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The human heart is continually operating as a muscular pump, contracting, on average, 80 times per minute to propel 8000 liters of blood through body tissues each day. Whereas damaged skeletal muscle has a profound capacity to regenerate, heart muscle, at least in mammals, has poor regenerative potential. This deficiency is attributable to the lack of resident cardiac stem cells, combined with roadblocks that limit adult cardiomyocytes from entering the cell cycle and completing division. Insights for regeneration have recently emerged from studies of animals with an elevated innate capacity for regeneration, the innovation of stem cell and reprogramming technologies, and a clearer understanding of the cardiomyocyte genetic program and key extrinsic signals. Methods to augment heart regeneration now have potential to counteract the high morbidity and mortality of cardiovascular disease.

Heart failure is a major source of morbidity and mortality. Replacing lost myocardium with new tissue is a major goal of regenerative medicine. Unlike adult mammals, zebrafish and neonatal mice are capable of heart regeneration following cardiac injury. In both contexts, the regenerative program echoes molecular and cellular events that occur during cardiac development and morphogenesis, notably muscle creation through division of cardiomyocytes. Based on studies over the past decade, it is now accepted that the adult mammalian heart undergoes a low grade of cardiomyocyte turnover. Recent data suggest that this cardiomyocyte turnover can be augmented in the adult mammalian heart by redeployment of developmental factors. These findings and others suggest that stimulating endogenous regenerative responses can emerge as a therapeutic strategy for human cardiovascular disease.

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program. Heart attacks—which are a major cause of death in humans—occur when a blocked blood vessel stops blood from flowing to the heart. This causes many heart muscle cells to die, which can result in permanent damage that makes survivors more susceptible to heart failure in the future. A major goal of regenerative medicine is to develop therapies that can improve the recovery of heart muscle cells after a heart attack and restore normal heart activity to patients with heart failure. Unlike the human heart, the heart of an adult zebrafish is able to regenerate even after extensive damage. After an injury, the remaining heart muscle cells divide to replace the lost heart muscle, but it is not clear how this works. A protein called Neuregulin1 (or Nrg1 for short) can stimulate heart muscle cells to divide. Gemberling et al. investigated the role of this protein in the regeneration of the heart in adult zebrafish. The experiments show that when the heart is injured, the gene encoding the Nrg1 protein is switched on in cells of the outer layer of the heart wall. When Nrg1 is deliberately activated in uninjured adult zebrafish hearts, it causes the muscle cells to divide, leading to many new layers of heart muscle forming over the course of several weeks. Along with promoting cell division, Nrg1 also makes the heart muscle cells return to an immature state more like stem cells. Gemberling et al. found that Nrg1 also supports regeneration of the heart by changing the environment surrounding the muscle cells. For example, it stimulates the growth of new blood vessels and recruits non-muscle cells to the injury site. Gemberling et al.'s findings demonstrate that Nrg1 is sufficient to induce the growth of heart muscle growth in an adult animal, even in the absence of injury. To develop its therapeutic potential, future work will also need to identify how the gene that encodes Nrg1 is switched on by injury and identify the other molecules that interact with Nrg1.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals. Zebrafish can regenerate after paralyzing spine injuries and regain locomotor ability, unlike mammals. Here authors show that the neurogenic factor Hb-egf promotes spinal cord regeneration in zebrafish and is regulated by an enhancer that can similarly direct expression in the pro-regenerative setting of neonatal mice.

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b ( lepb ) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb -linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for ‘tissue regeneration enhancer elements’ (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs. An injury-dependent enhancer element is identified that activates gene expression in regenerating zebrafish tissues and can be engineered into DNA constructs that increase tissue regenerative capacity; the element is also active in injured mouse tissue. Identification of a tissue regeneration enhancer Ken Poss and colleagues identify an injury-dependent enhancer element that activates gene expression in regenerating zebrafish tissues. They find that the element, which they term a 'tissue regeneration enhancer element' (TREE), is divisible into tissue-specific modules that can each direct expression in zebrafish hearts or fins. The identified element can be used to direct the expression of pro- or anti-regenerative factors in zebrafish tissues and thus control the efficiency of regeneration. Finally, by engineering TREEs upstream of mitogenic factor genes, the authors demonstrate their ability to boost tissue repair in injured mouse tissue.

Heart regeneration offers a novel therapeutic strategy for heart failure. Unlike mammals, lower vertebrates such as zebrafish mount a strong regenerative response following cardiac injury. Heart regeneration in zebrafish occurs by cardiomyocyte proliferation and reactivation of a cardiac developmental program, as evidenced by induction ofgata4regulatory sequences in regenerating cardiomyocytes. Although many of the cellular determinants of heart regeneration have been elucidated, how injury triggers a regenerative program through dedifferentiation and epicardial activation is a critical outstanding question. Here, we show that NF-κB signaling is induced in cardiomyocytes following injury. Myocardial inhibition of NF-κB activity blocks heart regeneration with pleiotropic effects, decreasing both cardiomyocyte proliferation and epicardial responses. Activation ofgata4regulatory sequences is also prevented by NF-κB signaling antagonism, suggesting an underlying defect in cardiomyocyte dedifferentiation. Our results implicate NF-κB signaling as a key node between cardiac injury and tissue regeneration.

