A steroid receptor coactivator stimulator (MCB-613) attenuates adverse remodeling after myocardial infarction

Fig. 1.. The SRC-3 activator MCB-613 attenuates loss of cardiac function post-MI. (A) Ejection fraction was measured by echocardiography at the indicated times. Values shown are mean ± SEM; two-way ANOVA with repeated measures. n = 10 control and n = 11 MCB-613. Day 14 **P 0.0078. Day 80 **P = 0.008. (B) Control-treated hearts (n = 3) and MCB-613-treated hearts (n = 3) stained with picrosirius red. Quantification of percent fibrosis. Student’s t test for fibrosis (*P = 0.03). (C) Values shown are mean ± SEM; Student’s t test for HW/TL (n = 6); (*P = 0.03).

Fig. 2.. RNA transcriptional profiling of cardiac cells 24 h and 12 wk post-MI. (A) Schematic representation of isolation procedures to obtain noncardiomyocytes for single cell RNA-seq analysis from control-treated and MCB-613-treated mice 24 h and 12 wk post-MI. n = 2 hearts/group. (B) UMAP plot of all cell populations identified by unsupervised clustering of single cell transcriptional data. Each dot indicates a single cell. Nonmacrophage immune cells (DC-like 1to 3), B cells, T cells, NK cells, granulocytes, macrophages (M1 to 4), endothelial cells (Endo1 to 3), cardiac fibroblasts (CF1 to 2), and epicardial cells. (C) Heat map of top differentially expressed genes in each cell cluster. (D) UMAP plot of subclustered cells at 24 h and 12 h. (E) The χ2 cluster composition analysis of cells from MCB-613 compared to control at 24 h and 12 wk post-MI.

Fig. 3.. Single cell analysis of fibroblast cells 24 h post-MI reveals that the MCB-613 protective response is associated with decreased fibroblast differentiation, decreased fibrosis, and Il-1 response. (A) UMAP plot of fibroblasts from control (salmon) and MCB-613 (aqua) 24 h post-MI. (B) χ2 cluster composition analysis of subclustered fibroblasts from MCB-613 compared to control at 24 h post-MI (Left). UMAP plot of fibroblast subclusters 24 h post-MI (Right). (C) Heat map showing top five differentially expressed genes. (D) Pseudotime trajectory for cardiac fibroblasts 24 h post-MI (Left). Pseudotime trajectory colored according to subcluster identity (Right). Density plots according to fibroblast subclusters and heatmap showing dynamics of gene expression for subclusters across the trajectory (Bottom). (E) Pseudotime trajectory colored according to χ2 cluster composition (Left) and treatment condition (Right). Density plot according to treatment condition. Gene expression trends for individual genes across the pseudotime trajectory (Bottom). (F) GO term analysis of differentially expressed genes from fibroblasts in control compared to MCB-613.

Fig. 4.. Single cell analysis of macrophages 24 h post-MI reveals that the MCB-613 protective response is associated with increased anti-inflammatory macrophages and macrophage and granulocyte functions. (A) UMAP plot of macrophages from control (salmon) and MCB-613 (aqua) 24 h post-MI. The χ2 cluster composition analysis of subclustered macrophages from MCB-613 compared to control at 24 h post-MI (Top Right). UMAP plot of macrophage subclusters 24 h post-MI (Bottom Right). (B) Heat map showing top five differentially expressed genes. (C) Pseudotime trajectory and density plot of Ccr2n macrophages across the pseudotime trajectory. Heatmap showing dynamics of gene expression for Ccr2n macrophages across the trajectory. (D) GO terms analysis of differentially expressed genes from macrophages in control compared to MCB-613. (E) Gene expression trends for individual genes across the pseudotime trajectory. (F) Feature plot showing Tsc22d3 expression in macrophages for control and MCB-613.

Fig. 5.. MCB-613 promotes long-lasting fibroblast differentiation and enrichment of Cbr2 macrophages. (A) χ2 cluster composition analysis of subclustered fibroblasts 12 wk post-MI (Left). UMAP plot of fibroblast subclusters (Right). (B) Heat map showing top differentially expressed genes. (C) Pseudotime trajectory and corresponding density ridge plot across the pseudotime trajectory colored according to cluster identity (Top). Heatmap showing dynamics of gene expression for fibroblasts across the trajectory (Bottom). (D) The χ2 cluster composition analysis of subclustered fibroblasts across the trajectory. Density ridge plot according to treatment condition. (E) GO terms analysis of differentially expressed genes from fibroblasts in control compared to MCB-613. (F) UMAP plot of macrophage subclusters 12 wk post-MI. (G) The χ2 cluster composition analysis of subclustered macrophages from MCB-613 compared to control 12 wk post-MI. (H) Cbr2 expression feature plot.

Fig. 6.. MCB-613 promotes granulocyte functions and reduces proinflammatory signaling in macrophages and fibroblasts. (A) mRNA expression in granulocytes isolated from bone marrow 24 h post-MI and MCB-613 treatment. Gene expression changes in S100a9, Tlr7, and Lcn2 were measured by qPCR and 18s RNA expression was used as a control. Values shown are mean ± SEM (n = 10 control group, n = 12 MCB-613 group); Student’s t test (Tlr7 *P = 0.01, Lcn2 *P = 0.04). (B) mRNA expression in RAW 264.7 macrophages stimulated with M1 reprogramming factors for 8 h followed by vehicle control or MCB-613 for an additional 24 h. Values shown are mean ± SEM (n = 3 control group, n = 3, 5 μM MCB-613 group, n = 3, 10 μM MCB-613 group); Student’s t test (Il1B *P = 0.01, ***P 1.3 × 10−5; Il6 ***P = 2.9 × 10−5, ***P = 3.1 × 10−6; Spp1 *P = 3.0 × 10−3, ***P = 2.4 × 10−4, Tnf-α ***P = 3.3 x 10−5, ***P = 5.0 × 10−4; Ccl4 *P = 0.03, ***P = 4.8 × 10−5; Cxcl9 *P = 0.02, ***P = 6.9 × 10−8. (C) Cardiac fibroblasts were treated with dimethylsulfoxide (DMSO) or MCB-613 for 24 h and protein was immunoblotted for SRC-1, -2, and -3. Cardiac fibroblasts were transfected with a GAL4 DNA binding site-luciferase reporter (pG5-luc)…