MLL4 protects cardiomyocytes against ischemia-reperfusion injury through STAT3-mediated mitochondrial function

Figures

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  Fig. 1. MLL4 is upregulated during MIRI and NRCMs treated with H/R. A. The heatmap showing the normal H3K4me1 and H3K27ac flag thickness relative to translation begin destinations (TSS) with ±3 kb flanking locales gotten from the CUT&Tag in the heart of MIRI mice. B. The genomic dispersion of H3K4me1 and H3K27ac alteration crests over chromosomes. C. Heatmap illustrating relative gene expression for histone methylation modifiers, comparing non-failing (NF) and ischemic cardiomyopathy (ICM) tissue samples ((n = 3 for every group). D. Representative plots of immunohistochemical recoloring of heart tissue from MIRI and pretense mice, scale bar = 100 μm, (n = 6 for each group). E. Representative immunoblotting pictures appearing MLL4 protein levels within the heart of MIRI mice (n = 6 for every group) and NRCMs treated with H/R (n = 6 for every group). Fand G. Measurement of E. H. Measurement of D. Factual contrasts were decided by One Independent-sample t-test (two-tailed), *P < 0.05 and ***P < 0.001.

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  Fig. 2. Mll4 deletion in adult cardiomyocytes accelerates acute MIRI. A. The inquiry about flowchart. (1-7) B. Serum levels of phosphocreatine kinase (CK), C, CK-MB, and D, LDH in MIRI and pretense mice (n = 4 for each group). E-G. Representative images of Evans blue/TTC (triphenyl-2H-tetrazolium chloride)-staining and averaged infarct size in Mll4 transgenic (KO) mice after MIRI, (n = 4 for each group). H and I. Apoptosis of cardiomyocytes within the border zone by TUNEL recoloring in wild sorts and Mll4-cKO mice after MIRI, scale bar = 100 μm, (n = 8 for each group). J. The protein levels of Cleaved-caspase3 in Mll4-cKO mice after MIRI, (n = 6 for every group). K. Evaluation of (1-2) J. Data are shown as the mean ± SD. Statistical differences were determined by One-way ANOVA with Tukey test. Column bars represent mean, error bars indicate the SD. (B-D, I, K), One Independent-sample t-test (two-tailed) (F and G). AAR demonstrates region at hazard; and LV, left ventricle. *P < 0.05, **P < 0.01 and ***P < 0.001.

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  Fig. 3. MLL4 deficiency aggravates cardiac dysfunction, myocardial fibrosis and mitochondrial damage. A-I. Cardiac work was assessed by echocardiography Mll4-cKO mice at 28 days post-reperfusion (n = 6 for each group). J. Masson trichrome dyeing of WT and Mll4-cKO hearts 28 days post-surgery, scale bar = 1000 μm. K. Quantification of fibrotic area (%) in mice hearts treated as shown (n = 6 for each group). L. Representative transmission electron microscopy pictures of WT and Mll4-cKO hearts, scale bar = 1 μm. M. Measurement of mitochondria-related variables from L (n = 6 for each group). Mitochondrial region alludes to the proportion of mitochondrial range to picture area. N. Representative immunohistochemical staining images of TOMM20 in WT and Mll4-cKO mice, scale bar = 100 μm, (n = 6 for each group). O. Quantification of the TOMM20-positive area shown in panel N. Data are shown as the mean ± SD. Statistical differences were determined by One-way ANOVA with Tukey test, *P < 0.05, **P < 0.01 and ***P < 0.001. Column bars represent mean, error bars indicate the SD.

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  Fig. 4. MLL4 deficiency aggravated oxidative stress and mitochondrial damage, and promoted apoptosis in cardiomyocytes induced by H/R. A. Representative pictures of ROS in cardiomyocytes watched by confocal microscopy, scale bar = 100 μm. B. Evaluation of A (n = 6 for every group). C. Representative pictures of JC-1 staining in cardiomyocytes treated with H/R, scale bar = 100 μm. D. Detection of ROS release in cardiomyocytes by flow cytometry. E. The cell viability in H/R treated cardiomyocytes (n = 6 for each group). F. The discharge level of Malondialdehyde (MDA) in H/R treated cardiomyocytes (n = 6 for every group). G. Measurement of mitochondria-related proportion of green/red fluorescence (n = 9 for each group). H. Representative immunofluorescence staining images for TOMM20 in cardiomyocytes, scale bar = 10 μm. I. Corresponding quantification for TOMM20, (n = 6 for every group). J, M. Representative pictures of cardiomyocyte apoptosis identified by stream cytometry (n = 6 for every group). K. Representative immunoblotting pictures showing the BCL-XL protein levels in cardiomyocytes with MLL4 inhibition after H/R treatment. L. Quantification of K (n = 6 for every group). Data are…