Spinal cord injury is a devastating condition in which massive cell death and disruption of neural circuitry lead to long-term chronic functional impairment and paralysis. In mammals, spinal cord tissue has minimal capacity to regenerate after injury. In stark contrast, the regeneration of a completely transected spinal cord and accompanying reversal of paralysis in adult zebrafish is arguably one of the most spectacular biological phenomena in nature. Here, we review reports from the last decade that dissect the mechanisms of spinal cord regeneration in zebrafish. We highlight recent progress as well as areas requiring emphasis in a line of study that has great potential to uncover strategies for human spinal cord repair.

Certain lower vertebrates like zebrafish activate proliferation of spared cardiomyocytes after cardiac injury to regenerate lost heart muscle. Here, we used translating ribosome affinity purification to profile translating RNAs in zebrafish cardiomyocytes during heart regeneration. We identified dynamic induction of several Jak1/Stat3 pathway members following trauma, events accompanied by cytokine production. Transgenic Stat3 inhibition in cardiomyocytes restricted injury-induced proliferation and regeneration, but did not reduce cardiogenesis during animal growth. The secreted protein Rln3a was induced in a Stat3-dependent manner by injury, and exogenous Rln3 delivery during Stat3 inhibition stimulated cardiomyocyte proliferation. Our results identify an injury-specific cardiomyocyte program essential for heart regeneration.

While the heart regenerates poorly in mammals, efficient heart regeneration occurs in zebrafish. Studies in zebrafish have resulted in a model in which preexisting cardiomyocytes dedifferentiate and reinitiate proliferation to replace the lost myocardium. To identify which processes occur in proliferating cardiomyocytes we have used a single-cell RNA-sequencing approach. We uncovered that proliferating border zone cardiomyocytes have very distinct transcriptomes compared to the nonproliferating remote cardiomyocytes and that they resemble embryonic cardiomyocytes. Moreover, these cells have reduced expression of mitochondrial genes and reduced mitochondrial activity, while glycolysis gene expression and glucose uptake are increased, indicative for metabolic reprogramming. Furthermore, we find that the metabolic reprogramming of border zone cardiomyocytes is induced by Nrg1/ErbB2 signaling and is important for their proliferation. This mechanism is conserved in murine hearts in which cardiomyocyte proliferation is induced by activating ErbB2 signaling. Together these results demonstrate that glycolysis regulates cardiomyocyte proliferation during heart regeneration. Heart attacks are a common cause of death in the Western world. During a heart attack, oxygen levels in the affected part of the heart decrease, which causes heart muscle cells to die. In humans the dead cells are replaced by a permanent scar that stabilizes the injury but does not completely heal it. As a result, individuals have a lower quality of life after a heart attack and are more likely to die from a subsequent attack. Unlike humans, zebrafish are able to regenerate their hearts after injury: heart muscle cells close to a wound divide to produce new cells that slowly replace the scar tissue and restore normal function to the area. It remains unclear, however, what stimulates the heart muscle cells of zebrafish to start dividing. To address this question, Honkoop, de Bakker et al. used a technique called single-cell sequencing to study heart muscle cells in wounded zebrafish hearts. The experiments identified a group of heart muscle cells close to the site of the wound that multiplied to repair the damage. This group of cells had altered their metabolism compared to other heart muscle cells so that they relied on a pathway called glycolysis to produce the energy and building blocks they needed to proliferate. Blocking glycolysis impaired the ability of the heart muscle cells to divide, indicating that this switch is necessary for the heart to regenerate. Further experiments showed that a signaling cascade, which includes the molecules Nrg1 and ErbB2, induces heart muscle cells in both zebrafish and mouse hearts to switch to glycolysis and undergo division. These findings indicate that activating glycolysis in heart muscle cells may help to stimulate the heart to regenerate after a heart attack or other injury. The next step following on from this work is to develop methods to activate glycolysis and promote cell division in injured hearts.

Gene regulatory mechanisms that underlie tissue regeneration have been largely studied at the level of transcription. Here, proximity labeling methods identify increased presence of the RNA helicase and P-body marker Ddx61 in adult zebrafish cardiomyocytes induced to divide by injury or mitogens. Ddx61 molecules form complex condensates in cardiomyocytes during cardiogenic settings in zebrafish, developing mice, and cultured human cells. ddx61 mutations disrupt cardiomyocyte proliferation and heart regeneration indices in adult zebrafish, and DDX6 knockdown reduces proliferation of cultured human cardiomyocytes. During heart regeneration, Ddx61 associates with and is required to restrain expression of mRNA encoding Chordin, a secreted BMP inhibitor that impedes regeneration if present at high levels. Our experiments indicate that mRNA sorting by context-dependent condensates can impact tissue regenerative capacity. Pronobis et al . show that Ddx61 localizes to P-bodies and regulates heart muscle proliferation during cardiac regeneration in zebrafish. Ddx61 is required is required to restrain expression of Chordin, a secreted BMP inhibitor that impedes regeneration if present at high levels.