Certain miRNAs can attenuate hypoxia/re-oxygenation-induced autophagic cell death reported in our previous studies, but how these miRNAs regulate the autophagy-related cellular signaling pathway in preventing cell death is largely unknown. In the current study, the autophagy-related miRNAs of hsa-miR-20b were investigated in an in vitro model of hypoxia/re-oxygenation-induced endothelial autophagic cell death. Of these, miR-20b was found to be the most important miRNA which targeted on the key autophagy kinase ULK1 and inhibited hypoxia/re-oxygenation injury-induced autophagy by decreasing both autophagosomes and LC3I to II transition rate and P62 degradation. These processes were reversed by the transfection of an miR-20b inhibitor. Re-expression of ULK1 restores miR-20b-inhibited autophagy. Propofol, a commonly used anesthetic, promoted miR-20b and METTL3 expression and attenuated endothelial autophagic cell death. The inhibited endogenous expression of miR-20b or silenced METTL3 diminished the protective effect of propofol and accentuated autophagy. Additionally, METTL3 knockdown significantly inhibited miR-20b expression but up-regulated pri-miR-20b expression. Together, our data shows that propofol protects against endothelial autophagic cell death induced by hypoxia/re-oxygenation injury, associated with activation of METTL3/miR-20b/ULK1 cellular signaling.

N6-Methyladenosine (m6A) plays important roles in regulating mRNA processing. Despite rapid progress in this field, little is known about the role and mechanism of m6A modification in myocardial development and cardiomyocyte regeneration. Existing studies have shown that the heart tissues of newborn mice have the capability of proliferation and regeneration, but its mechanism, particularly its relation to m6A methylation, remains unknown. Methods. To systematically profile the mRNA m6A modification pattern in the heart tissues of mice at different developmental stages, we jointly performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) of heart tissues of mice, respectively, aged 1 day old, 7 days old, and 28 days old. Results. We identified the linkages and association between differentially expressed mRNA transcripts and hyper or hypomethylated m6A peaks in C57BL/6J mice at different heart developmental stages. Results showed that the amount of m6A peaks and the level of m6A modification were the lowest in the heart of mice at 1 day old. By contrast, heart tissues from 7-day-old mice tended to possess the most m6A peaks and the highest global m6A level. However, the m6A characteristics of myocardial tissue changed little after 7 days old as compared to that of 1 day old. Specifically, we found 1269 downmethylated genes of 1434 methylated genes in 7-day-old mouse heart tissues as compared to those in 1-day-old mice. Hypermethylation of some specific genes may correlate with the heart’s strong proliferative and regenerative capability at the first day after birth. In terms of m6A density, the tendency shifted from coding sequences (CDS) to 3 ′ -untranslated regions (3 ′ UTR) and stop codon with the progression of heart development. In addition, some genes demonstrated remarkable changes both in methylation and expression, like kiss1, plekha6, and megf6, which may play important roles in proliferation. Furthermore, signaling pathways highly related to proliferation such as “Wnt signaling pathway,” “ECM-receptor interaction,” and “cardiac chamber formation” were significantly enriched in 1-day-old methylated genes. Conclusions. Our results reveal a pattern that different m6A modifications are distributed in C57BL/6J heart tissue at different developmental stages, which provides new insights into a novel function of m6A methylation of mRNA in myocardial development and regeneration.

Myocardial infarction that causes damage to heart muscle can lead to heart failure. The identification of molecular mechanisms promoting myocardial regeneration represents a promising strategy to improve cardiac function. Here we show that IGF2BP3 plays an important role in regulating adult cardiomyocyte proliferation and regeneration in a mouse model of myocardial infarction. IGF2BP3 expression progressively decreases during postnatal development and becomes undetectable in the adult heart. However, it becomes upregulated after cardiac injury. Both gain- and loss-of-function analyses indicate that IGF2BP3 regulates cardiomyocyte proliferation in vitro and in vivo. In particular, IGF2BP3 promotes cardiac regeneration and improves cardiac function after myocardial infarction. Mechanistically, we demonstrate that IGF2BP3 binds to and stabilizes MMP3 mRNA through interaction with N6-methyladenosine modification. The expression of MMP3 protein is also progressively downregulated during postnatal development. Functional analyses indicate that MMP3 acts downstream of IGF2BP3 to regulate cardiomyocyte proliferation. These results suggest that IGF2BP3-mediated post-transcriptional regulation of extracellular matrix and tissue remodeling contributes to cardiomyocyte regeneration. They should help to define therapeutic strategy for ameliorating myocardial infarction by inducing cell proliferation and heart repair.

Long non-coding RNAs (lncRNAs) were reported to potentially play a regulatory role in the process of myocardial regeneration in the neonatal mouse. N6-methyladenosine (m6A) modification may play a key role in myocardial regeneration in mice and regulates a variety of biological processes through affecting the stability of lncRNAs. However, the map of m6A modification of lncRNAs in mouse cardiac development still remains unknown. We aimed to investigate the differences in the m6A status of lncRNAs during mouse cardiac development and reveal a potential role of m6A modification modulating lncRNAs in cardiac development and myocardial regeneration during cardiac development in mice. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) of the heart tissue in C57BL/6 J mice at postnatal day 1 (P1), P7 and P28 were performed to produce stagewise cardiac lncRNA m6A-methylomes in a parallel timeframe with the established loss of an intrinsic cardiac regeneration capacity and early postnatal development. There were significant differences in the distribution and abundance of m6A modifications in lncRNAs in the P7 vs P1 mice. In addition, the functional role of m6A in regulating lncRNA levels was established for selected transcripts with METTL3 silencing in neonatal cardiomyocytes in vitro. Based on our MeRIP-qPCR experiment data, both lncGm15328 and lncRNA Zfp597, that were not previously associated with cardiac regeneration, were found to be the most differently methylated at P1-P7. These two lncRNAs sponged several miRNAs which further regulated multiple mRNAs, including some of which have previously been linked with cardiac regeneration ability. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that differential m6A modifications were more enriched in functions and cellular signalling pathways related to cardiomyocyte proliferation. Our data suggested that the m6A modification on lncRNAs may play an important role in the regeneration of myocardium and cardiac development.