Journal: Nature Cardiovascular Research

Article Title: Oxidative phosphorylation is required for cardiomyocyte re-differentiation and long-term fish heart regeneration

doi: 10.1038/s44161-025-00718-x

Figure Lengend Snippet: a , Representative images of immunofluorescence staining showing Mef2 + PCNA + double-positive proliferating cardiomyocytes in TU and WIK at 7 dpci. Framed areas highlight the wound border zone (red) (scale bar, 300 μm). b , Quantification of Mef2 + PCNA + double-positive cells showing differences in proliferating cardiomyocytes in the border zone at 7 dpci but not at 21 dpci. c , Positive correlation of percentage of proliferating cardiomyocytes in the border zone between 7 dpci and 21 dpci. d , No correlation between 7 dpci border zone proliferation and 90 dpci wound length. e , Venn diagram displaying complete lack of overlap between genes correlating to 7 dpci border zone proliferation and 90-dpci wound length. f , g , No correlation between border zone proliferation and OXPHOS ( f ) or Glycolysis ( g ) at 7 dpci. h , Quantification of Mef2 + PCNA + cells showing no difference in proliferating cardiomyocytes in the border zone of 7 dpci KCL adult treated with inhibitor PF-04859989 or rotenone compared to DMSO control. i , Temporal wound length reduction of all strains between 1, 7, 21 and 90 dpci. Arrow highlighting the strong decrease in wound length in WIK between 7 dcpi and 21 dpci. j , Percentage of hearts completely regenerated at 90 dpci or with closed compact wall but remaining internal scar or with open compact wall and internal scar remaining. b , 7 dpci: AB, NA, TU n = 7; SAT, WIK n = 6; TL n = 5; KCL n = 3, 21 dpci: AB, SAT, TL, TU n = 5; NA n = 6; WIK n = 7; KCL n = 8 (biological replicates); h , PF-04859989, DMSO n = 6, rotenone n = 5 (biological replicates); i , AB: 1, 90 dpci n = 7; 7 dpci n = 8; 21 dpci n = 6. NA: 1, 7, 90 dpci n = 7; 21 dpci n = 11. SAT: 1, 7, 21, 90 dpci n = 7. TL: 1, 7, 90 dpci n = 7; 21 dpci n = 9. TU: 1, 7 dpci n = 7; 21 dpci n = 5; 90 dpci n = 6. WIK: 1, 7, 21, 90 dpci n = 7. KCL: 1, 7, 21, 90 dpci n = 8 (biological replicates); j , AB, NA, SAT, TL, WIK n = 7; TU n = 6; KCL n = 8. b , h , One-way ANOVA with Tukey’s test. c , d , f , g , Simple linear regression. h , Two-way ANOVA with Tukey’s test. i , Data presented as mean ± s.e.m. Mef2, myocyte enhancer factor 2; PCNA, proliferating cell nuclear antigen.

Article Snippet: Primary antibodies Mef2c (Biorbyt, orb256682), PCNA (clone PC10; Dako, M0879), GFP (Abcam, ab13970), MF20 (Developmental Studies Hybridoma Bank (DSHB), AB_2147781) and embcmhc N2.261 (DSHB, AB_531790) and secondary antibodies Alexa Fluor 488 (Invitrogen, anti-mouse A11001, anti-chick A11039 and anti-rabbit A21206) and Alexa Fluor 555 (Invitrogen, A31570 ) were prepared using TNB buffer at a ratio of 1:200.

