Identification of Hub Genes in the Remodeling of Non-Infarcted Myocardium Following Acute Myocardial Infarction

(1) Background: There are few diagnostic and therapeutic targets for myocardial remodeling in the salvageable non-infarcted myocardium. (2) Methods: Hub genes were identified through comprehensive bioinformatics analysis (GSE775, GSE19322, and GSE110209 from the gene expression omnibus (GEO) database) and the biological functions of hub genes were examined by gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Furthermore, the differential expression of hub genes in various cell populations between the acute myocardial infarction (AMI) and sham-operation groups was analyzed by processing scRNA data (E-MTAB-7376 from the ArrayExpress database) and RNA-seq data (GSE183168). (3) Results: Ten strongly interlinked hub genes (Timp1, Sparc, Spp1, Tgfb1, Decr1, Vim, Serpine1, Serpina3n, Thbs2, and Vcan) were identified by the construction of a protein–protein interaction network from 135 differentially expressed genes identified through comprehensive bioinformatics analysis and their reliability was verified using GSE119857. In addition, the 10 hub genes were found to influence the ventricular remodeling of non-infarcted tissue by modulating the extracellular matrix (ECM)-mediated myocardial fibrosis, macrophage-driven inflammation, and fatty acid metabolism. (4) Conclusions: Ten hub genes were identified, which may provide novel potential targets for the improvement and treatment of AMI and its complications

Master regulator genes and their impact on major diseases

Master regulator genes (MRGs) have become a hot topic in recent decades. They not only affect the development of tissue and organ systems but also play a role in other signal pathways by regulating additional MRGs. Because a MRG can regulate the concurrent expression of several genes, its mutation often leads to major diseases. Moreover, the occurrence of many tumors and cardiovascular and nervous system diseases are closely related to MRG changes. With the development in omics technology, an increasing amount of investigations will be directed toward MRGs because their regulation involves all aspects of an organism’s development. This review focuses on the definition and classification of MRGs as well as their influence on disease regulation

SMYD1, an SRF-Interacting Partner, Is Involved in Angiogenesis

<div><p>Previous studies have demonstrated that Smyd1 plays a critical role in cardiomyocyte differentiation, cardiac morphogenesis and myofibril organization. In this study, we uncovered a novel function of Smyd1 in the regulation of endothelial cells (ECs). Our data showed that Smyd1 is expressed in vascular endothelial cells, and knockdown of SMYD1 in endothelial cells impairs EC migration and tube formation. Furthermore, Co-IP and GST pull-down assays demonstrated that SMYD1 is associated with the Serum Response Factor (SRF). EMSA assays further showed that SMYD1 forms a complex with SRF and enhances SRF DNA binding activity. Our studies indicate that SMYD1 serves as an SRF-interacting protein, enhances SRF DNA binding activity, and is required for EC migration and tube formation to regulate angiogenesis.</p></div

SMYD1 expressed in vascular endothelial cells.

<p>(A) RT-PCR analysis of SMYD1 gene expression in HUVEC and HMEC-1 cells. (B) Western blot analysis of endogenous SMYD1 and SRF expression in HUVEC and HMEC-1 cells. Over-expressed Flag-SMYD1 and HA-SRF were used as a positive control to indicate the sizes of SMYD1 or SRF. (C) and (D) Immunohistochemical expression of SRF and SMYD1 in mouse limb buds at E12.5 was shown. Both SMYD1 and SRF were expressed by vascular endothelial cells. Sections were slightly counterstained with hematoxylin.</p

SMYD1 interacts with SRF <i>in vitro</i>.

