M6a demethylase FTO regulates the oxidative stress, mitochondrial biogenesis of cardiomyocytes and PGC-1a stability in myocardial ischemia-reperfusion injury

Figures

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  Figure 1.. Construction of myocardial ischemia-reperfusion injury rats. (A)The MIRI rats showed a higher ST-segment elevation than the sham group (N = 6). (B) Echocardiography was performed to test heart function of rats in sham group, MIRI model group (N = 6). (C–D) Ejection fraction, fractional shortening of the left ventricular diameter (N = 6). (E) Heart sections of rats from the two groups were stained with TTC staining (N = 3). (F) The infarct size differences between the MIRI model group and sham group rats (N = 3). (G) H&E staining of the heart sections of the sham and MIRI rats (N = 3) (scale bar = 100 µm). ***p < 0.001. All the data were presented as the means ± SD.

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  Figure 2.. FTO was down-regulated in MIRI rats and H/R-induced cardiomyocytes. (A) FTO mRNA expression in MIRI and Sham rats (N = 3). (B) Western blot was conducted to access the protein expression of MIRI and Sham rats (N = 3). (C) FTO mRNA expression in H/R-induced and normal H9C2 cells (N = 3). (D) Western blot was conducted to access the protein expression of FTO in H/R-induced and normal H9C2 cells (N = 3). ***p < 0.001. All the data were presented as the means ± SD.

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  Figure 3.. Overexpression of FTO inhibits excessive oxidative stress and restores mitochondrial function in H/R cells. (A–B) RT-PCR and western blot were used to detect the mRNA and protein expression level in H/R (Vector) and H/R + FTO-OE cardiomyocytes (N = 3). (C) CCK8 assay showed the cell proliferation of H/R (Vector) and H/R + FTO-OE cardiomyocytes (N = 3). (D)The ROS level in H/R (Vector) and H/R + FTO-OE cardiomyocytes (N = 3). (E) RT-PCR showed the expression of SOD2 (N = 3). (F) The expression of mitochondrial transcription factor TFAM and mtDNA marker gene COXI (N = 3). (H) The protein expressions of SOD2, TFAM and COXI determined by Western blot assay (N = 3). (I–K) Quantitative analysis of expression level of SOD2, TFAM and COXI (N = 3). **p < 0.01, ***p < 0.001. All the data were presented as the means ± SD.

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  Figure 4.. Overexpression of FTO inhibits excessive oxidative stress and restores mitochondrial function in MIRI rats. (A) Electrocardiogram was performed to test heart function of the rats (N = 6). (B) Echocardiography was performed to test heart function of sham, MIRI, and MIRI + FTO-OE rats (N = 6). (C–D) Ejection fraction, fractional shortening of the left ventricular diameter (N = 6). (E) H&E staining of the heart sections of the rats (N = 3) (scale bar = 100 µm). (F) Heart sections of rats from the three groups were stained with TTC staining (N = 3). (G) The infarct size differences among the three group rats (N = 3). (H)The ROS level in H/R (Vector) and H/R + FTO-OE cardiomyocytes (N = 3). (I) RT-PCR showed the expression of SOD2 (N = 3). (J) The expression of mitochondrial transcription factor TFAM (N = 3). (K) RT-PCR detected the expression of COXI mRNA level (N = 3). (L) The protein expressions of SOD2, TFAM and COXI determined by Western blot assay (N = 3). (M-O) Quantitative analysis of expression level of SOD2, TFAM and COXI (N = 3). ***p < 0.001. All the data were presented as the means ± SD.

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  Figure 5.. Identification of PGC-1a as a downstream target of FTO. (A) FTO and PGC-1a mRNA expression in FTO overexpression and normal H/R induced H9C2 cells (N = 3). (B) FTO and PGC-1a protein expression in FTO overexpression and H/R induced H9C2 cells (N = 3). (C) FTO and PGC-1a mRNA expression in sham, MIRI, and MIRI + FTO-OE rats (N = 3). (D) FTO and PGC-1a protein expression in sham, MIRI, and MIRI + FTO-OE rats (N = 3). (E) Silver staining of the gel after SDS‒PAGE separation of the proteins that were immunoprecipitated with the 3′ biotin-labeled probe for PGC-1a and the biotinylated antisense probe in RNA pull-down assays (N = 3). The line shows the position of FTO. (F)Western blot analysis following the RNA pull-down assay indicated the interaction between FTO and PGC-1a (N = 3). (G) RT‒PCR analysis following the RIP assay confirmed the interaction between FTO and PGC-1a (N = 3). **p < 0.01, ***p < 0.001. All the data were presented as the means ± SD.

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  Figure 6.. FTO enhanced the stability of PGC-1a mRNA through uninstalling the m6A modification of PGC-1a mRNA. (A) m6A dot blot analysis detected the m6A content in total RNA of H/R (Vector) and H/R + FTO-OE cardiomyocytes (N = 3). (B) m6A dot blot analysis detected the m6A content in total RNA of sham, MIRI, and MIRI + FTO-OE rats (N = 3). (C) Online predictive tool SRAMP showed the potential m6A modification sites and the m6A modification sites of PGC-1a mRNA were identified in the genomic sequence (GGACA). (D) MeRIP-PCR assay indicated the m6A modification level of H/R-induced cardiomyocytes upon FTO overexpression (N = 3). (E) MeRIP-PCR assay indicated the m6A modification level of sham, MIRI, and MIRI + FTO-OE rats (N = 3). (F) RNA stability analysis detected PGC-1a mRNA expression in H/R-induced cardiomyocytes treated with actinomycin D (N = 3). *p < 0.05, **p < 0.01. All the data were presented as the means ± SD.