Actin‐related protein 5 suppresses the cooperative activation of cardiac gene transcription by myocardin and MEF2

Figures

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  Fig. 1. Change in Actr5 expression level in the hearts. (A) Changes in expression of the mouse Actr5 gene during heart development. RNA‐seq data were obtained from the Gene Expression Omnibus (GEO) database (accession no. GSE79883, left panel; GSE58455, right panel). (B) Expression of human ACTR5 gene in failing hearts. RNA‐seq data were obtained from GEO (GSE116250, left panel; GSE46224, right panel). (C) Hematoxylin and eosin staining images of murine hearts infected with control AAV6 (control‐AAV) or ARP5‐AAV6 (ARP5‐AAV) vectors (upper panels). Bar = 2 mm. Actr5 expression in these hearts was measured by real‐time RT‐PCR (lower left panel). The ratio of the heart weight to body weight (HW/BW) was calculated and statistically analyzed (lower right panel). (D) Masson's trichrome staining images of control AAV6‐ and ARP5‐AAV6‐infected hearts (upper panels). Bar = 100 μm. The blue‐stained fibrotic areas were measured and statistically analyzed (lower panel). (E) Expression of ARP5, COL1A1, ACTA2, and GAPDH proteins in control AAV6‐ and ARP5‐AAV6‐infected hearts (left panel). The band intensities were measured and statistically analyzed (right panels). All statistical data in Fig. 1 are…

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  Fig. 2. Upregulation of fibrotic genes in the ARP5‐overexpressing heart. (A) GSEA enrichment plot of the top 1000 genes whose expression was increased by ARP5‐AAV6 infection in the DNA microarray analysis for the gene set CUI_DEVELOPING_HEART_C3_FIBROBLAST_LIKE_CELL. (B) MA plot of the DNA microarray data between control AAV6‐ and ARP5‐AAV6‐infected hearts. The red dots indicate the position of the fibrotic genes. (C) Expression of fibrotic genes in control AAV6‐ (cont) and ARP5‐AAV6‐infected (OE) hearts was validated by real‐time RT‐PCR. Statistical data are presented as the mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01 (Student's t‐test).

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  Fig. 3. Downregulation of muscle‐related genes in the ARP5‐overexpressing heart. (A) GSEA enrichment plot of the top 1000 genes whose expression was decreased by ARP5‐AAV6 infection in the DNA microarray analysis for gene set DESCARTES_FETAL_MUSCLE_SKELETAL_MUSCLE_CELLS. (B) MA plot of the DNA microarray data between control AAV6‐ and ARP5‐AAV6‐infected hearts. The blue dots indicate the position of the muscle‐related genes. (C) GO analysis of the top 1000 genes whose expression was decreased by ARP5‐AAV6 infection in the DNA microarray analysis. (D) Expression of the muscle‐related genes in control AAV6‐ (cont) and ARP5‐AAV6‐infected (OE) hearts was validated by real‐time RT‐PCR. Statistical data are presented as the mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01 (Student's t‐test).

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  Fig. 4. Upregulation of muscle‐related genes by ARP5 knockdown in P19CL6 cells. (A) Enrichment analysis in ChIP Atlas using the top 1000 genes whose expression was decreased by ARP5‐AAV6 infection in the DNA microarray analysis. (B) Real‐time RT‐PCR analysis of Mef2c and Myocd genes in P19CL6 cells cultured in the presence and absence of DMSO. (C) Identification of the alternative spliced isoform of Myocd in murine hearts, aorta, and P19CL6 cells based on the presence or absence of Myocd exon 2a by RT‐PCR. White triangles indicate the position of PCR products from smooth muscle type (SM) and cardiac type (Card) isoforms of Myocd. (D) Real‐time RT‐PCR analysis of muscle‐related genes in P19CL6 cells transfected with the DOX‐regulated Actr5 shRNA expression vector. The cells were incubated with DMSO in the presence and absence of DOX for 14 days. All statistical data in (B) and (D) are presented as the mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01 (Student's t‐test).

