The MEF2A transcription factor interactome in cardiomyocytes

Fig. 1. Affinity purification and qualitative proteomic strategy to identify the Flag-MEF2A protein complex in PCMs. A PCMs are first isolated and then transfected with a 3xFlag-MEF2A construct. The Flag-MEF2A complex was pulled down and purified using α-Flag magnetic beads. The beads were washed several times and the Flag-MEF2A complex was eluted with Acetonitrile/TFA pH 1.9. B Six independent samples of Flag-MEF2A lysates and non-transfected lysates were used to run six IPs. The elutes were trypsin digested and then labelled with iTRAQ reagents. Isobaric labelling reagents that produce reporter ions at m/z 117 and 118 were not used. The labeled peptides were separated using HPLC and then identified using MS/MS by relative quantification. C 3xFlag MEF2A and non-transfected lysates were subjected to Western blotting to indicate protein expression (Top panels). Six separate IP experiments were performed with α-Flag-magnetic beads then eluted and blotted with anti-Flag and MEF2A antibodies to confirm the AP step. IP with lysates from non-transfected PCMs were used as negative controls. M is an indication of the loading of the marker lane (Bottom panels). D 56 proteins in the Flag-MEF2A…

Fig. 2. Protein networks associated with 56 enriched MEF2A interactors. A The network between identified proteins is built using the STRING database using confidence level of 0.4 (default setting) and with no additional interactors. The edge color corresponds to the confidence score depending on the database, as defined in the legend. The gray edges are added to the network and represent the novel protein-protein interaction network identified by our study. Node position and edge length and width are created arbitrarily. B The top scoring IPA protein networks ‘Gene expression, RNA Damage and Repair, RNA Post-Transcriptional Modification’ is depicted for the highly enriched proteins in MEF2A complex in PCMs. The shape of the node represents the molecular type of the proteins as defined in the legend. The red color intensity represents the magnitude of enrichment of the protein in the MEF2A complex. The protein interaction networks were generated with IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/). STAT3 gene is marked by a black star in both networks.

Fig. 3. Validation of selected putative interactions. A PCMs were transfected with a Flag-MEF2A construct and lysates were assessed for expression by Western blotting. Flag-MEF2A lysates were used for IP using α-Flag magnetic beads and the eluates were blotted with anti-Flag and STAT3 antibodies. IP with lysates from non-transfected PCMs were used as negative controls (Left panel). Non-transfected PCM lysates were used for IP using MEF2A antibody to confirm the interaction between endogenous MEF2A and RBPMS. IP with IgG was used as controls (Top right). Non-transfected PCM lysates were used for IP using a STAT3 antibody IP to confirm the interaction between endogenous STAT3 and MEF2A. IP with IgG was used as control (Bottom right). Number of biological replicates carried out for the Western blot and IP data is n = 3. B Confocal immunofluorescence analysis of endogenous MEF2A and STAT3 indicates a nuclear localization in PCMs. PCMs were fixed and stained for MEF2A in red and counterstained for STAT3 in green. Slides were analyzed by confocal microscopy. The scale bars are 2 and 10 μm. The intensity blot of MEF2A and STAT3 signals is shown over the region shown by the yellow line in the…

Fig. 4. STAT3 regulates MEF2A-dependent transcription. A PCMs were transfected with a 4xMEF2 luciferase reporter gene along with two independent siRNAs, siSTAT3#1 and #2, to deplete the endogenous STAT3 levels or a scrambled siRNA was used as control. Renilla luciferase was used to normalize for transfection efficiency. Lysates were collected at 48 h. The luciferase activity under each condition was measured and normalized to Renilla values to determine the fold change (Top panel). Corresponding western blot analysis using equal amounts of total protein of the cell lysates were used to confirm siRNA mediated STAT3 depletion as compared to scrambled controls (Bottom panel). Number of biological replicates for western blotting was n = 3. A schematic of 4xMEF2A-Luc construct is shown below the luciferase results. B PCMs were transfected with an α-MHC-Luc reporter gene with two independent siRNAs, siSTAT3#1 and #2, or scrambled siRNA as control. Luciferase activity was assessed after 48 h and normalized to Renilla activity (Top panel). Corresponding western blot analysis of cell lysate to confirm the depletion of STAT3 protein level as compared to the control is shown (Bottom panel). Number…

