleviates Cardiomyopathy Phenotypes Caused by Short-Chain Enoyl-Coa Hydratase 1 Deficiency.

Short-chain enoyl-CoA hydratase 1 (ECHS1) deficiency plays a role in cardiomyopathy. Whether ECHS1 deficiency causes or is only associated with cardiomyopathy remains unclear. By using Echs1 heterogeneous knockout (Echs1 +/-) mice, we found that ECHS1 deficiency caused cardiac dysfunction, as evidenced by diffuse myocardial fibrosis and upregulated fibrosis-related genes. Mechanistically, ECHS1 interacts with the p300 nuclear localization sequence, preventing its nuclear translocation in fibroblasts. ECHS1 deficiency promotes p300 nuclear translocation, leading to increased H3K9 acetylation, a known risk factor for cardiomyopathy. Nicotinamide mononucleotide-mediated acetylation targeting suppressed ECHS1 deficiency-induced cardiomyopathy phenotypes in Echs1 +/- mice. Thus, enhancing p300-mediated H3K9ac is a potential interventional approach for preventing ECHS1 deficiency-induced cardiomyopathy.

miR-29b-3p Inhibitor Alleviates Hypomethylation-Related Aberrations Through a Feedback Loop Between miR-29b-3p and DNA Methylation in Cardiomyocytes.

As a member of the miR-29 family, miR-29b regulates global DNA methylation through target DNA methyltransferases (DNMTs) and acts as both a target and a key effector in DNA methylation. In this study, we found that miR-29b-3p expression was inversely correlated with DNMT expression in the heart tissues of patients with congenital heart disease (CHD), but whether it interacts with DNMTs in cardiomyocytes remains unknown. Further results revealed a feedback loop between miR-29b-3p and DNMTs in cardiomyocytes. Moreover, miR-29b-3p inhibitor relieved the deformity of hypomethylated zebrafish and restored the DNA methylation patterns in cardiomyocytes, resulting in increased proliferation and renormalization of gene expression. These results suggest mutual regulation between miR-29b-3p and DNMTs in cardiomyocytes and support the epigenetic normalization of miRNA-based therapy in cardiomyocytes.