C/EBP transcription factors mediate epicardial activation during heart development and injury

Abstract

The epicardium encapsulates the heart and functions as a source of multipotent progenitor cells and paracrine factors essential for cardiac development and repair. Injury of the adult heart results in reactivation of a developmental gene program in the epicardium, but the transcriptional basis of epicardial gene expression has not been delineated. We established a mouse embryonic heart organ culture and gene expression system that facilitated the identification of epicardial enhancers activated during heart development and injury. Epicardial activation of these enhancers depends on a combinatorial transcriptional code centered on CCAAT/enhancer binding protein (C/EBP) transcription factors. Disruption of C/EBP signaling in the adult epicardium reduced injury-induced neutrophil infiltration and improved cardiac function. These findings reveal a transcriptional basis for epicardial activation and heart injury, providing a platform for enhancing cardiac regeneration.

Fig. 1

Functional screen and identification of epicardial enhancers. (A) Epicardial lacZ expression in E11.5 mouse hearts 1 to 3 days after transfection of a CMV-lacZ plasmid. Whole-mount and transverse-section views are presented. (B and C) Enhancer activity of each conserved region (CR) in the epicardium [(B), n = 2 to 4 hearts] and in HEK293 cells [(C), n = 3] (mean ± SEM). (D) Relative activity. The data for Raldh2 CR2 and Wt1 CR14 are highlighted in red. (E and F) Raldh2 CR2 and Wt1 CR14 are sufficient and necessary to direct epicardial gene expression. Displayed are transgenic hearts that express a nuclear lacZ (nlacZ) driven by an enhancer (left), an EGFP reporter in a control BAC (middle), or an EGFP reporter in an enhancer-deleted BAC (right). The number of embryos that show epicardial reporter activity out of the total number of transgenic embryos is shown. Scale bars, 200 μm. *P < 0.05; **P < 0.01.

Fig. 2

Dynamic activity of Raldh2 and Wt1 epicardial enhancers. (A) Epicardial enhancer activity of Raldh2 CR2 maps to a 160-bp region (mean ± SEM, n = 2 to 4 hearts). (B) The Raldh2 CR2 minimal enhancer directs epicardial expression in vivo. The staining pattern represents six out of seven F₀ transgenic embryos analyzed. (C) Temporal and spatial expression of the 160-bp Raldh2 CR2-nlacZ transgene in a stable mouse line with an emphasis on the activity in the heart. (D) Dynamic activity of the 635-bp Wt1 CR14 enhancer in a stable mouse line. (E) Mapping of the epicardial enhancer activity of Wt1 CR14 to a 53-bp sequence (n = 2 to 4 hearts). (F) Epicardial expression of a 53-bp Wt1 CR14-nlacZ transgenic heart, which represents three out of six transgenic embryos analyzed. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bars, 200 μm.

Fig. 3

A C/EBP-dependent combinatorial code for Raldh2 and Wt1 activation in the embryonic epicardium. (A and B) Quantitative analyses of mRNA abundance and relative enrichment of C/EBP family members in E11.5 epicardial cells (n = 3). (C) Binding of C/EBPβ proteins to predicted sites. (D) Activation of the wild-type (wt) but not mutant (m) enhancers by C/EBP proteins in HEK293 cells (n = 3). (E) Mutations of C/EBP sites reduce epicardial enhancer activity in vivo. (F) X-gal stain of a transgenic embryo. H, heart; L, Liver. (G) Immunostaining of E12.5 transgenic hearts. DAPI, 4′,6-diamidino-2-phenylindole (H and I) Three–base-pair deletion scanning analyses of minimal enhancers (n = 2 to 6 hearts). The numbers on the top denote the positions of the critical regions (red) revealed by functional mapping. (J to M) Point mutations in either HOX, MEIS, or CP2 binding sites abolish epicardial enhancer activity in heart organ cultures [(J and L), n = 3 hearts] and transgenic embryos (K and M). For (E), (K), and (M), the number of epicardial lacZ⁺ embryos out of the number of transgenic embryos analyzed is presented in the upper right corner. Scale bars: 2 mm (F), 200 μm [(E), (K), and (M)], 100 μm (G). All error bars are SEM. In statistical analyses, all mutants were compared with wild-type controls. *P < 0.05; **P < 0.01; ***P < 0.005.

Fig. 4

Epicardial C/EBP signaling regulates injury-induced epicardial gene activation and cardiac remodeling in adult hearts. (A and B) Reactivation of Raldh2 and Wt1 enhancers after myocardial infarction (MI). Magnified views of boxed areas are shown below. Arrowheads mark coronary ligatures. (C and D) Diminished injury responses in transgenic mice carrying C/EBP-binding mutant enhancers. The number of epicardial lacZ⁺ hearts out of the number of transgenic hearts analyzed is shown. (E) Epicardial induction of C/EBPβ proteins 3 days after MI. (F) Reduced RALDH2 and WT1 expression in AdACEBP-infected epicardial cells 3 days after MI. White arrowheads point at infected cells (GFP⁺) and yellow arrowheads mark uninfected epicardial cells (GFP^–). (G) Magnetic resonance images (left) and ejection fraction (EF) measurements (right) of AdGFP- versus AdACEBP-injected hearts at different weeks (w) after IR surgery. (H) Analysis of fibrotic area (stained in blue in the trichrome stain) of heart sections from AdGFP- versus AdACEBP-injected mice 12 weeks after IR surgery. (I) Immunohistochemical stains of GR-1 on heart sections reveal enrichment of neutrophils around the epicardium 1 day after IR injury. (J) Neutrophil counts in the infarct area of AdGFP- versus AdACEBP-injected hearts 1 day after IR. LV, left ventricle. For (G), (H), and (J), the number in the column denotes the total number of animals analyzed. Scale bars, 200 μm for (A) to (D) and (H); 20 μm for (E), (F), (I), and (J). *P < 0.05; ** P < 0.01.