Fig. 5. Identification of differential expressed genes in STAT3 knock down in PCMs. A Workflow of RNA sequencing in PCMs. PCMs were transiently transfected by siRNA targeting STAT3 (siSTAT3#1) to deplete the endogenous STAT3 level or a scrambled siRNA used as controls at 48 h. RNAs were extracted for next gen-sequencing. B PCMs were transiently transfected with siSTAT3#1 or scrambled siRNA as control. Lysates were collecting at 48 h. Equal amounts of proteins were assessed by western blotting analysis to confirm siRNA mediated STAT3 depletion as compared to scrambled controls. Data are representative of n = 3 biological replicates. C Genes identified by RNA-seq analysis that are upregulated (Red) or downregulated (Blue) in STAT3 depleted PCMs (FDR < 0.05). D A volcano plot indicates upregulated and downregulated genes in siSTAT3 RNA-seq data. Black data point if FDR value >0.01. Red data points if FDR value < 0.01. Labels of the genes are shown if −1.35 > logFC > 2 and FDR value <0.01.

Fig. 6. Comparison between siMEF2A and siSTAT3 datasets. A 9 genes are common between DEGs in siMEF2A and siSTAT3 datasets in PCMs that are indicated in the Venn diagram (Top left). Table represents logFC of the 9 common genes in response to MEF2A and STAT3 depletion in PCMs (Top right). A Venn diagram represents the common genes between the three data sets: MEF2A DNA binding associated genes, siMEF2A DEGs, and siSTAT3 DEGs (Bottom left). Table represents logFC of the 11 common genes between MEF2A DNA binding associated genes and siSTAT3 DEGs (Bottom right). B The enriched GO BP terms for each dataset have been clustered using an Autoannotation plugin in Cytoscape software. The blue clusters identify the specific processes related to MEF2A in PCMs. The red clusters indicate the specific processes related to STAT3 in cardiac cells. The green cluster shows the common processes between MEF2A and STAT3 in primary cardiomyocytes. The edges between clusters show the shared genes between clusters. The cluster size and position are created arbitrarily. C 24 GO BP terms are common between siMEF2A and siSTAT3 datasets in PCMs. The node size represents the gene size. The edge represents the common…

Fig. 7. MEF2A/STAT3 complex regulates the MMP9 expression in cardiomyocytes. A PCMs were transiently transfected with an MMP9 luciferase reporter gene along with siRNA targeting the endogenous MEF2A and STAT3 levels individually and in combination. Scrambled siRNA was used as a control. Renilla luciferase was used to normalize for transfection efficiency. Lysates were collected at 48 h. The luciferase activity under each condition was measured and normalized to Renilla values to determine the fold change. N = 3 biological replicates per condition (Top panel). A representative western blot for three biological replicates for MEF2A, STAT3, MMP9, and β-actin is presented. Corresponding western blot analysis was used to confirm siRNA mediated MEF2A and STAT3 depletion as compared to scrambled controls (Bottom panel). A schematic of the MMP9-Luc construct is shown below the representative western blot. The expression of MEF2A, STAT3, and MMP9 are assessed using western blot analysis. The dot graphs represent the level of MEF2A, STAT3, and MMP9 proteins after normalization to β-actin (right panel). Both prominent bands (hyper and hypophosphrylated) for MEF2A are included together in the…

Fig. 8. MEF2A/STAT3 complex is involved in biological processes in myocardial cells. A MEF2A and STAT3 are involved in the increase of cardiomyocyte surface area in response to phenylephrine. Immunofluorescence analysis of α-Actinin and DAPI was performed in PCMs transfected with siRNA targeting MEF2A and STAT3 individually and in combination for 48 h then treated with PE (200 μM) or vehicle (H2O) for a further 48 h. The figure illustrates 2 representative images for each condition. The scale bar is 20 μm. B The scatter plot represents the quantification of the surface area of α-Actinin-positive cells using Image J software. The blue dots represent PCMs treated with H2O and purple dots represent the PCMs treated with PE. The data presented are derived from the surface areas of 100 cells that were randomly selected from four biological replicates (25 cells per biological replicate). Each cell surface area measurement is represented as a dot in each condition and the mean is indicated by a horizontal line. Tukey’s Multiple comparisons test in one-way ANOVA using GraphPad Prism 8.0 was used to test for statistical significance. * represents statistical significance from control…