Platyfish possess a heart type lacking a compact vascularized myocardium.

(A) Simplified phylogenetic lineages of the relevant fish species, based on 11. The clade of teleosts diversified into two main branches 250 million years ago (mya). The Otophysi clade contains more than one-third of fish species, including zebrafish and tetras 41. Percomorphaceae is a hyperdiverse clade described as “the bush at the top” of the fish phylogenetic tree 100, which encompasses 55% of extant teleost diversity, including perciforms, cichlids and poeciliids 101. (B) AFOG staining of longitudinal heart sections of platyfish and zebrafish hearts. Col., collagen (blue). (C-H) Fluorescence staining of hearts shown in (B). The myocardium is F-actin-positive. Note the presence of the compact myocardium in zebrafish (D), and its absence in the platyfish (G). The N2.261 immunoreacts with a specific myosin isoform that is nearly absent in the uninjured zebrafish heart (C-E), except for a few fibers at the outflow tract (oft). In platyfish, this antibody immunolabels the atrium, suggesting evolutionary changes of myosins between the species. Fibronectin is an extracellular matrix protein present in the bulbus arteriosus. (I-L) The activity assay for alkaline phosphate reveals a dense network of blood vessels in zebrafish, but not in platyfish hearts. Frames depict the magnified areas shown on adjacent panels, labeled with the corresponding letter. avc, atrioventricular canal; at, atrium, ba, bulbus arteriosus; oft, outflow tract; v, ventricle. These conventions and abbreviations apply throughout all figures.

Cryoinjured ventricles in Xiphophorus fish display transient wound bulging and permanent scarring.

(A) Schematics of heart cryoinjury in platyfish. (B) Hearts from uninjured fish and at 7 days post-cryoinjury (dpci) stained with Phalloidin (green). The damaged area of the heart is revealed by a weak fluorescence signal in the absence of contractile cells. Arrowheads indicate the border between the intact myocardium and the wound. ba: bulbus arteriosus, at: atrium, v: ventricle. (C) AFOG staining of transversal ventricle sections of zebrafish, platyfish and swordtail, collected at different time points after cryoinjury. Intact myocardium (orange); fibrin and other protein deposits (red); collagen (blue). The arrowheads indicate the edge of the wound; double-ended arrows depict the myocardial (myo) bridge; the dashed line encircles the wound tissue that has expanded beyond the normal circumference of the heart (i.e. a pseudoaneurysm).

Partial restoration of the heart in platyfish and swordtail after cryoinjury.

(A) Classification of injury phenotypes based on AFOG staining, representatively shown in Figure 2. The bar plots display the percentage of each category at different time points after injury in platyfish and swordtail. Numbers at the bottom of each bar correspond to biological replicates (fish). Pearson’s chi-squared test with Holm’s post hoc correction: ns, not significant; *, p < 0.05. (B) Fluorescence staining of transversal sections of platyfish hearts. Fibronectin-positive wound (red) contrasts with intact myocardium labeled by F-actin (green). Sham ventricles at 30 days post-thoracotomy serve as control. (C-D) Quantification of regeneration based on wound size and fibronectin deposition, as represented in (B). Kruskal-Wallis test followed by Dunn’s test with Holm’s post-hoc correction. Adjusted p-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. For C and D, n: Sham: 24; 7 dpci: 24; 14 dpci: 22; 30 dpci: 32; 60 dpci: 13; 90 dpci: 12. For E, n: Sham: 24; 7 dpci: 15; 14 dpci: 20; 30 dpci: 17.

Comparison of bulk-RNA sequencing between zebrafish and platyfish reveals different tissue responses to cryoinjury.

