Spatiotemporal il11a expression during heart regeneration in adult zebrafish.

a Representative cardiac section images of il11a^EGFP. Uninj, Uninjured. dpa, days post-amputation. b Quantification of EGFP expression area near the wound area. Biological replicates = 4, 3, 3, and 3 for uninjured and 3, 7, and 14 dpa hearts, respectively. Comparison of il11a^EGFP expressing cells with kdrl:mCherry (c) and tcf21:mCherry (d) in 3 dpa hearts. kdrl:mCherry and tcf21:mCherry represent endocardium and epicardium, respectively. Dash boxes correspond to the region magnified in the panels below. Dotted lines demarcate the amputation plane. Scale bars, 100 µm in (a), 50 µm in (c) and (d). Data are mean ± SEM in (b). p-values were determined by one-way ANOVA with Dunnett’s multiple comparison test in (b).

Myocardial il11a overexpression (OE) triggers CM proliferation in the absence of injury.

a Schematic of tamoxifen (Tam) or 4-HT (4-Hydroxytamoxifen) inducible il11a overexpression (OE). Tg(actb2:loxp-TagBFP-STOP-Loxp-il11a); cmlc2:CreER were administered with Tam or 4-HT to overexpress il11a (il11aOE). CreER-negative littermates were used as controls. b Representative cardiac section images stained with Mef2 (Red) and PCNA (Green) from control and il11aOE at 10 days post-treatment (dpt). Arrows indicate proliferative CMs. c Representative cardiac sections images stained with α-Actinin (green) from control and il11aOE uninjured hearts at 30 dpt. Insets correspond to higher magnifications of dashed boxes. d The percentage of PCNA⁺ CMs of uninjured hearts from control (Con) and il11aOE (OE) at 7 dpt. Biological replicates = 11 for control and il11aOE. e Quantification of disorganized sarcomere structures in the ventricles of Con and il11aOE at 30 dpt. Biological replicates = 3 and 4 for control and il11aOE, respectively. f Whole-mount images of 3- and 7-months post treatment (mpt) hearts from Con and il11aOE. g Quantification of the ventricle size from 3 mpt Con and il11aOE. Biological replicates = 8 for control and il11aOE. h Representative cardiac section images of 30 dpt control and il11aOE stained with Mef2 (red) and DAPI (blue). i Quantification for CM numbers in the ventricles from 30 dpt Con and il11aOE. Biological replicates = 5 for control and il11aOE. j Representative images of dissociated ventricular CMs stained with α-Actinin (green) from 2 mpt control and il11aOE. Quantification of the width (k) and the length (l) of individual CMs. Biological replicates = 92 and 234 for control and il11aOE, respectively. Scale bars, 50 µm in (b), (c) and (h), 0.5 mm in (f), 25 µm in (j). Data are mean ± SEM in (d), (e), (g), (i), (k), and (l). p-values were determined by unpaired two-tailed t-test in (d), (e), (g), (i), (k), and (l).

il11a induction stimulates vascularization in the absence of injury.

a Representative whole-mount images of 30 dpt fli1a:EGFP hearts from control and il11aOE. Insets correspond to higher magnifications of dashed boxes. b Quantification of fli1a:EGFP⁺ vessel area in the ventricles for control Con and il11aOE. Biological replicates = 5 for control and il11aOE. c Representative cardiac section images of 30 dpt control and il11aOE hearts. Green and red indicate fli1a:EGFP and myosin heavy chain (MHC), respectively. Arrows indicate expanded EGFP⁺ region in the cortical layer. d Quantification of EGFP⁺ area in the cortical muscle layer from Con and il11aOE. Biological replicates = 6 and 5 for control and il11aOE, respectively. e Gene expression plots of angptl2a, sulf1, and thsd7aa induced in epicardium at 3 days post injury (dpi). Cells expressing the indicated gene are colored purple, and the relative intensity indicates relative expression levels. Red dashed lines indicate epicardial expression of indicated genes. uniform manifold approximation and projection (UMAP) clustering with annotated cell-types are in Supplementary Fig. 6. f Representative images of in situ hybridization (ISH) for angiogenic factors (angptl2a, sulf1, thsd7aa) on cardiac sections of control and il11aOE. The number in the lower right corner of each image represents the fraction of the analyzed hearts with displayed phenotype. Biological replicates = 6 and 8 for control and il11aOE, respectively. g Representative cardiac section images of control and il11aOE expressing tcf21:mCherry; col12a1b:EGFP. Top, 10 dpt. Bottom, 30 dpt. Blue, DAPI. Green, EGFP. Red, mCherry. Arrows indicate tcf21:mCherry and col12a1b:EGFP double positive cells. Arrowheads indicate tcf21:mCherry^-; col12a1b:EGFP⁺ cells. h Representative cardiac section images of 30 dpt control and il11aOE hearts. Green indicates hapln1b:EGFP. i Quantification of hapln1b:EGFP⁺ area in the cortical muscle layer from Con and il11aOE. Biological replicates = 4 and 6 for control and il11aOE, respectively. Scale bars, 500 µm in (a), 50 µm in (c), (f), (g), and (h). Data are mean ± SEM in (b), (d), and (i). p-values were determined by unpaired two-tailed t-test in (b), (d), and (i).

