Heart repair and regeneration: recent insights from zebrafish studies

Abstract

Cardiovascular disease is the leading cause of death in the U.S. and worldwide. Failure to properly repair or regenerate damaged cardiac tissues after myocardial infarction is a major cause of heart failure. In contrast to humans and other mammals, zebrafish hearts regenerate after substantial injury or tissue damage. Here, we review recent progress in studying zebrafish heart regeneration, addressing the molecular and cellular responses in the three tissue layers of the heart: myocardium, epicardium, and endocardium. We also compare different injury models utilized to study zebrafish heart regeneration and discuss the differences in responses to injury between mammalian and zebrafish hearts. By learning how zebrafish hearts regenerate naturally, we can better design therapeutic strategies for repairing human hearts after myocardial infarction.

Figure 1. Summary of events during zebrafish heart regeneration

The process begins at amputation; time points of 3 hours post amputation (hpa) and 1–3, 7, 14 and 30 days post amputation (dpa) are illustrated. The amputation plane is marked by a red dashed line. Right after amputation, a blood clot (red) forms. Within hours, the endocardium (brown) is activated and shows morphological and gene expression changes. At 1–3 dpa, the blood clot becomes a fibrin clot (yellow). The activated raldh2 expression in endocardium becomes localized to the injury site (brown). At the same time, the epicardium (blue) is activated and expresses embryonic markers. At 7 dpa, the epicardium encloses the apex and starts to invade the fibrin clot while a population of gata4:EGFP positive cardiomyocytes appears at the sub-epicardium and begins to proliferate. At 14 dpa, the gata4:EGFP positive cardiomyocytes localize to the apex and newly formed blood vessels vascularize the newly formed myocardium. By 30 dpa, the myocardium is almost fully regenerated. The new blood vessels vascularize the new myocardium. A, atrium; V, ventricle; BA, bulbus arteriosus.

Figure 2. Summary of the timing of events during zebrafish heart regeneration

dpa, days post amputation; dpc, days post cryoinjury. Time course of events are marked with horizontal bars. The relative level of activity is shown as a gradient; darker colors indicate stronger activity. The peak of activity is marked with triangles.

Figure 3. Strategy for Cre-lox based lineage tracing of the source of newly formed cardiomyocytes during zebrafish heart regeneration

Cre recombinase coding sequence is fused to the estrogen receptor coding sequence (cre-ert2) to make an inducible Cre protein (orange), which is then induced using the estrogen agonist 4-OHT (brown) prior to amputation; this fusion protein is specifically expressed in pre-existing cardiomyocytes using the cardiacmyosin light chain 2 (cmlc2) promoter. Expression of a gfp reporter (green) is driven by the constitutive β-actin promoter, but expression is blocked by a stop sequence (STOP) flanked byloxP sites. A. Expected result if newly regenerated cardiomyocytes are derived from existing cardiomyocytes. The stop sequence is specifically excised via Cre-mediated site-specific recombination in the cardiomyocyte lineage when 4-OHT is added and gfp is therefore expressed in newly formed cardiomyocytes (this is the result reported in ref (32, 33)), even if the cardiomyocytes transiently dedifferentiate into a non-cmlc expressing cell. B. Theoretical result if newly regenerated cardiomyocytes were derived from stem/progenitor cells. Since these cells would not express cmlc2-driven cre-ert-2 prior to amputation, 4-OHT would have no effect on the stop sequence, which would therefore remain intact and continue to block gfp expression in this lineage, even after differentiation to form new cardiomyocytes. The blue line indicates that there is no expression from the promoter.