(A-B) Volcano plots of transcriptomes in zebrafish (A) and platyfish (B) cryoinjured ventricles at 7 dpci, compared to uninjured control. The log2 fold change (log2FC) values show the ratio of gene transcript abundance in cryoinjured versus uninjured conditions. An adjusted p-value (padj) threshold of less than 0.05 was used to identify genes showing significant changes in transcript levels (decreased in blue and increased in orange). (C) Scatter plot comparing the log2FC of the two RNA-seq analyses. Green represents gene transcripts that are more abundant in platyfish than in zebrafish upon cryoinjury, while purple represents the opposite situation. Highlighted genes are at least twice as abundant after cryoinjury in one species than the other. (D) A comparison of the Gene Set Enrichment Analysis obtained from the two species. Color intensity reflects the statistical significance of reduction (blue) or enrichment (orange) at 7 dpci, compared to uninjured control. Dot size corresponds to the magnitude of difference for each gene set. Selected gene sets have been organized into broader functional categories. (E) Transcript abundance comparison of orthologous genes between conditions (uninjured, dark color vs. cryoinjured, light color) and species (zebrafish, ZF, purple vs. platyfish, PF, green). Each point in the bar plot corresponds to the DESeq2-based TPM-like values (so-called Normalized read counts, see Methods) of the gene in one replicate. The log2FC and -log10(padj) are those shown in (A) and (B). The orthologue genes are grouped into general categories.

Delayed recruitment of immune cells during platyfish heart repair.

(A) A comparison of the abundance of macrophage-specific transcripts. For details, see legend to Figure 4 and Methods. (B-C) Immunofluorescence analysis of neutrophils (Mpx-positive cells, B) and phagocytes (L-plastin-positive cells, C) following cryoinjury at 7, 14, and 30 dpci. Control sections are from uninjured ventricles of sham-operated fish at each time point. The sham image corresponds to 30 dpt. (D-E) Quantification of Mpx-positive area (D) and L-plastin-positive area (E), corresponding to the images representatively shown in B and C. In the cryoinjured ventricles (CI), quantifications were assessed separately in the intact myocardium (CI: Intact) and the wounded area (CI: Wound). Statistical comparisons of Sham vs. CI: Intact were done with unpaired Wilcoxon tests, as different animals were in each group, whereas comparisons between the intact myocardium and the wound of the same cryoinjured hearts were based on a paired Wilcoxon test. Holm’s post-hoc correction was applied for multiple comparisons. Adjusted p-value: * P< 0.05, ** P< 0.01. (F-G) Recruitment kinetics of neutrophils (grey, left Y-axis) and phagocytes (purple, right Y-axis) in the intact myocardium (F) and wounds (G) of ventricles at 7, 14, and 30 dpci. ‘Sham’ corresponds to all data from the sham-operated ventricle at different time points and represents the baseline. Kruskal-Wallis statistical tests were used to compare the different time points for each marker.

Ventricular cryoinjuries trigger transient activation of cardiomyocytes.

(A) Schematic illustration of the three analyzed areas: tropomyosin-negative wounded area, a border zone myocardium within 100 μm from the injury border, and a remote myocardium distant from the border zone. (B) Comparison of the abundance of orthologous transcripts defined as markers of CM proliferation and dedifferentiation. For further details, see legend to Figure 4 and Methods. (C) PCNA localization analysis of sham-operated platyfish (7 dpt and 14 dpt) and cryoinjured platyfish (7 dpci and 14 dpci). The orange frame of the top images depicts the area magnified in the middle images. The bottom images show the same magnification, displaying only PCNA. bz: border zone. (D) Quantification of proliferating cells in the Tropomyosin-positive myocardium at different time points and different regions of the heart. Sham vs. CI: Remote Myoc. and CI: Remote Myoc. vs. CI: Border zone Myoc. were compared using unpaired and paired Wilcoxon tests, respectively, followed by Holm’s post-hoc correction for multiple comparisons. Adjusted p-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (E-F) BrdU labeling of zebrafish (E) and platyfish (F) for 23 days (from day 7 to 30 days post-surgery) and 27 days (from day 3 to 30 post-surgery), respectively. Ventricular sections were counterstained with DAPI and Tropomyosin. In zebrafish, the initial wounded area is defined based on myocardium appearance and the high concentration of BrdU-positive nuclei. The myocardium bridge (m.b.) is shown with the double-ended arrow. A myocardial bridge is not observed in platyfish. For C, E, and F, dashed red lines encircle the outer border wound, whereas dashed yellow lines surround the border zone (bz).