Fibrotic roles of il11a in adult zebrafish hearts.

a Representative AFOG staining images of uninjured control and il11aOE hearts at 30 dpt (top), 3 mpt (middle), and 7 mpt (bottom). b Thickness of the collagen⁺ layer from Con and il11aOE at 3 and 7 mpt. Biological replicates = 8, 9, 9, and 8 for 3 mpt control, 3 mpt il11aOE, 7 mpt control, and 7 mpt il11aOE, respectively. c Differential expression of genes associated with regeneration (top), cardiac fibroblasts and pathological fibrosis (bottom) between 7 dpt and 7 mpt. Gene associated with fibrosis and fibroblasts are significantly upregulated in 7 mpt il11aOE hearts, compared to control, while those genes are not induced in 7 dpt il11aOE hearts. Expanded list of genes is in Supplementary Fig. 7a. d Representative cardiac section images of control and il11aOE hearts. Green and blue indicate Periostin (Postn)⁺ fibroblasts and DAPI, respectively. e Thickness of the Postn⁺ layer from Con and il11aOE at 10 and 30 dpt and 3 and 7 mpt. Biological replicates = 4 for all conditions, except 3 mpt control (n = 3). f Representative images of cardiac sections labeled with Postn (green) and tcf21:mCherry (red) at 30 dpt. Arrows indicate tcf21⁺ epicardial cells expressing Postn. g Colocalization assay of Postn⁺ cells with tcf21:mCherry⁺ layer. Biological replicates = 4 for control and il11aOE. h Representative cardiac section images stained with mCherry (red) and pERK (green) of 30 dpt Con and il11aOE hearts expressing tcf21:mCherry. i Quantification of pERK⁺ area in tcf21⁺ epicardium in the ventricles for Con and il11aOE. Biological replicates = 4 for control and il11aOE. j Dual roles of IL11 in the hearts. IL11 triggers cardiac regeneration programs, including CM proliferation and revascularization. Simultaneously, IL11 contributes to fibrosis by stimulating quiescent epicardial cells to generate epicardial progenitor cells (EPCs), which give rise to cardiac fibroblasts. Created in BioRender. Kang, J. (2024) BioRender.com/g25e465. Scale bars, 25 µm (top and middle) and 50 µm (bottom) in (a), 50 µm in (d), (f), and (h). Data are mean ± SEM in (b), (e), (g) and (i). p-values were determined by two-stage step-up multiple t-test in (b) and (e) and by unpaired two-tailed t-test in (g) and (i).

Combinatorial treatment enables to harness the regenerative potential of il11a in injured hearts.

a Representative cardiac section images of 14 dpi control and il11aOE hearts stained with Mef2 (red) and PCNA (green). Cryoinjury was performed one day after administering four daily injections of 4-HT. Arrowheads indicate proliferative CMs. b The percentage of PCNA⁺ CMs in the remote and border zone from Con and il11aOE at 14 dpi. Biological replicates = 14 for control and il11aOE. c Representative AFOG staining images of control and il11aOE hearts at 30 dpa. d Quantification of collagen⁺ area in the ventricle of control Con and il11aOE at 30 dpa. Biological replicates = 7 and 9 for control and il11aOE, respectively. e Dual roles of il11a in injured hearts. il11a promotes CM proliferation while inducing fibrosis via Extracellular signal-regulated kinase (ERK) activity. f Experimental scheme to harness the regenerative potential of il11a while mitigating fibrosis by treating U0126, an ERK inhibitor, starting at 14 dpi. g Representative AFOG staining images of vehicle (Veh, 0.1% DMSO) or U0126 (10 µM) treated hearts from control and il11aOE. Hearts were collected at 45 dpi/55 dpt. The arrow indicates the presence of scar tissue near the injury site in Veh-treated il11aOE hearts. h Quantification of scar area in the ventricles of Veh or U0126-treated control and il11aOE hearts. Biological replicates = 6, 7, 6, and 7 for control_DMSO, control_U0126, il11aOE_DMSO, and il11aOE_U0126, respectively. i Representative cardiac section images of Veh- or U0126-treated control and il11aOE hearts stained with Mef2 (green) and PCNA (red) at 45 dpi. Arrowheads indicate proliferative CMs. j The percentage of PCNA⁺ CMs in the ventricles of Veh or U0126-treated control and il11aOE hearts at 45 dpi. Biological replicates = 6, 7, 6, and 7 for control_DMSO, control_U0126, il11aOE_DMSO, and il11aOE_U0126, respectively. Scale bars, 25 µm in (a) and (g), 50 µm in (c) and (i). Data are mean ± SEM in (b), (d), (h) and (j). p-values were determined by unpaired two-tailed t-test in (b), (d), (h) and (j).