Cardiac regenerative capacity and mechanisms.

The heart holds the monumental yet monotonous task of maintaining circulation. Although cardiac function is critical to other organs and to life itself, mammals are not equipped with significant natural capacity to replace heart muscle that has been lost by injury. This deficiency plays a role in leaving millions worldwide vulnerable to heart failure each year. By contrast, certain other vertebrate species such as zebrafish are strikingly good at heart regeneration. A cellular and molecular understanding of endogenous regenerative mechanisms and advances in methodology to transplant cells together project a future in which cardiac muscle regeneration can be therapeutically stimulated in injured human hearts. This review focuses on what has been discovered recently about cardiac regenerative capacity and how natural mechanisms of heart regeneration in model systems are stimulated and maintained.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Pharmacological Enhancement of Regeneration-Dependent Regulatory T Cell Recruitment in Zebrafish.

Regenerative capacity varies greatly between species. Mammals are limited in their ability to regenerate damaged cells, tissues and organs compared to organisms with robust regenerative responses, such as zebrafish. The regeneration of zebrafish tissues including the heart, spinal cord and retina requires foxp3a+ zebrafish regulatory T cells (zTregs). However, it remains unclear whether the muted regenerative responses in mammals are due to impaired recruitment and/or function of homologous mammalian regulatory T cell (Treg) populations. Here, we explore the possibility of enhancing zTreg recruitment with pharmacological interventions using the well-characterized zebrafish tail amputation model to establish a high-throughput screening platform. Injury-infiltrating zTregs were transgenically labelled to enable rapid quantification in live animals. We screened the NIH Clinical Collection (727 small molecules) for modulators of zTreg recruitment to the regenerating tissue at three days post-injury. We discovered that the dopamine agonist pramipexole, a drug currently approved for treating Parkinson's Disease, specifically enhanced zTreg recruitment after injury. The dopamine antagonist SCH-23390 blocked pramipexole activity, suggesting that peripheral dopaminergic signaling may regulate zTreg recruitment. Similar pharmacological approaches for enhancing mammalian Treg recruitment may be an important step in developing novel strategies for tissue regeneration in humans.

Myocardial NF-κB activation is essential for zebrafish heart regeneration.

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 of gata4 regulatory 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 of gata4 regulatory 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.

Transcriptional components of anteroposterior positional information during zebrafish fin regeneration.

Many fish and salamander species regenerate amputated fins or limbs, restoring the size and shape of the original appendage. Regeneration requires that spared cells retain or recall information encoding pattern, a phenomenon termed positional memory. Few factors have been implicated in positional memory during vertebrate appendage regeneration. Here, we investigated potential regulators of anteroposterior (AP) pattern during fin regeneration in adult zebrafish. Sequence-based profiling from tissues along the AP axis of uninjured pectoral fins identified many genes with region-specific expression, several of which encoded transcription factors with known AP-specific expression or function in developing embryonic pectoral appendages. Transgenic reporter strains revealed that regulatory sequences of the transcription factor gene alx4a activated expression in fibroblasts and osteoblasts within anterior fin rays, whereas hand2 regulatory sequences activated expression in these same cell types within posterior rays. Transgenic overexpression of hand2 in all pectoral fin rays did not affect formation of the proliferative regeneration blastema, yet modified the lengths and widths of regenerating bones. Hand2 influenced the character of regenerated rays in part by elevation of the vitamin D-inactivating enzyme encoded by cyp24a1, contributing to region-specific regulation of bone metabolism. Systemic administration of vitamin D during regeneration partially rescued bone defects resulting from hand2 overexpression. Thus, bone-forming cells in a regenerating appendage maintain expression throughout life of transcription factor genes that can influence AP pattern, and differ across the AP axis in their expression signatures of these and other genes. These findings have implications for mechanisms of positional memory in vertebrate tissues.

Zebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function.

Second heart field (SHF) progenitors perform essential functions during mammalian cardiogenesis. We recently identified a population of cardiac progenitor cells (CPCs) in zebrafish expressing latent TGFß-binding protein 3 (ltbp3) that exhibits several defining characteristics of the anterior SHF in mammals. However, ltbp3 transcripts are conspicuously absent in anterior lateral plate mesoderm (ALPM), where SHF progenitors are specified in higher vertebrates. Instead, ltbp3 expression initiates at the arterial pole of the developing heart tube. Because the mechanisms of cardiac development are conserved evolutionarily, we hypothesized that zebrafish SHF specification also occurs in the ALPM. To test this hypothesis, we Cre/loxP lineage traced gata4(+) and nkx2.5(+) ALPM populations predicted to contain SHF progenitors, based on evolutionary conservation of ALPM patterning. Traced cells were identified in SHF-derived distal ventricular myocardium and in three lineages in the outflow tract (OFT). We confirmed the extent of contributions made by ALPM nkx2.5(+) cells using Kaede photoconversion. Taken together, these data demonstrate that, as in higher vertebrates, zebrafish SHF progenitors are specified within the ALPM and express nkx2.5. Furthermore, we tested the hypothesis that Nkx2.5 plays a conserved and essential role during zebrafish SHF development. Embryos injected with an nkx2.5 morpholino exhibited SHF phenotypes caused by compromised progenitor cell proliferation. Co-injecting low doses of nkx2.5 and ltbp3 morpholinos revealed a genetic interaction between these factors. Taken together, our data highlight two conserved features of zebrafish SHF development, reveal a novel genetic relationship between nkx2.5 and ltbp3, and underscore the utility of this model organism for deciphering SHF biology.

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

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.

Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.

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.

tcf21+ epicardial cells adopt non-myocardial fates during zebrafish heart development and regeneration.

Recent lineage-tracing studies have produced conflicting results about whether the epicardium is a source of cardiac muscle cells during heart development. Here, we examined the developmental potential of epicardial tissue in zebrafish during both embryonic development and injury-induced heart regeneration. We found that upstream sequences of the transcription factor gene tcf21 activated robust, epicardium-specific expression throughout development and regeneration. Cre recombinase-based, genetic fate-mapping of larval or adult tcf21(+) cells revealed contributions to perivascular cells, but not cardiomyocytes, during each form of cardiogenesis. Our findings indicate that natural epicardial fates are limited to non-myocardial cell types in zebrafish.

The regenerative capacity of zebrafish reverses cardiac failure caused by genetic cardiomyocyte depletion.

Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.

HOPX-associated molecular programs control cardiomyocyte cell states underpinning cardiac structure and function.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.

A dual role for ErbB2 signaling in cardiac trabeculation.

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Regulatory T cells regulate blastemal proliferation during zebrafish caudal fin regeneration.

The role of T cells in appendage regeneration remains unclear. In this study, we revealed an important role for regulatory T cells (Tregs), a subset of T cells that regulate tolerance and tissue repair, in the epimorphic regeneration of zebrafish caudal fin tissue. Upon amputation, fin tissue-resident Tregs infiltrate into the blastema, a population of progenitor cells that produce new fin tissues. Conditional genetic ablation of Tregs attenuates blastemal cell proliferation during fin regeneration. Blastema-infiltrating Tregs upregulate the expression of igf2a and igf2b, and pharmacological activation of IGF signaling restores blastemal proliferation in Treg-ablated zebrafish. These findings further extend our understandings of Treg function in tissue regeneration and repair.

Zebrafish Heme Oxygenase 1a Is Necessary for Normal Development and Macrophage Migration.

Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes is known to be responsive to oxidative stress suggesting a conserved physiological function. In this study, we generate a knockout allele of zebrafish hmox1a and characterize the effects of hmox1a and hmox1b loss on embryonic development. We find that loss of hmox1a or hmox1b causes developmental defects in only a minority of embryos, in contrast to Hmox1 gene deletions in mice that cause loss of most embryos. Using a tail wound inflammation assay we find a conserved role for hmox1a, but not hmox1b, in normal macrophage migration to the wound site. Together our results indicate that zebrafish hmox1a has clearly a partitioned role from hmox1b that is more consistent with conserved functions of mammalian Heme oxygenase 1.

Haem oxygenase limits Mycobacterium marinum infection-induced detrimental ferrostatin-sensitive cell death in zebrafish.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary cofactor for host immunity. Haem oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of haem. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here, we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish haem oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from M. marinum infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against M. marinum infection by reducing infection-induced iron accumulation and ferrostatin-sensitive cell death.

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish.

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

IL-7 receptor signaling is necessary for stage transition in adult B cell development through up-regulation of EBF.

Cytokine receptor signals have been suggested to stimulate cell differentiation during hemato/lymphopoiesis. Such action, however, has not been clearly demonstrated. Here, we show that adult B cell development in IL-7(-/-) and IL-7R alpha(2/-) mice is arrested at the pre-pro-B cell stage due to insufficient expression of the B cell-specific transcription factor EBF and its target genes, which form a transcription factor network in determining B lineage specification. EBF expression is restored in IL-7(-/-) pre-pro-B cells upon IL-7 stimulation or in IL-7R alpha(-/-) pre-pro-B cells by activation of STAT5, a major signaling molecule downstream of the IL-7R signaling pathway. Furthermore, enforced EBF expression partially rescues B cell development in IL-7R alpha(-/-) mice. Thus, IL-7 receptor signaling is a participant in the formation of the transcription factor network during B lymphopoiesis by up-regulating EBF, allowing stage transition from the pre-pro-B to further maturational stages.

Cardiac Resection Injury in Zebrafish.

The zebrafish (Danio rerio) possesses a spectacular capacity for cardiac regeneration. Zebrafish have been used in cardiac regeneration research for nearly two decades, contributing to the identification of signals and cellular mechanisms as potential targets for human heart repair. Investigations into cardiac regeneration in zebrafish have been facilitated by multiple methods of inducing cardiac tissue damage. Among the established methods, cardiac resection injury is a relatively simple, yet robust approach traditionally used to induce cardiac tissue damage in a reproducible manner. Here, we describe a detailed protocol to perform a cardiac resection injury in adult zebrafish and discuss potential complications for researchers who are new to this technique.

Krüppel-like factor 1 is a core cardiomyogenic trigger in zebrafish.

Cardiac regeneration requires dedifferentiation and proliferation of mature cardiomyocytes, but the mechanisms underlying this plasticity remain unclear. Here, we identify a potent cardiomyogenic role for Krüppel-like factor 1 (Klf1/Eklf), which is induced in adult zebrafish myocardium upon injury. Myocardial inhibition of Klf1 function does not affect heart development, but it severely impairs regeneration. Transient Klf1 activation is sufficient to expand mature myocardium in uninjured hearts. Klf1 directs epigenetic reprogramming of the cardiac transcription factor network, permitting coordinated cardiomyocyte dedifferentiation and proliferation. Myocardial expansion is supported by Klf1-induced rewiring of mitochondrial metabolism from oxidative respiration to anabolic pathways. Our findings establish Klf1 as a core transcriptional regulator of cardiomyocyte renewal in adult zebrafish hearts.