Techniques: Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) Schematic of cardiac progenitors and their contributions to linear heart tube development from cardiac crescent (E7.75) to looped heart tube (E9) stage. B) Immunofluorescent staining of MEF2C (cyan) and cardiac Troponin T (cTnT, magenta) in E7.75, E8.5, and E9 WT embryos. C) Representative images of WT and MEF2C KO embryos at E7.75, E8.5 and E9. Cardiac progenitors are marked by the Smarcd3 -F6-eGFP reporter transgene (green). BF, brightfield. D) Schematic of the methodology and biological insights presented in the current study. Elements of this panel were created in BioRender. B, B. (2024) https://BioRender.com/i72e213 . Scale bars = 200 μm. FHF, first heart field; aSHF, anterior second heart field; pSHF, posterior second heart field; LV, left ventricle; RV, right ventricle; V, ventricle; IFT, inflow tract; OFT, outflow tract.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Staining

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A-C) UMAPs of snRNA-seq data for cardiac progenitors, cardiomyocytes, and related mesoderm subtypes from E7.75 (A), E8.5 (B), and E9 (C) embryos labeled by cell type (left) and genotype/sample ID (right). D-F) Bar plots displaying the number of up-regulated and down-regulated genes (MEF2C KO-vs-WT) for cell types of interest at E7.75 (D), E8.5 (E), and E9 (F). G-I) Dot plots displaying gene expression of key CM genes and anterior/posterior (A/P) markers at E7.75 (G), E8.5 (H), and E9 (I). J) Fluorescence in situ hybridization of key CM genes and A/P markers in E8-E8.5 WT and MEF2C KO embryos. Scale bars = 200 μm. CMs, cardiomyocytes; FHF, first heart field; SHF, second heart field; JCF, juxtacardiac field; CrM, cranial mesoderm; PrxM, paraxial mesoderm; LPM, lateral plate mesoderm; SoM, somitic mesoderm; NMPs, neuromesodermal progenitors; KPs, kidney progenitors; ExM, extraembryonic mesoderm; HSCs, hematopoietic stem cells; V-CMs, ventricular cardiomyocytes; IFT-CMs, inflow tract cardiomyocytes; OFT-CMs, outflow tract cardiomyocytes; aSHF, anterior second heart field; pSHF, posterior second heart field; PostM, posterior mesoderm; PhM, pharyngeal mesoderm; MixM, mixed mesoderm; A-CMs, atrial cardiomyocytes; AVC-CMs, atrioventricular canal cardiomyocytes; Pe, proepicardium; VP, venous pole; *, Genes known to be associated with CHDs ; #, Direct targets of MEF2C based on MEF2C ChIP-seq data .

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Labeling, Gene Expression, Fluorescence, In Situ Hybridization, ChIP-sequencing

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) UMAP of integrated snRNA-seq and snATAC-seq data for cardiac progenitors, cardiomyocytes, and related mesoderm subtypes at E8.5 labeled by cell types determined from snRNA-seq clustering (left) and genotype/sample ID (right). B) Bar plots displaying the number of gained and lost DARs (MEF2C KO-vs-WT) for cell types of interest at E8.5. C) Venn diagrams displaying unique and overlapping DARs in the three heart tube segments. D) Scatter plots displaying the relationship between the Log2 fold change (Log2FC) values of the DEG and DAR analyses for all identified Peak2Gene (P2G) links in the three heart tube segments. Dots are colored by the P2G correlation score. E-F) Genome browser tracks displaying snATAC-seq accessibility profiles at the Myh6 / Myh7 (E) and Wnt2 (F) loci for the indicated pseudobulked cell types. DARs are highlighted and the peaks are indicated by red bars (MEF2C KO-vs-WT IFT-CMs). Loops indicate P2G links, colored by the P2G correlation score. V-CMs, ventricular cardiomyocytes; IFT-CMs, inflow tract cardiomyocytes; OFT-CMs, outflow tract cardiomyocytes; aSHF, anterior second heart field; pSHF, posterior second heart field; LPM, lateral plate mesoderm; PostM, posterior mesoderm; PhM, pharyngeal mesoderm; NA, cells not available in the snRNA-seq dataset.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Labeling