<p>GST pull-down assay for determination of SMYD1-SRF interaction. (A) The GST control protein, GST-SRF and its deletion mutants were incubated with SMYD1 protein that was produced by <i>in vitro</i> translation in the presence of [³⁵S] methionine. Bound proteins were analyzed by autoradiography. (B) GST control protein, GST-SMYD1-C, GST-SMYD1-N and GST-Nkx2.5-H (positive control) were incubated with SRF protein, produced by <i>in vitro</i> translation in the presence of [³⁵S] methionine. Bound proteins were analyzed by autoradiography. (C) SMYD1 protein that was produced by <i>in vitro</i> translation in the presence of [³⁵S] methionine. SRF protein, which was produced by <i>in vitro</i> translation in the absence of [³⁵S] methionine. The Co-IP experiment was carried out as described in the Materials and Methods section. Anti-HA antibody (negative control) and anti-SRF antibody were used for IP, and the immunoprecipitates were examined by autoradiography. The results represent the averages ± mean from three independent experiments.</p

SMYD1 knockdown impairs angiogenesis <i>in vitro</i>.

<p>(A) and (B) Capillary tube formation of HMEC-1 cells infected with sh control or SMYD1 shRNAS (sh1SMYD1 or sh2SMYD1) seeded onto Matrigel. After 4 to 6 h, HMEC-1 cells were fixed and tubular structure was quantified by calculating the tube length of high-power fields (200×). All error bars represent SD. A value of *<i>p</i><0.05 was compared with the control.</p

SMYD1 forms complex with SRF and enhances SRF DNA binding activity.

<p>Nuclear protein was isolated from 293T cells. (A) EMSA was performed using purified GST-SMYD1-N fusion protein, SRF- <i>in vitro</i> trans-protein and ³²P-labeled oligonucleotide probes containing a consensus binding motif for SRF. Lane 1 was vector control, lane 3 contained 100× cold as a competitor. SMYD1 dose-dependently enhances SRF DNA binding activity (lanes 2, 5, 6). The anti-SRF antibody (lane 4) and anti-SMYD1 antibody (lane 7) were used for the supershift assay. (B) EMSA was performed using purified GST-SMYD1-N fusion protein, SRF-transfected-protein and ³²P-labeled oligonucleotide probes containing a consensus binding motif for SRF. Lane 1 was vector control, lane 3 contained 100× cold as a competitor. SMYD1 dose-dependently enhances SRF DNA binding activity (lanes 4, 5). The anti-SRF antibody (lane 6) and anti-SMYD1 antibody (lane 7) were used for the supershift assay. The anti-His antibody was used as a control (lane 8). (C) EMSA was performed using purified GST-SMYD1-N fusion protein, SRF fusion protein and ³²P-labeled oligonucleotide probes containing a consensus binding motif for SRF. The anti-SRF antibody (lane 2, 4) and anti-SMYD1 antibody (lane 3) were used for the supershift assay. The IgG was used as a control (lane 5).</p

SMYD1 interacts with SRF in cells.

<p>(A) Co-IP experiment in 293T cells. Flag-SMYD1, HA-SRF or null vector (pCMV-HA or pCMV-tg2B) were transfected into 293T cells as indicated and cell lysates were then immunoprecipitated using anti-Flag or anti-HA antibody. The immunoprecipitates were examined by western blotting using anti-HA or anti-Flag antibodies. Input represented 10% of cell lysates used in the Co-IP experiment. (B) SMYD1 co-localization with SRF. Hela cells were transiently transfected with Flag-SMYD1 and HA-SRF. Then, cells were fixed and stained for anti-Flag and anti-HA antibodies. (C) and (D) Mapping of SMYD1 and SRF to identify the SRF or SMYD1-binding region. A total of 293T cells were transfected with Flag-SMYD1 in addition to different HA-SRF deletion mutants as indicated, or cells were transfected with HA-SRF and different Flag-SMYD1 deletion mutants as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody. The immunoprecipitates and cell lysates were then analyzed by western blotting separately using anti-HA antibody, anti-flag antibody for Flag-SMYD1 and its deletion mutants as indicated, and anti-HA antibody for HA-SRF and its deletion mutants as indicated.</p