Sepsis is a life-threatening condition involving organ dysfunction that is caused by a dysregulated host response to infection (Cohen et al., 2015), It has been reported that approximately 50% of patients with septic shock are diagnosed with septic cardiomyopathy, which causes myocardial dysfunction, and these patients tend to have poor prognoses (Zaky et al., 2014). Myocardial dysfunction is an important factor that contributes to the high mortality rate of sepsis. Therefore, it is necessary to elucidate the pathogenesis of myocardial dysfunction in order to develop new treatment strategies to reduce the mortality rate of sepsis.

To date, a series of reversible posttranscriptional modifications located in RNAs have gained increasing attention. More than 140 chemical modifications have been discovered in RNAs. The modifications range from simple methylation or isomerisation, such as N6-methyladenosine (m⁶A), 5-methylcytosine (m5C), N1-methyladenosine (m¹A), pseudouridine (Ψ), 5-methyluridine (m⁵U),1-methylguanosine, and 7-methylguanosine (m¹G and m⁷G, respectively) and inosine (I), to complex multistep chemical modifications, such as N⁶-threonylcarbamoyladenosine and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s²U) (Pan, 2018). Among them, the m⁶A is one of the most abundant and influential modifications in eukaryotes. M⁶A modification is involved in regulating a variety of posttranscriptional events, including pre-miRNA processing and RNA stability, translation and alternative splicing (Chokkalla et al., 2019). M⁶A modifications occur via the m⁶A methyltransferases called “writers”; they are removed by the demethylases called “erasers” and are recognized by m6A-binding proteins called “readers”. The orchestrated interplay among writers, erasers, and readers drives the dynamics and outcomes of the m⁶A modification of RNAs. An increasing number of studies have reported that m⁶A modification plays important roles in biological and pathophysiological processes, such as tumorigenesis, embryonic stem cell differentiation, and viral infection (Kennedy et al., 2016). In mouse GC-1 SPG cells, meclofenamic acid inhibits spermatogonial proliferation by affecting CDKs expression through a m⁶A-dependent mRNA degradation pathway (Huang et al., 2019). In liver cancer, overexpression of METTL3 leads to the degradation of suppressor of cytokine signalling two and promotes tumor growth (Lin et al., 2019).

M⁶A modification is also associated with the pathophysiological processes of cardiac differentiation and a variety of cardiovascular diseases. Methyltransferase-like 3 (METTL3) is a major factor involved in abnormal m⁶A modification. Silencing METTL3 enhances the autophagic flux and represses apoptosis in hypoxia/reoxygenation-treated cardiomyocytes (Song et al., 2019). Overexpression METTL3 increased m⁶A levels in mRNAs isolated from transverse aortic constriction (TAC) mice hearts. TAC inducd pathological hypertrophic cellular growth was attenuated in hearts of METTL3-overexpressing mice, as evidenced by the crosssectional area of myocytes (Kmietczyk et al., 2019). In addition, aging caused METTL3/METTL14 down-regulation in aorta and atria in male animals while the differentiation-induced increased level of METTL16 (Arcidiacono et al., 2020). Prabhu M et al. found that downregulation of fat mass and obesity associated protein (FTO) expression in failing mammalian hearts and hypoxic cardiomyocytes increased the m⁶A modification of RNAs and reduced the contractile function of cardiomyocytes (Mathiyalagan et al., 2019). FTO also played a vital role in cardiac contractile function during homeostasis and remodeling. FTO overexpression attenuated the ischemia-induced elevation in m⁶A modification and significantly improved cardiac function of post-myocardial infraction (Mathiyalagan et al., 2019). Berulava et al. reported that differently expressed genes of m⁶A methylation are involved in heart failure development. Bioinformatics analysis has revealed that the differentially m⁶A are mainly involved in metabolism and cardiac signaling (Berulava et al., 2020). However, the regulatory role of the m⁶A modification in cardiac function is still unclear.

The contraction of the ventricle transports blood to the capillaries of the body or lungs, while the contraction of the atria send blood into the ventricle. In addition, the right ventricle mainly supplies blood to the lungs, and the blood in the right ventricle is venous blood. The left ventricle mainly supplies blood to the organs and tissues of the body, and the blood in the left ventricle is arterial blood. Therefore, the function of the left ventricle is very important to maintain organ perfusion. If the left ventricular function declines, it will leads to organ perfusion insufficiency and, eventually, organ failure. In this direction, we focused on the mechanism underlying left ventricle injury in sepsis to find any potential clinical significance for improving organ perfusion. The overall goal of this study was to explore a new layer of epigenetic alterations through the genome-wide screening of the altered m⁶A-tagged transcript profiles in lipopolysaccharide (LPS)-induced myocardial dysfunction. We successfully mapped m⁶A transcripts in left ventricle tissue in LPS-induced sepsis. Some potential roles of the m⁶A modification in the physiological and pathological mechanisms of sepsis were revealed.