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) Schematic of selection process to identify candidate MEFC-dependent enhancers from integrated snRNA-seq and snATAC-seq data. B) Genome browser tracks displaying snATAC-seq accessibility profiles, MEF2C ChIP-seq occupancy profiles , and H3K27ac ChIP-seq occupancy profiles at example loci containing IFT-specific (left), V-specific (middle), and OFT-specific (right) candidate enhancers (yellow highlights). C) Genome browser tracks displaying snATAC-seq accessibility profiles, MEF2C ChIP-seq occupancy profiles , and H3K27ac ChIP-seq occupancy profiles at the Myh6 / Myh7 locus, which contains two candidate enhancers (yellow highlights, MVEB1 and MVEB2) with IFT-specific altered accessibility that overlap with regions found in the VISTA Enhancer Browser database . D) Images of E11.5 mouse embryos from the Vista Enhancer Browser database demonstrating positive enhancer activity of regions that overlap with candidates MVEB1 and MVEB2. E) Schematic of the Tol2 Transgenesis assay used to screen candidate enhancers in zebrafish. Elements of this panel were created in BioRender. B, B. (2024) https://BioRender.com/h83r503 . F) Representative ventral view images of zebrafish embryos at 72 hpf injected with candidate enhancers that demonstrated positive activity in the heart. Boxed area in the representative brightfield image (left) indicates the anatomical region of interest captured in the fluorescent images. G) Schematic representation of the observed onset of enhancer activity for candidate enhancers that demonstrated positive activity in the heart. H) Representative ventral view images of zebrafish embryos at 24 and 72 hpf injected with MVEB2:eGFP or MVEB6:eGFP reporter constructs. Scale bars = 100 μm.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Selection, ChIP-sequencing, Activity Assay, Injection, Construct

Journal: bioRxiv

Article Title: Heart tube morphogenesis is regulated by segment-specific gene regulatory networks controlled by MEF2C

doi: 10.1101/2024.11.01.621613

Figure Lengend Snippet: A) TF binding motif enrichment analysis for gained DARs (MEF2C KO-vs-WT) in IFT-CMs at E8.5. B) Pie charts showing the proportion of gained or lost DARs (MEF2C KO-vs-WT) containing NR and MEF2C motifs or only NR motifs (top), and those containing GATA and MEF2C motifs or only GATA motifs (bottom). C) Odds ratio analysis for NR2F2 target genes (left) and GATA4 target genes (right) amongst DEGs up-regulated in MEF2C KO IFT-CMs. p-values were calculated using Fisher’s exact test. D) Ridge plots displaying module scores for up-regulated NR2F2 targets (left) and GATA4 targets (right) in MEF2C KO and WT heart tube segments. E) Inferred GRNs constructed for MEF2C KO and WT IFT-CMs at E8.5 and E9. F) Schematic of GRN validation by predicting gene expression with Mef2c knockdown in the E8.5 WT network and comparing to measured gene expression changes at E9. G) Results of GRN validation displaying predicted and measured gene expression changes. H) Visualizations of cardiac subnetworks within the WT and MEF2C KO E8.5 IFT-CM GRNs. I-J) Visualizations of direct NR2F2 (I) and GATA4 (J) interactions in the WT and MEF2C KO E8.5 IFT-CM GRNs. Direct interactions that occur upon MEF2C KO are highlighted. #, up-regulated DEG in MEF2C KO IFT-CMs at E8.5; *, Direct target of NR2F2 or GATA4 ( , ) . K-L) Immunofluorescent staining of cardiac Troponin T (cTnT, green) in representative E9.5 embryos (18-24 somites) collected from Mef2c del/+ ; Nr2f2 del/+ to Mef2c del/+ crosses. Boxed regions in (K) are shown at higher magnification in (L). Arrows indicate more expanded ventricle phenotype in Mef2c del/del ; Nr2f2 del/+ embryos. Asterisks indicate better developed and looping atria in Mef2c del/del ; Nr2f2 del/+ embryos. n=5 Mef2c del/del and n=4 Mef2c del/del ; Nr2f2 del/+ embryos from 5 independent litters. Scale bars = 200 μm.

