METTL3 boosts mitochondrial fission and induces cardiac fibrosis after ischemia/reperfusion injury

Abstract

METTL3, an RNA methyltransferase enzyme, exerts therapeutic effects on various cardiovascular diseases. Myocardial ischemia-reperfusion injury (MIRI) and subsequently cardiac fibrosis is linked to acute cardiomyocyte death or dysfunction induced by mitochondrial damage, particularly mitochondrial fission. Our research aims to elucidate the potential mechanisms underlying the therapeutic actions of METTL3 in MIRI, with focus on mitochondrial fission. When compared with Mettl3^flox mice subjected to MIRI, Mettl3 cardiomyocyte knockout (Mettl3^Cko) mice have reduced infarct size, decreased serum levels of myocardial injury-related factors, limited cardiac fibrosis, and preserved myocardial ultrastructure and contractile/relaxation capacity. The cardioprotective actions of Mettl3 knockout were associated with reduced inflammatory responses, decreased myocardial neutrophil infiltration, and suppression of cardiomyocyte death. Through signaling pathway validation experiments and assays in cultured HL-1 cardiomyocytes exposed to hypoxia/reoxygenation, we confirmed that Mettl3 deficiency interfere with DNA-PKcs phosphorylation, thereby blocking the downstream activation of Fis1 and preventing pathological mitochondrial fission. In conclusion, this study confirms that inhibition of METTL3 can alleviate myocardial cardiac fibrosis inflammation and prevent cardiomyocyte death under reperfusion injury conditions by disrupting DNA-PKcs/Fis1-dependent mitochondrial fission, ultimately improving cardiac function. These findings suggest new approaches for clinical intervention in patients with MIRI.

Keywords: DNA-PKcs; Fis1; METTL3; cardiac ischemia-reperfusion injury.; mitochondrial fission.

Figure 1

Mettl3 knockdown reduces reperfusion-related myocardial dysfunction. Mettl3^flox and Mettl3^Cko mice were subjected to 30-min ischemia via temporary LAD ligation, followed by 6-h reperfusion to induce MIRI. (A, B) TTC and Evans blue stainings were used to examine the extent of myocardial infarction. (C-F) ELISA was used to measure serum concentrations of myocardial injury biomarkers, including Troponin T (TnT), creatine kinase MB (CK-MB), lactate dehydrogenase (LDH), and brain natriuretic peptide (BNP). (G) Representative ECG signals from mice subjected to MIRI. (H) Ultrastructural analysis of the myocardium using EM. (I-J) Sirius Red staining of cardiac fibrosis. *p<0.05 vs. sham group, #p<0.05 vs. MIRI group.

Figure 2

Mettl3 deletion maintains heart function following cardiac I/R. (A-F) Echocardiography was utilized to assess MIRI-related changes in heart function in Mettl3^flox and Mettl3^Cko mice. (G-L) Analysis of the contractile properties of cardiomyocytes isolated from Mettl3^flox and Mettl3^Cko mice. *p<0.05 vs. sham group, #p<0.05 vs. MIRI group.

Figure 3

Mettl3 deficiency reduces myocardial inflammation and cardiomyocyte death after MIRI induction. (A-C) RT-qPCR analysis of the transcription of pro-inflammatory genes, including IL-6, MM9, and TNFα, in mouse cardiac tissue. (D-E) Gr-1 immunofluorescence was conducted in heart tissues to determine neutrophil infiltration rates. A TnT antibody was used to stain myofibrils and DAPI was used for nuclear counterstaining. (F) ELISA was used to observe changes in caspase-3 activity in cultured HL-1 cardiomyocytes exposed to hypoxia/reoxygenation (H/R). (G, H) TUNEL staining was used to detect apoptosis in H/R-treated HL-1 cells. (I) Results of CCK-8 assays, used to determine the effect of Mettl3 deletion on the viability of H/R-exposed HL-1 cells. (J) The concentration of LDH in cultured media from HL-1 cells was determined by ELISA. *p<0.05 vs. sham group or Ctrl group, #p<0.05 vs. MIRI group or H/R group.

Figure 4

Mettl3 deletion attenuates mitochondrial abnormalities in cardiomyocytes exposed to H/R injury. (A). EM was used to observe ultrastructural changes in mitochondria from H/R-treated HL-1 cells. (B, C) Mitochondrial membrane potential was evaluated in HL-1 cells loaded with JC-1 by quantification of the red-to-green fluorescence ratio. (D) Quantification of ATP production by ELISA in HL-1 cells. (E, F). The activities of Bax and Bcl-2 in HL-1 cells were measured through ELISA. *p<0.05 vs. Ctrl group, #p<0.05 vs. H/R group.

Figure 5

Mettl3 ablation attenuates I/R- and H/R-mediated mitochondrial fission in cardiomyocytes. (A-D) RT-qPCR-based transcriptional analysis of Drp1, Mff, Mfn2, and Opa1 expression in heart tissues. (E-G) Mitochondrial immunofluorescence was used to monitor mitochondrial fission. Mitochondrial length as well as the ratio of fragmented to tubular mitochondria were recorded. *p<0.05 vs. sham group or Ctrl group, #p<0.05 vs. MIRI group or H/R group.

Figure 6

Mettl3 deficiency prevents I/R-mediated DNA-PKcs phosphorylation and activation. (A) ELISA was used to assess the activity of DNA-PKcs in HL-1 cells exposed to H/R injury. (B-D) Western blots were conducted to analyze DNA-PKcs and Fis1 phosphorylation status in H/R-treated HL-1 cells. *p<0.05 vs. Ctrl group, #p<0.05 vs. H/R group.

Figure 7

Mettl3 deficiency improves mitochondrial performance and cardiomyocyte viability through interrupting the DNA-PKcs/Fis1/mitochondrial fission pathway. FCCP was added to cultured HL-1 cells 30 min before H/R exposure to stimulate mitochondrial fission. (A, B) Mitochondrial membrane potential was measured in HL-1 cells loaded with the JC-1 probe by quantifying the relative red-to-green fluorescence ratio. (C) ATP production was measured via ELISA in cultured HL-1 cells. (D, E) The activities of Bax and Bcl-2 were measured through ELISA in cultured HL-1 cells. (F, G). TUNEL staining was used to estimate apoptosis in cultured HL-1 cells. (H) Results of CCK-8 assays, conducted to quantify HL-1 cell viability. *p<0.05 vs. Ctrl group, #p<0.05 vs. H/R group, **p<0.05 vs. H/R+sh/Mettl3+FCCP group.