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  Fig. 5. Cooperative action between cardiac MYOCD and MEF2C in muscle‐related gene expression. Real‐time RT‐PCR analysis of muscle‐related genes in P19CL6 cells exogenously expressing MEF2C, smooth muscle type (SM), or cardiac type (Card) isoforms of MYOCD, and ARP5 in the indicated combinations. Statistical data are presented as the mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01 (Student's t‐test).

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  Fig. 6. Interaction between ARP5 and distal N‐terminal region of cardiac MYOCD. (A) Schematic diagram of smooth muscle and cardiac MYOCD and the sequence of N‐terminal functional motifs. ++, basic domain; LZ, leucine zipper; Q, glutamine‐rich domain; RPEL, RPEL motifs; SAP, SAP domain; TAD, transactivation domain. Bold letters in the sequence indicate the RPxxxEL residues in the RPEL motifs. The locations of MEF2‐binding motif (MBM) and RPEL fragments used in the co‐immunoprecipitation analysis are also indicated. (B) Co‐immunoprecipitation analysis of FLAG‐tagged ARP5 (FLAG‐ARP5) with GFP‐tagged fragments of the RPEL motifs. (C) Co‐immunoprecipitation analysis of FLAG‐ARP5 with GFP‐tagged fragments of the RPEL1 and MBM. (D) Co‐immunoprecipitation analysis of competitive binding of myc‐ARP5 and MEF2C‐HA to the RPEL1‐GFP. (E) Promoter luciferase assay using the MEF2‐binding element promoter in P19CL6 cells exogenously expressing cardiac MYOCD, MEF2C, and ARP5 in the indicated combinations. Statistical data are presented as the mean ± standard error of the mean (SEM). *P < 0.05, **P < 0.01 (Student's t‐test).

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  Fig. 7. N‐domain requirement for ARP5 to bind to and inhibit cardiac MYOCD. (A) Schematic diagram of the domain‐truncated fragments of ARP5 protein. N‐domain (N) and C‐domain (C) are regions with similarities to other ARPs and conventional actins, while N‐terminal (S1), central (S2), and C‐terminal (S3) regions are unique to ARP5. (B) Promoter luciferase assay using the MEF2‐binding element promoter in P19CL6 cells exogenously expressing cardiac MYOCD and MEF2C together with the indicated truncated ARP5 fragments. Statistical data are presented as the mean ± standard error of the mean (SEM). **P < 0.01 (Student's t‐test). (C) Co‐immunoprecipitation analysis of truncated FLAG‐ARP5 fragments with RPEL1‐GFP.

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  Fig. 8. RNA‐seq analysis of cardiac MYOCD‐target genes. (A) Venn diagrams showing the number of genes whose expression was increased more than 4‐fold by the combination of cardiac MYOCD and MEF2C in RNA‐seq analysis. Also shown are the numbers of genes whose increased levels were suppressed by ARP5‐full or ARP5‐N to less than half. logFC represents log2 (fold‐change in gene expression level). (B) Heatmap representing the gene expression profiles from the RNA‐seq analysis. The left panel shows the logFC in the expression levels of the upregulated genes in cardiac MYOCD‐ and MEF2C‐expressing cells relative to mock‐transfected cells (control). The right panels show the logFC in gene expression for the cells expressing ARP5‐full or ARP5‐N in addition to cardiac MYOCD and MEF2C relative to the cardiac MYOCD‐ and MEF2C‐expressing cells. The representative gene names are listed in the center. (C) GO analysis of the genes whose levels of induction by MYOCD and MEF2C were suppressed by ARP5‐N (logFC < −1, upper panel) and those that were not (logFC > −1, lower panel). (D) Real‐time RT‐PCR analysis of cardiac genes in P19CL6 cells exogenously expressing cardiac MYOCD, MEF2C, ARP5‐full, and ARP5‐N…