Article Snippet: Primary antibodies used were sheep polyclonal MEF2C (R&D Systems, AF6786, used at 1:250) and rabbit polyclonal cardiac Troponin T (Thermo Fisher Scientific, 15513-1-AP, used at 1:250).

Techniques: Binding Assay, Construct, Biomarker Discovery, Gene Expression, Knockdown, Staining

Journal: Medicine

Article Title: LncRNA MCM3AP-AS1 is downregulated in atherosclerosis and sponges miR-448 to suppress vascular smooth muscle cell proliferation

doi: 10.1097/MD.0000000000033731

Figure Lengend Snippet: Overexpression of MCM3AP-AS1 increased the expression levels of MEF2C. To test whether MCM3AP-AS1 can serve as the endogenous sponge of miR-448, the effects of overexpression of miR-448 and MCM3AP-AS1 on the expression of MEF2C were analyzed by RT-qPCR (A) and Western blot analysis (B). Western blot data were also quantified (C). All experiments were repeated 3 times and mean values were presented and compared. *, P < .05. MEF2 = myocyte enhancer factor 2.

Article Snippet: After gel transfer and blocking, the membranes were incubated sequentially with primary antibodies against MEF2C (ab211493, Abcam) and GAPDH (ab9485, Abcam), followed by HRP-conjugated goat anti-rabbit secondary antibody (IgG) (ab97051, Abcam).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot

Journal: Medicine

Article Title: LncRNA MCM3AP-AS1 is downregulated in atherosclerosis and sponges miR-448 to suppress vascular smooth muscle cell proliferation

doi: 10.1097/MD.0000000000033731

Figure Lengend Snippet: MCM3AP-AS1 regulated the miR-448/MEF2C axis to suppress the proliferation of HAOSMCs. Cell proliferation assay was performed to analyze the effects of MCM3AP-AS1, miR-448 and MEF2C overexpression on the proliferation of HAOSMCs. All experiments were repeated 3 times and mean values were presented and compared. *, P < .05. MEF2 = myocyte enhancer factor 2.

Article Snippet: After gel transfer and blocking, the membranes were incubated sequentially with primary antibodies against MEF2C (ab211493, Abcam) and GAPDH (ab9485, Abcam), followed by HRP-conjugated goat anti-rabbit secondary antibody (IgG) (ab97051, Abcam).

Techniques: Proliferation Assay, Over Expression

Journal: Medicine

Article Title: LncRNA MCM3AP-AS1 is downregulated in atherosclerosis and sponges miR-448 to suppress vascular smooth muscle cell proliferation

doi: 10.1097/MD.0000000000033731

Figure Lengend Snippet: LncRNA MCM3AP-AS1 sponges miR-448 to suppress the proliferation of vascular smooth muscle cell through MEF2. MEF2 = myocyte enhancer factor 2.

Article Snippet: After gel transfer and blocking, the membranes were incubated sequentially with primary antibodies against MEF2C (ab211493, Abcam) and GAPDH (ab9485, Abcam), followed by HRP-conjugated goat anti-rabbit secondary antibody (IgG) (ab97051, Abcam).

Techniques:

Journal: Oncology Reports

Article Title: MicroRNA-190b expression predicts a good prognosis and attenuates the malignant progression of pancreatic cancer by targeting MEF2C and TCF4

doi: 10.3892/or.2021.8223

Figure Lengend Snippet: Primers and DNA segments used for vector construction.

Article Snippet: The membranes were incubated at room temperature for 2 h with 5% non-fat dry milk in Tris-buffered saline-0.5% Tween-20 prior to immunoblotting with primary antibodies against MEF2C (1:1,000; cat. no. YT2702, ImmunoWay Biotechnology Company) and TCF4 (1:1,000; cat. no. YT4580; ImmunoWay Biotechnology Company) at 4°C overnight.