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  Fig. 5. Inhibition of MLL4 reshapes the chromatin landscape and gene expression profiles in NRCMs. A. The pie chart outlining the genomic dispersion of H3K4me1 alteration crests over chromosomes (top), and a heatmap showing the normal H3K4me1 flag thickness relative to translation begin destinations (TSS) with ±3 kb flanking locales (bottom) gotten from the CUT&Tag measures for H3K4me1 in control H/R and Mll4-KO H/R treated NRCMs. B. Top translation figure themes distinguished by HOMER from regions with differential H3K4me1 adjustment within the Mll4-KO H/R group compared to the control H/R group in NRCMs. C. The number of related genes clarified in regions (crests), differentially adjusted for H3K4me1 levels utilizing the HOMER suite device, annotates Crests. D. Relationship heatmap of histone marks over all tests. Each group had three biological replicates. E. GO examination of genes with differentially H3K4me1 modified.

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  Fig. 6. MLL4 acts as a transcriptional activator of Stat3 through H3K4me1 and H3K27ac. A. Representative IGV browser tracks appearing MLL4 authoritative crests at the Stat3 locus in NRCMs (top). Tracks showing H3K4me1 and H3K27ac (middle) improvement at the Stat3 locus in Mll4-knockdown and control NRCMs taken after H/R treatment, and representative IGV browser tracks of Stat3 mRNA reads in Mll4-knockdown and Control NRCMs upon H/R treatment (bottom). Different colored boxes mark the candidate intronic fragments (En1-En4). Each track speaks to a combined flag from three organic duplicates. B. Representative immunoblotting pictures of MLL4, STAT3, p-STAT3 (Try705) and STAT3(Ser727) expression levels in Mll4 transgenic (KO) mice after MIRI. C, D Evaluation of B, (n = 6 for each group). E. Mice with or without MLL4 knockdown were treated with MIRI and arranged for immunofluorescence recoloring of p-STAT3 and WGA in heart tissues, scale bar = 50 μm. F, G. RT-qPCR investigation of Mll4 and Stat3 mRNA expression in NRCMs treated with H/R (n = 6, n = 8 for each group). Data are shown as the mean ± SD. Statistical differences were determined by One-way ANOVA with Tukey test, *P < 0.05, **P <…

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  Fig. 7. MLL4 overexpression promotes STAT3 expression and further alleviates cardiomyocytes apoptosis. A. Representative pictures of lentiviral transfection effectiveness in NRCMs were assessed by surveying EGFP and mCherry expression utilizing fluorescence microscopy. Cells showing the double inspiration for EGFP and mCherry were considered effectively transfected, scale bar = 100 μm. B. Representative immunoblotting pictures showing expanded expression of MLL4 in NRCMs through co-transfecting with sgRNAs and dCas9 lentivirus. C. Representative pictures of cardiomyocyte apoptosis identified by flow cytometry. D. Measurement of B (n = 6 for every group). E. Representative immunoblotting pictures showing the overexpression of MLL4, STAT3, p-STAT3, and Cleaved-caspase3 in NRCMs through co-transfecting with sgRNAs and dCas9 lentivirus. F. Measurement of C (n = 6 for every group). G-J. Measurement of E (n = 6 for every group). K. Representative images of MitoTracker staining in NRCMs using confocal microscopy, scale bar = 10 μm. L. Quantification of K (n = 8 for each group). Data are shown as the mean ± SD. Statistical differences were determined by One-way ANOVA with Tukey test, *P < 0.05,…

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  Fig. 8. STAT3 inhibition abrogates MLL4 overexpression induced protective effect. A. Representative immunoblotting images of MLL4, STAT3 and p-STAT3 in NRCMs via co-transfecting with sgRNAs and dCas9 lentivirus and siStat3 in NRCMs treated as shown. B–C. Quantification of A. D and E. Representative pictures of ROS in cardiomyocytes watched by flow cytometry (n = 8 for every group). F. The discharge level of MDA in H/R treated cardiomyocytes (n = 8 for every group). G and H. Representative images of cardiomyocyte apoptosis detected by flow cytometry (n = 6 for every group). I. Representative immunoblotting images of apoptosis-related proteins in NRCMs treated as shown. J-L. Quantification of I, (n = 6 for every group). Data are shown as the mean ± SD. Statistical differences were determined by One-way ANOVA with Tukey test, *P < 0.05, **P < 0.01 and ***P < 0.001. Column bars represent mean, error bars indicate the SD.