N6-methyladenosine (m6A) methyltransferase METTL3 regulates sepsis-induced myocardial injury through IGF2BP1/HDAC4 dependent manner

Abstract

Recent studies have identified that N⁶-methyladenosine (m⁶A) extensively participates in the myocardial injury pathophysiological process. However, the role of m⁶A on sepsis-induced myocardial injury is still unclear. Here, we investigated the functions and mechanism of m⁶A methyltransferase METTL3 for septic myocardial injury. Results illustrated that the m⁶A modification level and METTL3 up-regulated in the lipopolysaccharide (LPS)-induced cardiomyocytes (H9C2 cells). Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed the m⁶A profile of the septic myocardial injury cellular model. Functionally, METTL3 knockdown repressed the inflammatory damage of cardiomyocytes induced by LPS. Mechanistically, we found that HDAC4 had remarkable m⁶A modification sites on its 3'-UTR genome, acting as the downstream target of METTL3. Besides, m⁶A reader IGF2BP1 recognized the m⁶A modification sites on HDAC4 mRNA and enhanced its RNA stability. In conclusion, the findings illustrated a role of METTL3/IGF2BP1/m⁶A/HDAC4 axis on sepsis-induced myocardial injury, which might provide novel therapeutic strategy for septic myocardial injury.

Fig. 1. The m⁶A profile in LPS-administrated cardiomyocytes.

A Cardiomyocytes (H9C2) was induced by LPS (10 μg/mL) to construct myocardial cell injury for septic myocardial cell injury investigation. B RT-PCR indicated the mRNA level of m⁶A methyltransferase METTL3 in cardiomyocytes with the increasing doses of LPS (0, 0.5, 1, 2, 5, 10 μg/mL) for 24 h. C Western blot analysis for the protein level in cardiomyocytes with the increasing doses of LPS (0, 0.5, 1, 2, 5, 10 μg/mL) for 24 h. D The m⁶A modification distribution in myocardial cell injury cardiomyocytes revealed by methylated RNA immunoprecipitation sequencing (MeRIP-Seq). E m⁶A metagene showed the m⁶A peak density in integral genome. F The candidate motifs. Biological experiments were performed in triplicate. Data were exhibited as Mean ± Standard Deviation (SD). **p < 0.01, *p < 0.05.

Fig. 2. METTL3 knockdown repressed the inflammatory damage of cardiomyocytes induced by LPS.

A The silencing efficiency was detected using RT-qPCR and (B) western blot for cardiomyocytes transfected with METTL3 knockdown (sh-METTL3) or controls (sh-NC). C The lactate dehydrogenase (LDH) release analysis was performed using microplate reader, including control, LPS, LPS + sh-NC, LPS + sh-METTL3. D ELISA analysis was performed to detect the levels of proinflammatory cytokines (interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α)) for cardiomyocytes (H9C2), including control, LPS, LPS + sh-NC, LPS + sh-METTL3. E Proliferative ability using CCK-8 assay for the proliferation ability of cardiomyocytes, including control, LPS, LPS + sh-NC, LPS + sh-METTL3. F Flow cytometry for apoptosis analysis revealed the apoptosis of cardiomyocytes transfected with METTL3 knockdown (sh-METTL3) or controls (sh-NC) upon LPS administration. Biological experiments were performed in triplicate. Data were exhibited as Mean ± Standard Deviation (SD). **p < 0.01, *p < 0.05.

Fig. 3. HDAC4 acted as a target of METTL3 in cardiomyocytes.

A The predictive tools using sequence-based N⁶-methyladenosine (m⁶A) modification site predictor (SRAMP, http://www.cuilab.cn/sramp) revealed the potential m⁶A modification sites on HDAC4 genome. B The binding motif of METTL3 on HDAC4 m⁶A site was GGACU sequences. C RT-PCR detected the HDAC4 mRNA was detected in cardiomyocytes with the increasing doses of LPS (0, 0.5, 1, 2, 5, 10 μg/mL). D Knockdown of METTL3 decreased the m⁶A methylation level of HDAC4 mRNA using m⁶A MeRIP-qPCR analysis. E RIP assays reflected the interaction of METTL3 and HDAC4 mRNA in cardiomyocytes. Relative enrichment of HDAC4 mRNA associated with METTL3 relative to input was calculated. IgG antibody acted as a control. Biological experiments were performed in triplicate. Data were exhibited as Mean ± Standard Deviation (SD). **p < 0.01, *p < 0.05.

Fig. 4. METTL3/IGF2BP1 enhanced the stability of HDAC4 mRNA.

A RT-PCR detected the expression of candidate m⁶A readers (YTHDF1, YTHDF2, IGF2BP1, IGF2BP2, IGF2BP3) in myocardial injury cardiomyocytes (H9C2) induced by LPS (10 μg/mL). B Western blot analysis detected the level of HDAC4 protein. C RNA immunoprecipitation analysis (RIP-qPCR) showed the enrichment of HDAC4 mRNA precipitated by anti-IGF2BP1 or anti-IgG in cardiomyocytes transfected with shRNA-METTL3. D RIP-qPCR was performed to detected the HDAC4 mRNA level precipitated by anti-IGF2BP1 or anti-IgG in cardiomyocytes. E shRNA-METTL3 or F si-IGF2BP1 was transfected into Act D treated cardiomyocytes and the HDAC4 mRNA level was detected using RT-qPCR. Biological experiments were performed in triplicate. Data were exhibited as Mean ± Standard Deviation (SD). **p < 0.01, *p < 0.05.

Fig. 5. METTL3/IGF2BP1/HDAC4 axis regulates the inflammatory damage of cardiomyocytes induced by LPS.

A Western blot analysis detected the HDAC4 protein level in cardiomyocytes (H9C2) transfected with HDAC4 overexpression (HDAC4 OV) or IGF2BP1 knockdown (si-IGF2BP1) or METTL3 knockdown (sh-METTL3) respectively. B CCK-8 analysis was performed to detect the proliferative ability of cardiomyocytes. C ELISA analysis detected the levels of proinflammatory cytokines, including interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α). Biological experiments were performed in triplicate. Data were exhibited as Mean ± Standard Deviation (SD). **p < 0.01, *p < 0.05.