Techniques: Plasmid Preparation

Journal: Oncology Reports

Article Title: MicroRNA-190b expression predicts a good prognosis and attenuates the malignant progression of pancreatic cancer by targeting MEF2C and TCF4

doi: 10.3892/or.2021.8223

Figure Lengend Snippet: MEF2C and TCF4 are direct targets of miR-190b in pancreatic cancer. (A) Potential target genes of miR-190b were predicted by using bioinformatic analyses. (B) Luciferase activities of MEF2C, TCF4, MEF2C-MUT and TCF4-MUT in AsPC-1 cells transfected with miR-190b mimics or NC. (C) Western blot analyses of MEF2C and TCF4 in transfected and parental AsPC-1 cells. Levels were normalized to those of GAPDH, and endogenous MEF2C and TCF4 levels were notably reduced in miR-190b-transfected AsPC-1 cells. Values are shown as mean ± SD. n=3. *P<0.05. miR, microRNA; MEF2C, myocyte enhancer factor 2C; TCF4, transcription factor 4; NC, negative control; WT, wild-type; MUT, mutated.

Article Snippet: The membranes were incubated at room temperature for 2 h with 5% non-fat dry milk in Tris-buffered saline-0.5% Tween-20 prior to immunoblotting with primary antibodies against MEF2C (1:1,000; cat. no. YT2702, ImmunoWay Biotechnology Company) and TCF4 (1:1,000; cat. no. YT4580; ImmunoWay Biotechnology Company) at 4°C overnight.

Techniques: Luciferase, Transfection, Western Blot, Negative Control

Journal: Oncology Reports

Article Title: MicroRNA-190b expression predicts a good prognosis and attenuates the malignant progression of pancreatic cancer by targeting MEF2C and TCF4

doi: 10.3892/or.2021.8223

Figure Lengend Snippet: High expression of MEF2C and TCF4 is associated with reduced miR-190b expression. (A and B) MEF2C and TCF4 expression in PDAC tissues was determined using immunohistochemistry. Positive expression is shown as brown-yellow particles distributed in the cell nucleus and cytoplasm. The cellular staining was classified using a scale of 0-2 as follows: 0, negative; 1, weakly positive; and 2, moderately positive. Scale bar, 200 µm. Magnification, ×100 (main panels) and ×200 (insets). (C) The non-parametric test demonstrated that miR-190b levels were significantly lower in samples with high expression of MEF2C and TCF4. (D) Pearson's correlation analysis demonstrated that miR-190b levels were significantly negatively correlated with MEF2C and TCF4 levels in pancreatic cell lines. Values are shown as mean ± SD. n=3. *P<0.05. miR, microRNA; MEF2C, myocyte enhancer factor 2C; TCF4, transcription factor 4; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: The membranes were incubated at room temperature for 2 h with 5% non-fat dry milk in Tris-buffered saline-0.5% Tween-20 prior to immunoblotting with primary antibodies against MEF2C (1:1,000; cat. no. YT2702, ImmunoWay Biotechnology Company) and TCF4 (1:1,000; cat. no. YT4580; ImmunoWay Biotechnology Company) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: A balanced circadian glycolytic rhythm drives cardiomyocyte cell cycle progression during fish heart regeneration