Our study suggested that the m⁶A modification level was decreased in septic heart tissues. Our genome-wide screening of m⁶A-tagged transcript profiles indicated that the m⁶A modification levels in 39 mRNAs and 7 lncRNAs were significantly decreased and that the m⁶A modification levels in 23 mRNAs and four lncRNAs were significantly increased.

Pathway analyses of mRNAs with altered m6A modification were conducted in our study. The results showed that the mRNAs with decreased m⁶A modification levels were enriched in many pathways related to immune and inflammatory responses. In recent years, the roles of m⁶A modification in autoimmune and inflammatory diseases have attracted substantial attention (Rubio et al., 2018). METTL3 is a key enzyme of m⁶A methylation modification and is involved in immune and inflammatory regulation. Feng et al. found that METTL3 inhibits inflammation by affecting the alternative splicing of MyD88 (Feng et al., 2018). METTL3 expression is significantly upregulated in blood samples from patients with rheumatoid arthritis. In addition, METTL3 attenuates LPS-induced macrophage-mediated inflammation through the NF-κB pathway (Wang et al., 2019a). The m⁶A “reader” YT521-B homology domain family 2 (YTHDF2) inhibits HCC cells and tumor vasculature by degrading the mRNAs of IL11 and serpin family E member 2 (Wang et al., 2019b). These findings indicate the important role of m⁶A modification in inflammatory and immune disorders. Sepsis is mainly caused by the host’s unbalanced response to infection. In sepsis, microbial infections or necrotic tissues release a large amount of harmful substances, leading to the activation of the systemic immune response and the excessive activation of immune cells. The excessive release of cytokines is destructive. There is currently no effective treatment for sepsis because in the past few decades, attempts to use anti-inflammatory treatments to limit the tissue damage caused by excessive inflammation have failed (Zeni et al., 1997). Our study suggests that alterations in m⁶A modification in sepsis are closely related to inflammatory and immune responses. Since we study cardiac tissue, we speculate that m⁶A modification plays an important role in sepsis-induced cardiac function dysfunction.

We selected four lncRNAs and six mRNAs for the qPCR validation of single-base m⁶A sites in our study. The results showed that among the lncRNAs, the m⁶A modification levels were significantly increased in XR_346771. Among the mRNAs, the m⁶A modification levels were significantly increased in Clec1b and Tnfrsf26 and significantly decreased in Stk38l. These results are consistent with those of high-throughput sequencing; therefore, we attempted to conduct an in-depth analysis of this lncRNA and the three mRNAs.

First, we performed PPI analysis of Clec1b, STK38L, and Tnfrsf26 to investigate the downstream possible regulatory genes and biological functions of these proteins. The Clec1b-interacting proteins are enriched in the pathway of “platelet activation”. C-type lectin-like receptor 2 (CLEC-2) is a protein encoded by the Clec1b gene. Platelets play a critical role in innate and adaptive immunity (Semple et al., 2011). Platelets can alleviate LPS-induced septic shock by regulating the ability of macrophages to engulf and kill bacteria (Xiang et al., 2013). In two mouse models of sepsis (intraperitoneal LPS injection and cecal ligation and puncture), platelet CLEC-2 reduced the severity of sepsis by controlling the migration of monocytes/macrophages to the infection site, the expression of inflammatory mediators, and damage to organs (Rayes et al., 2017). In our sepsis model, we found that the m⁶A modification level of Clec1b mRNA was significantly increased. Considering that the degree and pattern of m⁶A modification may affect the transport, splicing, storage, translation, stability, and decay of mRNAs, we speculated that the elevated level of Clec1b m⁶A modification affects the functions of Clec1b and associate with activation of platelets. The interacting proteins of Clec1b were glycoprotein VI (Gp6), lymphocyte cytosolic protein 2 (Lcp2), and spleen tyrosine kinase (Syk). These proteins might be used as target proteins for in-depth research.

Our study suggests that the m⁶A modification level of Tnfrsf26 mRNA is increased and of Stk38l mRNA is decreased. According to PPI network analysis, Stk38l-interacting proteins are enriched in the “Hippo signaling pathway” pathway. Hippo signaling pathway may promote cell differentiation and death and inhibit cell proliferation (Chang et al., 2019). Tnfrsf26-interacting proteins are mainly enriched in the pathway of apoptosis. Sepsis-induced cardiac dysfunction has been reported by many studies to be closely related to apoptosis. When LPS stimulates H9C2 cells (rat embryonic cardiomyoblasts), the overexpression of miR-146a suppresses apoptosis and helps attenuate myocardial depression (An et al., 2018). Chao et al. found that the overexpression of Tid1-S can enhance ER-α to activate p-PI3K/p-Akt, attenuating LPS-induced apoptosis in H9C2 cells (Chao et al., 2019). In recent years, as the understanding of m⁶A modification has gradually deepened, studies have found that m⁶A modification plays an important role in the process of apoptosis. In the human glioma cell line U251, lower level of m⁶A modification can reduce apoptosis and promote cell proliferation (Li et al., 2019). Moreover, METTL3 can regulate the expression of several important proteins that regulate the survival, apoptosis, invasion, and proliferation of lung cancer cells (Lin et al., 2016). We hypothesized that the m⁶A modification of Tnfrsf26 and Stk38l might be related to cardiomyocyte apoptosis.

