Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes

Abstract

Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life. Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart.

Figure 1. Cardiomyocytes marked by gata4:EGFP are activated by injury and proliferate at the injury site

a, gata4:EGFP is induced throughout cells in the compact muscle by 7 dpa (right). b, c, gata4:EGFP (arrowheads in (c)) co-labels with cardiomyocyte markers Mef2 (b) and Myosin heavy chain (MHC) (c). d, gata4:EGFP (arrowheads) co-stained with β-catenin, indicating EGFP restriction within the myocardial wall. e, gata4:EGFP does not co-localize with epicardial Raldh2 protein (arrowheads). f–i, gata4:EGFP expression (arrowheads) in uninjured and regenerating ventricles. Dotted line indicates approximate plane of resection. j, k, BrdU labeling and immunofluorescence of 7 (j) and 14 (k) dpa gata4:EGFP ventricles. Arrows indicate co-labeling. (b–e, insets in j and k): single confocal slices. (j, k): confocal projections of 7 (j) or 8 µm (k) z-stacks. Scale bars, 50 µm.

Figure 2. Major contribution of gata4⁺ cardiomyocytes to heart regeneration

a, gata4:ERCreER; β-act2:RSG animals injected once daily with vehicle (left) or 4-HT at 5–7 dpa, before collection at 9 (center; n = 5) or 14 dpa (right; n = 6). EGFP fluorescence (arrows) is observed at injury borders by 9 dpa and within the injury site by 14 dpa. b, cmlc2:CreER; β-act2:RSG animals were injected prior to injury with vehicle (left) or 4-HT once daily for 3 days. 4-HT labeled the vast majority of cardiomyocytes in uninjured ventricles (center) and 30 dpa regenerates (right; n = 10). (Inset) DsRed channel used for calculation of labeling efficiency. Single confocal slices shown. Scale bars, 50 µm.

Figure 3. Electrical coupling of regenerated cardiomyocytes

a, Two-millisecond isochronal density maps of surface myocardium near the apex of explants of uninjured, 7, 14, and 30 dpa ventricles. b, Mean conduction velocities measured from local velocity vectors, indicating slowed velocities at 7 and 14 dpa. Mean ± s.e.m, n = 4 to 7 ventricles for each timepoint. One-way ANOVA, *P < 0.05. c, Representative traces of surface action potentials, indicating a slowing of the maximum depolarization rate at 7 dpa. Scale bar, 100 µm.

Figure 4. Restoration of gata4:EGFP expression and a new ventricular wall after scarring

a, b, Typical wound after resection and 30 days Fgfr inhibition. c, d, gata4:EGFP fluorescence (arrowheads) induced by a regimen of injury, 30 days Fgfr inhibition (heat-shock, HS), and 14 days recovery at room temperature (RT; n = 8). Dotted line indicates wound. e, f, Normal regeneration (brackets) after injury and RT incubation for 60 days. g, h, 60 days RT recovery, following resection and 30 days Fgfr inhibition, improves wall anatomy (brackets) without removing scar (arrowheads; n = 10). Green = EGFP-tagged Dnfgfr1; red = myocyte nuclei via cmlc2:nucDsRed2 transgene in (a, e, g). Acid fuchsin Orange G stain (red = fibrin, blue = collagen) in (b, f, h). Animals were heat-shocked 4 hours before heart collection in (e, g), inducing EGFP. Scale bars, 50 µm.