doi: 10.1101/2025.09.15.675600

Figure Lengend Snippet: a , Frequency histogram of DNA intensity of cardiomyocyte nuclei at 14 dpci in Pachón and surface hearts. Images of dissociated cardiomyocytes DNA content, showing G1/2n, S, and G2M/4n nuclei, stained with MF20 and DAPI. b , S:G2M ratio comparison of Mef2c+/PCNA+ BZ CM between Pachón and surface at 10 dpci (unpaired two-tailed Student’s t-test). c , RNAscope analysis of slc2a1a in 7 dpci Pachón and surface heart sections, co-stained with MF20. d , e , Glycolysis (ECAR) and ATP levels comparison before injury and at 7 dpci between Pachón and surface (one-way ANOVA with Tukey). f , S:G2M ratio comparison of Mef2c+/PCNA+ BZ CM between surface treated with 2-Deoxy-d-glucose and control hearts at 10 dpci (unpaired two-tailed Student’s t-test). g - i , Rhythmic expression of genes representing the glycolytic process , slc2a1a and per1a in Pachón and surface hearts at 7 dpci. ( g to i ) dryR analysis. Scale bars 100 μm. BZ, border zone cardiomyocytes; W, wound.

Article Snippet: Primary antibodies, Mef2c (Biorbyt Cat# orb1336562), MF20 (DSHB), PCNA (Clone PC10, Dako), BrdU (Abcam Cat# ab6326), and secondary antibodies, Alexa Fluor® 488 and Alexa Fluor® 568 (Invitrogen), were prepared in TNB buffer at a ratio of 1:200.

Techniques: Staining, Comparison, Two Tailed Test, RNAscope, Control, Expressing

Journal: bioRxiv

Article Title: A balanced circadian glycolytic rhythm drives cardiomyocyte cell cycle progression during fish heart regeneration

doi: 10.1101/2025.09.15.675600

Figure Lengend Snippet: a , RNAseq experimental design schematic. Horizontal bars indicate light (14h) and dark (10h) periods, before and during sampling of 7 dpci WT and insra mutant hearts. b , Dot plot showing phase and amplitude of core clock genes in WT vs. insra hearts at 7 dpci. c , Rhythmic expression of per1b in WT and insra hearts at 7 dpci. d , Heatmap of genes identified as rhythmic exclusively in WT 7 dcpi hearts (biological replicates ≥ 3 sampled at 4-hour intervals over 20 hours). e , f , Rhythmic expression of genes indicative of insulin signaling and glycolysis between WT and insra hearts at 7 dpci. g , Glycolysis (ECAR) in WT vs. insra hearts at 7 dpci (unpaired two-tailed Student’s t-test). h , Expression levels of SLC2A1 at ZT0 in WT and insra hearts at 7dpci (unpaired two-tailed Student’s t-test). i , Rhythmic expression of pcna in WT and insra hearts at 7 dpci. j , PCNA/Mef2-positive BZ CM quantification in WT vs. insra hearts at 7 dpci. k , AFOG staining showing collagen (blue) and fibrin (red) scars in WT vs. insra zebrafish hearts at 14 dpci with scar area quantification (unpaired two-tailed Student’s t-test). l , Circadian heatmap of g lycolysis and OXPHOS subset genes in WT and insra at 7 dpci heart. m , Immunofluorescence for γH2a.x in MF20+ BZ CMs at 7 dpci (unpaired two-tailed Student’s t-test). n , Rhythmic expression of genes representing cellular stress in WT and insra hearts at 7 dpci. o , Circadian heatmap of mitotic subset genes in WT and insra hearts at 7 dpci. p , q , Rhythmic expression of cdk1 and anln in WT and insra hearts at 7 dpci. r , S:G2M ratio comparison in Mef2c+/PCNA+ BZ CM between WT and insra hearts at 14 dpci (unpaired two-tailed Student’s t-test). ( b to f , i , l , n to q ) dryR analysis. Scale bars 100 µm. V, ventricle; W, wound.

Article Snippet: Primary antibodies, Mef2c (Biorbyt Cat# orb1336562), MF20 (DSHB), PCNA (Clone PC10, Dako), BrdU (Abcam Cat# ab6326), and secondary antibodies, Alexa Fluor® 488 and Alexa Fluor® 568 (Invitrogen), were prepared in TNB buffer at a ratio of 1:200.

Techniques: Sampling, Mutagenesis, Expressing, Two Tailed Test, Staining, Immunofluorescence, Comparison