LncRNAs are a class of noncoding RNAs that are more than 200 nucleotides in length. In recent years, m⁶A-modified lncRNAs have received extensive attention. M⁶A modification may control gene expression by regulating the translation efficiency and stability of lncRNAs (Coker et al., 2019). For example, the m⁶A-modified lncRNA MALAT1 is implicated in ischemia reperfusion injury-induced inflammation (Yang et al., 2020). LncRNAs contain miRNA-responsive elements, which function as ceRNAs, interact with miRNAs and indirectly regulate mRNAs. In the present study, we established and analyzed a potential ceRNA network of XR_346771 through bioinformatics analysis. We performed GO and KEGG analyses of the predicted mRNAs to further explore the function of XR_346771.

GO analysis showed that “inorganic cation import across plasma membrane”, “inorganic ion import across plasma membrane”, “transmembrane transport”, and “import across plasma membrane” were significantly enriched. Cardiac contraction requires Ca²⁺, so abnormal Ca²⁺ homeostasis may play a key role in the pathogenesis of common cardiovascular diseases. Studies have reported that m⁶A modification promotes neurite extension and neuronal differentiation by acting as a Ca²⁺ channel (Mukobata et al., 2002). In 6-OHDA-induced PC12 cells and the cerebral striatum of rats with Parkinson’s disease, decreased m⁶A modification induces the expression of N-methyl-d-aspartate receptor 1 and increases oxidative stress and Ca²⁺ influx, leading to apoptosis of dopaminergic neurons (Chen et al., 2019). These studies indicate that m⁶A modification is involved in Ca²⁺ transport. We speculated that the m⁶A modification of XR_346771 might associate with abnormal Ca²⁺ homeostasis in cardiac tissues.

It is interesting that even though the methyltransferase complex was downregulated upon sepsis induced myocardial dysfunction; there were still 27 transcripts that were significantly hypermethylated. Chokkalla et al. found that, compared with sham group, global m⁶A increased significantly at 12 and 24 h of reperfusion in transient middle cerebral artery occlusion in C57BL/6J mice. The FTO decreased significantly after stroke compared with sham. While 139 transcripts (122 mRNAs and 17 lncRNAs) were hypermethylated, 8 transcripts (5 mRNAs and 3 lncRNAs) were hypomethylated in the ischemic brain at 12 h reperfusion (Chokkalla et al., 2019). Wang et al. found that METTL3 was downregulated after traumatic brain injury in mice. In total, 922 m⁶A peaks were differentially expressed as determined by m⁶A-RIP-seq, with 370 upregulated and 552 downregulated methylated mRNA (Wang et al., 2019b). These studies are consistent with our research. Previous research indicated that the methylation modification of m⁶A has been proved to be reversible, as it modulated by RNA methyltransferases, RNA-binding proteins and demethylases. The m⁶A methyltransferase, also known as “Writers”, including METTL3/14, WTAP, KIAA1429 and RNA binding motifs protein 15/15B (RBM15/15B), E3 ubiquitin-protein ligase Hakai (HAKAI) and zinc finger CCCH-type containing 13 (ZC3H13). The RNA-binding proteins, also known as “readers”, including: YTHDF1/2/3, YTH domain-containing reader proteins 1/2 (YTHDC1/2), insulin-like growth factor 2 mRNA-binding proteins 1/2/3 (IGF2BP1/2/3), epithelium-specific ETS (ESE) transcription factors (ELF3), heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1). The m⁶A demethylase, also known as “eraser”, including: FTO, α-ketoglutarate-dependent dioxygenase alkB homologue 5 (ALKHB5). In our research we tested mRNA level of some enzymes, but not all enzymes. Therefore, there may be other enzymes involved in m⁶A modification upon sepsis induced myocardial dysfunction. The m⁶A modification status of specific RNA was catalyzed by different enzymes. METTL3 positively regulates expression of MYD88, a critical upstream regulator of NF-κB signaling, by facilitating m⁶A methylation modification to MYD88-RNA in mesenchymal stem cells (Yu et al., 2020). However, reduced expression of FTO increased Nanog mRNA m⁶A methylation under TNF-α stimulation, decreased Nanog mRNA and protein expression, and significantly inhibited mesenchymal stem cells capacity for differentiation to sweat gland cells (Wang et al., 2020). In our research, these transcripts that have undergone m⁶A modification might have been modified by different enzymes.

In conclusion, our experiments revealed that the level of m⁶A modification is significantly decreased in septic cardiac tissue. Through the genome-wide profiling of m⁶A-tagged mRNAs and lncRNAs and subsequent bioinformatics analysis, we revealed some potential functions of transcripts with altered m⁶A modification.